Studies in Natural Product Chemistry, Volume 25: Bioactive Natural Products, Part F

Studies in Natural Products Chemistry Volume 25 Bioactive Natural Products (Part F)

studies in Natural Products Chemistry edited by Atta-ur-Raliman

Vol. 1 Vol. 2 Vol. 3 Vol. 4 Vol. 5 Vol. 6 Vol. 7 Vol. 8 Vol. 9 Vol. 10 Vol. 11 Vol. 12 Vol. 13 Vol. 14 Vol. 15 Vol. 16 Vol. 17 Vol. 18 Vol. 19 Vol. 20 Vol. 21 Vol. 22 Vol. 23 Vol. 24 Vol. 25

Stereoselective Synthesis (Part A) Structure Elucidation (Part A) Stereoselective Synthesis (Part B) Stereoselective Synthesis (Part C) Structure Elucidaton (Part B) Stereoselective Synthesis (Part D) Structure and Chemistry (Part A) Stereoselective Synthesis (Part E) Structure Chemistry (Part B) Stereoselective Synthesis (Part F) Stereoselective Synthesis (Part G) Stereoselective Synthesis (Part H) Bloactive Natural Products (Part A) Stereoselective Synthesis (Part I) Structure and Chemistry (Part C) Stereoselective Synthesis (Part J) Structure and Chemistry (Part D) Stereoselective Synthesis (Part K) Structure and Chemistry (Part E) Structure and Chemistry (Part F) Bloactive Natural Products (Part B) Bloactive Natural Products (Part C) Bloactive Natural Products (Part D) Bloactive Natural Products (Part E) Bloactive Natural Products (Part F)

Studies in Natural Products Chemistry Volume 25 Bioactive Natural Products (Part F)

Edited by

Atta-ur-Rahman H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

2001 ELSEVIER Amsterdam - London - New York - Oxford - Paris - Shannon - Tokyo

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ISBN:

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FOREWORD The present volume of Studies in Natural Product Chemistry covers many important classes of bioactive natural products. The article on bloactive indole alkaloids by Reija Jokela et. al., describes the acid-catalysed epimerization of these compounds and resulting pharmacological activities. Toshihiro Akihisa & Ken Yasukawa present the antitumor-promoting and anti-inflammatory activities of triterpenoids and sterols from plants and fungi, many of which are of medicinally importance. A number of articles on bioactive terpenes Include oleanene glucuronides (James Kinjo et. al.,) biotransformation of terpenoids by microorganisms (Jan C.R. Demyttenaere), cycloartane and oleanane saponins (Luisella Yerotta et, al.,) and labdane-type diterpenes (Cost as Demetzoo et. al.,) as well as metabolism of the tomato saponin alpha-tomatine by phytopathogenic fungi (Manuel Ruiz-Rubio et. al.,). An interesting article has been written by James Zlegler, et. al.,) on heme aggregation inhibitors. Intercellular communication in higher plants continues to be of interest and in addition to the known phytoharmones i.e. auxins, cytokinins, gibberellins, abscisic acid and ethylene, certain bioactive peptides have been identified in signal transductions involved In plant defence, growth and development which is discussed by Andreas Schaller. The articles on the biosynthesis of brassinosteroids by Jochen Winter, immunopotentlating effects of a glycoprotein from Chlorella vulgaris by Kuniaki Tanaka et. al. and hepatoprotective plants components by Shiegeoshi Kadota et. al., should also be of considerable interest. The induction and regulation of biosynthetic activity of phytoalexins in carrot cells is reviewed by Fumiya Kurosaki. The plant inhibition of eukaryote signal transduction induced by secondary metabolites is another important area which is comprehensively described by Gideon M. Polya. Two articles written by Juan Duarte et. al., & J. Galvez et. al., cover the important effects offlavonoidson cardiovascular and gastrointestinal disorders respectively, while the bioactivity of phenolic componds in higher plants is described by Juan M. Ruiz et. al. Marine substances continue to be important sources of bioactive natural products. Recent advances in marine natural products are covered by M.J. Abad et. al., in detail. Biologically active halogen compounds described by Gerhard Laus and marine sulfurcontaining natural products reviewed by Carolos Jimenez and biological activities chlorogenic acids discussed by Motoyo Ohnishi et. al., should also be of interest to the readers. Ethanolic extracts of Crocus sativus and Its components in learning behaviour and long term potentiation of memory processes is another recent area of interest which has been reviewed by Y. Shoyama et. al.,). It is hoped that Volume-25 of "Studies in Natural Products Chemistry" would prove to be of considerable interest to the readers and represent important another additions this growing series books natural product chemistry. I would like to express my thanks to Dr. Shakeel Ahmad for his assistance in the preparation of the index. I am also grateful to Mr. Muhammad Asif and Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.

Atta-ur-Rahman

Ph.D. (Cantab), SaD. (Cantab)

April, 2001

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PREFACE When Professor Atta-ur-Rahman informed me, many years ago, of his desire to launch a new series which would become 'Studies in Natural Products Chemistry', I enthusiastically encouraged him to do so but pointed out what a difficult task it would be. First, the expert authors able to write the very numerous chapters of such a work would have to be found. Then, a logical sequence to the series would have to be determined. Finally, because a large number of books, reviews etc. already exist which dealt with particular areas of the chemistry, biology and therapeutic applications of natural products (in particular, Sir Derek Barton's posthumous 'Comprehensive Natural Products Chemistry'), something new would have to be offered. So we have now arrived at Volume 25 and I am very pleased to see that our colleague Atta-ur-Rahman has once more realised a Herculean feat. Taken together, this still uncompleted work Is colossal In breadth and, especially, extremely useful for ail scientists, not only those working in the natural products field. At a time when much (too much?) is being said about combinatorial chemistry, here we are face-to-face with part (and only a small part at that) of the products of the Creator's own combinatorial chemistry, which, it is believed, took 4.8 billion years to produce. Thus, sincere thanks are due to our colleague Atta-ur-Rahman who has since become Minister of Science and Technology in his mother country, Pakistan. And heartfelt thanks also to the authors of this Volume 25 as well as of ail preceding volumes for the considerable contribution they have made to our entire scientific community. And we now impatiently await the coming volumes. P.Potier April. 2001

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ix

CONTENTS Foreword

v

Preface

vii

Contributors

xi

Acid-catalysed epimerization of bioactive indole alkaloids and their derivatives

M. BERNER, A. TOLVANEN AND R. JOKELA

3

Antitumor-promoting and anti-inflammatory activities of triterpenoids and sterols fi'om plants and fungi T. AKIHISA AND K. YASUKAWA

43

Bioactive oleanene glucuronides obtained from fabaceous plants J. KINJO AND T. NOHARA

89

Biotransformation of terpenoids by microorganisms JAN C.R. DEMYTTENAERE

125

Cycloartane and oleanane saponins from Astragalus sp. L. VEROTTA AND N.A. EL-SEBAKHY

179

Labdane-type diterpenes: Chemistry and biological activity C. DEMETZOS AND K.S. DIMAS

235

Metabolism of the tomato saponin a-tomatine by phytopathogenic fungi M. RUIZ-RUBIO, A. PEREZ-ESPINOSA, K. LAIRINl, T. ROLDAN-ARJONA, A. DIPIETRO AND N. ANAYA

293

Heme aggregation inhibitors: Antimalarial drugs targeting an essential biomineralization process J. ZIEGLER, R. LINCK AND D.W. WRIGHT

327

Bioactive peptides as signal molecules in plant defense, growth and development A. SCHALLER

367

Enzymes involved in the biosynthesis of brassinosteroids J. WINTER

413

Immunopotentiating effects of a glycoprotein from Chlorella vulgaris strain CK and its characteristics K. TANAKA, Y. SHOYAMA, A. YAMADA, K. NODA, F. KONISHI AND K. NOMOTO

429

Hepatoprotective effect of plant components: Inhibition of tumor necrosis Factor-ot-dependent inflammatory liver injury K. HASH, Q. XIONG AND S. KADOTA

459

Induction and regulation of biosynthetic activity of phytoalexin in carrot cells F. KUROSAKl

483

Inhibition of Eukaryote Signal Transduction Components by Plant Defensive Secondary Metabolites G.M. POLYA

513

Flavonoids and cardiovascular diseases J. DUARTE, F. PEREZ-VIZCAINO, J. JIMENEZ, J. TAMARGO AND A. ZARZUELO

565

Effects of flavonoids on gastrointestinal disorders J. G A L V E Z , F. S A N C H E Z D E MEDINA, J. JIMENEZ AND A. ZARZUELO

607

Bioactivity of the phenolic compounds in higher plants J.M. RUIZ AND L. ROMERO

651

Bioactive natural products from marine sources M.J. ABAD AND P. BERMEJO

683

Biological activities of natural halogen compounds G. LAUS

757

Marine sulfur-containing natural products C.JIMENEZ

811

Absorption, metabolism and biological activities of chlorogenic acids and related compounds H. MORISHITA AND M. OHNISHI

919

Effects of ethanol extract of Crocus sativus L. and its components on learning behavior and long-term potentiation H. SAITO, M. SUGIURA, K. ABE, H. TANAKA, S. MORIMOTO, F. TAURA AND Y. SHOYAMA

955

Subject Index

971

CONTRIBUTORS MJ. Abad

Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain

K.Abe

Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033,Japan

Toshihiro Akihisa

College of Science and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan

Nuria Anaya

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

P. Bermejo

Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain

Mathias Berner

Laboratory of Organic Chemistry, Helsinki University of Technoloy, Espoo, Finland

Costas Demetzos

School of Pharmacy, Department of Pharmacognosy, Panepistimiopolis Zografou 15771, University of Athens, Athens, Greece

Jan C.R. Demyttenaere

Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

Konstantinos S. Dimas

International Institute of Anticancer Res., Kapandriti, 19014, Attiki, Greece

Antonio Dipietro

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

Juan Duarte

Department of Pharmacology, School of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain

Nadia A. El-Sebakhy

Faculty of Pharamcy, Alexandria, Egypt

J. Galvez

Department of Pharmacology, School of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain

Koji Hase

Institute for Consumer Healthcare, Yamanouchi Pharmaceutical Co. Let., Tokyo 174-8612, Japan

University

of

Alexandria,

Carlos Jimenez

Departamento de Quimica Fundamental, Universidade de A Corunia, 15071 A Coruiia, Spain

Jose Jimenez

Department of Pharmacology, School of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain

Reija Jokela

Laboratory of Organic Chemistry, Helsinki University of Technoloy, Espoo, Finland

Shigetoshi Kadota

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

Junei Kinjo

Faculty of Pharmaceutical Sciences, University, Kumamoto 862-0973, Japan

Fumiko Konishi

Research Laboratories, Chlorella Industry Co. Ltd., 1343 Hisatomi, Chikugo City, Fukuoka 833-0056, Japan

Fumiya Kurosaki

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan

Khalid Lairini

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

Gerhard Laus

Immodal Pharmaka GmbH, Bundestrasse 44, A-6111 Volders, Austria

Rachel Linck

Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282-1530, USA

F. Sanchez de Medina

Department of Pharmacology, School of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain

S. Morimoto

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Hideko Morishita

Faculty of Education, Wakayama University, Sakaedani Wakayama 640-8510, Japan

Kiyoshi Noda

Research Laboratories, Chlorella Industry Co. Ltd., 1343 Hisatomi, Chikugo City, Fukuoka 833-0056, Japan

Toshihiro Nohara

Faculty of Pharmaceutical Sciences, University, Kumamoto 862-0973, Japan

Kumamoto

930

Kumamoto

Kikuo Nomoto

Department of Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-0054, Japan

Motoyo Ohnishi

Department of Pharmaceutical Science, Institute of Medical Science, Kansai Shinkyu Medical College, 1-11-2 Wakaba Kumatori Sennan Osaka 590-0482, Japan

Alonso Perez-Espinosa

Departamento de Genetica, Facuhad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

Francisco PerezVizcaino

University Complutense of Madrid, Department Pharmacology, School of Medicine, Madrid, Spain

Gideon M. Polya

Department of Biochemistry, La Trobe University, Bundoora, Melbourne, Victoria 3083, Australia

Teresa Roldan-Arjona

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

Luis Romero

Departamento de Biologia Vegetal, Facultad de Ciencias, Universidad de Granada, 18071-Granada, Espafia

Juan M. Ruiz

Departamento de Biologia Vegetal, Facuhad de Ciencias, Universidad de Granada, 18071-Granada, Espafia

Manuel Ruiz-Rubio

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071, Cordoba, Spain

H. Saito

Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033,Japan

Andreas Schaller

Institute of Plant Sciences, Federal Institute of Technology Zurich, CH-8092 Zurich, Switzerland

Y. Shoyama

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Yikihiro Shoyama

Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-0054, Japan

M. Sugiura

Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033,Japan

of

Juan Tamargo

University Complutense of Madrid, Department Pharmacology, School of Medicine, Madrid, Spain

H. Tanaka

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Kuniaki Tanaka

Research Laboratories, Chlorella Industry Co. Ltd., 1343 Hisatomi, Chikugo City, Fukuoka 833-0056, Japan

F. Taura

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Arto Tolvanen

Laboratory of Organic Chemistry, Helsinki University of Technoloy, Espoo, Finland

Luisella Verotta

Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via Venezian 21, 20133 Milano, Italy

Jochen Winter

Max-Planck-Institut fur Zuchtungsforschung, Carl-vonLinne-Weg 10, D-50829, Koln, Germany

David W. Wright

Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282-1530, USA

Quanbo Xiong

Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

Akira Yamada

Kurume University Research Center for Innovative Cancer Therapy, and Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume City 830-0011, Japan

Ken Yasukawa

College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan

Antonio Zarzuelo

Department of Pharmacology, School of Pharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada, Spain

James Ziegler

Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282-1530, USA

of

Bioactive Natural Products

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 25 © 2001 Elsevier Science B.V. All rights reserved.

ACID-CATALYSED EPIMERIZATION OF BIOACTIVE INDOLE ALKALOIDS AND THEIR DERIVATIVES MATHIAS BERNER, ARTO TOLVANEN AND REIJA JOKELA* Laboratory of Organic Chemistry, Helsinki University of Technology, Espoo, Finland ABSTRACT: The acid-catalysed epimerization reaction of bioactive indole alkaloids and their derivatives is reviewed. The three mechanisms, which have been proposed for the P-carboline-type indole alkaloids, are discussed. Through recent developments, evidence for all three mechanisms has been obtained, which shows the complexity of the epimerization reaction. The epimerization seems to depend on structural features and reaction conditions making it difficult to define one universal mechanism. On the other hand, the isomerization mechanism of oxindole alkaloids has been widely accepted. The acid-catalysed epimerization reaction provides a powerful tool in selectively manipulating the stereochemistry at the epimeric centre and it can also have a marked effect on the pharmacology of any epimerizable compound. Therefore, examples of this reaction in the total synthesis of indole alkaloids are given and pharmacological activities of some C-3 epimeric diastereomers are compared. Finally, literature examples of acid-catalysed epimerization reactions are presented.

INTRODUCTION It was suggested back in the early 1950s that yohimbine-type alkaloids epimerize at the centre attached to the 2-position of indole [1], which corresponds to C-3 in the biogenetic numbering [2]. Interest in the acidcatalysed epimerization reaction was aroused during the structure elucidation of the pharmacologically important indole alkaloid reserpine (1) in the 1950s [3]. The equilibration of reserpine (1) and its derivatives in acid provided important stereochemical information on the reserpine skeleton. The epimerization of reserpine (1) resulted in the more stable C3 epimer, isoreserpine (2). When 1 was, for example, refluxed in acetic acid (AcOH) for 3 days, isoreserpine (2) was obtained in 60% yield [4], Scheme (1). Due to inversion of the configuration at C-3, reserpine (1) and isoreserpine (2) differ in sterical shape, and the pharmacological properties of the two compounds are dissimilar: isoreserpine (2) is completely inactive [5]. Thus, acid-catalysed epimerization can have a marked effect on the pharmacology of any epimerizable compound.

H CH30

CH3O H' C H 3 0 0 C ^ ^ 6 \ x ^ ^ OTMB J

6CH3

CHaOOC*^ " Y " ^OTMB ^

6CH3

TMB = 3,4,5-trimethoxybenzoyl Scheme (1). Epimerization of reserpine (1).

The epimerization reaction has been surveyed earUer by three different groups [6-8]. Kametani and Ihara [6] dealt with the epimerization and racemization of isoquinohne and indole alkaloids. In a review of the Pictet-Spengler condensation, Cook and Cox [7] included a short paragraph on the epimerization of natural products (indole alkaloids), and, more recently, Lounasmaa and co-workers [8] published a review of the epimerization reaction of reserpine (1) and other indoloquinolizidines. Our purpose here is to give in-depth information on the C-3 epimerization reaction in the synthesis of bioactive indole alkaloids and their derivatives, including oxindole alkaloids and 2-substituted indoles, and serve as a guideline for predicting the epimerization behaviour of indole alkaloids in general. We also compare the pharmacological activities of some C-3 epimeric diastereomers. Articles dealing with the epimerization reaction are not easy to find. The term epimerization is often not mentioned in the abstract or title of an article and hence the discovery of a specific publication is sometimes pure coincidence. Furthermore, some authors use the term isomerization instead of epimerization, which naturally makes the search even more complicated. By definition, epimerization is the alteration of one asymmetric centre (the given compound has more than one asymmetric centre) but isomerization is the process whereby a compound is converted into an isomer [9]. Isomerization is therefore a more general term, resulting in an abundance of references and making it virtually impossible to track down all the publications of interest. We therefore apologize if we have omitted any crucial publications from this review.

EPIMERIZATION OF p-CARBOLINE-TYPE INDOLE ALKALOIDS AND THEIR DERIVATIVES Mechanistic Discussion In 1955, Wenkert and Liu [10] proposed a mechanism for the acidcatalysed C-3 equihbration of alio- and epialloyohimbane (Mechanism 3). Three years later, in 1958, two other mechanisms (Mechanisms 1 and 2) were introduced by Woodward and co-workers in connection with the first total synthesis of reserpine (1) [11]. In Mechanism 1, Scheme (2), protonation takes place at C-7 (the 6position of indole) and is then followed by enamine formation via proton abstraction at C-3. The change of configuration at C-3 is completed by enamine protonation and subsequent proton cleavage at C-7.

Scheme (2). Presentation of Mechanism 1.

Mechanism 2, Scheme (3), involves initial protonation at C-2 and the formation of an intermediate enabling cleavage of the C-2 - C-3 bond to give an iminium ion. Acid-induced recyclization of the iminium species gives rise to the epimerized product. In Mechanisms 1 and 2 the reactions are considered to proceed via an equilibrium concentration of the free base. Mechanism 3, Scheme (4), which was first proposed by Wenkert and Liu [10], starts with protonation at Nb, the most basic site and so the most likely to be protonated first. After protonation, the C-3 - Nb bond is cleaved due to participation of the indole nitrogen lone pair, giving a carbocation intermediate. Ring reclosure is assisted by the Nb lone pair to effect the inversion at C-3.

Scheme (3). Presentation of Mechanism 2.

S

1 H ,

f^^N

ii

n

f-^

i»H

Scheme (4). Presentation of Mechanism 3.

A feature common to all mechanisms is the necessity of the Na nitrogen lone pair and aromatic n electrons. Recently, Lounasmaa and coworkers [12] proved the vital role of the Na nitrogen lone pair in the epimerization reaction by chosing c/^-deethylebumamonine (3) as a target molecule. Compound 3 possesses a lactam moiety, which blocks the lone pair of Na. Deethylebumamonine (3) was not therefore expected to epimerize; nor did it, as was experimentally proven. When, however, the amide system was removed, c/.y-deethyldihydroebumamenine (4) epimerized readily when treated with refluxing trifluoroacetic acid (TFA) ovemight, resulting in an equilibrium ratio of 20:80 (4:5), Scheme (5). By analogy, it has been reported that Na-sulfonamido p-carbolines also failed to epimerize under acidic conditions [13].

— ^

no epimerization

20h

ii refl* 16h

Scheme (5). Epimerization experiments with compounds 3 and 4.

To test the necessity of aromatic n electrons in the epimerization reaction, indolo[2,3-a]quinoUzidines with different substituents at C-10 were prepared [14], Subjecting these compounds to epimerization conditions (TFA, 90°C, 60 min) gave the following results, Table 1. Table 1. Epimerization experiments with 10-substituted indolo[2,3-a]quinolizidines.

: l r RXJCJU^N

+ ^XXJC^N

H CHsOOC^

Starting ester 6

R H

Transicis ratio 74:26 (6:7) No epimerization

8

NO2

9

NH2

No epimerization

10

NHAc

Traces of cw-epimer

11

OH

32:68(11:12)

13

Br

Traces of c/5-epimer

CHaOOC^

Deactivating groups such as the nitro or bromo group hinder or slow down epimerization whereas activating groups such as the hydroxy group accelerate it. The amino group, originally electron donating and strongly activating, protonates immediately in acidic solution and becomes electron withdrawing and strongly deactivating; this hinders the epimerization reaction. An increase or decrease in the electron density of the aromatic A ring is therefore crucial for the epimerization reaction, thus underlining the vital role of the aromatic n electrons. Further proof was provided by MacPhillamy and co-workers [4] who in the 1950s had already demonstrated that reserpine (1) epimerizes with greater ease than deserpidine (11-demethoxyreserpine). Proton Abstraction at C-3 (Mechanism 1) In general. Mechanism 1 has not attracted much support. Lately, however, evidence in its favour has started to accumulate. Enamine formation by hydrogen abstraction in Mechanism 1 provides a basis for deuterium incorporation/abstraction experiments. In their pioneering work, Gaskell and Joule [15] prepared 3-deuteroisoreserpine and subjected it to epimerization conditions (AcOH, IIS'^C). Samples withdrawn from the reaction mixture showed that epimerization had occurred without loss of label. Only more vigorous conditions (AcOH, 140°C, 3 d) resulted in totally unlabelled reserpine (1) and isoreserpine (2). On the other hand, when 3-deuteroisodeserpidine was treated with AcOH (118°C), deuterium was lost more rapidly than the epimerization advanced. Hence, Gaskell and Joule concluded that deuterium abstraction occurs via Mechanism 1 and that reprotonation of the enamine intermediate takes place stereospecifically, leading to the original base and not to epimerization. Reports have recently been published of protonation at C-7 (a prerequisite for Mechanism 1) under strongly acidic conditions [16,17]. While investigating the protonation site of Rauwolfia alkaloids, Balon and co-workers [16] showed through ^^C NMR studies that protonation takes place at C-7 in 18M H2SO4. Experiments by Royer et al. [17] indicated that reserpine (1) and isoreserpine (2) are transformed into the corresponding 2,7-dihydro compounds (14) (55%) and (15) (87%) by NaBH3CN in TFA at room temperature. Scheme (6). This is only possible through protonation at C-7. Further evidence was provided by deuterium incorporation at C-7 when TFA-d was used.

NaBHgCN CH30

TFA, rt

CH3O

OTMB

CH3OOC

CH300C^ ^ V ^ ^OTMB 6CH3

OCH3

14 (H-2P, H-3p, H-7P) 15 (H-2a, H-3a, H-7a)

1 (H-3p) 2 (H-3a)

Scheme (6). Reduction of reserpine (1) and isoreserpine (2) with NaBH3CN.

Deuterium incorporation into reserpine (1) with a strong deuterated acid (TFA'd) was investigated by Lounasmaa et al. [18]. Treating reserpine (1) with refluxing TFA-J overnight yielded hexadeuterated reserpine (16) and isoreserpine (17) (ratio 15:85), Fig. (1). When the reaction was repeated with a shorter reaction time (5 min), only 10,12dideuterated reserpine and isoreserpine were obtained, proving that, even with a strong acid, epimerization and deuteration at the epimeric centre occur at different rates. This finding imphes, at the least, that Mechanism 1 is not primarily responsible for the epimerization reaction of reserpine (1). Interestingly, in a review of Woodward's [11] reserpine synthesis, Nicolaou and Sorensen recently suggested that the acid-catalysed epimerization reaction might occur via Mechanism 1 [19].

CH30

0CH3 0CH3

16(p.D) 17 (a-D) Fig. (1). Hexadeuterated reserpine (16) and isoreserpine (17).

0CH3

10

Other examples of deuterium incorporation at the epimeric centre have also been reported. When treated with a deuterated acid (DCl/MeOD and TFA-J, respectively), both P-carboline derivative 18 [20] and vinylogous urethane 19 [21] resulted in deuterium incorporation at C-1 and C-12b (both correspond to C-3), respectively. Fig. (2). Mechanisms similar to that of Mechanism 1 were suggested to explain the epimerization. It should be noted, however, that, as pointed out above, mere deuterium incorporation is not sufficient evidence for Mechanism 1. Were Mechanism 1 alone responsible for the epimerization, both deuterium incorporation and epimerization would have to happen at the same rate.

COOH

CHaCHzOOCr H^v; "COOCH2CH3 H

18

19

I

H

''

Fig. (2). P-Carboline derivative 18 and vinylogous urethane 19.

Another way to test the operation of Mechanism 1 is to subject C-12b alkyl substituted indolo[2,3-a]quinolizidines to epimerization conditions. Should Mechanism 1 be operative, these compounds could not epimerize. Thus, C-12b methyl substituted indoloquinolizidines 20 - 25 with different structural features were prepared by Lounasmaa and co-workers [22], Fig. (3). As well as testing Mechanism 1, they investigated the effect of different structural features on the epimerization reaction in general.

cc

][CH3

COOCH3

COOCH3

20 (a-CHs)

22 (a-CH3)

24 (a-CH3)

21 (P-CH3)

23(P-CH3)

25(p-CH3)

Fig. (3). Indolo[2,3-«]quinolizidine derivatives 20 and 21, lactams 22 and 23 and vinylogous urethanes 24 and 25.

11

The "normal" indoloquinolizidines 20 and 21 resemble compounds such as reserpine (1). Considering the results of the deuterium experiments (see above), one could expect compounds 20 and 21 to epimerize under acidic conditions. And indeed, refluxing 20 or 21 in TFA ovemight yielded an equilibrium ratio of 55:45 (21:20). Lactams 22 and 23 were also expected to epimerize, since prior results (see below) suggested that Mechanism 3 is active in the epimerization reaction of these compounds. Compounds 22 and 23 epimerized in deaired TFA resulting in an equilibrium ratio of 65:35 (23:22). Winterfeldt and co-workers [23] have reported epimerization of similar compounds under acidic conditions. Not only were vinylogous urethanes known to epimerize with great ease but deuterium was also known to be incorporated at room temperature at the epimeric centre (see above) [21]. Furthermore, Wenkert and co-workers [24,25] have suggested an epimerization mechanism for vinylogous urethanes analogous to Mechanism 1, Scheme (7). Epimerization of 24 and 25 was therefore expected to produce interesting results. When different mixtures of vinylogous urethanes 24 and 25 were treated with TFA, the ratios remained unchanged. Thus, the methyl group at C-12b had hindered epimerization, which meant that vinylogous urethanes epimerize via Mechanism 1. Systematic acidcatalysed study of compounds 20 - 25 proved the marked effect of structural features on the epimerization reaction.

C00CH3

C00CH3

C00CH3

C00CH3

Scheme (7). Epimerization mechanism for vinylogous urethanes.

C-2 - C'3 Bond Cleavage (Mechanism 2) Mechanism 2, which constitutes a retro Pictet-Spengler type process, has long been held responsible for the acid-catalysed epimerization reaction

12

of reserpine (1) [15,26]. The main reason for this is the outcome of the trapping experiments of Gaskell and Joule [15], who obtained two bond scission products 26 and 27 when reserpine (1) was refluxed in Zn/AcOH for 24 h, Fig. (4). 2,3-Secoreserpine (26) suggests Mechanism 2 but 3,4secoreserpine (27) points to Mechanism 3.

CH30

OTMB

OTMB 0CH3

26

0CH3

27

Fig. (4). 2,3-Secoreserpine (26) and 3,4-secoreserpine (27).

Ahhough compound 27 was obtained in a much higher yield than was 26, Gaskell and Joule concluded that Mechanism 2 is active in the epimerization reaction of reserpine (1). They discredited Mechanism 3 because of the incapability of the metho salts 28 and 29 to epimerize. Instead, treatment of 28 and 29 with AcOH (140°C, 3 d) resulted in inversion of Nb to yield 30 and 31, respectively. Fig. (5). It was concluded that the inversion probably occurs via C-3 - Nb bond scission. 91 ^CH3 CH30

CH3O H' CHsOOC^ > ^

C H 3 0 0 C ' ' ^ ^ V ^ ' ^ OTM B

°^"^

28

CH3O

"OTMB

^^"'

29

CH3O H""|

H'

CHsGOC" "^Y'^^^OTMB

CH300C^ ^ V ^

6CH3

30 Fig. (5). Metho salts 28-31.

^OTMB

6CH3

31

13

Moreover, in the case of reserpine (1), the methoxy group at C-11 increases the electron density at C-2 and hence supports Mechanism 2, Fig. (6) [27].

Fig. (6). Resonance stabilization of the methoxy group.

In conjunction with their studies on electrophiUc substitution in indoles, Jackson and co-workers [28] suggest that the initial protonation occurs at C-7 as in Mechanism 1, followed by hydride rearrangement of the indole P-hydrogen to the a-position. The epimerization reaction would then take place as depicted in Mechanism 2. Hence, Gaskell and Joule and Jackson et al. differ only in the matter of initial protonation. In 1989, Cook and co-workers [29] reinvestigated the epimerization reaction in connection with reserpine (1). One of their key observations, based on the results of Martin et al. [30] and of Sakai and Ogawa [31], is that Mechanism 2 cannot be primarily responsible for the epimerization reaction of reserpine (1). Both Martin and co-workers and Sakai and Ogawa report that the iminium species 32, Fig. (7), cyclizes under acidic conditions mainly to reserpine (1) and not to isoreserpine (2). If Mechanism 2 alone were responsible for epimerization, then isoreseipine (2), not 1 should be the main product. Mechanism 2 was accordingly discredited.

CH30

£00H

H" CHaOGC^ " Y ^ ^OTMB 22 Fig. (7). Iminium species 32.

6CH3

14

C-5 - A^^ Bond Cleavage (Mechanism 3) If only three mechanisms are considered and two of them are discredited, then the third must be active. And indeed, a substantial body of evidence has built up in favour of Mechanism 3. Cook and co-workers have thoroughly investigated the acid-catalysed epimerization reaction with P-carboline derivatives [13,32,33]. Their conclusive work has provided strong evidence for Mechanism 3. When compound 33 was treated with TFA-J (2.9 equiv.), the more stable C-1 epimer 34 was obtained in high yield. Moreover, no deuterium had incorporated at C-1, thus ruling out Mechanism 1. A reduction experiment with NaBH4 on 33 yielded the C-1 epimer 34 and a ring cleavage product 35. Compound 35 provides strong evidence for Mechanism 3. In a control experiment, compound 34 was subjected to reductive conditions similar to those used for 33. The experiment resulted only in starting material, proving that intermediate 35 truly arises from the epimerization reaction. Scheme (8) [13]. Cook and co-workers had earlier suggested that reserpine (1) epimerizes analogously to p-carboline derivatives and hence via Mechanism 3 [29]. H

COOCH3

kjCI^^

H ^."v. I^COOCHa

^.vTs.

NaBH4 KJ^J CH2CI2, rt

H

35

KJ COOCH3 Ph

TFA NaBH4

+ 34

O

no reaction

CH2CI2, rt

Scheme (8). Epimerization experiments conducted by Cook and co-workers.

15

Lounasmaa et al also investigated the epimerization behaviour of lactams [12]. They found that lactams such as 36 epimerize with great ease. Epimerization of 36 with TFA at room temperature resulted in an equilibrium ratio of 70:30 (36:37) within a reaction time as short as 2 h, Scheme (9).

CH3

Scheme (9). Epimerization of lactam 36.

Being protonated at the carbonyl oxygen in strongly acidic media, lactams enable delocahzation of the Nb lone pair, Fig. (8).

Fig. (8). Delocalization of the Nb lone pair in a protonated lactam.

The canonical forms displayed above show a similarity with Mechanism 3, namely, the positive charge at Nb. Proof of the involvement of Mechanism 3 was therefore sought. When 36 was refluxed in a Zn/TFA solution for 3.5 h the secolactam 38, Fig. (9), was obtained in 22% yield. Compound 38 indicates that Mechanism 3 is active in the acid-catalysed epimerization of such lactams, a finding in accordance with the previously reported result that C-12b methyl substituted lactams epimerized under acidic conditions (see above).

16

Fig. (9). Secolactam 38.

Racemization of P-Carbolines In connection with the preparation of (+)- and (-)-tetrahydroharmine (compounds 39 and 40 in Fig. (10), respectively) the racemization of these compounds was studied by Chrisey and Brossi [34]. They demonstrated that, under acidic conditions, the pure enantiomers racemized with relative ease, and suggested that the racemization resembled epimerization of reserpine (1) and 1,3-disubstituted tetrahydrop-carbolines. Therefore, the mechanism responsible for the racemization would be one of those mentioned above. CH3O CH30 H CH3

39

u

40

NH -> H CH3

NH

CH3O

H CH3

41

Fig. (10). (+)-Tetrahydroharmine (39), (-)-tetrahydroharmine (40), and (-)-tetrahydroroeharmine (41).

Cook and co-workers suggested that partial racemization had occurred in the acid/base mediated isolation of (-)-tetrahydroroeharmine (41), Fig. (10) [35]. Proof was obtained by treating 41 with TFA/CH2CI2 at room temperature, which resulted in racemization of 41. In an experiment with TFA'd, deuterium was incorporated only at C-5 and C-8, not at the epimeric centre, indicating that a mechanism analogous to Mechanism 1 was not active. Reddy and Cook, in contrast, proposed that the mechanism depicted in Scheme (10) was responsible for the racemization of (-)-tetrahydroroeharmine (41). This mechanism is analogous to Mechanism 3. Interestingly, compounds with more than one asymmetric

17

centre, e.g. reserpine (1) and 1,3-disubstituted tetrahydro-P-carbolines, do not racemize under epimerization conditions [29,33].

CH

"V^^JI ^Q A ^

i l l ^ A s ^ NH

bond rota and and reclosure redo ^, CH36

Scheme (10). Racemization mechanism for (-)-tetrahydroroeharmine (41).

Synthesis of Bioactive Indole Alkaloids and Their Derivatives by Utilizing the Acid-Catalysed Epimerization Reaction Obtaining the correct stereochemistry for a given compound is of vital importance for the pharmaceutical industry because pharmacological properties often depend on it. For indole alkaloids and their derivatives, the acid-catalysed epimerization reaction provides, contrary to the more traditional oxidation/reduction procedure, a convenient tool to influence selectively the configuration at C-3 (biogenetic numbering). The following examples demonstrate the high utility of the epimerization reaction. Woodward's Total Synthesis of Reserpine Reserpine (1) with its remarkable pharmacological properties remains an attractive synthetic target. The first total synthesis of reserpine (1), by

18

Woodward and co-workers [11] in the 1950s, constitutes one of the most classical acid-catalysed epimerizations in indole alkaloid chemistry. After the key intermediate 43 had been prepared, it was reacted with 6methoxytryptamine (42) in benzene, Scheme (11).

.OrTi 42 CH3OOC

I.POCI3 •

2. NaBH4, MeOH

L " ^,,'T ^

CHaOGC^ ^Y"^ ^ OAc

CHsGOC^^^Y^ ^ OAc

43 ^^"^

44 . KOH,

OCH3

f ^

MegCCOOH

MeOH ^ „ ^ J k . J k

•"

2. NaBH4, MeOH

H H'

2.DCC, pyridine

46 . NaOMe,

'•

xylene, A

OCH3

\ \ i

MeOH ^ „ ^ J k s J k

[..•"

47

2.TMBC1, pyridine

OCH3 ^^"'

H H H"' CHaOOC^ ^ S ^ "*OTMB 6CH3 (±)-l

Scheme (11). The final stages of Woodward's reserpine synthesis.

Immediate sodium borohydride (NaBH4) reduction gave lactam 44. Bischler-Napieralski cycHzation of 44 followed by NaBH4 reduction yielded (±)-methyl-0-acetyl-isoreserpate (45). The correct stereochemistry at C-3 was obtained by first lactonizing compound 45; epimerization with pivalic acid then resulted in (±)-reserpic acid lactone (47). Treatment with base followed by acylation with TMBCl yielded racemic reserpine. The stereochemical considerations involved in the epimerization reaction will be discussed later.

19

Synthesis of Deethyleburnamonines by Lounasmaa and Co-workers An efficient route to both cis- and ^ra«5-deethylebumamonines is a further example of the efficacy of the epimerization reaction [36], These unnatural compounds are close derivatives of the well-known, pharmacologically important indole alkaloid, ebumamonine. For the synthesis of c/^-deethylebumamonine (3), Scheme (12), trans-QSi^x 6 was epimerized in refluxing TFA to give a readily separable mixture of starting material and m-ester 7 (ratio 22:78).

^

OgCii

THF*

H CHaOGC

7

N

pyridine P^"^^"'

k^N^si^N

DMF*

H TsO-

49

k A H^ ^

M^o" kJsAl^''

NC-

50 Scheme (12). Lounasmaa et al synthesis of deethylebumamonine (3).

After the correct stereochemistry at C-12b had been obtained, the methyl ester group was reduced with lithium aluminium hydride to yield alcohol 48. Tosylation of the alcohol and subsequent replacement of the tosylate with cyanide resulted in nitrile 50. Finally, acid treatment of 50 resulted in the target compound, deethylebumamonine (3).

20

Synthesis of the Tangutorine Skeleton by Jokela and Co-workers Tangutorine (51), Fig. (11), an indole alkaloid recently isolated from the leaves of Nitraria tangutorum [37] constitutes an interesting synthetic target for pharmacological evaluation. The carbon framework of 51 was therefore prepared to investigate, for the first time, the conformational and stereochemical features of this ring system [38]. The epimerization reaction served as a tool in the studies.

OH

Fig. (11). Tangutorine (51).

Preparation of the dodecahydro benz[/]indolo[2,3-a]quinolizidine ring structure relied on the Fry reaction [39] of salt 52, which, after subsequent acid-induced cyclization, yielded tlu*ee compounds, 53 - 55, Scheme (13).

OTX. „ e

l.KCN,6NHCl, NaBH. 2. 50% AcOH

52

Scheme (13). Preparation of compounds 53-55.

The unexpected a-amino nitrile 53 could easily be converted via AgBF4 treatment followed by reduction to the tangutorine model 56, Scheme (14).

21

l_AgBF4 2. NaBH.

Scheme (14). Conversion of a-amino nitrile 53 to the tangutorine skeleton 56.

After the Fry reaction, the tangutorine skeleton 56 could also be obtained via an epimerization sequence, Scheme (15). Since the catalytic hydrogenation of 54 yielded predominantly the wrong isomer (H-3, H-19 and H-20, all p), an alternative approach was sought to exploit compounds 54 and 55. Refluxing 54 in TFA overnight resulted in a ratio of 40:60 for compounds 55 and 54. Compound 57 was obtained by catalytic hydrogenation of 55, after which the tangutorine skeleton was formed via acid-catalysed epimerization of 57 (ratio 60:40, 56:57). Hence, by an epimerization sequence, compounds 54 and 55 can be converted into the desired target molecule 56.

+ 54

55 refl.*

Scheme (15). Epimerization sequence to obtain tangutorine model 56.

54

140:601

+

57

22

Stability and Conformational Analysis of Epimers The different conformations of indolo[2,3-^]quinolizidines play an important role in predicting the thermodynamically more stable epimer (see below). The indolo[2,3-a]quinolizidine system can exist in three main conformations: one C/D trans ring juncture (conformation a) and two C/D cis ring junctures (conformations b and c). These are in equilibrium by nitrogen inversion and c/^-decalin type ring interconversion. Ring C is considered to be in a half chair conformation and ring D in a chair conformation, Scheme (16) [40]. c/5-decalin type ring interconversion

a

C Scheme (16). The three conformations of indolo[2,3-<2]ciuinolizidines.

The stability of indolo[2,3-a]quinolizidine epimers can be determined by conformational analysis, bearing in mind three important factors: 1) conformation a is more stable than conformation c\ 2) substituents, that are equatorial in conformation a become axial in conformation c, leading to steric interactions between them; and 3) hydrogen bonding between substituents and Na/Nb. These three factors must be weighed in each case separately to establish the thermodynamically more stable epimer. The following are representative examples. Reserpine (1) epimerizes to isoreserpine (2) with great ease to give a ratio of 15:85 [18]. Hence, isoreserpine is the more stable epimer. The empiric result can be verified by stereochemical and conformational analysis. In reserpine (1) the conformational equilibrium is shifted to

23

conformation c (C-6: 16.9 ppm [30]) but in isoreserpine to conformation a (C-6: 21.8 ppm [30]). In both compounds all substituents are in the preferred equatorial position. Therefore, the stability of each compound is resolved not by substituents but by conformation. Since conformation a is more stable than c, isoreserpine (2) is obtained as the major product in the epimerization reaction. The destabilizing 1,3-diaxial interactions between H-15 and H-21 and the indole residue at C-3, which are present in 1, are therefore non-existent in 2.

Fig. (12). Stereoformulas of reserpine (1) and isoreserpine (2).

In the course of the first total synthesis of reserpine (1), Woodward and co-workers [11] obtained an isoreserpine derivative instead of a reserpine derivative (see above). Thus, the problem arose of how to epimerize with good yield the more stable isoreserpine derivative to a reserpine

24

derivative. The problem was ingeniously solved by Woodward in converting (±)-methyl-0-acetyl-isoreserpate (45) into isoreserpic acid lactone (46), Fig. (13). The lactone ring forces compound 46 into conformation c, which exhibits remarkable steric strain between the lactone and indole substructure. In the C-3 epimer, reserpic acid lactone (47), these steric interactions are relieved because the conformational equilibrium is shifted to the more stable conformation a. Hence, epimerization of 46 to 47 could be expected to succeed well. And indeed, reserpic acid lactone (47) was formed in 79% yield when 46 was refluxed in a pivalic acid/xylene solution.

47

46

Fig. (13). Stereoformulas of isoreserpic acid lactone (46) and reserpic acid lactone (47).

As a last example, the epimerization of corynantheidol (58) is examined. Refluxing 58 in AcOH resulted in a mixture of C-3 epimers 58 and 59 in a ratio of 27:73, Fig. (14) [41]. Both compounds possess one equatorial and one axial substituent and they are in conformation a, as can be verified by ^^C NMR data. The question arises as to which is the lesser evil: an axial ethyl group or an axial hydroxy ethyl group. The empiric result shows that the latter is preferred. A plausible explanation is that, in axial position, hydrogen bonding may exist between Nb and the alcohol proton.

58

59

Fig. (14). Stereoformulas of corynantheidol (58) and 3-epicorynantheidol (59).

25

ISOMERIZATION OF OXINDOLES An isomerization reaction closely similar to that observed with indole alkaloids has been noted with oxindole alkaloids. Due to their facile isomerization, it is pharmacologically difficult to test the individual oxindole isomers expected to have different activities. Instead of epimerization the term isomerization has been used with oxindole alkaloids since inversion of configuration can occur in more than one asymmetric centre. Isomerization was employed mainly to provide structural proof of different oxindole epimers isolated in nature. As early as 1959, Wenkert and co-workers [42] proposed a mechanism for the isomerization of oxindole alkaloids, Scheme (17). Almost simultaneously, Seaton et al. [43] reported analogous findings.

£?;

,N—:

tf4—i

N—i

H

N^O

Scheme (17). The isomerization mechanism of oxindole alkaloids.

As Scheme (17) demonstrates, the isomerization of oxindoles may occur, at least in theory, at C-3 or C-7, or both. Originally, Wenkert and co-workers [44] suggested that it took place only at C-7, a conclusion they arrived at by examining the equilibration of formosanine (60), isoformosanine (61), mitraphylline (62) and isomitraphylline (63), Fig. (15). Equilibration of any of these compounds resulted in a mixture of C7, not C-3, epimers. Hence, it was assumed that isomerization occurs with inversion of configuration at C-7 and, due to the bulk of the group, with retention at C-3. In 1966, Johns and Lamberton [45] showed that equilibration of uncarine C and uncarine D in acidic and basic solutions resulted in C-3 isomerizations. Further investigations [46,47] revealed that equilibration of e.g. uncarine C in an aqueous acetic acid solution (50%) resulted in four isomers, namely uncarine C (64), D (65), E (66) and F (67), Fig. (16). Ratios of 40:40:10:10 were obtained for uncarine C, D, E and F. These were demonstrated to be true equilibrium ratios by treating each isomer under analogous conditions: the ratios were the same.

26

H CH3

H

60

H V=/ CH3OOC

H N^^ H \^\l^ H

62

H CH3

H

61 CH3

H CH3OOC

H y=^ CH3OOC

H CH3 Uv y\^

f^ H

63

H CH3OOC

Fig. (15). Formosanine (60), isoformosanine (61), mitraphylline (62) and isomitraphylline (63).

H CH3

H

64

H y==J CH3OOC

H

65

H y=zj CH300C

H N - ^ _H CH3

H

66

H CH3OOC

H

67

H CH3OOC

Fig. (16). Uncarine C (64), D (65), E (66) and F (67).

Hence, it was concluded that epimerization of oxindole alkaloids with a trans D/E ring junction results in only C-7 epimers, whereas that of oxindole alkaloids with a cis D/E ring junction can result in C-7 and C-3 epimers [47], The conclusion was based on steric and conformational considerations. Studies with corynantheine-type oxindole alkaloids have also been conducted [48]. Since rhynchophylline (68) and isorhynchophylline (69), Fig. (17), have an H-15 and H-20 trans relationship, epimerization would be expected to occur only at C-7. And indeed, this proved to be the case: acetic acid treatment yielded a ratio of 70:30 (68:69). Even though

27

corynoxine (70) and corynoxine B (71) have an H-15 and H-20 cis relationship, only the alio epimers were produced under acidic conditions. The result was attributed to the higher stability of the alio compounds. Acidic treatment of an analogous 9-hydroxy substituted alio oxindole alkaloid resulted in four epimers (2 alio and 2 epiallo) due to the stabilizing intramolecular hydrogen bonding between N-4 and phenolic hydrogen [49].

0CH3

H

69

H CH3OOC

0CH3

jlT^N--. H 0CH3

H

71

H CH3OOC

OCH3

Fig. (17). Rhynchophylline (68), isorhynchophylline (69), corynoxine (70) and corynoxine B (71).

Further evidence in favour of the mechanism depicted in Scheme (17) was obtained by showing that quatemization inhibits the isomerization of oxindole alkaloids [50]. The result is plausible enough, since participation of the Nb lone pair is a prerequisite for the mechanism. Of interest is that base- and acid-catalysed reactions give rise to different product ratios. The most likely explanation is that in an acidic medium the protonated Nb can, in certain conformations, possess hydrogen bonding to the lactam oxygen, which contributes to considerable stabilization. On the other hand, in a basic medium the Nb lone pair and the lactam oxygen exert electrostatic repulsion, thus favouring other conformations [50]. Recently, rigorous kinetic investigations of the isomerization of oxindole alkaloids were conducted by Laus and co-workers [51,52]. Their findings clearly show that the isomerization process is not acid-catalysed. On the contrary, the use of acid as a solvent slows down the isomerization rate because it inhibits the formation of the iminium ion in the mechanism involved. It has, however, a marked effect on the product composition.

28

EPIMERIZATION OF C-2 SUBSTITUTED INDOLES A closely related example of compounds not possessing the indoloquinolizidine skeleton is worth noting. Repke and co-workers [53] found that indoles appropriately substituted at the 2-position, compounds 72 and 73 in Scheme (18), epimerized when treated with HCl/EtOH. With DCI/CD3OD, deuterium incorporated immediately at the 3-position of indole and after a while at the epimeric centre of the starting material. Full deuterium incorporation at the epimeric centre of the thermodynamically more stable epimer was also observed. That the incorporation of deuterium atoms occurred more rapidly than the epimerization was attributed to the presence of a higher energy intermediate, which generally converts to the product but occasionally gives back the starting material. The epimerization rates were very similar for 72 and 73, but the deuterium exchange at the C-3 of indole was significantly faster with the tropane derivative 72.

reflux, 0.5 h

72 (X=NCH3) 73 (X=CH2)

^ ^ ' - ^ N - ' ^ y ^ ^ CH3 H N 74 (X=NCH3) 75 (X=CH2)

Scheme (18). Epimerization of compounds 72 and 73.

Thus, in this case, deuterium incorporation at the 3-position of indole and at the epimeric centre suggests that the mechanism depicted in Scheme (19) is active. This mechanism is analogous to Mechanism 1 as reported above in the context of indole alkaloids. While working on the synthesis of dihydrocinchonamine, Sawa and Matsumura [54] clarified the correct configuration at the epimeric centre via the acid-catalysed epimerization reaction. They easily obtained both epimers of the skeleton by equilibrating methoxy-substituted dihydrocinchonamine 76 in HCl/EtOH, Scheme (20), thus providing a basis for structure determination.

29

CH3

CH3

Scheme (19). Suggested mechanism for epimerization of the tropanyl compounds.

CH3O

CH2OH

•"xx^

HCl/EtOH reflux 22 h

CH20H

+

76

51:49 Scheme (20). Epimerization of compound 76.

COMPARISON OF THE PHARMACOLOGICAL PROPERTIES OF SOME C-3 EPIMERIC INDOLE ALKALOIDS The acid-catalysed epimerization reaction often contributes to the change of conformation that ahers the sterical shape of a compound. This may have a severe effect on pharmacological properties as with reserpine (1) and isoreserpine (2). The same seems to apply to the C-3 epimers yohimbine (78) and pseudoyohimbine (79). Yohimbine (78) blocks a2receptors, whereas pseudoyohimbine (79) has little affinity for this

30

receptor [55]. Similar behaviour can be observed in the allo/epiallo series: alloyohimbine (80) is a selective a2-antagonist but epialloyohimbine (81) is a very weak a2-adrenoceptor antagonist. In other words, normal and alio compounds are potent a2-adrenoceptor blockers but the pseudo and epiallo series are weak ones. This can be explained by the fact that compounds 78 and 80, but not compounds 79 and 81, have the indole nucleus, Nb, and the oxygen of the methyl ester group in the same medium plane [56].

79

OH

Fig. (18). Yohimbine (78), pseudoyohimbine (79), alloyohimbine (80) and epialloyohimbine (81).

In general, it has been suggested that in the yohimbine series the planarity of the A, B, C and D rings is necessary for the affinity to aadrenoceptors [57]. The selectivity between ai- and a2-adrenoceptor blocking activities is determined by the orientation of the carbomethoxy or the hydroxy group in ring E. Pseudoyohimbine has been found much less potent than yohimbine to inhibit the stimulation-evoked release of labelled GABA [58]. Some early studies on the stereochemical and pharmacological properties of yohimbine and its isomers have been reviewed [59]. The above structure/activity tendency is seen again in heteroyohimbine alkaloids. Tetrahydroalstonine (83) exhibits considerable selectivity towards presynaptic a2-adrenoceptors but raubasine (ajmaUcine) (84)

31

preferentially acts at postsynaptic sites. On the other hand, akuammigine (82), the C-3 epimer of tetrahydroalstonine (83), is a very weak antagonist at both sites [60-62].

..CH3

..CH3

Fig. (19). Akuammigine (82), tetrahydroalstonine (83) and raubasine (84).

Hirsutine (85) is a corynantheine-type indole alkaloid with a C/D cis ring juncture (pseudo stereochemistry). This compound has recently been found to exhibit highly potent inhibition of the replication of the strains of influenza A (subtype H3N2) [63]. The EC50 of hirsutine was 11- to 20fold more potent than that of the clinically used ribavirin. Exploration of the important structural features of this molecule revealed that the stereochemistry at C-3 {R) and C-20 {R) as well as the presence of the Nb lone pair were essential for the anti-influenza A activity. Thus, the C-3 epimer, dihydrocorynantheine (86) (normal stereochemistry), was much less active than hirsutine (85).

0CH3

0CH3

Fig. (20). Hirsutine (85) and dihydrocorynantheine (86).

Dihydrocorynantheine (86), in tum, along with other structurally related alkaloids, has been found to decrease specific [•^H]-5 hydroxytryptamine (5-HT) binding to membrane preparations from rat

32

brain and from in vitro experiments on guinea-pig ileum [64]. In these studies, hirsutine (85) (and some derivatives with the same stereochemistry at C-3) was ineffective. Dihydrocorynantheine might be useful in the treatment of diseases originating from disorders of 5-HT metabolism, such as migraine or other headaches. Recently, both hirsutine (85) and dihydrocorynantheine (86) were found to be active when the effects of these compounds on the action potentials of sino-atrial node, atrium and ventricle tissues were studied with standard microelectrode techniques [65]. In sino-atrial node preparations, both compounds concentration-dependently increased cycle length, decreased the slope of the pacemaker depolarization, decreased the maximum rate of rise and prolonged action potential duration. Thus, it was for the first time shown that hirsutine and dihydrocorynantheine have direct inhibitory effects on the cardiac pacemaker. In atrial and ventricular preparations, both compounds concentration-dependently decreased the maximum rate of rise and prolonged action potential duration. Although stereochemically different, these two alkaloids exhibited no difference in their effects on various myocardial action potential parameters. Dihydrocorynantheine also displays potent a-adrenoceptor blocking activity, while hirsutine is inactive [66]. Experiments with ion channels indicate that the mechanisms for these two phenomena probably differ. The direct effects of hirsutine and dihydrocorynantheine on the action potential of cardiac muscle through inhibition of multiple ion channels may explain the negative chronotropic and antiarrhythmic activities of these two alkaloids. Biotransformation studies of hirsutine (85) and dihydrocorynantheine (86) reveal an interesting difference between these two isomers [67]. Dihydrocorynantheine and some of its derivatives in the normal, alio and epiallo series are metabolized principally by 0-demethylation, whereas hirsutine or other pseudo isomers are metabolized by a different process yielding unidentified products. Oxindole alkaloids exhibit interesting pharmacological properties. However, due to their isomerization behaviour, it is extremely difficult to compare the pharmacological effects of pure oxindole alkaloid isomers, and therefore an equilibrated mixture of isomers is often used instead [68]. CONCLUSIONS Obtaining the correct stereochemistry at an asymmetric centre is crucial in the total syntheses of natural products. Moreover, pharmacological properties are closely related to the correct stereochemistry. Thus, the epimerization reaction, which enables equilibration of C-3 epimers.

33

remains a powerful tool in indole alkaloid synthesis. The efficacy of the acid-catalysed epimerization reaction has been demonstrated in the preparation of, among others, reserpine (1) and the tangutorine skeleton (56). Care must, however, be taken, especially if electron-donating and activating groups are present in the aromatic A ring, to avoid unnecessary epimerization/racemization during isolation or cyclization procedures. The mechanistic studies of the epimerization reaction still cause confusion. For the first time, direct evidence for Mechanism 1 has been presented based on the incapabiUty of the C-12b methyl substituted vinylogous urethanes to epimerize. Further evidence for Mechanism 1 was provided by deuterium incorporation at the epimeric centre of various compounds (see above), a process most likely due to a mechanism analogous to Mechanism 1. The difference in epimerization rate and deuterium incorporation states merely that Mechanism 1 is not primarily responsible for the acid-catalysed epimerization reaction and hence does not completely discredit it. Evidence for all three mechanisms therefore now exists, revealing the complexity of the epimerization process. The results with p-carbolines and the trapping of 3,4-secoreserpine (27) and secolactam 38 provide strong evidence for Mechanism 3. Mechanism 2, which was earlier considered to be responsible for the epimerization reaction, has since been discredited. Nevertheless, the presence of 2,3secoreserpine (26) in the trapping reaction remains undisputed and indicates that Mechanism 2 is active under the conditions employed. Thus, several mechanisms may be active simultaneously in the epimerization reaction, so further complicating the matter. Structural features, reaction conditions and acid strength also influence the acid-catalysed epimerization reaction. For example. Mechanism 1 requires protonation at C-7, which seems to occur under strongly acidic conditions. When a p-carboline derivative was treated with a weakly acidic solution (TFA-J, 2.9 equiv., rt), deuterium incorporation did not occur, whereas refluxing of a similar compound in a DCl/MeOH solution resulted in deuterium incorporation at the epimeric centre. Therefore, it is impossible to define one universal mechanism to explain the epimerization reaction for any given compound. On the contrary, each compound type must be separately investigated under different conditions. Clearly, then, the acid-catalysed epimerization reaction of indole alkaloids is a fruitful research area. The isomerization mechanism of oxindole alkaloids, in contrast, has never caused confusion. The mechanism, proposed independently by Wenkert and Seaton, has been accepted throughout the literature. Finally, we provide a list of literature examples of acid-catalysed epimerization reactions. Table 2, that can be used as a guideline in epimerizing similar compounds. We emphasize, however, that all data presented here are valid only under the conditions applied.

34

Table 2. Examples of Acid-Catalysed Epimerization Reactions. Entry/ Conditions

Starting Material

Epimer

Ratio or Yield

J 18,30

15:85 a,b

TFA, reflux 5 min

CHaOOC^ ^ Y ^ ^OTMB

CH3(X)C^ ^Y''^ ^OTMB

6CH3

6CH3

»n

79% CHaO*

Pivalic acid, xylene OCH3

reflux 13 h >^69,70

16.4*

17%

48% HBr/ AcOH(l:l) reflux 3 h 6CH3

OCH3

471,72

21.8

22:78'

48% HBr/ AcOH(l:l) reflux 6 h

5^^

25% iCHsO'

CH3O'

AcjO/AcOH

CH3

CH3

(7.5:1), reflux 4 d

CHsOOi

CH3OOC'

The C NMR chemical shift of C-6 (biogenetic numbering; CDCI3 used as solvent except where otherwise stated) is given when available and necessary to determine the conformation at nand. In the case of lactams and vinylogous urethanes, the chemical shift is omitted because these compounds do not possess conformations in the classical sense due to their structural features. To determine the stability of different epimers of lactams and vinylogous urethanes, the steric relations of each epimer must be examined and thus must be considered in each case separately, a) Verified by backwards epimerization; b) determined by H NMR integration; c) determined by TLC and/or specific rotation; *) DMSO-^^j used as solvent.

35 r39

22.1

16.2

40:60

TFA, reflux overnight

739

60:40

TFA, reflux overnight g23

90%

TFA, rtl5h >23

90%

TFA, rtl5h .oAa,b

17.2

10^^

20:80'

TFA, reflux 16 h

H

1174 TFA,

OgQ

H H' H

reflux

37:63*^

H H""'|

'^'^^.^^O

overnight

j24l,25

18.1

I

21.

H

AcOH, reflux 15 h OH

•sn*"' 20:80'

36 J34..75

21.6

80:20'

AcOH, reflux 18 h

^ 1441,76

OH

Co3

OJ0

AcOH,

.'H'ja^C

27:73

21.6

reflux 18 h OH

OH

15'^

40:60

18%HC1/

H

H

AcOH (2:1) reflux 24 h

CHaOOC^

"COOCH3

CHaOOC^

OuCvl

16^«

COOCH3

29:71

cone. HCl, reflux

1779

24:76 H

TFA,

H

CH3OOC

reflux 18 h

18^^

CH3OOC'

22.0

TFA, reflux 16 h

22:78

CHsOOcH^^ H

I9I4

32:68' H

TFA, reflux 1 h

HO' CH3OOC'

.

37

20'°

20.0

21.5

.<<:».'> 45:55'

19.3

21.7

15:85

TFA, reflux 16 h

2^80 TFA

^

reflux 16 h

22^^

.a,b

21.7

19.1

II TFA, reflux 16 h

20:80'

II y H

Cos

24^^ aired),

15:85

n^)

reflux 16 h

TFA (de-

.Qca,b

21.7

TFA,

238O

a,b

COOCH3

45:55^''

C00CH3

reflux overnight

25^^ TFA (deaired), rt2h 12

26

TFA (deaired), rt2h

OJ

35:65"'^

ifcHri CH3

CH3

.n(\^h

riTi

30:70'

OJ4

CH3

38

If' TFA (de-

cu^

OOj

20:80''''

aired), rt2h

28^' TFA, rtl.5h

70% CH3CH200C^i><; COOCH2CH3 H I H Ph

29^^ TFA (deaired), rt2h

C H 3 C H 2 0 0 C ^ i ^ ^ < ^COOCHzCHs H I H Ph

99%

ri H COOCH3

<.

NC'^nsCH2CH3

XOOCH3

NC^nSCH2CH3 H

30^' TFA (deaired), rt2h

80%

H THI COOCH3

COOCH3

"^COOCH2CH3

COOCH2CH3

31^^

48%

AcOH, reflux 10 d ..COOCH3

_...C00CH3

0:100'

' ^ - ^ 1 Ph

1%HC1/ MeOH, reflux 6 h

COOCH3

COOCH3

33^^

...COOCH3

k

1%HC1/ MeOH, reflux 20 h

Ph

COOCH3

^

C00CH3

0:100"

39

34'^ TFA(2.1

003

.COOCHa

94%

eq.) in CH2CI2, rtl7h 82

35

30:70

CH3

10%AcOH CH3OOC

CH3OOC

36^^

CH3

H

CH3

50:50'

10%AcOH reflux 1 h

CH3OOC

CH3OOC

37^^

70:30'

50% AcOH

__ ^ 0 C H 3

^

^QCH3

reflux 48 h

3848

20:80

AcOH

^^OCHa CH3OOC

39^^ HCl/EtOH, reflux 0.5 h

40" HCl/EtOH, reflux 0.5 h

Co

a

I CH3

83%

90%

40

41sr

COOCH2CH3

CH30,

COOCH2CH3

49:51'

sat. HCl/ EtOH,

-: H H

reflux 4 h

sat. HCl/

XXXi

CH20H

CH30,

CH20H

49:51'

EtOH, reflux 22 h

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Cookson, R. C ; Chem. Ind. (London), 1953, 337. Biogenetic numbering: Le Men, J.; Taylor, W. I.; Experientia, 1965, 27, 508. For a review, see: Schlittler, E. In The Alkaloids', R. H. F. Manske, Ed.; Academic Press: New York, 1965; Vol. 8, pp. 287-334. MacPhillamy, H. B; Huebner, C. F.; Schlittler, E.; St. Andre, A. F.; Ulshafer, P. R.; J. Am. Chem. Soc, 1955, 77, 4335. Hakkesteegt, T. J.; Pharm. Weekblad, 1970,105, 801. Kametani, T.; Ihara, M.; Heterocycles, 1976, 5, 649. Cox, E. D.; Cook, J. M.; Chem. Rev., 1995, 95, 1797. Lounasmaa, M.; Bemer, M.; Tolvanen, A.; Heterocycles, 1998, 48, 1275. Parker, S. P., Ed.; Dictionary of Chemistry, McGraw-Hill: New York, 1997, p. 141 and 207. Wenkert, E.; Liu, L. H.; Experientia, 1955,11, 302. Woodward, R. B.; Bader, F. E.; Bickel, H.; Frey, A. J.; Kierstead, R. W.; Tetrahedron, 195S, 2, 1. Lounasmaa, M.; Bemer, M.; Brunner, M.; Suomalainen, H; Tolvanen, A.; Tetrahedron, 1998, 54, 10205. Cox, E. D.; Hamaker, L. K.; Li, J.; Yu, P.; Czerwinski, K. M.; Deng, L.; Bennett, D. W.; Cook, J. M.; J. Org. Chem., 1997, 62, 44. Lounasmaa, M.; Bemer, M.; Tolvanen, A.; Heterocycles, 1999, 50, 243. Gaskell, A. J.; Joule, J. A.; Tetrahedron, 1967, 23,4053. Muiioz, M. A.; Carmona, C ; Hidalgo, J.; Balon, M.; Heterocycles, 1989, 29, 1343. Royer, D.; Doe de Maindreville, M.; Laronze, J.-Y.; Levy, J; Wen, R.; Tetrahedron, 1996, 52, 9069. Lounasmaa, M.; Bemer, M.; Tolvanen, A; Jokela, R.; Heterocycles, 2000, 52, 471.

41

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Nicolaou, K. C ; Sorensen, E. J. Classics in Total Synthesis, VCH Publishers, Inc.: New York, 1996, pp. 62-63. Saxena, A. K.; Jain, P. C ; Anand, N.; Indian J. Chem., 1974,12, 892. Rosentreter, U.; Bom, L.; Kurz, J.; J. Org. Chem., 1986, 51, 1165. Lounasmaa, M.; Bemer, M.; Mansus, A.; Tolvanen, A.; Heterocycles, 1999, 51, 501. Benz, G.; Riesner, H.; Winterfeldt, E.; Chem. Ber., 1975,108, 248. Wenkert, E.; Moeller, P. D, R.; Shi, Y.-J.; J. Org. Chem., 1988, 53, 2383. Wenkert, E.; Guo, M.; Pestchanker, M. J.; Shi, Y.-J.; Vankar, Y. D.; J. Org. Chem., 19S9, 54, 1166. Schiffl, E.; Pindur, U.; Arch. Pharm. (Weinheim), 1986, 319, 443. Hester, J. B., Jr.; J. Org. Chem., 1964, 29, 2864. Jackson, A. H.; Naidoo, B.; Smith, P.; Tetrahedron, 1968, 24, 6119. Zhang, L.-H.; Gupta, A. K.; Cook, J. M.; J. Org. Chem., 1989, 54, 4708. Martin, S. F.; Rlieger, H.; Williamson, S. A.; Grzejszczak, S.; J. Am. Chem. Soc, 1987, 109, 6124. Sakai, S.; Ogawa, M.; Heterocycles, 1978,10, 67. Zhang, L.-H; Cook, J. M.; Heterocycles, 1988, 27, 1357. Zhang, L.-H.; Bi, Y.-Z.; Yu, F.-X.; Menzia, G.; Cook, J. M.; Heterocycles, 1992, 3^,517. Chrisey, L. A.; Brossi, A.; Heterocycles, 1990, 30, 267. Reddy, M. S.; Cook, J. M.; Tetrahedron Lett., 1994, 35, 5413. Lounasmaa, M.; Miikki, L.; Tolvanen, A.; Tetrahedron, 1996, 52, 9925. Duan, J.-A.; Williams, L D.; Che, C.-T.; Zhou, R.-H.; Zhao, S.-X.; Tetrahedron Lett., 1999, 40, 2593. Bemer, M.; Tolvanen, A.; Jokela, R.; Tetrahedron Lett, 1999, 40, 7119. Fry, E. M.; J. Org. Chem., 1964, 29, 1647. Gribble, G. W.; Nelson, R. B.; J. Org. Chem., 1973, 38, 2831. Dastoor, N. J.; Gorman, A. A.; Schmid, H.; Helv. Chim. Acta, 1967, 50, 213. Wenkert, E.; Udelhofen, J. H.; Bhattacharyya, N. K.; /. Am. Chem. Soc, 1959, 81, 3763. Seaton, J. C ; Nair, M. D.; Edwards, O. E.; Marion, L.; Can. J. Chem., 1960, 38, 1035. Wenkert, E.; Wickberg, B.; Leicht, C ; Tetrahedron Lett., 1961, 822. Johns, S. R.; Lamberton, J. A.; Tetrahedron Lett, 1966, 4883. Hart, N. K., Johns, S. R.; Lamberton, J. A.; Chem. Commun., 1967, 87. Beecham, A. F.; Hart, N. K.; Johns, S. R.; Lamberton, J. A.; Aust J. Chem., 1968,27,491. Trager, W. F.; Lee, C. M.; Phillipson, J. D.; Haddock, R. E.; Dwuma-Badu, D.; Beckett, A. H.; Tetrahedron, 1968, 24, 523. Hemingway, S. R.; Houghton, P. J.; Phillipson, J. D.; Shellard, E. J.; Phytochemistry, 1973,14, 557. Finch, N.; Taylor, W. I.; J. Am. Chem. Soc, 1962, 84, 3871. Laus, G.; Brossner, D., Senn, G.; Wurst, K.; J. Chem. Soc, Perkin Trans. 2, 1996,1931. Laus, G.; J. Chem. Soc, Perkin Trans. 2,1998, 315. Repke, D. B.; Artis, D. R.; Nelson, J. T.; Wong, E. H. F.; /. Org. Chem., 1994, 59, 2164.

42

[54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66\ [67] [68] [69] [70] [71]

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Sawa, Y. K.; Matsumura, H.; Tetrahedron, 1970, 26, 2923. Hedler, L.; Stamm, G.;Weitzell, R.; Starke, K.; Eur. J. Pharmacol, 1981, 70,43. Ferry, N.; Goodhardt, M.; Hanoune, J.; Sevenet, T.; Br. J. Pharmacol, 1983, 78, 359. Ito, Y.; Yano, S.; Watanabe, K.; Yamanaka, E.; Aimi, N.; Sakai, S.; Chem. Pharm. Bull, 1990, 38, 1702. Maurin, Y.; Arbilla, S.; Langer, S. Z.; Eur. J. Pharmacol, 1985, 111, 37. Lambert, G. A.; Lang, W. J.; Friedman, E.; Meller, E.; Gershon, S.; Eur. J. Pharmacol, 1978, 49, 39. Demichel, P.; Roquebert, J.; Br. J. Pharmacol, 1984, 83, 505. Roquebert, J.; Demichel, P.; Eur. J. Pharmacol, 1986,106, 203. Roquebert, J.; Arch. Int. Pharmacodyn. Ther., 1986, 282, 252. Takayama, H.; limura, Y.; Kitajima, M.; Aimi, N.; Konno, K.; Inoue, H.; Fujiwara, M.; Mizuta, T.; Yokota, T.; Shigeta, S.; Tokuhisa, K.; Hanasaki, Y.; Katsuura, K.; Bioorg. Med. Chem. Lett, 1997, 7, 3145. Kanatani, H.; Kohda, H.; Yamasaki, K.; Hotta, L; Nakata, Y.; Segawa, T.; Yamanaka, E.; Aimi, N.; Sakai, S.; J. Pharm. Pharmacol, 1985, 37,401. Masumiya, H.; Saitoh, T.; Tanaka, Y.; Horie, S.; Aimi, N.; Takayama, H.; Tanaka, H.; Shigenobu, K.; Life ScL, 1999, 65, 2333. Yano, S.; Watanabe, W.; Ito, Y.; Horiuchi, H.; Yamanaka, E.; Aimi, N.; Sakai, S.; J. Pharmacobio-Dyn., 1987,10, s-56. Beckett, A. H.; Morton, D. M.; Biochem. Pharmacol, 1967,16, 1609. Wurm, M.; Kacani, L.; Laus, G; Keplinger, K.; Dierich, M.; Planta Med., 1998, 64,101. Wenkert, E.; Roychaudhuri, D. K.; J. Am. Chem. Soc, 1958, 80, 1613. Wenkert, E.; Chang, C.-J.; Chawla, H. P. S.; Cochran, D. W.; Hagaman, E. W.; King, J. C ; Orito, K.; /. Am. Chem. Soc, 1976, 98, 3645. Aldrich, P. E.; Diassi, P. A.; Dickel, D. F.; Dylion, C. M.; Hance, P. D.; Huebner, C. F.; Korzun, B.; Kuehne, M. E.; Liu, L. H.; MacPhillamy, H. B.; Robb, E. W.; Roychaudhuri, D. K.; Schlittler, E.; St. Andre, A. F.; van Tamelen, E. E.; Weisenbom, F. L.; Wenkert, E.; Wintersteiner, O.; J. Am. Chem. Soc, 1959, 81, 2481. Lounasmaa, M.; Jokela, R.; Tetrahedron, 1990, 46, 615. Shamma, M.; Richey, J. M.; J. Am. Chem. Soc, 1963, 85, 2057. Lounasmaa, M.; Din Belle, D.; Tolvanen, A.; Tetrahedron, 1998, 54,4673. Lounasmaa, M.; Jokela, R.; Tirkkonen, B.; Miettinen, J.; Halonen, M.; Heterocycles, 1992, 34, 321. Lounasmaa, M.; Jokela, R.; Heterocycles, 1990, 31, 1351. Wenkert, E.; Halls, T. D. J.; Kunesch, G.; Orito, K.; Stephens, R. L.; Temple, W. A.; Yadav, J. S.; J. Am. Chem. Soc, 1979,101, 5370. Ono, K.; Kawakami, H.; Katsube, J.; Heterocycles, 1980,14, AW. Gmeiner, P.; Feldman, P. L.; Chu-Moyer, M. Y.; Rapoport, H.; J. Org. Chem., 1990, 55, 3068. Lounasmaa, M.; Miikki, L.; Tolvanen, A.; Tetrahedron, 1997, 53, 5349. Hammer, H.; Rosner, M.; Rosentreter, U.; Winterfeldt, E.; Chem. Ber., 1979,112, 1889. Yeoh, G. B.; Chan, K. C ; Morsingh, F.; Tetrahedron Lett., 1966, 931.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

43

ANTITUMOR-PROMOTING AND ANTIINFLAMMATORY ACTIVITIES OF TRITERPENOIDS AND STEROLS FROM PLANTS AND FUNGI TOSHIHIRO AKIHISA' and KEN YASUKAWA^ ^College ofScience and Technology, Nihon University, 1-8 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan ^College of Pharmacy Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan ABSTRACT: Triterpenoids and sterols occur in all major groups of organisms from fungi to humans as secondary metabolites. During our search for active cancer chemopreventive agents in edible plants and fungi, and in medicinal herbs, we have found that various triterpenoids, including triterpene alcohols, and sterols isolated from these sources to possess inhibitory activities on inflammation induced by 12-(9tetradecanoylphorbol-13-acetate (TPA), a well known tumor promoter, and for tumor promotion in two-stage carcinogenesis in mice. In this review we discuss the antitumor-promoting activities of triterpenoids and sterols, isolated from plants and fungi and their derivatives, resulting from our own and other research groups' investigation. Triterpenoids and sterols are minor but ubiquitous components of our diet, and are considered to be relatively non-toxic to humans. These compounds, therefore, constitute possible candidates for cancer chemopreventive agents. Anti-inflammatory and anti-allergic properties of triterpenoids and sterols evaluated in various assay systems are also summarized.

INTRODUCTION Cancer prevention is now one of the most urgent projects in public health. It is evident that an understanding of the mechanisms of the carcinogenesis process is essential for cancer chemoprevention. Most cancer prevention research is based on the concept of multistage carcinogenesis: initiation—> promotion^progression [1-3] [Fig. (1)]. Among these stages, in contrast to both the initiation and progression stage, animal studies indicate that the promotion stage takes a long time to occur and may be reversible, at least early on. Therefore, the inhibition of tumor promotion in the multiple stages is expected to be an efficient approach to cancer control [4-7]. Cancer chemoprevention is defined as the use of specific natural and synthetic chemical agents to reverse or suppress carcinogenesis and prevent the development of invasive cancer. There is a growing awareness in recent years that dietary non-nutrient compounds can have important effects as chemopreventive agents, and there has been considerable work

44

on the cancer chemopreventive effects of such compounds in animal models. Several classes of natural products are now known as chemopreventive agents which included carotenoids, curcumin (turmeric yellow), limonene, quercetin (4), retinoids, tea tannins, and terpenoids [3,6,8-14].

Initiation epidermis

Promotion

' MMWBw.«MuwmaMU4Uwiiiwiiiiiwiwaiitti]yi)iiiiyiii»i^^

Death

^cxX3r)P3P"'o*^3Sc^::v?5rd^^ carcinomas

"• apoptosis no threshold irreversible short-lerm

I Progression

- threshold - reversible - long-term

- invasion - metastasis no threshold irreversible

Fig. (1). Critical steps and characteristics in multistage carcinogenesis [6]

In the course of our research on potential antitumor-promoters (cancer chemopreventive agents) from edible plants and fungi, and from crude herbal drugs, we have found that various triterpene alcohols and sterols and their oxygenated derivatives showed inhibitory effects on inflammation in mouse ear induced by 12-(9-tetradecanoylphorbol-13-acetate (TPA; 1), an in vivo primary screening assay for antitumor-promoters, and on tumor promotion in two-stage carcinogenesis in mouse skin initiated by 7,12-dimethylbenz[a]anthracene (DMBA, 2) and promoted by TPA. In this review, we summarize the inhibitory activities of triterpenoids and sterols (mostly from plants) on TPA-induced inflammation and on tumor promotion during carcinogenesis with DMBA and TPA, as well as in several other assays for antitumor-promoters. In addition, we have summarized anti-inflammatory and anti-allergic activities of these compounds evaluated using various assay systems described in the literature. We will not refer to activities of triterpenoidal and steroidal saponins because they have been subject to recent reviews [15-18]. The

45

Strategy of chemoprevention is different from that of cancer chemotherapy where the agents are directed against cancerous or fully promoted cells and seek to selectively kill the cell based on some aspect of its aberrant biochemical equilibrium [19-21]. BIO ASSAY FOR ANTITUMOR-PROMOTERS Bioassay systems used for screening antitumor-promoting compounds are listed in Table 1. They are classified into in vitro assays using subcellular structures, in vivo anti-inflammation assays induced by the tumor promoters, i.e,, croton oil, 12-0-hexadecanoyl-16-hydroxyphorbol-13aceate (HHPA), and TPA (1), and in vivo experimental carcinogenesis assays. The following in vitro assays are used usually as primary screening assays: Epstein-Barr virus early antigen activation inhibition (EBV) [22-27], TPA-stimulated *^^Pi (radioactive inorganic phosphate) incorporation in HeLa cells (human cervical cancer cells) inhibition (HeLa) [28,29], TPA-induced ornithine decarboxylase inhibition (GDC) [30,31], and protein kinase C (Ca^"*^- and phospholipid-dependent protein kinase) inhibition (PKC) [25,32]. Many compounds that active in EBV and HeLa assays have been proved to be inhibitors of tumor promotion on two-stage carcinogenesis tests in vivo. Assays for the inhibition of croton oil induced edema (CRO) [33,34], HHPA induced edema (HPA) [35], and TPA induced edema (TPA) [31,36,37] are used as rapid in vivo preliminary tests for screening antitumor-promoting substances. Among the existing models for experimental carcinogenesis studies, two-stage carcinogenesis in mouse skin with DMBA and TPA is being performed most frequently [8,37-42] since skin proves to be a unique target to differentiate between the effect of a test agent as either an anti-initiator or an antipromoter under appropriate experimental conditions [8]. Further studies using animal models have been undertaken for carcinogenesis of skin [28,38,41-43], colon [44], liver [45], lung [46], and mammary [42,47] as shown in Table 1. Assay for linhibition of TPA-induced Inflammatory Ear Edema in Mice For screening antitumor-promoting agents, we used TPA-induced inflammatory ear edema in mice [Fig.(2)] [36,37,48]. TPA (l|Lig/ear) dissolved in acetone (20 |LI1) was applied to the right ear of ICR mice by means of a micropipette. A volume of 10 |al was delivered to both the inner and outer surfaces of the ear. The samples or their vehicles, methanol-chloroform-water (2:1:1,20 |Lil) or chloroform (20 |a,l), as control, were applied topically about 30 min before TPA treatment. For ear

46

Table 1. Bioassay Systems for Screening of Compounds with Antitumor-Promotion, Anti-Inflammatory and Anti-Allergic Activities Described in This Review Code Bioassay system Assay for Antitumor Promoters In vivo primary screening assay CRO Croton oil induced edema inhibition HP A 11-0 -Hexadecanoyl-16-hydroxyphorboI-13-acetate (HHPA) induced edema inhibition TPA 12-0 -Tetradecanoylphorbol-13-acetate (TPA; 1) induced edema inhibition In vitro primary screening assay Inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced EBV by TPA TPA-Stimulated ^^Pi incorporation in HeLa cell inhibition HeLa TPA induced ornithine decarboxylase inhibition ODC Protein kinase C inhibition PKC In vivo assay Colon Inhibition of A^-methyl-A^-nitrosourea (MNU) induced colon tumors Liver Inhibition of A^-diethylnitrosamine/phenobarbital induced hepatic tumors Inhibition of 4-(methylnitrosoamino)-l-(3-pyridyl)-l-butanone (NNK) Lung induced lung tumors Mammary I Inhibition of MNU induced mammary tumors Mammary 11 Inhibition of spontaneous mammary tumors Skin I Inhibition of 7,12-dimethylbenz[fl ]anthracene (DMBA; 2)/TPA induced skin tumors (Topical application) Skin II Inhibition of DMBA/TPA induced skin tumors (Oral application)

References"

Skin III application) Assay for Anti-Inflammatory and Anti-Allergic Compounds In vivo assay AA Arachidonic acid induced edema inhibition AIP Adjuvant induced polyarthritis inhibition BRA Bradykinin induced edema inhibition CAR Carrageenan induced edema inhibition CPS Capsaicin induced edema inhibition CTN Cotton pellet granuloma inhibition DEX Dextran induced edema inhibition Delayed-type allergy suppressant activity DTA Ethyl phenylpropiolate (EPP) induced edema inhibition EPP FAA Formaldehyde induced arthritis inhibition HSI Histamine induced edema inhibition ITA Immediate-type allergy suppressant activity PAF Platelet aggregation factor (PAF) acether induced edema inhibition SER Serotonin induced edema inhibition In vitro assay ACM Anticomplementary activity cAK Cyclic AMP-dependent protein kinase inhibition

[28,38]

" Only some representative references were given.

[33,34] [35] [31,36,37,48] [22-27] [28,29,39] [30,71] [25,32] [44] [45] [46] [47] [42] [8,37,39-42] [43]

[56,89,93,104] [105,106] [107] [64,89,108,109] [110] [89] [108] [111,112] [62] [105,108] [107] [112] [107] [64] [113,114] [115,116]

47

Table 1. Continued. Code

COX COX2

HLE HPT HSR HYA iNOS 5L0X 12L0X 15L0X PLA2

SOF

Bioassay system Cyclooxygenase inhibition Inducible cyclooxygenase inhibition Human leucocyte elastase inhibition Hepatoprotective activity Histamine release inhibition Hyaluronidase inhibition Inducible nitric oxide synthase inhibition 5-Lipoxygenase inhibition 12-Lipoxygenase inhibition 15-Lipoxygenase inhibition Phosphoslipase A2 inhibition Inhibition of A^ -formylmethionyl-leucyl-phenylalanine (FMLP) induced superoxide formation from rat neutrophils

Sample Img/ear

TPA l^g/ear

1

Ear Edema Measurement

i 30 min

References'* [93,102,117] [118] [93,119] [115,120] [91,121-123] [124] [97,99-101] [93,102,117] [117] [102,125] [126,127] [122]

1

^~-"—

m

6hr

Fig. (2). Assay of TPA-induced inflammation in mice [37]

thickness determinations, a pocket thickness gauge with a range of 0-9 mm, graduated at 0.01 mm intervals and modified so that the contact surface area was increased to reduce the tension, was applied to the tip of the ear. The ear thickness was measured before treatment (a), and 6 h after TPA treatment (b =TPA alone; b' =TPA plus sample). The following values were then calculated: Edema A is induced by TPA alone (b - a). Edema B is induced by TPA plus sample (b' - a). Edema A ~ Edema B Inhibition ratio (%) = x 100 Edema A Each value was the mean of individual determinations from 4-5 mice. The 50% inhibitory dose (ID50) values were determined by the method of probit-graphic interpolation for four dose levels. This assay is

48

advantageous since it requires only few milligrams of sample, and it can be performed within one day. The activities of inhibitors on TPA-induced inflammation almost parallel their inhibitory activities on tumor promotion [49]. Assay for linhibition of DMBA/TPA Induced Skin Tumors in Mice For the highly inhibitive compounds in the TPA-induced inflammatory assay, we evaluated their antitumor-promoting activity by a standard initiation-promotion protocol [Fig. (3)] [37,40]. Initiation was achieved by a single application of 50 |ig of DMBA (2), a well known tumor initiator, to the skin of backs of 8-week-old female ICR mice. From one week after initiation, 1 |Lig of TPA (1) was applied twice a week until week 20. The test compound (2.0 or 0.2 |iM) dissolved in acetone-dimethyl sulfoxide (DMSO) (9:1, 100 |il) was applied 30 min before each TPA treatment. Groups of 15 mice were used. The number of tumors was counted weekly. Initiation

Promotion

i{

DMBA DMSO-acetone (195 nM) (,.9)

DMSO-acetone TPA TPA (1.7 nM) (1.7 nM) (1:9) in acetone in apetone

DMSO-acetone (1:9)

t/ 30 min

30 min . Twice a week

1 week

1

1-

DMSO-acetone TPA TPA (1:9) (1.7 nM) (1.7 nM) in Acetone in ^etone

30 min

{ Twice a week

y30 min , 1

20 weeks

i+

DMBA Test compound TPA Test compound TPA Test compound TPA Test compound TPA (195 nM) (2.0 or 0.2 nM) (1.7 nM) (2.0 or 0.2 ^M) (1.7 nM) (2.0 or 0.2 ^iM) (1.7 nM) (2.0or0.2^M) (1.7 nM) in acetone in DMSO-acetone in acetone in DMSO-acetone in acetone in DMSO-acetone in acetone in DMSO-acetone in acetone

1 week

•J-/4 30 min

1

30 min . Twice a week

1

30 min

20 weeks Fig. (3). Method of two-stage carcinogenesis assay in mouse skin [37]

•1: Twice a week

y. 30 min .

49 C13H27COQ

IV. Lanostane

II. Ergostane

(17) 3P-OH. 5:6. 7:8. 9:11. 22:23Etetraene (18) 3P-0H. 5:6, 7:8, 22:23E-triene (19) 3P-0H. 6:7, 22:23E-diene, 5a,8a-epidioxy (20) 3P-0-FER, 5:6-ene, 24R+S Stigmastane

(42) 3a-0Me. 7:8-ene, 23-oxo; 26-COOMe (43)3p-OH. 7:8. 9:11, 24:25triene, 21-COOH (44) 3P-0H, 8:9-ene (45) 7p,15a-diOH, 3, 11, 23-trioxo. 8:9-ene, 26-COOH (46)3p,7p-diOH. 11.15-dioxo, 8:9-ene. 26-COOH (47)3p,7p,15a-triOH. 11.23dioxo, 8:9-ene. 26-COOH (48)7p,12p.diOH. 3. 11,15, 23tetraoxo, 8:9-ene, 26-COOH (49)3p.16a-diOH, 7:8, 9:11, 24:25-triene, 21-COOH (50) 3p,16a-diOH, 8:9, 24:25diene, 21-COOH (51) 3p-0Ac. 16a-0H, 8:9, 24:25-diene. 21-COOH (52) 3P-0H. 8:9. 24:25-diene

(21) 3p,7a-diOAc, 5:6-ene (22) 3p,7p-diOAc, 5:6-ene (23)3p-OH, 5:6, 7:8.9:11. MeO ^ ^ ^^ (5) O ^ 22:23E-tetraene. 24p(R) (24) 3P-0H. 4a-Me. HO,^>S1AAOH 7:8,24:24^Z-diene (25) 3P-0H, 5:6. 7:8. 22:23Etriene.24p(R) (26) 3P-0H. 6:7, 22:23E-diene, O^^v^^^v^ (6) 5a,8a-epidioxy, 24p(R) I. Cholestane (27) 3P-OH, 5:6. 24:24^E-diene (53) 3-0X0, 7:8.9:11-diene. 21-COOH (28) 3p,4p-diOH. 5:6-ene (54)3p-OAc,16a-OH,7:8.9:11-diene. (29) 3p.7a-diOH, 5:6-ene 21-COOH (30) 3p.7p-diOH, 5:6-ene (55) 3p,16a-diOH. 7:8, 9:11-diene, (31) 3P-0H, 5:6-ene, 7-oxo (7) 3P-0H, 5:6-ene 21-COOH (8) 3P-0H, 4.4-diMe, 5:6-ene (32) 3p-0H. 7:8-ene (56) 3a.16a-diOH. 7:8. 9:11-diene. (9) 3-0X0, 4,4-diMe, 5:6-ene (33) 3B.0H, 5:6-ene 21-COOH (10)3p-OH,4,4-diMe, (34) 3P-0-FER. 5:6-ene (57) 3p-0-Bz-p.0H. 16a-0H.

rrr

5a,6a-epoxy

(35) 3P-0H. 7:8, 22:23E-diene (11) 3-0X0. 4,4-diMe. (36) 3p.0H. 5:6. 22:23E-diene 5a,6a-epoxy )^J. 00 ^o L.^L. ^ r (12) 3a,5a-diOH. 4.4-diMe (37) 3p.6p-diOH. 4:5-ene (13) 3P-0H. 4.4-diMe, 7:8-ene(38) 3p,6a-diOH (14) 3p.0H, 4a-Me, 7:8-ene (39) 33 ep-diOH (15) 3-OH, 4-Me. 3:4-ene ) ' J^' ^.^ 16 3p.0H, 5:6-ene, 7-0X0 ^ ^ J ' ^ ' f ^° ^ ^ (41)3,6-dioxo, 4:5-ene

Fig. (4). Structures of compounds described in this review

7:8. 9:11-diene. 21.C00H (^^l f'^^^ ^(11)^ne (59) 3p-0Ac. 16a-0H. 8:9-ene. 21-CO6H (60)3-0X0. 16a,25-diOH, 7:8. 9:11-diene. 21-COOH (61) 3p.16a-diOH.8:9-ene.21-COOH

50

(81) 24:25-ene (82) 24,25-epoxy (24R+S) (83) 20:21-dlhydro(20R). 13:17, 24:25-dJene (84) 20:21-dihydro(20S). 13:17. 24:25-dlene (85) 24-methylene

HOOC/,

ROOC

(62) (63) (64) (65)

R=H, 24-methylene R=Me, 24-methylene R=H, 24:25-ene R=H, 24-methylene, 25-OH

VIII. Euphane

7C^^^ 'h

(99) 8:9, 24:25-dlene (100)7:8, 24:25-diene (101) 7:8-ene, 24,25-epoxy (24R+S)

V. Cycloartane

IX. Tirucallane

21//,.

HOOC

VII. Cucurbitane (66) 3 P - 0 H . 24:25-ene (102) R=oxo. 7:8, 24:25-diene, (67) 3P-0-FER, 24:25-ene 27-COOH (68) 3-0X0, 21-COOH, 24,25(103) R = a - O H . 7:8, 24,25-diene, epoxy (24R+S) 27-COOH (69) 3 P - 0 H . 21-CHO, 24:25-ene (104) R=p-OH. 7:8. 24:25-diene (70) 3 P - 0 H , 21-COOH. 24:25-ene (105) R=p-OH, 8:9, 24,25-diene (71) 3-0X0, 21-COOMe, 24,25epoxy (24R+S) RO^ (72) 3 P - 0 H . 24-methylene X. Quasslne QH (88) R=H (73) 3P-0-FER, 24-methylene H04^x%,N\C00Me (89) R=H, 24:25-dihydro (74) 3 P - 0 H . 21-COOMe, (90) R=H, 7-0X0 24:25-ene (75) 3-0X0, 21-COOMe, 24:25-ene (91) R=Ac, 7-0X0 (92) R=H, 7-0X0, 24:25-dihydro (76) 3-0X0, 21-CHO, 24:25-ene

xffix: H

(77) 3-0X0, 2 1 - C O O H , 24:25-ene (78) 3-0X0, 2 I - C H 2 O H , 24:25-ene

(106)3:4-ene. R=Bz ^R^107)3:4-ene,R=Q

2V/,.

CHO

(93) R = a - O H , R^=H,

R2=AC

(94) R=oxo R^=H, 23:24.ene, R2=AC

(95) R=a-OH, R^=H,23:24-ene, R2=H

(96) R=a-OH,

=H. R^=H

(97) R=a-OH.

=H, 15-0X0,

2=L 23:24-ene. R''=H

( 9 8 ) R = a - O H , R ^ = H , 15-0X0, R2=H

Fig. (4). Continued-1

(108)3:4-ene,R=^

"^

(109)3:4-ene, R=^

"^

(IIO)ip-OH,

"^

(111)ip-0H, (112)3-OH. 3:4-ene

-•or

(113)R=Ac,3:4-ene

OAc

51 (137) 3p,16p-diOH, 28-CH2OH (138)3|3-OH, 16P-0H (139) 3P.O-OCC13H27. 16P-0H (140)3p-O-OCCi5H3i, 16P-0H (141) 2a.3p-diOH. 28-COOH (142)3p-OH,28-COOH (143)3-0X0, 28-COOH (144)3p.16p-diOH (145) 3P-0H, 28-COOH, 3O-CH2OH (160)R=H,R^=P-OH, R2= Me '2=1Me (161)R=H,R^=oxo, R' 2=r (162) R=H, R'=oxo, R^=CH20H 3l=, (163) R=H, R'=:p-OH, R^=CH20H

COOR (146) R=H, 2a-0H. R^=COOH (116)3p-OAc,27-COOH R^=Me (117)3p-OAc,28-CH20H (118) 3P-0AC. 28-COOH (147) R=H, R^=Me. R 2 = C 0 0 H 164) R=H,2-CN. 12:13-ene (119)3p-OH 165) R=H.2-CN, 9:11-ene, (148) R=0-CCi7H35, R^=Me. (120)3p-OAc 12-0X0 R2=COOH (121)3p-OH,30.CH2OH 166)R=H. 11-0X0.12:13-ene (149) R=PHT-Na. R^=Me, (122)3p-0-PHT-Na, 167)R=H. 11-0X0.13:18-ene R2=COOH 30-CH2O.PHT-Na 168) R=H, 12-0X0 (123)3p-0-SUC-Na, (150)R=PHT-Na,R^=Me, 169) R=H, 9:11-ene, 12-0X0 30-CH2O-SUC-Na R2=C00Na 170)R=H.9:11-epoxy (124)3p-0-P03Na2, 171) R=H,2-0H. 12:13-ene (161)R=H, R^=Me, 30-CH2OPO3Na2 172)R=H,2-OMe, 12:13-ene (125)3p-0-PHT-Na, 173) R=Me, 12:13-ene 3O-CH2OH 174) R=H (126) 2a,3p-diOAc,28-COOH 175)R=H.9:11-ene (127)1,3-dloxo 176)R=H, 12:13-ene (128) 3p,16p-diOH. 28-COOH 177) R=H,2-CHO. 12:13-ene (129)3p,15a,16a-triOH. ;i78)R=H, 11:12, 13:18-diejjie 28-COOH (130)3p-OH,28-CH2OH (131) 3-oxo,22p-OH.28-COOH (152)R=H,2a-OH, R'=CH20H. (132) 3-0X0. 22P-0-TIG. R2=C00H 28-COOH (153)R=H. 2a-0H. R^=CH20H. (133)3p-OH.22p-0-TIG. R2=COOMe 28-COOH (154)R=Ac,2a-OAc, R^=CH20Ac. R 2 = C 0 0 H (179) R=H. Me, 13:18-ene >1. (155)R=Ac.2a-OAc, (180) R=H, R'=Me, 18:19-ene (134) 3-0X0.22pR^=CH20Ac, R2=C00Me (181) R=H. R^=CH20H. 28-COOH (156) R = H , R ^ = C H 2 0 H . R 2 = C O O H 9:11. 12:13-diene (135)3p-OH.22p-i (157)R=H. R ^ = R 2 = C H 2 0 H (182) R=PHT-Na, R^=CH2028-COOH (158)R=PHT-Na, R^=R2=: PHT-Na. 9:11.12:13-diene CH20-PHT-Na (136) 3-0X0.22p-CL^^S^ (183) R=H, R^=CH20H, 28-COOH ^ (159)R=H,2a-OH, R^= 11:12,13:18-dlene CH2OH. R2=Me (184) R=PHT-Na,R^=CH20PHT-Na,11:12.13:18-diene Fig. (4). Continued-2

rr

'TV

52 (137) 3p,16MiOH. 28-CH2OH (138)3p-OH, 16P-0H (139) 3P-O-OCC13H27. lep-OH (140)3p-O-OCCi5H3i, 16P-0H (141) 2a.3p-diOH. 28-COOH (142) 3P-0H. 28-COOH (143) 3-0X0, 28-COOH (144)3p.16p-diOH (145) 3P-OH. 28-COOH. 3O-CH2OH (160) R=H, R^=p-OH. R2=Me (161)R=H, R^=oxo, R^=Me (162) R=H. R^=oxo. R 2 = C H 2 0 H (163) R=H. R^=p-OH. R 2 = C H 2 0 H

COOR (146) R=H. 2a-0H. R^=COOH. (116)3p-OAc.27-COOH R2=Me (117)3p-OAc.28-CH20H (118) 3p-0Ac, 28-COOH (147) R=H, R^=Me, R 2 = C 0 0 H 164)R=H.2-CN, 12:13-ene (119)3p-OH 165) R=H.2-CN, 9:11-ene. (148) R=0-CCi7H35. R'=Me (120)3p-OAc 12-0X0 R^=COOH (121)3p-OH.30-CH2OH 166)R=H, 11-0X0.12:13-ene 1=11 (149) R=PHT-Na, R'=Me (122)3p-0-PHT-Na, 167)R=H. 11-0X0,13:18-ene R2=COOH 30-CH2O-PHT-Na 168) R=H, 12-0X0 (150)R=PHT-Na,R'=Me (123)3p-0-SUC-Na, 169) R=H, 9:11-ene, 12-0X0 30-CH2O-SUC-Na R2=C00Na 170)R=H.9:11-epoxy (124)3p-0-P03Na2. 171) R=H.2-0H, 12:13-ene (151)R=H.R^=Me, 30-CH2OPO3Na2 172)R=H,2-OMe. 12:13-ene R2=COO-CCI7H35 (125)3p-0-PHT-Na. 173) R=Me. 12:13-ene 3O-CH2OH 174) R=H (126) 2a.3p-diOAc.28-COOH 175) R=H. 9:11-ene (127)1.3-dioxo 176) R=H. 12:13-ene (128) 3p.16p-diOH, 28-COOH 177) R=H,2-CH0.12:13-ene (129)3p,15a,16a-triOH. 178) R=H. 11:12. 13:18-diejjie 28-COOH (130)3p-OH,28-CH2OH (131)3-oxo,22p-OH, (152) R=H. 2a-0H, R^=CH20H, 28-COOH R2=COOH (132)3-0X0. 22P-0-TIG. (153) R=H. 2a-0H. R^=CH20H. 28-COOH R2=C00Me (133)3p-OH.22p-0-TIG. (154)R=Ac,2a-OAc. 28-COOH 32^, R'=CH20Ac. R^=COOH (179) R = H 13:18-ene (155)R=Ac,2a-OAc. 18:19-ene (134) 3-0X0,22p. R'=CH20Ac. R^=COOMe ( I 8 I ) R=H. R^=CH20H 28-COOH 32=. (156) R=H.R'=CH20H.R^=C00H 9:11.12:13-diene (157)R=H, R ^ = R 2 = C H 2 0 H (135)3p-OH,22p-< (182) R=PHT-Na. R^=CH2028-COOH (158)R=PHT.Na. R^=R2= PHT-Na, 9:11, 12:13-diene CH20-PHT-Na (136) 3-0X0,22p-OLx'*v^/ (183) R=H, R^=CH20H, 28-COOH ^ (159)R=H,2a-OH, R^= 11:12.13:18-diene CH2OH, R2=Me (184) R=PHT-Na,R^=CH20PHT-Na.11:12.13:18-diene Fig. (4). Continued-2

Rcrys

'XT

^CY

53

COOH

(207) 3-0X0, 1:2-ene, 28-COOH XIV. Taraxastane (208) 2a.3p,19a-triOH, 28-COOH (209)3a.16p-OH (210) 3P-OH. 28-COOH (211)3p-OH,28-CH20H

(185)2a,3p-diOAc, 18P-0H, 5:6,12:13-diene (186) 2a,3p-diOAc, 5:6.12:13-diene (187)2p.3a-diOAc. 5:6, 12:13-diene (188) 2a,3a-diOH, 5:6,12:13-diene (189) 3-0X0, 11:12,13:18-diene (190)3a-OH, 11:12,13:18-diene

Multiflorane p (230) 3p.16p-diOH, 20:30-ene (231) 3P-O-OCC15H31, 16P-0H. 20:30-ene (232) 3p,16p-diOH. 20:21-ene (233) 3P-O-OCC13H25.16P-0H. 20:21-ene (234) 3P-O-OC15H31.16P-0H. 20:21-ene (235) 3p,16p-diOH, 28-CH2OH, 20:21-ene (236)3p,16p,22a-triOH, 20:21-ene (237)20:21-ene (238) 3P-0H, 20:21-ene (239) 3P-0H, 20:30-ene (240) 3P-0AC. 20:30-ene

(212)3p-OH, R=CH20H (213)3p-0-SUC-K, R=CH20H (214) 3P-0-SUC-K, R=CH20-SUC-K (215)3p-0-PHT-K, R=CH20-SUC-K (216)3p-OH, R=COOH (217) 3P-0-SUK-K, (191) R=H,R^=R2=Me,R^=CH20H R=COOK (192) R=PHT-Na. R^=R2=Me, (218)3p-0-PHT-K, R^=CH20-PHT-Na R=COOK (219)3a-OH. R=CH20H (193)R=H, R^=R2=Me, R^= XV. Taraxerane (220) R=Me COOH. 11-0X0 (221)3p-OH,R=Me. 7-0X0 (194) R=H,R^=R^=Me,R2=CH20H (222) 3a-0H. R=CH20H. 7- 0x0 (195) R=H,R^=R2=CH20H,R2=Me -. R^

XII. Ursane

~ (241) (242) 3P-0H

2^ 24

(196) 3p- OAc. 11-0X0, 24-COOH (197) 3p- OH (198)3p- OAc (199) 3p- O-OCC15H31 (200) 3p- O-OCC17H31 (201) 3a OH, 24-COOH (202) 3p, 16p-diOH (203) 3p- O-OCC13H27,16P-0H (204) 3p- O-OCCisHai.iep-OH (205) 3,1 1-dioxo, 1:2-ene, 28-COOH (206) 3 a-OH, 28-COOH

Fig. (4). Continued-3

XVI. Glutlnane (223)3p-OH, 7:8.9:11-diene, R=CH20H (224) 3P-0-SUC-K, 7:8, 9:11diene, R=COOK (225) 3a-0H, 7:8. 9:11-diene. R=CH20H (226) 3a-0Bz. 7:8. 9:11dlene, R=CH20H (227) 3P-0H, 7:8-ene. R=Me (228)9:11-ene. R=Me (229) 3a-0Ac. 7-oxo. 9:11-ene, R=CH20Ac

(243) 3P-0H. 5:6-ene. 10a-H (244)5:10-ene

54

XVII. Friedelane ROOq..

(259)3p-0-OCi5H3i. XXi. Arborinane 16P-0H (260) 3a-0H (261)3p,16p-dlOH, 28-CH2OH (262) 3P-0H. 3O-CH2OH (263) 3P-0H, 30-CHO (281) R=H (264) 3P-0H (282) R=Ac (265) 3p.0Ac (266) 3P.O-OCC15H31 XXII. Other Triterpenes (267) 3P.O-OCC17H31 (268) 3-0X0 (269) 3P-0-CAF, 28-COOH \^ (270) 3P.O-SO3K, 28-COOH (271) 3P-0-SUC, 28-COOH HO

(247) 3P-0H (248) 3-0X0 HOOa.

(272) 28-COOH (273) (274)12:13-ene, 18P-H

(249) R=H (250) R=Ac

HOO9.,

XIX. Hopane , H '

H I

OH

(251)

XVIII. Lupane

(275)17:21-ene, R=H OH OH

(276)R=^X^

OH OH

3 0 v ^

(277)13:18-ene (278)12:13-ene (252) 3p-0Ac, 30-CHO (253) 3p-0Ac. 28-COOH (254) 3P-0BZ, 28-COOH (255) 3P-0H, 28-CH2OH (256) 3-0X0, 28-CHO (257) 3P-0H, 28-COOH (258)3p, 16p-diOH

XX. Moretenane

H nr

^\H

(279) R = H (280)

Fig. (4). Continued-4

R=AC

(288)

^^

55 XXIII. Spirostane

XXV. Phytoecdysone

*

^

0

H OMe

FER (feruroyi) (289) 3P-0H, 12-0X0 (290) 3,12-dioxo, 4:5-ene (291)3|3-OH

(295) R=H.

Ritf^^_

ZQH\—0

XXIV. Cardiac steroid

(296) R=H, Ri="^^p^'^'Y^O (297)R=OH. R^=^1f^^^'^^H Structures of abbreviated moieties

o

P

PHT (pthalyl)

.OH O s u e (succinyl)

(292) R=H, R^=H, R2=Me (293) R=H, R^=OH, R 2 = C H 2 0 H ,

^

11a-0H (294) R=H, R^=OH. R^=CHO

Ac (acetyl) Bz (benzoyl)

OH CAP (caffeoyi)

. TIG (tigulyl)

Fig. (4). Continued-5

INHIBITORY EFFECTS OF TRITERPENOIDS AND STEROLS ON PRIMARY SCREENING ASSAYS FOR ANTITUMORPROMOTERS Table 2 lists all compounds discussed in this paper. Their structures are shown in Fig. (4). They are oxygenated compounds having the following skeletons: regular steroids [cholestane (I), ergostane (II), and stigmastane (III)], tetracyclic triterpenoids [lanostane (IV), cycloartane (V), dammarane (VI), cucurbitane (VII), euphane (VIII), tirucallane (IX), and quassine (X)], pentacyclic triterpenoids [oleanane (XI), ursane (XII), multiflorane (XIII), taraxastane (XIV), taraxerane (XV), glutinane (XVI), friedelane (XVII), lupane (XVIII), hopane (XIX), moretenane (XX), and arborinane (XXI)], other plant steroids [spirostane (XXIII), cardiac steroid (XXIV), and phytoecdysone (XXV)]. Most of the semi-synthetic compounds listed were preparedfromnatural products by simple chemical modification. The bioassay systems in which the compounds exhibited inhibitory effects, together with the major sources of the compounds, are included in the Table. Inhibitory Effects on TPA, HHPA, and Croton Oil-Induced Inflammatory Edema

56

The inhibitory effects of the sterols and triterpenoids on TPA-induced inflammatory ear edema in mice are shown in Table 2. The inhibitory effects of three reference compounds, quercetin (4), a known inhibitor of TPA-induced inflammation in mice, and of two commercially available anti-inflammatory drugs, indomethacin (5) and hydrocortisone (6), were included for comparison. As is evident from Table 2, most of the compounds examined exhibited activity almost equivalent to or higher than quercetin (4). Inhibitory effects on the other experimental models were also included in Table 2. Sterols and Their Derivatives Cholesterol (7), a representative animal-sterol, did not show an appreciable inhibitory effect, whereas its 24-ethyl homologues, sitosterol (33) and stigmasterol (36) [50], the most ubiquitous phytosterols, exhibited activity almost equivalent to that of 4. A^-Unsaturated sterols, schottenol (32) and spinasterol (35), were more active than their A^-isomers, 33 and 36, respectively [50]. Ergosterol (18), a typical fungal sterol possessing a A^* -conjugated-diene system in the nucleus, exhibited higher activity than those of the above animal- and phytosterols [36,51]. Oxygenation of the nucleus of sterol usually enhanced the activity. Thus, all of the oxygenated sterols assayed, with the exception of 7a-hydroxysitosterol (29) and its diacetate (21), markedly inhibited inflammation induced by TPA [51,52]. Acetylation of the hydroxyl group at C-3 exerted almost no influence on the activity of sterols (29/21 and 30/22) whereas esterification with ferulic acid enhanced the activity (34/33). Methylation at C-4 of sterol nucleus was one of the other factors affecting activity enhancement. Thus, in general, 4-methylsterols (14,15) and 4,4dimethylsterols (8,13) exhibited higher activity than 4-desmethylsterols. A similar structure-activity relationship was observed also in the HHPA-induced inflammation on mouse ear [35]. Whereas cholesterol (7) did not show inhibitory activity, several 4,4-dimethylcholestane derivatives, 8-12, exhibited activity. 4,4-Dimethylcholestane-3a,5a-diol (12) was the most potent inhibitor: its activity was comparable to that of ursolic acid (210) [35]. Compound 12 reduced also the inflammation induced by teleocidin B (3), one of the indole alkaloid-type of tumor promoters [53]. Triterpenoids Most of the tetracyclic and pentacyclic 3-monohydroxy triterpenoids

57

examined exhibited higher activity than 4-desmethylsterols. This is consistent with the above observation that C-4 methylation of a steroid or triterpenoid nucleus enhances the activity. Further oxygenation, e.g., hydroxylation, carbonylation, or carboxylation, in addition to at C-3 enhanced the inhibitory effect of triterpene alcohols as has been observed for lanostane-, cucurbitane-, oleanane-, ursane-, taraxastane-, and lupane-type compounds. Several lanostane-type compounds, viz., dehydroeburiconic acid (53), dehydropachymic acid (54), dehydrotrametenolic acid (43), 16a-hydroxytrametenolic acid 3-O-acetate (51), poricoic acid A (62), and poricoic acid B (64) [37,54-56] showed a strong inhibitory activity which was at the same level as hydrocortisone (6). Twenty-five triterpenoids from Compositae flowers, viz., twelve monohydroxy, seven dihydroxy, and four trihydroxy triterpenoids, were evaluated with respect to their anti-inflammatory activity induced by TPA [57-59]. It was observed that these triterpenoids examined markedly inhibited the inflammation with 0.03-0.8 mg/ear of the 50% inhibitory dose. There was a close relationship between the hydroxylation of triterpenoids and the inhibitory effects. Di- and trihydroxy triterpenoids always showed higher activity than their corresponding 3 p-monohydroxy compounds. In the case of A -taraxastenes, faradiol (232; 0.2 mg/ear), showed a higher inhibitory effect than its 3p-monohydroxy homolog, v|/-taraxasterol (238; 0.4 mg/ear). Further hydroxylation of 232 at C-22a to give heliantriol C (236; 0.03 mg/ear) enhanced the effect markedly. Heliantriol C (236), brein (202; ursane deriv.), and heliantriol B2 (261; lupane deriv.) showed a fairly strong inhibitory effect which was almost comparable with that of hydrocortisone (6). Esterification with a fatty acid or with ferulic acid at C-3 of triterpene alcohols exerted almost no influence on the activity as has been observed with lupane- (264/265) and cycloartane- (66/67, 72/73) type compounds and a C-3 monohydroxy triterpenoid (264/266). However, esterification at C-3 with a fatty acid reduced the inhibitory activity of some dihydroxy triterpenoids (138/139,140; 202/203, 204; 230/231; 232/233, 234). Three lanostane carboxylic acids, 16a-hydroxydehydrotrametenolic acid (49) [60], 16a-hydroxytrametenolic acid (50) [60], and dehydrotumulosic acid (55) [60,61], and dehydropachymic acid (54) [60] and pachymic acid (59) [61], have been reported to be active against topical anti-inflammatory activities induced by TPA. Ten pentacyclic triterpenoids, viz., six oleananes (119,130,142, 147,156, and 193), three ursanes (197,208, and 211), and one lupane (264) were considerably active against TPA-induced edema. Of those compounds, the triterpenoid acids (142,147,193, and 208) were the most active. However, erythrodiol (130) surpassed all others [62]. Three triterpenoids 210 [63], 255, and 257 have been evaluated by the TPA-induced inflammatory ear edema assay [64]. The anti-inflammatory effects of glycyrrhetic acid (147), and its

58

derivatives on TPA-induced mouse ear edema were studied [65]. Among the derivatives of 147 tested, six dihemiphthalate derivatives, i.e., di-O-hemiphthalates of olean-12-ene-3p,30-diol (deoxoglycyrrhetol; 120), oleana-9(ll),12-diene-3p,30-diol (181), and oleana-11,13(18)-diene3p,30-diol (183), and their disodium derivatives, 122, 182, and 184, respectively, inhibited most strongly ear edema on both topical (ID50, 1.6 mg/ear for 120,2.0 mg/ear for 181, and 1.6 mg/ear for 183) and oral (ID50, 88 mg/kg for 122, 130 mg/kg for 182, and 92 mg/kg for 184) administration. Compound 147 and deoxoglycyrrhetol (121), the parent compounds, produced little inhibition by oral administration at less than 200 mg/kg. In this study, the mechanisms of TPA-induced ear edema were investigated with respect to the chemical mediators. The formation of ear edema reached a maximum 5 h after TPA application (2 |ag/ear) and the prostaglandin E2 (PGE2) production of mouse ear increased with the edema formation. TPA-induced ear edema was prevented by actinomycin D and cycloheximide (0.1 mg/ear, respectively) when applied during 60 min after TPA treatment. It has been suggested that the dihemiphthalate derivatives of triterpenes derivedfi^om147 by chemical modification are usefiil for the treatment of skin inflammation by both topical and oral application [65]. Among five triterpenoids isolated from Calendula officinalis flowers, P-amyrin (119), faradiol (232), v|/-taraxasterol (238), taraxasterol (239), and lupeol (238), the diol 232 was the most active. It showed a dose-dependent effect with a potency that equals that of indomethacin (5) in the topical anti-inflammatory assay with croton oil [33]. Esterification at C-3 of 232 with a fatty acid reduced the activity by more than 50% [33] consistent with our observation in the TPA-induced assay described above. The anti-inflammatory properties, as determined by croton oil-induced edema of mouse ear, of faradiol-3-O-myristate (233) and its 3-0-palmitate (234), the main components of lipophilic extracts of C officinalis flowers, were shown to be contribute significantly to the pronounced antiphlogistic activity of the lipophilic extracts of C. officinalisflowers[34]. Inhibitory Effects on TPA-induced EBV Activation The in vitro EBV-EA activation inhibition assay uses EBV genome-carrying lymphoblastoid cells (Raji cells derived from Burkitt's lymphoma). Many compounds which inhibit EBV-EA induction by tumor promoters have been demonstrated to act as inhibitors of tumor promotion in vivo [16,22,42,66-68]. This assay has an advantage since it obtaines the information on tiie cytotoxicity from the viability of Raji cells. High viability of these cells is an important factor in developing a compound for the chemoprevention of cancer [16]. So far, a number of triterpenoids and steroids from plants and their derivatives have been shown to possess

59

inhibitory effects on EBV-EA activation induced by TPA. They are derivatives of cycloartane [24], cucurbitane [26,69,70], quassine [71], oleanane [22,23,27,42,72-74], ursane [22,42], multiflorane [67], taraxastane [42,67], taraxerane [42,67], glutinane [67], friedelane [75], lupane [42,72], hopane [67], cardiac steroid [76], and phytoecdysone [68], as shown in Table 2. Glycyrrhetic acid (147) and retinoic acid are known in vivo antitumor-promoters which inhibit EBV-EA induction by tumor promoters [77]. Oleanolic acid (142) and ursolic acid (210) significantly inhibit the activation induced by TPA and teleocidin B (3) as do 147 and retinoic acid [22]. Enhancement of the inhibitory activity was found in 3-oxo derivatives of 142 and 210, while either loss of oxygen functionality at C-3 of 210 or oxidation at C-3 of 147 led to the reduction of the activity [22]. Two 3-0-acetyl oleananes, 3-0-acetylerythrodiol (117; 68% inhibition at 1x10^ mol ratio compound/TPA; 80% cell viability) and acetyloleanolic acid (119; 64% inhibition; 80% viability), showed remarkable inhibitory effects with preserved higher viabilities of Raji cells than their free alcohols, erythrodiol (130; 81% inhibition; 50% viability) and 142 (70% inhibition; 60% viability) [72]. Under the same assay conditions, betulinic acid (257) exhibited complete inhibition of activation with 80% cell viability [72]. Arjunolic acid triacetate (154; 66% inhibition at 1x10^ mol ratio compound/TPA) and arjunolic acid triacetate methylester (155; 73%) [23], and lantadene B (134; 41%) and lantadene C (136; 45%), and the hydroxylated derivative of ketone 134, i.e., 135 (59%) [27], with always >80% cell viability at the assay conditions, appear to have valuable potency as antitumor-promoters. Three cucurbitanes [25-acetyl-23,24-dihydrocucurbitacin F (93; 42% inhibition), cucurbitacin F (95; 37%), and 23,24-dihydrocucurbitacin F (96; 45%)] [69], ten quassines (106 - 115; 100%) [71] (in which enhancement of the activity by a methylenoxy bridge and side chain was observed), a taraxastane (taraxasterol, 239; 64%), and a taraxerane (taraxerol, 242; 57%) [42], two friedelanes (2,3-dihydroxy-24norfriedela-l,3,5(10),7-tetraen-29-oic acid, 249; 57%; and its diacetoxy derivative, 250; 64%) [75], three phytoecdysones: decumbesterone A (296; 34%), cyasterone (297; 24%), and polypodine B (298; 34%) [68], all showed strong inhibitory effects on EBV-EA induction at 1x10 mol ratio compound/TPA with the preservation of high viability of Raji cells. Twenty-three triterpenoid hydrocarbons isolated from ferns were screened [67]. The following eight exhibited strong inhibitory effects at 1x10^ mol ratio compound/TPA with >80% viability of Raji cells [67]. They are: multiflor-8-ene (220; 45%), multiflor-9(ll)-ene (228; 41%), taraxastane (237; 43%), taraxerane (241; 37%), glutin-5(10)-ene (244; 52%), hop-17(21)-ene (275; 51%), neohop-13(18)-ene (277; 37%), and neohop-12-ene (278; 37%). It should be mentioned that the inhibitory

60

effects of these eight compounds were stronger than those of glycyrrhetic acid (147; 46% inhibition at 5x10^ mol ratio compound/TPA and >80% viability of Raji cells) and retinoic acid, which are known to be strong antitumor-promoters [68]. Inhibitory Effects on TPA Stimulated ''^Pi Incorporation in HeLa Cells The inhibitory effect on in vitro TPA-stimulated ^^Pi-incorporation into phospholipids of HeLa cells as the primary screening test is known to correlate well with antitumor-promoter effects in vivo [28,39,78.]. Seventeen triterpenoids isolated from cacti and ten derivatives have been examined for the inhibition of TPA stimulated "^^Pi-incorporation into phospholipids of HeLa cells [79]. Echinocystic acid (cochalic acid; 128; 50% inhibition at 50 |ig/ml of compound with 50 nM of TPA), erythrodiol (130; 42%), queretaroic acid (145; 41%), oleanolic acid (142; 48%), and betulinic acid (257; 100%) showed significant inhibitory activities with 257 being the most active. The conclusion was that, in the case of lupane and oleanane type triterpenoids, the presence of the free carboxyl, formyl, or hydroxymethyl group at C-28 is important for the inhibitory effect [79]. Several other triterpenoids have been reported to be active in this assay, viz., seven oleananes (entagenic acid, 129 [43]; 18a-olean-12-ene3p,28-diol, 194 [39,43]; glycyrrhetic acid, 147 [43]; hederagenin, 156 [43]; olean-12-ene-3p,23,28-triol, 159 [39,43]; and its 18a-epimer, 195 [39,80]; and 18a-deoxoglycyrrhetol, 191 [39,43]); and two other compounds, viz., abiesenonic acid methyl ester (42) [29] and ursolic acid (210) [43]. Inhibitory Effects on TPA-induced ODC Accumulation Induction of epidermal ODC is a characteristic biochemical alteration elicited by TPA and may be representative of the effects of phorbol esters with strong tumor promoting activity [81]. A single application of TPA (5 |ig) resulted in a substantial and transient increase of epidermal ODC activity in mice with a peak at about 4 h after TPA treatment, and the induction was potently inhibited by treatment (5 |LIM) of the mouse skin with sitosterol (33, 65% inhibition) and three lupane type triterpenoids: betulin (255; 79%), betulinic acid (257; 89%), and lupeol (264,96%) [30]. The inhibitory effect on TPA-induced ODC activity was further reported for ursolic acid (210; 45% inhibition at 2.0 |aM/5nM of TPA) [31]. Inhibitory Effects on Protein Kinase C

61

TPA-type tumor promoters can activate both phospholipid and Ca^^-dependent protein kinase C (PKC), an enzyme activated by endogeneous diacylglyceroi released by an activation of phospholipase C. PKC actually constitutes a family of several isozymes and is widely accepted as one of the major intracellular targets of TPA-type tumor promoters. Activated PKC undergoes phosphorylation of proteins regulating cellular differentiations and/or proliferation [6]. Glycyrrhetic acid (147) has been demonstrated to inhibit PKC activity (90% inhibition at 1 mM) [25]. The potent antagonism of tumor promotion in mouse skin by 147 may be a consequence of both its binding interactions with steroid receptors and its inhibition of PKC [25]. Nine different PKC isozymes have been identified recently by cDNA coding [6]. Several lupane type triterpenoids have been examined on the inhibitory effect against isozymes of PKC, such as piI-H, y-H, 5-H, and s-H. Dihydrobetulinic acid (272) and succinyl betulinic acid (271) showed inhibitory effect against 5-H [IC50: 272, 46|aM and 271, 49|iM] and y-H PKC [IC50 : 272, 74^M and 271, 37fiM] [32]. Inhibitory activities against y-H PKC were also observed with compound 254 (a lupane 3-benzoate) and 270 (a lupane 3-sulfate). None of the triterpenoids examined showed inhibition against pII-H and 8-H PKC(IC5o>150^M)[32]. ANTITUMOR-PROMOTING ACTIVITIES OF TRITERPENOIDS AND STEROLS Inhibitory Effects on Skin Tumors Glycyrrhetic acid (147) was the first triterpenoid shown to inhibit the tumor promotion with DMBA and TPA in mouse skin [38]. The inhibitory effect was also demonstrated for oleanolic acid (142) and ursolic acid (210). The activities of 142 and 210 were comparable to that of retinoic acid, a known inhibitor of tumor promotion [66]. For some of the compounds with highly inhibitory activities in the TPA-induced inflammatory assay (Table 2), we have evaluated their antitumor-promoting activity on two-stage carcinogenesis by DMBA and TPA in mouse skin and have found that all of the compounds evaluated possessed remarkable activity. They are two ergostanes: ergosterol (18) [36] and ergosterol peroxide (19) [51], two stigmastanes (sitosterol, 33 [30] and stigmasterol, 36 [50]), three lanostanes (16a-hydroxytrametenolic acid 3-0-acetate, 51 [54], pachymic acid, 59 [54], and poricoic acid B, 64 [54]), a cycloartane (cycloartenol ferulate, 67) [48], an euphane (euphol, 101) [82], a multiflorane (karounidiol, 225) [83], three taraxastanes (faradiol, 232 [40,84,85], heliantriol C, 236 [40,84], and taraxasterol, 239 [84,85]), and four lupanes (betulin, 255 [86], betulinic acid, 257 [30,86],

62 100

U

1 g

o

S

5 10 15 Weeks of promotion

5 10 15 Weeks of promotion

20

Fig. (5). Inhibitory effect of taraxasterol (239) and faradiol (232) on the promotion of skin papillomas by TPA in DMBA-initiated mice. Starting 1 week after initiation by a single topical application of 50 ng of DMBA, 1 ^g of TPA was applied twice weekly. Topical application of 239 (2.0 fimol), 232 (2.0 nmol) and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). • = +TPA with vehicle alone; O = +TPA with 239; A = +TPA with 232 [85].

lupeol, 264 [86], and lupeol acetate, 265 [86]). Of the seventeen compounds evaluated, two 3p-acetoxy-16a-hydroxylanost-8-enes with a carboxyl group at C-21 (51 and 59) and a 3p,16p,22atrihydroxytaraxastene (236), exhibited the highest activity. Fig. (5) A shows the time dependence of skin tumor formation in the groups treated with DMBA plus TPA with or without 2.0 |iM each of taraxasterol (239) and faradiol (232). The first tumor appeared at week 11 in the group treated with DMBA/TPA. In the groups treated with DMBA/TPA and 239, the first tumor appeared at week 13. Of mice treated with DMBA/TPA, 73% were tumor-bearing at week 20, as compared 20% in the group treated with DMBA/TPA and 239. Fig. (5) B shows the average number of tumors per mouse. The group treated with DMBA/TPA produced 7.1 tumors per mouse at week 20, whereas the group treated with DMBA plus TPA and 239 had 1.0 tumor per mouse. The treatment with 232 caused an 80% reduction in the average number of tumors per mouse at week 20. In the group treated with DMBA/TPA and 232, no tumors had appeared at week 20 [85]. Fig. (6) A shows the time dependence of skin tumor formation in the group treated with DMBA/TPA, with or without 0.2 |aM each of 232 and heliantriol C (236). The first tumor appeared at week 8 in the group treated with DMBA/TPA. In the group treated with DMBA/TPA and 232 and 236, the first tumor appeared at weeks 10 and 13, respectively. The percentage of tumor-bearing mice treated with DMBA/TPA was 93% at week 20,

63

100

e

10 o E

50

S

i

*a

S

ON

O

10

15

Weeks of promotion

20

5 10 15 Weeks of promotion

20

Fig. (6). Inhibitory effect of faradiol (232) and heliantriol C (236) on the promotion of skin papillomas by TPA in DMBA-initiated mice. Starting 1 week after initiation by a single topical application of 50 ^g of DMBA, 1 fig of TPA was applied twice weekly. Topical application of 232 (0.2 fimol), 236 (0.2 nmol) and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). • = +TPA with vehicle alone; A = +TPA with 232; O = +TPA with 236 [54].

whereas the percentages in the groups treated with DMBA/TPA and 232 and 236 were 40% and 20%, respectively. Fig. (6) B shows the average number of tumor per mouse. The group treated with DMBA/TPA produced 8.6 tumors per mouse at week 20, whereas the group treated with DMBA/TPA and 232 and 236 had 2.9 and 0.9 tumors per mouse, respectively. The treatment with 232 and 236 caused 66% and 90% reductions, respectively, in the average number of tumors per mouse at week 20 [40]. Of the three compounds tested, the trihydroxy compound 236 was the best anti-tumor promoter, followed by the dihydroxy compound 232 and monohydroxy compound 239 of which activity was paralleled with the inhibitory effect on TPA-induced inflammation (Table 2). Tests of variousflavonoidsdescribed in the literature [87] also showed a close relationship between the inhibitory activities on tumor promotion and on TPA-induced inflammation. Fig. (7) A and shows the time dependence of skin tumor formation in the groups treated with DMBA/TPA, with or without 0.2 ^iM each of three lanostanes, pachymic acid (59), 16a-hydroxytrametenolic acid 3-0-acetate (51), and poricoic acid B (64) [54]. Thefirsttumor appeared at week 11 in the group treated with DMBA/TPA. In the groups treated with DMBA/TPA and 59, 51, and 64, the first tumor appeared at week 13, 14, and 13, respectively. The percentage of tumor-bearing mice treated with DMBA/TPA was 73% at week 20, whereas the percentages in the groups

64 100

^

JB 08

o B 5 10 15 Weeks of promotion

20

5 10 15 Weeks of promotion

20

Fig. (7). Inhibitory effect of pachymic acid (59), 16a-hydroxytrametenoIic acid 3-O-acetate (51) and poricoic acid B (64) on the promotion of skin papillomas by TPA in DMBA-initiated mice. Starting I week after initiation by a single topical application of 50 fig of DMBA, 1 ^g of TPA was applied twice weekly. Topical application of 59 (0.2 ^mol), 51 (0.2 ^mol), 64 (0.2 ^mol) and vehicle was performed 30 min before each TPA treatment. Data are expressed as percentage of mice bearing papillomas (A), and as average number of papillomas per mouse (B). # = +TPA with vehicle alone; O = +TPA with 59; A = +TPA with 51; A = +TPA with 64 [40].

treated with DMBA/TPA and 59, 51, and 64 were 27, 33, and 33%, respectively. Fig. (7) B shows the average number of tumors per mouse. The group treated with DMBA/TPA produced 7.1 tumors per mouse at week 20, whereas the groups treated with DMBA/TPA and 59, 51, and 64 had 1.2, 0.6, and 2.3 tumors/mouse, respectively. The treatments with 59, 51, and 64 caused a 83, 92, and 68% reduction, respectively, in the average number of tumors per mouse at week 20 [54]. The inhibitory effects of 0.2 |LiM of 51,59, and 64, as well as of the two taraxastanes (232 and 236) [40], corresponded to those of 2.0 |LIM of the other sterols (18,19,33, and 36) and triterpenoids (67,101, 225, 239, 255, 257, 264, and 265) described above, and, therefore, compounds 51, 59, and 64 had about ten times the activity of the other compounds on tumor promotion induced by TPA in mouse skin. The following compounds have further been revealed to possess activity on tumor promotion by TPA in two-stage carcinogenesis initiated with DMBA in mouse skin: a lanostane (abiesnonic acid methyl ester, 42) [29]; two cucurbitanes (cucurbitacin F, 95, and 23,24-dihydrocucurbitacin F, 96) [69]; nine oleananes (arjunolic acid triacetate, 154, and arjunolic acid triacetate methylester , 155 [74], erythrodiol, 130 [39], lantadene A, 132, and lantadene B, 134 [45], olean-12-ene-3p,23,28-triol, 157, and 18a-glycyrrhetinic acid, 193 [88], and 18a-olean-12-ene-3p,28-diol, 194, and 18a-olean-12-ene-3p,23,28-triol, 195 [39,43]); a taraxerane (taraxerol,

65

242) [42]; two hopanes [hop-17(21)-ene, 275, and neohop-13(18)-ene, 277] [67]; and a phytoecdysone (cyasterone, 297) [68]. It must be pointed out that the 18a-oleanane triterpenoid 195, prepared from glycyrrhetic acid (147), was 100 times more active than 147 [39]. 01ean-12-ene-3p,23,28-triol tri-0-hemiphthalate sodium (158), on oral administration, has been proved to suppress carcinogenesis in mouse skin induced by DMBA and TPA [43,89]. This is the first report of an effective oral administration of triterpenoid suppressing skin tumor promotion in mice. Glycyrrhetic acid (147) has also been demonstrated to noticeably suppress the promoting effect of teleocidin B (3) on skin tumor formation in mice induced by DMBA [28,38]. Inhibitory Effects on Colon, Liver, Lung, and Mammary Tumors Several sterols and triterpenoids have been shown to possess preventive activities, both on skin tumors and on tumors of internal organs, v/z., colon, liver, lung, and mammary tumors. The compounds that inhibit nitrosamine formation in the assays described below have been suggested to be inhibitors of carcinogen formation and they inhibit 4-(methylnitrosamino)-1 -(3 -pyridyl)-1 -butanone (NNK) induced tumors by acting as blocking (anti-initiating) agents [3]. The inhibitory effect of sitosterol (33) on colon tumor formation in rats treated with the carcinogen A'^-methyl-A^-nitrosourea (MNU) has been studied, and it has been demonstrated that compound 33 nullified in part the effect of this direct-acting carcinogen on the colon [44]. This suggested that phytosterols may have a protective dietary action to retard colon tumor formation. Squalene (287) is an acyclic triterpenoid and a key biosynthetic intermediate for the other cyclic triterpenoids and sterols. Its chemopreventive efficacy on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) has been assessed [90]. Oral administration of 287 inhibited total ACF induction and crypt multiplicity by 46%, and at a level of 1% 287 did not show any significant effect on serum cholesterol (7) levels. Squalene (287) is a characteristic constituent of olive oil, and epidemiologic and laboratory studies have suggested a cancer protective effect and/or lack of a tumor promoting effect by dietary olive oil as compared with other types of non-marine oils. Compound 287 significantly suppressed ACF formation and crypt multiplicity in the colon. This strengthened the hypothesis that 287 has chemopreventive activity against colon carcinogenesis [90]. In addition, compound 287, as well as olive oil, by oral administration, has been revealed to possess inhibitory effect on NNK-induced lung tumorigenesis thus demonstrating that dietary olive oil and 287 can effectively inhibit NNK-induced lung tumorigenesis

66

[46]. The inhibitory effect of olean-12-ene-3p,23,28-triol tri-0-hemiphthaiate sodium (158), ursoHc acid (210), and betulinic acid (257) on lung tumors have been reported [43]. Lantadene B (134), on oral administration, has an inhibitory effect on mouse hepatic tumors induced by A^-nitrosodiethylamine and phenobarbital [45]. The effect of dietary cholesterol (7) on mammary tumor development was examined in female Sprague-Dawley rats exposed to the carcinogen MNU [47]. Tumor incidence in the compound 7 group (67%) was significantly lower than in the control group (96%) which suggested that dietary 7 inhibits mammary tumor development in this model. Taraxasterol (239), on oral administration, showed remarkable inhibitory effects on mouse spontaneous mammary tumors using C3H/0iJ mouse [42]. THE MECHANISMS OF ANTITUMOR-PROMOTION The mechanisms by which antitumor-promoters suppress the tumor promotion are not Imown, but may be due to the following effects: (i) inhibition of polyamine metabolism; (ii) inhibition of arachidonic acid metabolism; (iii) protease inhibition; (iv) induction of differentiation; (v) inhibition of oncogene expression; (vi) inhibition of PKC; and (vii) inhibition of oxidative DNA damage [3,6,91]. The polyamine content of cells is correlated to their proliferative, and often, their neoplastic capabilities. A key enzyme in the polyamine biosynthetic pathway, ornithine decarboxylase (ODC), catalyzes the convertion of omithine to putrescine. Phorbol ester promoters such as TPA cause increased ODC activity and accumulation of polyamines in affected tissues. Diacylglycerol activated PKC, and the potent tumor promoter, TPA, binds to, and activates PKC, in competition with diacylglycerol. PKC stimulation results in phosphorylation of regulatory proteins that affect cell proliferation. Some chemopreventive agents have inhibitory activity towards PKC. Refer to recent review articles for further discussion [3,6,91]. A TRITERPENOID AS A TUMOR-FROMOTER As discussed in the previous sections, a number of triterpenoids with great structural diversity have antitumor-promoting activities. In contrast, 28-deacetylbelamcandal (288), a spiroiridal-type triterpenoid, has recently been reported to possess tumor promoting activity [92]. Compound 288, which stimulated differentiation of human promyelocytic leukemia (HL-60) cells (a fast method for screening TPA-type tumor promoters).

67

bound to and activated PKC, and induced tumor necrosis factor-a release from HL-60 cells in the same manner as TPA. In an in vivo study, groups treated with 100 jag DMBA plus 400 nM of 288 showed 64.3% tumor incidence by week 20. Compound 288 represents a new structural class of mouse skin-tumor promoters. Iridal-type triterpenoids are characteristic constituents of plants in the genera of Iris and Belamcanda, and it has been suggested that other congeners of 288 may be potential mouse skin-tumor promoters. ANTI-INFLAMMATORY AND ANTI-ALLERGIC ACTIVITIES OF TRITERPENOIDS AND STEROLS Inflammation is one of physiological responses of organisms when they suffer physically or chemically induced stress, and comprises complex processes influenced by chemical mediators [35]. The mediators belong to different chemical classes, such as biologically active amine (histamine, serotonin), proteins and peptides (hydrolytic enzymes, cytokines, growth factors, colony stimulating factors, complement factors, antibodies, kinines), activated oxygen species (superoxide anion, hydroperoxide, hydroxyl radicals), and lipids (PAF, prostanoids, leukotrienes) [93]. Anti-inflammatory compounds can suppress the inflammation by inhibiting activity or by interaction with one or several of the above cited chemical mediators. Many triterpenoids have been used as antiinflammatory remedies in folk medicines and have been reported to possess anti-inflammatory and anti-allergic activities in various experimental models. In Table 1 are listed selected fourteen each of in vivo and in vitro bioassay systems, among a number of assay systems reported in the literature, applied to triterpenoids and sterols for their anti-inflammatory and anti-allergic activities. Table 2 includes the bioassay systems in which individual triterpenoids and sterols exhibited significant activities. The concept that inflammation and carcinogenesis are related phenomena has been the subject of many studies that have attempted to link these two processes in a mechanistic fashion [94-99]. In connection with this, as is evident in some triterpenoids such as oleanolic acid (142), glycyrrhetic acid (147), and ursolic acid (210) in Table 2, those possessing anti-inflammatory activity in various experimental models exhibited antitumor-promoting activity as well. This suggests that most of the other anti-inflammatory triterpenoids and sterols listed in Table 2 might have antitumor-promoting properties which have not been evaluated, yet. Two inflammatory enzymes, inducible nitric acid oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) have critical roles in the response of tissues to injury or infectious agents. These inducible enzymes are

68

essential components of the inflammatory response, the ultimate repair of injury, and carcinogenesis [97,99]. More than eighty novel triterpenoids have been synthesized from 142 and 210, and have been tested for their ability to suppress the de novo formation of two enzymes, iNOS and COX-2 [97]. Two synthetic oleananes, 3,ll-dioxoolean-l,12-dien-28-oic acid (166) and 3,12-dioxoolean-l-en-28-oic acid (168), were found to be highly active. Then, new derivatives with electron-withdrawing substituents at the C-2 position of 3-oxoolean-l-en-28-oic acid were synthesized [97,100]. Among them, 2-cyano-3,12-dioxooleane-l,9dien-28-oic acid (164) was 400 times potent than previous compounds prepared as an inhibitor of iNOS (IC50, 0.4 nM). The potency of 164 was similar to that of dexamethasone, although 164 does not act through the glucocorticoid receptor [98,101]. CONCLUSION Even though the antitumor promoting activities of many of the naturally occurring triterpenoids and sterols from plants and fungi described in this review are not remarkably high, use of these compounds might be advantageous because they are considered to be non-toxic or less toxic and to show fewer side effects [91,102,103]. Thus steroids and triterpenoids are possible candidates for cancer chemopreventive agents. In the future, more mechanistic-oriented basic research is needed to elucidate the mechanisms of action. Studies of derivatives of these naturally occurring triterpenoids and sterols are also necessary to elucidate the structure-activity relationship and to guide the development of novel antitumor-promoters. ABBREVIATIONS Refer to Table 1 for abbreviations. ACKNOWLEDGEMENT We thank Dr. W. C. M. C. Kokke (ChiRex Cauldron, Malvern, Pennsylvania, U.S.A.) for his help during the preparation of this review.

Table 2. Triterpenoids, and Sterols and Their Oxygenated Derivatives from Plants and Fungi and the Bioassay Systems in which the Compounds Exhibited inhibitory Activities Compound Reference Compound Quercetin lndomethacin Hydrocortisone 1. Cholestane Cholesterol 4,4-Dimethylcholesterol 4,4-Dimethylcholest-5-en3-one 4,4-Dimethyl-5,6aepoxycholestan-3~-ol 4,4-Dimethyl-5,6a-epoxycholestan-3-one 4,4-Dimethylcholestane-3a,5a-diol 4,4-Dimethyllathosterol Lophenol 4-Methylcholest-4-en-3-01 7-Oxochoiesterol 11. Ergostane 9(11)-Dehydroergosterol Ergosterol Ergosterol peroxide 24-Methylcholesterol ferulate Ill. Stigmastane 7a-Acetoxysitosteryl acetate 7B-Acetoxysitosteryl acetate 7,9(1 I)-Bisdehydroporiferasterol Citrostadienol 'See Table 1 for the assay system. IDSO(mgtear).

Code Source and Occurrence 4 5 6

The most common flavonoid in higher plants Commercial Commercial

Animal fats; Minor sterols of many higher plants 8 Prepared from 7 9 Prepared from 7 10 Prepared from 7 11 Prepared from 7 12 Prepared from 7 13 Prepared from 7 14 Liliaceae; Solanaceae; Cactaceae 15 Prepared from 7 16 Woolwax 7

Bioassay Systema TPAD Other Assays 1.6

136,371

0.3 0.03

[36,371 [36,371

>2.0 Mammary I; HPA

[35,37,47]

0.3

[30,35,37] [351 I351 [351 [351 [30,371 [30,371 130,371 [37,5 11

>2.0 0.3

20 21 22 23 24

Prepared from 33 Prepared from 33 Chlorella vulgaris Widespread occurrence in higher plants

>2.0 0.5 >2.0

19

HPA HPA HPA HPA HPA

0.4 0.2 0.3 0.2

Chlorella vulgaris (Chlorella) Major sterol of fungi and yeast; Chlorella vulgaris Chlorella vulgaris Rice bran

17 18

References

0.2 0.3

Skin I Skin I

A CM

[37,851 [36,371 [36,37,5I] [481 [37,521 137,521 [37,851 [I 141 OI

\O

Table 2. Continued-1 Compound 7-Dehydroporiferasterol . . 7-Dehydroporiferasterol peroxide Fucosterol 4$-Hydroxysitosterol 7a-Hydroxysitosterol 7$-Hydroxysitosterol 7-(;brositosterol Schottenol Sitosterol Sitosterol ferulate Spinasterol Stigmasterol Stigmast-4-ene-3$,6$-diol Stigmastane-3$,6a-diol Stigmastane-3$,6$-diol Stigmastane-3,6-dione Stigmast-4-ene-3,6-dione N. Lanostane Abiesenonic acid methyl ester Dehydroeburiconicacid Dehydropachymic acid Dehydrotrametenolic acid Dehydrotumulosic acid 24,25-Dihydrolanosterol 3-Epidehydrotumulosicacid Ganodelic acid A Ganodelic acid B Ganodelic acid C Ganodelic acid D 30-u -Hydroxvbenzoyldehydrotumulosicacid

2 0

Code Source and Occurrence TPAD Other Assays 25 ChloreNa vulgaris 0.5 26 Chlorella vulgaris 0.7 27 Brown algae; Solanaceae; Olive; Rice bran >2.0 28 Trichosanrhes kirilowii (Cucurbitaceae) 0.8 seeds >2.0 29 Trichosantheskirilowii seeds 30 Trichosantheskirilowii seeds 0.6 31 Cucurbitaceae 1.0 1.0 32 Theaceae; Cucrubitaceae; Amaranthaceae 33 Major sterol of most higher plants 1.8 Colon, Skin I, CAR, ODC 0.2 34 Rice bran 1.1 35 Theaceae; Cucurbitaceae; Spinach 36 Major sterol of most higher plants 1.9 Skin I 0.6 37 Prepared from 33 38 Trichosantheskirilowii seeds 0.5 0.6 39 Prepared from 33 40 Polygonum chinensis (Polygonaceae) HSR, SOF HSR, SOF 41 Polygonum chinensis 42 53

54 43 55 44 56 45 46 47 48 57

Prepared from abieslactone isolated from Abies mariessi Fomes oficinalis (Polyporaceae) Poria COCOS Poria cocos Poria cocos Solanaceae seeds; Animal fats Poria cocos Ganodenna lucidum (Polyporaceae) Ganoderma lucidum Ganoderma lucidum Ganoderma lucidwn Poria cocos (Hoelen; Polyporaceae)

Skin I, HeLa 0.03 0.02 AA 0.03 AA TPA AA, CAR, PLA2 0.2 0.09 HSR HSR HSR HSR 0.3 AA

References 137.511 . [37,511 [30,371 137,521 [37,521 [37,521 137,521 [37,50 [20,30,44,50,128,129] [481 [20,501 [30,37,50] [37,52] [37,521 [37,521 [I221 [I221 [29,130] [37,551 [37,55,56,60] [37,55,56] [60,61,126] [30,371 [37,551 11231 [ 1231 [I231 [I231 [56]

Table 2. Continued-2 Compound

16a-Hydroxydehydrotrarnetenolicacid 16a-Hydroxytrametenolicacid 16a-Hydroxytrametenolicacid 3-0 -acetate Lanosterol 24-Methylenelanost-9(1 l)-en-3P-ol Pachymic acid Polyporenic acid C Poricoic acid A Poricoic acid AM Poricoic acid B Poricoic acid D Tumulosic acid V. Cycloartane Cycloartenol Cycloartenol ferulate 1-oic (24R ,S)-24,25-Epoxy-3-oxocycloartan-2 acid 3B-Hydroxycycloart-24sn-21-al 30-Hydroxycycloart-24en-21-oic acid Methyl (24R ,S)-24,25-epoxy-3-oxocycIoartan2 1+ate 24-Methylenecycloartanol 24-Methylenecycloartanol ferulate Methyl 25,26,27-Trisnor-3-oxocycIoartan-24-al21-oate Methyl 3B-hydroxycycloart-24-en-2I-oate Methyl 3-oxocycloart-24en-2l-oate 3-0xocycloart-24en-214 3-Oxocycloart-24-en-21sicacid 3-0xocycloart-24-en-21-01

Code 49 50 51 52 58 59 60 62 63 64 65 61

Source and Occurrence Poria COCOS Poria cocos Poria COCOS Solanaceae seeds; Yeast; Animal fats Theaceae seeds Poria cocos and other fungi Polyporus spp. and other fungi Poria COCOS Poria COCOS Poria COCOS Poria COCOS Poria COCOS

66 67 68

Widespread occurrence in higher plants Rice bran, y-Oryzanol Prepared from 77

69 70 71

Prepared from 77 Prepared 6om 77 Prepared from 77

72 73 79

Widespread occurrence in higher plants Rice bran Prepared from 77

74 Prepared from 77 75 Prepared from 77 76 Prepared from 77 77 Lansium domesticum (Meliaceae) 78 Prepared from 77 25,26,27-Trisnor-3-oxocyc10artane-21,24-dial 80 Prepared 6om 77 VI. Dammarane Dammaradienol 81 Dammar resin; Shea butter; Theaceae

TPAD Other Assays TPA TPA 0.02 Skin I 0.2 0.4 0.04 Skin 1, AA, CAR, PLA2 0.1 0.03 AA 0.08 0.02 Skin 1, AA 0.1 0.2 0.3 0.2

CAR Skin I EBV

[37,59,133] 1481 1241

EBV EBV EBV

1241 [241 1241

EBV

[20,37,59] [48] 1241

EBV EBV EBV EBV EBV EBV

1241 1241 [241 [24,134] [241 1241

0.2 0.2

0.8

References 1601 [601 [37,54,55] [20,37,55] [37,131] [37,54,55,61,126,132] 137,551 [37,55,56] [37,55,56] [37,54,55,56] 137,551 137,551

137,591

c .I

Table 2. Continued3 Compound (24R ,S)-24,25-Epoxydammaradienol Isoeuphol Isotimcallol Mansumbinone Mansumbinoic acid 24-Methylenedammarenol V11. Cucurbitane 25-Acetyl-23,24-dihydrocucurbitacinF Cucurbitacin B Cucurbitacin F I Oa-Cucurbitadienol 23,24-Dihydrocucurbitacin F 24-Dihydrel Oa-cucurbitadienol 7-0~0-1Oa-cucurbitadienol

74x0-10a-cucurbitadienol acetate 7-0~0-24-dihydro- 1Oa-cucurbitadienol 15-OxocucurbitacinF 15-0~0-23,24dihydrocucurbitacinF VIII. Euphane Butyrospermol (24R ,S)-24,25-Epoxybutyrospermol Euphol IX.Tirucallane Masticadienoic acid Shinol (3B-Masticadienolic acid) A'-~imcallol Timcallol

4

Code Source and Occurrence 82 Theaceae seeds 83 Theaceae seeds 84 Theaceae seeds 86 Commiphora incisa (Burseraceae) resin 87 Commiphora incisa resin 85 Shea butter Hernsleya carnosflora (Cucurbitaceae) rhizomes 94 Wilbrandia ebracteata (Cucurbitaceae) 95 Cowania mexicana (Rosaceae) leaves and branches 88 Cucurbitaceae seeds 96 Hemrleya carnosflora rhizomes 89 Prepared from 88 90 Trichosanthes kirilowii (Cucurbitaceae) seeds 91 Prepared from 88 92 Prepared from 88 97 Cmvania mexicana leaves and branches 98 Co~vaniamexicana leaves and branches

TPAD Other Assays 0.5 0.3 0.3 CAR CAR 0.5

93

99 Theaceae seeds 100 Theaceae seeds 101 Euphorbia spp. latex; Theaceae seeds

[691

CAR EBV

11351 [26,691

Skin I, EBV >2.0 0.7 0.7 0.4

102 Schinus terebinrhifolius (Anacardiacee)

berries 103 Schinus rerebinrhifolius berries 104 Theaceae seeds 105 Euphorbia spp. latex; Theaceae seeds

EBV

>2.0

0.6 0.5 0.2

0.8 0.4

N

References [37,131] [37,131] [37,131] [log] [ 1091 [37,131]

[37,136] [26,691 [37,136] [37,136]

EBV EBV

[37,136] [37,136] [261 [261

Skin l

[37,131] [37,131] [37,82,131]

PLAZ

11261

PLA2

[ 1261 [37,591 11311

Table 2. Continued-4 Compound X. Quassine Bruceanol-A Bruceanol-B Bruceanol-C Bruceanol-D Bruceanol-E Bruceanol-G Bruceantin Isobruceine-B Dehydrobruceantinol Dehydrobruceantin XI. Oleanane 3p-Acetoxylean-l2en-27-oicacid 3 - 0 -Acetylerythrodiol Acetyloleanolic acid B-Amyrin B-Amyrin acetate &Amyrin Arjunolic acid Arjunolic acid methylester Arjunolic acid triacetate Arjunolic acid triacetate methylester Crategolic acid

Code Source and Occurrence

TPA' Other Assays

References

106 107 108 109 110 111 112 113 114 115

Brucea antidysenterica (Simaroubaceae) Brucea antidysenterica Brucea antidysenrerica Brucea antidysenterica Brucea anridysenterica Brucea antidysenterica Brucea antidysenrerica Brucea antidysenterica Brucea anridysenierica Brucea antidysenterica

EBV EBV EBV EBV EBV EBV EBV EBV EBV EBV

1711 1711 1711 ~711 1711 [711 [711 [711 [711 [711

116 117 118 119 120 179 152

Vitex negundo (Verbenaceae) seeds Eupteleapolyandra bark Prepared from 142 by acetylation Widespread occurrence in higher plants Prepared from 119 by acetylation Theaceae seeds Cochlorspermumtinctorium (Cochlorspermaceae) rhizomes Prepared from 152 Prepared from 152 Prepared from 152 Boswellia serrata resin Prepared from 142 Prepared from 142

CAR EBV EBV CAR, CRO,EPP, 5LOX 5LOX EBV

[I371 [721 1721 [33,37,59,62,138] 11381 [37,131] 123,741

EBV Skin I, EBV Skin I, EBV ACM iNOS C O X , iNOS

123,741 [23,741 [23,741 11131 [lo31 [98,101]

2-Cyano-3-oxooleana-l,12-dien-28-oic acid 2-Cyano-3,12dioxoolana-1,9(11)-dien-28-oic

153 154 155 146 164 165

acid Deoxoglycyrrhetol Deoxoglycynhetol di-PHT 2Na

121 Prepared from 147 122 Prepared from 147

Deoxoglycyrrhetol-di-0-SUC 2Na

123 Prepared from 147

0.4 0.3

SLOX, l2LOX TPA AA.BRA,CAR,COX,CPS, DTA, HPT, HSI, 5LOX 12LOX, PAF COX, 5LOX l2LOX

[I171 [43,65,107,110,117, 1391 [I 171

Table 2. Continuedd Compound Deoxoglycyrrhetol-di-0-phosphate 4Na Dwxoglycyrrhetol3-0-PHT Na 18a-Deoxoglycyrrhetol 18a-Deoxoglycyrrhetol-di-0 -PHT 2Na

Code Source and Occurrence TPAD Other Assays References 124 Prepared from 147 I2LOX [I171 125 Prepared from 147 SLOX, I2LOX [I171 191 Prepared from 147 HeLa [431 192 Prepared from 147 AA [I391 2a,3~-Diacetoxy-l8-hydroxyoleana-5,12-dien-185 Vitex negundo (Verbenaceae) seeds CAR 11371 28-oic acid 2a,3B-Diacetoxyolean-5,12-dien-28-oicacid 186 Vitex negundo seeds CAR 11371 2~,3a-Diacetoxyolean-5,12-dien-28-oic acid 187 Vitex negundo seeds CAR 11371 126 Prepared from 141 by acetylation EBV 1721 2,3-Di-0 -acetylmaslinic acid 2~,3a-Dihydroxyoleana-5,12-dien-28-oic acid 188 Vitex negundo seeds CAR 11371 127 Eupteleapolycndra (Eupteleaceae)bark EBV 1721 1,3-Dioxoolean-12-ene acid 166 Prepared from 142 COX2, iNOS [97,100,101] 3,l I-Dioxoolean-1,12-dien-28-oic 3,11-Dixoxooleana-l,13(18)-dien-28-oicacid 167 Prepared from 142 iNOS 11001 3,12-Dioxoolean-1-en-28-oic acid 168 Prepared from 142 COX2, iNOS [97,100] 3,12-Dioxooleana-l,9(11)-dien-28-oicacid 169 Prepared from 142 iNOS [100,101] 128 Sapogenin from Echinocystis spp., 0.2 EBV, HeLa [16,30,37,43,140-1421 Echinocystic acid (Cochalic acid) Albizzia spp. Entagenic acid 129 Entada phaeseolides (Leguminosae) HeLa [431 acid 170 Prepared from 142 9 0 1K-Epoxy-3-oxoolean-l-en-28-oic iNOS 11001 Erythrodiol 130 Olive, grape, Compositae, and other plants TPA Skin l,CAR DTA, EPP, HeLa [39,43,62,79,11 I] 180 Theaceae seeds Germanicol 0.9 [37,131] 147 Sapogenin from GIycyrrhiza glabra and Glycyi-rhetic acid (Glycyrrhetinic acid) [16,25,28,35,38,43,62, 0.1 Skin I, 111, AA, ACM, cAK, C A R , EBV, EPP, HeLa, 67,68,86,88,89,93,107, some other plants HPA, HSI, PKC 113,115,1431 Glycynhetinyl stearate 148 Prepared from 147 CTN 1891 149 Prepared from 147 Glycyrrhetinic acid 3-PHT Na TPA 143,651 I2LOX 11171 150 Prepared from 147 Glycyrrhetinic acid 3-PHT 2Na 193 Commercial; Prepared from 147 TPA Skin I,cAK, CAR, EPP, HPT [62,88,115] 18a-Glycyrrhetinic aicd 156 Sapogenin from Clematis, Holboellia , 0.1 AA. ACM, CAR, CRO, EPP, [20,37,43,59,62,93,124 Hederagenin Hedera spp. HeL& HYA ,144, 1451 131 Prepared from 132 by alkaline hydrolysis EBV 1271 228-Hydroxyoleanonicacid 2-Hydroxy-3-oxooleana-1,12-dien-28-oic acid 171 Prepared from 142 iNOS fl00,lOll

2

Table 2. Continued-6 Com~ound Lantadene A "Lantadenol A" Lantadene B "Lantadenol B" Lantadene C Longispinogenin Maniladiol Maniladiol3-0 -myristate Maniladiol3-0 -palmitate Maslinic acid 2-Methoxy-3-oxooleana-1,12-dien-28-oicacid Methyl 3-oxooleana-1,12-dien-28-oate Oleanonic acid Olean-12ene-3a, 168-diol Oleanolic acid

Code Source and Occurrence TPAD Other Assavs 132 Lantana camara (Verbenaceae)leaves Liver, Skin I, EBV EBV 133 Prepared from 132 by NaBH4 reduction Liver, Skin I, EBV 134 Lantana camara leaves EBV 135 Prepared from 134 by NaBH, reduction 136 Lantana camara leaves EBV 137 Compositae flowers 138 Compositae flowers 139 Edible chrysanthemum flower 140 Edible chrysanthemum flower 141 Eupteleapolyandra bark EBV 172 Prepared from 142 iNOS iNOS 173 Prepared from 142 143 Commerical; Prepared from 142 EBV, iNOS HPT 144 Canarium album (Burseraceae) Skin 1, ACM, AIP, CAR, cAK, 142 Occurs as glycosides in olive leaves, sugar beet, panax rhizomes, etc. COX2,DEX,EBV,EPP, FAA, HeLa, HLE, HPT, HYA, iNOS Skin I, HeLa 157 Prepared from 156 Olean-12-ene-38,23,28-trio1 Lung, Skin 11 Olean-l2-ene-3P,23,28-trioltri-PHT Na 158 Prepared from 156 159 Commiphora merkeri (Burseraceae) roots CAR Olean-12-ene-2a,3~,23-triol TPA 181 Prepared from 147 Oleana-9( 1I), 12-diene-313.30-diol TPA AA,BRA,CAR,COX,CPS. Oleana-9(11),12-diene-3P,30-diol di-PHT 2Na 182 Prepared from 147 DTA, HPT, HSI, 5LOX I2LOX PAF TPA Oleana-11,13(18)-diene-3~,30-diol 183 Prepared from 147 TPA AA, BRA, CAR, COX, CPS, Oleana-1 1,13(18)-diene-3P,30-diol di-PHT 2Na 184 Prepared from 147 DTA, HPT, HSI, 5LOX, 12LOX PAF Skin I, HeLa 18a-Olean-l2-ene-3D,28-diol 194 Prepared from 142 Skin I, HeLa 18a-Olean-l2-ene-3(3,23,28-triol 195 Prepared from 156 3-Oxoolean-1-en-28-oic acid iNOS 174 Prepared from 142

References 127,451 1271 [27,451 [271 1271 137,581 [37,581 [ 1461 11461 1721 [100,101] [loo] [22,100] [ 1201 [22,30,31,37,43,62,66, 72,93,103,108,113,115, 118,124,147,148] [39,431 [89,130] 11491 [651 [43,65,80,89,107,110, 1171 1651 [43,65,80,89,107,110, 117,1391 [39,431 [39,431 [I 001

Table 2. Continued-7 Compound 3-Oxooleana-1,9(1 I)-dien-28-oic acid 3-Oxooleana-1,12-dien-28-oicacid 3-Oxooleana-1,12-dien-2-al-28-oic acid 3-Oxooieana-I, 11,13(18)-trien-28-oic acid Papyriogenin A

Papyriogenin C Queretaroic acid Soyasapogenol B Soyasapogenol E Stearyl glycyrrhetinate Wistariasapogenol A Wistariasapogenol B XII. Ursane Acetyl-l I-keto-S-boswellic acid (AKBA) a-Amyrin a-Amyrin acetate a-Amyrin palmitate a-Amyrin linoleate D-Boswellic acid Brein Brein 3 - 0 -myristate Brein 3-0-palmitate 3,ll-Dioxoursa-I, 12-dien-28-oic acid 3-Epiursolic acid 3-Oxoursa-I, 12-dien-28-oic acid Tormentic acid Urs-l2ene-3a, 16p-diol(3-Epibrein)

Code Source and Occurrence 175 Prepared from 142 176 Prepared from 142 177 Prepared from 142 178 Prepared from 142 189 Aglycone of papyrioside L-Ila from Tefrapannrpapyrifenrm(Araliaceae) leaves 190 Aglycone of papyrioside L-IIb 145 Sfenocereussfellatus (Cactaceae) 160 Wistaria brachybotrys (Leguminosae) knots 161 Sapogenin of wistariasaponin D 151 Prepared from 147 162 Wistaria brachyborrys knots 163 Wistaria brachybortys knots

TPAD Other Assays iNOS iNOS iNOS iNOS CAR

CAR HeLa EBV

EBV CTN EBV EBV

Boswellia serrafa resin 5LOX HLA Widespread occurrence in higher plants 0.2 cAK, CAR, EPP Prepared from 197 by acetylation 5LOX Synthesis; Alstonia boonei (Apocynaceae) AIP, cAK Prepared from 197 cAK, 5LOX Gum resin of BosweNia serrata ACM Compositae flowers; Canarium album 0.05 HPT fruits 0.2 Edible chrysanthemum flower 0.2 Edible chrysanthemum flower iNOS Prepared from 210 Prepared from 210 COX2 Prepared from 210 iNOS Poferiumancisfroides (Rosaceae) TPA CAR, EPP 209 Canarium album (Burseraceae) h i t and HPT 196 197 198 199 200

References [I 001

Table 2. Continued-8

Rhododendron spp. (Ericaceae)

Uvaol

211 Olive oil; Ilex latifolia (Aquifoliaceae);

0.1

CAR,COX2,DTAA,EBV 72,91,93,102,111,113, HeLa,HLE,HPA,HPT, 115,118,119,130,158HSR, 5LOX ISLOX, O X , 1601 SER CAR, EPP, ELE, HPT [37,58,62,93,115]

Compositae

X I I I . Multiflorane Bryonolol Bryonolol3-SUC K Bryonolol di-SUC 2K Bryonolol di-PHT 2K Bryonolic acid Bryonolic acid 3-SUC 2K Bryonolic acid 3-PHT 2K 3-Epibryonolol 3-Epikarounidiol 3P-Hydroxylmultiflora-7,9(1I)-dien-29-oic acid 3-SUC 2K Karounidiol Karounidiol3-benzoate Multiflor-8-ene Multiflorenol Multiflor-9(11)-ene 7-Oxoisomultiflorenol 7-Oxodihydrokarounidiol 7-0xo-8P-multiflor-9(I1)-ene-3a,29-diol diacetate XIV. Taraxastane Amidiol Amidiol3-0 -palmitate

212 213 214 215 216 217 218 219 223 224

Trichosanthes kirilowii seeds Prepared from 212 Prepared from 212 Prepared from 212 LuJa cylindrica cell suspension culture; Bryonia dioica roots Prepared from 216 Prepared from 216 Trichosanthes kirilowii seeds Trichosanthes kirilowii seeds Prepared from 216

>2.0 DTA, ITA DTA, ITA DTA, ITA DTA, ITA DTA, ITA

Trichosanthes kiriloivii seeds Trichosanthes krriloivii seeds Fern rhizomes Benincasa hispida (Cucurbitaceae)fruits Fern rhizomes Trichosanthes kirilowii seeds Trichosanthes kiriloivii seeds Trichosanthes kirilowii seeds

0.4 0.2

230 Compositae flowers 231 Edible chrysanthemum flower

DTA, ITA DTA, ITA 0.2 0.6 DTA, ITA Skin l EBV HSR EBV 0.2 0.3 0.8

Table 2. Continued-9 Compound Faradiol Faradiol3-0 -myristate Faradiol3-0 -palmitate Heliantriol Bo Heliantriol C Taraxastane v-Taraxasterol Taraxasterol Taraxasterol acetate XV. Taraxerane Taraxerane Taraxerol XVI. Glutinane Glutinol (Alnusenol) Glutin-5(lO)ene XVII. Friedelane Celastrol 2,3-Dihydroxy-24-norfriedera-1,3,5(10),7tetraen-29-oic acid 2,3-Diacetoxy-24-norfriedera-1,3,5(10),7tetraen-29-oic acid Epifriedelanol Friedelin Pristimerin Regenol B XVIII. Lupane

3~-Acetoxylup-20(29)-en-30-al Acetyl betulinic acid Benzoyl betulinic acid

w 4

Code Source and Occurrence 232 Compositae flowers 233 Edible chrysanthemum flower 234 Edible chrysanthemum flower 235 Compositae flowers 236 Compositae flowers 237 Fern rhizomes 238 Compositae 239 Compositae

TPA" 0.2 1.0 0.9 0.1 0.03 0.4 0.3

240 Echinops echinatus (Compositae) 241 Fern rhizomes 242 Compositae; Rhododendron spp.; Euphorbia spp.

Other Assays Skin I, CRO CRO CRO Skin I EBV CRO

Mammary 11, Skin I, CRO, EBV AIP, CAR. FAA

References [33,34,37,40,58,85] [33,146] [33,146] [37,581 [37,40,58] [671 [33,34,37,59] [33,37,42,59,85] [lo51

EBV Skin I, EBV

[671 [37,42,59]

243 Benincasa hispida fruits 244 Fern rhizome

HSI EBV

[I211 [671

245 Triptetygium wiflordii (Celastraceae) 249 Prepared from 245

EBV EBV

[751 1751

250 Prepared from 245

EBV

[751

CAR CAR

[I631 [20,30,37,163]

iNOS EBV

1991 [751

EBV PKC

[37,861 [72] [32]

247 Calyophyllumapetalum 248 Cork; Lingnania chungii ;Calyophyllum apetalum 246 Celastraceae; Hippocrateaceae 251 Tripterygium wilfordii (Celastraceae) 252 Prepared from 264 253 Prepared from 257 by acetylation 254 Prepared from 257 by benzoylation

0.4

0.9

0.8

Table 2. Continued-10 Compound Betulin (Betulinol) Betulonaldehyde Betulinic acid Calenduladiol Calenduladiol 3-0-palmitate Dihydrobetulinicacid 3-Epilupeol Heliantriol B2 30-Hydroxylupeol

3~-Hydroxylup-20(29)-en-30-al Lupeol

Code Source and Occurrence 255 Birch bark; Corylus avellana (Betulaceae); Vicia.faba 256 Prepared from 264 257 Cornusflorida (Cornaceae); Ziryphus vulgaris (Rhamnaceae) 258 Compositae flowers 259 Edible chrysanthemum flowers 272 Prepared from 257 260 Prepared from 264 261 Compositae flowers 262 Flourensia heteroleptis and other higher plants. 263 Prepared from 264 264 Widespread occurrence in higher plants

269 270 271

Vernonia cinerea and many other higher plants Acacia dealbata ;Stevia Prepared from 264 Acacia dealbata ; Cneorum tricoccon Prepared from 264 Pluchea lanceolata (Compositae) stem and leaves Pyracantha crenulata (Rosaceae) Prepared from 257 Prepared from 257

275 277 278 276

Fern rhizomes Fern rhizomes Fern rhizomes Bacterium Zymomonas mobilis

Lupeol acetate

265

Lupeol palmitate Lupeol linoleate Lupenone Lupanol Neolupenol (lup-12-en-3P-01)

266 267 268 273 274

Pyracrenic acid Sulfonyl betulinic acid Succinyl betulinic acid XIX.Hopane Hop-1 7(2 1)-ene Neohop-13(18)-ene Neohop-12-ene Tetrahydroxybacteriohopane

TPA" Other Assays References 0.2 Skin I , cAK, CAR, ODC,SER [30,37,64,86,115] 1.O 0.3

Lung, Skin I, CAR, EBV,HeLa, ODC, SER

0.2 0.3 PKC 0.4 0.05 0.6 0.6 0.6 0.6 0.6

137,861 [30,37,64,72,79,130, 164) 137,581 [I461 [321 137,861 137,581 [37,861

137,861 Skin I, AIP, cAK, CAR. CRO, [30,33,37,62,86,116, EPP ,ODC 165,1661 Skin 1 [30,37,86] cAK AIP, cAK

CAR

[30,37,116] [115,165,166] 137,861 [37,861 11671

CTN PKC PKC

1891 1321 1321

Skin I, EBV Skin I, EBV EBV AA, ISLOX

1671 [671 1671 1104,1261

1.7 0.2

I \O .

Table 2. Continued-11 Compound XX. Moretenane Moretenol Moretenol acetate XXI. Arborinane Sorghumol Sorghumol acetate XXII. Other Triterpene Bacchara-12,2 I-dien JS-ol Helianol Limonin Sasanquol Squalene 28-Deacetylbelamacandal XXIII. Spirostane Hecogenin 24R -Spirost-4-ene-3,12-dione Tigogenin XXIV. Cardiac Steroid Digitoxigenin Ouabagenin Strophanthidin Decumbesterone A Cyasterone Polypodine B

-

Code Source and Occurrence

TPAD Other Assays

279 Pluchea lanceolata stem and leaves 280 Pluchea lanceolafa stem and leaves

CAR CAR

281 Pluchea lanceolata (Compositae) roots 282 Pluchea lanceolata roots

CAR CAR

Theaceae seeds Compositae flowers; Theaceae seeds Evodia rutaecarpa (Rutaceae) fruits Camellia sasanqua (Theaceae) seeds Fish liver oils; Yeast lipids and higher ~lants 288 Iris tectorum (Iridacee) rhizomes 283 284 285 286 287

289 Polygonurn chinensis ;Commercial 290 Polygonurn chinensis (Polygonacae) 291 Commercial 292 293 294 295 296 297

Commercial Commercial Commercial Ajuga decumbens (Labiatae) Ajuga decumbens Ajuga decumbens

0.8 0.1 AA, BRA, CAR 0.4 >2.0 Colon, Lung

Skin tumor promoter CAR, HSR, SOF HSR. SOF CAR

EBV EBV EBV EBV Skin I, EBV EBV

References

[37,13 I] [37,591 [20,169] [I701 [30,37,46,89]

81

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B .V. All rights reserved.

89

BIOACTIVE OLEANENE GLUCURONIDES OBTABVED FROM FABACEOUS PLANTS

JUNEI KINJO and TOSHfflIRO NOHARA Faculty of Pharmaceutical ScienceSy Kumamoto University, Kumamoto 862-0973, Japan ABSTRACT: Oleanene-glucuronide (OG) is defined as an clean-12-ene type triterpene with a C-28 methyl group and a glucuronic acid moiety linked at C-3 of the triterpene. Soyasaponin I is a representative OG. It has been revealed that OGs are widely distributed in fabaceous plants and show several biological activities. As a part of our study on the chemical constituents of fabaceous plants, we have obtained over 191 OGs from 40 fabaceous plants. Furthermore, in the course of our study on hepatoprotective drugs, we devised the conditions for an in vitro assay method using immunologically induced liver injury on primary cultured rat hepatocytes and confirmed hepatoprotective actions of more than 40 OGs and the related compounds. Structure-activity relationships for the sapogenol moiety suggested that the p-hydroxy group at C-21 would enhance hepatoprotective activity; on the contrary, the hydroxy group at C-23, 24, 29 and 30 could reduce the activity. On the other hand, the free carboxylic acid group at C-28 may mediate cytotoxicity toward liver cells. The structure-hepatoprotective relationships of the sugar moiety suggested that the skeleton with glucuronic acid linked at C-3 was a crucial unit in mediating hepatoprotective activity. In the case of a disaccharide chain bound at C-3, an oxygen-bearing group at C-5" seems to enhance the hepatoprotective activity. The terminal rhamnopyranosyl group of fabatrioside seems not to be necessary for the activity. Since antiviral activities of OGs against herpes simplex virus type 1 were reported, we also examined antiherpetic activity of some OGs and related compounds. A trisaccharide group shows greater action than a disaccharide group. Monoglucuronide did not show any antiherpetic activity. Further, the sapogenols showed more potent antiherpetic activity than those of their saponins. This structure-activity relationship was completely different from that obtained from hepatoprotective and antiherpetic activities. The mechanism of antiherpetic activitiy of sapogenols might be different from that of saponins. Furthermore, since OGs were known to have not only anti-complementary but also anti-nephritic activities, we tested some OGs toward the classical pathway. Monoglucuronides and diglycosides were most potent then followed by triglycosides, whereas the aglycones exhibited increase of hemolysis. These results indicate that the glucuronic acid moiety is important for expression of anti-complementary activity. The anti-complementary activity of the OGs with a free acid form of glucuronic acid was more potent than that of sodium salt or methylester forms. Furthermore, reduction of the glucuronic acid moiety decreased significantly their activity. The free acid form of the glucuronic acid moiety seemed to contribute to the potency. The hydroxy group at C-24 did not affect the anti-complementary activity except for the methylester forms.

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INTRODUCTION Saponins are glycosidic compounds present in many edible and inedible plants. Structurally, they are composed of a lipid-soluble aglycone consisting of either a sterol or, more commonly, a triterpenoid and watersoluble sugar residues differing in type and amount of sugars [1]. Because of their amphiphilic nature, they are highly surface-active. Their biological activity is closely related to the chemical structures that determine the polarity, hydrophobicity and acidity of compounds [1]. A recent review by Safayhi and Sailer shows that pentacyclic triterpenes, including the oleanane skeleton, might be a rich natural resource of lead compounds for anti-inflammatory drug development [2]. Some oleanane-type triterpene saponins are known to exhibit anti-hepatic (hepatoprotective) action. Among them, glycyrrhizin [3-7] and saikosaponins [8, 9] are the most well-known. Oleanene-glucuronide (OG) would be defined as an olean-12ene type triterpene with a C-28 methyl group and a glucuronic acid moiety linked at the C-3 of the triterpene. Soyasaponin I (1) [10, 11] is a representative OG, and glycyrrhizin also belongs to the OG species. It was been revealed that OGs are widely distributed in leguminous plants and show several biological activities. For example, anti-hepatitis [12, 13], antihypercholesteremia[14], anti-urolithiasis [15], anti-inflammatory [16] and anti-nephritic [17] activities were confirmed in experimental in vivo models. Furthermore, antiviral

[18], anti-complementary

[17] and calcium-

dependent potassium channel-opening [19] activities were examined using in vitro models. In view of the fact that leguminous plants are widely distributed and used as foodstuff and folk medicines, we have focused on these plants and are trying to develop natural medicines after proving the effectiveness of these crude drugs and to find the lead compounds among these natural sources. Herein, we describe the structures of OGs and some of their biological activities, discussing the structure-activity relationships.

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EXTRACTION, ISOLATION AND DISTRIBUTION Puerariae Flos (the flowers of Pueraria lobata) is a crude drug used for counteraction to alcohol intoxication in traditional Japanese and Chinese therapeutic systems [20]. We have found that total OGs were effective for alcohol intoxication [21]. It was also effective for an experimental in vivo model of hepatic injury [22]. That is, the total OG decreased the alanine aminotransferase (ALT) level in high fatty food-induced and CCI4-induced experimental liver injury. After repeating some chromatographic technique, we obtained three OGs together with several isoflavones [20]. On the other hand, Abri Herba, the whole plant of Abrus cantoniensis, is used as a folk medicine for infectious hepatitis in China. Its efficacy has been substantiated clinically [23]. Chiang et d. confirmed the efficacy of the total saponin fraction in a pharmacological experimental model [24]. We also reported that the total saponin fractions of this plant were effective for experimental liver injuries in an in vivo model induced by CCI4, Fig. (1) [25]. 4000^ (IU/1) AST

3000 J

ALT

2000 J 1000 J

control

CCI4

Soya I

OG-I

OG-II

Rg. (1). Effects of Extract of Abri Herba in Mice Treated with CCI4 Effects of Soyasaponin I (Soya I), total OG-l (CXJ-I) and total CXJ-D (OG-H) on CCI4 induced liver injury by oral administration. These doses were each 500mg/kg. Each column represents mean of 10 mice. *p<0.05, **p<0.01. AST (aspartate aminotransferase), ALT (alanine aminotransferase)

92

Especially, the lower polar fraction (OG-II) was more potent than the higher fraction (OG-I). By means of many isolation procedures of both fractions, we isolated twenty-three saponins from the active fraction and elucidated their structures [26, 27]. As the result of our continuing study on the chemical constituents of leguminous plants, so far we have obtained over 191 OGs from 40 plants (Table 1). Of these, 73 OGs are new glycosides, including 21 novel sapogenols. Since many leguminous plants include OGs, they seemed to be ubiquitous ingredients in the leguminous family. However, their distribution is localized to Faboideae in Leguminosae, /.e., Fabaceae in the present botanical taxonomy, Fig. (2) [28]. TRITERPENOIDAL SAPONINS

Fabaceae

28-Me Type Saponins (Oleanene glucuronides)

Mimosaceae

Caesalpiniaceae

Mimosoideae

Caesalpinioideae

Leguminosae

Fig. ( 2 ) . Distribution of Characteristic Saponins in Leguminosae

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Table 1. Characteristic Oleanene Glucuronides (OGs) Isolated from Fabaceous Plants Plants

References

Abrus cantoniensis Abrus precatorius Aeschynomene indica Amphicarpaea edgeworthii Arachis hypogaea Astragalus complanatus Astragalus sinicus Baptisia australis Campylotropis hirtella Canavalia gladiata Cicer arietinum Crotalaria albida Dalbergia hupeana Desmodium styracifolium Glycine max Glycine max cv. Kuromame Glycine soya Melilotus officinalis Melilotus officinalis [Kumamoto] Melilotus officinalis [Rotenburg] Medicago polymorpha Mucuna sempervirens Lathyrus palustris vaT.pilosus Lens esculenta Lotus corniculatus Lupinus polyphyllus hybrid Lupinus polyphyllus hybrid Pachyrhizus erosus Phaseolus coccineus cv. Murasakihanamame Phaseolus coccineus cv. Murasakihanamame Phaseolus coccineus cv. Ooshirobananamame Phaseolus vulgaris cv. Uzuramame Phaseolus vulgaris cv. Taishokintoki Phaseolus vulgaris cv. Toramame Phaseolus vulgaris cv. Toramame Phaseolus vulgaris cv. Torosuku Pisum sativum Pueraria lobata Pueraria lobata Pueraria thomsonii Pueraria thomsonii Robinia pseudo-acacia Rudea aurea Sophora subprostrata Sophora flavescens Vicia faba Vicia sativa Vigna angulariscv. Dainagon Vigna unguiculata cv. Chuguro Wisteria brachybotrys Total

40

[26,27] [63] [64] [65] [66] [83] [28] [67] [68] [69] [70] [64] [37] [71] [72]

[47] [73] [67] [74] [67] [67] [64] [67] [67] [67] [67] [64] [64] [20] [39,40] [75] [49] [76,77] [78-80] [81] [64] [64] [64] [82]

Parts whole seed whole aerial seed seed seed root root root seed whole bark whole root seed aerial root aerial aerial aerial leaf aerial seed aerial root seed tuber seed aerial seed seed seed aerial root seed seed flower root root flower bark seed root root seed seed seed seed bark 50

Total OGs

New Aglycones

New OGs

23 1 1 1 1 6 6 2 2 4 1 6 3 2 4 2 4 4 4 4 1 2 4 1 2 7 4 2 1 2 3 2 1 2 3 3 1 3 12 5 3 13 1 11 4 1 1 2 2 9

10 1 0 0 0 1 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 4 0 0 0 0 0 0

11 0 0 0 0 4 1 1 0 2 0 2 2 0 0 0 0 1 1 0 0 0 2 0 0 5 0 0 0 0 0 0 1 0 1 0 0 1 10 3 0 11 0 7 3 0 0 0 0 5

191

21

73

94

STRUCTURAL DETERMINATION a. Structures The structures of OGs were determined by both chemical and physicochemical methods. The *^C-NMR data of a representative OG, Soyasaponin I (1) was assigned using HMBC spectra [26]. Further, all carbon signals were assignable in comparison with *^C-NMR spectral data of each OG. The absolute configurations of the sugar moieties were determined according to the procedure developed by Hara et al [29]. Figure (3) shows some examples of OGs which were obtained during our studies. These saponins have two common features: one, a methyl group at C-28 of sapogenol; the other, glucuronic acid linked at C-3. Further, most of them possess someoxgen functional group. Since the sugar moiety (S,) is widely distributed and characteristically comprises glucuronic acid in fabaceous plants, we called it p-fabatriose [26]. b. HPLC Profiles Although OGs are widely distributed in the fabaceous family, fabaceous crude drugs used as anti-hepatitis were limited. Therefore, a qualitical analysis seemed to be important. In order to confirm the constitution of OGs, HPLC profile analyses of the total OG fraction for some crude drugs were performed, Fig. (4). The test samples were classified into two groups: one is used as anti-hepatitis (Abri Herba and Puerariae Flos) and the other one is not for such use (the roots of Lupinus polyphyllus and Campylotropis hirtella). The HPLC profile analysis showed that the crude drugs used as anti-hepatitis were a rich source of sophoradiol OGs. On the other hand, the crude drugs not used for anti-hepatitis included substantial amount of soyasapogenol B OGs. Therefore, sophoradiol OG seemed to be more potent than soyasapogenol B OG in terms of anti-hepatitis action.

95

soyasapogenol B Ri8F\2sH

:H20H Soyasaponin I (1) Azukisaponin V (2) Soyasaponin II (3) Astragaloslcle VIII (4) Soyasaponin V (5) Soyasaponin III (6) Azukisaponin II (7) Soyasaponin IV (8) Abrisaponin SB (9) Albioside II (10) Meliiotus-saponin Oi (11)

Si 52 53 54 Sg SB S9 S10 Sl3 Sl4 Sis

H SBMG(12)Gk:A H Acetylsoyasaponin I (13) Si H Kudzusaponin SBi (14) S^ H Comploside II (15) Si H Lupinoside PA4 (16) S^ H Comploside I (17) S4

R2 H Ac -ara -glc -rha -glc

Lupinoside PAs (18) S^ -rha - glc Subproside VII (19)

Si

-xyl^ glc ^ Phaseoside I (20) Se -ara2-glc

Kalkasaponin III (21) Kakkasaponin 1 (22) Baptisiasaponin 1 (23) Phaseoside V (24) Albioside 1 (25) Kalkasaponin 1 (26) Abrisaponin S01 (27) Acetylkaikasaponin III (28)

Ri Si S3 S4 Ss S7 Se S13

Si Abrisaponin S02 (29) S13

R2 H H H H H H H Ac -xyl

H2OR2 abrisapogenol E Ri*R2«H

kudzusapogenol A RisF^ SR3S

R1O

R1O

DHaOH Kudzusaponin A3 (30) Kudzusaponin As (31) Subproside II (32) Kudzusaponin A2 (33) Kudzusaponin A4 (34) Subproside VI (35) Lupinoside PA3 (36) Kudzusaponin Ai (37)

Ri R2 R3 Si H H H S2 H H S4 H H Ss H S9 H H S^ -rha H Si -xyl H S3 H -xy

Soyasaponin A3 (38) Kudzusaponin SAi (39) Lupinoside PA^ (40) Lupinoside PAg (41) Kudzusaponin SA3 (42) Kudzusaponin SA2 (43) Kudzusaponin SA4 (44)

Si Se S, Se Si Sa Si2

R2 R3 H H M H -xyl H -xyl H H -ara H -ara H -ara

R2 H H -glc Subproside V (47) -glc Wistariasaponin B3 (48) S4 6 Robiniosidel (49) S^ -gto" spi c Robinioside J (50) S2 -glc - api Wistariasaponin B2 (45) Palustroside II (46)

Ri Si S9 Si

Si = (^fabatrlosyl)

OH

Fig. (3). The Structures OGs Obtained from Some Fabaceous Plants

96

oxytrogenin R^sRg = H

Hooa

HOHzCs abrisapogenoi B

abrisapogenoi D Rl=:R2«H

R1O

^^CHgOH Ri Sophoraflavoside II (51) Si Robin!oside B (52) S2 Robinioside C (53) Si RobinlosideD(54) S2 Sophoraflavoside III (55) Si Sophoraflavoside IV(56)

R2 H H -rha -rha -ara

^XH20H Ri Si

Robinioside E (57) Robinioside F (58) Robinioside G (59)

R2 H H

S2 Si -rha S2 -rha

Robinioside H (60)

Abrlsaponin D, (61) Abrisaponin Dg (62) Subproside IV (63) Abrisaponin D3 (64)

R, S^ S7 S, S7

Rg H H -glc -glc

S, -ara-glc

cantoniensistriol R=H

H

kudzusapogenol C RisRgsH

R1O' XH2OH Abrisaponin Ca (65) 8^

CH2OH Ri Si

Kudzusaponin Ci (66)

HOH2C

Ffe -glc

Ri R2 Wistariasaponin YC2 (67) Si -glc Wistariasaponin YCi (68) S4 -glc

H0H2C^

abrisapogenoi A R«H

CH2OH Abrisaponin A (69) S,

Subproside I (70)

S13 =

R

Si

Abrisaponin L (71) S^

Sl4 =

Sl5 =

OHOH

Fig. ( 3 ) . Continued.

97 HOOC^ 24-deoxyoxytrogenin R*H

soyasapogenol E R=H

RO 3H2OH Dehydrosoyasaponin I (72) Palustroside I (73) Wistariasaponin D (74)

p S^ S9 S4

Phaseoslde IV (75) Si

Compound 2 (77) Si Robinioside A (78) S2

Abrisaponin F (76) S7

H2OR2

HOOC, kudzusapogenol B R>:H

:H20H

H2OH

R

Comploside IV (80) Si Comploside III (81) S4 Comploside V (82) S3

Kudzusaponin 6^ (79) S^

R,

Subproside 111(83) 8^ Wistariasaponin A2 (84)

S4 -glc

Wistariasaponin A3 (85)

Se -glc

HOOC^

RoOoa

,COOH

azukisapogenol B R,=R2 = H

R1O

Azukisaponin VI (86)

6

S9 -glc-glc

Melilotus-saponin O2 (87)

R2 -glc

S,

Phaseoside III (89) S^

Fig. ( 3 ) . Continued.

Abrisaponin I (88)

S,

98 (CHaCN) 50%-

Abri Herba (the Roots of Abrus cantoniensi^

Puerariae Flos (the Flowers of Pueraria lobata)

Fig. ( 4 ) . HPLC Profiles for Total OG Fractions of Some Fabaceous Crude Drugs HPLC conditions were as follows; column, Nova-Pak CI8 (4 mm, 8 x 100 mm) with Radial-Pak RCM 8 x 10 module, solvent A, H20:TFA= 100:0.05 (v/v); solvent B, CH3CN:H2O:TFA=60:40:0.05(v/v). Elution was done with the following process: 0-»83% solvent B (5 min), 83% solvent B (5 min), 83-^100% solvent B (5 min), 100% solvent B (5 min). Flow rate was 1 ml/min. Detection was done by UV at 205 nm. Sample of 50 |Xl of total 0 0 (1 mg/ml in MeOH) were injected.

99

HEPATOPROTECTIVE EFFECTS a. Antihepatotoxic Effects of OGs (using cytotoxicity)

CCI4 induced

In order to prove the hypothesis obtained from HPLC profile analysis, antihepatotoxic effects of OGs obtained from Abri Herba were tested [30]. The tested OGs were representative ones, soyasaponin I (soyaspogenol B OG, 1) and kaikasaponin III (sophoradiol OG, 21). The antihepatotoxic activity was studied on liver injury induced by CCI4 in primary cultured rat hepatocytes [31], Fig. (5). AST (lU/l) 300

500 (^g/ml) Fig. ( 5 ) , Effects of Some OGs on Primary Cultured Rat Hepatocytes injured with CCI4 Effects of glycyrrhizin (GL, triangles), kaikasaponin HI (2 1, Kaika HI, circles) and soyasaponin I (1, Soya I, squares) on CCI4 (5niM)-induced cytotoxicity in primary cultured rat hepatocytes. The marker of liver injury is the aspartate aminotransferase (AST), measured in lU (International Units)/1. Data are the mean ± S.D. for three independent cell preparations.

Soyasaponin I (1) inhibited the elevation of aspartate aminotransferase (AST) activity, which was comparable to that of glycyrrhizin (positive control). On the other hand, Kaikasaponin III (21) was more effective than 1. Compound 21 showed antihepatotoxic activity at less than 100|Lig/ml. Furthermore, the highest activity was observed even at lower doses (50, 100|ig/ml). Therefore, sophoradiol OGs were concluded to be the anti-hepatotoxic principle in both crude drugs (Abri Heba and Puerariae Flos).

100

b. Assay Method Antiserum)

for

Hepatoprotective

Effects

(using

Higuchi et d. reported an assay method using in vitro immunological liver injury and confirmed the hepatoprotective effects of total OG from soybeans [32]. They used an antiserum against rat plasma membranes as a lesion model of liver cells. In a series of studies on hepatoprotective drugs, Liver Plasma Membranes + FIA

Hepatocytesl <=

J

Rat FIA: Freund's incomplete adjuvant

^ci::>c:>cz:>/

I

Incubated for 24 h in Eagle's MEM Medium

/c::>cz:>c::>y

Rabbit

Sample (10.30.90. 200, 500 jiM)

Antiserum against Rat Hepatocytes

\ Assay ALT Activities I

Fig. ( 6 ) . Assay Method for Hepatoprotective Drugs Using Primary Cultured Rat Hepatocytes Injured with Antiserum against Rat Liver Plasma Membranes

we also devised a similar in vitro assay method [33], Fig. (6). Liver cells were isolated according to a procedure developed by Berry and Friend [34]. The detailed procedure was described in a previous paper [33]. The rat liver plasma membranes were prepared according to a procedure developed by Loten et d. [35]. The detailed procedure was also described in the previous paper [33]. One day after the isolated rat hepatocytes were plated, the cultured cells were exposed to the above-prepared medium (300 |LI1) containing the antiserum against rat plasma membranes (40 |il/ml) and DMSO solution (4 ml) of the tested compounds [final concentration 0

101

(reference), 10, 30, 90, 200, 500 ^iM]. Forty min after the rat plasma membranes were treated with the antiserum, the medium was taken for determination of enzyme activity (ALT). The percent of protection is calculated as {1-(Substance-Control) / (Ref.-Control)} x 100. The ALT activity was assayed by autoanalyzer COBAS MERA (Roche) using commercial kits based on the ALT assay method [36]. Data are the mean ± SD (n=4). After analysis of variances, Sheffe's test was employed to determine the

significance

of

differences

between

reference

and

experimental samples. c. Hepatoprotective Activity of Soyasaponins I - IV Glycine soya is a wild-type relative to the cultivated soybean (Glycine max).Wc isolated four known OGs, soyasaponins 1(1), II (3), III (6) and IV (8) from the aerial parts of this plant [37]. The results of hepatoprotective actions of these OGs are shown in Fig. (7) [37]. The action of 3 was almost compalable to that of 1, whereas those of 6 and 8 were much more effective than that of 1 and 3. Moreover, 6 was significantly active even at 30 (XM. Soyasaponins I -- IV have the same aglycone, soyasapogenol B. Therefore, the differences in action among these saponins seem to arise from the differences in the sugar units linked at C-3. Since the order of potency for these saponins was 6 > 8 » 1 > 3, it meant that the potency of the sugar moieties was in the order of Sg > S,o » Sj > S3. In a detailed comparison between disaccharide and trisaccharide groups, the former group shows greater action. Also, from a comparison study of each disaccharide or trisaccharide group, the OG having a galactosyl unit in the central sugar moiety shows greater action anytime than the arabinosyl unit. Hence, the hydroxymethyl group of the galactosyl unit would enhance the hepatoprotective activity.

102

(A)

90,

(B)

80,

701

601

I I 40 I 30 # I 20 f I 10 0 I II 50

<

<

Com. Ref.

II II II II II II

II II II II II II

II II II II II II

II II II II II II

I in II I II I II I II I

< <

90. 80. 70. 60. 50. 40. 30 4

201 10 4 0

10 30 90 200 500 OiM)

rini irii i I II II II II m

I II II II II II II I

Com. Ref.

10 30

90

200 500 OiM)

(D) ?0 80 70

I II II II I I II II II I I II II II I •0 II II II 30 20 I II II II II 10 4 I II II II II II I 0 om. Ref. 10 30 90 200 500 I II II II II II II(fiM)I

50

50

<

I II II II II II I 11 II II II II II II I Com. Ref.

Fig.

10 30 90 200 500 (JiM)

<

( 7 ) . Hepatoprotective Actions of Soyasaponins I - IV toward in vitro

Immunological Liver Injury on Primary cultured Rat Hepatocytes Control (Cont.) is the value of hepatocytes which were not administered the antiserum. Reference (Ref.) value were administered the antiserum and not treated with Soyasaponins I (A), II (B), III (C), IV (D). Significantly different from Ref., effective *p < 0.01.

d. Hepatoprotective Activity of Soyasapogenol B Analogs In order to obtain more information on the structure-activity relationship, soyasapogenol B monoglucuronide (SBMG, 12) was prepared by partial hydolysis of 1 [38]. SBMG (12) was significantly effective even at 30 |IM (Table 2) [38]. Since 12 showed strong hepatoprotective activity, the actions of soyasapogenol B and glucuronic acid were tested. The action of soyasapogenol B was almost compalable to

103

that of 1. In contrast, glucuronic acid did not show any action even at the highest dose (500 |1M). When the two compounds were mixed, the hepatoprotective action did not change compared to that of soyasapogenol B. Therefore, we concluded that the linkage between glucuronic acid and soyasapogenol B could enhance hepatoprotective activity. e. Hepatoprotective Activity of OGs from Puerariae Lobatae Radix Puerariae Lx)batae Radix, the roots of Pueraria lobata, is an important oriental crude drug used as a perspiration, antipyretic and antispasmodic agent. During the course of our studies on the constituents of this crude drug, we have reported eleven new oleanene-type triterpene saponins together with two known ones [39, 40]. Furthermore, in a series of studies on hepatoprotective drugs, we reported the preventive effect of the total saponin fraction of the titled plant against immunological liver injury [33]. A survey of hepatoprotective OGs in this drug gave the following results (Table 2) [41]. The tested saponins were classified into three groups, namely, soyasapogenol A glycosides (3 8, 4 3 and 4 4), kudzusapogenol A glycosides (30, 3 1 , 3 3 , 34 and 37) and kudzusapogenol C glycoside (6 6). On the other hand, these saponins were also divided into six groups, the dervatives of Sj (38, 30 and 66), S^ (31), S3 (37), Sg (33 and 43), S9 (34) and S,2 (44). With special attention to the above-mentioned classifications, the relationships between the hepatoprotective activity and their structures were investigated. Within the Sj derivatives group, 3 8 is significantly effective at 90 |IM, while the effect of 30 was very weak, even at 500 |IM. Moreover, all kudzusapogenol A glycosides except for 34 were less effective than glycyrrhizin, although soyasapogenol A glycosides were the most effective. Therefore, the hydroxy group at C-29 would reduce the hepatoprotective activity of these types of OGs. On the other hand, the preventive effect of 3 8 is twice or more than that of 1. Hence, the

104

Table 2. Hepatoprotective Activity of Some Oleanene Glucuronides and Related Compounds Isolated from Fabaceous Plants Substance

References

Glycyrrhizin Soyasaponinl(l) SoyasaponinII(3) Soyasaponin 111(6) Soyasaponin IV (8) SBMG (12) Soyasapogenol B Glucuronic Acid Soyasapogenol B+ Glucuronic Acid Soyasaponin A3 (3 8) Kudzusaponin S Aj (4 3) Kudzusaponin S A4 (4 4) Kudzusaponin A3 (3 0) Kudzusaponin A5 (31) Kudzusaponin Aj (3 3) Kudzusaponin A4 (3 4) Kudzusaponin Aj (3 7) Kudzusaponin Cj (66) Dehydrosoyasaponin I (7 2) Lupinoside PA4 (16) Comploside II (15) Lupinoside PAj (4 0) Subproside V (4 7) Wistariasaponin YC2 (6 7) Astragalloside VIE (4) Wistariasaponin YCj (6 8) AzukisaponinII(7) Azukisaponin V (2) Palustroside I (7 3) Palustroside II (4 6) Kaikasaponin HI (21) Kakkasaponin I (2 2) Phaseosidel(20) Soyasaponin V (5) Azukisaponin VI (8 6) Palustroside HI

[41] [38] [37] [38] [37] [38] [38] [38] [38] [41] [41] [41] [41] [41] [41] [41] [41] [41] [53] [53] [53] [53] [53] [53] [53] [53] [47] [47] [47] [47] [49] [49] [84] [84] [84] [49]

Dose (|IM) 200 90

10

30

. . -

18% 27% -

. . . -

55% 54% 40% 13% 43% 46% 26% -

500

33% 17% 57% 22% 68% 73% 68% 85% 80% 79% 8% 31% 32% 87% 100% 82% 94% 24% 94% 12% 24% 21% 23% 16% 40% 9% 25% 19% 78% 22% 60% 22% 54% 30% 17% 25% 17% 36% 55% 18% 89% 90% 35% 86% 83% 54% 81% 37% 76% 30% 66% 8% 11% 22% 18% 26% 62%

105

hydroxy group at C-21 could enhance the hepatoprotective activity. Among kudzusapogenol A glycosides, 3 0 and 3 3 have a galactosyl unit, whereas 3 1 and 3 4 have a glucosyl unit. When the hepatoprotective activities of 3 0 and 3 1 were compared, 30 was slightly more effective than 3 1 . In contrast, when the hepatoprotective activities of 3 3 and 3 4 were compared, 34 was more effective than 33. Therefore, the configuration of the hydroxy group at C-4" could be less important. Similarly, as regards soyasapogenol A glycosides, the hepatoprotective activity of 4 3 having a terminal galactosyl unit was almost equal to that of 4 4 having a terminal glucuronyl unit. Since the OG having a hexosyl unit shows greater action than that of the pentosyl unit [37], the oxygen-bearing group at C-5" seems to be the factor to enhance the hepatoprotective activity. f. Hepatoprotective Activity of Other Miscellaneous OGs Dehydrosoyasaponin I (72) having a carbonyl group at C-22 shows hepatoprotective activity equal to that of 1 (Table 2) [53]. The action of lupinoside PA4 (16) which was a 22-0-rhamonopyranosyl derivative of 1 was also comparable to that of 1. Therefore, the free hydroxy group at C22 would be not essential for the activity. On the other hand, the action of lupinoside PAj (40) which was a 21-(9-xylopyranosyl derivative of 3 8 was almost completely depressed compared to that of 3 8. Therefore, the free hydroxy group at C-21 might be more important for the activity than that at C-22. Furthermore, since the hepatoprotective action of 6 7 was less than one- half of that of 6 6, the p-configuration of the hydroxy group at C-21 is more effective than the a-configuration in regard to the hepatoprotection. Similarly, the action of the hydroxy derivative (46) at C-30 of Azukisaponin II (7) was depressed compared with that of 7 [47]. Since we reported a similar effect for the hydroxy] group at C-29 in the preceding section, the hydroxymethyl group at C-20 seems to reduce the hepatoprotective action, regardless of configuration.

106

g. Hepatoprotective Glucuronides

Effects

of

Oleanolic

acid-Type

Some oleanolic acid-type saponins were known to exhibit similar hepatoprotective activity [4, 7, 32, 42, 43, 44-46]. We also reported the hepatoprotective activity of Palustroside III which has a glucosyl carboxy group at C-28 [47]. As a part of our continuing study, we examined the preventive effects of five oleanolic acid-type glucuronides (90-95), Fig. (8), isolated from Dwnasia truncata (Table 3) [48]. Oleanolic acid (Ri, R2, R3=H) Hederagenin (Ri, R2=H, R3 =0H)

COOR2 GIcA

v-»n2n3 R2 Ri Chikusetsusaponin iVa (90) GIcA Glc

R3 H

Dt-C(91)

S2

Glc

H

Dt-B (92) Dt-D (93)

Si Si

Glc H

H

Dt-E (94)

S2

Dt-A (95)

S2 Oleanolic Acid 3-O-glucuronicle (96) GIcA Oleanolic Acid 28-0-glucoslde (97)

H

Rha

H

H

H

Glc

OH

H

H

Glc

H

Fig. ( 8 ) . Oleanolic-Acid Type Saponins Obtained from Dumasia truncata

Furthermore, since one of the saponins showed strong cytotoxicity in these experiments, we also examined the cytotoxicity toward hepatocytes without antiserum. Moreover, from the standpoint of the structure-hepatoprotective and -hepatotoxic relationships for the carboxyl group at C-28, oleanolic acid 3-0-glucuronide (96) and oleanohc acid 28-0-glucoside (97) were prepared and tested.

107 Table 3. Hepatoprotective and Hepatotoxic Activity of Some Oleanolic acid-Type Glucuronides and Related Compounds [52] Substances

Dose (^M)

Chikusetsusaponin IVa (9 0)

10 30 90 200 500 10 Dt-C (91) 30 90 200 500 Dt-B(92) 10 30 90 200 500 Dt-D(93) 10 30 90 200 500 Dt-E(94) 10 30 90 200 500 Dt-A(95) 10 30 90 200 500 Oleanolic Acid 3-0-glucuronide (96) 10 30 90 200 500 Oleanolic Acid 28-0-glucoside (9 7]) 10 30 90 200 500 Oleanolic Acid 10 30 90 200 500

Protection (%) "^ Cytotoxicity (%) ^^ -1 -2 9 10 20 * 5 0 -2 -2 30 * -4 -4 4 29 ** 4-7 *«* 8 10 11 1 22 * -6 0 -8 -4 t -298 ^ 1 -2 2 7 19 * 24 21 39 *** 24 1 20 -3 -2 -14 -22 14 15 17 9 30 *

104 106 104 134., 408'' 111 105 106 103 100 96 113 96 94 102 100 106 96 116 . 176 ' 110 118 tt 339, 383,' 373 90 92 91 83 80 148 157 939,; 2075 !! 1970^^ 110 105 130 110 130,, 229,; 300,, 304 313,! 267"

a) Hepatoprotective activity toward in vitro immunological liver injury on primary cultured rat hepatocytes. Significantly different from Reference, effective *p<0.05, **/7<0.01, ***p < 0.001, toxic tp < 0.001. b) Hepatotoxicity in primary cultured rat hepatocytes. Significantly different from Reference, toxic tp<0.01, t t p < 0.001.

108

A known antihepatotoxic saponin (9 0) [43] also showed hepatoprotective activity in this model. Although both a rhamnosyl derivative (91) and a rhamnosyl xylosyl derivative (9 2) of 9 0 similarly showed hepatoprotective activity, the levels of activity depended upon the number of sugar molecules linked at C-3. In a similar manner, the hepatoprotective activity of saponin 9 3, which was a prosapogenin of 9 2, was less effective than that of 9 2. Furthermore, not only did 94 lack hepatoprotective activity, but also it showed strong hepatotoxicity. Since Saito et al also reported a similar result [46], the monodesmosyl saponin would be less effective than the bisdesmosyl saponin in an in vitro model. On the other hand, the action of the hydroxyl derivative (95) at C-23 of 9 1 was slightly depressed compared with that of 9 1 . Since we reported a similar effect for the hydroxyl group at C-24 [30, 49], the hydroxymethyl group at C-4 seems to reduce the hepatoprotective action, regardless of configuration. Since 94 showed strong cyototoxicity at the highest dose, the cytotoxicity toward liver cells was also examined without antiserum (Table 3). Both monodesmosyl saponins (93, 94) showed hepatotoxicity. In contrast, the bisdesmosyl saponins (91, 92, 95), except for 90, did not show any hepatotoxicity. When the hepatotoxicity of 9 3 and 9 4 was compared, 9 4 was much more toxic than 9 3. Since 9 0 also showed some hepatotoxicity, even though it was bisdesmoside, the hepatotoxicity might depend on the number of sugar molecules. In order to clarify the effects of the sugar residues at C-3 and C-28 responsible for hepatoprotective and hepatotoxic actions, oleanolic acid 3-0-glucuronide (96) and oleanolic acid 28-0-glucoside (97) were prepared and tested [52]. Oleanolic acid 28-0-glucoside (97) showed neither hepatoprotective action nor hepatotoxicity. In contrast, oleanolic acid 3-(9-glucuronide (96) was slightly effective at 90 |iM for hepatoprotection, although it showed the strongest hepatotoxicity at higher dose. Similarly to the results of Hikino et al [42], oleanolic acid itself showed both hepatoprotective activity and weak hepatotoxicity. Since some oleanolic acid-type saponins were known

109

to exhibit preventive effects toward in vivo liver injury model [4, 6, 45], the hepatoprotective activity of these types of saponins could represent a balance between hepatoprotective action and hepatotoxicity. h. Structure-Hepatoprotective Relationships of OG Some structure-hepatoprotective relationships were obtained through our experiments. Figure (9) shows the structure-hepatoprotective relationships of the aglycone moiety. That is to say, the p-hydroxy group at C-21 would enhance hepatoprotective activity; on the contrary, the hydroxy group at C-23, 24, 29 and 30 could reduce the activity. On the other hand, the free carboxylic acid group at C-28 may mediate cytotoxicity toward liver cells. Reduce Activity

Enhance.. ; Activity

R'=OH.=0 No Contribution

HO" 23 Reduce Activity

R = COOH To^

Fig. ( 9 ) . Structure-Hepatoprotective Relationships of Aglycone Moiety

Figure (10) shows the structure-hepatoprotective relationships of the sugar moiety. The skeleton with glucuronic acid linked at C-3 was a crucial unit in mediating hepatoprotective activity. In the case of a disaccharide chain bound at C-3, an oxygen-bearing group at C-5" seems to enhance the hepatoprotective activity. The terminal rhamnopyranosyl fabatrioside does not seem to contribute to the activity.

group of

110

Enhance Activity ~0 Me 1

HO N

HO-

k _

m 1 _ HO

1

Sufficient for Mediating Activity Reduce Activity

J

Fig. ( 1 0 ) . Structure-Hepatoprotective Relationships of Sugar Moiety

As the result of our structure-hepatoprotective relationship study of OGs, we prepared Fig. (11) summarizing the obtained relationships. The mechanism of our immunological liver injury is regarded as being caused by complement-mediated cell damage [50, 51]. Therefore, OGs seem to prevent the complement system from injuring the hepatocyte membrane. HydroPhilic Region (HR) Lipophilic Region (LR)

. ^

^..

ALT

Fig. ( 1 1 ) . Structure-Hepatoprotective Relationships of Oleanene Glucuronides

At that time, OGs could recognize the hepatocyte membrane, that is, the hydrophobic area around at C-4, C-20; hydrophilic area around at C-21,

Ill

C-5". In view of its strong affinity toward hepatocyte membranes, free carboxylic acid at C-28 might induce transaminase (ALT) leaking, i.e., hepatotoxicity. i. Hepatoprotective Effects of SoMG As described above, since the hydroxyl group at C-24 reduces the hepatoprotective activity, sophoradiol glucuronides having a methyl group at C-24 was much more effective than soyasapogenol B glucuronides [30, 49]. In order to clarify in more detail the structure-hepatoprotective relationship of OGs, we investigated the hepatoptotective effects of the hydrolytic products of kaikasaponin III (21), i.e., kaikasaponin I (26), sophoradiol monoglucuronide (SoMG) and sophoradiol. The

information

supported

previously

obtained

structure-

hepatoprotective relationship data (Table 4), namely, that the methyl group at C-24 enhances the hepatoprotective activity. Furthermore, 2 6 was more effective than 2 1 . Compound 26 showed hepatoprotective activity even at 30 |iM. On the other hand, not only did SoMG reduce hepatoprotective activity, but also it showed strong hepatotoxicity at the highest dose (500 |LiM). In contrast, the aglycone (sophoradiol) showed hepatoprotective activity at the same dose although less potent. Since SoMG showed strong cyototoxicity at the highest dose, their cytotoxicity, together with that of soyasapogenol B analogs (1, 6 and 12), toward liver cells was also examined without antiserum (Table 4). Only 3 showed hepatotoxicity at doses of 200 |LiM and 500 |J.M. This is the first example of OG revealing hepatotoxicity although some glucuronides of oleanolic acid have showed hepatotoxicity [52]. Since SoMG showed hepatoprotective activity at 200 |LiM, the hepatoprotective activity of OG could represent a balance between hepatoprotective action and hepatotoxicity.

112 Table 4. Hepatoprotective and Hepatotoxic Activity of for Hydrolytic Products of Kaikasaponin III and Soyasaponin I Substances Kaikasaponin III (21)

Kaikasaponin I (2 6)

SoMG

Sophoradiol

Soyasaponin I (1)

Soyasaponin III (6)

SBMG (12)

Soyasapogenol B

Dose (|LlM) 10 30 90 200 500 10 30 90 200 500 10 30 90 200 500 10 30 90 200 500 10 30 90 200 500 10 30 90 200 500 10 30 90 200 500 10 30 90 200 500

Protection (%)"' 5 3 10 34** 81 ** 3 17 * 76** 93** 94 ** 4 6 0 18 * -49 t 0 3 2 0 13 *

Cytotoxicity (%)*' 95 90 80 95 75 113 144 113 138 156 115 105 115

350 I

660 82 82 91 95 100 121 95 95 105 89 82 100 106 100 141 90 85 80 85 110 118 82 106 112 100

a) Hepatoprotective activity toward in vitro immunological liver injury in primary cultured rat hepatocytes. Significantly different from Reference, effective *p<0.01, **p< 0.001, toxic tp< 0.001. b) Hepatotoxicity in primary cultured rat hepatocytes. Significantly different from Reference, toxic tp< 0.001.

113

j . Structure-Hepatoprotective Relationships of SoMG The structure-activity relationship of OGs obtained from the results of Sophoradiol derivatives was divided into two models. One is for 2 1 and 26 which accorded with structure-hepatoprotective relationships obtained from the previous results, Fig. (12). Lipophilic Region Hydrophilic Region

^

R i , R 2 = H Enhanced Activity

I Ri = OH, R2 = Rha Reduced Activity

Fig. ( 1 2 ) . Structure-Hepatoprotective Relationships except for SoMG

They seemed to show hepatoprotective action due to the suitable distance they keep from hepatocyte membranes. The other model for SoMG is completely different, Fig. (13). Lipophilic Region Hydrophilii Region

CH3

Transaminase Leal
Fig. ( 1 3 ) . Structure-Hepatotoxic Relationship for SoMG

Because of its strong affinity toward hepatocyte membranes, SoMG appeared to be too close to the membranes for protection. Hence, it showed hepatotoxicity, i,e,, injured hepatocytes, although it also showed protective

114

action against antiserum. Consequently, the transaminase (ALT) leaked out of the hepatocytes. A similar observation has been obtained from the experiments with oleanolic acid-type glucuronides, Fig. (11). In the meantime, Shiraki reported that glycyrrhizin inhibited the replication of hepatitis B virus after penetrating through hepatocyte membranes [54]. Since soyasaponins I and II also showed antiviral effects against several viruses [18], OG might show not only protective action on hepatocyte membranes but also antiviral action after penetrating through the membranes. Therefore, we tried to elucidate the antiviral effects of OGs in the next section. ANTIVIRAL ACTIVITY OF OG There are some reports concerning the antiviral activites of triterpenoidal saponins [55-58]. Especially, anti-herpes simplex virus type 1 (HSV-1) activities are well-known [59, 60]. Further, some sapogenols showed in vivo efficacy [61]. As mentioned above, Shiraki have elucidated the unique mechanism of glycyrrhizin action on HBsAg processing, intracellular transport and secretion [54]. Glycyrrhizin suppressed the intracellular transport of HBsAg at the trans-Golgi area after O-linked glycosylation and before its sialylation. Hayashi et d, reported antiviral activities of two OGs (1 and 3) against human cytomegalovirus, influenza virus, HIV-1 together with HSV-1 [18]. They reported that antiviral action was not due to inhibition of virus penetration and protein synthesis, but might involve a virucidal effect. From the standpoint of the structure-activity relationship, we also examined antiherpetic activity of some OGs and related compounds against HSV-1. The tested samples were soyasapogenol B glucuronides (1 - 4, 6, 7 and 12), sophoradiol glucuronides (21, 22, 26 and SoMG) and soyasapogenol E glucuronides (72 and 74). On the other hand, these saponins were also divided into seven groups which have different sugar

115

moieties linked at C-3. It was, therefore, the aim of our study to investigate the relationships between the anti-HSV-1 activity and the structures of the saponins, with special attention to the above-mentioned classifications. Among sophoradiol glycosides ( 2 1 , 2 6 and SoMG), kaikasaponins III (21) and I (2 6) are a trisaccharide and disaccharide, respectively, whereas SoMG is a monosaccharide. When the anti-HSV-1 activities of 2 1 , 2 6 and SoMG were compared (Table 5), the order of potency was 2 1 > 26 » SoMG. It was suggested that the trisaccharide group shows greater action than the disaccharide group. Similar observation was obtained from the comparison with azukisaponins V (2) and II (7). Not only SoMG but also SBMG (12) showed no activity. Within the trisaccharide group of soyasapogenol B (1 - 4), the order of anti-HSV-1 activity was azukisaponin V (2) > soyasaponin II (3) > astragaloside VIE (4) » soyasaponin I (1). Therefore, the potency of the sugar moieties was in the order of Rha-(l->2)-Glc-(1^2)-GlcA > Rha-(l->2)-Ara-(l->2)-GlcA > Rha- (l-^2)-Xyl-(l-^2)-GlcA » Rha-(l-^2)-Gal-(l->2)-GlcA. Similarly, within the disaccharide group of soyasapogenol B (6 and 7), the potency of the sugar moieties was also in the order of Glc-(l-^2)-GlcA » Gal-(l->2)GlcA. The saponin having a glucosyl residue seemed to show greater action. When the anti-HSV-1 activities of 1, 2 1 and 72 which have the same trisaccharide group (Sj) were compared, the order of potency was dehydrosoyasaponin I (72) > kaikasaponin III (21) » soyasaponin I (1). This meant that the potency of the sapogenol moieties was in the order of soyasapogenol E > sophoradiol » soyasapogenol B. Similarly, in the case of another trisaccharide group (S4, 4 and 7 4), the potency of the sapogenol moieties was also in the order of soyasapogenol E » soyasapogenol B. Furthermore, within the same disaccharide group (S5, 6 and 26), the potency of the sapogenol moieties was in the order of sophoradiol » soyasapogenol B. Hence, the carbonyl group at C-22 would be more effective than the hydroxyl group in regard to the anti-HSV-1 activity, while the hydroxy group at C-24 could reduce the activity.

116

Table 5. Anti-HSV-1 Activity, Cytotoxicity and Selectivity Index of Some OGs Sample

Anti-HSV-1 Activity (IC5,,; ^M)

Soyasaponin I (1) Azukisaponin V (2)

>75.0 21.0 ± 1.0

Soyasaponin II (3)

27.4 ± 1.6

Astragaloside Vin (4)

43.0 ± 2.0

Kaikasaponin in (21)

43.2 ± 2.5

Kakkasaponin I (2 2) Soyasaponin III (6) Azukisaponin II (7) Kaikasaponin I (2 6) SBMG (12)" SoMG' Dehydrosoyasaponin I (7 2)

22.3 ± 1.3 >75.0 54.0 ± 2.5 64.1 ± 3.8

Cytotoxicity

Selectivie Index

(CC50; HM)

(CCjo/ICjo)

-

-

>332 115 ± 2 9 >343

> 15.8 4.2 > 13.7

119 ± 2 0

2.8

698± 117

31.3

>393 641 ± 108

>7.3 10.0

>75.0 >75.0 19.1 ± 0 . 9

>332

> 17.4

Wistariasaponin D (7 4)

25.1 ± 1.2

>343

> 13.7

Soyasapogenol B Sophoradiol Acyclovir (positive control)

5.6 ± 0.6 37.8 ± 2.2 1.11 ±0.09

116±29 > 141 >444

20.7 >3.7 >400

On the other hand, when the sapogenols themselves were tested, soyasapogenol B showed the most potent activity (5.6 |iM), although sophoradiol was slightly more effective than its trisaccharide (21). Since both monoglucuronides showed no activity, the mechanism of the aniti-HSV activity for sapogenol itself might be different from that for its glycoside. Further investigation to confirm the anti-HSV activity of some sapogenols obtained from various fabaceous plants is now being planned. In Fig. (14), the obtained structure-antiherpetic activitiy relationships are shown.

117

\H0|

Enhance HO \

1 SNS"i HO-
Enhance

' 11

[=0 > -OH]

HO

Fig. ( 1 4 ) . Structure-Antiherpetic Activity Relationships of Oleanene Glucuronides

ANTICOMPLEMENTARY ACTIVITY OF OG The complement system is a humoral effector of inflammation which is activated by a cascade mechanism through the classical and/or alternative pathway [62]. Activation of the system is nomially beneficial for the host. However, excessive activation may evoke pathological reaction in a variety of immunological and degenerative diseases and hyperacute rejection in transplantation. Therefore, the modulation of complement activity should be useful in the therapy of inflammatory diseases. As described previously, the mechanism of our immunological liver injury is regarded as being caused by complement-mediated cell damage [50, 51]. In connection with this view, Shinohara et cd, reported anticomplement actions of soyasaponin I analogs [17]. The order of potency was soyasaponins III (l-'2 |Llg/ml) > IV (3-^5 |Xg/ml) > SBMG (5 |ig/ml) » I (125 |Lig/ml) > II (100-150 |J.g/ml). The order was very similiar to the order for hepatoprotective activity obtained from our experiments. Shinohara et al also revealed anti-nephritis action of those analogs on an experimental in vivo model. Since the antibody-complement system plays an important role in humoral immunity, we were interested in the structure-hepatoprotective relationships of the individual OGs. At first, we tested sophoradiol

118

glucuronides together with soyasapogenol B glucuronides (Table 6). Table 6. Anti-complementary Activity of Soyasaponin I, Kaikasaponin III and Related Compounds Substance

Classical Pathway (CP) IC50

Alternative Pathway (AP) % inhibition (32^lg/ml)

Soyasaponin I (1)

67 ^M

14%

Soyasaponin HI (6)

32^lM

15%

3|XM

-19 %* 1% -18 %* -7%* -2%* 15%

SBMG(12) Soyasapogenol B Kaikasaponin EQ (21) Kaikasaponin I (2 6) SoMG Sophoradiol Azukisaponin V (2) Dehydrosoyasaponin I (7 2)

-50% (32|Lig/ml)* 62|iM 13 ^M 13 ^M -47% (32|Lig/ml)* 14 ^M 50 ^iM

-

Soyasaponin 11(3)

26 ^M

-

Wistariasaponin D (7 4)

15 ^M

-

Astragaloside VIII (4) Lupinoside PA4 (16) Azukisaponin II (7)

21 ^IM 8^M 3^M

-

Kudzusaponin A3 (3 0) Lupinoside PA, (4 0) Lupinoside PA3 (3 6) Dt-E(94)

120|ilM

-

29|IM 61 |LlM

-

*

-

Dt-C (91)

niiM

-

Dt-B(92)

28 ^IM

-

-

-

* hemolytic

Among them, diglycosidic glucuronides (6 and 21) showed the most potent anti-complementary activity, followed by monoglucuronides (12 and SoMG) and triglycosidic glucuronides (1 and 21). Sophoradiol and soyasapogenol B did not show the inhibition of hemolysis, but rather promoted it under the presence of serum on the classical pathway, whereas

119

all of them showed very weak or no hemolytic effects on the alternative pathway of the complement system (Table 6). The anti-complementary activity of each saponin was influenced by the nature of glucuronic acid (Table 7), where the free acid (COOH) showed much more potent activity than the sodium salt (COO"Na"*") or methylester (COOCH3). Reduction of the acid (glucuronic acid) to the alcohol (glucose) of saponins decreased significantly their activity. The free acid form of the glucuronic acid moiety seemed to contribute to the potency. These results indicate that the glucuronic acid moiety is important for expression of the anti-complementary activity. In contrast to hepatoprotective action, the hydroxy group at C-24 did not affect the anticomplementary activity. However, in the case of the methyl ester of sophoradiol glucuroides (Table 7), the anti-complementary activity was significantly depressed. Therefore, the hydroxy group at C-24 of soyasapogenol B glucuronides might enhance anti-complementary activity instead of the methoxy carbonyl moiety at C-6 of glucuronic acid. Table 7. Anti-complementary Activity of Various Analogs for the Glucuronic Acid Moiety of Oleanene Glucuronides on CP Substance

IC50

Free acid (-COOH) Soyasaponin I Soyasaponin lU SBMG Kaikasaponin III Kaikasaponin I SoMG

10 JIM 6.8 iiM 7.9 jlM 44|iM 2.6 JIM 9.9 JIM

Sodium salt (-COONa^ -11%(35|LIM)* 42|LlM 15|IM 50 ^M 14 ^M 30|LtM

% inhibition Methyl ester (-COOCH3) 30|XM 40 ^M 35|XM 180 jlM 210 |IM 660|LlM

Reduced (-CH2OH) -5%(100^M)* -2%(100^M/

4%(100|IM) 29%(100|iM) 12%(100jiM) -20%(100|IM)*

hemolytic

Various other oleanene glucuronides having a characteristic functional group were also tested and the structure-anti-complementary activity

120

relationships were discussed (Table 6). Within the trisaccharide group of soyasapogenol B, the order of anticomplementary activity was azukisaponin V (2) > astragaloside Vni (4) > soyasaponin II (3) > soyasaponin I (1). Therefore, the potency of the sugar moieties was in the order of Rha-(l-^2)-Glc-(l->2)-GlcA > Rha(l-^2)-Xyl-(l->2)-GlcA > Rha-(l-^2)-Ara-(l-^2)-GlcA > Rha-(1~>2)Gal-(l-»2)-GlcA. Similarly, within the disaccharide group of soyasapogenol B (6 and 7), the potency of the sugar moieties was also in the order of Glc-(l->2)-GlcA > Gal-(l-^2)-GlcA. The saponin having an a-hydroxy group at C-3" (Glc or Xyl) seemed to show greater action. When the anti-complementary activities of 1 and 7 2 which have the same trisaccharide group (S,) were compared, the order of potency was dehydrosoyasaponin I (72) > soyasaponin I (1). This meant that the potency of the sapogenol moieties was in the order of soyasapogenol E > soyasapogenol B. Similarly, in the case of another trisaccharide group (S4, 4 and 7 4), the potency of the sapogenol moieties was also in the order of soyasapogenol E > soyasapogenol B. The information that the hydroxy group at C-29 reduced activity was similar to that on the structure-hepatoprotective relationship. In contrast, the sugar moiety in the E-ring enhanced anti-complementary activity. Further, we tested some oleanolic acid-type glucuronic acids. Although bisdesmosidic saponins showed moderate anti-complementary activity, a monodesmosidic saponin (94) showed cyototoxicity. Since some inflammation, including hepatitis and nephritis, is caused by excessive immunoreaction, it might be possible that OGs in the edible fabaceous plants play an important role for suppression of some inflammation. In Fig. (15), the obtained structure-anticomplementary activity relationships are shown.

121 Hemolytic ? Essential

Not Important

COOH

Reduce Enhance ? COOH* > COONa* > COOMe » CH2OH

Reduce

Fig. ( 1 5 ) . Structure-Anticomplementary Activity Relationships of OGs

ABBREVIATIONS OG: ALT:

= Oleanene-Glucuronide = Alanine Aminotransferase

AST: SBMG: SoMG: HSV-1:

= Aspartate Aminotransferase = Soyasapogenol B Monoglucuronide = Sophoradiol Monoglucuronide = Herpes Simplex Virus Type 1

ACKNOWLEDGEMENTS I express my appreciation to Dr. H. K. Lee of Korea Research Institute of Bioscience and Biotechnology for measurement of anti-complementary activity. I am grateful to Dr. K. Yokomizo and Prof. M. Uyeda in this faculty for measurement of antiherpetic activity.

122

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Prod., 1995, 55, 1372-1375. [59] Ushio, Y.; Abe, H. Planta Medica, 1992, 58, 171-173. [60] Sindambiwe, J. B.; Calomme, M.; Geerts, S.; Pieters, L.; Vlietinck, A. J.; Vanden Berghe, D. A. J, Nat. Prod, 1998, 61, 585-590. [61] Kurokawa, M.; Basnet, P.; Ohsugi, M.; Hozumi, T.; Kadota, S.; Namba, T.; Kawana, T.; Shiraki, K. J. Pharmacol. Exp. Ther., 1999, 289, 12-1%. [62] Park, S.-H.; Oh, S.R.; Jung, K.Y.; Lee, I.S.; Ahn, K.S.; Kim, J.H.; Kim, Y.S.; Lee, J.J.; Lee, H.-K. Chem. Pharm. Bull, 1999, 47, 1484-1486. [63] Kinjo, J.; Matsumoto, K.; Inoue, M.; Takeshita, T.; Nohara, T. Chem. Pharm. Bull, 1991,39, 116-119. [64] Kinjo, J.; Hatakeyama, M.; Udayama, M.; Tsutanaga, Y.; Yamashita, M.; Nohara, T.; Yoshiki, Y.; Okubo, K. BioscL Biotech. Biochem., 1998, 62, 429-433. [65] Cui, B.; Sakai, Y.; Takeshita, T.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992,40, 136-138. [66] Cui, B.; Inoue, J.; Takeshita, T.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992, 40, 3330-3333. [67] Kinjo, J.; Yamashita, M.; Nohara, T. In Food Factors for Cancer Prevention; Ohigashi, H.; Osawa, T.; Terao, J.; Watanabe, S.; Yoshikawa, T., Eds.; SpringerVerlag: Tokyo, 1977, pp. 323-327. [68] Ding, Y.; Kinjo, J.; Yang, C ; Nohara, T. Chem. Pharm. Bull, 1991, 39, 496498. [69] Yahara, S.; Emura, S.; Feng, H.; Nohara, T. Chem. Pharm. Bull, 1989, 37, 2136-2138. [70] Kubo, T.; Hamada, S.; Nohara, T.; Wang, Z.-R.; Hirayama, H.; Ikegami, K.; Yasukawa, K.; Takido,M. Chem. Pharm. Bull, 1989, 37, 2229-2231. [71] Udayama, M.; Kinjo, J.; Yoshida,N.; Nohara, T. Chem. Pharm. Bull, 1998, 46, 526-527. [72] Hirakawa, T.; Okawa, M.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 2000, 48, 286-287. [73] Kinjo, J.; Kishida, F.; Watanabe, K.; Hashimoto, F.; Nohara, T. Chem. Pharm. Bull, 1994,42, 1874-1878. [74] Yahara, S.; Irino, N.; Takaoka, T.; Nohara, T. Natural Medicines, 1994, 48, 312313. [75] Arao, T.; Idzu, T.; Kinjo, J.; Nohara, T.; Isobe, R. Chem. Pharm. Bull, 1996, 44, 1970-1972. [76] Cui, B.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992, 40, 2995-2999. [77] Cui, B.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1993, 4], 553-556. [78] Takeshita, T.; Yokoyama, K.; Ding, Y.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1991,59, 1908-1910. [79] Ding, Y.; Takeshita, T.; Yokoyama, K.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992,40, 139-142. [80] Ding, Y.; Tian, R.; Takeshita, T.; Kinjo, J.; Nohara, T. Chem. Pharm. Bull, 1992,40, 1831-1834. [81] Ding, Y.; Tian, R.; Kinjo, J.; Nohara, T.; Kitagawa, I. Chem. Pharm. Bull, 1992,40,2990-2994. [82] Kinjo, J.; Fujishima, Y.; Saino, K.; Tian, R.; Nohara, T. Chem. Pharm. Bull, 1995, 43, 636-640. [83] Udayama, M.; Kinjo, J.; Nohara, T. Phytochemistry, 1998, 48, 1233-1235. [84] Kinjo, J.; Udayama, M.; Hatakeyama, M.; Ikeda, T.; Sohno, Y.; Yoshiki, Y.; Okubo, K.; Nohara, T. Natural Medicines, 1999, 5i, 141-144.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry', Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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BIOTRANSFORMATION OF TERPENOIDS BY MICROORGANISMS JAN C.R. DEMYTTENAERE Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, 8-9000 Gent, Belgium; e-mail: Jan. Demvttenaere(^rug. ac. be ABSTRACT: In recent years, theflavourmarket has undergone a tremendous 'back-tonature' demand, which is illustrated by the consumers' preference for 'natural' flavourmg substances instead of synthetic 'artificial' compounds. These natural products can be obtained by extraction from plant material, but they can also be produced by biotechnological processes using micro-organisms. De novo synthesis of 'bioflavours', such as volatile esters, and biotransformation of monoterpenes are fields of investigation that gain a growing interest. Terpenes are obtained from the essential oil of many plants and are relatively cheap. They are usually isolated from the oils by rectification. This renders abundant monoterpenes, such as a-pinene and limonene, inexpensive starting materials for chemical and biochemical transformations. Thefiingalbiotransformation of these natural precursors to more valuable aroma compounds offers a very interesting alternative source of naturalflavours.This review article deals with the biotransformation of some monoterpenoids and sesquiterpenoids by both fiingi and bacteria. As substrates myrcene, ocimene, geraniol, nerol, citronellol, citral, citronellal, linalool, limonene (and related compounds), pinenes, menthol, camphor, pulegone, ionones, nerolidol, famesol, caryophyllene, valencene, patchoulol, etc... are discussed.

INTRODUCTION 1 Definition of 'Natural Flavours' As consumers want more and more 'natural flavours' instead of synthetic ones, there is a trend to focus on the production of these natural flavour substances [1,2]. In the United States of America the Code of Federal Regulations (CFR 101.22.a.3) defines the term 'natural flavour' to include not only animal or plant derivatives but also products obtained from enzymatic and fermentative processes. According to the regulations of the US FDA (Food and Drug Administration) guidelines (1958), a natural flavour must be produced from natural starting materials and the endproduct must be identical to a product already known to exist in nature. Thus, biocatalytic, but not chemical transformation of natural substances can be legally labelled as natural [3]. The European guidelines (88/388/EWG of June 22, 1988; 91/71/EWG and 91/2/EWG of January 16, 1991) define natural aroma compounds as isolated by 'physical, enzymatic, or microbiological processes or traditional food preparation

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processes solely or almost solely from the foodstuff or the flavouring source concerned' [4]. However, there is a difference between natural, nature-identical and artificial products. Natural products are directly isolated from plant or animal sources, by physical processes, such as extraction, maceration, distillation,... Microbial production of flavours is also a source of natural products. Nature-identical compounds are produced synthetically, but are chemically identical to their natural counterparts. Artificial flavour substances are compounds that have not yet been identified in plant or animal products for human consumption. They are made synthetically, have the same or similar smell and other properties as some natural flavours, but are chemically totally different. For example, vanillin can be obtained via at least five different ways: (i) by isolation from the orchid (Vanilla planifolia), which is a very expensive method; (ii) by tissue culture followed by extraction; (iii) by microbial transformation of eugenol, the main compound of clove; (iv) from lignine by synthesis, and (v) from guaiacol, a natural aroma compound, with comparable molecular structure. Only the vanillin obtained via the first three methods is natural. The other routes afford a nature-identical vanillin. Since not only the isolation from nature but also biotechnological processes (the use of microorganisms and enzymes) are a source of 'natural flavours' [5], the term 'bioflavours' will be used. 2 Bioflavours In 1987, the term 'bioflavours' was used for the first time as the title of an intemational symposium at Wiirzburg University (Bioflavour *87). In his introductionary lecture, Drawert defined 'bioflavours' as 'natural* and 'naturally produced' flavours [6]. Many important food aromas originate from biochemical pathways. These pathways comprise microbial reactions, endogenous and exogenous enzymatic action, and plant metabolism. In the past, flavour research concentrated on characterising the important chemicals in foods responsible for their specific aroma. Less information is therefore available on the biogeneration of flavours. At present, however, a renaissance of studies of natural flavours, including their biogeneration can be observed [1]. A number of factors appear to be responsible for the renewed interest in bioflavour research, e.g,: - consumers increasingly reject artificial flavours and demand natural ones;

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- bioflavour formation may provide possibilities for producing industrially important secondary metabolites which are not available by conventional procedures; - advances in genetic research and genetic engineering permit speculations to explore bioflavour production. Within the field of biotechnology, food technology is the oldest and economically most important part. Ten years ago, about 90% of the industrially produced flavours and fragrances were of synthetic origin, the rest was mainly derived from agricultural sources [6]. In contrast to repeated predictions, the trend to natural flavours is unbroken. Today, food flavours share about 2 billion US $ in the world-wide 5 billion US $ market for volatile flavours and fragrances [4]. Natural aromas account for 60% and 40%, respectively, of the total sales in Europe and in the United States of America. The respective figures for Japan and the remaining countries are 10% and 5%. In Germany however, about 70% of all food flavours used in 1990 were natural [7]. 3 Scope and Production of Bioflavours Some advantages of the biotechnological production of flavours are [6]: - the products may possess the legal status of a natural compound; - the high substrate and reaction specificity of enzymes guarantee a defined stereochemistry; - optimised reaction conditions lead to complex, uniform products and to constant productivity; - multiple-step reactions, which are not possible in aqueous solution by chemical means, proceed under mild conditions; - adverse extemal influences such as unfavourable climate, pest infestations, economical or ecological drawbacks can be ignored. There are many different ways to produce bioflavours: plant tissue and cell cultures can be used, microorganisms, such as bacteria, yeasts, fungi and algae can produce flavours de novo and through bioconversions, and enzymes can be applied. 3.1 Plant Tissue and Cell Culture

A large number of various fine chemicals is derived from plants, e.g., drugs, pigments, and other biologically active substances. In the past, their production by means of plant cell cultures has attracted the interest of many researchers. Although most plant cell cultures have been unable to

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produce an adequate yield of flavour substances, a few examples exist where tissue cultures exhibited an increased production compared to that of the plant. On the other hand, a variety of volatiles, different from those occurring in the plant, has been isolated from cell cultures [8]. As de novo synthesis has been proven unsuccessful in most cases, biotransformation of added precursors has been studied extensively. There is evidence that plant cell cultures retain an ability to transform specifically exogenous substrates administered to the cultured cells. Therefore, plant cell cultures are considered to be useful for transforming cheap and plentiful substances into rare and expensive compounds by using the cell culture as a bioreactor. For instance, cofactor dependent specific conversions of terpenoids in suspension cultures of aromatic plants often proceed with high yields and negligible amounts of byproducts. In Fig. (1), three examples of biotransformations of terpenes by plant cell cultures are shown (after [6]). I Salvia officinalis

_ _

^

Borneol (1)

CHO

r v ^

1 ^

Conversion rate no 0/

78/.

Camphor (2)

Melissa officinalis • 2h

Citronellal (3)

I I Is^ CHjOH

99.5%

Citronellol (4)

66%

Valencene (5)

Nootkatone (6)

Fig. (1). Biotransformation of terpenes by plant cell cultures (after [6])

3.2 Use of Microorganisms

A very efficient way to obtain bioflavours is by biotechnological processing using microorganisms [5]. The microbial production of aromas offers many advantages: the circumstances under which the reactions occur are generally mild: e.g., neutral pH, ambient temperature. There are

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two important ways to produce microbial flavours: de novo production of aromas, also called biosynthesis and the transformation of natural precursors into interesting flavour compounds, called bioconversion [9]. An extensive overview on the biotranformation of terpenoids by microorganisms will be given in the next chapter. The capacity of microorganisms to produce pleasant odours has been known at least since the beginning of this century [10]. Terpenoids are natural compounds responsible for the characteristic smell of essential oils. Many of the terpenoid producing microorganisms are fungi growing on decaying fir-wood and belonging to the ascomycetes or basidiomycetes. One of the most important examples is the genus Ceratocystis, producing a variety of terpenoid alcohols with a very attractive smell [11]. Esters also constitute a group of important flavour compounds. They are the main aroma components found in fruits (apples, pears, ...). For example, bananas contain 12-18 ppm acetates. The price of the pure flavour compounds, when isolated from fruit, can range between 10,000 and 100,000 US $/kg! In the past, research has been carried out by our group about the microbial production of fruity esters by the yeast Hansenula mrakii and the fimgus Geotrichum penicillatum [10]. A fermentation was developed whereby fusel oil was continuously converted into a mixture of 3-methylbutyl acetate (isoamyl acetate) and 2-methylbutyl acetate, the 'character impact compounds' of banana flavour. A very well known dairy product is Roquefort cheese, its flavour is generated by mould action. This so called 'Blue cheese flavour' is attributed to methyl ketones and is formed by the degradation of fatty acids by Penicillium roquefortii. The production of these bioflavours has also been investigated by our group [12,13] and will not be further discussed here.

BIOTRANFORMATION OF TERPENOIDS BY MICROORGANISMS Introduction Terpenoids (often referred to as isoprenoids) constitute the largest group of natural products. They belong to the most important flavour and fragrance compounds, and are found in the microbial, plant and animal kingdoms [14]. They offer a very wide variety of pleasant and floral scents. Living organisms synthesize a remarkable diversity of isoprenoids [15]. More than 23,000 different compounds have been isolated and all contain one or more isoprene unit, the building block of these compounds [16]. Thus, the

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Structure of terpenoids is built by combining these isoprene (7) units, usually joined head to tail. Terpenes are classified according to the number of isopentenyl units (rule of Ruzicka, Fig. (2)), e,g,, 2: monoterpenes (C10H16), 3: sesquiterpenes (C15H24), 4: diterpenes (C20H32), 6: triterpenes (C30H48X and higher: polyterpenes.

or

Isoprene (7)

Myrcene (8)

Ocimene (9)

Fig. (2). Rule of Ruzicka: head to tail coupling of two isoprene units, giving myrcene

Until 1993, all terpenes were considered to be derived from the classical acetate/mevalonate pathway involving the condensation of three units of acetyl CoA to 3-hydroxy-3-methylglutaryl CoA, reduction of this intermediate to mevalonic acid and the conversion of the latter to the essential, biological isoprenoid unit, isopentenyl diphosphate (IPP) [17,18,15]. Recently, a totally different IPP biosynthesis was found to operate in certain eubacteria, green algae and higher plants. In this new pathway glyceradehyde-3-phosphate (GAP) and pyruvate are precursurs of isopentenyl diphosphate, but not acetyl-CoA and mevalonate [19,20]. So, an isoprene unit is derived from isopentenyl diphosphate, and can be formed via two alternative pathways, the mevalonate pathway (in eukaryotes) and the deoxyxylulose pathway in prokaryotes and plant plastids [16,19]. An extensive overview of the biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants is given by Chappell [17]. Abundant sources of terpenoids are the essential oils. They consist of a complex mixture of terpenes or sesquiterpenes, alcohols, aldehydes, ketones, acids and esters [21]. The monoterpenes are subdivided into three groups: acyclic, monocyclic and bicyclic (there is only one tricyclic terpene: tricyclene). Each group contains hydrocarbon terpenes, terpene alcohols, terpene aldehydes, ketones, oxides etc... The isolation of terpenes from plants entails several problems (e.g., very low concentrations). Therefore other sources of these flavour compounds are searched for: microorganisms for example (especially bacteria and fungi) are used for the production of terpenoids [22]. Since terpenoids are very important flavour and fragrance compounds, the biotransformation of terpenes offers a very interesting source of novel

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flavours, the so-called bioflavours. A recent review of the biotechnological production of flavours and fragrances is given by Krings and Berger [7]. This literature review will deal with the biotransformation of terpenoids by microorganisms, more specifically fungi and bacteria. 1 Monoterpenes

LI Acyclic Monoterpenes 1.1.1 Acyclic hydrocarbon monoterpenes

Myrcene, ocimene As can be seen from Fig. (2), myrcene (8) is the most simple terpene: in nature, it is formed directly by head to tail linking of two Cs-biological isoprene units, isopentenyl pyrophosphate [14]. Rearrangement of one double bond gives its isomer ocimene (9). The biotransformation of these two acyclic hydrocarbon terpenes however, is not very well documented. One of the earliest reports [23] describes the degradation of )ff-myrcene (8) by a strain of Pseudomonas putida, commencing with the oxidation of the terminal methyl group. In 1985, the bioconversion of acyclic terpenes and related structures with a terminal isoprenoid group was described [24]. The preferred microorganism for this reaction was Diplodia gossypina ATCC 10936. The main reactions were hydroxylation reactions, Fig. (3). On oxidation, myrcene (8) gave next to the diol (10) (yield up to 60%) also a side-product (11) that possesses one carbon atom less than the parent compound, in yields of 1-2%.

Diplodia gossypina

Myrcene (8)

I X^-OH

10

+

I ^,CH

11

I k,^

Myrcene (8)

G. applanatum Pleurotus sp.

Myrcenol(12)

Fig. (3). Hydroxylation of myrcene by Diplodia gossypina (after[24]) and by Ganoderma applanatum and Pleurotus sp. (after [25])

One of the most recent publications dealing with the bioconversion of myrcene [25] described its transformation to a variety of oxygenated metabolites, with Ganoderma applanatum, Pleurotus flabellatus and Pleurotus sajor-caju possessing the highest transformation activity. The extracted metabolites represented a complex mixture of numerous acyclic and monocyclic metabolites of which myrcenol (12) (2-methyl-6-

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methylene-7-octen-2-ol), with a fresh-flowery impression, dominated the yield and sensory impact of the mixture, Fig. (3). 1.1.2 Acyclic monoterpene alcohols and aldehydes

Geranioly nerol citronellol citral citronellal Geraniol (3,7-dimethyl-(£)-2,6-octadien-l-ol) (20) occurs in nearly all terpene-containing essential oils, frequently as an ester. Palmarosa oil contains 70-85% geraniol; geranium oils and rose oils also contain large quantities. Geraniol is a colourless liquid, with a flowery-rose-like odour [26]. Nerol (14) is the Z-enantiomer of geraniol and occurs in small quantities in many essential oils where it is always accompanied by geraniol. Its name originates from its occurrence in neroli oil. It has a fresh, sweet rose-like odour and a bitter flavour [27]. Citronellol (3,7dimethyl-6-octen-l-ol) (4) has been found in about 70 essential oils and in the oil of Rosa bourbonia. The Bulgarian rose oil contains more than 50% L-citronellol, whereas East African geranium contains more than 80% of the D-isomer [27]. L-Citronellol, which is also called rhodinol, has a sweet, peach-like flavour and is more delicate than D-citronellol which has a bitter taste. Citral (3,7-dimethyl-2,6-octadien-l-al) (26) occurs as Z(neral) and E- (geranial) isomers, analogous to the corresponding alcohols, nerol and geraniol. Natural citral is nearly always a mixture of the two isomers. It is found in lemongrass oil (Cymbopogon flexuosus (Nees.) Stapf.) in concentrations of up to 75% [27], in Lit sea cubeba oil (up to 75%) and in small amounts in many other essential oils. The citrals are colourless to slightly yellowish liquids, with an odour reminiscent of lemon [26]. (-f)-Citronellal (3,7-dimethyl-6-octen-l-al) (3) occurs in citronella oil at concentrations of up to 45%; Backhousia citriodora oil contains up to 80% (~)-citronellal. Racemic citronellal is found in a number of Eucalyptus citriodora oils at concentrations of up to 85%. It is a colourless liquid with a refreshing lemon-, rose-type odour, similar to balm mint [26]. The first detailed work on the microbial degradation of the acyclic monoterpene alcohols and aldehydes was reported in the early part of 1960 [28-31]. They studied the metabolism of citronellol, citronellal, geraniol and geranic acid by the soil bacterium, Pseudomonas citronellolis. They observed that the metabolism of these acyclic monoterpenes is initiated by the oxidation of the primary alcohol group to the carboxyl group, followed by carboxylation of the C-10 methyl group Off-methyl) by a biotindependent carboxylase [31]. The carboxymethyl group is eliminated at a later stage as acetic acid. Further degradation follows the jff-oxidation pattern. The details of the pathway are shown in Fig. (4) (after [32]).

133

CHO

Citronellol (4)

Citronellal (3)

Citronellic acid (13)

C02

10

CH-OOOH

CH2OH

Nerol (14)

CHjOH OOOH

Geraniol(20)

Geranial (21)

Geranic acid (22)

19

Fig. (4). Microbial degradation of citronellol, nerol and geraniol by Pseudomonas citronellolis (after [32])

The microbial transformation of the aldehydes citronellal (3) and citral (26) by Pseudomonas aeruginosa was also reported [33]. This bacterium, capable of utilising citronellal or citral as the sole carbon and energy source, has been isolated from soil by the enrichment culture technique. It metabolised citronellal (3) to citronellic acid (13) (65%), citronellol (4) (0.6%), dihydrocitronellol (23) (0.6%), 3,7-dimethyl-l,7-octanediol (24) (1.7%) and menthol (25) (0.75%), Fig. (5). The metabolites of citral (26) were geranic acid (22) (62%), l-hydroxy-3,7-dimethyl-6-octen-2-one (27) (0.75%), 6-methyl-5-heptenoic acid (28) (0.5%), and 3-methyl-2-butenoic acid (29) (1%). In a similar way, citral was converted to geranic acid by P. convexa [34]. The biotransformation of citronellol and geraniol by P. aeruginosa, P. citronellolis and P. mendocina was also reported by another group [35]. In 1977, a Pseudomonad was isolated from soil by enrichment culture technique with linalool as the sole source of carbon and energy [36]. The bacterial strain was later identified as Pseudomonas incognita and given the name 'linalool strain'. It was also capable of growing on geraniol, nerol and limonene. The biotransformation of geraniol by this

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microorganism was studied. Shake cultures were treated with geraniol (20). At the end of the incubation period (40 hr), the contents were pooled, acidified with 3 N HCl and extracted with ether and separated in their neutral and acid components. In the neutral fraction (1.4 g) the following metabolites were found: 3-(4-methyl-3-pentenyl)-3-butenolide (30) (25 mg), geranial (21) (130 mg), unreacted geraniol (20) (900 mg), a hydroxyketone (40 mg): l,3-dihydroxy-3,7-dimethyloct-6-ene-2-one (32), which upon treatment with sodium metaperiodate yielded the oxidation product 6-methyl-5-hepten-2-one (33), and a triol (20 mg): 3,7dimethyloct-6-ene-l,2,3-triol (31). The acidic fraction (2.0 g) yielded as major metabolite geranic acid (22) (1.75 g), and a keto acid (20 mg): 7methyl-3-oxo-6-octenoic acid (19), Fig. (6).

Citral(26)

22

27

28

29

Fig. (5). Degradation of citroncllal and citral by Pseudomonas aeruginosa (after [33])

An analogous experiment was run by the same group to study the bioconversion of nerol. It was degraded in essentially the same way as geraniol. Identical products as from geraniol were recovered as major metabolites, together with neral and neric acid [37]. Finally the biotransformation of limonene was carried out with the same microorganism in the same fashion as with geraniol and nerol. The details of this research will be discussed in the chapter of cyclic monoterpenoids (1.2.1). Based on these data, two pathways for the degradation of geraniol (20) were proposed by Madyastha [32], Fig. (7). Pathway A involves an oxidative attack on the 2,3-double bond resulting in the formation of an epoxide. Opening of the epoxide yields the triol (31) which upon oxidation forms a ketodiol (32). The ketodiol (32) is then converted to 6methyl-5-hepten-2-one (33) by an oxidative process. Pathway B is

135

initiated by the oxidation of the primary alcoholic group to geranic acid (22) and further metabolism follows the mechanism as proposed earlier for P, citronellolis [28,31]- In the case of nerol (the Z-isomer of geraniol) degradative pathways analogous to pathways A and B as in geraniol are observed. It was also noticed that P. incognita metabolises acetates of geraniol, nerol and citronellol much faster than their respective alcohols [38]. oo-o

CHjOH

CHO

CHjOH

Geraniol (20) Periodate OOCH

22

CDOH

19

Fig. (6). Degradation of geraniol by Pseudomonas incognita (after [36])

CO2 + H2O 9^ 7

Geraniol (20)

Triol (31)

Ketodiol (32)

6-Methyl-5-hepten-2-one (33)

B

GOGH

)S-Oxidation

Geranial (21)

Geranic acid (22)

y9-Keto acid (19)

Fig. (7). Pathways of degradation of geraniol by Pseudomonas incognita (after [32])

136

In the eighties, the bioconversion of monoterpene alcohols by fungi had not been studied intensively [32]. However, a strain of Aspergillus niger was isolated from garden soil, able to transform geraniol, citronellol and linalool to their respective 8-hydroxy derivatives. This reaction was called 'cy-hydroxylation' [39,40]. Fermentation of citronellyl acetate with A. niger resulted in the formation of a major metabolite, 8-hydroxycitronellol accounting for approx. 60% of the total transformation products, accompanied by 38% citronellol. Fermentation of geranyl acetate with A. niger gave geraniol and 8-hydroxygeraniol (50 resp. 40% of the total transformation products). However, in the case of linalyl acetate, besides linalool and 8hydroxylinalool which were formed to the extent of 25 and 45% of the total products resp., small amounts of geraniol and a-terpineol (together 25%) were also formed. Nearly 40% of the acetates were metabolised in 72 hr. One of the most important examples of fungal bioconversion of the monoterpene alcohols citronellol, geraniol and nerol and the terpene aldehyde citral is the biotransformation by Botrytis cinerea. Fig. (8). B. cinerea is a fungus of high interest in winemaking [41]. In an unripe state of maturation the infection of grapes by B, cinerea is very much feared, as the grapes become mouldy ('grey rot'). With fully ripe grapes however, the grovrth of 5. cinerea is desirable; then the fungus is called 'noble rot' and the infected grapes deliver famous sweet wines, such as, e.g., Sautemes of France, Tokay Aszu of Hungary or Trockenbeerenauslese wines of Germany [42]. Several key components among the volatiles of grapes and wines are terpenoid compounds. The last years, the influence of 5. cinerea on the monoterpene fraction of wine aroma has been studied intensively, especially the biotransformation of geraniol, nerol, citronellol, linalool and citral. One of the first reports in this area dealt with the biotransformation of citronellol (4) by B. cinerea [43], Fig. (8). The substrate was metabolised predominantly to the co-hydroxylation product (J?)-2,6-dimethyl-2-octene1,8-diol (34) and its reduction product 2,6-dimethyl-l,8-octanediol (35). Minor amounts of (Z)-2,6-dimethyl-2-octene-l,8-diol (36), 3,7-dimethyl1,7-octanediol (37),p-menthane-3,8-diol (38), isopulegol (39), 7-hydroxy6-methylhept-5-en-2-one-l-ol (40) and 2-methyl-y-butyrolactone (41) were found. It was also found that with grape must, citronellol (4) was completely metabolised, while using a synthetic medium an incomplete transformation was noticed, also yielding 6-methyl-5-hepten-2-one (33) and citronellic acid (13) as degradation products [42]. The same group also investigated the bioconversion of citral (26) [44], Fig. (8). A comparison was made between grape must and a synthetic medium. When using grape must, no volatile bioconversion products were found. With a synthetic medium, biotransformation of citral (26) was observed yielding predominantly nerol (14) and geraniol (20) as reduction products and as

137

.CH,OH

Fig. (8). Biotransformation of citroneliol, geraniol, nerol and citral by Botrytis cinerea (after [4245])

138

minor compounds the co-hydroxylation products (2£',6Z)-2,6dimethylocta-2,6-diene-1,8-diol (47), (2E, 6jE)-2,6-dimethylocta-2,6-diene1,8-diol (43), 6-methyl-5-hepten-2-one (33), 6-methyl-5-hepten-2-ol (52), 7-hydroxy-6-methylhept-5-en-2-one-l -ol (40) and 2-methyl-ybutyrolactone (41). Finally the bioconversion of geraniol (20) and nerol (14) was described by the same group [45], Fig. (8). When using grape must, a complete bioconversion of geraniol (20) was observed yielding as predominant product from co-hydroxylation (2jB,6£)-2,6-dimethyl-2,6octadiene-l,8-diol (43) and its reduction product (£)-3,7-dimethyl-2octene-l,8-diol (44). The following minor compounds were found: (2Z, 6^-2,6dimethylocta-2,6-diene-l,8-diol (42), 2,6-dimethyM,8-octanediol (35),/?menth-l-en-9-ol (45), 7-hydroxy-6-methylheptan-2-one (46), 7-hydroxy-6methylhept-5-en-2-one (40). (Z)-2,6-Dimethyl-2,7-octadiene-1,6-diol (48), (j5)-2,6-dimethyl-2,7-octadiene-l,6-diol (49), (Z)-3,7-dimethyl-2octene-l,8-diol (50) and 3,7-dimethyM-octene-3,8-diol (51) were bioconversion products formed after rearrangement of geraniol to nerol and linalool. With a synthetic medium, geraniol was not completely metabolised. Analogous results were obtained with nerol (14): complete transformation took place with grape must, yielding predominantly the cohydroxylation product (2£,6Z)-2,6-dimethylocta-2,6-diene-l,8-diol (47) and its reduction product (Z)-3,7-dimethyl-2-octene-l,8-diol (50). On synthetic medium, the substrate (14) was not completely metabolised, resulting in low yields of (2£',6Z)-2,6-dimethylocta-2,6-diene-l,8-diol (47) and /7-menth-l-en-9-ol (45). The most important metabolites from citronellol (4), geraniol (20), nerol (14) and citral (26) are displayed in Fig. (8). In the same year the biotransformation of these monoterpenes by B. cinerea in model solutions was described by another group [41]. Although the major metabolites found were co-hydroxylation compounds, it is important to note that these authors only identified the JJ-isomers in the extracts and that some new compounds were detected that were not described by the previous group, Fig. (9). Geraniol (20) was mainly transformed to (2£,5£r)-3,7-dimethyl-2,5-octadiene-l,7-diol (53), (£)-3,7dimethyl-2,7-octadiene-1,6-diol (54) and (2£:,6£)-2,6-dimethyl-2,6octadiene-l,8-diol (43), nerol (14) to (2Z,5£)-3,7-dimethyl-2,5-octadiene1,7-diol (55), (Z)-3,7-dimethyl-2,7-octadiene-l,6-diol (56), and (2£,6Z)2,6-dimethyl-2,6-octadiene-l,8-diol (47). Furthermore a cyclisation product (57) was formed which was not previously described. Finally citronellol (4) was converted to trans- (60) and cis-vosQ oxide (61) (a cyclisation product not identified by the other group), (£)-3,7-dimethyl-5octene-1,7-diol (58), 3,7-dimethyl-7-octene-1,6-diol (59) and (£0-2,6-dimethyl-2-octene-l ,8-diol (34). The preparation of menthol from citronellal, pulegol or isopulegol by the fungus Penicillium digitatum was patented as early as 1955 [46]. To a

139

culture of P. digitatum propagated for 48 hr at 22°C on 1.5% brewer's wort, 2% citronellal was added and the culture was cultivated for 28 days at 22°C. Menthol was then separated by steam distillation, freezing and centrifugation and obtained in 93% yield. Q\p\\

X Geraniol(20)

53

Nerol(14)

55

56

47

57

58

59

34

60

Citronellol(4)

54

43

61

Fig. (9). Biotransformation of geraniol, nerol and citronellol by Botrytis cinerea (after [41])

One of the latest reports in this area described the biotransformation of citronellol by the plant pathogenic fungus Glomerella cingulata to 3,7dimethyl-1,6,7-octanetriol [47]. In all those examples submerged liquid fungal cultures and mycelia were used. Indeed, fungal spores are generally considered as a dormant stage in the life-cycle of fungi. However, also fungal spores are well known for their biocatalytic activity, e.g. the degradation of fatty acids to methyl ketones has been known as early as the late fifties. In 1958, Gehrig and Knight [48] were the first to describe the transformation of organic compounds by fungal spores: the conversion of octanoic acid to 2heptanone by Penicillium roqueforti spores. Another very important transformation known to be carried out by fungal spores is the biotransformation of steroids (triterpenoids) [49-51]. An example of this conversion is the hydroxylation of progesterone by Aspergillus ochraceus spores [52-54].

140

The ability of fungal spores of Penicillium digitatum to biotransform monoteq^ene alcohols, such as geraniol and nerol and the mixture of the aldehydes, i.e, citral by has only been discovered very recently by our group [55,56,13]. Spores of Penicillium digitatum were inoculated on solid media. After a short incubation period, the spores germinated and a mycelial mat was formed. After two weeks, the culture had completely sporulated and bioconversion reactions were started. Geraniol, nerol or citral were sprayed onto the sporulated surface culture. After one or two days, the period during which transformation took place, the cultures were extracted. Geraniol and nerol were transformed into 6-methyl-5-hepten-2one by sporulated surface cultures. Spores retained their activity for at least two months. An overall yield of up to 99% could be achieved. The bioconversion of geraniol and nerol was also performed with sporulated surface cultures oi Aspergillus niger, Geraniol was converted to linalool, a-terpineol and limonene, and nerol was converted mainly to linalool and a-terpineol [57]. Linalool and linalyl acetate Linalool (3,7-dimethyl-l,6-octadien-3-ol) (62) occurs as one of its enantiomers in many essential oils, where it is often the main component. (i?)-(~)-Linalool for example occurs at a concentration of 80-85% in Ho oils from Cinnamomum camphora; rosewood oil contains ca 80%. (iS)(+)-Linalool makes up 60-70% of coriander oil [26]. The first data on the biotransformation of linalool date back to the seventies. The bioconversion of linalool to camphor by a newly isolated Pseudomonas pseudomallei (strain A) was described [58]. Other products were (£)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid (8-carboxylinalool), 2-methyl-2-vinyltetrahydrofixran-5-one and (£)-4-methyl-3-hexenoic acid. As mentioned before, the group of Madyastha isolated a soil Pseudomonad, Pseudomonas incognita by enrichment culture technique with linalool as the sole carbon source [36]. This organism, the iinalool strain' as it was called, was also capable of utilising limonene, citronellol and geraniol but failed to grow on citral, citronellal and 1,8-cineole. Fermentations were carried out with shake cultures containing 1% linalool as the sole carbon source. Fig. (10). It was suggested by the authors that linalool was metabolised by at least three different pathways of biodegradation. One of the pathways appeared to be initiated by the specific oxygenation of the C-8 methyl group of linalool (62), leading to 8hydroxylinalool (49), which was fiirther oxidised to linalool-8-carboxylic acid (63), probably via the aldehyde. The presence of furanoid linalool oxide (65) and the unsaturated lactone, 2-methyl-2-vinyltetrahydrofuran-5one (66) in the fermentation medium suggested another mode of utilisation of linalool. The formation of these compounds was believed to proceed

141

through the epoxidation of the 6,7-double bond giving rise to 6,7epoxylinalool (64), which upon further oxidation yielded (65) and (66). The presence of oleuropeic acid (74) in the fermentation broth suggested a third pathway. Two possibilities were proposed: (3a) water elimination giving rise to a monocyclic kation (72), yielding a-terpineol (73), which upon oxidation gave oleuropeic acid (74); (3b) oxidation of the C-10 methyl group of linalool (62) before cyclisation, giving rise to oleuropeic acid (74). This last pathway was also called the 'prototropic cyclisation' [32].

V^oX~~^ o^oX~

CDOH

3a^

7^8 Linalool (62)

67

68

69 OOOH

-< >-

Fig. (10). Bioconversion of linalool by Pseudomonas incognita (after [36])

Later it was found that P. incognita accepted linalyl acetate better than linalool as the sole source of carbon [59]. A microbial degradation of linalyl acetate leaving the acetoxy group intact was suggested. Only few literature data are available about the fungal biotransformation of linalool and its acetates. As mentioned before, the biotransformation of linalyl acetate by Aspergillus niger isolated from garden soil was studied [39,40]. Part of the unmetabolised substrate was

142

recovered from the cultures, together with linalool, 8-hydroxy- linalool, aterpineol, geraniol and some unidentified products in trace amounts. The biotransformation of linalool by Botrytis cinerea has also been described [60]. After addition of linalool to botrytised must, a series of transformation products was identified: (£)- (49) and (Z)-2,6-dimethyl-2,7octadiene-l,6-diol (48), trans- (76) and cw-fiiranoid linalool oxide (77), trans- (78) and c/^-pyranoid linalool oxide (79) and their acetates (80, 81), 3,9-epoxy-p-menth-l-ene (75) and 2-methyl-2-vinyltetrahydrofuran-5-one (66) (unsaturated lactone), Fig. (11). Quantitative analysis however, showed that linalool was predominantly (> 90%) metabolised to (jEr)-2,6-dimethyl-2,7-octadiene-l,6-diol (49) by B. cinerea. The other compounds were only found as by-products in minor concentrations. Another example of fimgal bioconversion of linalool was described in literature: the biotransformation by Diplodia gossypina ATCC 10936 [61]. A conversion scheme for the bioconversion of both (if)-(-)- and (S)-(+)linalool was proposed.

^XQX,

a^o^ 66

78

79

80

81

Fig. (11). Biotransformation products of linalool by Botrytis cinerea (after [60])

The bioconversion of linalool was also investigated by our group [62]. Biotransformation of (±)-linalool with submerged shaking cultures of Aspergillus niger^ particularly A. niger ATCC 9142 yielded a mixture of cis- and /raw^-furanoid linalool oxide (yield 15-24%) and cis- and transpyranoid linalool oxide (yield 5-9%). Biotransformation of (/?)-(•-)linalool (62a) with the same strain yielded almost pure trans-fyxxd^oid (76) and /rara-pyranoid (78) linalool oxide (ee > 95), Fig. (12). These conversions were purely biocatalytic, since in acidified water (pH < 3.5) almost 50% linalool was recovered unchanged, the rest was evaporated. The biotransformation was also carried out with growing surface cultures.

143 HO,

:^:K HO

A. niger |]ATCC9142 '^ O HO "

(R)-(-)-LinaloI (62a)

Linalool epoxide (64)

/rfl[W5-furanoid linalool oxide (76)

/ra/?5-pyranoid linalool oxide (78)

Fig. (12). Biotransformation of (/?)-(-)-iinaiool by Aspergillus niger ATCC 9142 (after [62]) 1.1.3 Acyclic monoterpene ketones

The literature about the bioconversion of acyclic monoterpene ketones is very limited, since only few monoterpene ketones are known, such as tagetone (82a,b) and ocimenone (83a,b), Fig. (13). The bioconversion of these examples will not be discussed here. However, 6-methyl-5-hepten2-one (33), although not a terpene, but a very important degradation product of geraniol, nerol and citral (see 1.1.2), is also a possible precursor for interesting chiral compounds. Indeed, microbial asymmetric reduction of the prochiral 6-methyl-5-hepten-2-one (33) yields 6-methyl-5-hepten-2ol, sulcatol (84), an important aggregation pheromone of the ambrosia beetle, Gnathotricus sp. [63]. The chirality of the molecule is involved in its biological activity: one species, Gnathotricus sulcatus, responds to a mixture of 65% of isomer (5)-(+)-sulcatol and 35% of isomer (i?)-(-")sulcatol and does not respond to either one of these isomers alone. Another species, G. retusus is sensitive only to the iS-isomer and its response seems to be inhibited by the i?-enantiomer. Baker's yeast {Saccharomyces cerevisiae) and the anaerobic bacterium Clostridium tyrobutyricum for example gave the iS-enantiomer from 6-methyl-5hepten-2-one, while the two fungi Geotrichum candidum and Aspergillus niger gave the i?-enantiomer of sulcatol from the same ketone. The enantioselective synthesis of (S)-(+)-6-methyl-5-hepten-2-ol by asymmetric reduction of 6-methyl-5-hepten-2-one mediated by baker's yeast in 64% yield with 90% enantiomeric excess (ee) was also reported by another group [64]. 6-Methyl-5-hepten-2-one is a valuable precursor for microbial epoxidations and hence the production of chiral ethers with high optical purities. The biotransformation of 6-methyl-5-hepten-2-one (33) by Botryodiplodia malorum CBS 13450 to (i?)-sulcatol (84) was described [61], which is then epoxidised to the (55)-epoxide (85) and opened intramolecularly to c/5'-(2i?,57?)-2-(2'-hydroxyisopropyl)-5-methyltetrahydrofuran (86) and c/5'-(3iS',6^)-3-hydroxy-2,2,6-trimethyltetrahydropyran (87). Reduction of 6-methyl-5-hepten-2-one (33) with baker's yeast to (5)-sulcatol (88) which was used as substrate for Kloeckera corticis

144

yielded the rraw^-tetrahydrofuran, derivative (91), Fig. (14).

Z-Tagetone (82a)

£-Tagetone (82b)

(2i?,55)-pityol (90) and -pyran

Z-Ocimenone (83a)

£-Ocimenone (83b)

Fig. (13). Acyclic monoterpene ketones: tagetone and ocimenone

HoV^ 86 Botryodiplodia ^ malorum 84

85

"KX 87

HOY^

yeast

90

'OH Kloeckera^ corticis

89

"

» 91

Fig. (14). Bioconversion of 6-methyl-5-hepten-2-one (after [61])

Comparable results were also reported by another group [65]. Controlled conversion of 6-methyl-5-hepten-2-one by Botrytis cinerea resulted in the formation of (5)-(+)-6-methyl-5-hepten-2-ol (sulcatol) of 90% ee. In addition, {2R,5Ry, (25,55)-, (2i?,5S)- and (25,5i?)-pityol and the four enantiomers of 3-hydroxy-2,2,6-trimethyltetrahydropyran were found as biotransformation products of 6-methyl-5-hepten-2-one. A complete synthesis of optically pure (2i?,55)-pityol (90), a pheromone of the bark beetle Pityophtorus pityographus using a

145

chemoenzymatic route was also described [66]. The conversion consisted of a baker's yeast asymmetric reduction of 6-methyl-5-hepten-2-one (33) to (S)-sulcatoi (88), which was then submitted to an epoxidation carried out by A. niger. The last step of this reaction was a cyclisation [67] yielding (2i?,55)-pityol (90). L2 Cyclic Monoterpenes 1.2.1 Monocyclic hydrocarbon monoterpenes

Limonene and other compounds with ap-l-menthene skeleton Limonene (92) is the most widely distributed terpene in nature after a-pinene [68]. The (+)-isomer is present in Citrus peel oils at a concentration of over 90%; a low concentration of the (-)-isomer is found in oils from the Mentha species and conifers [26]. The first data on the microbial transformation of limonene date back to the sixties. A soil Pseudomonad was isolated by enrichment culture technique on limonene as the sole source of carbon [69]. This Pseudomonad was also capable of growing on a-pinene, jff-pinene, 1-p-menthene and/^-cymene. The optimal level of limonene for growth was 0.3-0.6% (v/v) although no toxicity was observed at 2% levels. Fermentation of limonene by this bacterium in a mineral-salts medium resulted in the formation of a large number of neutral and acidic products. Dihydrocarvone, carvone, carveol, 8-pmenthene-1,2-cz5'-diol, 8-p-menthen-1 -ol-2-one5 8-p-menthene-1,2-transdiol and l-p-menthene-6,9-diol were among the neutral products isolated and identified. The acidic compounds isolated and identified were perillic acid, jff-isopropenyl pimelic acid, 2-hydroxy-8-/?-menthen-7-oic acid and 6,9-dihydroxy-l-p-menthen-7-oic acid. Based on these data three distinct pathways for the catabolism of limonene by the soil Pseudomonad were proposed by the same group [70], involving allylic oxygenation (pathway 1), oxygenation of the 1,2-double bond (pathway 2) and progressive oxidation of the 7-methyl group to perillic acid (pathway 3) (see Fig. (15), after [68]). A fourth pathway, yielding a-terpineol and carried out by fungi such as Penicillium digitatum, P. italicum and Cladosporium and several bacteria will be discussed later. Also a fifth (hydroxylation in C-3) and sixth pathway (hydroxylation in C-4) will be mentioned later. The first pathway gives c/5-carveol (93), D-carvone (94) (an important constituent of caraway seed and dill-seed oils [27,71]) and 1-p-menthene6,9-diol (95). (+)-(5)-Carvone is a natural potato sprout inhibiting, fungistatic and bacteristatic compound [72,73]. It is important to note that L-(~-)-carvone (the 'spearmint flavour') was not yet described in microbial transformation [68]. However, the biotransformation of limonene to L-carvone was patented by a Japanese group [74]: a Corynebacterium species grown on limonene was able to produce about 10 mg/L of 99%

146

Pathway

\/

Carveol (93)

95

Pathway 2 ^

Limonene (92)

Limonene epoxide (96)

Dihydrocarvone (97)

COOH

COOH HOOC

HO' Limonene-l,2-diol (98)

OH

103

106

Pathway 3 (main pathway)

CHjOH

CHO

COOH

^ss

^

Perillyl alcohol (100) Perillaldehyde (101)

Perillic acid (102)

COOH OH

104

Fig. (15). Pathways for the degradation of limonene by a soil Pseudomonad (after [68])

COOH

105

147

pure L-carvone in 24-48 hr. Pathway 2 yields (-f)-dihydrocarvone (97) via intermediate limonene epoxide (96) and 8-p-menthen-l-oi-2-one (99) as oxidation product of limonene-1,2-diol (98). The third and main pathway leads to perillyl alcohol (100), perillaldehyde (101), perillic acid (102), constituents of various essential oils and used in the flavour and fragrance industry [27], 2-hydroxy-8-/7-menthen-7-oic acid (104), 2-oxo-8-/7menthen-7-oic acid (105), )ff-isopropenyl pimelic acid (106) and 4,9dihydroxy-l-/7-menthen-7-oic acid (103). As mentioned before, a Pseudomonas incognita was isolated by enrichment technique on the monoterpene alcohol linalool that was also able to grow on geraniol, nerol and limonene [36]. The metabolism of limonene by this bacterium was also investigated [37]. After fermentation the medium yielded as main product a crystallic acid, perillic acid, together with unmetabolised limonene, and some oxygenated compounds: dihydrocarvone, carvone, carveol, />-menth-8-en-l-ol-2-one, /?-menth-8ene-l,2-diol or/>-menth-l-ene-6,9-diol (structure not fiilly elucidated) and finally jff-isopropenyl pimelic acid. The same group has also isolated a strain of Pseudomonas putidaarvilla (PL-strain) from limonene and (+)-a-pinene as the sole carbon source that was capable of growing on (+)-limonene, (-+-)-a-pinene, (~)-apinene, jff-pinene, 1-p-menthene, 3-p-menthene andp-cymene as substrates [75]. Limonene was degraded to perillyl alcohol, perillaldehyde and perillic acid. More recently the biotransformation of limonene by another Pseudomonad strain, P. gladioli was reported [76,77]. P. gladioli was isolated by an enrichment culture technique from pine bark and sap using a mineral salts broth with limonene as the sole source of carbon. Fermentations were performed during 4-10 days in shake flasks at 25°C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene. Major conversion products were identified as (+)-a-terpineol and (+)-perillic acid. This was the first time that the microbial conversion of limonene to (+)-a-terpineol was reported, see pathway 4. The conversion of limonene to a-terpineol was achieved with an enzyme, a-terpineol dehydratase (a TD), by the same group [78]. The enzyme, purified more than tenfold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4if)-(-f)-limonene to (4i?)-(+)-a-terpineol or (4S)-(+)-limonene to (45)(+)-a-terpineol. a-Terpineol is widely distributed in nature and is one of the most commonly used perfimie chemicals [27]. The first data onfimgalbioconversion of limonene date back to the late sixties [79,80]. Three soil microorganisms were isolated on and grew rapidly in mineral salts media containing appropriate terpene substrates as sole carbon sources. The microorganisms belonged to the class Fungi Imperfecti, and two of them had been tentatively identified as Cladosporium species. A Cladosporium designated Ti was isolated fi*om terpene-soaked soil using l-menthene (107) as the sole carbon source.

148

The major catabolic product isolated from the growth medium of this organism was found to be a cyclic 1,2-diol, identified as trans-pmenthane-l,2-diol (108). A similar but biochemically distinct Cladosporium sp. designated as T7 was isolated on D-limonene (92). The growth medium of this strain contained 1.5 g/L of the analogous product, /ra«5'-limonene-l,2-diol (109), Fig. (16). Minor quantities of the corresponding c/5-l,2-diol were also isolated. The third organism, designated as laboratory culture Tg, was isolated on 3-menthene and yielded /ra«5'-/7-menthane-3,4-diol. The same group [81] isolated a fourth microorganism from a terpene-soaked soil on mineral salts media containing D-limonene as the sole C-source. The strain, Cladosporium, designated T12, was capable of converting D-limonene into an optically active isomer of a-terpineol in yields of approx. 1.0 g/L.

Cladosporium ^ p

1-Menthene(107)

108

y'^

I ^

Cladosporium

Limonene(92)

109

Fig. (16). Biotransformation of 1-menthene and limonene by Cladosporium (after [79,80])

The fungal bioconversion of limonene was further studied [82]. Penicillium sp. cultures were isolated from rotting orange rind that utilised limonene and converted it rapidly to a-terpineol. Bowen [83] isolated two common citrus moulds, Penicillium italicum and P. digitatum, responsible for the postharvest diseases of citrus fruits. Fermentation of P. italicum on limonene yielded cis- and trans-c^rvtoX (93) (26%) as main products, together with cis- and /ra«5-p-mentha-2,8-dien-l-ol (110) (18%), (+)carvone (94) (6%), p-mentha-l,8-dien-4-ol (111) (4%), perillyl alcohol (100) (3%),/7-menth-8-ene-l,2-diol (98) (3%), Fig. (17). Conversion by P. digitatum yielded the same products in lower yields. The two alcohols /7-mentha-2,8-dien-l-ol (110) andp-mentha-l,8-dien-4-ol (111) were not described in the transformation studies where soil Pseudomonads were used [69]. The biotransformation of limonene by Aspergillus niger is a very important example of fungal bioconversion. Screening for fungi capable of metabolising the bicyclic hydrocarbon terpene a-pinene (see 1.2.2) yielded a strain of ^4. niger NC^IM 612 that was also able to transform limonene [75]. This fungus was able to carry out three types of oxygenative rearrangements. Fig. (18). The conversion of limonene (92) to a-terpineol (73) is an example of pathway 4 (cfr. supra).

149

110

111

carveol (93)

carvone (94)

perillyl alcohol (100)

98

Fig. (17). Biotransformation products of limonene by Penicillium digitatum and P. italicum (after [83])

• niger ,

Limonene (92)

a-terpineol (73)

c/5-carveol (93)

110

Fig. (18). Oxygenative rearrangements carried out by Aspergillus niger NCIM 612 on limonene (after [75])

More recently, the production of glycols from limonene and other terpenes with a 1-menthene skeleton was reported [84]. An extensive screening of 1000 different microorganisms showed that limonene was attacked by a large number of strains (320 strains). Accumulation of glycols during fermentation with several fungi was observed. The most appropriate strains were Corynespora cassiicola DSM 62475 and Diplodia gossypina ATCC 10936. An extensive overview on the microbial transformations of terpenoids with a l-p-menthene skeleton was published by Abraham et aL [85]. In 1985, the same group [24] investigated the biotransformation of (i?)(+)-limonene by the fungus Penicillium digitatum, A complete transformation of the substrate to a-terpineol by P. digitatum DSM 62840 was obtained with a yield of 46% pure product. The bioconversion of (4i?)-(-)-limonene to (4i?)-(-)-a-terpineol by immobilised fungal mycelia of Penicillium digitatum was described more recently [86]. The fungi were immobilised in Calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4°C. a-Terpineol production by the fungus was 12.83 mg/g beads per day, producing a 45.81% bioconversion of substrate. The optimum conversion temperature was 28°C and the optimum pH was 4.5. The highest

150

concentration of product was formed with a contact time of between 1 and 2 days [87]. A Japanese group also studied the biotransformation of limonene and related compounds (1-methylcyclohexene and cyclohexene) by Aspergillus cellulosae M-77 [88]. It is important to note tiiat (+)-limonene (92) was mainly converted to (-f)-isopiperitenone (112) (19%), (+)-limonene-l,2trans-6xo\ (109) (21%), (+)-c/5-carveol (93) (5%) and (+)-perillyl alcohol (100) (12%), Fig. (19). Although these alcohols (93,100,109) have been found by other authors in the past, the production of isopiperitenone (112) from limonene (92) had not been published before. The conversion of limonene to isopiperitenol by hydroxylation in the C-3 position and further oxidation of isopiperitenol to isopiperitenone is an example of the fifth pathway of limonene biotransformation. Only very recently, it was published by another group [89] that two unclassified strains of the basidiomycetes, Trichosporon, transformed (+)-limonene to isopiperitenone (0.05 and 0.4 g[L\ yield 2% and 20%), and transA,!dihydroxylimonene (0.6 g/L; yield 30%). This group also reported the conversion of (+)-limonene by the yeasts Arxula adeninivorans and Yarrowinia lipolytica to perillic acid.

Aspergillus cellulosae

92

112

100

93

109

Fig. (19). Biotransformation of (+)-limonene hy A. cellulosae (after [88])

Very recently, the purification and characterisation of an epoxide hydrolase, catalysing the conversion of limonene-1,2-epoxide to limonene1,2-diol has been described [90]. The enzyme was isolated from Rhodococcus erythropolis DCL14 and is induced when the microorganism is grown on monoterpenes. The authors found evidence that the enzyme, limonene-1,2-epoxide hydrolase is the first member of a new class (the third class) of epoxide hydrolases [91]. In a recent extensive overview on the biotransformation of terpenoids by Aspergillus spp., Noma and Asakawa [92] also mentioned a sixth pathway of limonene bioconversion: the hydroxylation at the C-4 position to give /7-mentha-l,8-dien-4-ol (111), Fig. (20), a compound also identified earlier as one of the bioconversion metabolites of limonene with Penicillium italicum [83]. In this review, the fifth pathway, leading to isopiperitenol (113) which isfiirtheroxidised to isopiperitenone (112) and its rearrangement product, piperitenone (114) is also discussed.

151

Hydroxylation products of this fifth pathway are 5-hydroxyisopiperitenone (115), 10-hydroxyisopiperitenone (116), 4-hydroxyisopiperitenone (117) and 7-hydroxyisopiperitenone (118), Fig. (20).

116

117

118

Fig. (20). Metabolic pathways 5 and 6 of limonene by Aspergillus spp. (after [92]) 1.2.2 Bicyclic hydrocarbon monoterpenes

a-Pinene The most abundant terpene in nature is a-pinene (119) which is industrially obtained by fractional distillation of turpentine [68]. (+)-aPinene occurs, for example, in oil from Pinus palustris Mill, at concentrations of up to 65%; oil from Pinus pinaster Soland. and American oil from Pinus caribaea contain 70% and 70-80% resp. of the (-)-isomer [26]. One of the earliest publications described the biotransformation of apinene by A, niger [93,94] A 24-hr shake culture of this strain metabolised 0.5% a-pinene (119) in 4-8 hr. After the fermentation the

152

culture broth contained a ketone, verbenone (121) (2-3%), an alcohol, cisD-verbenol (120) (20-25%), a diol, D-(+)-rraAi^-sobrerol (122) (2-3%), and a hydroxyketone, hydroxycarvotanacetone (123), Fig. (21). A. niger NCIM612

a-Pinene(119)

cw-Verbenol (120)

Verbenone (121)

trans-Sohrerol (122)

Hydroxycarvotanacetone (123)

Fig. (21). Bioconversion of a-pinene by Aspergillus niger NCIM 612 (after [93])

The degradation of a-pinene and other A^-menthene skeletons by Pseudomonas (PL-strain) was first investigated by Hungund et al [95]. A terminal oxidation pattern was proposed, leading to the formation of organic acids through ring cleavage. Shukla et al [96] described the fermentation of a-pinene and )ff-pinene in shake cultures by a soil Pseudomonas sp. (PL-strain) able to grow on a-pinene as the sole carbon source. A complex metabolite mixture was obtained composed of neutral as well as acidic compounds. Fig. (22).

Pseudomonas (PL-strain)

a-Pinene(119)

OOOH

(Jr™ Borneol (1)

Myrtenol (124)

Myrtenic acid (125)

OOOH

Phellandric acid (126)

Fig. (22). Bioconversion of a-pinene by Pseudomonas (after [96])

More recently, the degradation of a-pinene by Pseudomonas fluorescens NCIMB 11671 was described [97,98]. A novel pathway for the microbial breakdown of a-pinene (119) was proposed. Fig. (23). The attack is initiated by enzymatic oxygenation of the 1,2-double bond to form the epoxide (127). This epoxide then undergoes rapid rearrangement to produce a novel diunsaturated aldehyde, occurring as two isomeric forms. The primary product of the reaction (Z)-2-methyl-5-isopropylhexa2,5-dien-l-al (trivial name isonovalal) (128) can undergo chemical isomerisation to the jE-form (novalal) (129). Isonovalal, the native form of

153

the aldehyde, possesses citrus, woody, spicy notes, whereas novaial has woody, aldehydic, cyclene notes. The same bioconversion was also carried out by another bacterial strain, Nocardia sp. strain PI8.3 [99,100]. Also the biotransformation of a-pinene derivatives and other pinane monoterpenoids by Cephalosporium aphidicola has been described [101]. The best conversion was the oxidation of verbenol to verbenone (yield 61%). CHO mono-oxygenase

a-Pinene (119)

fC/^

dccyclising enzyme

a-Pinene epoxide (127)

f^ Ci\0 L ^

chemical isomerisation

Isonovalal = Z-isomer (128)

Novaial = £-isomer (129)

Fig. (23). Bioconversion of a-pinene by Pseudomonas fluorescens NCIMB 11671 (after [97])

j^Pinene jff-Pinene (130) is found in many essential oils. Optically active and racemic j9-pinenes are present in turpentine oils, although in smaller quantities than a-pinene [26]. Only very little is known about microbial transformations of )ff-pinene, which is an abundantly occurring natural terpene [68]. Shukla et aL [96] obtained a similarly complex mixture of transformation products from /?-pinene as from a-pinene through degradation by a Pseudomonas sp. (PL-strain). On the other hand, Bhattacharyya and Ganapathy [102] indicated that fungi such as A, niger NCIM 612, act differently and more specifically on the pinenes by preferably oxidising /?-pinene (130) in the allylic position to form the interesting products pinocarveol (131) and pinocarvone (132), besides myrtenol (124), Fig. (24). The same group [75] also described the conversion of ^-pinene by Pseudomonas putida-arvilla (PL-strain): a degradation pathway for the conversion of j5-pinene to bomeol was proposed. By enrichment culture technique, a bacterium was isolated from local sewage sludge, utilising caryophyllene as the sole source of carbon and energy [103]. Fermentation of ^-pinene by this culture in a mineral salt medium (Seubert's medium) at 30°C with agitation and aeration for four days yielded a few neutral and acidic transformation products. The metabolites isolated and identified were camphor (2), bomeol (1),

154

isobomeol (133), a-terpineol (73) and jff-isopropyl pimelic acid (134), Fig. (25). The organism \vsis idQntifiQd SiS Pseudomonas pseudomallai. Using modified Czapek-Dox medium and keeping the other conditions the same, the pattem of the metabolic products was dramatically changed. The metabolites then recovered were trans-pinocarveol (131), myrtenol (124), a-fenchol (135), a-terpineol (73), myrtenic acid (125) and two unidentified products. CHjOH Aspergillus niger NCIM612

Pinocarveol (131)

P-Pinene (130)

Pinocarvone (132)

Myrtenol (124)

Fig. (24). Bioconversion of ^-pinene by ^. niger (after [102])

COOI f'u^^

P. pseudomallai ^ Seubert's medium

f^^^^ ^ i ^

(^X^ ^Ax^

Camphor (2)

Isobomeol (133)

p-Pinene(130)

.OH f r i ' ^ix^

Bomeol(l)

COOH

OH

a-Terpineol (73)

COOH

CH,OH P. pseudomallai

^

"°;l)

.OH

modified Czapek-Dox medium

P-Pinene(130)

/ra«5-Pinocarveol (131)

p-Isopropyl pimelic acid (134)

Myrtenol (124)

a-FenchoI (135)

a-Teipineol (73)

Fig. (25). Bioconversion of j8-pinene by Pseudomonas pseudomallai (after [103])

Myrtenic acid (125)

155

1-2.3 Cyclic monoterpene alcohols

Menthol Together with a-terpineol, menthol (25) is one of the few terpene alcohols occurring widely in nature that have physiological properties making them important fragrance or flavour compounds [26]. There are in fact 8 isomers with a menthol (p-menthan-3-ol) skeleton, (-)-menthol (138) is the most important one, because of its cooling and refreshing effect. It is the main component of peppermint and commint oils obtained from the Mentha piperita and Mentha arvensis species. Many attempts have been made to produce (-)-L-menthol from inexpensive terpenoid sources, but these sources unavoidably also yielded the (±)-isomers: isomenthol, neomenthol, and neoisomenthol [68]. Especially Japanese researchers have been active in this field, maybe because of the large demand for L-menthol in Japan itself: 500 t/year [104]. Indeed, most literature deals with the enantiomeric hydrolysis of (±)-menthol esters to optically pure (~)-menthol. The asymmetric hydrolysis of DL-menthyl chloroacetate by an esterase of Arginomonas non-fermentans FERM-P-1 924 has been patented by the Japanese Nippon Terpene Chemical Co. [105,106]. Moroe et al [107] from the Takasago Perfumery Co. Ltd. claim that certain selected species of Absidia, Penicillium, Rhizopus, Trichoderma, Bacillusy Pseudomonas and others asymmetrically hydrolyse esters of (±)-menthol isomers such as formiates, acetates, propanoates, caproates, and esters of higher fatty acids. Fig. (26). Besides the hydrolysis of menthyl esters, the biotransformation of menthol and its enantiomers has also been published. The microbial transformation of menthol was studied by Shukla et al [108]. More recently Asakawa et al, [109] described the fungal biotransformation of (-)- and (+)-menthols by Aspergillus niger and A. cellulosae. A, niger converted (-)-menthol to 1-, 2-, 6-, 7- and 9-hydroxymenthols and the mosquito repellent-active 8-hydroxymenthol, whereas (+)-menthol was smoothly biotransformed by the same fungus to give 7-hydroxymenthol. A. cellulosae on the other hand, biotransformed (-)-menthol specifically to 4-hydroxymenthol. The bioconversion of (+)- and (--)-neomenthol and (+)-isomenthol by A. niger was studied later by the same group [110], mainly giving a hydroxylation. For a very detailed schematic overview of these reactions, we refer to the review given by Noma and Asakawa [92]. More recently, the fungal transformation of (-)-menthol by Cephalosporium aphidicola was reported [111]. Incubation of (-)menthol with this fungus for 12 days yielded four new metabolites, 10acetoxymenthol, 4a-hydroxymenthol, 3a-hydroxymenthol, and 10hydroxymenthol and two known compounds identified as 7hydroxymenthol and 9-hydroxymenthol.

156

Microorganism ^ O-CDCHj

(-)-Menthyl acetate (136)

^Y^'0-CDCH3

(+)-Menthyl acetate (137)

\ ^ O H

(-)-Menthol (138)

^^''O-COCHj

(+)-Menthyl acetate (137)

Fig. (26). Asymmetric hydrolysis of (±)-menthyl acetate to obtain pure (~)-menthol 1.2.4 Cyclic monoterpene ketones and norterpenoids

Camphor Both optical isomers of camphor (2) are found widely in nature, (+)camphor being more abundant. It is the main component of oils obtained from the camphor tree Cinnamomum camphora [26]. The hydroxylation of D-(-H)-camphor by Pseudomonas putida C\ was described [112]. The substrate was hydroxylated exclusively in its 5-exo- and 6-exo-positions. The earliest investigation of the degradation of (•f)-camphor dates back to the late fifties [113 and references cited therein]. Although only limited success was achieved in understanding the catabolic pathways, key roles for methylene group hydroxylation and biological Baeyer-Villiger monooxygenases in ring cleavage strategies were established [113]. A degradation pathway of (+)-camphor by Pseudomonas putida ATCC 17453 and Mycobacterium rhodochorus Ti was proposed [113]. Pulegone (i?)-(+)-Pulegone (139), a mint-like odour monoterpene ketone, is the main component (up to 80-90%) of Mentha pulegium essential oil (Pennyroyal oil) which is sometimes used in beverages and food for human consumption and occasionally in herbal medicine as an abortifacient drug. The biotransformation of (i?)-(+)-pulegone by fungi was investigated [114]. Most fungal strains tested, grown in a usual liquid culture medium, were able to metabolise (i?)-(+)-pulegone to some extent in a concentration range of 0.1-0.5 g/L; higher concentrations were generally toxic, except for one of the strains {Aspergillus sp.) isolated from mint leaves inftision, which was able to survive to concentrations of up to 1.5 g/L. The predominant bioconversion product was generally 5hydroxypulegone (140) (20-30% yield). Other metabolites were present in lower amounts (5% or less). Fig. (27). The formation of 5hydroxypulegone (140) was explained by hydroxylation at a tertiary

157

position. Its dehydration to piperitenone (114), even under the incubation conditions, during isolation or derivatisation reactions precluded any tentative determination of its optical purity and absolute configuration. This metabolism of (i?)-(+)-pulegone is very similar to the bioconversion pathway that was very recently published by another group [115]. Using the fungal strain Mucor piriformis, eight metabolites were isolated from the fermentation medium after conversion of (i?)-(-f)-pulegone (139), namely 5-hydroxypulegone (140), piperitenone (114), 6-hydroxypulegone (144), 3-hydroxypulegone (141), 5-methyl-2-(l-hydroxy-l-methylethyl)-2cyclohexen-1-one (142), 3-hydroxyisopulegone (146), 7hydroxypiperitenone (145), and 7-hydroxypulegone (147), Fig. (28).

Piperitenone (114)

141

142

143

Fig. (27). Bioconversion of pulegone hy Aspergillus sp. (after [114])

The biotransformation of (i?)-(+)-pulegone was also studied by a Japanese group [116]. The major bioconversion metabolite of this substrate with Botrytis allii was (-)-(lif)-8-hydroxy-4-/7-menthen-3-one. The secondary major product from this biotransformation was isolated and its structure established as piperitenone [117]. It is interesting to note that the same group also investigated the bioconversion of piperitone, the dihydrogenation product of piperitenone: a strain of Rhizoctonia solani was found able to hydroxylate the substrate preferentially at the 6-position [118,119]. Recently, the biotransformation of (+)-pulegone and other monoterpenoid ketones, like (-)-piperitenone, (+)- and (-)-carvone, (-)menthone and (-)-verbenone by yeasts and yeast-like fungi was also

158

Studied [120]. Only one organism, a Hormonema isolate (UOFS Y-0067), quantitatively reduced (-)-menthone and (-f-)-pulegone to (+)-neomenthol.

hydroxylation

(i?)-(+)-Pulegone(139)

hydroxylation

dehydration

5-HydroxypuIegone (140)

shydroxylation

Piperitenone(114)

allylic methyl oxidation

CHoOH

^ 3-Hydroxypulegone (141)

allylic alcohol rearrangement

142

6-Hydroxypulegone (144)

7-Hydroxypiperitenone (145)

.isomerisation

reduction of double bond

3-Hydroxyisopulegone (146)

7-Hydroxypulegone(147)

Fig. (28). Transformation of (7?)-(+)-pulegone by Mucor piriformis (after [115])

Carvone Carvone (94) occurs as (+)-carvone, (~)-carvone or racemic carvone. (AS)-(+)-Carvone is the main component of caraway oil (ca 60%) and dill oil and has a herbaceous odour reminiscent of caraway and dill seeds. (R)(-)-Carvone occurs in spearmint oil at a concentration of 70-80% and has a herbaceous odour similar to spearmint [26]. (S)-(H-)-Carvone (94) was used as substrate for bioconversions by selected microorganisms: five

159

bacteria and one fungus [121]. The substrate was reduced predominantly to both dihydrocarvones (97a, 97b) and to neo-isodihydrocarveol (148), Fig. (29). Sensitivity of the microorganisms to (5)-(+)-carvone and some of the products prevented yields exceeding 0.35 g/L in batch cultures. The fungus Trychoderma pseudokoningii gave the highest yield of neoisodihydrocarveol. O

(5)-(+)-Carvone (94)

97a

97b

OH

148

Fig. (29). Bioconversion of carvone (after [121])

(5)-(+)-Carvone is known to inhibit fungal growth of Fusarium sulphureum when the substrate was administered via the gas phase [73]. Under the same conditions, the related fungus, F. solani var. coeruleum was not inhibited. In liquid medium, both fungi were found to convert (5)(+)-carvone with the same rate, mainly to isodihydrocarvone, isodihydrocarveol and neo-isodihydrocarveol. As mentioned before, the biotransformation of (-f)- and (-)-carvone and other monoterpenoid ketones by yeasts and yeast-like fungi has been reported recently [120]. ^lonone j?-Ionone (149) and its derivatives are widely distributed in nature and represent important constituents of many essential oils [68]. The hydroxylation of jff-ionone by Aspergillus niger JTS 191 was reported [122]. Two major metabolites were isolated and the structures proposed as (iS)-2-hydroxy-j5-ionone (150) and (jf?)-4-hydroxy-^-ionone (151), Fig. (30). The complex was found to be very effective for tobacco flavouring at ppm level. For this research, more than 1000 microorganisms from various culture collections were tested for their abilities to convert ionones (a- and )5-ionones) to other aroma compounds. These microorganisms comprised over 150 fungi, 28 yeasts and more than 800 terpene utilising bacteria which were isolated from plants and soils [123]. Many of the bacterial isolates consumed the substrate without accumulation of interesting flavouring substances. Some fungi however {Aspergillus, Phialophora, or Rhizopus) successfully converted a- and ^-ionones to other aroma compounds. A, niger JTS 191 was selected as the most

160

suitable strain for the production of new aromatic metabolites from jiionone and ^-methylionone. O

0

0

6H P-Ionone (149)

O

a-Ionone(152)

(5)-2-Hydroxy-p-ionone (150)

O

3-Hydroxy-a-ionone (153)

(/?)-4-Hydroxy-p-ionone (151)

O

3-Oxo-a-ionone (154)

Fig. (30). Bioconversion of j5-ionone and a-ionone by Aspergillus niger JTS 191 (after [122,129])

A considerably different type of ^-ionone transformation was reported by Krasnobajew and Helmlinger [124]. They described the biotransformation of )ff-ionone by pregrown mycelia of Lasiodiplodia theobromae ATCC 28570. The fermentation of )ff-ionone had to be performed with pregrown cultures because the substrate inhibited the growth and led to lysis of the cells. Therefore mycelia cultures at the end of their logarithmic growth phase (after 20 to 50 hr) were employed. After 3 to 4 days the transformation capacity of the mycelia decreased considerably, and the fermentation broth was extracted. Under optimal conditions mycelia of L theobromae ATCC 28570 transformed \h g Pionone per litre of culture fluid. An essential-oil-type product with tobacco flavour could be obtained from the culture fluid. The biotransformation of j9-ionone by the same microorganism, L theobromae IFO 6469 was later patented by the Japan Tobacco, Inc. [125]: the metabolites were used as cigarette flavour improvers. More recently, the fed-batch biotransformation of )8-ionone by Aspergillus niger was described [126]. A commercially available strain, A. niger IFO 8541 was selected and was found to be an efficient biocatalyst for the biotransformation of ^-ionone into 2- and 4-hydroxy-)ffionone and 2-oxo-^-ionone. 4-Hydroxy-^-ionone was the main product with a mass yield close to 90%. It is interesting to note that the metabolism of )9-ionone involves a lag phase, which is a function of the

161

biomass concentration. Therefore, substrate addition was carried out after 100 hr of preliminary fungal growth in conical flasks. In a fed-batch operation, product formation was noticed about 40 hr after the first ^8ionone addition. 2- And 4-hydroxy-j5-ionone were the first compounds detected, 4-oxo-^-ionone appeared far later, after 320 hr cultivation. a-Ionone In 1978 Givaudan patented a transformation of a-ionone [127]: a fermentation of Botryodiplodia theobromae IFO 6469 with a-ionone (152) yielded a mixture of compounds (155 -157) with honeysuckle aroma. Fig. (31). The same fungi which efficiently biotransformed )ff-ionone also transformed a-ionone [128]. As observed for jS-ionone, a-ionone (152) transformations with Lasiodiplodia theobromae ATCC 28570 also suggested an oxygenase-type enzyme system to be responsible for the degradation of the molecule by loss of one C2-unit. The bioconversion of a-ionone, a-methylionone and a-isomethylionone by Aspergillus niger JTS 191 was described [129]. The major products from a-ionone (152) were cis- and rra«5-3-hydroxy-a-ionone (153) and 3oxo-a-ionone (154), Fig. (30). The biotransformation of a- and jff-ionones by ten kinds of Aspergillus spp., one of which was A. niger JTS 191, and other microorganisms was also studied later by another group [130]. The results with A. niger JTS 191 were essentially the same as the ones obtained by the previous group [129]. A complete schematic overview of all metabolites produced from a-ionone and ^-ionone is given by Noma and Asakawa [92]. o PK^'^^::^^ \^SJ^

Botryodiplodia (^^''^^T^^^^ ^ P'S''"^^^^" ^. (^^ 1 eo romae ^^X^^^^ O^^^X^^ G^^^^O^

a-Ionone(152)

155

156

157

Fig. (31). Bioconversion of a-ionone by Botryodiplodia theobromae IFO 6469 (after [127])

2 Sesquiterpenes Sesquiterpenes and their derivatives are found together with monoterpenes in many essential oils. Many of them are important flavour and perfume compounds, some are of considerable importance for pharmaceutical applications [131]. As sesquiterpenoids contain one more isoprene unit than monoterpenes, a greater variety of structures is possible which is

162

manifested in nature by a tremendous diversity of this group of compounds [68]. Only around 30 different sesquiterpenes were known 20 years ago and these had nearly 15 different carbon skeletons. There are now almost 1000 known sesquiterpenoid compounds belonging to about 200 different skeletal families [132]. 2.1 Acyclic Sesquiterpenes 2.2.1 Acyclic sesquiterpene alcohols

Nerolidol Nerolidol (3,7,ll-trimethyM,6,10-dodecatrien-3-ol) (158, 162) is the sesquiterpene analogue of linalool (62). Because of the double bond at the 6-position, it exists as E- (158) and Z- (162) isomers. (+)-£^-Nerolidol occurs in cabreuva oil; (~)-nerolidol has been isolated from Dalbergia parviflora wood oils [26]. Arfmann et aL [133] investigated the hydroxylation of acyclic terpenes and analogues such as Z- and jE-nerolidol, famesol, nerylacetone and geranylacetone. Aspergillus niger ATCC 9142 and Rhodococcus rubropertinctus DSM 43197 were found to be the best suited strains. The bacteria exclusively oxidised the terminal methyl group to the primary alcohol (159) and further to the carboxylic acid (161), Fig. (32). The fungus on the other hand attacked parallel the double bond of the terminal isoprenyl group resulting in glycols (160) analogous to the transformation of 1-menthenes (see 1.2.1). The best yields with nearly 25% of primary alcohol (a)-hydroxy-£-nerolidol) (159) were obtained with A, niger ATCC 9142 and the substrate ^-nerolidol (158). Apart from the terminal oxidation and co-hydroxylation, hydroxylations in the chain of acyclic terpenes were also reported [112]. In a limited screen some microorganisms were found capable of introducing a hydroxy group in 8- or 9-position of the substrate JS-nerolidol. The epoxidation of the isoprenyl double bond of J?-nerolidol by Nocardia alba DSM 43130 was reported by the same group [112]. Terminal oxidations yielding both the co-hydroxylated product and the 10,11-diol were observed as parallel reactions. The same group also studied the bioconversion of Z- and £-nerolidol with three more fungal species: Diplodia gossypina, Corynespora cassiicola and Gibberella cyanea [112]. It was found that all strains hydroxylated the substrates to their respective vicinal diols (glycols). The highest yield was obtained with the strain G. cyanea (79.5%) and the substrate jE-nerolidol. Also hydroxyketones were found in lower yields (0.5-5%) and in some cases traces of epoxides were produced. More recently a very interesting report describing a completely different degradation pathway of nerolidol (158) by Alcaligenes eutrophus was

163

published [134]. This bacterial strain was isolated from soil by enrichment culture technique with nerolidol as the sole source of carbon and energy. Instead of the usual epoxidation of the 10,11-double bond, an epoxidation of the 1,2-double bond, followed by reduction of 166 to a triol (167) which was cleaved to a C2-ketol (glycolaldehyde) (168) and geranylacetone (169) was noticed, Fig. (33). This pathway is quite similar to the degradation of nerol and geraniol to 6-methyl-5-hepten-2-one (see Fig. (7)). To the best of our knowledge an analogous pathway for the degradation of linalool to 6-methyl-5-hepten-2-one has not been published before. The authors as well [134] claim that their proposed pathway for the biotransformation of nerolidol is hitherto unknown. Geranylacetone (169) was further reduced to (5)-(+)-geranylacetol (170), the "norsesquiterpene"-analogue of sulcatol. p

I

A. Niger

£-NerolidoI(158)

^y^^^::^^^^^.^^

+ "''ilT''^^^

159 (20%)

160

+ "^v>^

159 (16%)

161 (16%)

163 (1.5%)

164

163 (0%)

165 (25%)

A. niger

Z-Nerolidol(162) R. rubropertinctus

Fig. (32). a>-Hydroxylation of £- and Z-nerolidol (after [133])

These findings were also in contrast to the results they obtained carrying out the bioconversion of nerolidol with Aspergillus niger [135,136]. In this case, the two main metabolites identified were 12hydroxynerolidol (159) and 10,11-dihydroxynerolidol (160). The same reactions were noticed when dihydronerolidol was used as substrate: comethyl hydroxylation and dihydroxylation of the remote double bond.

164

H20 + C02 (5)-(+)-Geranylacetol (170)

Geranylacetone (169)

Glycolaldehyde (168)

Fig. (33). Proposed pathway for the degradation of nerolidol by Alcaligenes eutrophus (after [134])

Finally, a Japanese group also studied the biotransformation of Z- and £-nerolidol [137,138] with the plant pathogenic fungus Glomerella cingulata. Both Z- and £-nerolidol were mainly oxidised at the remote double bond. Z-Nerolidol (162) was transformed into (Z)-3,7,lltrimethyl-l,6-dodecadiene-3,10,ll-triol (164) while ^-nerolidol (158) was mainly oxidised to (£)-3,7,ll-trimethyl-l,6-dodecadiene-3,ll-diol (171); only small amounts of (£)-3,7,11-trimethyl-l,6-dodecadiene-3,10,11-triol (160) were obtained. Fig. (34).

Z-NerolidoI (162)

164

Fig. (34). Hydroxylation of £-nerolidol and Z-nerolidol by Glomerella cingulata (after [137,138])

165

Farnesol Farnesol (3,7,11 -trimethyl-2,6,10-dodecatrien-1 -ol) (181) is the sesquiterpene analogue of geraniol (20) and nerol (14), depending on its 2E' resp. 2Z-configuration. It is a component of many blossom oils. It is a colourless liquid with a linden blossom odour, which becomes more intense when evaporated, possibly due to oxidation [26]. The levels found in essential oils are generally low (0.5-1.0%) with the exception of cabreuva, which contains up to 2.5% famesol, and the distillate from flowers of Oxystigma buccholtzii Harms., which contains up to 18% famesol [27]. The first data about the bioconversion of famesol date back to the sixties: its degradation pathway is similar to that of geraniol and nerol. Seubert [139] showed that the degradation of famesol by Pseudomonas citronellolis proceeds through the oxidation of C-1 to give famesic acid, followed by carboxylation of the ^-methyl group. Subsequently, the 2,3double bond of the dicarboxylic acid is hydrated to a 3-hydroxy acid which is then acted upon by a lyase resulting in the formation of a j9-keto acid and acetic acid. The jff-keto acid readily enters the fatty acid oxidation pathway [29]. When famesol (181), as a mixture of the four isomers, was incubated with Rhodococcus rubropertinctus DSM 43197 for 23 hr, two products were obtained: a low yield (3%) of an 11-hydroxylated compound, 11hydroxygeranylacetone (182) and a higher yield (12%) of nerylacetone (174), Fig. (35) [133,140]. A common feature of both was that the famesol skeleton was shortened by a C-2 unit. It is interesting to remark that the transformation of famesol (181) to nerylacetone (174) is analogous to the conversion of geraniol or nerol to 6-methyl-5-hepten-2one. Aspergillus niger ATCC 9142 did not hydroxylate famesol, but reaction of the substrate with A. niger DSM 63263 yielded, after 48 hr, 4.5% 12-hydroxyfamesol (183), Fig. (35). HO.

J^^Z JL^^ Rhodococcus ^ ^ ^ ^^^ ^''^^'^"'^^OH rubropertinctu^ ^^^^^ ^"^^ ^^'^^^ ^ DSM 43197 Famesol (181) 182 Mixt. of isomers Aspergillus niger 183 Fig. (35). Microbial transformation products of famesol (after [140])

174

166

The bioconversion of famesol by A. niger was investigated by Madyastha and Gururaja [135]: analogous transformation products as in the case of nerolidol were identified: 12-hydroxyfamesol and (5)-(-)10,11 -dihydroxyfamesol. More recently, the biotransformation of (2jE',6^-famesol (181a) was also carried out by a Japanese group [141] using the fungus Glomerella cingulata. At the first step, oxidation proceeded at the remote double bond to give (2£,6jEr)-3,7,ll-trimethyl-2,6-dodecadien-l,ll-diol (184) and (2£,6£)-3,7,ll-trimethyl-2,6-dodecadien-l,10,l 1-triol (185). In the second step, the diol (184) was further hydroxylated to (2£',6£:)-3,7,lltrimethyl-2,6-dodecadien-l,5,ll-triol (186), which was further isomerised to its (2Z,6£)-isomer (187), Fig. (36). In the course of this work, the same group [142] also investigated the biotransformation of (2Z,6Z)-famesol (181b) by this fungus, Glomerella cingulata. Oxidation of the remote double bond and isomerisation of the 2,3-double bond gave (2Z,6Z)-3,7,11-trimethyl-2,6-dodecadiene-l, 10,11triol (188) and (2J5:,6Z)-3,7,ll-trimethyl-2,6-dodecadiene-l,10,ll-triol (189) as major metabolites. Further degradation to (Z)-9,10-dihydroxy6,10-dimethyl-5-undecen-"2-one (177) was also observed, Fig. (36). 2.1.2 Acyclic sesquiterpene ketones

As in the discussion of the acyclic monoterpene ketones (see 1.1.3) instead of the sesquiterpene ketones, the structurally related compounds neryl(176) and geranylacetone (169) will be reviewed. Indeed, as mentioned before, neryl- and geranylacetone are the "norsesquiterpene"-analogues of 6-methyl-5-hepten-2-one, which is the j(?-oxidation product of nerol and geraniol, just like neryl- and geranylacetone are the bioconversion products of famesol (see 2.1.1). As mentioned before (2.1.1) Arfmann et aL [133] studied the cohydroxylation of the sesquiterpenes nerolidol and famesol and the related compounds neryl- and geranylacetone. This was done because cohydroxylated sesquiterpenes are important intermediates in the synthesis of industrially used fragrances and flavours. Aspergillus niger ATCC 9142 and Rhodococcus rubropertinctus DSM 43197 were unable to hydroxylate nerylacetone or its ^-isomer geranylacetone, at the co-position of the molecule. Incubation of nerylacetone with Mucor circinelloides CBS 27749 for 25 hr resulted in seven transformation products, including one co-hydroxylation compound, in 3% yield [140]. The group of Abraham et aL [112] expanded their studies of the hydroxylation of the sesquiterpenes nerolidol and famesol to the ketones neryl- and geranylacetone. Again similar results were obtained: Diplodia gossypina, Corynespora cassiicola and Gibberella cyanea were able to hydroxylate the substrates to the corresponding glycols (9,10-diols) with

I-

Fig. (36). Possible pathways for the metabolism of (2E,6E)-farnesol and (2562)-farnesol by Glomerella cingulata (after [141,142])

?i

168

high yields {ca 50-60%). In some cases smaller amounts of the epoxide and hydroxyketones were also noticed. The bioconversion of geranylacetone WiXh Aspergillus niger yielded the co-hydroxylation product 11-hydroxygeranylacetone and the vicinal diol (S)-(-)-9,10-dihydroxygeranylacetone as in the case of nerolidol and famesol [135]. The same reactions were noticed in the bioconversion of geranylacetol. More recently the group of Miyazawa et al. [137,138] investigated the biotransformation of neryl- and geranylacetone by the plant pathogenic fungus Glomerella cingulata. This research was undertaken in parallel with their studies of the sesquiterpene alcohol nerolidol (see 2.1.1). The results obtained with nerylacetone (176) were similar to those with Znerolidol (162): formation of a ketodiol (Z)-9,10-dihydroxy-6,10-dimethyl5-undecen-2-one (177). From geranylacetone (169) however, the main reaction obtained was a monohydroxylation yielding the hydroxyketone (£)-10-hydroxy-6,10-dimethyl-5-undecen-2-one (173), comparable with the transformation of ^-nerolidol (158), Fig. (37), resp. Fig. (34). 2.2 Cyclic Sesquiterpenes 2.2.1 Cyclic hydrocarbon sesquiterpenes

Caryophyllene and humulene Caryophyllene (190) is the main constituent (> 50%) of Copaiba (balsam) oils, which are obtained by steam distillation of the exudate (balsam) from the trunk of several species of Copaifera L. {Fabaceae), a genus of trees growing in the Amazon basin [26]. Copaiba balsam oils and balsams are used mainly as fixatives in soap perfumes. In 1979, Rama Devi [143] isolated a strain of Pseudomonas cruciviae by enrichment culture on caryophyllene. The major metabolite of this bioconversion was a product hydroxylated at the bridgehead and at the allylic position. The yield, however, was very low. Abraham et al [144-146] studied the biotransformation of caryophyllene (190) and humulene (196) by Diplodia gossypina (ATCC 10936) and two strains of Chaetomium cochliodes (DSM 63353 and ATCC 10195), Fig. (38) and Fig. (39). Sixty three products, including 49 that had never been described previously, were obtained and tested for their biological activity [147]. More recently, the bioconversion of (-)caryophyllene by Chaetomium cochliodes IFO 30576 was also studied by another group [148]. The substrate was first epoxidized at the C-C double bond, producing (-)-caryophyllene-4,5-oxide (191), which was then hydroxylated at the gem-dimethyl group and C-7 position giving 193.

Geranylacetone (169)

Nerylacetone (176)

178

Fig. (37). Hydroxylation of geranylacetone and nerylacetone by Glomerella cingulata (after [137,138])

170

Caryophyllene (190) D.

gossypina

CO(M

HO*"*'

194 (5%)

195

193 (12%)

Fig. (38). Main biotransformation products of caryophyllene by Diplodia gossypina and Chaetomium cochliodes (after [144,145])

OH HO

j3 Chaetomium cochliodes

Humulene(196)

HO'"

197

II i

^^ 198

'^

OH

199

Fig. (39). Hydroxylation of humulene by Chaetomium cochliodes (after [145])

Valencene Valencene (5), a sesquiterpene hydrocarbon isolated from orange oils is used as starting material for the synthesis of nootkatone (6), which is used for flavouring beverages [26] and which is a much sought-after aromatic substance [131]. Two bacterial strains, onefi-omsoil and the other from infected local beer, which utilised calarene as the sole source of carbon and energy have been isolated by enrichment culture techniques [149]. Both these bacteria were adapted to grow on valencene as the sole carbon source. Fermentations of valencene (5) by these bacteria of the genus Enterobacter in a mineral salts medium yielded several neutral metabolic products: dihydro alpha-agarofiiran (200) (7.5%), nootkatone (6) (12%), another ketone (201) (18%) and a-cyperone (202) (8%), Fig. (40).

171

Enterobacter

Valencene (5)

CO

Dihydro alpha-agarofuran (200) 7.5%

Ketone C14H22O (201) 18%

Nootkatone (6) 12%

a-Cyperone (202) 8%

Fig. (40). Biotransformation of valencene by Enterobacter sp. (after [149])

Very recently, the chemoenzymatic preparation of nootkatone from valencene was described [150]. Nootkatone was prepared from valencene by copper(I) iodide catalysed oxidation with tertAmtyX hydroperoxide and hydroxylated at C-9 by Mucor plumbeus and Cephalosporium aphidicola, 2.2.2 Cyclic sesquiterpene alcohols and ketones Patchoulol

Patchouli alcohol or patchoulol (203) is a major constituent (30-45%) of the patchouli essential oil which is extensively used in perfumery [68]. The essential oil is obtained by steam distillation of the dried leaves of Pogostemom cablin (Blanco) Benth. (Lamiaceae), Although it is the main component of the patchouli oil, this compound contributes less to the characteristic odour of the oil than nor-pachoulenol (206), present at a concentration of only 0.3-0.4%. A process for the production of the latter compound, via the microbial 10-hydroxylation of patchoulol (203) and subsequent oxidation of the 10hydroxypatchoulol (204) was published [151]. From the 350 microorganisms screened, four strains of Pithomyces species carried out regio-selective hydroxylation of patchoulol to 10-hydroxypatchoulol: Pithomyces sp. NRJ201 and P. chartarum NRJ210, isolated from soil situated in the neighbourhood of Kamakura, Japan, the most important ones. A method was developed by which 10-hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-litre fermentation jars at 2 to 4 g of patchoulol per litre and isolated as pure material in 30% yields. The

172

obtained hydroxylated product can easily be converted chemically to the industrially more important nor-patchoulenol (206), Fig. (41). An overview was given by Seitz [152]. ^^^^^2^^^>^

^^\.A^^^^V^

/yC>J

/K^J-

LL

1 Pifhomyces s p ^

Patchoulol (203)

02.Pt02^

^^^i^ pT ^

\1

\ CHjCH

10-Hydroxypatchoulol (204)

Pb(0Ac)4^

yT^-^P^ OOOH 4-Carbohydroxypatchoulol (205)

"°C^C^ ^ i^KP Nor-patchoulenol (206)

Fig. (41). Regioselective lO-hydroxylation of patchoulol by Pithomyces sp. and subsequent chemical conversion to nor-patchoulenol (after [151,152])

Germacrone Hikino et aL [153] have investigated by enzymatic means the stereospecific epoxidation reactions of olefinic double bonds in the plant Curcuma zedoaria Roscoe. They studied the bioconversion of germacrone (207), a constituent of C zedoaria, by microorganisms in the hope of obtaining stereoselective epoxidation as in the case of the plant. Cunninghamella blakesleeana yielded three major products (208 - 210) from germacrone, Fig. (42). More recently, Asakawa et aL [154] described the biotransformation of germacrone by Aspergillus niger. A very unstable allylic alcohol (211) was obtained from the metabolite of germacrone along with (213), Fig. (42).

173

R = H(211) R = Ac(212) Fig. (42). Biotransformation of germacrone by Cunninghamella blakesleeana (after [153]) and by Aspergillus niger (after [154])

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

179

CYCLOARTANE AND OLEANANE SAPONINS FROM ASTRAGALUS SP. LUISELLA VEROTTA,*^ NADIA A. EL-SEBAKHY^ Dipartimento di Chimica Organica e Industriale, Universitd degli Studi di Milano, via Venezian 21, 20133 Milano-Italy; ^Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt ABSTRACT: Astragalus spp. (Leguminosae) represent a valid rural crop of ecological importance in many countries, and some species serve as foodstuffs and pharmaceutical emulsifiers. Astragalus species are used in Chinese traditional medicine as antiperspirant, antihypertensive, antidiabetic, diuretic and tonic. The pharmacologically active constituents of these Astragalus are of two different types, polysaccharides and saponins, and the most interesting pharmacological properties are hepatoprotective, inmiunostimulant and antiviral. Astragalus species are a source of cycloartane type saponins, derived from cycloartenol by oxidation at C-6, C-16, C-20, C-23, C-24, followed by possible ring closures with the formation of a 20,24-epoxide (cycloastragenol or cyclogalegenin) or a 20,25epoxide, or a 16,24- 20,24-diepoxide (cycloalpigenin), or a 16,23- 16,24-diepoxide (cycloorbigenin B). Approximatively 100 saponins mainly derivatives of the 20(R\24(S) form of cycloastragenol [3p,6a,16p,25-tetrahydroxy-20(/?),24(5)-epoxy9,19-cyclolanostane], (astragalosides or astraversianins), and more rarely of the 20{S), 24(R) form (cyclogalegenin), have been isolated. Some species also contain trihydroxyolean-12-ene saponins. Details on the isolation, purification and structural elucidation, along with recent results on the pharmacological properties of Astragalus cycloartane and oleanane saponins, will be discussed.

INTRODUCTION Saponins are widely distributed in the plant kingdom, and occur in many food plants (soybeans, chick peas, peanuts, mung beans, broad beans, kidney beans, lentils, garden peas, spinach, oats, aubergines, asparagus, fenugreek, garlic, sugar beet, potatoes, green peppers, tomatoes, onions, tea, cassava, yams), as well as in forage species (alfaalfa). Saponins are also contained in numerous herbal remedies. The useful biological applications of saponins, generally based on theii* membrane-disrupting properties, range from fish and snail poisons [1, 2], to potentially interesting anti-cancer agents [3] and ion channel-blockers [4, 5]. Other interesting biological applications for various specific

180

saponins include their uses as antiinflammatory, hypocholesterolemic, immune-stimulating, andflavor-modifyingsubstances [4, 6]. Saponins consist of a terpenoid core (the aglycone), having oxygenated positions bound to sugar moieties (up to ten monosaccharidic units). In water they form colloidal solutions which foam on shaking and precipitate cholesterol. When saponins are near cell membranes, their interaction with cholesterol may create pore-like structures that eventually cause the membrane to burst. Hemolysis is an example of this phenomenon (i.e. the distruction of erythocyte membranes, but not hemoglobin). Occasionally, they cause hypersecretion, which could explain their expectorant activities and also their toxicity to fish. Table 1. Plants containing cycloartane saponins.

Plant (family) Ranunculaceae

Genus

Distribution

Actaea

Asia

nd

Beesia

Asia

anti-inflammatory, analgesic

[71

Cimicifuga

Asia

antipyretic, analgesic, anti-inflammatory

[7]

Souliea

Asia

anti-inflammatory, analgesic

[7]

Thalictrum

fevers, hypertension, diarrhea, irritant, stomach ache

[81

anti-inflammatory, antip>Tetic, contraceptive sedative

[91

chest coughs, sypliilis, aphrodisiac, diarrhoea, bilharziasis, fever, anthelmintic, headaches, dysentery, dressing wounds, snake bites, vaginal orifice reduction digestive disorders, sweetener

[8, 10-131

antiperspirant, diuretic, tonic, anticancer, antidiabetic nd

[151

analeptic for the treatment of decline

[171

1 Rubiaceae

Mussaenda

Southern Africa, Europe, Asia, North America Asia

1 Passifloraceae

Passiflora

South America

1 Combretaceae

Combretum

Southern Africa

1 Leguminosae

Abrus

Oxytropis

Tropical Africa, South-East Asia Northern Africa, Asia, Europe Asia

Curculigo

Asia

Astragalus

Hypoxidaceae

nd. No details

Ref

Uses

[81

[141

[161

p lupenyl cation

Fig (1). Biogenelic pathway to cycloartane and oleanane saponins

I@ ti

oleanyl cation

182

Biogenetic pathway to cycloartane and oleanane saponins Tran^-squalene oxide, suitably positioned and folded on the enzyme surface (i.e. in a chair-boat-cliair-boat conformation), gives a polycyclic triterpene structure through a concerted carbocation-mediated cyclization. The resulting protosteryl cation then undergoes a series of Wagner-Meerwein 1,2 shifts. In plants, the final triterpene alcohol is cycloartenol. Should rran^-squalene oxide be folded in a chair-chairchair-boat conformation, then an identical carbocation mechanism ensues, and the transient dammarenyl cation (precursor of dammarane "ginseng" saponins) can evolve to oleanyl systems through ring expansion. Fig. (1)

"COOH

R'

R^

H

R^ mollic acid

a-L-arabinosyl

p-D-glucosyl

moilic acid glycoside

H

p-D-xylosyl

moliic acid xyloside

a-L-arabinosyl

P-D-4ACxylosyl

mollic acid 4Acxyloslde

p-D-xyloside

R^ mollic acid arabinoside

CH3 CH2

Jessie acid

CH2

Jessie acid arabinoside CH2

Vyis^CH3 0

a-Larabinosyl

CH3

moilic acid arabinoside

Fig. (2). Cycloartane saponins isolated in Combretum sp.

CH3

Jessie acid xyloside

183

Cycloartane saponins are relatively rare when compared to the many oleanane saponins (Table 1) and their occurrence is limited to herbs, shrubs and twinning plants (Astragalus, Abrus, Mussaenda, Passiflora), growing in dry, moderate climates. An exception is represented by Combretum which are trees diffused in tropical regions. Combretum genus, as well as Astragalus, contain both oleanane and cycloartane saponins (mollic acid and imberbic acid derivatives), thus possessing both the enzymatic pathways [10,11], Fig. (2).

R^

R^

R'

R*

R*

R«

R^

OH

H

OH

CH3

CH3

H

COOH

OH

H

OH

CH2-0-4AcRha

CHa

H

COOH

OH

H

O-Rha

CH2-0-4AcRha

CH3

H

COOH

OH

H

0-Rha

CH2-0-Rha

CH3

H

COOH

H

O-Rha

CH2-0-Rha

CH3

H

CH2OH

OH

H

O-Rha

CH2-0-4AcRha

CH3

H

CH2OH

OH

H

OH

CH2-0-Rha

CH3

H

COOH

OAc

H

OH

CH2-0-Rha

CH3

H

COOH

H

OH

OH

CH3

COOGIc

OH

Fig. (2). Oleanane saponins isolated in Combretum sp.

OH

CH3

Table 2. Astragalus species studied for their cycloartane and oleanane saponins Source

adsurgens alexandrinus Boiss. alopecurus amarus Pall babaagi basineri Trautv. brachyprerus cephalores Banks & Sol. var brevicalyx Eig. chrysopterus coluteocarpur cornplanatus R. Br. dissecrur dasyanrhus Pall.

ernesrii Comb exilis L. galegiformis L. glycyphyllos L hamosus L. kuhirangi (Nevski) Boriss. kulabensis illiensis L nlelanophrurius Boiss. rnembranaceus Bunge microcepllalus Willd.

Aglycone

China Mediterranean Coastal strip Russia Russia Russia Russia Turkey Turkey

cycloastragenol; 6a,25 dihydroxy, 3,164ioxo-9P,19cyclolanostane cycloastragenol: 3P,6a,16P,24(R),25 pentahydroxy 9~,19cyclolanostane

China Russia China Russia Russia

cycloastragenol, soyasapogenol B cycloastragenol cycloastragenol; soyasapogenol B; complogenin cycloastragenol, cyclocanthogenin cycloastragenol, 3P.7422 hydroxy-16,23epoxy-24.25.26.27tetranor-9p, 19cyclolanostane cycloalpigenin A cycloastragenol cycloastragenol cyclogalegenin cycloastragenol, soyasapogenol B, 19-keto-soyasapogenol B soyasapogenol B cycloastragenol cycloastragenol cycloastragenol 9~,19cyclolanostane cycloastragenol; 3P,6a,16P,24a,25-pentahydroxy, cycloastragenol, soyasapogenol B, huangqiyenin, cyclocanthogenin cycloastragenol, 3P,6cr, 16P,24a-tetrahydroxy-20,25-epoxy 9P,19-cyclolanostane

Russia Japan Russia Russia Russia Russia Russia Russia China Turkey China Turkey

cycloalpigenins cycloastragenol cycloastragenol cycloastragenol cycloastragenol, cyclocanthogenin cyclocanthogenin

Ref

nwngholicus Bunge oleijolius DC. orbiculatus Ledeb. pamirensis Ovcz. tk Rassulova peregrinus Vohl.

Turkey Russia Russia

pterocephalus Bunge pycnanthus

Russia Russia

quisqualis Bunge schachirudensis Bunge sieberi DC sieversianus Pall. sinicus L spinosus Vahl. taschkendicus Bunge tornentosus Lam.

Russia Russia Egypt China China Egypt Russia Egypt

tragacantha Habl. trigonus DC

Russia Egypt

trojantcs Stev. itninodus verrucosus Moris villosissinlrrs

Turkey Russia Italy Russia

cycloastragenol; la,3P,16P,27-tetrahydroxy9P, 19 cyclolanost-24Eene. cyclocanthogenin 1a,7P,24&25 tetrahydroxy-9P,19cyclolanostane cycloorbigenin B cycloastragenol cycloastragenol cycloastragenol 6dehydroxycycloastragenol, 24Rcycloartan-3P,6a,16P,24,25-pentaol, cycloastragenol

L. Verotta et al., unpublished [181 [921

cycloastragenol cyclogalegenin cycloastragenol soyasapogenol B; complogenin cycloastragenol cycloastragenol, cycloasgenin C 38 hydroxy[6a-acetoxy,23-ethoxy,16~,23(R)-epoxy-24,25,26,27-tetranor]-9~,19cyclolanostane cycloastragenol; cyclocanthogenin 25ene; cycloastragenol; 3~,6~,16~-trihydroxy-9~,19~yclolanost-24x0, 3 P . 6 ~16P-trihydroxy-9.19-cyclolanost-24ene, soyasapogenol B cycloastragenol; cyclocanthogenin, soyasapogenol B cycloastragenol cycloastragenol cycloastragenol

186

Astragalus genus is one of the richest source of cycloartane saponins. Oleanane type saponins are also found in Astragalus sp., but their occurrence is limited to structures common to Leguminosae. Fig (3) shows the naturally occurring cycloartanes and oleananes which have been isolated from different Astragalus species. The largest body of work on Astragalus saponins is found in the Russian literature [18]. The Tashkent group has been involved for years in the study of saponins from native plants. Unfortunately, most of their publications in the Russian literature were not available until recently as an English translation. In addition, confusion arose regarding the configuration at the centres involved in the formation of the tetrahydrofiiran ring (cycloastragenol and the less frequent cyclogalegenin are the peculiar skeletons of Astragalus sapogenins). A paper [19] eventually shed light on this misunderstanding, describing the X-ray analysis of the two diastereoisomers and solving the diatriba. Since this publication appeared in Russian, many authors remained unawai'e of these data for years. R = GH2CH3I H

soyasapogenol B

complogenin

Fig. (3). Tetranor-cycloartane and oleanane triterpenoids isolated from Astragalus sp.

No rational and corrected review on the so far described Astragalus saponins is presently available. Recently, a paper describing the pharmacological properties of Astragalus species has been published [20]. It reports an overview of the biological properties of the different metabolites present in the genus.

187

Fig.(3). Structures of the aglycones common to Astragalus sp.

29

28

OH

cycloastragenol

OH cyclogaiegenin

R = O; a-OH. H; ^-OH, H cycloalplgenlns 2^.0H

OH cycloorbigenin B

"

OH

cyciocanthogenin

ChfeOH

R = H. H; OH, H

188

The word Astragalus is derived from two Greek words "Astron" means a star and "Gala" means milk, for the belief that the presence of Astragalus plants in grass-land improved milk yield of livestock [21]. Genus Astragalus (Leguminosae) is one of the largest and most widely distributed genera of flowering plants [22, 23], it includes 2000 species, grouped into more than 100 subdivisions [24, 25]. The North American species (384) are chiefly perennial herbs or subshrubs while Old World species are more often shrubby. Many species in western and central Asia are spiny and develop phylloides or modified leaves in xerophytic habitats. Astragalus is the largest genus of the Russian flora with 889 species. It is represented in the mediterranean area by over 70 species, varied from small herbs to spiny shrubs [26]. Astragalus spinosus and A, trigonus are the most common species of the genus Astragalus in the western Mediterranean coastal region. While few of these species are native to Tropical Africa, many, if not most, still grow today in Egypt and Israel, on both sides of the Suez Canal. In fact, the Biblical World, bridging Africa via Egypt through Israel and Palestine to Europe and the Middle East, is at the crossroads of the African and the European Continent. Singular are these citations from ancient texts: "A little balm and a little honey, gum {Astragalus bethlehemiticus Boiss. ''Bethlehem)

Astragalus sieberi DC

Astragalus membranaceus Bunge

Tragacanth" "Gum"), myrrh, pistachio nuts, and almonds." Genesis 43:11. " I have gathered my myrrh with my spice..." (Astragalus gummifer Labill "Tragacanth", "Spice"). Song of Solomon 5. Reported ancient uses are for cancer, burns, cough, diarrhea. The medicinal properties oi Astragalus species are also described in the Chinese Pharmacopoeia [7]. Astragalus species are used in Chinese

189

Astragalus alexandrinus Boiss.

Astragalus bethlehemiticus Boiss.

Astragalus spinosus (Forssk.) Muschl..

Astragalus kahiricus DC.

190

traditional medicine as antiperspirant, antihypertensive, antidiabetic, diuretic and tonic [15]. In China, many pharmaceutical preparations containing Astragalus extracts or isolated compounds have been used. Water decoctions of the roots of A. membranaceus and A. sinicus were incorporated into pharmaceutical preparations for treatment of toothache or for oral hygiene. These extracts were found to remove tobacco stains, plaque and food residues from teeth. Many Astragalus species have attracted interest because of their apparent cytotoxic constituents and have been used for treatment of patients with leukemia and uterine cancers [27]. The most common use oi Astragalus is as forage for livestock and wild animals, although 32 sp have been recognized as of use in foods, medicines, cosmetics, as substitutes for tea or coffee, or as sources of vegetable gums [28]. However, a number of species are toxic for livestock and in many cases the toxins could be transferred to humans through meat or milk. Thus, there are two groups of Astragalus species that are closely related to health: the toxic species and the medicinal plants. The genus Astragalus appears highly uniform from a chemical point of view, with two kinds of pharmacologically active principles and three different kinds of toxic compounds. In the former group the polysaccharides and the saponins stand out, and, in the second, the indolizidine alkaloids (swansonine and its N-oxide derivative, and lentiginosine) [29-31], the nitrocompounds endecaphyllins (nitropropionic acid-glucose derivatives) and 3-nitropropyl-glucosides [32-34], and the seleniferous derivatives (seleno-cysteine, -cystathionine, -cystine, and methionine), are found [29, 35]. The triterpenes and saponins are the most widely studied secondary metabolites. Astragalus species are a source of cycloartane type saponins. They are characterized by a 9j8, 19 cyclolanost-24-ene-3j^ol skeleton, whose oxygenated positions can be linked through acetal bondings to sugar moieties. All derive from the parent cycloartenol which undergoes oxidations (typical are at C-6, C-16, C-20, C-23, C-24), followed by possible ring closures with formation of a 20,24-epoxide (cycloastragenol or cyclogalegenin) or a 20,25-epoxide, or a 16,24-, 20,24-diepoxide (cycloalpigenin), or a 16,23-, 16,24-diepoxide (cycloorbigenin B). The 1oxidation is very rare. Biogenetically, the configuration at C20 is /?, although, rarely, a 20 (5) configuration has also occasionally been found. Fig. (4).

191

Fig. (4). 9.19 CYCLOLANOST-24-EN-3p-OL

Cycloartanes are found in the plants in free states as well as in the glycosidic form. Over than 100 saponins, mainly derivatives of the 20(R),24(S) form of 9p, 19 cyclolanost-24-.ene-3p-ol (cycloastragenol), named astragalosides or astraversianins, and more rarely the 20(5^, 24(R) form (cyclogalegenin), named cyclogaleginosides and sieberosides have been so far isolated (Table 3). Some species also contain trihydroxyolean12-ene saponins (Table 4). Isolation and purification Oleanane and cycloartane saponins can be isolated and structurally elucidated using similar techniques. A single chromatographic step is rarely sufficient to isolate a pure saponin from an extract. As a general rule, several preparative techniques are required to obtain the pure product. Saponins are generally extracted from plants through an alcoholic extraction of the defatted vegetable material. Due to the possible contemporary presence of acidic components (phenols and their acids, flavonoids, etc.) care should be taken about the pH of the alcoholic solution, which, if too low, can produce undesidered chemical modifications. Acidic methanol can hydrolize glycosidic bonds or produce transesterification. A subsequent useful step is the partition of the total dried alcoholic extract between n-butanol and water. This operation is important to eliminate mono- and disaccharides which complicate further separations.

rable 3. Cycloartane saponins found in Astragalus species

Rz

Et

Name

Astragalus sp. [Rel]

H

H

cycloastragenol

H

H

H

j3-D-xyl

H

H

trigonoside I

brachypterus [54], dissectus [60], kuhitangi [18], membranaceus [77], microcephalus [50], mongholicus [18], pamirensis [18], pterocephalus [18], sieversianus [18, 951, tragacantha [I 81, lminodus [ l 091 dissectus [60], rnembranaceus [79], pomirensis [I 81, tragacanrha [I 81, uninodus [I091 trigonus [I041

j3-D-glc

H H

brachyoside B

brachypterus [54], spinoslcs [98]

COCH3

H H

COCH3

I1

H

huangqiyenin D

membranaceus [80]

j3-D-xyl

kI

H

astraversianin X, cyclosieversioside E

alexandrinus [39], tnelanophrurius [71,72], pterocephalus [18] sieversianus [18, 961 , schachirudensis [18,93], uninodus [I091

boeticus [* *]

cyclosieversioside C astraversianin VI astraversianin V

schachirudensis [18, 931, sieversianus [18, 391, uninodus [109] sieversianus [96]

cyclosieversioside A, astraversianin I1 astraversianin III

melanophrurius [72], schachirudensis [18, 931 , sieversianus [18, 961, uninodus [I091 sieversianus [96] sieversianus [96]

astragaloside IV, cyclosieversioside F, astraversianin XTV, astramembrannin I astragaloside lI, astraversianin W isoastragalosideIt, astraversianin VIII cyclocephaloside I1 astragaloside I, astraversianin IV

alexandrinus [39], dissectus [60], kuhitangi [18], melanophrurius [71.72], membranaceus [76,79, 821, brachypterus [54], pterocephalus [18], p y c m t h u s [92], shachirudensis [18], sieversianus [95], spinosus [98], tragacantha [18], uninodus [log] brachypterus [54], melanophrurius [71,721, membranaceus [76, 791, mongholicus* [85], sieversianus [96], spinosus [98] membranaceus [76], sieversianus [96] microcephalus [54]

isoastragaloside I

brachypterus [54], membranaceus [76], melanophrurius [71.721. mongholicus* [85], spinosus [98], sieversianus [95], trigonus [lo41 membranacerrs [76, 821, spinosus [98]

acetylastragaloside I

membranaceus [76, 821

trojanoside A

rrojanus [ 1081

cyclocarposide

colureocarpus [581

cyclocarposide B

cycloaraloside A, astraverrucin I astraverrucin I1

amarus [45], membranaceus , peregrinus [u], verrucosus [1 lOa] verrucosus [1 lOa]

astraverrucin III

verrucosus [1 lOa]

cyclosieversioside D

basineri [53], schachirudensis [18, 931

cyclosieversioside B

basineri [53], schachirudensis [93]

cycloaraloside E

amarus [49]

trojanoside B

trojmus [log]

astragaloside W

kuhitangi [18], membranaceus [74],

astragaloside III

membranaceus [75,82], mongholicus* [85]

astrachysoside A

chrysopterus [56]

cyclosieversioside G , astraversianin XV astraversianin I X

sieversianus, [95, 961 chrysoprerus [56]

astraversianin XJ

sieversianus [95]

trigonoside II, askendoside D trigonoside Ill

lashkendicus [ l o l l , trigonus [104]

askendoside B

astragaloside VI

kulabensis [60],sieversianus [95]

C

P

astraversianin W

sieversianus [96]

astraversianin Xm

sieversianus [96]

cycloaraloside C, astrailienin A cycloaralosideD

illiensis [70],amarus [50],villosissimus [51] m r u s [48],peregrinus [u]

cycloaraloside B

amarus [46]

astraverrucin IV

peregrinus [u],verrucosus [llob]

astraverrucin V

verrucosus [l lob]

astraverrucin VI

verrucosus [ 1 lob]

cycloaraloside F

amarus [511, villosissimus [511,

astragaloside V

membra~ceus[75]

asernestioside A

ernesrii [61]

asernestioside B

ernestii [61]

asernestioside C

ernestii [61]

huangqiyegenin I

membranaceous [82]

huangqiyegenin A

membranaceous [811

R2

Name

Astragalus sp. [Ref]

H H H H H

cyclogalegenin

galegifonnis 1641. sieberi [94]

cyclogaleginosideB

galegifonnis [65]

cyclogaleginoside A

falcatus, galegifomus [65]

sieberoside I

sieberi [94]

sieberoside IJ

sieberi [94]

Rz

H

R3

H

Name

Asfragalus sp. [Ref]

cyclocanthogenin

tragacantha [ I 81

cyclocanthoside A

cephalotes [55]

cyclocanthoside E agroastragaloside I1

dissect us [60],cephalotes[55],microcephalus [54],melanophurius [71,72],tragacantha [I031 nronbranaceus* [82b]

agroastragaloside I

membranaceus* [82a]

cyclocanthoside D brachyoside C

cephalotes [55],kuhitangi [18],tragacantha [I81 brachypterus [54]

agroastragaloside N

membranaceus* [82c]

agroastragaloside V

membranaceus* [82c]

cephalotoside A

cephalotes [55]

brachyoside A

brachypterus [54]

cyclocanthoside G

rragacantha [103]melanophrurius [71,72]

5

74

75 76 77

a-L-rha(1--2)P-D-xyl PD-glc a-L-rha(1--2)PD-xyl a-L-ara(1--2)PD-xyl

H

H

P-D-glc

trojanoside C

trojanus [I081

P-D-glc

H

PD-glc

trojanoside D

trojanus [I081

P-D-glc

H

PD-glc

trojanoside E

trojanus [I081

P-D-glc

H

PD-glc

trojanoside F

trojanus [I081

Rz

R3

Name

Astragalus sp. [Refl

H H H

H H H

cycloasgenin

raschkendicus [I81

alexandroside I

alexandrinus [39]

askendoside C

taschkendicus [18]

H

H

askendoside A

taschkendicus [18]

P-D-glc

H

cyclopycnanthoside I pycnanrhus [92]

H

PD-&

askendoside G

taschkendicus [loo]

RI

Rt

Name

Asfragalus sp., [Refl

P-D-xyl

H H

macrophyllosaponin B

okifoiius [86]

macrophyllosaponin A

oleifolius [86]

P-D-xyl(4Ac) H

P-D-xyl(l--2)P-D-xyl

PD-glc

macrophyllosaponin C

oleifolius [86]

H

macrophyllosaponin D

olcifolitrs [86]

Astrngalus sp., [Refl

tomentoside I

H

CH2CH3

CH3

OAc

H

tomentosidc IJ

OH

H H

CH3

H

H

dayanthopenin

OAc

200

R

Name

Astragalus sp., [Ref]

6-oxo-cycloartan 3,16 diglucoside

trigonus [ 107]

91

j3-D-glc

O

92

p-D-g\c

a-OH

trigonus [106]

93

j8-D-glc

P-On

synthetic [106]

RO

R

Ri

94

^-D-glc

O

95

p-D-g\c

OH

Name

Astragalus sp,, [Ref] trigonus [105] trigonus [u]

!\.OH

Astragalus sp,, [Ref]

R 96

j8-D-xyl

)3-D-glc

cyclocephaloside I

microcephalus [83]

201

#

R

97

Ri

Name

Astragalus sp.^ [Ref]

H

0

cycloalpigenin A

alopecurus [41]

98

/3-D-xyl

0

cycloalpioside A

alopecurus [41]

99

H

a-OH, H

cycloalpigenin B

alopecurus [42]

100

P-D-xy\

a-OH, H

cycloalpioside B

alopecurus [42]

101

H

i3-OH, H

cycloalpigenin C

alopecurus [43]

102

j3-D-xyl

i3-0H, H

cycloalpioside C

alopecurus [43]

R

Name

Astragalus sp.^ [Ref]

103

H

cycloalpigenin

alopecurus [44]

104

/3-D-xyl

cycloalpioside

alopecurus [44]

202

Name

Ri

Astragalus sp.^ fRcf)

105

II

H

huangqiycgcnin 11

membranaceous (81)

106

)3-D.glc

H

huangqiycgcnin B

fnettibranaceous (81 ]

CH2OR2

Ri

Name

Astragalus sp.^ [Rcf]

107

H

H

)3-D-glc

mongholicosidc I

nionghoUcus (84)

108

CH3CO

OH

/J-D-glc

mongholicoside I

nwngholicus (84)

R 109

H

OH

R2

Name

Astragalus sp.^ (Ref)

H

cycloorbigenin B

orbiculatus [^9]

110

/3-D-xyl

H

H

cycloorbicoside A

orbiciilatus [SI]

111

/3-D-xyI

OH

H

cycloorbicoside B

orbiculatus [SS]

112

j3-D-xyl

OH

/3-D-gic

cycloorbicoside G

^rf7icw/arM5 (90)

* hairy roots; ** Assad, AM. PhD Pharm. Sci. Thesis, Faculty of Pharmacy, University of Alexandria (1984); (u): Verotta, L unpublished.

203

Table 4. Oleananane saponins found in Astragalus

23

24

species

CH2OH

R

Ri

R2

Name

Astragalus sp,, [Ref]

113

H

H

H

soyasapogenol B

glycyphyllos [66]

114

i3-D-glc-UA

H

H

115

/3-D-gal(l—2)-)3-Dglc-UA Me ester j3-D-ara(l-~2)i3-D-glc )3-D-glc(l—2)-j3-Dglc-UA Me ester a-L-rha(l-2)^-D-gal(l-2)-/3-Dglc-UA and Me ester

H

H

H

116 117 118

119

120

121

122

123

124

a-L-rha(l-2)/3-D-glc(l—2)-i3-Dglc-UA Me ester a-L-rha(l--2)a-L-ara(l—2)-i3-Dglc-UA Me ester a-L-rha(l"2). j3-D-xyl(l—2)-j3-Dglc-UA and Me ester a-L-rha(l-2)^-D-xyl(l—2)-j3-Dglc-UA Me ester a-L-rha(l-2)j3-D-gal(l-~2)-)3-D. glc-UA Me ester a-L-rha(l-2)i3-D-xyl(l—2)-i3-Dglc-UA

sinicus [97] sinicus [97]

H

soyasaponin III Me ester soyasaponin FV

H

H

azukisaponin 11

H

H

soyasaponin I

H

H

azukisaponin V

trigonus [105], tribuloides [u] membranaceus [75], chrysopterus [56], sinicus [97], complanatus [59] trigonus [105]

H

H

soyasaponin n Me ester

sinicus [97]

H

H

astragaloside

complanatus [59], membranaceous [74]

P-D-g\c

H

j3-D-glc

H

H

0-/3-D-glc

vm

sinicus [97]

complanatus [59]

comploside 11

complanatus [59]

astrojanoside A

trojanus [iOS]

204

R

Name

125

H

complogenin

126

a-L-rha( 1 --2)-/3-D-xyl( 1—2)-i3D-glc-UAand Me ester a-L-rha( 1 -2)-j3-D-gal( 1—2)-j3D-glc-UA and Me ester

127

Astragalus sp,, [Ref]

siniciis [97], complancuus [59] sinicus [97], complanatus [59]

[u]: Verotta et. Al., unpublished

A typical isolation strategy is the preliminary purification of the nbutanol extract over dextran supports like Sephadex LH20 or Fractogel TSK, followed by further fractionation of the crude saponin mixtures [111]. A new generation of polymers has been exploited for the initial purification steps. They are highly porous polymers (Daion HP-20, MCI gel CHP-20P (both from Mitsubishi Chemical Industries, Tokyo), Amberlite XAD-2) [112]. Methanol-water or acetone-water solvent gradients are used. The polar characteristics of saponins suggest to avoid unmodified silica gel stationary phases, which, if used, require water containing mobile phases to desorb the glycosides. Nevertheless, silica gel chromatography with chloroform-methanol-water as eluent is still the most popular and inexpensive method and is used in most of separations [51, 94]. A limited number of applications of centrifugal thin-layer chromatography for the separation of saponins have also been reported. The technique is a planar method earned out on a centrifugally accelerated inclinated plate, coated with a suitable sorbent. Solvent elution produces concentric bands across the plate which are spun off at the edges of the plate together with separated solutes and collected for subsequent analysis. Examples of

205

separations of cycloartane and oleanane saponins have been reported. Astragalus membranaceus saponins have been isolated through CTLC eluted with chloroform-methanol-water (100:30:3) [75]. The solvent system ethyl acetate-ethanol-water (8:2:1 or 16:3:2) was used on a starchbound plate to isolate cycloartane saponins from Passiflora quadrangularis [11, 113], Fig. (5). Chemically derivatized silica packings have obtained increasing popularity due to their chemical stability and good separation efficiency. RP8 and RP18 sorbents are the most frequent packings used. The commercially available particle sizes from 3 microns to 60 microns allow the use of vacuum chromatography (VLC) or medium to high pressure (MPLC or HPLC) chromatography depending on the desidered load/resolution result. Thus, analytical or preparative separations can be performed, the analytical method being easily tranferred onto a preparative separation, if the chemistry of the sorbents is similar. The solvents of choice are mixtures of methanol-water or acetonitrile-water using gradient conditions [55, 111, 114, 115]. Liquid-liquid partition methods have proved ideal for application to the field of saponins. Very polar saponins are amenable to counter-cunent chromatographic separation, especially as there is no loss of material by irreversible adsorption to packing materials. Counter-current chromatography (CCC) describes liquid-liquid chromatography without a sorbent, requiring two immiscible solvent phases. In most variants of CCC, one phase remains stationary while the second phase is passed through the stationary solvent component. The principle of separation involves the partition of a solute between the two immiscible solvents, the relative proportions of solute passing into each of the two phases being determined by the respective partition coefficients. Initially, the techniques profited of a gravitational stationary phase (Droplet Counter Current Chromatography, DCCC) where droplets of a mobile phase flows through an immiscible stationary liquid phase present in a series of vertical glass columns. Ternary or quaternary solvents are seldom used to produce two unmiscible phases, conveniently chosen through the profile of separation obtained by running a silica gel TLC plate with the water-saturated organic phase of the two phase aqueous solvent system [116]. The system chloroform-methanol-water in different ratios has been involved in the greatest number of applications [113, 117, 118]. The resolution is not high and separations require days, nevertheless the lack of irreversible adsorption and the consistences of loadings (up to 5 g) increased exponentially the study of bioactive saponins in the eighties.

206

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E

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X

f

9. X X

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O)

E

E CO CO CO

CO CO

|± CCS

£

8-

CM CO CD

X

I

I-

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x:

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207

Centrifugal partition chromatography (CPC) relies on centrifugal force rather than gravitation for the retention of the stationary phase and solvents can be pumped at higher speeds through the instruments. In addition, no need for droplets formation is required. This allov^s shorter separation times, without loss of resolution, and an infinite choice of solvents with the only requirement of forming two immiscible phases, stable with the time. Chloroform-based systems have been mostly used for the separation of saponins due to their favourable partition coefficient towards such solvents. [116, 119,120]. Structure elucidation Saponins are constituted of a triterpenoid core to which, in one or more positions, a number of sugar unities are bound. Due to their biological activity their structures have been extensively studied and, at present, several hundreds structures have been elucidated. Such structural investigations often turned out to be very time consuming and tiresome, mainly due to the presence of the sugar moieties for which the site(s) of binding to the triterpenoid, the inter-glycosidic bonds and the conformation of them had to be assessed. This was generally achieved by chemical degradation leading to partial or total cleavage of the sugar moieties, which could be identified by classical methods. The NMR analysis of the intact molecules has been often bumpered by the extensive overlapping of most sugar signals. A relatively easier task was the identification of the terpenoidic portion of the saponins, for which a plenty of NMR information are reported in the literature. The conventional methods include hydrolytic studies followed by the characterization of the aglycone (sapogenin) and oligosaccharide moieties. The drawback of this procedure is the loss of information about the glycosilation site and sometimes about the anomeric configuration owing to anomerization of the reducing monosaccharide. The hydrolytic studies are usually performed under acidic conditions, the major disadvantage with cycloartane saponins being cyclopropane ring opening and rearrangement. In most oleanane-type saponins no degradation occurs during the acidic treatments, and the sapogenins can be extracted from the reaction mixture. Partial hydrolyses are of crucial interest, leading to the isolation of partially glycosidated saponins, for which comparison of the ^^C NMR spectra allow to determine interglycosidic linkages. Mixture of prosapogenins are usually obtained during controlled hydrolytic

208

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CO £ V> w © O

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209

210

conditions, causing accumulation of problems in products separations. Alkaline hydrolyses with methanolic sodium hydroxide are also performed when ester linked olisaccharidic chains are hypothesized to be present in the saponin [121], Fig. (6). The enzymatic hydrolysis is the method of choice when undesired reactions are to be avoided. j8-glucosidases (or /^-glucuronidases) from Helix pomatia digestive juice are now commercially available. They usually show a good hydro lytic performance, even if sometimes need long time reactions (days). Other enzymes are reported to have been used to specifically hydrolize saccharidic bonds like hesperidinase, j5-xylosidase, /?-galactosidase, and mixed crude enzymes like cellulase. Sugar identification and interglycosidic linkages require two steps: first the acid hydrolysis, and subsequent analysis (GC) of the silanized monosaccharidic units allow to determine number and type of sugars (if compared with appropriate standards) [117]; permethylation followed by methanolysis of the saponins identifies interglycosidic linkages if one is in possess of the partially methylated monosaccharides [122]. Sugar sequences can be interpreted through the NMR and mass of the permethylated alditol acetate [123], Fig. (7). This combination of chemical reactions and NMR analysis, anyway, requested some hundreds milligrams of pure saponin which often are not available. The numerous biological activities of saponins, as well as their widespread occurrence in the plants and the hundreds structures described so far, prompted the development of a strategy for their rapid, highly sensitive and, if possible, non degradative, structure elucidation. Mass spectrometry, obtained through the use of soft techniques (FAB, ESP MS) gives information about molecular weight (pseudomolecular ions [M+H]^ or [M-H]") and some very limited sequence and branching confirmation. [124-127]. Recourse to innovations in NMR spectroscopy is essential for further advances in the investigation of complex saponins. The use of a 600 MHz spectrometer gives the required sensitivity, consistent with the low amounts available by modern isolation techniques, while the use of sequential ID and inverse-detected 2D NMR techniques, couples the short time necessary to perform the experiments with the selfconsistency of the obtained results and, thus, the unambiguous structure assignment [128]. An essential prerequisite for deducing the structures of saponins by NMR spectroscopic studies is the unambiguous assignment of ^H and ^^C

211

o

•a '-3

c o •a o

^

c

s

•

212

resonances, which, in principle, involves three main steps: homonuclear correlations, heteronuclear correlations, tridimensional relationships. Recording of the broad-band decoupled ^^C NMR spectrum and DEPT experiments allows to obtain the number of carbon resonances (usually, even a non-quantitative spectrum gives good agreement between line intensities and number of carbon atoms) and carbon multiplicities. Recording of the ^H-^^C COSY spectrum allows to determine protons chemical shifts. F2 slices also allow to read coupling constants. Due to complexity of the spin systems, the determination of both proton chemical shifts and coupling constants of the aglycone can be achieved with "exclusive correlation spectroscopy" (E-COSY) [129]. This experiment favors the diagonal peaks in the diagonal multiplets, allowing the analysis of cross-peak multiplets even closer to the diagonal than for 2D-filtered COSY spectra. The advantage of the E-COSY experiment is a reduction of multiplets lines in the cross peak. It allows easy measurement of active constants and a very accurate measurement of passive coupling constants, thus it falls among the most appropriate experiments in order to obtain the largest number of couplings with the greatest accuracy possible [130]. It becomes the experiment of choice when overlapped signals occure endowed with multiple couplings, but it has been rarely used for the assignment of triterpene protons [104,131]. Selective excitation experiments (HOHAHA and ID-, 2D-TOCSY) determine spin-spin connectivities in isolated spin systems. Complete analysis of COSY spectra of saponins is often difficult because of peak overlap. Therefore it is very useful to be able to relay coupling information from an isolated proton (like the anomeric hydrogen of saccharides), which, properly excited, transfers the relayed coherence to coupled protons. This tranfer is blocked by a quaternary carbon or an heteronucleus, thus, in a TOCSY experiment, each network of mutually coupled protons can be detected by tracing the cross peaks from certain specific protons, or, by reading each ID subspectrum as an isolated spin system, through a series of ID TOCSY experiments, as shown in Fig. (8). The experiments allow to extract: 1) ^H-NMR subspectra of the monosaccharides; 2) ^H-NMR subspectra of isolated spin systems in the aglycone. Moreover, the correct choice of the mixing times, together with the peculiar in-phase multiplet structure (which contrasts with the antiphase structure in COSY spectra) permits the use of ID TOCSY experiments to correctly read vicinal couplings, even the low values undetermined by the less resolved COSY experiment. TOCSY experiments are frequently used for the determination of the aminoacid

Fig. (8). 1D-TOCSY experiments [128]

Alexandroside 1 [39] (Astragalus alexandrinus Boiss.)

Fig. (9). 1D-TOCSY e.uperirnents.

^«

CO' U>

I 6

I' .9

I

I

'H' I

I

'ii'

O

r-r-n-T-TT-i

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V

J"

i

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215

216

constituents of peptides in which the aminoacid units are separated by amide carbonyl atoms. [39, 132-135]. The application to oligosaccharides is rarely reported [104, 131, 134, 135], Fig. (9). The subtraction from the total ^^C decoupled spectrum of the sugar ^^C resonances allows to determine the aglycone contribution. Through the ^H-^^C COSY (HMQC) spectrum, aglycone protons are identified and ^H-^H COSY (better E-COSY) or 2D-T0CSY experiments allow to assign all protons in the molecule (except for methyl groups). ^H-^^C long range COSY (HMBC, COLOC) allows to assign quaternary carbons, sugar interconnections and their linkages to the aglycone. At the end, 2D ROESY experiment assigns methyl groups, stereochemistry of the molecule and spatial arrangements. It also confirms sugar interconnections. Fig. (10). Thus, the rapid and unambiguous structure elucidation of complex saponins and their NMR full assignments can be performed with a combination of ID and 2D NMR techniques [136]. Since all the experiments can be run in an automated sequence, all the necessary information can be obtained during a "week end" acquisition. Moreover, the use of a 600 MHz spectrometer gives the necessary sensitivity to work with very low amounts of samples (few milligrams are enough), that, in natural product chemistry, often is a Umiting factor. 3.11, ddd, 11.2,9.0,12.3 3.80, tdd. 9.5.7.4,6.1 0.2r0.6,2d,4

/

H CH3 H 4.80, ddd. 7.9,8.0.6.9 3.55, dd, 11.2,5.0

2.55, d, 7.9

Fig (11). Typical *H NMR resonances of cycloastragenol.

217

31.2

H O '"•*' y^< ^^ 16.5

29X)

=68.3 O H

13C NMR signals of cycloastragenol l20(fl),24(S)-epoxy-9P.19 cyclolanostan-3p-16P,25-tetrolJ

For the identification of the skeletal type, the chemical shifts of cyclopropane protons are diagnostic of a cycloartane type saponin. They resonate between 0.2 and 0.6 ppm as doublets with ^J= 4 Hz. Characteristic skeletal protons are the protons on oxygen bearing carbons and olefinic protons (which are seldom undistinguishable from the anomeric protons, except for their multiplicities). A characteristic of the cycloastragenol skeleton is the deshielded C-22a proton which resonates at 3.11 ppm, a relatively *'free" spectral zone. Fig. (11). The ^^C resonances of cycloastragenol are also shown. They have been assigned through the strategy discussed before and are characteristic and diagnostic for the conformation described. As a comparison the resonances of the diastereoisomeric cyclogalegenin [20(5j,24(/?)] are reported in Fig. (12). Oleanane-type saponins present in Astragalus species are based on soyasapogenol B as the aglycone. The name clearly identifies its origin: soyasapogenol B is one of the aglycones found in soyabean saponins. Even the sugar chains have common characteristics: usually a /J-Dglucuronic acid is directly linked to the aglycone (at C-3) and carries (always bonded to C-2') a a-L-rhamno(l—2)-j8-D-gluco- or galacto- or xylopyranoside moiety, (see table IV). For the identification of the oleanane skeleton, diagnostic protons are at 5 5.51 (t, J= 4.5 Hz, H-12), and ca 5 3.30 H-18j3 (dd), which gives an NOE with H-12 and Me-30, and the protons of oxygenated carbons. A very recent review accurately describes the ^^C NMR spectra of glucuronide oleanane-type triterpenoid saponins [137].

218

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E

219

Pharmacological properties of Astragalus saponins. Several Astragalus species are used as medicinal plants, especially A, membranaceus, which is the one most widely used and studied. It is officially listed in the Chinese Pharmacopoeia [7], as are A. complanatus and A. mongholicus (A. membranaceus var. mongholicus). Radix Astragali is the dried roots of Astragalus membranaceus (Fisch.) Bge. or A. membranaceus var. mongholicus (Bge.) Hsiao, belonging to the non-toxic medicine. It can strenghthen general health and it is used as a common tonic for vital energy (Qi) and Yin. The "sweet" and *'warm" drug enters the lung and spleen meridians. Its functions are to replenish vital energy and cause Yang to ascend, to benefit vital energy and stabilize the exterior, to remove toxins, to promote healing and water metabolism and to reduce edema [138]. Recent pharmacological studies have shown that radix Astragali can inhibit the biosynthesis of glycogen and dilate coronary as well as renal vessels. It also serves as a cardiac tonic, diuretic and antibacterial agent. More interestingly, it possesses an antiinfluenza action and may prevent respiratory infections. Both effects are believed to be related to the enhancement of immunological functions [139]. The LD50 of Astragalus is approximately 40 g/kg when administered by intraperitoneal injection. Overall it is very safe and doses as high as 100 g/kg of the raw herb have been given to rats with no adverse effect. The pharmacologically active constituents of these Astragalus belong to two different kinds of chemical compounds, polysaccharides and saponins, and the most interesting pharmacological properties are hepatoprotective, immunostimulant and antiviral. Zang et al [140] reported the liver protective effects of the saponins isolated from A. membranaceus and A. sieversianus against chemical injury induced by CCI4, D-galactosamine and acetaminophen in mice. In all cases there were positive activities and the saponins inhibited the rise in SGPT levels, decreased the malondialdehyde (MDA) content and increased the glutathione reduced (GSH) concentration in mouse liver. The same compounds were also evaluated in cultured rat hepatocytes, and the results indicated that the activity may be due to to the antioxidative activity of the saponins, since the content of liver protein in treated mice was more than the control. Moreover, in all treated mice, the level of hepatic microsomal cytochrome P-450 was increased. The liver metabolism and immunoregulating action produced by saponins may be also involved in their hepato-protective effects. Similar results were obtained by Zhang et al[\A\] when they studied the activity in vitro and

220

in vivo of A. membranaceous extract and its component astragaloside IV on lipid peroxidation. Both inhibited the in vitro production of lipid peroxides, and restrained the lipid peroxidation increased by adriamycin in mice, with a potent effect for the triterpene derivatives [142], Antiinflammatory effects of astramembrannin I (astragaloside IV), isolated from A. membranaceus, were demonstrated in rats. This compound inhibited the increase of vascular permeability mduced by serotonin or histamine. Oral administration of astramembrannin I caused a dose dependent reduction in carrageenan-induced edema of the hind paw of rats [15].

R=

HO HO

OH Astragaloside iV or astravBrsianin XIV

R=

HO HO OAc Astragaloside II

Various extracts of A. siculus (from Italy) showed marked antiinflammatory, anti-pyretic and analgesic activity in rats and mice. The extracts also showed antimicrobial effects against Gram-negative and Gram-positive bacteria responsible for urinary tract infections. Among the active principles, saponins stand out [143]. Astraverrucin I and IV, from A. verrucosus also showed antibacterial activity against B. subtilis and E. CO//[144]. Among the many pharmacological properties of Astragalus, those on the cardiovascular system are of special interest. Astramembrannin I (astragaloside IV), produced a hypotensive effect after i.v. administration to anaesthesized cats and rats [15]. From A. membranaceous, Wang [145] obtained a saponin-enriched extract and studied its effect on the isolated heart of rats. At doses of more than 50 jig/ml, the extract showed a positive inotropic effect, which turned negative at 30 |ig/ml. The mechanism was similar to that of cardiotonic glycosides. By bioactivity-guided fractionation, the active constituent with positive inotropic activity was isolated and characterized as astragaloside IV [146].

221

A clinical study among nineteen patients with heart congestive failure treated with astragaloside IV injection was carried out. After two weeks of treatment, the symptoms of chest distress and dispnea were allieviated in 15 patients, and their capability of exercise was reinforced. Left ventricular end-diastolic and -systolic volume diminished [147]. 92 patients suffering from ischemic heart disease were successfully treated with Astragalus membranaceus. After administering the drug, the patients were markedly relieved from angina pectoris [148]. A recent study by Zhang et aL [149] reported the dose and time-dependent decrease in plasminogen activator inhibitor type 1 (PAI-1) and an increase in tissuetype plasminogen activator (t-PA) synthesis by astragaloside IV. Clinical studies reports the association of elevated PAI-1 activity in blood with thrombotic disorders, coronary heart disease and myocardial infarction. In general. Astragalus saponins exert a positive and direct effect on the function of heart. Alternatively, they help to treat related diseases. For instance, they inhibit the formation of lipid peroxides in the cardiac muscle or in the liver, influence the function of enzymes contained in them, decrease blood coagulation, cholesterol and sugar levels in blood, and stimulate the immunological system. They act either direct, blocking the transfer of Ca^"^ ions or modulatmg the function of Na^-K^-ATPase, or, alternatively, help resorb other active principles [150]. Several in vitro and in vivo studies have evaluated the cytotoxic effect of saponins against tumors. In general, it is difficult to separate the anticarcenogenic effects of saponins from their immune-modulatory effects. However, few studies have looked at these two events sequentially. Although the mechanism involved in the induction of cell proliferation by saponins is not clear, saponins seemingly affect the membrane environment by direct binding to its components and affecting changes in transmembrane signals. Interactions of saponins with cell membranes can vary depending on the type of saponin and the cell membrane. It is, therefore, difficult to make generalizations, although there is strong evidence to suggest that saponins act as immuno-stimulating agents. Astragaloside II, a cycloartane triterpene glycoside isolated from the Egyptian Astragalus spinosus, was tested at minimum of five concentrations 10-fold dilution against a total of 60 human tumor cell lines derived from seven cancer types (lung, colon, melanoma, renal, ovarian, brain and leukemia). The results indicated that the colon cancer (SW-620) and the leukemias (CCRF-CEM, HL-60) were the most sensitive cell lines [98]

222

Soyasapogenol B, soyasaponins I and II and wistariasaponins from Wistaria brachybotrys (wistariasaponin C corresponds to astragaloside VIII) decreased (20-30%) the Epstein-Barr Virus (EBV) activation induced by the tumor promoter TPA (12-0-tetradecanoylphorboH3acetate) in Raji cells at a concentration of 1x10^ mol ratio [151]. Soyasaponm I from the same plant exhibited remarkable inhibitory effects on mouse skin tumor promotion on the basis of the two-stage DMBATPA carcinogenesis test in vivo. Soyasaponin I reduced the number of papillomas per mouse at about 40% even at 20 weeks [152], HoX----^^^ Astragaloside Vin Wistariasaponin C

HOGG

HO

^OH

HO

HO' OR

HO

HO

Soyasaponin I

Soyasaponin II

Apart from the more direct property of saponins as anticarcinogenic agents, saponins may also act to delay the initiation and progress of cancer. Epidemiological studies have revealed a strong association between colon cancer and a high concentration of cholesterol metabolites and bile acids in the feces [153]. These observations are important since there is convincing evidence of an interaction between saponins and bile [154]. Saponins were shown to form in vitro large mixed micelles (1x10^ Da) with bile acids [154]. Similar interactions in vivo would reduce the free form of bile acids in the upper gastrointestinal tract and decrease the absorption of bile acids across the mucosa as well as the formation of secondary bile products from primary bile acids. The results suggest that saponins from different dietary sources reduce the availability of bile acids to form secondary bile acids by intestinal microflora and therefore may prevent the development of colon cancer. Hypocholesterolemic effects of soybean saponins have been dimonstrated. Isolated soybean saponins reduced diet-induced hypercholesterolemia in rats through an increase in bile acid excretion

223

[155]. They also form complexes with bile acids and reduce their absorption in vitro. The anticarcinogenic properties of soybean saponins are apparently related to their ability to bind with bile acids. In the intestine, saponins bind to mucosal cell membranes and change their physiology. Since the membranes of some cancer cells contain more cholesterol than do normal cells membranes [156], it is possible that saponins bind more to cancer cells and as a result induce their destruction. Since saponins are surface-active compounds that are not absorbed, their possible interaction with intestinal mucosal cell membranes must be emphasized. Because the average transit time of food is 24h, saponins can either in the intact or in the partly hydrolyzed form, remain in the intestine long enough to interact with free sterols and membrane Upids [157]. The effect of soyasaponins on tumor cells may be mediated by mechanisms other than membrane permeability, because of their weak hemolytic activity. Saponins actively interact with cell membrane components, resulting m changes in intracellular morphology and cell membrane permeability. However, the differences between types of saponins and their effects on cell membrane are evident. Two saponins from soybean seeds having soyasapogenol as aglycone were shown to have a partial inhibitory effect on HIV-induced cytopathology in infected human MT-2 lymphocytes cultures [158]. The major constituent of group of B saponins from soybean seeds completely inhibited HIV-induced cytophatic effects and virus-specific antigen expression 6 days after infection at concentration > 0.25 mg/ml. Saponins isolated from soybean seeds inhibited HIV-1 replication in MT-4 cells at 0.5 |ig/ml (Nakamura et al. 1992) [159]. These saponins had a narrow therapeutic index and did not inhibit HIV-1 RT. One of them was found to inhibit HIV-induced cell fusion in MOLT-4 cells. Soyasaponin I and II were studied in vitro against herpes simplex virus type I (HSV-1). Soyasaponin II was more potent than soyasaponin I in the reduction of HSV-1 production. Soyasaponin II was also found to inhibit the replication of human cytomegalovirus, influenza virus, and human immunodeficiency virus type 1. This activity was not due to the inhibition of virus penetration and protein synthesis, but might involve a virucidal effect. When acyclovir and soyasaponin II were evaluated in combination for anti-HSV-1 activity, additive antiviral effects were observed for this virus [160]. Astragaloside II afforded almost 100% protection of Tlymphocytes in vitro against the cytophatic effects of HIV infection. However, the EC50 of ca. 2.5 x 10"^ molar was difficult to achieve in vivo [98].

Fig (13). Lymphocyte transformation (mouse). Numbers represent proliferation increase (%) at 0.1 pg/rnl Astragalus saponlns 6-0-fi-D-xyloplranosldes

Astragalus saponlns 6-0-p-D-glucopyranosldes

R = H Trigomside l (A. trigonus Dc) + 136 H HO

O OH

R = H cycloastragenol-6-0-glwoside (A. spinosus Vahl.) 64

+

~

(+ 202 at 1 pglml)

HO OH

+ 104

-E H

OH astraversianinX (A. alexandrinus Boiss.)

+ 105 0Ac

astraversianin VI

y w +48 OAc astraversianln II

1 OAc

astraversianin XIV

or Astragaloside IV

(A. alexandrinus biss.)

+a2

OH

(A. alexandrinus Boiss A. spinosus Vahl.)

OAc

Astragaloside I1 (A. trigonus DC A. spinosus Vahl.)

Hoa HO

Astragaloside I Trigonoside II

Trigonoside Ill

astraversianin XV

(A. trigonus DC)

(A. trigonus DC)

(A. alexandrinus Boiss.)

(A. trigonus DC A. spinosus Vahl.)

OH

Astixgaloside

Fig (14). Lymphocyte transformation (mouse). Numbers represent proliferation increase (%) at 0.1 pglml

Astragalus peregrinus

R = cydoastragenol

+ 161 (-19)

HOCH 2

Sieberoside 1

- 51

OH Sieberoside ll

+ 246

Astragalus trigonus DC OH

Glc 0

226

A modification of the immune response could be responsible for the antiviral and anticancer activity of saponins. The mode of action of immunostimulants involves an increased phagocytosis by granulocytes and macrophages, an activation of T-helper cells and a stimulation of cell division and transformation in lymphocytes. Astragalus saponin I (astragaloside IV) isolated from A. membranaceus, when given subcutaneously to mice, increased phagocytosis, bactericidal activity, and acid phosphatase activity by peritoneal macrophages [161]. The natural killer (NK) activity of human peripheral blood lymphocytes was suppressed by Astragalus saponins, especially Astragalus saponin I (astragaloside IV) and astraversianin XI at high concentrations (250 ^ig/ml), but was stimulated at concentrations 0.05-5 M.g/ml. [162]. 115 cases of leucopenia were treated with pure Astragalus preparation (PAP) at different doses. One group was treated with 15g of PAP twice a day and the other group with 5 g of PAP twice a day, for 8 weeks. A dose dependent significant increase in the count of white blood cells was observed [163]. The effect of Astragalus membranaceus on lymphocytes was studied. It stimulated the proliferation of murine spleen cells in vitro. [164]. The efficacy of Astragalus membranaceus oral liquor combined with routine therapy on T-lymphocyte subset of peripheral blood in viral myocarditis patients has been reported [165].

CH300C O ^ HO'

Ri = CHzCHa Ri = H

- 22

^

^

./•..CH.OH

HO OR

+22 H

(Astragalus tomentosus Lam) R=

Li^^I^I^/ ^O-'^^'^Z^^ "° 6H Fig. (15). Lymphocyte transformation (mouse). Numbers represent proliferation increase (%) at 0.1 ^g/ml.

Azukisaponin li -2 {Astragalus trlgor)u$DC) AzuWsaponln V +3 {Astragalus trigonus DC)

227

The immunological function of Astragalus membranaceus hairy roots was found to be comparable to that of the dry roots. The content of crude saponins and astragaloside IV in the hairy roots were 5.81 and 0.14%, respectively [166]. Recently, lymphocyte stimulation tests were performed in order to evaluate the immunomodulatory properties of Astragalus saponins. Lymphocyte proliferation or transformation is a process whereby de novo DNA synthesis takes place in response to a mitogen or any other appreciable stimulator (concanavallin A). Astraversianins II, and X, astragalosides I, II, IV and VI and cyclocantosides E and G isolated from A. melanophrurius were able to stimulate mouse lymphocyte proliferation in the concentration range of 0.01-10 ^ig/ml. At higher concentration, inhibition of thymidine incorporation was observed [72]. In the same assay, a number of cycloartane and oleanane saponins isolated from Egyptian Astragalus were tested. As shown in the Figures (13-15), 20(/?),24(5) epoxycycloartane (cycloastragenol), 20(5),24(/?)epoxycycloartane (cyclogalegenin) and A^"* cycloartane saponins gave higher stimulation (at the same concentration) than tetranorcycloartane and oleanane saponins. (Verotta, L. et al unpublished results). Cycloartane saponins seemingly contribute to the immunomodulatory properties of Astragalus^ in fact, curculigosaponin G from Curculigo orchioides was reported to significantly promote the proliferation of spleen lymphocytes in mice compared to controls, without a marked influence on antibody formation [167]. HOCHz R=

HO HO Ho\-.---^-\/

cH-jr^O''

HO^^

O"

OH

Curculigosaponin G

ACKNOWLEDGEMENTS Work supported by Ministero deir University e della Ricerca Scientifica e Tecnologica of Italy.

228

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. Allrightsreserved.

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LABDANE -TYPE DITERPENES: CHEMISTRY AND BIOLOGICAL ACTIVITY COSTAS DEMETZOS^ and KONSTANTINOS S. DIMAS' ^School of Pharmacy, Department of Pharmacognosy, Panepistimiopolis Zografou 15771, University of Athens, Athens Greece ^International Institute of Anticancer Res,, Kapandriti, 19014, Attiki, Greece ABSTRACT: The terpenoids are a class of natural products with biological activity. The number of isoprene units from which they are biogenetically composed is used for their classification. The diterpenes are another group from which several compounds with high biological activity have been produced. The most studied plant families are Asteraceae and Labiatae, as well as Conifers, which are the main sources of diterpenes. The lack of, or insufficient, chromatographic data (RI, Rt) as well as of reference compounds for most labdanes is the main reason for their absence from the chemical fingerprints of plant extracts. Also their use as chemotaxonomic markers within the species in which they occur is limited. The configuration at the C-13 carbon atom has been investigated, especially in the case of manoyl oxide, as well as the various isomers, the ratio of which determines antimicrobial activity. A variety of biological activities have been encountered in labdane diterpenes such as antibacterial, antifungal, antiprotozoal, enzyme inducing, anti-inflammatory activities and modulation of immune cell functions. More recent studies have shown that labdane exhibits significant cytotoxic and cytostatic effects against leukemic cell lines of human origin and interferes with the biochemical pathways of apoptosis and the cell cycle phases, as well as with the expression of several protooncogenes such as c-myc and bcl-l. This report underlines the role of these compounds, not only as tools for the study of the biochemistry and regulation of biochemical and metabolic pathways of mammalian cellular systems, but also as potential pharmacological agents in the fight against diseases such as cancer and heart disorders.

1. SECONDARY METABOLITES OF PLANTS The processes generating plant compounds have been separated into primary and secondary metabolism. Primary metabolism produces the basic products for the life of the plant like carbohydrates, amino acids, fatty acids, polysaccharides, proteins, lipids, RNA and DNA. The primary metabolites are produced in relatively large quantities and their distribution is universal. On the contrary, the secondary metabolites are

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not as common as they may exist only in a single plant species or be characteristic of specific groups of plants (i.e, genus) [1]. However, they are recognised as vital for the life of the plant even if their production is limited. This view is supported by the fact that secondary metabolites create a defence mechanism against bacterial, viral and fungal attacks analogous to the immune system of animals [1, 2, 3]. Additionally, they are linked to hormones that are responsible for the growth of the plants or the healing of their wounds. Secondary metabolites may also be found in animals, but 80% of all such metabolites known are of plant origin. An explanation for this wealth of plant-originated secondary metabolites may be the fact that plants are rooted and immobile. Hence they do not have the ability, vital to animals, to move away from danger. Plants do not respond to the environment in the same way as animals do. Nevertheless, they are exposed to weather conditions, soil factors, gradual environmental pollution, herbivorous animals or symbiotic organisms and other competing plants [3]. Plants are significant to the diet of humans and animals since they provide most of the essential nutrients and vitamins. Vitamins C (ascorbic acid), E (a-tocopherol) and K (phylloquinone) are biosynthesized by plants, while P-carotene, the precursor of vitamin A and ergosterol, the precursor of vitamin D, are also secondary plant metabolites. These metabolites are used in folk medicine and for industrial purposes, as raw materials for pharmaceutical and other products [3]. On the other hand, plants may produce substances, which are toxic and/or irritant to man. The classification of plants is primarily based on the similarities and differences that are displayed by their morphological and anatomical characteristics. In some instances this does not suffice since the morphological differences may not be genetically defined but have been caused by local bio-climatic factors. Nevertheless is apparent that secondary metabolites can contribute to the taxonomy of plants and their systematic evolution. There are many examples of cases where the morphological features are not clear and secondary metabolites serve to clarify the morphological classification {e.g. classification of the tribes of the family Asteraceae). It has also been proved to be significant to use all the secondary metabolites for the above purpose and not only one of their chemical groups [4].

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2 TERPENOIDS The terpenoids are secondary metabolites that are found in essential oils, resins, tissues of higher plants and micro-organisms, whilst recently some have also been located in liverworts [5,6]. The terpenoids are formed from linear arrangements of isoprene units. Fig. (1), which are derived from acetate metabolism through mevalonic acid (MVA). This pathway was found to be common to the whole range of natural terpenoid derivatives

/v Isoprene unit

Isoprene

Fig. (1). Isoprene units

[7]. It is useful to divide terpenoids into classes according to the number of C5 isoprene units they contain, each class being derived from a primary metabolite precursor. They are classified as hemiterpenes (C-5); monoterpenes (C-10); sesquiterpenes (C-15); diterpenes (C-20); sesteterpenes (C-25); triterpenes (C-30); and tetraterpenes (C-40). Higher polymers are found in materials such as rubber. Geranyl pyrophosphate is the primary metabolite from which monoterpenes are derived. Farnesyl pyrophosphate gives rise to the sesquiterpenes and through its conversion to squalene, to the triterpenes and steroids (C 18-30), while geranylgeranyl pyrophosphate is the primary metabolite precursor of the diterpenes and carotenoids (C-40) [4], Fig. (2). The terpenoids usually play a role in the growth or the defence of the organism that contains them. That is why some terpenoids are toxic, irritant or allergenic and some are repellent smelling (small terpenes)

238 Irregular monoterpenes

Hemiterpenes

Diterpenes

t

Monoterpenes

Carotenoids

Phytosterols

Steroids

Fig. (2). Primary metabolites precursor in the biogenesis of various terpenoids.

whilst some taste bitter. Nevertheless, they also play a primary role in the growth of the plants, e.g the growth hormone giberelline belongs to the terpenoid group [3]. Their significance in ecological biochemistry and pharmacology has been proven by a number of studies conceming their pharmaceutical or healing qualities in vitro and in vivo. Examples of these qualities are anaesthetic, analgesic, anthelmintic, antiepileptic, antiinflammatory, antirheumatic, antitumour, diuretic, expectorant, hypotensive, insecticidal, organoleptic (odour, taste), spasmolytic, toxic and purgative [8]. The terpenoids may also be used for the identification of the various taxa [9]. They were initially used extensively as taxonomic markers of gymnosperms, mainly due to their abundance in the leaves of conifers. In contrast, the terpenoids of angiosperms have been poorly researched, possibly due to their irregular distribution in such families e.g, in a survey of 34 species of Plectranthus (Labiatae) [10], 18 were found to have excellent oil profiles, with up to 32 components, but the remainder of the species failed to give any leaf volatiles. Compared with other secondary

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constituents, terpenoids tend to show more intraspecific variation. Different classes of terpenoids have been detected in individual species of almost every plant genus that has been studied extensively. Some variations occur at population level. Such differences in pattern are often correlated with geographical or ecological factors, so that the results of such surveys have been important in extending our knowledge of plant population structure. From the taxonomic point of view, the terpenoids have been mainly useful as an aid in: 1. Defining the species; 2. Detecting hybridisation in natural populations, which is a universal phenomenon in conifers; 3. Confirming the presence of geographical races; 4. Confirming generic and tribal limits. The taxonomy of species is important, because it puts the ecosystem in an order, and it also provides information about species and life evolution [3]. Generally terpenoids exhibit a variety of chemical structure and complexity. The elaboration of Gas Liquid Chromatography (GLC) method has made the detection of plant volatiles such as terpenoids easier, offering both qualitative and quantitative data. 3. DITERPENES The diterpenes (C-20) are derived from geranyllinalyl pyrophosphate or its C-13 allylic isomer, geranyl-geranyl pyrophosphate (GGPP). After protonation of GGPP, a cyclization process can be initiated, yielding copalyl PP. An alternative pathway leads to labdadienyl PP, which is the enantiomeric product. Fig. (3),[11]. These are usually found in plants as mixtures with other related compounds. They occur in nature as both normal and antipodal stereochemical series. Fig. (4) shows the general precursor of cyclic diterpenes, le geranyl geraniol (1), as well as the main diterpene skeletons classified by Rowe et al [12]. Ponsinet [13] has noted that the transformation of the water soluble geranyllinalyl pyrophosphate. Fig. (3), requires a set of enzymes different from those, which bring about the cyclizations of the water insoluble hydrocarbon, squalene, into the various triterpenes. This may explain why diterpenes and triterpenes rarely occur together in the same plant tissues.

240

a

CH3

—^''

f*7« C

5

Geranyllinalyl pyrophosphate R=pyrophosphate

t'•T; OPP

OPP Copalyl PP

GGPP

OPP

OPP

OPP H ^

GGPP

labdadienyl PP

OPP

Fig. (3). Biosynthetic pathways to the bicyclic diterpenes.

Usually diterpenes are found in plant resins and latexes where they are involved in their sticky texture. Resins often exude from the wounds of a

241

plant, playing an antimicrobial role. Nevertheless, some leaves, stems or woods of plants are covered with resin and latex despite the absence of

gibberellane

kaurane

Fig. (4). Structures of important classes of diterpenes.

wounds and are hostile to animal and predators; e.g. leaf-cutting ants avoid attacking such plants [10].

242

The resin produced by conifers is rich in diterpenes. There is growing evidence that non-volatile diterpenes (e.g. kaurenic acid) inhibit the feeding of insects and hinder the growth of their larvae. This is achieved through the disturbance of the hormonal processes, especially of ecdysone synthesis [3]. On the other hand an adapted sawfly {Neodiprion seltifer) stores the chemical compound of the resin whilst still at the larval stage and uses it for its own chemical defence against birds. This diet of resin reduces the growth rate but the alternative of not feeding it from the tree would leave the insect without any chemical defence. Most other insects, which utilise diterpenes for defence, appear to make their own toxins from simple starting materials [3]. Toxicity to animals is especially associated with certain diterpene types, like grayanotoxin I, which occurs in some genera of the family Ericaceae [10]. The leaves and flowers oi Rhododendron sp. contain toxic diterpenes, able to contaminate the honey produced by bees [3,10]. Many Euphorbiaceae and Thymelaceae also contain toxic diterpenes, which have the additional property of being highly irritant and hence cocarcinogenic. The diterpene phorbol (1), which belongs to the tigliane skeleton. Fig. (5), was isolated after hydrolysis of the seed oil (croton oil) of Croton tiglium in 1931. Later it was also found in other plant species of the Euphorbiaceae family. The oil is still used in experimental cancer biology. Compound 2, Fig. (5), has been characterized as phorbol -12myristate-13-acetate (PMA) (2) and is the most potent tumor-promoting constituent of croton oil [14]. From the Euphorbiaceae family, various phorbol esters have been isolated from individual species of four genera: Croton, Sapium, Euphorbia and Ostodes. The natural occurring esters can be divided into two general types: the C-13 monoesters and the C-13, 20 diesters. The length of the C-13 ester chain varies from two - to sixteen carbons. Of interest is prostatin (3), Fig. (5), which has shown significant cytoprotective properties in human lymphocytic cells infected with the HIV - I virus [15]. A related family of compounds is the daphnane diterpenes. Resiniferatoxin (4), Fig. (5), is known for its potency as a proinflammatory agent [16]. Derivatives of compound 4 isolated from both the Euphorbiaceae and Thymelaceae families are well known for their antileukemic activity [17]. Ingenanes - Fig. (5) - are tetracyclic polyol esters isolated from the Euphorbia species. The parent compound is the polyol ingenol (5), derivatives of which have shown selective cytotoxic

243

activities against several human cancer cell lines derived from leukemia, non-small-cell lung cancer, colon cancer, melanoma and renal cancer [18]. The phorbol esters have proved to be most potent tumor promoters for two-stage mouse skin carcinogenesis [19]. The major phorbol ester receptor has been identified as protein kinase C [20]. Computer modeling of phorbol esters and other classes of tumor promoters has shown an interesting similarity in the relative positions of certain heteroatoms and hydrophobic groups [21].

daphnane

tigliane

ORi 0R2

O

OH

1: 2:

R,=R2=H Ri=CO(CH2)l2CH3 R2= COCH 3

PhCH2

O2CCH2 -

Fig. (5). Diterpenes from Euphorbiaceae and Thymelaceae families.

ingenane

244

Taxol (1), Fig (6), is another important diterpene with anticancer properties. It was isolated in 1971 from Tcaus brevifolia Nutt. (Taxaceae), which is a slow growing shrub/tree, found in the forests of N.W Canada and USA. Taxol is one of over one hundred taxanes, which have been

Fig. (6). Structures of Taxol and taxane derivatives

characterized from various Taxus species. It belongs to a group of compounds with a four-membered oxetane ring and an ester side-chain, both of which are essential for anticancer activity. Taxol was isolated

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from the bark off. brevifolia in low amounts (c. 0.01-0.02 %), but a content of up to 0.033 % was found in samples from leaves and twigs [22]. Taxol is derived from GGPP via cyclization reactions, employing the same mechanistic principles as in mono- and sesquiterpenes. Baccatin III (2), another taxane derivative, has been found (up to 0.2%) in the bark. A number of other taxane derivatives were isolated and characterized as 10 - deacetyltaxol, 10 - deacetylbaccatin III (3), cephalomannine (4) and 10 - deacetylcephalomannine (5) while some of them have been microbiologically transformed into taxol, Fig. (6). Cell cultures of T. baccata as well as microorganisms and enzymes, also offer excellent potential for the production of structurally related compounds and thus improve the yields of e.g 10-deacetylbaccatin III [23] in crude extracts. Taxol (Paclitaxel) is an important new anticancer drug for the treatment of ovarian cancer and is currently in clinical trials against metastatic breast cancers. It is also a potential drug for lung, head and neck cancer. Docetaxel (Taxotere) (6), (Fig. (6), is an analogue of taxol, which has been prepared from 10-deacetylbaccatin III, by semisynthesis. This compound has better water solubility than taxol and is being tested clinically against ovarian and breast cancer. The mechanism of action of Taxol involves the stabilization of ordinary cytoplasmic microtubules and the formation of abnormal bundles of microtubules [24, 25]. Various other biologically active diterpenes have been isolated from plants and are used by man for the treatment of a variety of diseases. The bark of Pinus strobus (Pinaceae), rich in diterpenes, among them manoyl oxide, is used in anticough syrups, whilst traditionally it has been used for the treatment of coughs, colds, congestion, injury, rheumatism and swelling by Indian Americans [26]. The antiinflammatory and antimicrobial activity of various diterpenes has been reported [27], as well as anti-fungal activity [28] and remarkable cytotoxic activity for some of them [27,29]. The ability to synthesize diterpenes is universal to plants, since phytol, the acyclic parent compound of the series, is present in ester attachment in the chlorophyll molecule and hence occurs in all green plants. Gibberellic acid is also widespread in the plant kingdom as a growth hormone. Besides phytol and gibberellic acid, the remaining diterpenoids are very restricted in occurrence and usually occur within one or only a few plant

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families. The families in which the diterpenoids are regular constituents include among the gymnosperms, Pinaceae, Cupressaceae and Podocarpaceae and among the angiospemis, Ericaceae, Euphorbiaceae, Labiatae, Leguminosae and Thymelaceae. There are also records of diterpenes in individual species in many other angiosperm families [10]. This may be the reason why taxonomists use diterpenes as chemotaxonomic markers [30]. The diterpenes have been extensively used as taxonomic tools for the classification of various taxa mostly among the gymnosperms. The study of their diterpene content promoted the taxonomic reassessment of Tetraclinis articulata and Chamaecyparis obtusa (Cupressaceae) [10]. However the absolute stereochemistry must be examined carefully because the same species have been found to contain one or other enantiomeric series of diterpene. For example, the leaves of Podocarpus macrophyllus contain (-) kaurene and those of P. ferrugineus contain its enantiomer [31]. The relative configuration of diterpenes at carbon atoms 5, 8, 9 and 10 is derived from a concerted trans-anti-trans addition to the double bonds of the "all trans" acyclic precursor, geranyllinalyl pyrophosphate, folded in the most stable all chair conformation, Fig. (3). The two decalin rings have a trans configuration because this is the most stable conformation since only 2.7 kcal/mole are required, as opposed to the cis configuration, which requires 6-8.8 kcal/mole. It has been established though, that the trans is the active configuration since phorbol esters, as well as ingenane and daphnane derivatives are biologically active as trans isomers, while the cis conformation is inactive. Within a given plant source, the configuration at C-13 in the diterpenes varies with skeleton type. In fact, diterpenes of the same skeleton may have a different configuration at C-13. Sclareol and 13-£?/7/-sclareol from Salvia sclarea are examples of this phenomenon [32]. The same phenomenon has been observed in manoyl oxide isolated from several parts of Cistus creticus. The configuration at C-13 has been found to be different in different parts of the plant. The antibacterial activity was also found to be related to the C-13 configuration [33]. 4. LABDANE DITERPENES A very large number of diterpenoids possessing a labdane skeleton (1), Fig. (7), occur in nature [34]. The interest in studing labdanes is

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r;6?

c?5^ 7 R=OH 8 R=OAc

Fig. (7). Labdane diterpenes

heightened due to the wide range of biological activities of these compounds [35]. They comprise a decalin system and a C-6 ring, which

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may be open or closed with an oxygen atom, as in manoyl oxide and its derivatives. In the ring system of labdanes the substituents below the plane of the ring are drawn with a dashed bond indicating a-configuration, while when the substituents are drawn above the ring with a wedge bond, the p-configuration is designated. Usually, a-hydrogen atoms found at ring junctions are omitted for clarity, if they have the natural configuration. The antipodal of the normal series is indicated by the prefix ent' before the complete name of the compound. Labdane diterpenes have five chiral carbon atoms and they occur in nature in the two enantiomeric series. It has been observed that, in a single plant species, both enantiomeric labdanes co-occur [36,37]. The normal and the enantiomeric carbocation are produced from the achiral geranylgeranyl pyrophosphate (1), Fig. (8). The co-occurence of normal labdadienoic acid and antipodal

OPP

H ^ ^

OPP OPP

Fig. (8). The biosynthetic precursors of normal and enantiomeric labdanes

dihydroeperutic acid in the same plant was reported in 1967 [38]. A plant biosynthesizes the normal as well as the enantiomeric labdane - type diterpenes via 2 or via 3 intermediates, respectively. Fig. (8). From the leaves of Mimosa hostilis (Leguminoseae) both normal and antipodal labdanes have been isolated and their absolute configuration confirmed by x-ray crystallographic analysis [39]. The optical rotation of labdanes has been correlated to their structures and a total of 143 labdanes have been examined. The conclusion of this study was that this correlationship could provide information about the structure, as well as the stereochemistry at C-13. This study can also be used as a tool in order to correlate the optical

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rotation and the structure for monoterpenoids, sesquiterpenoids and triterpenoids, which have comparable structures [40]. 4.1 THE ORIGIN OF LABDANES Labdanes have been isolated from several plant families, such as Asteraceae, Labiateae, Cistaceae, Pinaceae, Cupressaceae, Taxodiaceae, Acanthaceae, Annonaceae, Caprifoliaceae, Solanaceae, Apocynaceae, Verbenaceae and Zingiberaceae. In addition they have been isolated from marine algae of the genus Laurence, from Taonia atomaria and from the red alga Chondria tenuissima, from which has been isolated the bromoditerpene entA3-epi-concirmdiol, the structure of which was determined by x-ray crystallography [41]. The conifers are an important source of diterpenoids. Several labdanes have been detected in the neutral fraction of the oleoresin of Araucaria excelsa, including manool as well as nor-labdanes [42]. Some neutral diterpenoids, such as eA7^1abd-8, 13(E)-dien-15-ol and its acetate, were obtained after extraction of the resin of Araucaria bidwillii [43], while the normal series of the above labdanes have been isolated from Cistus creticus subsp. creticus [44]. From Pinus sylvestris, 3P-hydroxybiformene has been obtained, while from the needles of the American red wood pine {Pinus resinosa), 8,13-epoxy-labd-14-en-19-oic acid, was isolated. In Pinus nigra, labdane acids have been found to be the major acid components of its needles [45,46]. The occurrence of diterpenoids including labdanes in conifers has been published as a review of the chemistry of the order Pinales [47]. The genus Sideritis (Labiatae) has been an important source of novel diterpenoids, thus extensive studies have been done on this genus and a large number of labdanes have been isolated and identified in the past few years [48]. From Sideritis arborescens, andalusol was isolated and x-ray analysis was used to determine its absolute stereochemistry [49]. From another Sideritis species, i.e S. gomerae, gomeraldehyde (ent-S, 13,epoxylabd-15-al) and gomeric acid as well as their 13-epimers were isolated [50]. The chemical investigation of the aerial parts of S. nutans afforded, in addition to the known labdanes also isolated from S. gomeae, some new entAabdanQ oxides, such as gomerol, 13-epi gomerol, sidnutol and 3a-hydroxy-gomeric acid [51]. A derivative of manoyl oxide, namely

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borjatriol, has been isolated from S. mugronensis, while from S. arborescens, 6-deoxyandalusol and barbatol have been obtained. Borjatriol has been synthesized, using the diterpene larixol as the starting material [52]. The structures of borjatriol and of barbatol were determined considering their interrelationship with manoyl oxide [53, 54,55]. A series of andalusol derivatives have been obtained from S. foetens [56], while from the hexane extract of S, javalambrensis, eA7f-16-hydroxy-13-epimanoyl oxide was isolated [57]. From Sideritis canariensis and S. varoi (Labiateae), ribenol (^«f-3ahydroxy-13-e/7/-manoyl oxide) has been obtained and it was converted into 13-e/7/-manoyl oxide [58,59,60]. Ribenol was converted to its thiomidazolide (45% yield), by treatment with N, N'thiocarbonyldiimidazole [61]. This new ^-em/'-synthetic compound has been extensively studied against human leukemic cell lines [62]. Ribenol and its acetyl derivative have been isolated from the fruits of C. creticus subsp. creticus, in addition to other labdanes, and their percentage content in the extracts and in the essential oils of the leaves and fruits of the plant have been compared to that of C. creticus subsp. eriocephalus for chemotaxonomic purposes [33]. Ribenol and its acetyl derivative have first been identified as volatiles in the essential oil of the resin 'Ladano' and their chromatographic data using Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS) were obtained [63]. In the same paper, a comparative study was carried out concerning the antistaphylococcal activity of these compounds and of manoyl oxide as a mixture of isomers. The results showed that ribenol had a better Minimum Inhibitory Concentration (MIC = 0.1 mg/ml) in contrast to the other compounds tested. From the plants Cistus ladaniferus, C. laurifolius, C. palinhae, C. clusii, C, symphytifolius and C. libanotis (Cistaceae) several labdanes have been obtained [64-71], while from C hirsutus (Cistaceae), 6p-acetoxylabd-8 (17) en-15-oic acid has been isolated and its structure determined considering its interrelationship with 6-oxocativic acid [43]. Isolation of several labdanes has also been reported from the genus Halimium of the Cistaceae family, their structures being determined by spectroscopic methods and by chemical transformations [72,73,74]. In the family of Asteraceae the ent- series of labdanes have been identified as the common components [75]. The following species of Asteraceae have yielded labdanes: Leyssera guaphaloides, Brickellia

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lemmonii, Baccharis petiolata, B. pedunculata, Aristeguetia buddleaefolia, Blepharisperum zanguebaricum, Stevia seleriana, Happlopappus pulchellus, H. arbutoides, H. parvifolius [76-82], Corymbium villosum [83], Chrysocephalum ambiguum [84] and Waitzia acuminata [85]. From Haplopappum parviflorus, twenty-one labdanes have been isolated which belong to seco-, nor-, and normal series. Sclareol (2), Fig.(7), however, was not isolated and as suggested in that report, it could be the common precursor of the isolated compounds [86]. Ent- labdanes have been isolated from Gutierrezia grandis, from Baccharis scoparia, from Oxylobus arbutifolius and from the Indian plant Phlogacanthus thyrsiflorus. The latter is used to treat bronchial conditions, while a similar action has also been reported for Andrographis paniculata (Acanthaceae) [87,88,89]. (-) Ozic acid has been isolated from the wild sunflower Helianthus occidentalis (Asteraceae) as cis- and transisomers [90]. In Austroeupatorium chaparense (Asteraceae) several furanoid 7p-acetoxylabdanes have been found [91], while other furan labdanes have been isolated from Galeopsis angustifolia (Labiatae) [92,93] and from Xanthocephalum linearifolium (Asteraceae) [94]. Dimeric labdanes have been obtained from the cones of Cunnighamia lanceolata [95] while dimeric labdanes, esters of malonic acid, have been previously obtained from the resin Ladano [96,97]. A number of labdane glycosides have been isolated and identified from the family of the Asteraceae. Gutierrezia sphaerocephala has given several glycosides, while from Aster spathulifolius a number of labdane 13-O-glycosides have been obtained [98,99]. Ent- labdanes of the same family have been isolated from Gutierrezia spathulata, from Haplopappus species and from Grindella species [98]. Glycosides derived from labdan-8(17), 13-dien3b,15,18-triol have been obtained from Rubus foliolosus which is used in Chinese traditional medicine [100]. From the root stalks of Gleichenia japonica some labdane glycosides have been obtained and they were found to be growth inhibitors to other plants [101]. From Viburnum suspensum (Caprifoliaceae) the gomojosides A-J have been obtained [102], while mitrariosides A-D have been obtained from Mitraria coccinea (Gesneriaceae) [103]. Gaudichaudioside F has been isolated as a bitter-tasting arabinoside of Baccharis gaudichaudiana. The phlomosides, a series of labdane glycosides isolated from the roots of the Tibetan plant Phlomis medicinalis (Labiateae), have also been used in folk medicine

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[101]. The aerial parts of Conyza steudellii (Asteraceae) have given new labdane xylosides, while from the leaves of Conyza trihecatactis, a xylopyranoside of 13-epi-sclareol has been isolated among other labdanes [104,105]. Havardic acids A-F (as methyl esters), have been isolated from another genus of the Asteraceae family, i.e Grindelia havardii [106]. 11oxo-manoyl oxide derivatives and coleosol, which is also a manoyl oxide derivative, have been obtained from Coleus forskohlii (Labiateae) [107,108] while from another plant of the Labiatae family, Roylea calycina a tumor inhibitory compound, namely precalyone, as well as calyone have been isolated [109]. From the plant Agathis robusta a group of labdane isomers at C-13 have been isolated and related to 13-e/?/-manool after interconvertion using lithium in diaminoethane [110]. The Hymenaea species, le H. ablongifolia and H. parvifolia, have been studied and enantiopinifolic acid as well as guamaic acid have been isolated, while enantio-lS-e/?/labdanolic acid has been isolated from Trachylobium verrucosum [111]. One of the well known labdanes is sclareol (syn. Labd-14-ene-8, 13diol) (2), Fig. (7), a ditertiary alcohol widely distributed in nature. It was first isolated from clary sage oil {Salvia sclarea, Labiatae) [112]. This oil is used in soaps, as a fragrance in cream and lotions, in food and beverages as a flavoring component [113] as well as in folk medicine [26,114]. Sclareol is an epimeric mixture at C-13 (ratio 9:1), where predominates the 13 R-epimer [115]. GC-MS analysis of the n-hexane extract of the liverwort Pleurozia acinosa (Jungermanniaceae), revealed a large peak of the compound which then was isolated and identified as 8,13-c//-ep/-sclareol. The spectral data of this compound was not shown to be close to those obtained from sclareol, from 13-ep/-sclareol, from ent-^epi -sclareol or from ent- 8,9 -di-epi- sclareol [116]. The percentage content of sclareol is up to 2% in clary sage oil, while in the concrete its concentration is up to 70% [114]. The Flavor and Extract Manufactures Association of the U.S.A [117] generally recognize Sclareol as a safe material. The use of clary sage oil in fragrances in the U.S.A amounts to about 10,000 lb/year. Sclareol is used on a commercial scale for preparing a series of Ambra odorants [118]. Cistus ladaniferus [119] is referred to as a plant having an amber odour and has been used as a perfumery raw material. Another plant which also belongs to the genus Cistus, namely Cistus creticus subsp. creticus (Cistaceae) contains sclareol in its leaves

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[44] and resin named Ladano [63]. Ambrox (3), Fig. (7), is reported as a key component of ambergris and it well known as an important ingredient for perfumery [119]. Ambrox is a degradation compound of sclareol and has been isolated from the oil of clary sage (Salvia sclarea), while it has also been shown to be present in Labdanum oil from C. ladaniferus (Cistaceae), as well as in cypress oil {Cupressus sempervirens) [118]. In the search for products for perfumery with ambergris odor, sclareol and manoyl oxide (4), Fig. (7), have been investigated and an oxidation process leading to the production of acetals has been examined and evaluated. The relationship between the structure of manoyl oxide and ambergris - type odour has also been examined and manoyl oxide was converted to its ketone or ethers [120]. 4.2, MICROBIAL TRANSFORMATION OF LABDANES Sclareol as well as several labdanes have been used as substrates of microbial cultures in order to produce compounds with higher potency and efficiency against various diseases. Microbial transformation of sclareol with Mucor plumbeus gave a mixture of triols from which the labd-14-en-3p, 8a, ISp-triol yielded in high amounts [121]. Sclareol has been referred to as a starting material to obtain interesting biologically active drimanes [122]. Sclareol possess antimicrobial properties against Gram positive and Gram negative bacteria [123,124]. Its \2-epi isomer has also shown an MIC of 250 |ig/ml, against Staphyloccoccus aureus, while sclareol was found to be more active against Staphylococci [63]. Manool (5), Fig. (7), which has also been obtained from Salvia sclarea as sclareol [125], is also a raw material for the commercial preparation of amber-type perfumes. In a biotransformation process that was carried out using Mucor plumbens, manoyl oxide, sclareolide and a 7a-hydroxy derivative of manool were obtained [126]. The oxidation of manool in order to produce ambergris-type perfumes has been examined and several known derivatives have been obtained [127]. The microbial transformation of entA3-epi'VCidinoy\ oxide by Rhizopus nigricans has been utilized to produce biologically active derivatives [128]. Incubation of eA7/-19-hydroxy 13-^j9/-manoyl oxide as well as e^/-3p-hydroxy IS-epimanoyl oxide with the fungus Gibberelafujikuroi rendered derivatives of

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manoyl oxide, the structure of which were determined using spectroscopic methods [129]. Ent -3p-hydroxy 13-£?p/-manoyl oxide (7) (ribenol), Fig. (7), has been used as starting material for a biotransformation process by Curvularia lunata. The in vitro micropropagation of the Sideritis foetens species has been described and several ^«/-manoyl oxide derivatives, as well as other labdanes, have been isolated from micropropagated plants. Recently, biotransformation by Curvularia lunata of some e«^13-e:/>/-manoyl oxides functionalized at C-3 or C-3 and C-12 produced derivatives, which inhibited the growth of the pathogenic protozoa, Leishmania donovani [130].

4.3. THE STEREOCHEMISTRY AT C-13 OF LABDANES The configuration at C-13 of the diterpenes has been a problem for many years. NMR spectroscopy using chiral shift reagents has been suggested as a method to differentiate manool from 13-ep/-manool [131]. Most of the diterpenes with a saturated side chain were present as mixtures of C13 epimers. Small differences in chemical shifts in the ^H and *^C - NMR spectra did not allow assignement of the stereochemistry at C-13 [132]. The absolute configurations of sclareol (2) and manool (5), Fig. (7), at C-13 have been determined [133]. As Hanson reported [134], the absolute stereochemistry assigned to some labdanes should be reexamined due to the enantiomers of labdanes. The biosynthetic pathways of sclareol and manool start from a geranyllinalool - type skeleton which cyclizes in a similar fashion as that described for cativic acid [135] and, via the intermediate 6, Fig. (7), sclareol is formed by hydration of 6, or manool by the loss of a proton [112,136]. Manoyl oxide (4), Fig. (7), has been isolated as a pure compound or identified via analytical techniques, in several plant species [33,44,63,137, 138,139]. Ohloff has shown that manoyl oxide can be prepared from sclareol [140]. The physical and chromatographic data of the synthetic and of the natural manoyl oxides have been compared and discussed [141]. Hodges and Reed have substantially contributed to the knowledge of the stereochemistry of manoyl oxide [141]. Manoyl oxide occurs in nature in both normal and antipodal configurations [58]. The 13-6?p/-manoyl oxide has been isolated after

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extraction of jack pine bark {Pinus banksiana Lamb.) and from Haplopappus parvifolius (Asteraceae) [142] as normal (a^ positive), while its antipode (a^ negative), also referred to as olearyl oxide, has been reported to occur in Olearia paniculata, the fungus Gibberella fujikuroi and the herbs Beyeria sp. [139]. The synthesis of 13-e/7/-manoyl oxide from sclareol has been reported [143]. Its derivatives have been isolated from natural sources, during microbial transformation [144-146] or from in vitro micropropagation of plants [147]. Synthetic derivatives of manoyl oxides have been synthesized for pharmacological purposes [148]. The circular dichroism curves of manoyl oxide and of its 13-epimers have been examined [149]. In studies related to antiinflammatory activity, [56] the \l>-epi isomer derivatives of manoyl oxide have been reported to be are active; however manoyl oxide and its isomers have not yet been studied. Recent studies on the two subspecies of Cistus creticus (Cistaceae) {i.e C.creticus subsp. creticus and C creticus subsp. eriocephalus) concerning manoyl oxides, have proven that the percentage content of the isomers of manoyl oxide varies, depending on the part of the plant and the polarity of the solvent which was used [33]. These results correlate with the antimicrobial activity and the percentage content of isomers of manoyl oxide in the mixture. No other pharmacological data are available up to now concerning the percentage content of the isomers of manoyl oxide except those for their antimicrobial activity. Hence, in order to elucidate the structures and the percentage content in the mixture of manoyl oxide isomers, an analytical approach like GC - MS was selected as a simple and rapid methodology [33]. The analysis by GC-MS of manoyl oxide as a mixture of isomers as well as the study of extracts and essential oils of the two C creticus subspecies, revealed the existence of more than one isomer which was difficult to separate and distinguish. The results showed different peaks which are indicative of the presence of isomers with different chromatographic data (RI: Retention Indices and Rt: Retention time). The fragmentation of manoyl oxide at the chiral center C-13 and the speed of the removal of CH3-I6 showed that there was different intensity for the peak with m/z 275 and 257 for the isomers [33]. Manoyl oxide is referred, to without any clarification, for the presence of isomers but never for their proportion [137,138]. This results in errors during the pharmacological evaluation of manoyl oxide and also in its use as a

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reference compound, irrespectively of its origin. In its ^H-NMR spectrum when manoyl oxide exists as a mixture of isomers, the aliphatic as well as the olefmic regions exhibited more than one initial signal [44]. The study of manoyl oxide derivatives i.e. 7 and 8 in. Fig (7), (i.e enthydroxy and ^w/-acetoxy-3p-manoyl oxides) isolated from Cistus creticus, by GC-MS resulted in only one peak indicative of the purity of the products [33]. From the ^H-NMR data it is clear that the 13-epz isomer was present in both derivatives [58,139]. The chromatographic data of the compounds 7 and 8 were recently published [33,63]. Hence, investigations have proven that, apart from the \l)-epi isomer, there are more isomers with varying intensities, which correspond to isomers that arise from the different configuration of C-8 chiral center [33]. This isomer showing a different configuration at C-8 has been isolated from the volatile leaf oil of Alaska (yellow) cedar and its structure has been confirmed using spectroscopic methods as well as chemical reactions [150]. The most important manoyl oxide derivative is forskolin (9), Fig. (7), (7p-acetoxy-8, 13-epoxy-la, 6p, 9a-trihydroxylabd-14-en-ll-one) [151153]. It belongs to the labdane series of diterpenes and was isolated from the Indian herb Coleus forskohlii fWilld.) Briq. (Labiatae). Since ancient times it has been used in Hindu and Ayurvedic traditional medicine [154]. The plant Coleus forskohlii (Willd.) Briq. has been extensively studied, and from its extracted roots a group of diterpenoids, with the basic skeleton of 11-oxo-manoyl oxide, have been isolated. The main compound, forskolin, presented remarkable chemical and biological properties [155]. Analogues of forskolin were then prepared by semisynthesis [156] or obtained by microbial transformations [157]. New analogues, more soluble than forskolin have shown activities comparable to and even higher than forskolin [158]. 4.4. LABDANES AS VOLATILE COMPOUNDS The lack of or the insufficience of chromatographic data (RI, Rt) and reference compounds for most of the labdanes [159], present basic problems in studies concerning the qualitative and quantitative analysis of plant extracts using GC or/and GC-MS analysis. Also their use as chemotaxonomic markers within species where they occur is limited. GC

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and GC-MS methods have been appUed to the detection and analysis of labdanes using capillary chromatographic columns, HP-5MS and CP-Wax [33]. Additionally, this analysis can be useful in finding out the fingerprints of extracts where labdanes exist, using GC or/and GC-MS, for standardizing plant extracts, which are used in phytotherapy. Anastasaki et aL, [33] have studied some labdanes isolated from Cistus creticus. The results obtained from GC and GC-MS analyses of these labdanes have contributed to a better understanting of the chemotaxonomic relationship between the two selected Cistus subspecies, le C. creticus subsp. creticus and C.creticus subsp. eriocephalus. [33]. 5. BIOLOGICAL ACTIVITIES OF LABDANES A variety of biological activities have been associated with labdane diterpenes including antibacterial, antifungal, antiprotozoal, enzyme induction, anti-inflammatory modulation of immune cell functions, as well as cytotoxic and cytostatic effects against human leukemic cell lines. The leaves of Cryptomeria japonica (Taxodiaceae) are traditionally used in Japan for the treatment of eczema. In Tibet the roots of Phlomis younghushbandii and of P, medicinalis (Labiateae) are used as an antifebrile and as a cough medicine. In East Africa the twigs of the shrub Premna oligotricha (Verbenaceae) are used as chewing sticks against the Gram positive bacteria responsible for dental caries {Streptococcus sp., Lactobacillus sp.), while the smoke formed by burning the plant is used to sterilise milk containers. On the island of Crete (Greece), Cistus creticus (Cistaceae) is used as a dermis malady, in arthritis and stomachache. Antifungal activity has also been shown by labdanes isolated from the seeds of Alpinia galanga and Aframomum daniellii (Zingiberaceae). Labdanes from Cistus creticus and from its resin 'Ladano', from Viburnum suspensum (Caprifoliaceae), Juniperus procera (Cupressaceae), Premna oligotricha (Verbenaceae) and from the sponge belonging to the genus Mycale, also exhibit strong antimicrobial activity. In 1998 the antiinflammatory activity of a product isolated from Cryptomeria japonica (Taxodiaceae) with labdane skeleton, was reported [35].

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5.1. ANTIBACTERIAL, ANTIFUNGAL AND ANTIPROTOZOAL EFFECTS Although an initial study by Soederberg et ai failed to show any activity against Staphylococcus aureus, subsequent studies have established significant antibacterial, antifungal and antiprotozoal activity for many labdane type diterpenes, especially for the labdanes of the manoyl oxide series [160]. In the study mentioned above, manoyl oxide acid and pinifolic acid, two resin acids isolated from Scots pine needles, were tested against 20 strains ofS. aureus using the disc diffusion method, but no significant activity was observed. Two other diterpenes of the manoyl oxide series, epigomeric acid and gomeric acid, tested by Darias et aL [27] against three Gram (+) bacteria {Bacilus subtilis. Micrococcus luteus and S. aureus) and two Gram (-) {Escherichia coli and Pseudomonas aeruginosa) exhibited a remarkable activity against the Gram (+) bacteria. As mentioned elsewhere in this text, the Cistacae is a family of plants from which a significant number of labdane diterpenes have been isolated. From the dried leaves of the Cistus incanus subsp creticus seven labdane type diterpenes have been isolated [44]. All the compounds were tested for antibacterial activity against S. aureus, S. epidermidis [Gram (+) bacteria] and P. aeruginosae, Enterobacter cloacae, Klebsiela pneumoniae and E, coli [Gram (-) bacteria] as well as for their antifungal activity against Candida albicans, Torulopsis glabrata and the opportunistic infectious fungus Saccharomyces cerevisiae [123]. These studies were carried out using the disc diffusion method, and with the exception of one diterpene, which was completely inactive, the other diterpenes showed significant activity against S. aureus, P. aeruginosae and K. pneumoniae. They exhibited zones of inhibition comparable to netilmicin, ceftazidine and cefriaxon, while they were more toxic than ampicillin. One of them {[(5R, 8R, 9R, 10R)-labdan-13 (E)-ene-8a, 15diol]} was also found to be active against C albicans [123]. Antibacterial activity against the Gram (+) bacteria S. aureus, S. epidermidis, B. subtilis, S. hominis and M luteus, and against C. albicans was also exhibited by the essential oil of the same subspecies, extracted either from the leaves or from the resin of the plant [63,161]. Analysis of the composition of these oils revealed that manoyl oxide isomers were the

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main compounds. However, no activity against the Gram (-) bacteria, (E. coli and P. aeruginosae), tested in the same studies was observed. In a subsequent study [162], it was reported that the essential oil obtained from the resin of another subspecies, Cistus creticus subsp. eriocephalus, where manoyl oxide isomers also predominated, exhibited significant antibacterial activity against four Gram (+) bacteria (S. aureus^ S. epidermidis, Str.faecilis, B. cereus and B, subtilis). Two Gram (-) bacteria {E. coli, P. aeruginosae) also tested in that study were found to be resistant. Furthermore the oil was active against C albicans. Manoyl oxide was also reported to be the main compound in the essential oil of Helichrysum rupestre [163], which exhibited remarkable antibacterial activity against S. aureus but moderate activity against the Gram (-) strains, P. aeruginosae, E. cloacae, K. pneumoniae and E. coli. Taking into account the above studies, it may be concluded that the manoyl oxide isomers exhibit major antibacterial activity, while a specificity of these labdane diterpenes against Gram (+) is also highlighted. Two other labdanes tested for antibacterial activity are sclareol and manool. They were reported by Ulubulen et aL to be active against S. aureus in a study performed using the disc diffusion method as well as the tube dilution test, exhibiting a minimum inhibiting concentration of 48.25 and 13.75 |ig/ml, respectively. These two compounds were, however completely inactive against C albicans and Proteus mirabilis [164]. Sclareol was tested and was found to exhibit antibacterial activity against S. aureus, P. aeruginosae and K, pneumoniae [123] while it also seemed to control well rust fungi on different kinds of bean [165]. A series of other labdane diterpenes isolated from various sources have also been reported to exhibit antimicrobial activity. Aulacocarpinolide and aulacocarpines A and B, labdane diterpenes isolated from the Cameroonian spice Aframonum aulacocarpos (Zingiberaceae), exhibited weak antibacterial activity against B. subtilis and fungus Mucur miehei [166]. Labdanes of the series of gomojosidae, isolated from the leaves of Viburnum suspensum (Caprifoliaceae) exhibited antibacterial activity against E.coli, Aeromonas salmonishida and B. subtillis [102]. Cryptotrienolic and isocupressic acid isolated from the bark of Juniperus procera were found to exhibit weak antibacterial activity when tested against B. subtillis, S. aureus, Str. durans, E. faecilis, and Mycobacterium

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itracellular [167]. Moderate antifungal activity was also attributed to labdane diterpenes from the seeds of Alpinia galanga (Zingiberaceae) [168] and the perennial herb Aframonum daniellii (Zingiberaceae) [169]. In addition labdane diterpenoids exhibiting antimicrobial activity have been isolated from aquatic environments. Mycaperoxides isolated from the Thai sponge [170] and a sulphated sesterterpene, hydroquinone halisulphate from the dark brown sponge (Halichondriidae) [171] were found to exhibit antibacterial and antifungal activity. Also a new furanoid labdane diterpene isolated from the ethanolic extract of Potomogeton nodosus (Potamogetonaceae) was reported to exhibit moderate inhibitory activity against Gram (-) and Gram (+) bacteria, such as B, subtillis, S. aureus, Str. faecilis, B. cereus, Shigella boydii, S. sonnei and S. shiga [172]. Some investigators have reported not only antibacterial and/or antifungal activity, but also a significant antiprotozoal activity. Ent 13epi'kQio manoyl oxides, as well as their derivatives produced through biotransformation from the fungus Curvularia lunata [144, 173], exhibited significant activity against the pathogenic protozoa Leishmania donovani. Biological activity against the same promastigote was also reported for another diterpene [(4S, 9R, lOR) methyl 18-carboxy-labda8, 13 (E)-diene-15-oate] isolated from the stem barks of Polyalthia macropoda (Annonaceae) [ 174]. 5.2. EFFECTS OF LABDANES ON MAMMALIAN

ENZYME

SYSTEMS 5.2.1 ADENYLATE CYCLASES The adenylate cyclases (AC) are a family of enzymes, which catalyze the synthesis of cyclic AMP (cAMP), from ATP. Cyclic AMP, a ubiquitous molecule in mammalian cells, plays a key role in controlling a vast number of biological processes, functioning as a major second messenger. The ACs are present in bacteria, where c-AMP plays a key role in the regulation of transcription in fungi, parasites and mammalian cells. The mammalians ACs (at least nine enzymes) are structurally unrelated to the bacterial ones consisting of 12 transmembrane helices and two cytoplasmic catalytic domains. They differ from each other in their

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activation or inhibition by Ca^V calmodulin, phosphorylation by protein kinases etc [175]. Among the labdane type diterpenes, forskolin, the manoyl oxide which is isolated from the roots of the herb Coleus forskohlii, exhibits a unique biochemical property. As Seamon and Daly [176,177] first demonstrated, this manoyl oxide can activate adenylate cyclase by interacting directly with the catalytic subunit or a closely associated protein of the adenylate cyclase system. The latest findings suggest that the forskolin regulatory site is located near the catalytic site at the dimer interface of the catalytic subunits, in a single deep cleft [175], Fig (9). Forskolin may act by promoting the catalytically optimum juxtaposition of the two domains, by forming a hydrogen bond between the oxygen of the first hydroxyl group and the first carbon atom of an aspartate. The most important difference between the catalytic and the forskolin binding sites, which otherwise are very similar, is the replacement of aspartate at the catalytic site and of serine at the forscolin-binding site. The forskolin-binding site is sterically closed and covers most of the solvent-accessible surface area of forskolin. The uniqueness of the mechanism of its action, marked out forskolin as an invaluable tool for the investigation of the role of c-AMP in biological processes. This manoyl oxide derivative is now used widely in most studies related to the role of c-AMP in cellular machinery as a powerful adenylate cyclase activator. Apart from forskolin, a number of other manoyl oxides have been shown to interact with the AC enzyme system. Biotransformation of certain e«r-13-£77/-manoyl oxides by Curvularia lunata resulted in compounds fiinctionalized in C-3 or in C-3 and C-12, which exhibited an AC stimulatory effect, although milder than that of forskolin (about 30 times less) [173]. The same activity was also ascribed to some synthetic derivatives of ^wr-8a-hydroxy-13 (16), 14 dien-18-oic acid methyl ester [178,179]. The biotransformation of ^^/-manoyl oxide-16-hydroxy 18-oic acid methyl ester with Rhizopus nigricans, however, resulted in carbomanoyl oxide which showed a selective inhibitory action on the activity of adenylate cyclase depending on the material initially used to stimulate the enzyme. This manoyl oxide inhibited the activity of the enzyme previously stimulated by forskolin but not by glucagon. A manoyl oxide {ent-^P, 6)ff-dihydroxy-13-e/7/-manoyl oxide) which also inhibited the activity of AC, was produced from the biotransformation of

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Forskolin

\«A^-Ay'

VwA^Ay'

V-A.-AY'

Transmembrane helices CI domain

VWA^>V-/

es

. V«A-AY'

V«/yA«/

Membrane

C2 domain

Fig. (9). A) Schematical representation of the activation of intact adenylate cyclase (AC) b forskolin and Gsa (GTP- bound stimulatory G protein a subunit). Mammalian AGs consists o 12 transmembrane helices and two cytoplasmic catalytic domains (referred to as Ci and C2 represented as lightly shaded and black respectively), (a) Hypothetical basal state, (b) Th suggested forskolin-activated state, (c) Forskolin and GSa-activated state. B) Forskolin-binding site (f) in C1-C2 heterodimer. With S is represented the serine pai (Ser891-942), with which the Oi hydroxy 1 of forskolin makes hydrogen bond. (Modified from Ciirr. Opin. Struct. Biology, 1998, 8, 770. With the kind permission of J.H Harley).

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e«^3yff-hydroxy-13-e'p/-manoyl oxide, by Curvularia lunata [147]. The above studies show a clear structure-activity relationship, where the type and/or the position of substitutes may lead to compounds with either stimulatory or inhibitory activity, ahhough the pharmacophore part of the labdane diterpenes, concerning AC related activity, has not really been identified. S22. B-GLUCURONIDASE B-glucuronidase catalyzes the hydrolysis of various )ff-D-glucuronides, liberating free glucuronic acid. Extracts from Scoparia dulcis, a perennial herb of tropical and subtropical regions, have been reported to significantly inhibit the activity of y5-glucuronidase [180]. Further fractionation of the extract led to the isolation of three labdane type diterpenes, namely scoparic acids A, B and C.The most potent inhibitory diterpene was scoparic acid A showing an IC50 of 6.8x10"^ M (4 times less than that of glucosaccharo-1, 4-lactone, a well known yff-glucuronidase inhibitor) [181]. 5.2.3. PHOSPHOLIPASE A^ Phospholipase A2 (PL A2) catalyzes the hydrolysis of phospholipids esterified at the second carbon in the glycerol backbone. Arachidonic acid is commonly esterified in this position and the action of PLA2 releases arachidonic acid for subsequent metabolism via the cyclooxygenase and lipoxygenase pathways. Halisulphate isolated from the marine dark brown sponge (Halichondriidae) exhibits a 100% inhibition of PLA2at 16|j,g/ml [171]. Another natural labdane diterpene isolated from the Spanish herb Sideritis javalambrensis, ent-8 alpha-hydroxy-lambda-13 (16), 14-dien, was also found to inhibit non-pancreatic secretory phospholipase A2 [182] and human secretory synovial PLA2 at a concentration of 10'^ M [183]. 5.2.4. ALDOSE REDUCTASE Aldose reductase has been implicated in the pathogenesis of cataract in diabetic and galactosaemic animals. The enzyme catalyzes the reduction

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of glucose and galactose to their polyols, which then accumulate in large quantities in the lenses and ultimately lead to mature lens opacities. Preliminary investigations have shown that some metabolites isolated from the marine sponge Dysidea sp. could inhibit the activity of the enzyme aldose reductase [184].

5.2.5. PROTEIN KINASES Although originally discovered as an activator of phosphorylase kinase, it soon became apparent that protein kinase A (PKA) had a much wider role in eucaryotic metabolism. PKA is a c-AMP dependent enzyme, consisting of two regulatory (R) and two catalytic subunits (C), which dissociate when each of the R subunits binds two molecules of c-AMP. Agonistic c-AMP analogs, such as forskolin (and most probably other entmanoyl oxides, reported to affect c-AMP production), can activate PKA by increasing c-AMP levels and interfering in the modulation of the cAMP/PKA pathway [185]. Apart from PKA, some other protein-kinases were found to be controlled by forskolin, such as cytosolic sphingosine kinase in rat periosteal cells [186] and protein kinase B (PKB) [187]. The latter was found to be stimulated by the activation of PKA through a PI3 (phosphatidylinositol 3)-kinase-independent pathway. Furthermore, a distinct activation mechanism was suspected, other than that normally observed by growth factors such as insulin, since substitution of the serine at the S473 position of PKB with alanine could not prevent activation by forskolin. The JAK family of protein kinases in T lymphocytes can also be regulated by forskolin through the activation of PKA [188]. Thus it seems obvious that many other enzymes could be susceptible to control by forskolin. 5.3. ENDOCRINE EFFECTS OF LABDANES As mentioned earlier, the adenylate cyclase system is hormone sensitive and many hormones are capable of regulating the enzymes involved in either a stimulatory or an inhibitory manner, thus modulating

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the levels of intracellular c-AMP and eliciting the appropriate physiological responses. Forskolin has been shown to activate almost all hormone-sensitive adenylate cyclases in intact cells, tissues, or solubilized preparations of adenylate cyclase, with Effective Concentration (EC50) values between 5 and 15 |aM [175,189,190]. Low concentrations of forskolin (
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cytotoxic activity, but two of the latter (coronarins A and B) exhibited a much stronger activity. 8(17), 12(E)-labdadiene-15, 16-diai shov^ed a moderate cytotoxicity when tested against KB cells (epidermoid carcinoma) ehxibiting an IC50 at 40|xg/ml [194]. Recently, it was also isolated from the leaves of Renealmia alpinia (belonging also to Zingiberacae) and tested along with two other diterpenes (ll-OH-8 (17), 12(E)-labdadiene-15-16-dial 11,15-hemiacetal and I6-OXO-8 (17), 12(E)labdadiene -15-oic acid) for cytotoxic activity against the yeast Sc-7 and the Ml09 (Madison Lung Carcinoma) murine cell lines [195]. The compound was weakly cytotoxic for the yeast but completely inactive against the Ml09 cell line. However, the ll-hydroxy-8 (17), 12(E)labdadien-15, 16-dial 11, 15-hemiacetal exhibited good cytotoxic activity against Ml09. Aulacocarpinolide and aulacocarpines A and B are two other labdane diterpenes isolated from another species of the family of Zingiberacae, Aframonum aulacocarpus, which exhibited a moderate growth inhibiting activity against the L1210 murine lymphocytic leukemia cell line. They exhibited an Inhibiting Concentration (IC50) at 12.5 and 25 p-g/ml respectively against L1210, but were found inactive against Ehrlich ascites tumor cells [166]. As has already been mentioned, labdane diterpenes are predominant compounds in parts of the species of Cistus, as well as in the resin of the same species. A preliminary screening of the chromatographic fractions of Cistus incanus subsp. creticus extracts, where seven labdane diterpenes were included, revealed that most of them showed strong cytotoxic activity [123]. The in vitro tests were conducted using the 3,-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, while the murine leukemia P-388 (macrophage - monocyte like), KB and NSCLC-N6 (non-small cell bronchopulmonary carcinoma) cell lines were used. The seven diterpenes tested exhibited strong cytotoxic activity, while the mixture of sclareol isomers isolated, exhibited IC50S ^t least as good as 6-mercaptopurin (the drug used as control). In a subsequent study, natural metabolites of ^/7/-13-e?p/-manoyl oxides isolated from the resin of C. creticus were tested using the -^//-thymidine method [61] in three leukemia cell lines (RAJI, M0LT3 and H9). Six out of seven of the diterpenes tested exhibited a significant cytotoxic activity, not far from that exhibited by methotrexate, which was used as control drug. Recent extensive studies were carried out on a number of labdane type diterpenes.

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semisynthetic or naturally occurring in Cistus regarding their activity against a panel of 14 human leukemic cell lines [29, 62, 196] and peripheral blood mononuclear cells (resting and phytohemaglutinin (PHA) activated). In these studies the MTT method was used to estimate the IC50S of the compounds that were tested. Labd-13 (E)-en-8a, 15-diol showed a significant cytotoxic activity, exhibiting IC50S below 20 |Lig/ml in most leukemic cell lines and was found to be inactive only against one (the B cell leukemic cell line CCRF-SB). It was also found to have an antiproliferative effect, in some of the leukemic cell lines tested affecting DNA synthesis in a dose and time dependent manner. This diterpene was further tested by the National Cancer Institute (NCI), U.S.A in a one dose primary anticancer test. The assay was performed using the sulforhodamine B (SRB) method [197]. In this primary assay, the compound was tested against NCI-H460 (Lung), MCF-7 (Breast) and SF268 (CNS) cell lines at a single dose of l.OOE-04 M. The diterpene was found to be considerably active as it caused the death of 80%, 89% and 88% of the cell population respectively ^*\ Furthermore, the diterpene was tested in an in vitro model by the NCI, consisting of 60 human tumor cell lines, where it exhibited a mean growth inhibition (GI50) of 2.04E-05 M, a mean TGI of 4.00E-05 and a mean lethal concentration (LC50) of 7.29E05 M [44]. The dehydrated derivative of labd-13 (E)-en-8a, 15-diol however was found to be inactive against all but one cell line up to 50|ig/ml [29]. Among other diterpenes, ent'l3'epi-manoyl oxide was found to be inactive up to 50 |Lig/ml while two derivatives were found to be active against two of the 14 cell lines used [29] (the effects on the DNA synthesis of these diterpenes were not studied). Two other labdane type diterpenes were tested against the same panel of leukemic cell lines [62, 196] i.e. sclareol, and the thiomidazolide derivative of ent'3/]'hydroxy'l3' epi-manoyl oxide (ribenol). The study led to extremely interesting results, since both diterpenes exhibited significant cytotoxic activity against all but one cell line (B cell line NAMALWA). Because of their significant cytotoxic activity (thiomidazolide derivative exhibited IC^Q^ as low as 2 and 3.8 |ig/ml against H9 and HUT78 T-leukemic cell lines, respectively) these two diterpenes were more extensively studied, revealing a (*) Data not published; personal communication. The authors provided the compound to NCI where it was further tested according to the Institute methodology.

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remarkable cytostatic activity too. Most interesting however, were the morphological, DNA electrophoresis and flow cytometric studies in two T-leukemic cell lines (MOLTS and H33AJ-JA13) and the promyelocytic cell line HL60, which revealed that both diterpenes killed cells by activating the apoptotic machinery in a dose and time dependent manner. Moreover, a flow cytometric study revealed a possible phase specific action of the two diterpenes. Although not very clear, the results from those studies led to the conclusion that possibly the mechanism of action is GO/1-phase specific. Additionally, further studies concerning the effect of those two diterpenes against two protooncogenes, c-myc and bcI-2 ^**\ showed that both diterpenes could down-regulate the expression of c-myc oncogene but not of bcI-2. From the above studies, a structure-activity relationship could be established, since some substitutes, especially those of C-3, C-7 and C-8, as well as the substitutes of the side chain, seem to affect the activity of the diterpenes. Darias et al [27] tested the manoyl oxides epigomeric acid and gomeric acid, against Hela 229 cells (cervical carcinoma) and found them to exhibit IC50S at 10 |ig/ml (lO^fold higher than 6-mercaptopurin, used as control). Several studies concerning the cancer-related properties of forskolin have also been performed. In a study by Miyamoto et aL [198] the combined effect of forskolin with mitomycin C, a well-established anticancer drug, was reported. The effect of this combination was studied by using two cell lines AH66 and AH66F. The results showed that forskolin significantly enhanced the cytotoxicity of mitomycin C, increasing in parallel the uptake of the drug and of the intracellular cAMP in AH66 but not in AH66F cells, while the authors suggested that forskolin may be suitable for antitumour combination chemotherapy. In accordance with these results, in another recent study by Mann et al [199], forskolin was found to cause a 2.1-fold increase in the short-term accumulation (and subsequently the cytotoxicity) of another anticancer drug, cis-diamminedichloroplatinum (II) (DDP) in DDP-sensitive 2008 human ovarian carcinoma cell lines. Forskolin also increased accumulation in A2780 cells but not in DDP-resistant 2008 cells. (**) Data submitted for publjcation. The study concerns the effect of the thiomidazolide derivative of enf-3p-hydroxy manoyl oxide and of sclareol on the expression of the two oncogenes in two leukemic cell lines, where an apoptotic effect was also observed

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However the results concerning the effect of forskolin (and derivatives) on programmed cell death (apoptosis) are controversial. Galli et al, [200] reported that forskolin blocked apoptosis in cerebellar granule cells through a mechanism which did not require RNA synthesis. Machwate et al. [186] reported that forskolin demonstrated an antiapoptotic effect in the RP-11 (rat periosteal) cell line, by transiently up-regulating cytosolic sphingosine kinase activity. In the same study, however, forskolin failed to suppress apoptosis in two other osteoblastic cell lines. Forskolin was also reported to effectively suppress H2O2 -induced apoptosis in PC 12 cells [201] by a glutathione independent mechanism, whilst no new protein or RNA synthesis was required. An inhibitory effect on apoptosis was also reported for dideoxyforskolin [202]. This derivative of forskolin strongly inhibited cell death induced by ricin, modeccin, Pseudomonas and diptheria toxin in MDCK cells. On the other hand Keren-Tal I et al [203] reported that forskolin induced very rapid and massive apoptosis in primary granulosa cells expressing low amounts of SV40 T antigen incubated at 32^C, which could not be abolished by the presence of serum. Moreover Chen et al [185] reported that forskolin decreased proliferation, increased differentiation and induced apoptosis in A-172 cells, probably through an up-regulation of the c-AMP/PKA pathway. Forskolin was also implicated in the expression of certain protooncogenes. It was able to transiently induce c-fos and c-myc expression (and more slightly the expression of ras-h) in normal but not in w-ras transformed rat thyroid FRTL5 cell line [204]. In another study [205,] however, forskolin was found to reduce the expression of c-myc messenger RNA (mRNA) in cultured human foreskin fibroblasts, but had no effect on c-fos mRNA expression. Forskolin was also found to reduce c-myc expression in U937 monocytic cells [206]. Cytotoxic labdane type diterpenes have also been isolated from Baccharis gaudichaudiana (Asteraceae) [207]. A labdane type diterpene from the series of gaudichaudol was found to exhibit significant cytotoxic activity against P-388 and KB-VI (KB cell line resistant to vinblastine), but not against the parental KB cell line (which does not express multidrug resistance (MDR)) and only moderately in five other human cancer cell lines [207]. Two active nitrogenous labdanes were also isolated from the marine species Lissoclinum voeltzkowi Michaelson (Urochordata) [208]. These compounds (dichlorolissoclimide and

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chlorolissoclimide) were studied in vitro on NSCLC-N6 (non-small cell bronchopulmonary carcinoma cell line) and were found to exhibit an antiproliferative effect as a result of a blockade at the Gl phase. Finally forskolin, as other labdanes too can induce differentiation in different types of cells e.g. in epithelial cells [209] and in U937 (monocytic) cells through a sustained rise in c-AMP levels [206]. Cell differentiation-inducing diterpenes were also isolated from the methanol extract of the aerial part of Andrographis paniculata NEES (Acanthaceae) [210]. This activity was determined by observation of the inducibility of phagocytosis of polystyrene latex particles Ml by mouse myeloid leukemia cells. Eight monomeric, four dimeric diterpenes and five diterpene glycosides were tested. Most of them showed potent differentiation as well as growth-inhibitory activities. Dimers were generally more potent than monomers, while glucosides showed the weakest activity [210]. 5.5. MODULATION OF IMMUNE AND INFLAMMATORY CELL FUNCTIONS The immune system consists of a highly complex, intricately regulated group of cells whose integrated function is essential to health. Cells of the immune system may interact in a cognate cell-cell manner and may also respond to intracellular messages, including hormones and cytokines elaborated by various cells. Some effects of labdane type diterpenes on the function of T-cells, B-cells, macrophages, NK-cells, basophils, mast cells, neutrophils, eosinophils and platelets are described below. Each of these cell types is involved in immunity and inflammation and the effects of labdanes on these cells will be considered in this broad context. As noted, the labdanes display a broad spectrum of biochemical and pharmacological activities, suggesting that they may significantly affect the function of the immune system and inflammatory cells. The labdanes may affect critical enzymes such as adenylate cyclase, protein kinases and phospholipase A2, which are intimately involved in signal transduction and cell activation processes. Much of the information on labdane-type diterpene effects has been provided by forskolin and mainly in in vitro systems.

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Forskolin has demonstrated an ability to alter human peripheral blood lymphocyte activation by antigens and mitogens [211]. Shenker and Matt found that forskolin could cause a dose dependent inhibition of human peripheral blood lymphocyte response to concavalin A (con A), PHA, pokweed mitogen (PWM) and Staphylococcus aureus protein (SpA) mitogens. Additionally, the diterpene reduced the ability of the cells to recall antigens such as tetanus toxoid and streptokinase/streptodornase. Inhibition was reflected in altered DNA, RNA and protein synthesis, including immunoglobulin production. Cell viability was not affected while 12-0-tetradecanoyl phorbol 13-acetate, diacylglycerol (DAG) and ionomycin, could completely reverse the inhibition, suggesting a PKC involvement. More recently, an inhibiting dose-dependent effect of forskolin on mitogen-induced mouse splenocyte proliferation was also observed by Zhong et al [212]. In another study [213] it was reported that exposure of intact lymphocytes to forskolin induced loss in the betaadrenergic receptor density of these cells. T lymphocytes are involved in a vast number of major functions among cells of the immune system. They help antibody production, kill target cells, induce inflammation, and produce effector molecules (interleukins, lymphokines) etc. Forskolin either interferes with the production and signalling pathways of interleukins or directly affects T cells proliferation. IL2 production is inhibited by forskolin [214]. Janus Kinase 3 (JAK 3) is a kinase playing an important role in interleukin 2-dependent (IL2) signal transduction and the proliferation of T lymphocytes. Forskolin was able to inhibit (along with prostaglandin E2 and dibutyryl c-AMP) up-regulation of JAK3 protein in naive T cells (but not of IL2 receptors) and the expression of the common gamma chain (gammac) that associates with JAK3 [215]. The proliferation of T cells was also inhibited, while reduced induction of c-myc and c-jun pathways, but not of the IL2 -dependent induction of 6c/2 were also found. According to the authors, this suppression of JAK3 may represent a mechanism by which forskolin can inhibit T-cell proliferation. Forskolin was also found capable of suppressing the generation of class I specific cytolytic T lymphocytes (CTL) [216]. However, the generated CTL population retained its normal proliferative response to alloantigens and to lyse nonspecific targets. Forskolin has been reported to modify the voltage dependent K+ conductance in quiescent human peripheral blood T-lymphocytes, an effect found to be

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independent of c-AMP [217]. In contrast to forskolin, which mainly inhibits T cell proliferation, prehispanolone, a diterpene isolated from Leonurus hereterophyllus, has been reported to induce proliferation of this lymphocyte subpopulation [218]. This diterpene has been reported to act synergistically with conA, inducing a 5 to 8-fold greater proliferation than that induced by conA alone in mouse T-lymphocytes. Several effects of forskolin on B-lymphocytes, the cells of the immune system responsible for the production of immunoglobulins, have further been reported. This diterpene was found to inhibit cellular proliferation of B cells stimulated either by antibodies to surface immunoglobulins (antimu), and an antibody to CD20 antigen or 12-0-tetradecanoyl phorbol 13acetate [219]. There was also a clear inhibition of Gl entry and DNA synthesis, and forskolin maintained its inhibitory effect even when added later after anti-mu stimulation. Additionally, no differences were found in the inhibitory effect of forskolin on neoplastic B cells, as compared to the responses of normal cells. Growth inhibition associated with an accumulation of cells in Gl was later found when cells of the B-lymphoid precursor cell line Reh were incubated with forskolin [220]. In that study, a delay of cells in G2/M prior to Gl arrest was observed, suggesting that important restriction points located in the Gl and G2 phases of the cell cycle may be controlled by forskolin (due to cAMP levels elevation). In a subsequent study [221], it was found that the arrest of Reh cells was accompanied by rapid dephosphorylation of retinoblastoma protein, which was suggested to be a prerequisite for the forskolin mediated arrest of these cells inGl. Cross-linking of surface immunoglobulines (sigs) is known to promote proliferation of follicular mantle B cells, rescue germinal center B cells from apoptosis but induce apoptosis in susceptible Burkitt's lymphoma (BL). Signals transduced through CD40 induce resting follicular mantle B cells to enter the cell cycle while promoting germinal center B cells and Burkitt's lymphoma cell survival. Forskolin-triggered growth arrest in Ramos (Burkitt lymphoma cell line) could not, however, be reversed by anti-CD40 [222]. Forskolin also potentiated the proliferative response of follicular mantle B cells cultured with anti-sigs and anti CD40 together, but failed to affect spontaneous apoptosis and anti sig and antiCD40promoted survival of germinal center B cells. Finally the addition of forskolin in B cell cultures from atopic patients resulted in a dose-

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dependent inhibition of m vitro production of spontaneous IgE [223]. On the contrary no such effect was observed with the U266 myeloma cell line. The effect of prehispanolone on B cell proliferation was also studied, but no activity was observed either when the diterpene was used alone or after lipopolysaccharite (LPS) treatment [218]. Natural killer cells (NK cells) are nonadherent lymphocytes which share some properties of T cells and macrophages. They also share some similar functional activities, such as their cytolytic mechanisms, with cytolytic (CTL) lymphocytes. Killer cells (K cells) are also a lymphocyte subpopulation which present receptors for the Fc of IgG, able to bind IgG antibodies and to lyse target cells to which the antibody is directed (antibody dependent cell mediated cytotoxicity, (ADCC). Both subpopulations are affected by forskolin, although in different ways. Forskolin exerts a stimulating effect (at 2 jiM) on the induction of cytotoxic activity of IL2- activated Killer cells (K cells) (issued from murine spleen cells cultured with high leves of IL-2) [224]. On the other hand forskolin treatment of NK cells caused decreased lysis of K562 (target cells) by up to 45%, while forskolin was also found to diminish the ability of NK cells to bind K562 [225]. When forskolin was tested on antibody-dependent cell-mediated cytotoxicity (ADCC), which as mentioned is the major function of K cells, an inhibitory effect correlating with increased levels of c-AMP in the effector cells, was observed [226]. The assay was performed by using Chang liver cells as the target cells, immune rabbit serum as antibody and healthy human peripheral blood mononuclear cells as effector cells. Macrophages are the primary phagocytic cells of the immune system. In the blood they are called monocytes whilst in tissue, histiocytes. Macrophages nonspecifically process the antigen so that, specific antigenreactive cells may recognize it. They also invade sites of inflammation and serve to clear the site from necrotic debris. Forskolin was found to inhibit the adenylate cyclase activity of membrane preparations from J774 macrophage cells in concentrations between lOnM and lOOfiM [227]. Two diterpenes prepared from the hexane extracts of the Spanish herb, Sideritis javalambrensis, were tested for their effect on mouse peritoneal macrophages. £/2/-13-e/7/-12a-acetoxymanoyl oxide and ent-^a -hydroxylabda-13 (16), 14-diene inhibited, in a dose dependent manner, prostaglandin E2 generation in cultured mouse peritoneal macrophages

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Stimulated by zymosan, ionophore A23187, melittin and PMA. The latter labdane was more potent, exhibiting an IC50 of 3|iM in zymosan-activated macrophages, very close to aspirin, which exhibited an IC50 of 2|xM [228]. An interaction of these two diterpenes with the eicosanoid system in macrophages was also suspected. In a further study [229] ent-Sa hydroxy-labda-13 (16), 14-diene appeared to possess two mutually opposing actions on the eicosanoid system in macrophages: potentiation of delivery of substrate following cell activation, followed by an inhibition of the conversion of substrate to product. Inflammation is the primary process through which the body repairs tissue damage and defends itself against infection. It may be initiated either by immune or nonimmune pathways, but both employ similar effector mechanisms. Inflammation can be further divided into acute and to chronic categories. The cellular players in the process of acute inflammation include mast cells, neutrophils, platelets and eosinophils, which act in sequence. These cells are activated by a variety of chemical processes and, in turn, produce and release a number of chemical mediators (signaling molecules that act on smooth muscle cells endothelial cells or white blood cells to induce, maintain or limit inflammation). Major mediators of inflammation are the metabolic derivatives of arachidonic acid. The metabolism of arachidonic acid is believed to occur mainly in macrophages, but metabolites may also be synthesized by most, if not all, cells that take part in an inflammatory response, including mast cells. Metabolism of arachidonic acid occurs via cyclooxygenase and lypooxygenase pathways. The first pathway give rises to prostaglandins (PGs) and the second to leukotrienes. Most of the cells that take part in an inflammatory response as well as many chemical mediators seem to be affected by labdane type diterpenes. Fuller et aL [230] found that forskolin could inhibit not only macrophage activation, but also the release of thromboxane B2 (TXB2) and super oxide (SO) from alveolar macrophages stimulated by opsonised zymosan or IgE/anti IgE complexes. The diterpene however did not affect the release of leukotriene B4 or N-acetyl-beta-D-glucosaminidase (NAG). An antiinflammatory effect was also observed when an extract of Sideritis javalambrensis (Labiatae), containing an e«/-16-hydroxy-13-6'/7/-manoyl oxide derivative, was tested in vivo using the carrageenan mouse paw edema test [57]. The two diterpenes further isolated from S.

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javalambrensis (Labiatae) were found indeed to inhibit the generation of TXB2 and leukotriene B4 from A23287 ionophore-treated rat peritoneal leukocytes, at concentrations below 10'"^ M [183]. Neither of them however affected SO generation or scavenging and they did not affect granular enzyme secretion from activated human and rat neutrophils. Forskolin could also inhibit the arachidonic acid release by neutrophils activated by the chemotactic tripeptide formylmethionyl-leucylphenylalanine (fMLP), but not the release due to ionophore A23187 [231]. Forskolin inhibited, in a dose dependent manner, the SO production by human neutrophils treated with the same tripeptide [232] as well as the degranulation of these cells [233]. Other studies deal with the inhibitory effect of forskolin in the IgE-mediated release of histamine and peptide leukotriene C4 (LCT4) from human basophils and lung mast cells [234,235]. Antigen-induced prostaglandin D2 (PGD2), LTC and leukotriene B4 production were suppressed by forskolin in the PT-18 mouse spleen- derived mast cell line by 30-50% or more when the diterpene was combined with isobutylmethylxanthine (a phosphodiesterase inhibitor) [236]. In the same study, inhibition of the ionophore induced production of platelet activating factor and liberation of arachidonic acid were additionally observed. Anti-inflammatory activity was also exhibited by c/^'-communic acid, a diterpene isolated from the leaves of Cryptomeria japonica (Taxodiaceae) [237], in a study employing the carrageenan mouse paw edema test. The same compound was found to inhibit histamine induced contraction in ileum isolated from the guinea pig. Finally, a number of labdane diterpenes seem to affect the ability of platelets to form aggregates. Three diterpenes isolated from the methanolsoluble fraction of Shug Chher, a Bhutenese plant, inhibited platelet aggregation induced by the platelet activating factor (PAF) [238] while one of them, 3a-hydroxymanool, was found to act as a PAF antagonist. Moreover, in a study with manool-related synthetic labdane diterpenes it was found that 3a-hydroxylabdane skeleton is essential for the platelet aggregation inhibitory activity [239]. Pinusolide, a labdane type diterpene lactone, isolated from leaf extract of Biota orientalis, was tested for PAF receptor binding antagonistic activity using rabbit platelet receptor binding tests [240] and it was found to exhibit significant activity (IC50 2.52x10"^ M). Pinusolide also exhibited the same PAF antagonistic

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activity in in vivo studies [241]. Several pinusolid derivatives have been tested in recent studies [242, 243] and a structure activity relationship has been estabhshed. The carboxymethylester group at C-19, the integrity of a,p-unsaturated butenohde ring and the exocyclic olefinic function of pinusolide were found to be necessary for its maximum PAF receptor binding antagonistic activity. 5.6. OTHER EFFECTS OF LABDANES Labdane type diterpenes obviously exhibit a wide spectrum of actions. Primarily forskolin exhibits: (a) antihypertensive properties, lowering blood pressure in different animal species through a vasodilatory effect; (b) positive inotropic effects in cardiac muscle; and (c) bronchodilatory effects [155] and (d) pressure-lowering properties [244]. Cardiotonic activities were also exhibited by medigenin and medigenin acetate, which are genins isolated from Melodinus monogynus (Apocyanaceae), when tested in isolated frog and mammalian hearts [245] Compounds isolated from Dodonaea viscosa exerted smooth muscle relaxing properties [246]. Among them ent-XS, 16-epoxy-9 aH-labdanel3 (16) 14diene-3)5, 8a-diol inhibited the contractions of guinea-pig ileum evoked by acetylcholine, histamine, and barium chloride or electrically induced contractions. Sclareol glycol, a derivative of sclareol exhibited antihypoxic effects in mice [247] and also induced changes in core body temperature by interacting with dopamine receptors in rats [248]. Some labdane type diterpenes isolated from Hyptis spicigera (Labiatae) exerted insecticidal properties, inhibiting larval growth of the European corn borer [249] while, in another study, a glycocide of the manool series, isolated from the roots of Gleichenia japonica, showed a strong growth inhibition of lettuce [101]. On the contrary a diterpene alcohol of the same series of labdanes induced acceleration of the growth of the lettuce. Finally, sclareol inhibited the growth of wheat coleoptile [250] and reduced the severity of rust infection in French bean [251].

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CONCLUSION In conclusion it can be stated that the labdane type diterpenes have a variety of biological activities. Plants are important sources for discovering new drugs, or obtaining new lead compounds. Semisynthesis of labdanes or the biotransformation process using labdanes as starting materials, are important methodologies in order to obtain new and potent biologically active compounds. ABBREVIATIONS AC ACTH ADCC Con A CTL DAG DDP EC50

FMLP GC GI50

GLC GC-MS GGPP IC50

IL2 JAK3 K cells LC50

LCT4 LPS MDR MIC MTT NAG

Adenylate Cyclase Adenocorticotrophic hormone Antibody Dependent Cell mediated Cytotoxicity concavalin A Cytolytic T lymphocytes Diacylglycerol cis-Diamminedichloroplatinum (II) Effective concentration 50 formylmethionyl-leucyl-phenylalanine Gas Chromatography Growth Inhibition 50 Gas Liquid Chromatography Gas Chromatography-Mass Spectrometry Geranyl-geranyl pyrophosphate Inhibiting Concentration 50 Interleukin2 Janus kinase 3 Killer cells Lethal Concentration 50 Leukotriene C4 Lipopolysaccharite Multi drug resistance Minimum Inhibitory Concentration 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide N-acetyl-beta-D-glucosaminidase

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NCI NK PAF PGs PHA PLA2 PKA PKB PRL PMA PWA RI Rt SO SpA SRB STH TGI TXB2

= = = = = = = = = = = = = = = = = = =

National Cancer Institute Natural Killer Platelet Activating Factor Prostaglandins Phytohemaglutinin Phospholipase A2 Protein Kinase A Protein Kinase B Prolactin Phorbol-12-myristate-13 -acetate PokWeed Mitogen Retention Indices Retention time Super Oxide Staphylococcus aureus protein Sulforhodamine B Somatotropin Total Growth Inhibition Thromboxane B2

ACKNOWLEDGEMENTS The authors are thankful to Prof. H.E. Kaiser, D. Sc, (Department of Pathology School of Medicine, University of Maryland at Baltimore, Md 21201, U.S.A), and Dr. J.G. Delinassios (International Institute of Anticancer Res., Kapandriti, 19014 Attiki, Greece), for their valuable and critical suggestions. We are also grateful to T. Anastasaki, M.Sc. in Pharmacognosy, for her kind help in improving this manuscript.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. Allrightsreserved.

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METABOLISM OF THE TOMATO SAPONIN a-TOMATBVE BY PHYTOPATHOGENIC FUNGI MANUEL RUIZ-RUBIO, ALONSO PEREZ-ESPINOSA, KHALID LAIRINI, TERESA ROLDAN-ARJONA, ANTONIO DIPIETRO AND NURIA ANAYA Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071 Cordob a^ Spain ABSTRACT: a-Tomatine is a steroidal glycoalkaioid present in Solanaceoiis plants, principally in a number of Lycopersicon and Solanum species. High concentrations are found in leaves, stems, roots and green fruit of tomato plants, suggesting a possible role in resistance to pathogens. The toxic effects of a-tomatine are attributed to its ability to complex with membrane sterols, causing pore formation and leakage of cell contents. Pathogenic fungi to tomato are usually less sensitive to tomatine than pathogens of other plant species and saprophytes. Fungi have evolved two principal mechanisms to avoid the toxic effect of tomatine, either changing the composition of the cell membrane or producing specific tomatine-detoxifying enzymes, known as tomatinases. Tomatinases have distinct mechanisms of action, and molecular and catalytic properties in different pathogen species. Up to now, the knowledge of a-tomatine metabolism by phytopathogenic fungi is almost exclusively limited to the removal of sugar residues from the molecule, although recent studies in Gibberella pulicaris demonstrate that further hydroxylations of the aglycone moiety tomatidine occur, after the action of tomatinase. Tomatinase-encoding genes have been cloned from Septoria lycopersici and Fusariiim oxysporum. Both genes do not share sequence homology, suggesting that different tomato pathogens have independently developed the ability to cleave tomatine by convergent evolution. Expression of F. oxysporum tomatinase is fully repressed in the presence of glucose, indicating a possible role in nutrition besides detoxification of tomatine, although in BoUytis cinerea glucose repression of tomatinase is not observed. Other genes different to tomatinase are induced by a-tomatine, but the complete significance of this phenomenon is still unknown.

INTRODUCTION Plants have evolved different mechanisms to protect themselves against a great variety of invasive pathogens. As a part of the defense, plants produce a number of secondary metabolites with antimicrobial activity. Many of these compounds are produced constitutively, being present in healthy plants as normal metabolic products. VanEtten et al proposed the term "phytoanticipin" to define these preformed antimicrobial

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compounds as opposed to phytoalexins, which are synthesized de novo from separate precursors in response to pathogen attack [1]. In contrast to phytoalexins, phytoanticipins have received comparatively little attention by phytopatologists, although in many cases they may represent one of the first chemical barriers encountered by potential pathogens. Saponins are found in many major food crops and include a large number of examples of constitutive plant compounds with antimicrobial activity. Their name refers to the property of forming a stable foam when shaken with water, and they consist of triterpenoid, steroid or steroidal glycoalkaloid molecules bearing one or more sugar chains. Excellent general reviews on saponins have been published recently [2-6]. The steroidal glycoalkaloid a-tomatine (from hereon called tomatine) is the major saponin component of tomato (reviewed by Roddick [7]), and has also been found in other Solanaceous plants [7, 8], (reviewed by Maga [9]) The widespread distribution of tomatine in all parts of the tomato plant has stimulated searches for a function of this compound in the general plant metabolism, but apart from the suggestion that it may offer protection against herbivores and pathogens, little evidence exists for a biochemical or physiological function. Tomatine was first isolated from the wild tomato species Lycopersicon pimpinellifolium and from the cultivated variety Lycopersicon esculentum [10]. It is present in all organs of tomato plants: in leaves, stems, roots and green fruit where it is found at high concentrations (up to ImM), whereas in ripe fruit it is almost absent [11-14]. Tomatine is principally known for its inhibitory activity on fungal growth [10, 15-18], although it has also been reported to have insecticidal effects [16, 19-22], to inhibit the growth of nematodes [23], to be embryotoxic in Xenopus [24], to disrupt cell membranes in mammalian intestinal cells [25], and to induce the immune response in mammals [26]. The principal mechanism determining the toxic effect of tomatine is attributed to its interaction with sterols of cell membranes, the formation of complexes, and the resulting loss of membrane integrity and leakage of cells contents [2, 4,27-29]. Due to its antifungal properties it has been suggested that this glycoalkaloid may be important in resistance to fungal pathogens. Nevertheless, evidence for the role of tomatine as a barrier against microbial attack is inconclusive, and some confronting observations make

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it difficult assigning exclusively a direct involvement in plant resistance. However, one aspect that seems clear is the observation that fungal pathogens of tomato are generally more tolerant to tomatine than pathogens of other plant species or saprophytes [15, 17]. This suggests that tomato pathogens may have evolved specific mechanisms to avoid the toxic effect of this chemical. Tolerance to tomatine is based on two main mechanisms: some fungi are resistant to the compound because they have modifications in their membrane composition, while others produce specific tomatine-detoxifying enzymes, known as tomatinases. These enzymes carry out a number of reactions to cleave tomatine removing either all four sugars or a single sugar from the steroidal glycoalkaloid. Most of them remove all four sugars by cleaving the p,l-linked galactose, thus releasing the tetrasaccharide p-lycotetraose and the aglycone tomatidine. Other seems to degrade tomatine to tomatidine but releasing monosaccharides rather than the tetrasaccharide. Finally, others remove a single sugar, the terminal D-xylose, from tomatine yielding pi-tomatine, or the terminal p,l-2 linked glucose, forming p2"tomatine. In all these cases, deglycosylation appears to be sufficient to destroy the ability of tomatine to complex with membrane sterols and therefore to eliminate or reduce its toxic effect. Detoxification of tomatine may be how these tomato pathogens avoid the glycoalkaloid barrier. The mode of action and the importance of saponin-detoxifying enzymes from fungi in general has been reviewed elsewhere [6, 30, 31]. Two tomatinases-encoding genes have been cloned, one from Septoria lycopersici [32, 33] and the other from Fusarium oxysporum [34]. Unexpectedly, both genes do not share significant sequence homology, suggesting that these tomato pathogens have acquired the ability to cleave tomatine independently through a process of convergent evolution. Furthermore This is supported by the fact that both proteins employ different enzymatic mechanisms achieving the same goal: to detoxify tomatine. Convergent evolution may be extended to other tomatinases since an array of different molecular mass has been found in the enzymes characterized so far [33, 35-37]. The existence of tomatinases in fungal-tomato pathogens supports the idea that tomatine may play a role in resistance to fungal attack because these enzymes seem to act specifically on tomatine. Such a role is also supported by the finding that at least in F. oxysporum f. sp. lycopersici, tomatinase is produced during infection both in roots and stems

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throughout the complete disease cycle [34, 38]. However, the fact that expression of this tomatinase is fully repressed in the presence of glucose [34] indicates that besides a role in detoxification of tomatine, the enzyme might also have a nutritional purpose. Most tomatinases characterized so far are induced by tomatine, indicating that they may be part of a fungal defensive mechanism against the compound. The identification of other genes whose expression is activated in the presence of the glycoalkaloid may provide new insights into alternative mechanisms of resistance and further knowledge on the metabolism of tomatine. By applying mRNA differential display analysis to mycelium of F. oxysponim grown on minimal medium either in the absence or in the presence of tomatine, other genes selectively induced in response to the glycoalkaloid have been isolated. The significance and role of these novel genes are unknown, but they may represent adaptive metabolic changes of fungi to tomatine. The knowledge of their physiological role may contribute in the future to the development of new strategies to restrict pathogen propagation inside the plant. STRUCTURE OF TOMATINE Tomatine is a steroid alkaloid glycoside consisting of an aglycone moiety (tomatidine), and a tetrasaccharide moiety (p-lycotetraose) composed of two molecules of glucose and one each of galactose and xylose; the four monosaccharides form a branched structure that is attached at the C-3 position of the aglycone (reviewed by Roddick [7], see also references therein). The structure of tomatine is shown in Fig (1). Table 1.- Structural characteristics of partially hydrolyzed a-tomatine. Compound

Structure

a-tomatine

tomatidine + galactose + glucose + glucose + xylose

Pi-tomatine

tomatidine + galactose + glucose + glucose

p2-tomatine

tomatidine + galactose + glucose + xylose

7-tomatine

tomatidine + galactose + glucose

5-tomatine

tomatidine + galactose

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Partial hydrolysis of tomatine yields structures with modified sugar moieties [39]. The form whose sugar moiety is the tetrasaccharide is called a-tomatine (tomatine) and those forms lacking xylose, one glucose, xylose and one glucose, or having only galactose are called Pi-, P2"5 Y" ^^^ 6-tomatine respectively. Table 1 summarizes the glycosidic composition of the different structures originated by partial hydrolysis of tomatine.

CH,

p - LYCOTETRAOSE

TOMATIDINE

Fig. (1). Structure of the steroidal glycoalkaloid a-tomatine showing the aglycone moiety (tomatidine) and the tetrasaccharide moiety (p-lycotetraose).

Although some forms are detected when tomatine is extracted from the plant, these glycosides are probably either products of enzymatic hydrolysis during extraction or natural intermediates in the biosynthesis and/or degradation of tomatine. DISTRIBUTION OF TOMATINE IN TOMATO PLANTS Analysis of tomatine contents in tomato plants showed that the glycoalkaloid is present in all organs [7, 40-42]. There is a significant variation in the reported tomatine content of particular plant organs. For example, Tukalo (1958) found, in terms of dry weight, 0.86-1.9% tomatine in leaves, 0.3-0.6% in stems and roots and 0.93-2.2% in fully

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expanded flowers of tomato plants [43]. Much of this variation appears to be due to factors such as plant variety, the stage of plant development, time of season and culture conditions [44-47]. Recently, new methods have been developed for very precise quantification of tomatine in tomato plants [13, 48-50]. Thus, Friedman and Levin, used HPLC with a pulse amperometric detection method, for the direct analysis of tomatine in different parts of tomato plants. They found 0.134 % in roots, 0.287 % in stems, 0.567% in calyxes, 0.64% in leaves and 0.745% inflowers[13]. Tomatine is not transported into fruits from vegetative organs, but is synthesized by cells in the green fruit. The relative amount of tomatine varies from young green fruits (0.46 %) to red mature ones (0.002%), showing a decrease of 99.5% in the level of the glycoalkaloid. Therefore ripe fruit contained the lowest levels of tomatine relative to any other part of the plant [13]. This observation, that tomatine content decreases dramatically during ripening of the fruit, agrees with the reports of other authors. Thus, Sander showed that young developing fruits accumulated large amounts of tomatine, but when ripening begins alkaloid degradation occurred and concentrations declined [40]. Kajderowicz-Jarosinska recorded high levels of tomatine in green, yellowish and red (ripe) tomato fruits, but when ripe fruits were left on the plant for further 2-3 days, tomatine almost disappeared completely [51]. Finally, Bushway et al. found that only five of one hundred red tomatoes contained levels of tomatine detectable by HPLC [48]. Tomatine has not been detected in dormant seeds but it appears in the radicle during the early stages of germination. In the root, de novo synthesis of tomatine has been established by studies with cultured excised roots of L pimpinellifolium and L esculentum although, in the latter species, alkaloid levels per unit of dry weight were lower [52]. There are indications that the main sites of tomatine biosynthesis in the root are the actively growing regions [7, 52]. Generally it is recognized that the main organ of tomatine synthesis and accumulation is the shoot, and that synthesis takes place principally in meristematic region [40]. On the contrary to other steroids that are synthesized in the cell membrane, tomatine seems to be formed in the soluble phase of the cytoplasm of tomato cells [53]. In spite of the considerable variation in tomatine levels observed between different tomato cultivars [44-47], there is no formal

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demonstration that correlates saponin content to disease resistance. In fact, tomatine does not appear to be determinant in variety-specific resistance of near-isogenic tomato lines to the vascular wilt pathogen Verticillium albo-atrum, since no particular accumulation of tomatine in the resistant interaction w^as found as compared to the compatible one, both before and after infection with the pathogen [54]. Similar conclusions are obtained from studies with F . oxysporum f. sp. lycopersici. In this case, an increase in tomatine concentration in the xylem fluids was found in one cultivar after wounding and inoculating with the pathogen, but again no differences were observed between resistant and compatible interactions [55]. The interpretation of these experiments is complicated by the fact that both V. albo-atrum and F. oxysporum f. sp. lycopersici are able to degrade tomatine enzymatically. A more effective strategy to evaluate the importance of the glycoalkaloid in plant defense, might be the isolation of tomato-plant mutants defective in tomatine biosynthesis [56]. BIOLOGICAL ACTIVITY OF TOMATINE Mode of action of tomatine Tomatine is known to alter membrane integrity and cause lysis in fungal hyphae [57], animal cells [42] plant organelles [58] and plant tissues [28, 58]. The mode of toxic action of tomatine was earlier attributed to the detergent properties of the glycoalkaloid, but Shultz and Sander demonstrated that tomatine is able to complex in a highly specific manner with 3p-hydroxy-sterols in vitro [59]. Thus, it became evident that binding of the glycoalkaloid to membrane sterols could be the cause of destabilization of the membrane lipid bilayer and this explained the lytic and toxic activity observed. Nevertheless, the possibility that tomatine may have additional effects on fungi other than its membraneolytic action was not excluded. This primary mode of action of tomatine, that involves the formation of complexes with membrane sterols is similar to that described for polyene antibiotics [2, 4], and results in pore formation and loss of membrane integrity. This mode of action is supported by the reduced activity of tomatine on sterol-free bacteria and Oomycete fungi such as Pythium and Phytophthora [15, 28], and the strongly reduced toxicity of hydrolysis products of the glycoalkaloid which fail to bind sterols [57].

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In vitro studies by Roddick and Drysdale on the effect of tomatine on the leakage of peroxidases from Hposomes also support the idea that tomatine binds membrane sterols. They detected a significant disruption of the liposome by tomatine, only if the membranes contained sterol and additionally this destabilization was pH-dependent [27]. Later it was demonstrated that liposome membranes containing sterols but lacking 3(3hydroxy sterol were resistant to tomatine [28]. Since the mechanism of action of tomatine is relatively nonspecific, it is expected to affect all eukaryotic organisms which have free sterols in their membranes. Although it is not clear how plants protect themselves from the lytic effects of their own saponins, it seems that the content of p-hydroxy sterols in the membrane is likely to be a major mechanism [28, 29]. It has been demonstrated that the electrolyte loss from cells incubated in the presence of tomatine was less severe in tissue of tomato (10% free sterols) and potato (12% free sterols) than that of tobacco (50% free sterols) and Nicandra physaloides (54% free sterols) [28]. These observations suggest that tomato tissue is resistant to high concentrations of tomatine because the tomato cell membrane contains a low proportion of free sterol [28]. The same conclusion is drawn for potato tissues, which contain high quantities of a-solanine and achaconine [9], glycoalkaloids structurally related to tomatine and analogous in their mode of action [29]. In addition, plants may protect themselves from their own saponins by compartmentalizing them in the vacuole or in other organelles. In the case of tomatine it is stored in the vacuole of tomato cell as biologically active molecule [53]. The membranes of these organelles may avoid lysis due to a low sterol content or to a high proportion of sterols substituted at 3-p-hydroxy 1 position [28]. Carbohydrate side-chain and tomatine activity The precise way in which saponins becomes incorporated into membranes is still unclear and various models have been proposed [2]. The nature of the aglycone and the oligosaccharide moiety of the tomatine molecule are both likely to contribute to its membraneolytic properties [4]. Initial research led to the hypothesis that the inactivation process of tomatine involves removal of one or all the saccharides of the plycotetraose, and that tomatidine or other subproducts formed were

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nontoxic due to their inability to form a stable complex with membranebound sterol [57]. An alternative mechanism of action of tomatine was proposed in which the aglycone, rather than tomatine itself, would act as the membraneolytic agent [60]. According to this hypothesis, the hardly water-soluble aglycone would be the active part of the tomatine molecule and the sugars were regarded as a solubilizing group aiding delivery of the tomatine to the membrane, where membrane-bound glycosidases are then presumed to activate the saponin by converting it to the aglycone [61]. This view, however, has received little support because it is incompatible with evidence showing that tomatidine, the free aglycone of the tomatine molecule, does not bind cholesterol [57] and that tomatine affects the permeability of artificial membranes which lack hydrolytic enzymes [27, 28]. More recently, Blankemeyer et al studied the effect of tomatine and tomatidine on frog embryos and frog skin. They found that tomatine increased membrane permeability in frog embryos and decreased sodiumactive transport in frog skin, in contrast to the essentially negative results with tomatidine [62]. This reinforces the hypothesis that the carbohydrate side-chain is essential for the glycoalkaloid activity. Other studies show that partially hydrolyzed tomatine may be toxic to fungi. Thus, Sandrock et al obtained Aspergillus nidulans transformants, carrying the tomatinase gene from S, lycopersici. A transformed strain was as sensitive to tomatine as the fungus before transformation. Although such behavior could be due to poor expression of the tomatinase gene in Aspergillus it is more likely that p2-tomatine, one of the products originated from tomatine by tomatinase, may be toxic to A, nidulans. In fact, the authors showed that^. nidulans was quite sensitive to p2-tomatine and that the transformed strain was even more so [33]. In addition, it was observed that while p2-tomatine and tomatidine were less toxic to most tomato pathogens than tomatine, these partially hydrolyzed products were inhibitory to some saprophytes and nonpathogens of tomato [17]. The mechanism by which these breakdown compounds exert their toxic action is unknown.

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MECHANISMS OF RESISTANCE OF FUNGI TO TOMATINE Correlation between the capacity of pathogens to infect tomatinecontaining plants and their resistance to the glycoalkaloid in vitro, suggest that for many fungi, tomatine resistance is a prerequisite for successful infection [6, 17]. Fungi that invade tomato and other Solanaceous plants use different strategies for protecting themselves from the host tomatine. Avoiding release of tomatine An obvious way to escape the effect of tomatine is to avoid exposure to the compound. Tomatine is usually stored in the vacuole of tomato cells [53]. Stemphylium solani, is a leaf tomato pathogen very sensitive to tomatine [17], but it grows within the intercellular space and middle lamella of leaf [63], here the advancing hyphae may not come in contact with tomatine. A similar situation is true for the hemibiotrophic tomato pathogen Cladosporiumfulvum, also highly sensitive to tomatine but that avoids its release from the cells by restricting growth to the intercellular spaces of tomato leaves without causing cell damage [64]. In this case, it has been suggested that tomatine may play a role in the variety-specific resistance of tomato to incompatible races of C fulvum, through a release of the saponin from leaf cells in the incompatible interaction, thus killing the pathogen or stopping it from invading the plant [64]. The modes of pathogenicity of 5. solani and Cfulvum differ from other tomato pathogens, such as Septoria lycopersici [65] and Alternaria solani [66], which directly penetrate leaves. Other tomato pathogen very sensitive to tomatine are Verticillium dahliae and F. albo-atriim. These fungi are wilt pathogens and may avoid tomatine limiting their growth to the xylem tissue [17]. There is evidence, however, that avoidance of plant saponins is not a very frequent strategy in fungal pathogens. Changing ambient pH to avoid optimal action of tomatine The membraneolytic action of tomatine is strongly impaired at pH lower than five [27] and evidence exists that some fungi may be able to grow on tomatine-containing tomato tissue by lowering the pH at the infection site

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[42]. This is the case of A. solani, that may cope with the effects of tomatine by lowering the extracellular pH to levels at which the saponin is ineffective as an antifungal agent [60]. Altering membrane composition A number of lines of evidence indicate that membrane composition appears to be one of the main mechanisms of resistance of fungi to tomatine. First, fungal species which not produce 3p-hydroxy sterols are more tolerant to tomatine. Hence, Oomycetes such as Pythium and Phytophthora that lack this kind of membrane sterols are highly resistant to the glycoalkaloid. However, they can incorporate exogenous sterols into their membranes when these are added to the growth medium, acquiring then increased sensitivity to tomatine [28, 67]. Thus, Steel and Drysdale studied electrolyte leakage in Phytophthora megasperma in the presence of tomatine, measuring electrolyte loss due to membrane damage in mycelium grown in the presence of increasing concentrations of cholesterol. The sterol-containing mycelium was definitely more susceptible to tomatine than sterol-free mycelium [28]. Accordingly, fungi that normally contain sterols in their membranes become more resistant to saponins when they are grown in the presence of inhibitors of sterol biosynthesis [68]. A detailed sterol-binding studies with tomatine confirm that binding requires membrane sterols with free 3p-hydroxyl groups [28]. Therefore, susceptibility of membranes to tomatine appears to be mainly influenced by sterol composition. Second, the importance of membrane composition in determining the ability of fungi to infect tomatine-containing plant, is further emphasized by experiments involving a tomato-attacking isolate of Fusarium solani. The wild-type isolate was pathogenic only to ripe tomato fruits which contain very low levels of tomatine. However, sterol-deficient mutants of a tomato-attacking isolate of F. solani showed increased resistance to the steroidal glycoalkaloid and gained the ability to infect green fruits of tomato, which are particularly rich in tomatine [69, 70]. Analysis of the progeny from crosses between mutant and wild-type fungi showed that pathogenicity to green tomato fruits, low sterol content, and insensitivity to tomatine were always inherited together, revealing the importance of membrane composition in pathogenicity [70].

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Third, it has been demonstrated, studying tomatine sensitivity of ergosterol mutants of Neurospora crassa, that changes in the type of membrane sterol composition can increase resistance to tomatine [17]. Ergosterol, a 3p-hydroxy sterol, constitutes 80% of the total sterol component of N, crassa [71]. Several mutants affecting sterol biosynthesis and accumulating different sterol intermediates have been isolated [71, 72]. All the mutants became less sensitive to tomatine, and although the interaction of the sterol intermediates of each mutant with the glycoalkaloid is unknown, clearly the diverse types of sterols might interact differently with it, because the different mutants show specific grades of resistance to tomatine [17]. Recently, the erg-S gene from S. lycopersici coding the ergosterol biosynthetic enzyme sterol C-14 reductase has been cloned and has been confirmed that the gene complements the phenotype tomatine-resistant of a N. crassa erg-S mutant [73]. Other research has revealed that the increase of the concentration of glycolipids in cholesterol-containing membrane results in an increase of tomatine sensitivity [29]. Therefore it seems that different alterations of the membrane, not only sterol content, may protect tomato pathogens from the toxic effect of tomatine and other glycoalkaloids. Tomatine-detoxifying enzymes A second major mechanism of resistance to tomatine involves enzymatic detoxification by tomatinases. Although saponins are very numerous and widely distributed in the plant kingdom, detailed studies on saponin detoxification by fungi have been restricted to pathogens of a few plant species, principally to oat and the Solanaceous tomato and potato. This is because structures and antifungal properties of oat, tomato and potato saponins are well established, and that saponin profiles of these plants are relatively simple, in contrast to other plants like alfalfa, where over 20 predominant different saponins have been identified. The importance of saponin-detoxifying enzymes in determining the host range of fungal pathogens has been demonstrated for oat- and wheatattacking isolates of the soil borne fungus Gaeumannomyces graminis. Isolates of G. graminis var. avenae are resistant to avenacin A-1, a saponin predominant in oats. This fungus produces a saponin-detoxifying enzyme, called avenacinase, consisting in a p-glycosyl hydrolase that removes (J, 1-2- and p,l-4-linked terminal D-glucose molecules from avenacin A-1 [74-76]. Isolation of the gene encoding avenacinase allowed

305

the production, by targeted gene disruption, of specific fungal mutants defective in avenacin A-1 activity. Mutants lacking the ability to detoxify avenacin A-1 were more sensitive to the saponin and lost the ability to infect oats, thus demonstrating that avenacinase is necessary for pathogenicity on oat [77]. Interesting, the mutants maintained normal pathogenicity on the alternative host, wheat, which does not produce avenacin A-1. These results show that the ability of the pathogen to detoxify saponins is a determinant of host range, at least for the interaction of G. graminis var. avenae with oat. Furthermore this was the first unequivocal proof that saponins may be important in plant resistance to pathogenic fungi [77].

H

QH> H

/

^ H

CH^OH

0H>

OH

r

H O

y

H

CH^OH

H>H

f H OH

CH,OH

4l OH H

OH

Fig. (2). Different mechanisms to detoxify tomatine by fungal pathogens. 1: F. oxysponim, A. alternata, C. coccodes, F. solani, G. pulicaris, S. botryosum, S, solani, V. dahliae. 2: A. solani, V. albo-atrum. 3: B. cinerea. 4: A. solani.

It is well documented that potato plants produce several saponins, principally a-solanine and a-chaconine, which represent up to 95 % of all the glycoalkaloids in the plant [9]. A pathogen of potato. Gibberella piilicaris, metabolizes both glycoalkaloids by removing the 1,2-bound Lrhamnose [78]. The enzyme hydrolyzing a-chaconine, known as achaconinase, has been purified [79]. a-Chaconinase is inducible by at least three different glycoalkaloids from potato, tomatine, a-solanine and achaconine, although it is only able to use the latter as substrate [79].

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In contrast to other saponins, in tlie case of tomatine an important number of fungal pathogens of tomato is known to produce specific enzymes known as tomatinases, able to hydrolyze the compound (Table 2). These glycosyl hydrolases remove one or more sugars from the tetrasaccharide moiety of tomatine thus impairing its binding to 3phydroxy sterols and avoiding its toxic effect [29, 57]. The metabolites originated by the hydrolysis of tomatine have little or no antifungal activity to most tomato pathogens [17]. Thus, for many phytopathogenic fungi the production of tomatine-detoxifying enzyme may be a determinant to successfully infect tomatine-containing hosts. The mechanisms of action of tomatinases differ, Fig. (2). Whereas some remove a single sugar, most of them release the intact p-lycotetraose group, yielding tomatidine (Table 2). TomatinasefromSeptoria lycopersici S. lycopersici is a foliar pathogen of tomato. Arneson and Durbin demonstrated that crude mycelial extract or culture filtrates from this fungus converted a-tomatine to p2-tomatine. Fig (2), by hydrolysis of the p-l,2-D glucosyl bond on the tetrasaccharide moiety, removing a single D-glucose from p-lycotetraose [80]. The enzyme was partially purified and classified as p-l,2-D-glucosidase [81]. Purification to homogeneity was carried out by Sandrock et aL who named the enzyme P2-tomatinase [33] to distinguish it from other fungal tomatinases that release the entire tetrasaccharide moiety or other sugar molecules from tomatine [17, 82, 83]. Although mainly present in extracellular extracts, the enzyme was purified from intracellular extracts to overcome the problem of extracellular melanin-like pigments that hampered the purification. Avenacinase, an enzyme from G. graminis var. avenae, is related to tomatinase from 5. lycopersici because is able to deglucosilate tomatine by identical mode of action. However, the activity is very low and corresponds to approximately 2% of its activity towards avenacin A-1 [32]. Tomatinase form S, lycopersici, also can cleave avenacin A-1 but has less than 0.01% of activity towards it in comparison to its activity towards tomatine [32]. Therefore, the two enzymes are highly specific for their respective host plant saponins. Purification and characterization of 5. lycopersici tomatinase revealed that this enzyme shares many properties (including immunological cross-reactivity) with avenacinase

307

Table 2.- Fungal pathogens with tomatinase activity. Species

Final enzymatic degradation product

Reference

Altemaria alternata

tomatidine

p-^j

Alternaha alternata f. sp. lycopersici

tomatidine

[17]

Altemaria solani

tomatidine, p2-tomatine, 8-tomatine

[17]

Botrytis cinerea

Pl-tomatine, tomatidine

[17, 36]

Colletotrichum coccodes

tomatidine

[17]

Fusarium solani

tomatidine

[37]

Fusarium oxysporum f. sp. gladioli^

tomatidine

[89]

Fusarium oxysporum f. sp. lycopersici

tomatidine

[17, 35, 83]

Fusarium oxysporum f. sp. melonis^

tomatidine

[89]

Fusarium oxysporum f. sp. niveum^

tomatidine

[89]

Fusarium oxysporum f. sp. radiciS'lycopersici

tomatidine

[89]

Fusarium oxysporum f. sp. tuberosi^

tomatidine

[89]

Gibberella pulicaris^

tomatidine

[91]

Septoria lycopersici

P2-tomatine

[33, 80, 81]

Stemphylium botryosum

tomatidine

[17]

Stemphylium solani

tomatidine

[17]

Verticillium dahliae

tomatidine

[17]

Verticillium albo-atrum

P2-tomatine

[17, 54]

^ Not pathohenic on tomato.

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from G. graminis var. avenae [32, 33]. In fact, a cDNA clone encoding tomatinase from S. lycopersici was isolated using avenacinase cDNA as a heterologous probe [32]. The sequence of 5. lycopersici tomatinase revealed that this enzyme shares 68% amino acid similarity with avenacinase. Glycosyl hydrolases have been grouped into approximately 40 families on the basis of sequence similarity [84, 85]. Within this classification the majority of p-glucosyl hydrolases (EC 3.2.1.21) fall into two families: 1 and 3. The deduced amino acid sequence of the gene encoding tomatinase from 5. lycopersici reveals a high degree of similarity to several other microbial p-glucosidases belonging to family 3 of glucosyl hydrolases [85]. Avenacinase from G. graminis var. avenae belongs to the same family. The members of family 3 most closely related to avenacinase and tomatinase are p-glucosyl hydrolases from fungi that have importance in cellobiose degradation (BGLl from Trichoderma reesei, BGLl and BGL2 from Saccharomyces fibuligera, and BGLS from Candida pelliculosa). The degree of amino acid identity between tomatinase and these other p-glucosidases is 40 to 45%. Avenacinase and tomatinase are more closely related to each other than to other members of family 3, and therefore may represent a subgroup within this family that has the additional ability to degrade saponins [30]. Despite the homology between microbial p-glucosidases of family 3 and tomatinase, it is unknown whether other enzymes from this family, except avenacinase, posses activity towards tomatine or other saponins. The substrate specificity of tomatinase was evaluated by incubating the enzyme with other a- or p-D glycosides. Tomatinase showed a strong preference towards tomatine, having a small activity towards F-gitonin and digitonin, almost undetectable on para-nitrophenyl-p-D-glucopyranoside and no activity at all towards nine other glycosides and/or steroid or triterpenoid compounds similar to tomatine [32, 33]. Many organisms with pglucosidases found within family 3, such as soil-inhabiting saprophytes, may come in contact with saponins in nature and thus possess these enzymes for detoxification or degradation as a food source. More extensive studies on substrate specificity of these enzymes are required in order to establish whether this is the case. Tomatinase from S. lycopersici has been introduced in the hemibiotrophic tomato pathogen C.fulvum, that is very sensitive to tomatine. Tomatinase-producing transformants reveal increased

309

sporulation on cotyledons of susceptible tomato cultivars, and they also cause more extensive infection of seedling of resistant tomato lines [86]. These observations suggest that tomatine may contribute to restrict the growth of C.fulvum to some extent in both compatible and incompatible interactions, but indicate that tomatinase activity is not sufficient for pathogenicity. Tomatinase from Fusarium oxysporum The tomatinase from the vascular wilt pathogen F. oxysporum f. sp. lycopersici has a different mechanism of action than the S. lycopersici tomatinase, releasing the intact p-lycotetraose group to give the aglycone tomatidine, Fig. (2) [35, 83]. The enzyme is extracellular and inducible by tomatine whereas breakdown products (tomatidine and sugars) fail to increase the level of tomatinase activity [35]. When grown in the presence of tomatine, F. oxysporum f. sp. lycopersici secrets a unique protein species with tomatinase activity. The enzyme is a monomer of 50 kDa. In native conditions consists of at least five isoforms with pis ranging from 4.8 to 5.8, though the same molecular mass. Treatment of pure tomatinase with A^-glycosidase F gives a single band of 45 kDa indicating that the different isoforms are glycosylated [35]. The presence of glycosylation in extracellular enzymes is frequent, it aids to protein folding and to the stabilization of protein conformation, and additionally glycosilation facilitates protein transport and confers resistance to proteolytic degradation [87]. The optimum pH range of tomatinase activity is between 5.5 and 7, in agreement with the fact that tomatine reacts optimally with membrane sterols at pH 7 [27]. Thus, in the plant cellular environment with a pH around 7, the enzyme has maximal activity avoiding presumably membrane disruption. Tomatinase activity in infected tomato plants has been determined at different stages of infection [38]. No tomatinase activity is detected in leaves at any stage of infection. In stems, activity is higher during the final wilt stage compared to previous stages of infection. In roots, enzyme activity appeared with the first symptoms and is maintained until wilting. Tomatinase activity is not detected in protein extracts from infected plants before appearance of symptoms (1 to 5 days after infection). This may be due to the low quantity of pathogen in the plant and to the inhibitory effect of tomatine on spore germination and mycelial growth. It

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may also explain the very low tomatinase activity observed in stems of infected tomato plants at stages previous to wilting, where the fungus reaches and proliferates in the stem. Tomatinase activity is also absent in protein extracts from stems and roots of healthy plants, thus ruling out the presence of enzymes with tomatinase activity in non-infected plants. In all cases, the tomatinase activity detected converted the tomatine in tomatidine and p-lycotetraose [38]. Immunoblotting assays using rabbit polyclonal antibodies raised against tomatinase detected a 50 kDa band corresponding to the tomatinase enzyme in protein extracts from roots and stems of plants during the wilt stage of infection [35, 38]. These results demonstrate that the same tomatinase enzyme is induced both in vitro and in vivo. This conclusion is supported by experiments of reverse transcription-polymerase chain reaction (RT-PCR) in planta. Tomatinase gene was indeed expressed throughout the entire disease cycle of F. oxysporum f sp lycopersici both in roots and stems [34]. On the basis of these results it seems clear that expression of tomatinase gene is specifically controlled by the interaction with the host plant. Although several authors claimed that the glycoalkaloid tomatine is not present in sufficient concentrations in roots or stems to play a major role in resistance to Fusarium oxysporum f. sp. lycopersici [7, 57, 88], the fact that tomatinase is induced in tomato plants during infection, strongly suggests that the enzyme may have a specific role in pathogenicity of tomato plant by F. oxysporum f sp lycopersici. Tomatinase activity has been traditionally associated to fungi pathogenic on tomato [17, 30]. Recently, Lairini et al. [89], studying tomatinase induction in various ybrma^ speciales of F. oxysporum, found inducible tomatinase activity not only in the tomato pathogens F. oxysporum f sp. lycopersici and radicis-lycopersici, but also in formae speciales not pathogenic to tomato, such as tuberosi, melonis, niveum and gladioli (Table 2). These formae speciales are natural pathogens of other plant species. Some of these are phylogenetically related to tomato, such as the Solanaceous potato, pathogenized by f. sp. tuberosi, whereas others, are not closely related, such as the Cucurbitaceae muskmelon and watermelon pathogenized by f. sp. melonis and f. sp. niveum, respectively, and the Iridaceous monocot Gladiolus sp pathogenized by f sp. gladioli. The tomatinases found in these formae speciales have very similar characteristics to tomatinase from f sp. lycopersici, including the same mode of action hydrolyzing tomatine into tomatidine and p-

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lycotetraose, the same molecular mass (50 kDa), and they are recognised by polyclonal antibodies raised against f. sp. lycopersici tomatinase [38]. These results suggest that the tomatinases from different formae speciales of F. oxysporum are probably the same enzyme. The function of these tomatinase in formae speciales that do not pathogenise tomato is unknown. One possible explanation could be the presence of tomatine or similar saponins in their host plant species. However, (i) tomatine has not yet been reported in these plants [2, 7, 9] and (ii) although some of these species contain small amount of tomatine and other saponins structurally related to tomatine (e.g. potato contains a- solanine and a-chaconine [2,4, 9, 90]), these are inactive as inducers of tomatinase and, moreover, tomatinase cannot use any of these glycoalkaloids as substrate [89]. In addition, it is clear that tomatinase is not required for pathogenicity in these isolates, at least in the case of F. oxysporum f. sp. melonis, where some strains that are fully pathogens on muskmelon lack tomatinase activity [89]. If they do not have a function in pathogenicity, the presence of tomatinases in different formae speciales of F. oxysporum and in other fungi not pathogenic to tomato such as G.pulicaris [91], points out a close phylogenetic relationship between these formae speciales and may indicate common forms of evolution for fungal pathogens. Two possible models of evolution of formae speciales in F oxysporum and fungal tomato pathogens can be envisaged in general. Fig. (3). In one model, fungal saprophytes that have evolved into fungal pathogens of tomato may have acquired the ability to degrade tomatine as prerequisite for pathogenicity. Fig. (3A). Once the fungus has gained this ability, it may have evolved further into forms pathogenic to other host plants, maintaining (or not) the ability to degrade tomatine. In the second model. Fig. (3B), fungal pathogens with a broad host range could have started as "general pathogen" such as today are V. dahliae and V. albo-atrum; both of these pathogens cause vascular wilt on a wide variety of host plants range throughout the world, without the occurrence of formae speciales [92]. Thus, a hypothetical pathogen with a wide host range should have acquired several pathogenicity traits such as the ability to detoxify tomatine. On the other hand, hosts may have developed new resistance genes to overcome plant resistance mechanisms, and strains of the pathogen have increasingly evolved to forms with a remarkable degree of host specialization. During this process, the strains may have maintained

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(or not) some enzymatic characteristics from the ancestral pathogen, such as the ability to detoxify tomatine. These two models are not incompatible and could both have originated the different formae speciales of F, oxysporum and other fungal pathogens that maintain tomatinase activity and are not able to infect tomato. FUNGAL SAPROPHYTE

tomatinase acquisition

FUNGAL TOMATO PATHOGEN maintains or not tomatinase activity

FUNGAL PATHOGEN OF OTHER HOST PLANT

FUNGAL PATHOGEN WITH BROAD HOST RANGE, HAS TOMATINASE AND OTHER PHYTOANTICIPIN-DEGRADING ENZYMES

plant recognises specific avirulence gene product of the pathogen and develops resistance

EVOLUTION TO PATHOGEN FORMS WITH HIGH DEGREE OF HOST SPECIALIZATION (FORMAE SPECIALES)

Fig. (3). Possible mechanisms of evolution of fungal tomato pathogens and formae speciales. A: Fungal tomato pathogen or formae speciales originated from a non-pathogen. B: Specific tomato pathogen or formae speciales originated from broad host range pathogen.

The tomatinase gene from F. oxysporum f. sp. lycopersici has been cloned recently [34]. This gene encodes a protein that has no sequence homology to any previously described saponinase but which is highly similar to xylanases (family 10 of glycosyl hydrolases) [84, 85, 93-95]. Although F. oxysporum tomatinase does not have any detectable xylanase activity, it remains to be determined whether any of the xylanases listed in this family possesses activity against a-tomatine. In any case, it is

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conceivable that even highly related enzymes, both structurally and functionally, may have very different substrate specificity. As mentioned before, the tomatinase gene from the tomato pathogen S. lycopersici [32] encodes a protein closely related to avenacinase saponindetoxifying enzyme, produced by G, graminis, and that both enzymes are closely related to family 3 of glycosyl hydrolases, which mainly includes p-glucosidases [32]. The fact that F, oxysporum tomatinase shares no sequence homology with tomatinase from S. lycopersici suggests that these different tomato pathogens have developed the ability to cleave tomatine by convergent evolution. This may also explain the different molecular mass of both native tomatinases, 110 kDa for S. lycopersici [33] and 50 kDa for F. oxysporum [35]. The tomatinase from F. oxysporum is encoded by a single gene whose expression is induced by tomatine and fully repressed when mycelium is grown in the presence of glucose. This observation is in contrast to that reported for tomatinase from B. cinerea which is not subject to carbon catabolite repression [36]. Analysis of the tomatinase gene revealed the presence of promoter sequence motifs involved in carbon catabolite repression in yeast and filamentous fungi. Two putative CREA-binding sites [96], suggest that glucose repression may be modulated by CREA. Therefore, it can be concluded that tomatinase production is subject to substrate induction and to catabolite repression. In addition, probably signals derived from the interaction with the plant must regulate expression of this activity since the amount of tomatine found in roots (14 mg/lOOg fresh weight) and in stems (54 mg/lOOg fresh weight) is apparently sufficient to inhibit the growth of F. oxyxporum to some extent [17] and to induce expression of the tomatinase gene [34, 35]. On the other hand, since there is a high sequence homology between tomatinase and glycosyl hydrolases, enzymes presumably required for nutritional purposes, and tomatinase expression is repressed by glucose, it is possible to suggest that, together with its role in detoxification of tomatine, tomatinase might also have a nutritional function. I any case to verify the importance of tomatinase from F. oxysporum f. sp. lycopersici in pathogenicity, the cloned gene is being used to obtain tomatinase-minus mutants by targeted gene disruption, to further study their ability to pathogenise tomato plant.

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TomatinasefromBotrytis cinerea The necrotrophic pathogen E. cinerea causes grey mould disease on many fruits, vegetables and flowers. Although some reports indicate that tomatinase from B. cinerea deglycosylates tomatine completely [17, 41], with the same mechanism that tomatinase of F. oxysporum, Quidde et al. analyzing twelve isolates from different geographic areas found that this fungus metabolizes tomatine by removal of the terminal p-1,3-linked Dxylose moiety, yielding Pi-tomatine [36]. Therefore, for most isolates of B. cinerea the mechanism of tomatinase in detoxifying tomatine differs from the tomatinases of S. lycopersici and F. oxysporum [36]. One of the isolates used by Quidde et aL, was unable to metabolize tomatine and was more sensitive to the glycoalkaloid. In addition, this isolate was not able to induce disease symptoms on detached tomato leaves, but could pathogenize bean leaves, indicating that the ability to degrade tomatine may be required for pathogenicity of B. cinerea on tomato [36]. It would be very interesting to transformed this strain with one tomatinase gene to confirm this hypothesis. The structural relationship of tomatinase from B. cinerea to other tomatinases is unknown, but its molecular mass (70 kDa) is different from the other two enzymes mentioned before, 50 kDa from F. oxysporum [35, 38] and 110 kDa from S. lycopersici [33]. Moreover, when Quidde et al, attempted cloning of the tomatinase gene from 5. cinerea using the tomatinase from S. lycopersici as a probe, they isolated a gene with high sequence homology, whose product had not tomatinase activity but was able to detoxify avenacin A-1 [97], the saponin from oats related to some extent to tomatine. TomatinasefromFusarium solani The occurrence of a tomatine-detoxifying enzyme in F. solani, was studied in liquid medium with or without tomatine. No tomatinase activity was detected in the absence of tomatine, whereas a remarkable increase of tomatinase activity was observed after 12 h of growth in the presence of the glycoalkaloid, the maximal tomatinase activity being observed after 72 h of incubation [37]. Detection of tomatinase activity in F. solani contrasts with the results reported by Defago and Kern [70],

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which found tomatinase activity in F, oxysporum f. sp. lycopersici but were unable to detect such activity in F. solani (strain ETH 4874). The mode of action of F. solani tomatinase was studied by thin layer chromatography (TLC) analysis. No degradation of tomatine was observed using as source of the enzyme the crude extract from fungi grown without tomatine. Crude extracts from F. solani grown in the presence of tomatine converted the glycoalkaloid tomatine into two products, tomatidine and p-lycotetraose. The same results were obtained for both F. solani isolates studied. Hence, the mode of action of tomatinase from F, solani is similar to that of tomatinase of F. oxysporum [35, 83]. In order to investigate whether tomatinases from F. oxysporum and F. solani share similar molecular characteristics, F. solani tomatinase was partially purified. Comparative SDS-PAGE analysis of the protein fractions with and without tomatinase activity showed the presence of a 32.5 kDa band in all positive fractions, while this band was absent in fractions without tomatinase activity The apparent molecular mass of tomatinase of F solani differs from that of F oxysporum (50 kDa), S. lycopersici (110 kDa) [33], and Botrytis cinerea (70 kDa)[36]. The F. solani tomatinase presents a very low activity compared with F. oxysporum enzyme [35, 89]. Western blot analysis showed that the two enzymes also differ in their immunological characteristics since the polyclonal antibody against tomatinase of F oxysporum f. sp. lycopersici did not recognize the tomatinase from F solani. These results suggest that the enzyme from F solani is a novel tomatinase species. When inoculated on tomato fruits, Fusarium solani produced severe rot on the surface of the green fruit one week after inoculation [37]. This result is in contrast to F. solani isolate 4874 used previously by Defago and Kern which did not rot green tomato [70]. To study the possible biological relevance of tomatinase, F oxysporum f sp. lycopersici, with a tomatinase activity five-fold higher than that of F. solani isolates, was used as control. F oxysporum f sp. lycopersici, was not able to grow on the green fruit probably due to the high level of tomatine present; nevertheless, when the fruit began to turn red and its tomatine content decreased, F oxysporum f sp. lycopersici was able to grow into the fruit and produce severe rot. F solani isolates also were more aggressive on red than on green fruit, but whereas F. solani was more aggressive on green

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fruit than F, oxysporum f. sp. lycopersici, this was more aggressive on the red fruit than F. solani [37]. In conclusion, F. solani isolates appear to be less sensitive to tomatine than F, oxysporum f sp. lycopersici, although they produce much lower tomatinase activity, indicating that they must possess other inherent mechanisms of resistance. One possibility is that F. solani may have a lower content of sterols in the membrane than F. oxysporum. Thus, although tomatinase activity may help F. solani to grow on green tomato tissue it does not seem to be determinant in the resistance to tomatine. Other fungal tomatinases The tomatinases known remove either a single sugar or all four sugars from the steroidal tomatine, Fig (2). In a recent study, Sandrock and VanEtten examined twenty-three fungal isolates (12 tomato pathogens, 7 pathogens of other plant hosts, and 2 saprophytic fungi) finding that most tomato pathogens have tomatinase activity [17]. Table 2 lists the fungal pathogens so far known to have the ability to hydrolyze tomatine. Most fungi yield tomatidine by cleaving the p,l-linked galactose and releasing the tetrasaccharide lycotetraose and tomatidine. The tomato pathogen Alternaria solani was reported to degrade also tomatine to tomatidine but releasing monosaccharides rather than a tetrasaccharide [82]. However, Sandrock and VanEtten have shown recently that culture filtrates from A. solani grown without tomatine were able to convert tomatine to tomatidine[17]. Tomatinase activity increased within the mycelium after exposure to tomatine and in this new activity degraded tomatine through sequential hydrolysis to |32-tomatine, 8-tomatine and tomatidine. This result does not suggest that A, solani has only one tomatinase which hydrolyzes more than one glycosidic bond, as other saponin-degrading enzymes do [98]. On the contrary, the different patterns of tomatine degradation observed in the TLC plate, obtained by culture filtrates and mycelium extracts, indicate that there is probably more than one enzyme involved in degrading tomatine. Therefore, further biochemical characterization is necessary to determine the number of enzymes used by A, solani to metabolize tomatine. A few tomato pathogens remove a single sugar from tomatine. Besides to S, lycopersici and B. cinerea, (sections 5.4.1 and 5.4.3.), that remove

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either one glucose to produce p2-tomatine (S. lycopersici) or the terminal D-xylose to yield p i-tomatine, Verticillium albo-atrum cleaves the terminal p,l-2-linked glucose from tomatine [54], originating also P2tomatine. Based on these data, it is clear that there is not a common enzymatic mechanism by which tomato pathogens metabolize tomatine. It even seems that the regulation of tomatinase expression follows a different pattern in specific pathogens. Thus, although tomatinase enzymes usually are inducible by tomatine, as reported for F. oxysporum [34, 35, 83], V. albo-atnim [54], B. cinerea [36, 41], 5. lycopersici [33] and F. solani [37], some of them are constitutive [17]. In B, cinerea, a detectable level of constitutive activity is found in culture supernatant of the fungus grown without tomatine, although a considerable increase of activity is detected when tomatine is added [36]. In addition, although it is unequivocal that tomatinase form F, oxysporum is completely repressed by glucose [34], this is not the case of B. cinerea where tomatinase induction is independent of the presence, or not of glucose [36]. All these observations, together with the fact that the molecular masses of native tomatinases show significant differences, and that the two genes sequenced so far (S. lycopersici and F. oxysporum), do not present similarity, suggest that tomatinases have been acquired by phytopathogenic fungi independently during evolution. Therefore they may represent important tools to both become pathogens of tomatineproducing plants and metabolize the glycoalkaloid as saprophyte. METABOLISM OF THE TOMATINE SUBPRODUCTS: THE AGLYCONE TOMATIDINE In contrast to the extensive knowledge on the metabolism of tomatine by phytopathogenic fungi, there are only few reports dealing with the final metabolism of the subproducts generated by glycoside cleavage of the glycoalkaloid. Initial reports by Sato and Hayakawa [99-101] showed that the fungus Helicostylium piriforme was able to hydroxylate tomatidine and other related steroids. More recently, Weltring et al studied the metabolism of tomatine by the potato pathogen Gibberella pulicaris (anamorph Fusarium sambucinum) [91]. This fungus is not pathogenic on tomato plants and it is suggested that probably uses tomatine as a source of nutrients.

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although it is possible that it evolved from an original tomato pathogen, see Fig. (3). Weltring et al followed both the deglycosilation of tomatine and the further modification of the aglycone moiety tomatidine. When tomatine was incubated in the presence of fungal mycelium, it was completely metabolized in two hours giving lycotetraose and tomatidine. The aglycone was then converted into two more polar products that could be detected after four hours. These products were originated by the introduction of one oxygen atom into tomatidine giving mainly 7-ahydroxy-tomatidine and the corresponding D^-dehydro 7-a-hydroxytomatidenole as secondary metabolite. The structures of these subproducts of tomatine are based on mass spectrometry and nuclear magnetic resonance. These polar compounds were further metabolized after eight hours into at least three compounds with increased polarity, as determined by their migration on TLC plates. All these metabolic products were detected after forty-eight hours of incubation, although at this time tomatidine had completely disappeared [91]. Znidarsic et aL showed that tomatidine induces steroidal hydroxylase activity in the filamentous fungus Rhizopus nigricans [102], and similar activity was proposed to be the cause of hydroxylation of tomatidine in G. pulicaris by a membrane-bound cytochrome P-450 monooxygenase [91]. Cytochrome P-450 monooxygenases are responsible of hydroxylation of progesterone at various positions by Aspergillus species [103, 104], R. nigricans [102, 105], Phycomyces blakesleeanus [106], Sepedonium ampullosporum [107], Botryospheria obtusa [108] and Cochliobolus lunatus [109, 110]. Steroid hydroxylation may reflect defense mechanisms consisting in the removal of hydrophobic steroids toxic to fungal mycelium that may have evolved due to the exposure of fungi to plant secondary metabolites. Further characterizations of fungal cytochrome P-450 monooxygenases by gene cloning and disruption are required to prove their physiological and evolutionary roles. OTHER GENES INDUCED BY TOMATINE Identification of other genes induced by tomatine may provide new insights into the mechanisms of resistance to this glycoalkaloid. The physiological adjustments that take place in the fungal cell during exposure to tomatine are largely unknown. Differential display analysis [111] of mRNA from mycelium of F. oxysporum f sp. lycopersici grown

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on minimal medium either in the absence or in the presence of tomatine led to the isolation of three genes whose expression is selectively induced in response to tomatine (Perez-Espinosa, Roldan-Arjona and Ruiz-Rubio, unpublished results). One of these encodes a predicted cytosolic protein of 313 amino acids with high similarity (about 40 %) to leguminous isoflavone reductases (IFRs). The IFR-like gene is expressed by F, oxysporum during infection of tomato plant, as revealed by RT-PCR analysis with gene-specific primers. Since phytopathogenic fungi are exposed to more than one antifungal metabolite when they invade the plant, it is possible that tomatine could act as a signal to induce not only tomatinase but a number of enzymes able to detoxify different antifungal compounds. This is the case of tomatine and a-solanine in the potato pathogen G. pulicaris. Both glycoalkaloids induce the synthesis of achaconinase, an enzyme that metabolizes the potato-saponin a-chaconine but is not active towards tomatine or a-solanine [79]. In tomato, a pathogen invading the plant may encounter for instance the phytoalexin rishitin, and not only tomatine [112, 113]. Thus, the induction of the IFR-like gene may play a role in detoxification of antifungal isoflavone compounds during pathogenesis. The differential display technique allowed the identification of a second tomatine-induced gene from F, oxysporum. The cDNA sequence predicts a cytosolic protein of 376 amino acids and a molecular mass of 41 kDa, with high homology to microbial and plant pantothenate synthetases. A complementation experiment using this cDNA in a panC auxotroph mutant of Escherichia coli confirmed the pantothenate synthetase gene function. The gene is also expressed constitutively, but its expression increases in the presence of tomatine. Pantothenate is a component of acetyl-coenzyme A. In living organisms, the citric acid cycle consists of a series of reactions that oxidize the acetyl group to two molecules of CO2 in a manner that conserves the liberated free energy for utilization in ATP generation. Acetyl groups enter the citric acid cycle as acetyl-coenzyme A, which is the common product of carbohydrate, fatty acid, and amino acid breakdown. Acetyl-coenzyme A has therefore a critical role in cellular bioenergetics. Besides being a precursor of the phosphopantheteine moiety in Coenzyme A, pantothenate is also part of the acyl carrier protein involved in the biosynthesis of fatty acids. According to the role of pantothenate in ATP generation and fatty acid biosynthesis, it could be important for the fungus to induce pantothenate

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synthetase during plant infection for two reasons. First, tomatine stimulates sporulation in vitro [55] which implies a high energy demand. Second, the toxic effects of tomatine, as mentioned before, are attributed to its ability to complex with membrane sterols [27-29, 64, 114, 115]. Thus it is reasonable to envisage a scenario where the esterol components of the membrane are being reduced and biosynthesis of fatty acids is increased in order to repair the damages in the cell membrane. A third tomatine-induced gene from F. oxysporum has been identified that encodes a polypeptide with some similarity to cereal storage proteins. The endosperm of cereal grains serves as a seed storage organ where carbohydrates and proteins are laid down in order to feed the embryo during germination. The putative storage protein from F. oxysporum, as other storage proteins from plant seeds, is rich in glutamine and proline, containing in some regions more than 50% of these amino acids. Genes encoding storage proteins have been isolated from several cereal species [116], but there are no reports on any fungal genes and no formal descriptions of storage proteins in fungal spores exist in the literature. VanEtten et al, described a possible storage protein in dormant spores of the pathogen Botryodiplodia theobromae, which produces fruit rot. [117]. Approximately 23% of the total protein isolated from the spores consisted of a single polypeptide that was degraded during germination [118] . A region of the protein encoded by the F. oxysporum gene has homology to glutenin and y-gliadin from wheat, and to y-hordein from barley. Convergent evolution between seed storage proteins from different plants has been reported [119], as indicated by same structure of reserve proteins coming from different origins. Since spore production is stimulated by tomatine in vitro [55], it is reasonable to consider that the gene induced by tomatine should encode a protein that is stored in the spore and used during germination. FUTURE PERSPECTIVES There is increasing evidence that tomatine and other saponins that inhibit fungal growth in vitro act as phytoprotectants in plants. Recent data show that mutants of oat deficient in the saponin avenacin A-1 are compromised in their resistance to a variety of fungal pathogens, suggesting that sensitivity is a direct consequence of saponin deficiency [56]. Fungal mechanisms to resist toxicity of these compounds thus

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appear to be important for pathogenicity. Among the mechanisms of saponin resistance, enzymatic degradation has received most attention, most likely because characterization of genes encoding degradative enzymes offers a more immediate route to genetic tests of function through targeted gene disruption [31]. In the case of tomatine, most of the data indicate that the capacity to actively degrade tomatine is indeed a prerequisite for pathogenicity on tomato, at least in most cases [17]. Strikingly, several lines of evidence strongly suggest that tomatinasedegrading enzymes have been acquired independently by different tomato pathogens. First, tomatinases of fungal origin differ in their mechanism to remove sugar moieties from the glycoalkaloid. Second, predicted amino acid sequences from the two tomatinase genes cloned so far suggest that these enzymes belong to divergent families of glycosyl hydrolases, sharing few structural features. These results, together with the fact that certain fungal pathogens of other plant species unrelated to tomato, also produce tomatinase activity [89], raise intriguing questions about the nature of the evolutionary mechanisms that may have lead to host plant specificity in pathogenic fungi. In contrast to enzymatic degradation, nondegradative mechanisms of tomatine tolerance have received relatively little attention so far. The importance in pathogenicity attributed to these mechanisms is likely to increase in the future, as the molecular bases of tolerance will become clearer. Tomatine offers a suitable model for such studies since it is commercially available, tolerated by a considerable number of fungal tomato pathogens and has a mode of action relatively well known. It appears clear that membrane sterol content is a major factor in tomatine resistance [28, 29], but other mechanisms may also play an important role in the capacity of fungi to tolerate high concentrations of the glycoalkaloid. A common phenomenon in fungi involves the appearance of resistance to multiple, structurally unrelated fungitoxic compounds. Thus, mutants of the saprophytic fungus Aspergillus nidulans that were resistant to the fungicide thiazole also showed increased tolerance to tomatine [120]. Resistance to a broad range of antifungal compounds is mediated by ATP-binding cassette (ABC) membrane transport proteins [121]. It is conceivable that phytopathogenic fungi may use this kind of efflux mechanism to prevent buildup of high intracellular concentrations of phytoanticipins [122]. A predicted ABC transporter gene in the saprophytic fungus A, nidulans has recently been isolated and shown to

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be induced by the pea phytoalexin pisatin [120]. More compellingly, a transformant of the rice blast pathogen Magnaporthe grisea carrying a disrupted copy of an ABC transporter gene has dramatically reduced pathogenicity towards rice plants [123]. It is likely that even more, currently unknown mechanisms exist in fungi to withstand the toxicity of tomatine. The recent identification of several tomatinase-induced genes from F. oxysporum, all of them encoding predicted intracellular polypeptides, suggests that the presence of the saponin triggers major changes in fungal metabolism. To understand these changes and how they contribute to tolerance of tomatine and pathogenicity to plants is an exciting challenge for future research. ABBREVIATIONS RT-PCR TLC

= =

Reverse transcription-polymerase chain reaction. Thin layer chromatography.

ACKNOWLEDGMENTS This work was supported by grants from The European Commission (BIOTECH, Contract No. BIO2-CT94-3001) and from Junta de Andalucia (group 3084).

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B. V. All rights reserved.

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HEME AGGREGATION INHIBITORS: ANTIMALARIAL DRUGS TARGETING AN ESSENTIAL BIOMINERALIZATION PROCESS JAMES ZIEGLER, RACHEL LINCK, DAVID W. WRIGHT Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282-1530, USA ABSTRACT: Malaria, resulting from the parasites of the genus Plasmodium, places an untold burden on the global population. As recently as 40 years ago, only 10% of the world's population was at risk from malaria. Today, over 40% of the world's population is at risk. Due to increased parasite resistance to traditional drugs and vector resistance to insecticides, malaria is once again resurgent. An emergent theme from current strategies for the development of new antimalarials is that metal homeostasis within the parasite represents an important drug target. During the intraerythrocytic phase of its life cycle, the malaria parasite can degrade up to 75% of an infected cell's hemoglobin. While hemoglobin proteolysis yields requisite amino acids, it also releases toxic free heme (Fe(III)PPIX). To balance the metabolic requirements for amino acids against the toxic effects of heme, malaria parasites have evolved a detoxification mechanism which involves the formation of a crystalline heme aggregate known as hemozoin. An overview of the biochemistry of the critical detoxification process will place it in the appropriate context with regards to drug targeting and design. Quinoline-ring antimalarial drugs are effective against the intraerythrocytic stages of pigment-producing parasites. Recent work on the mechanism of these compounds suggests that they prevent the formation of hemozoin. Evidence for such a mechanism is reviewed, especially in the context of the newly reported crystal structure of hemozoin. Additionally, novel drugs, such as the hydroxyxanthones, which have many of the characteristics of the quinolines are currently being investigated. Recent work has also highlighted two classes of inorganic complexes that have interesting antimalarial activity: (1) metal-N402 Schiff base complexes and (2) porphyrins. The mechanism of action for these complexes is discussed. The use of these complexes as probes for the elucidation of structure-activity relationships in heme polymerization inhibitor design and the loci of drug resistance is also detailed. As the biochemistry of the complicated interactions between host, parasite, and vector become better understood, the rationale for new antimalarial drug treatments will continue to improve. Clearly, the homeostasis of metal ions is a complicated biochemical process and is not completely understood. For the immediate future, it does, however, provide a clear target for the development of new and improved treatments for malaria.

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INTRODUCTION As recently as forty years ago, only 10% of the world's population was at risk from malaria. Today, due to increased parasite resistance to traditional antimalarial drugs and vector resistance to insecticides, over 40% of the world's population is at risk. There is little doubt that malaria is once again resurgent. The disease, endemic in over 100 countries, infects 300-500 miUion people and results in the death of over 2 million children annually. The economic strain placed on developing countries reached 2.0 billion dollars (US) annually in 1995 and is predicted to increase [1,2,3]. The discovery of new therapies for malaria is complicated by the fact that any potential drug must satisfy two criteria: (1) the targeted process must be absent in the human host or significantly different between the parasite and human counterparts to ensure that drugs will be parasite specific and (2) the targeted process must be essential for parasite growth and survival in vivo [4]. Thus, unique and essential biochemical targets must first be identified. Research to date indicates that biochemical pathways in Plasmodium falciparum which may be viable targets for drug development include: the shikimate pathway, dihydrofolate reductase, lactate dehydrogenase, aspects of electron transport, hemoglobin catabolism and heme detoxification [5]. While the development of new treatments to combat drug resistant strains of Plasmodium is critical, significant progress can only be made with a substantial improvement in our understanding of the biochemistry of the parasite. Of the four species of parasites (Plasmodium falciparum, Plasmodium vivax, Plasmodium berghei, and Plasmodium malariae) responsible for human disease, P. falciparum is the major cause of malaria deaths, accounting for 90% of the cases in sub-Sahara Africa and 50% of the cases in Asia and Latin America. The life cycle of the parasite consists of three general stages. Upon infection of a human host by the female Anopheline mosquito vector, the malaria parasites move into the liver (sporozoite/hepatic stage) and rapidly reproduce for approximately five days. The parasites then burst firom the liver, enter the bloodstream, and within minutes invade the host's erythrocytes (merozoite/ intraerythrocytic stage), where they grow and divide passing through several morphological changes (ring stage, trophozoite stage, and schizont stage). Every 48-78 hours, the red blood cells rupture.

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dispersing more parasites in the merozoite stage along with waste products and toxins into the bloodstream. This produces the tell-tale clinical signs of malaria: fever, chills, and anemia. Untreated, these symptoms escalate into vascular blockage, cerebral malaria, coma, and death. The released parasites then invade other red blood cells, launching the cycle again. Every cycle, some invading parasites develop into sexual forms (gametocyte/sexual stage) that are subsequently ingested by uninfected mosquitoes wherein the parasites reproduce. These parasites make their way to the salivary glands of the mosquito, ready to move into another victim [6]. Therapeutic agents targeted against the parasite's unique pathway for hemoglobin catabolism have received considerable attention [4,7]. The primary focus of this review will be an examination of recent developments in the elucidation of the molecular details of the efficacy of an important class of antimalarial drugs, the heme aggregation inhibitors. The scope of this review will include an examination of the biochemistry involved in hemoglobin catabolism, the process of heme detoxification, the mode of action of traditional quinoline antimalarials and how an improved understanding of the process of heme aggregation and the mode of action for these compounds is yielding new strategies for the development of antimalarial compounds. Hemoglobin Catabolism Hemoglobin Proteolysis

Given that the malaria parasite has only a limited capacity to synthesize amino acids de novo or scavenge them exogenously, the parasite must catabolize them from the host. The oxygen transport protein hemoglobin provides these needed amino acids. During the intraerythrocytic phase of its life cycle, the malaria parasite can degrade up to 80% of an infected erythrocyte's hemoglobin [8]. Thus, in an average patient with 750 g of circulating hemoglobin and a heavy malarial infection of 20% parasitemia, as much as 100 g of hemoglobin can be degraded during the trophozoite stage of the intraerythrocytic cycle. The catabolism of hemoglobin occurs within a specialized acidic lysosomal organelle called the digestive vacuole (pH 4.5-5.2) [9]. Although early investigations of hemoglobin catabolism were inconclusive, several possible

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hemoglobinase activities were noted. With improved methodologies, a suite of Plasmodial proteinases were subsequently identified, including two aspartic proteases, plasmepsin I and II, and one cysteine protease, falcipain. There is additional evidence for the presence of one or more metalloproteases in the digestive vacuole as well. Through a variety of studies, an ordered cataboHc process of hemoglobin has been elucidated. Scheme (I) [10]. Scheme I. Ordered Catabolic Pathway of Hemoglobin Degredation within the Digestive Vacuole oi P. falciparum. Hemot ;lobin

PLASMEPSIN I AND n Heme Release

^ ^

T •"

Large Fragmenis FALCIPAIN \f

AGGREGATION

Small Fragments

METALLOPROTEASES

^f Hemozom

r Cnvill P(

• TRANSPORT TO CYTOSOL

Amino Acids J

Hemoglobin degradation is initiated by the action of the aspartic proteases which make an initial cleavage between residues a33Phe and a34Leu. Laser desorption mass spectrometry experiments clearly detect fragments for peptides 1-33 and 34-141 in vitro and in vivo [11]. The cleavage site is located in the hinge region of the domain responsible for holding the hemoglobin tetramer together when oxygen is bound [12]. The scission of this hinge results in the unraveling of the protein, thereby facilitating subsequent proteolysis. Plasmepsin I and II subsequently cleave the resulting fragments at secondary sites. It should be noted that the enzymes have altemate

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Specificities for these cleavages [8,9]. Plasmepsin I prefers phenylalanine in the PI position, while plasmepsin II prefers hydrophobic residues, particularly leucine in the PI' position, flanking the scissile bond. Not surprisingly, the aspartic proteases also maintain different sensitivities to various peptidomimetic inhibitors [13]. These inhibition studies have definitively shown that blocking plasmepsin I is lethal to the parasite and that plasmepsin II can not compensate for the loss of plasmepsin I activity. Recently, a number of specific inhibitors, both peptide and nonpeptide based, have been found for plasmepsin I and II [14,15,16]. The large fragments produced by the action of plasmepsin I and II are substrates for the cysteine protease falcipain (Mr 28 kD) [17]. The enzyme has substrate specificity similar to that of cathepsin L [18], generating a number of smaller fragments fi-om the globin chains. Inhibition of falcipain with a variety of cysteine protease inhibitors results in the marked swelling of the digestive vacuole, accumulation of undigested hemoglobin, and parasite death [19,20]. Recent results of Goldberg et al. suggest that these smaller fragments are further acted on by a metalloprotease found in the digestive vacuole [21]. Their studies, combining HPLC and laser desorption mass spectrometry, reveal additional peptide fragment pattems resulting fi-om a chelate sensitive enzyme. They have proposed that the action of this enzyme is the final step of hemoglobin degradation prior to transport of the small peptides from the digestive vacuole into the cytoplasm where they are further degraded to individual amino acids. Such a mechanism is further supported by the observation that no single amino acids have been found in extracts of the digestive food vacuole [22]. Consequently, this also implies that there are no exoproteases in the digestive vacuole. Although the design, synthesis and pharmacokinetic properties of P. falciparum protease inhibitors is not the purview of this review, it should be emphasized that such compounds represent an important target for the development of antimalarial therapies. Heme Detoxification

While hemoglobin proteolysis yields needed amino acids, it also releases toxic free heme. Fig. (1) Upon proteolysis, heme is released into the digestive vacuole where the iron of the heme moiety is oxidized from the predominantly ferrous (+2) state to the ferric (+3) state. Estimates suggest

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that the heme concentration within the digestive vacuole can reach as high as 400 mM [10]. At such levels, heme can disrupt metaboHc function via peroxidation of membranes [23,24], inhibition of enzymes [25,26], and the generation of oxidative free radicals [27]. To balance the metabolic requirements for amino acids against the toxic effects of heme, malaria parasites have evolved a detoxification mechanism which involves the formation of a crystalline heme aggregate known as hemozoin (malaria pigment, p-hematin) [28], The storage and/or detoxification of metal-containing species is a critical component of metal ion homeostasis and one of the primary functions of a biomineral. Fig. (1). The structure of Fe(III)protoporphyrin IX.

N-Donor of Pynole Heterocycle

Propionate Group Fe(III) Atom

On a time scale relevant to the catobolism of hemoglobin (2-4 days) during erythrocytic residence, aggregates of heme do not spontaneously form from either free heme or hemoglobin under the physiological conditions of the digestive vacuole [31], suggesting a mediating process which facilitates the formation of hemozoin. Researchers have proposed three different causative agents: (1) a heme polymerase, (2) a lipidmediated process or (3) a nucleating template protein. A review of the possible mechanisms for hemozoin formation reveals an exquisitely redundant system designed to ensure that the potentially fatal oxidative damage of free heme is tightly controlled. Slater and Cerami reported the trophozoite extracts of P, falciparum could polymerize monomeric heme in a protein concentration-, time- and pH- dependent fashion. Further, this activity, attributed to a "heme

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polymerase," was reported to be heat labile, destroyed by SDS denaturation, and inhibited by known antimalarials (see below) [29]. Similar results were obtained by Chou and Fitch using extracts from P. berghei [30]. Subsequently, Dom et aL contested this interpretation [31]. They could not find a heat labile activity, but did demonstrate that both purified hemozoin and synthetic p-hematin could seed the polymerization reaction of the malaria pigment. They concluded that the "heme polymerase" activity was not an enzymatic process, but rather a chemical process. In response, Cerami and colleagues have posited that their "heme polymerase" activity, isolated in a new acetonitrile extract, is in some way mediated by lipids [32]. Extraction of parasite-derived hemozoin with acetonitrile followed by HPLC analysis revealed a fraction with heme polymerization activity. The fraction contained significant amounts of organically bound phosphorous which could be cleaved by phospholipase B. Following exhaustive saponification at high temperature, mass spectrometry revealed several fatty acids, including oleic, palmitic, and stearic acid. The efficacy of lipids to polymerize heme has been recently confirmed by Dom et aL in a comparative study of in vitro synthesis methods for hemozoin [33]. They have shown that seeding by hemozoin, an acetonitrile extract of trophozoites, and several lipids of undefined phase (L-a-phosphatidylethanolamine, L-aphosphatidyl-L-serine, L-a-phosphatidylcholine, and sphingomeline) were all capable of initiating hemozoin formation. Similar results have been obtained by Fitch et al. in studies of chloroform extracts from cell free preparations of erythrocytes infected with P. berghei [34]. Additionally, they demonstrated that the individual unsaturated fatty acids, oleic, arachidonic, linoleic, and palmitoleic acids, and mono- and di-oleolyglycerol, were active in the aggregation of p-hematin, while the unsaturated fatty acids, stearic and palmitic, as well as cholesterol, trioleolglycerol, di-oleoylphosphatidylethanolamine and dioleoylphosphatidylcholine were inactive or slightly active. While there is no doubt that free lipids can facilitate the formation of hemozoin in model systems, their potential biological role must be placed in the appropriate context. The vast majority of these lipids are involved in cellular structures (organism membrane, organelles, etc.), not freely soluble in the cytoplasm. The methods of extraction modified from Bligh and Dyer [35] by Cohen [36] were designed to extract all of the available

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lipids. Thus, fractionations from trophoizoite extracts contain a number of lipids not associated in any way with the locus of hemozoin formation, the digestive vacuole. Consequently, the observed activity may or may not be completely biologically relevant. Further, in preparative extracts from the digestive vacuole, many of these lipids comprise the membrane of the vacuole and are not available to participate in the aggregation of released heme. In order to investigate the biological relevance of lipids in the biomineralization of heme, more detailed examinations of the composition of Plasmodial organelles is required, as is a detailed analysis of the concentration of non-membrane associated lipids within the digestive vacuole. Further, model studies of hemozoin aggregation in the presence of defined lipid architectures, such as monolayers, bilayers, langmuir-blodgett films and various micellular organizations are needed. The experimental results will significantly enhance the understanding of the role of lipids in the detoxification of heme. Another possible participant in hemozoin nucleation is the histidinerich protein (HRP). Using monoclonal antibodies to probe the proteins of the digestive food vacuole of P, falciparum, Sullivan et al. identified two histidine-rich proteins, HRP II and HRP III, and demonstrated that these proteins could mediate the formation of hemozoin [37]. HRP II (Mr 35 kD) contains 51 repeats of the sequence Ala-His-His (76% of the mature protein is histidine and alanine) [38], while HRP III (Mr 27 kD) contains 28 Ala-His-His sequences (56% of the mature protein is histidine and alanine) [39]. While a clone lacking both HRP II and III has been shown to produce hemozoin, a third protein which cross reacted with anti-HRP monoclonal antibodies has also been found in the digestive vacuole of this strain. Although this protein has not been isolated or shown to mediate hemozoin formation, a candidate is HRP IV (Mr 10 kD) which is 31% histidine [40]. Biophysical characterization showed that a single HRP II protein bound 17 molecules of heme [35]. In an in vitro heme polymerization assay, HRP II promoted the synthesis of hemozoin, while controls, such as the proteins bovine serum albumin and lysozyme or the homopeptides polyhistidine, polylysine, and polyasparagine, did not. FT-IR analysis of the reaction product showed the characteristic vibrations of hemozoin. The polymerization activity had a pH maximum near 4.0, which dropped off precipitously near the pKa of histidine. The heme polymerization

335

activity of HRP II was inactivated by boiling and inhibited by chloroquine. While there is compelling evidence to suggest a physiological role for the HRP's in the detoxification of heme, what is the specific function of these proteins? Several research groups have suggested that the HRP's are likely to play a role in the nucleation of hemozoin. Furthermore, the presence of the Ala-His-His repeat motif is reminiscent of a variety of nucleating scaffold proteins used in biomineralization [41,42,43]. In these scaffold systems, the three-dimensional structure of the protein yields a pre-organized functionalized surface that serves as a template for nucleation. For example, many carbonate and phosphate biominerals are associated with carboxylate-rich (Glu and/or Asp) and phosophorylated (phospho-Ser) proteins, respectively. These groups act as surrogate oxyanions that simulate the inorganic stereochemistry in the first layer of the biomineral, thereby, acting as a nucleation site. A novel approach to investigating the role of HRP's as bionucleating templates has been used by Ziegler et aL They have synthesized a model template protein based on dendrimeric multiple antigenic peptides [44]. An examination of the Ala-His-His repeats within HRP II reveals that a common organizational element is the tandem 9-mer repeat AlaHis-His-Ala-His-His-Ala-Ala-Asp. It should be emphasized that the nucleation model does not represent a unique organization of the Ala-HisHis tripeptides in HRP II, simply the initial organization examined. The first generation bionucleating templates (BNT I and II) couple the putative nucleating domain of HRP II to a tetralysine dendrimer core yielding model proteins with 8 Ala-His-His and 16 Ala-His-His repeats, respectively. Fig. (2) The templates were capable of binding near stochiometric amounts of not only Fe(III)PPIX, but a variety of other substrates, such as protoporphyrin IX (PPIX), Zn(II)protoporphyrin IX (Zn(II)PPIX), tetrasulfantophthalocyanine (PcS) and Ni(II)tetrasulfonatophthalocyanine (Ni(II)PcS). The demonstrated lack of a metal dependence, or even a metal requirement, suggests that substrate recognition of the HRP's is not mediated by axial ligation of a histidine residue to a metal ion. Further, given that the pKa of the imidazole side chain of histidine is 5.8, rendering the moiety protonated under the relevant physiological conditions, axial ligation to a metal is expected to be unlikely. Therefore, binding of the substrate is most likely mediated via a combination of n-

336

Stacking and electrostatic interactions. A recent study of the pH behavior of HRP II substantiates our original hypothesis about the mode of heme binding [45]. Fig. (2). Dendrimeric peptide bionucleating template composed of putative minimal binding domain from HRP II. BNTI has one layer of nucleating domain, while BNT II has two layers [44].

Gly-C0NH2

Bionucleating Template Nucleating Domain Asp-Ala-Ala-His-His-Ala-His-His-Ala Q

Linkage

(^

Lys Anchor

Like HRP II, the BNT's were capable of mediating the formation of insoluble heme aggregates with the characteristic IR spectrum of hemozoin in a modified in vitro assay. Fig. (3) The amount of insoluble heme derivatives formed was dependent on the number of binding domains linked to the tetralysine core, the template concentration and the incubation period of the reaction. The templates had an optimal pH regime between 4.0 and 4.5, above which the polymerization activity decreased. Chloroquine inhibited the template-mediated synthesis of hemozoin-like material, consistent with previous reports of HRP II inhibition by this drug. The fact that a biologically active molecule such as the bionucleating templates could be constructed from a single consensus motif from HRP II composed of only nine amino acids strongly suggests that these molecules, and by analogy HRP's found in the digestive vacuole of P. falciparum, function as a nucleating scaffold for the initiation of heme polymerization to hemozoin.

337

Fig. (3). Hemozoin Production Mediated by Bionucleating Templates. Representative polymerization assay with 50 \xM of hemin in 2 ml acetate buffer (500 mM, pH 4.8) at 37° C. BNTI and BNTII were used in 1 and 2 nmol amounts. Chloroquine (CQ, 100 ^M) was included with BNT I and BNT II in inhibition reactions. Polyhistidine and bovine serum albumin in approximately 1 and 2 nmol amounts were used in protein control experiments. The blank control was the acetate buffer above. Base line amounts of insoluble aggregate are consistent with those previously reported under similar conditions.

• • 1 nmole Tenplate r ? ^ 2 nmole Template

a> 2 M


Mai JULL &

/ /

/

/

It is important to stress that the variety of reported methods for the production of malaria pigment are not in any way mutually exclusive. Considering the fact that heme accumulation is absolutely toxic to the organism, it is not surprising that there are multiple, redundant systems to initiate and propagate hemozoin formation. While the preponderance of evidence clearly establishes that an enzyme is not required for the catalytic polymerization of hemozoin, critical questions concerning the possible role of proteins or lipids in the initiation/nucleation of hemozoin remain. Further studies of the biochemistry of hemozoin aggregation w^ill continue to provide insight into a specific and essential Plasmodia biochemical pathway. The result will undoubtedly aid in the design of new antimalarial therapies.

338

Chemical and Structural Properties of Hemozoin Carbone first isolated malaria pigment from the autopsied spleen of a 33 year-old man in 1891 [46]. From that point, the literature is beset with controversy over the material's composition, the presence of constituent proteins, the linkages between the heme units and the charge and spin of the central iron atoms within the individual heme units. Scrutiny of the literature reveals that the disparate reports of hemozoin's composition is primarily the artifacts of various purification attempts [47]. The reported composition of hemozoin from P. falciparum has ranged from 40-45% Fe(III)PPIX and 60-55% protein [48] to 65.1% protein, 15.5% Fe(III)PPIX, and 6% carbohydrate [49] to 100% Fe(III)PPIX [50]. The most recent studies indicate that preparations involving the exhaustive solubilization of non-specifically associated protein components by sonication, SDS buffers, and overnight protease digestion yield a substance consisting of only Fe(III)PPIX [50]. The long-standing model of hemozoin's structure is that of a coordination polymer of Fe(III)PPIX's in which an oxygen from a propionate group from one unit serves as an axial ligand to the fivecoordinate ferric ion of another [50,51]. Fig. (4A) Analysis of the carboxylate stretching bands from the propionic acid by IR and Raman spectroscopies provides strong evidence for the proposed FeO(propionate) linkage [50,52,53,54]. Additionally, models for the local iron environment of hemozoin derived from extended X-ray absorption fine spectroscopy (EXAFS) are consistent with a five coordinate iron atom of four porphyrin nitrogens and an axial monodentate oxygen atom originating from the carboxylate linkage with an Fe-0(propionate) distance of 1.90 A [50]. The electronic nature of hemozoin has, until recently, been ambigious. EPR studies of either P. falciparum- or P, berghei-dQUYGd hemozoin revealed a number of signals attributable to high spin species with g = 5.8 and g// = 2.0 at 27 K [55] and 77 K [56]. Reminiscent of monomeric Fe(III)PPIX(Oac), such signals led Bremard et al [53] to propose that hemozoin is composed of high-spin monomeric Fe(III)PPIX linked to uncharacterized "biological residues." In another study, at 10 K, investigators reported signals at g = 3.80 and 1.95 which the authors attributed to a low spin state [50]. More recently, Bohle et al, have completed the first detailed temperature dependent analysis of both the

339

Fig. (4). The Emerging Structure of Hemozoin. (A) Spectroscopically based model of hemozoin as a linear coordination polymer of heme units linked by a propionate linkage. (B) Hydrogen-bonded model of hemozoin from X-ray powder diffraction data showing two strands of heme units hydrogen bonded via the other propionic acid groups. (C) Current X-ray model of hemozoin revealing a hydrogen-bonded network of heme dimers linked by reciprocating axial propionate linkages.

340

EPR and Mossbauer spectroscopy of hemozoin [57]. Their work shows strongly temperature-dependent EPR spectrum that below 21 K has a distinct rhombic pattern with g values of 5.79, 3.80, and 2.04. Further, the Mossbauer spectra in a weak appUed field is only consistent with a single high-spin S=5/2 iron environment, thereby, ruling out structural models consisting of heterotrimeric repeat units with multiple iron environments [58]. With strong spectroscopic evidence for a unique coordination polymers of five coordinate heme in a unique high spin S=5/2 environment linked by a monodentate carboxylate group, only the actual crystal structure of hemozoin remained uncertain. Although X-ray powder diffiraction had been used to compare hemozoin with synthetic p-hematin as early as 1991 [47], the small size of natural and synthetic crystallites and variable phase heterogeneity of samples stalled efforts for more complete crystallographic analysis. It was not until high resolution synchrotron X-ray radiation was applied to the challenge that significant progress was made on the structural characterization of hemozoin. A refined topological model by Bohle and coworkers [59] proposed a variation of the dogmatic linear coordination heme polymer introducing hydrogen-bonded propionic dimerization between polymer chains. Fig(4B) Further, this high resolution powder study unambiguously demonstrated that the heme aggregate in the digestive vacuole of P. falciparum parasites was identical to synthetic phematin. Recently, the archetypal model of hemozoin has been overturned. Using simulated annealing techniques to analyze powder data obtained fi-om a high-resolution synchrotron source, Pagola et al [60] have shown that the correct structure of hemozoin is that of a dimeric heme unit linked by reciprocating iron-carboxylate bonds to one of the propionic side chains of each porphyrin. Additionally, the Fe(III)PPIX dimers are hydrogen-bonded into chains forming an extended crystalline network. Fig. (4C) Relevant metrics of the inversion center-related dimeric pair show an Fe-0(propionate) distance of 1.86 A, the central Fe atom 0.4 A above the mean plane defined by the pyrole nitrogens and OO distances for the hydrogen-bonded propionic acid groups of 2.60-2.70 A. This stunning structure not only highlight our natural inclination to think in two dimensions, but has significant implications concerning currently proposed mechanisms of action for a number of antimalarial drugs.

341

Assays for.Drug Efficacy and Hemozoin Inhibition In Vivo, In Vitro and Chemical Assays for the Detection of Antimalarial and Heme Aggregation Activity.

As is often the case in multidisciplinary research, a number of different assays have been developed to monitor the inhibition of hemozoin aggregation (Table 1). Consequently, a great deal of confusion has arisen in the literature concerning the exact efficacy of a particular hemozoin inhibitor. For this reason, it is worthwhile to discuss the principal assays employed in the evaluation of antimalarial compounds and those assays specifically designed to evaluate heme aggregation inhibition. Table 1. Comparison of IC50 Values of Some Typical Antimalarial Agents from Typical Assay Systems. 1

Antimalarial Drug

IC50 (nM) for P.falciparum growth 3D7*

IC5o(nM)for heme aggreg. Hemozoin Seeded*"

Chloroquine Amodiaquine Quinine Mefloquine Halofantrine

14.0 7.8 34.2 23.4 5.8

45 (24.4) 60(15.1) 160(64.8) 120(46.9) 30(184)

ICsoC^M) for heme aggreg. Acetonitriie Troph. Extract' 80 73 78 140 22

IC50 (|iM) for heme aggreg HRPII Mediated'

ICsoC^M) for heme aggreg BNTII Mediated"

75 ND ND ND ND

38 135 ND ND

' Ref. 87; ^ Ref 33;' Ref. 37; ^Ref. 44.

Most of the reported in vivo data presented in the Uterature involves the infection of donor mice with a strain of P, berghe [61]. After a parasitemia of circa 30% is achieved in the donor mice, a blood sample is taken, diluted, and injected into experimental and control mice. The parasitemia load is regularly monitored for 3 to 7 days after infection by blood smear analysis as afimctionof drug dose. While in vivo efficacy assays reveal the true scope of a potential antimalarial's efficacy, the methods are very expensive. Consequently, additional assays have been developed to rapidly assess the effectiveness of lead heme aggregation inhibitors. An important in vitro assay used to evaluate antimalarials is the inhibition of P. falciparum growth in culture. In this assay, parasite cultures are exposed to serial dilution of the potential drugs over 48-72

342

hours. Growth is assessed by the level of incorporation of ["^H]ethanolamine [62] or [G-^H]hypoxanthine [63] by the parasites compared to an appropriate control. The concentration of drug resulting in 50% inhibition of label incorporation (IC50) is calculated by interpolation from a dose response curve. While such an assay does not focus on hemozoin inhibition, it takes into account the critical drug design parameters of biotransportation and localization. In contrast, the in vitro assays for hemozoin aggregation inhibition discussed below provide important insights about the possible target of the drug, but do not, a priori, have any correlation to the efficacy of the compounds against P. falciparum. Only when used together do the in vitro assays begin to provide a clear picture about the potential effectiveness of a specific hemozoin aggregation inhibitors. There are a number of variations of the in vitro heme aggregation assay in the literature [29,31,32,33,37,44,66]. The basis of these assays is the incubation of heme (10-500 JLIM) in the presence of an aggregating template, be it trophozoite extract (25-500 |ig of protein), digestive vacuole extract, parasite-derived or synthetic seed hemozoin (2-10 |ig/ml), histidine-rich proteins or analogs (2-6 nmole) or a variety of lipids (10 mg/ml) in acetate or phosphate buffer (50 mM-500niM) at pH 4.5-5.5 for 12-48 hrs at 37 °C. After incubation, the heme containing pellet is processed by a number of sequential washes including SDS, water, DMSO and sodium bicarbonate (pH 9.5). The remaining insoluble material is typically named "hemozoin." The material is typically characterized by FT-IR for fingerprint vibrations at 1664 cm"^ and 1210 cm'^ Quantification of hemozoin is accomplished spectrophotometrically at 400 nm after solubilization of the final pellet in 0.1 M NaOH or scintillation counting of the incorporation of ^"^C-heme into the insoluble pellet. Another assay commonly referred to as in vitro is the chemical synthesis assay of P-hematin. Developed by Egan et al. [64], a heme substrate solution, with or without inhibitors, is prepared in 0.1 M NaOH. The substrate is added to a pre-equilibrated (60 °C) acetate buffer (final concentration 4.5 M) reaction mixture and stirred for 10-30 minutes. After cooling on ice, the precipitate is filtered, washed with water and dried over silica gel or P2O5. The dried reaction mixture is analyzed by FT-IR and for solubility in 0.1 M sodium bicarbonate (pH 9.1). Several researchers have expressed concems about extrapolating biologically

343

relevant results from this assay system due to the high concentration of acetate [65,66], despite a relationship between intraerythrocytic antimalarial activity and inhibition of P-hematin aggregation in this assay system. It has also been suggested that this assay actually yielded a heme-acetate complex [65]. Recently, Egan and coworkers have definitively shown that there is absolutely no evidence for the formation of a heme-acetate complex, even in 11.4 M acetate solution [67]. Through the use of X-ray powder diffraction, heme aggregates isolated from the chemical synthesis assay of P-hematin are indistinguishable from hemozoin isolated from the parasite or prepared by the method of Slater et al [50]. Heme Aggregation Inhibitors Quinoline-Based Drugs

For over 300 years, the quinoline family of drugs, and chloroquine in particular, has been used as the primary treatment for malaria. Fig. (5) There is substantial evidence that suggests the mode of action for chloroquine and related 4-aminoquinoline compounds arises from disruption of the detoxification of free heme released during host hemoglobin cataboHsm [68,69,70]. Chou and Fitch demonstrated in a comparison of 4-aminoquinolines and quinolinemethanol derivatives that only the 4-aminoquinolines inhibited hemozoin formation in an in vivo mouse model [71]. Although thousands of quinoline-based compounds have been investigated, precise structure-activity relationships for optimal antimalarial activity remain elusive. The difficulty of determining exact structure-function relationships arises from the dual requirements that heme polymerization inhibitors not only disrupt the process of hemozoin formation, but concentrate within the parasite at the site of heme aggregation, the digestive vacuole. As the precise mechanism of both heme aggregation and quinoline transport is largely speculative, deconvoluting the exact functions being affected by the structure of any

344

Fig. (5). Common quinoline-based antimalarials OH

NH

0r

NH

k

NH

NH

J06

cr ^ ^ N Monodesethyl amodiaquine

Amodiaquine

NH

Chloroquine

Bidisethylamodiaquine OH

OH

NH

NH

NH OCH3

Monodesethyl chloroquine

CI

Bidesethyl chloroquine

Amopyroquine CI

OH NH

Pyronaridine CI

or tk

^ OCH3 NH

„ ^ Tebuquine

Mepacrine

OH NH

„ ^

4-

^ ^

4'-dehydroxytebuquine

NH

NH

oil

4'-dehyroxy-4fluoro tebuquine

"/is

H2C =

^O

HN^-^

a' ^

N'

^

N

HC

H

cr^^ N N-t-butyl amodiaquine

Chloroquine-pyroUidinyl

Ro 47-0543 Quinine

s

H2C=HQ HN

r^

HN

HN-^N^

HO^

w

HaCO^ CI

Ro41-3118

Ro 47-9396

Ro 48-0346

Quinidine

345

given compound has proven difficult. Nevertheless, two general trends for typical 4-aminoquinoline ring systems have been noted [72]. The dialkylaminoalkylamino side groups are important for optimal activity, particularly when n = 2 to 6. Significantly, Ridley et al, have shown that chloroquine analogs with shortened side chains retain their heme polymerization inhibitory activity against chloroquine resistant strains of P, falciparum [73]. Another noted reactivity trend is the importance of a chlorine group at C-7 (or in some instances C-6) to achieve maximal activity. Intriguing results have also been obtained by linking two quinoline groups via a bridge at the 4-amino group. Fig (6) Although linkedquinoline rings have long been known to possess antimalarial activity [74], interest in these compounds has been driven by their efficacy against chloroquine-resistant strains. The most successful of the bisquinolines is trans-^^, N^-bis(7-chloroquinolin-4-yl)cyclohexane-1,2diamine (WR 268668). Ridley et al [75] demonstrated that the S,Senantiomer was significantly more potent than the R,R-form in chloroquine-resistant strains. Further, it proved to be a potent antimalarial to P. vivax in vitro and P. berghei in vivo. Unfortunately, this compound was not developed as a drug candidate due to extreme phototoxicity, a significant disadvantage for a drug to be used in a tropical climate. Ismail et ai have developed a series of related compounds in which the cyclohexane-bridging unit is replaced with an aryl unit [76]. Several compounds have been shown to be very efficacious in an in vivo mouse model, comparing favorably to chloroquine. Although extensive toxicity studies have not yet been reported, it seems likely that the addition of another aromatic group will only exacerbate phototoxicity issues. Despite the efficacy of the 4-aminoquinolines and their myriad variations, the precise mode of action remains unclear. One of the earliest hypotheses was that the pharmacological effect of chloroquine was due to its binding to Fe(III)PPIX. In 1967, Macomber et al, proposed that chloroquine accumulation, pigment clumping, and antimalarial activity were the result of the formation of a drug-heme complex [77]. Early attempts to demonstrate chloroquine binding to hemozoin or the presence of fi'ee heme were unsuccessful. Consequently, Fe(III)PPIX was not considered a likely physiological target for

346

Fig. (6). Examples of bis-quinoline antimalarial compounds.

R QR

HN ^ - ^ N H

Ro 47-7737 N \N^-bis(7-chloroquinolin4-yl)[henylene-l ,3-diamine

W%

NH NH

^

o

N ' ,N^-bis(7-chloro-quinolin4yl)phenylene-1,2-diamine

N ' ,N^-bis(7-chloro-quinolin4-yl)-phenylene-l ,4-diamine

chloroquine. Nevertheless, some 12 years later Fitch and coworkers, using equilibrium binding techniques, demonstrated that chloroquine and other antimalarials could bind heme [78]. Recent studies have continued to refine our understanding of heme-drug complex formation (Table 2) [67,79]. Additional studies showed that Fe(III)PPIX and chloroquineFe(III)PPIX complexes could lyse isolated malaria parasites from infected mouse erythrocytes [80]. It should be emphasized that release of less than 0.1 % of fi-ee heme in hemoglobin is more than sufficient to produce lysis. Table 2. Drug-Heme Binding Data and Correlation with Antimalarial Activity. Antimalarial Drug

K., M *•

K„ M*" (drug: heme)

Inhibits P-Hematin Aggregation^

44.0X10' (1:2) Amodiaquine 2.45X10' 9.3 X 10* + (1:2) Quinine 1.26X10* 2.1 XIO* + (1:2) Quinidine 1.05X10' ND + + 7.94 X 10^ Mefloquine 1.2x10* (1:1.5) Halofantrine 1.95X10' 4.6 X 10* + (1:0.5) Desbutyl-halofantrine 1.41 XIO' ND + ' Ref. 67, Assumes interaction with monomeric heme;*'Ref.79;*^Ref64.

Chloroquine

3.31 X 10'

Intraerythrocytic Antimalarial Activity + + + + + + +

1

347

These results suggest that the formation of a heme-drug complex prevents heme sequestration and allows the subsequent level of damaging heme to rise until lysis occurs [81]. These results do not, however, explain the experimental observations that the binding constants of heme: quinine, quinidine, or epiquinine complexes, respectively, do not correlate with their relative efficacy as in vitro heme polymerization inhibitors [82]. Nor do they explain the observation that mefloquine, quinine, and quinidine inhibit in vitro heme polymerization in the presence of 7 to 20-fold excess heme [81]. Sullivan and coworkers attempted to reconcile this problem by proposing that chloroquine first forms a drugrheme complex which in turn is incorporated into a growing chain of hemozoin, effectively terminating polymer extension [83]. This proposal was predicated on the observation that sub-inhibitory doses of [^H]-chloroquine or quinidine were associated with hemozoin, as assessed by electron microscope autoradiography and subcellular fractionation. Additionally, their results showed that in vitro binding of labeled inhibitors to a preformed hemozoin polymer was dependent on the addition of added heme substrate. Unfortunately, a simple chain termination scheme no longer appears relevant in light of the refined crystallographic picture of hemozoin [60]. If the current structural picture of hemozoin invalidates this possible mode of efficacy, how then is the chloroquine localized with hemozoin in the digestive vacuole? The work of Sullivan et aL clearly eliminates non-specific association of chloroquine to hemozoin in the absence of free heme [79]. The recently determined structure of hemozoin suggests, however, another alternative. The surface of the malaria pigment is an array of Fe(III)PPIX's, providing a large Ti-dense region for interactions with excess quinoHnes. With the addition of free heme, a 1:2 quinoline:heme complex could be formed with the 7i-stacked quinoline drug between hemes from hemozoin and solution. Such an arrangement is similar to the solution interactions reported for quinolines and metalloporphyrins by NMR [84,85]. Furthermore, this arrangement would account for the requirement of free heme for the association of quinolines with hemozoin, the localization of chloroquine and hemozoin observed in electron microscopy studies and the observation that the quinoline drugs can be either displaced from the hemozoin by either extensive washing or exchange with excess drugs. It would also rationalize the observation

348

that quinoline.heme complex binding to hemozoin is specific, saturable, and that diverse quinoline analogs can compete for binding [86]. In formulating a mode of action for quinoline-based antimalarials, one of the most significant "red-herrings" has been attempts to make extrapolations of efficacy and mode of action from in vitro heme aggregation assays without serious consideration of biotransportation and mechanisms of localization of drugs to the digestive vacuole. The importance of such considerations is highlighted in a detailed study of the relationship between antimalarial drug activity, accumulation and heme polymerization inhibition by Ward and coworkers [87]. In an examination of 14 quinoline and 13 nonquinoline antimalarials, their studies demonstrated that only those compounds exhibiting structural similarities to quinolines inhibited heme polymerization over a relatively narrow range of concentrations (15-100 fiM). Further, analysis of the experiments showed no obvious correlation between inhibition of heme polymerization and the absolute IC50 of the drug for either chloroquine sensitive or resistant strains. In a chloroquine susceptible isolate, normalization for the extent of drug accumulation did, however, result in a strong correlation between the in vitro activity of the quinoline drugs and their inhibition of heme sequestration. Thus, the antimalarial activity of quinoline-based drugs is dependent on both their ability to inhibit heme aggregation and to accumulate significant concentrations within the digestive vacuole. These results highlight the dynamic process of biotransportation, localization and inhibition that are required to design an effective heme polymerization inhibitor. Detoxification of liberated hemefiromthe catabohsm of hemoglobin is a dynamic process of metal ion homeostasis. Intravacuolar levels of heme have been estimated to be as high as 400 mM [10,80]. Further, studies on the localization of quinoline antimalarial drugs show that the most efficacious compounds concentrate at the highest levels within the digestive vacuole [83]. Chloroquine and amodiaquine accumulate at concentrations as high as millimolar in quinoline-sensitive strains of P. falciparum. Considering the sensitivity of Plasmodia to free heme, only a small percentage of liberated heme would be required to be involved in an effective drug:heme complex in order to lyse the parasite [83,86]. Thus, while some of the excess heme is detoxified as hemozoin, the formation of a drugiheme complexes via 71-71 stacking will effectively hinder the formation of the reciprocating Fe-0(propionate) bonds of the

349

dimeric subunit of hemozoin. Consequently, heme accumulates and disrupts critical metabolic function via peroxidation of membranes, inhibition of enzymes, and the generation of oxidative free radicals. Such a process ultimately results in the observed lysis of the parasite. While current evidence supports a complicated interaction between hemozoin, quinoline animalarials, and free heme as the primary potentiator of antimalarial activity for these compounds, increased resistance to this class of compounds is becoming a critical problem in endemic regions of the world. Consequently, there is much interest in compounds that will disrupt the same critical detoxification pathway via the prevention of heme aggregation. Emerging research suggests that despite increased resistance to traditional quinolines, disruption of hemozoin formation remains a viable drug target. Hydroxyxanthone-Based Drugs

A class of compounds known as oxidant drugs have been cited for their effectiveness against multidrug resistant strains oi Plasmodia [88]. Fig. (7) These drugs result in the enhanced production of oxygen radicals inside parasitized erythrocytes or increase these cells' susceptibility to oxygen radicals. This class is represented by such structurally diverse representatives as primaquine [89] methylene blue [90], and artemisin [91]. Recently, one such oxidant drug, rufigallol (1,2,3,5,6,7hexahydroxy-9,10-anthraquinone), demonstrated a potent antimalarial synergistic interaction with the structurally related compound exifone (2,3,4,3',4',5'-hexahydroxybenzophenone) [92]. The analysis of the synergistic effect by Riscoe and coworkers has led to the development of a new class of antimalarial compounds, the hydroxyxanthones. The origin of the observed synergy between rufigallol and exifone was explained by the "xanthone hypothesis." (Scheme II) [92]. In this proposal, exifone, acting as a prodrug, is transformed inside the infected erythrocyte into a tricyclic xanthone via hydroxy radicals, generated in the futile redox cycling of rufigallol. This hypothesis was supported by additional in vitro studies of infected cells using ascorbic acid to drive the conversion of exifone [93]. When exifone was incubated with ascorbic acid in the presence of iron in a mildly acidic buffered solution, a model reaction yielded a hydroxyxanthone which was identified by GC/mass spectrometry to be 2,3,4,5,6-pentahydroxyxanthone (X5). Subsequently,

350

Riscoe and coworkers synthesized X5 and confirmed its remarkable in vitro activity with both chloroquine sensitive and resistant strains of P. falciparum [66].

Fig (7).£xampies of ^'oxidant" antimalarial compounds. 2H3PO4

OH O OH nifigallol

HO^ ^

"O'

^

"OH

OH OH 2,3.4,5,6-pentahydroxyanthone

With the identification of X5 as a potent antimalarial, Riscoe probed both this compound's mode of action and structure-activity relationships [66]. Through a variety of spectroscopic techniques (UV-vis, IR, NMR), their findings suggest that X5 forms a soluble heme:drug complex with heme monomers or oligomers, not unlike those proposed for the quinoline based drugs. Structure activity studies reveal that inhibitory activity was observed only when the 4- and 5-positions are hydroxylated and was enhanced with a higher degree of hydroxylation. From these studies, Riscoe and coworkers proposed a model for a hemeidrug complex that involves significant interactions between (1) the heme iron and the carbonyl oxygen, (2) the two planar aromatic rings, and (3) the propionate groups of the heme and the 4- and 5-position hydroxyls of the xanthone. Such a model predicts that modifications of the functionaUtites in the 4and 5- position that increase heme propionate interactions will improve the efficacy of the compound. Currently, Riscoe's group is exploring

351

several xanthone congeners containing positively charged alkylamines or amidines at these positions to test this hypothesis. Preliminary results are encouraging [94].

Scheme II. The "xanthone hypothesis" as proposed by Riscoe and coworkers [92].

Jl

ll O

OH

^J^

Rufigallol

OH Futile redox cycling generates oxygen radicals

Exifone

Hydroxyl radicals attack exifone

OH

0

OH

HO HO

H2O

-

OH

HO

Ji

Cyclodehydration/cyclo-activation event

ll OH

Putative intermediate: 2,3,4,5,2',3',4'-heptahydroxybenzophenone

"I ^'^OH OH

2,3,4,5,6-pentahydroxyxanthone

N4O2 Schiff-Based Metallodrugs An unusual class of antimalarial agents targeted against heme aggregation is the Schiff-base coordination complexes developed by Sharma and Piwnica-Worms. Stemming from their application of this type of complex against multidrug resistance in certain cancer cell lines [95], it was demonstrated that pseudo-octahedral complexes with an N4O2 donor set disrupted heme aggregation in P. falciaprum. Using the hexadentate

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ethylenediamine-N,N'-bis[2-hydroxy-R-benzylimino] ligand (ENBPI) and a reduced (R-benzylamino) analog (ENBPA), complexes of Al(III), Fe(III), Ga(III) and In(III) were synthesized [95,96]. Fig. (8) When the R of the ligand was 3-methoxy, only the Fe(III) complex demonstrated strong efficacy. In contrast, the [R-ENBPI]-A1(III), -Fe(III) and -Ga(III) complexes, when R of the ligand was 4,6-dimethoxy, inhibited both parasite growth and heme aggregation, while the In(III) complex did not [97]. Of these compounds, the [4,6-MeO-ENBPI]-Fe(III) complex was the most efficacious with an IC50 of 1.0 |LIM against the growth of P. falciparum in intraerythrocytic culture. Surprisingly, the reduced ligand [3-MeO-ENBPA]-Ga(III) or -Fe(III) complexes were specifically effective against chloroquine-resistant parasites. Comparisons of growth inhibition assays and heme aggregation assays demonstrated while the [3MeO-ENBPA]-Ga(III) complex was not susceptible to the chloroquineresistance mechanisms, it inhibited the same target as chloroquine. A detailed analysis of the crystal structure, which correlates well with NMR solution data, suggested that the spatial orientation of the ligand*s aromatic periphery were critical to imparting the favorable biotransport properties [98]. In an intriguing aside, the efficacy of this complex mapped in perfect linkage with the same 36 kilobase segment of chromosome 7 previously identified as the chloroquine-resistance determinant [99]. While the N4O2 complexes appear very promising, there has been little discussion as to the actual mode of action of these drugs. Goldberg et al. reported that the [4,6-MeO-ENBPI]-Fe(III) complex was a potent heme aggregation inhibitor under both the seeded hemozoin and HRP IImediated hemozoin synthesis assays [97]. Control experiments demonstrated that neither demetallation reactions, counter ions, nor free ligands were responsible for the observed inhibition. Further, the variable efficacy of the Al(III), Fe(III), Ga(III) and In(III) complexes strongly suggests that the active agent is the intact metal complex. Studies of the possible inhibition of digestive vacuole protease enzymes, plasmepsin and falcipain, showed no inhibition by these complexes, suggesting that the metal complexes were in fact disrupting the formation of hemozoin. The possible mode of action of these drugs also emphasizes the dual importance of both the mechanism of transport, as well as hemozoin disruption. Goldberg et al suggested that, in part, the molecular conformation of the metal complex may be important to pharmocological

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activity [97]. Crystal structures of the [R-ENBPI]-Fe(III) and -Ga(III) complexes reveal a rran^-configuration for the phenolic oxygens across the central metal ion [100,101]. Given the larger ionic radius for sixcoordinate In(III) versus Fe(III), Ga(III) or Al(III), molecular modeling suggested that steric constraints force the phenolic oxygens to adopt a cisconformation for [R-ENBPI]-In(III). The obvious ramification is that the trans conformation of these drugs is recognized by a Plasmodial analog of MDRl P-glycoprotein. Thus, the complexes of Fe(III), Ga(III) and Al(III) are transported into the organism, while the In(III) complex is transported less effectively.

Fig. (8). N4O2 Schiff Base Complex. R„ is H or -OCH3.

While these structural speculations explain the observed efficacy of N4O2 complexes in terms of biotransport and localization phenomena, little work has been directed at unraveling the molecular details whereby these complexes disrupt heme aggregation. Recently, Ziegler et al have suggested that the diffuse positive charge of the cationic complex forms a salt bridge with the one deprotonated propionate moiety of free heme [102]. Such a salt bridge would prevent the propionate from forming the

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requisite linkage to another heme in order to aggregate into hemozoin. In support of this hypothesis, they have shown that in several heme polymerization assays that the efficacy of the N4O2 complex is dramatically affected by the concentration of the acetate buffer used in the assays. Thus, heme polymerization assays performed at 25 mM yield ICso's comparable to literature reports, while the compounds are entirely ineffective at 500 mM acetate buffer. Further, the neutral [R-ENBPI]Mg(II) complex [103] does not prevent malaria pigment formation. Porphyrins and Metalloporphyrin Complexes

Following the maxim that a good starting place for the rational design of an inhibitor is the structure of the actual substrate, there are several reports of the use of porphyrins and metalloporphyins as inhibitors to hemozoin formation. Fig. (9) Basilico et al. demonstrated that the metalfree porphyrins, protoporphyrin IX and hematoporphyrin were capable of inhibiting the aggregation of P-hematin in a dose dependent manner [104]. Using the heme aggregation assay of Egan et aL, differential solubility studies and FT-IR spectroscopy, probing for the fingerprint vibrations of hemozoin, revealed that complete inhibition of p-hematin aggregation occurred at a heme to protoporphyrin IX ratio of 1:3 and a heme to hematoporphyrin ratio of 1:0.5 (Table 3). The authors suggest that the inhibition of hemozoin is the result of TI-TT stacking between the metal-free porphyrin and heme. Further, the increased effectiveness of hematoporphyrin over protoporphyrin IX was attributed to hydroxyl coordination to the heme iron. Alternately, the improved efficacy could be the result of propionate-OH interactions similar to those proposed by Riscoe for the hydroxyxanthones. While these interactions provide a likely explanation for the inhibition results, the authors provided little direct evidence for their speculation. In related work, Martiney et al showed that metalloporphyrins with zinc or tin were capable of inhibiting hemozoin formation in trophozoite extracts [105]. Zn(II) and Sn(IV) analogs of heme were selected for study as a result of their known competitive interactions with heme in a number of systems and their use as pharmacological photosensitizers. The order of efficacy for these metalloporphyrins was ZnDPIX>SnPPIX»ZnPPIX. This order is consistent with the proposed

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interactions of Basilico et al [98] since deuteroporphyrin IX (DPIX) has four hydroxyl moieties, any of which could coordinate to the iron of heme Fig (9). Porphyrins and metalloporphyrins examined for antimalarial activity. A. Protoporphyrin IX, B. Metalloprotoporphyrin IX, C. Hematoporphyrin IX, D. Dueteroporphyrin IX.

HOOC

HOOC

HOOC

HOOC

Table 3. Comparison of Heme Aggregation Efficacy of Selected Porphyrins and Metalloporphyrins. Porphyrin Inhibitor

IC5o(nM) BNTII Mediated Assay

ICioo (molar equiv.) P-Hematin Assay

Protoporphyrin IX Hematoporphyin IX Co(III)PPIX Mn(III)PPIX Zn(II)PPIX Sn(IV)PPIX

ND ND 40 30 125 (480)* 100(75)''

3 1 3

1 0.5 0.5 * IC50 from trophozoite extract heme aggregation assay, Ref 105.

or interact with heme propionates. An examination of the inhibitory kinetics for these complexes was complex. At low inhibitor concentrations, the kinetics suggest competitive inhibition, while at

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higher concentrations of inhibitor, a noncompetitive component is apparent. Unfortunately, the analysis of kinetics in a crude extract system is of limited value. Proper interpretation of these experiments awaits reexamination of the kinetics under a better defined trophozoite-derived hemozoin aggregation system in terms of requisite components and purity of said components. Despite effective inhibition in vitro in the micromolar concentration range, these complexes did not inhibit parasite growth. Analysis of the specific fate of ZnBG with blood smears and light microscopy showed that the metalloporphyrin could neither cross the erythrocyte membrane to accumulate in the digestive vacuole, nor transverse the pores, the parisitophorous duct, or ion channels of the parasite. Once again, this result emphasizes the important dual requirements for all hemozoin inhibitors: transport to the digestive vacuole as well as the ability to disrupt heme aggregation. Monti et al. demonstrated a novel application of a metalloporphyrin inhibiting p-hematin formation when they used Fe(II)PPIX to disrupt the process [106]. In this report, Fe(II)PPIX, produced chemically or electrochemically, did inhibit the aggregation of heme to hemozoin with an IC50 of 0.4 molar equivalent. Control experiments revealed no aggregation or formation of hemozoin via an autooxidative mechanism. The putative inhibited complex is formed between a central Fe(II)PPIX capped by two axially hydroxo-bridged Fe(III)PPIX units. The linear trimer is proposed to be stabilized by coplanar n-n interactions. While there is no doubt that Fe(II)PPIX represents an effective inhibitor of the heme detoxification process, the proposed inhibition species seems unlikely. The n-n interactions likely do play a critical role in the inhibition process (see below), but the deprotonation of coordinated water to form a trimeric hydroxo-bridged species at pH 4.8 is far less certain in the absence of any experimental evidence. These results present, however, a tantalizing approach to the production of an endogenous inhibitor to hemozoin aggregation. If an effector drug, localized to the digestive vauole, could effectively reduce a small percentage of the Fe(III)PPIX released during hemoglobin catabolism, the detoxification pathway would be derailed, resulting in the death of the parasite. Current efforts are focussed on the identification of possible physiologically compatible reductants that alone or in concert with artemisin derivatives could provide effective therapies .

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In a recent study, Cole et al. demonstrated that the central metal ion served a significant role in the efficacy of metalloprotoporphyrins to inhibit p-hematin formation [107]. Their study investigated Cr(III)PPIX, Co(III)PPIX, Mn(III)PPIX, Cu(II)PPIX, Mg(II)PPIX, Zn(II)PPIX and Sn(IV)PPIX as potential inhibitors of P-hematin formation. The results (Table III) show that using the in vitro assay of Egan et aL, the ability of a metalloporphyrin to inhibit P-hematin aggregation was fi"om most to least effective: Mg(II)PPIX, Zn(II)PPIX and Sn(IV)PPIX > Cu(II)PPIX > Mn(III)PPIX » Co(III)PPIX and Cr(III)PPIX. IR analysis showed that the most effective inhibitors yielded complex precipitates with evidence of Fe-(0)propionate linkages, while the less effective inhibitors did not. This suggested two modes of inhibition. The trend of hemozoin formation inhibition correlates well with the rates of water exchange for octahedral aqua complexes of the metalloporphyrin's central metal ion [108]. Thus, inhibitors Mg(II)PPIX, Zn(II)PPIX, Sn(IV)PPIX and Cu(II)PPIX are most effective, corresponding to central metal ions with the rapid water exchange rates observed for the alkaline earth metals, Cu(II) and Zn(II). On the other end of the scale, inhibitors Co(III)PPIX and Cr(III)PPIX with their half filled d-orbitals are the least effective with central metal ions whose water exchange rates have half-lives of several days. It should be noted that the reported efficacy of Fe(II)PPIX [106] places this metalloporphyrin between Cu(II)PPIX and Mn(III)PPIX, approximately where one would predict based on the rate of Fe(II) water exchange. This trend also correlates to the observed relative solubility of the metalloprotoporphyrins under the examined experimental conditions. These two factors readily explain the inhibitor effects of MPPIX complexes. Using Mossbauer spectroscopy to monitor the formation of p-hematin under in vitro reaction conditions, Adams et al. have demonstrated that the reaction is a psuedo-zero-order process [109]. Such a process is consistent with a mechanism whereby a small concentration of heme is kept soluble via acetate, functioning as a phase-transfer catalyst, in a heme-saturated solution. In the rate limiting step, the soluble heme aggregates to p-hematin, which in turn grows until it precipitates from solution. There are clearly complicated heterogeneous reaction equilibria involved in the aqueous chemical formation of p-hematin. Consequently, it should be emphasized that the detailed mechanistic analysis of the complex solubilization of the species involved in the chemical synthesis

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of p-hematin under these experimental conditions must include consideration of the heterogeneous kinetics of the precipitate/solution interface which will be markedly influenced by factors such as crystallite morphology, size, the presence of inhibitor, and the solute/solution interactions. Nevertheless, to a first approximation under the reaction conditions, the more soluble metalloporphyrin inhibitors will maintain a higher effective concentration, increasing the potential for critical nstacking interactions with solublized heme, and thereby preventing hemozoin formation. Although all of the metalloprotoporphyrins appear to form n-n adducts to the heme substrate, the IR analysis of the precipitate suggests two different modes of binding. For Mg(II)PPIX, Zn(II)PPPIX and Sn(IV)PPIX, it appears that the inhibitors do not completely prevent the formation of Fe-(0)propionate linkages. In contrast, Co(III)PPIX, Cr(III)PPIX and Mn(III)PPIX, which are less effective inhibitors, apparently do prevent the formation of such linkages. A possible interpretation of these observations is that the Fe-(0)propionate bonds may in fact not be the dominant oligomerizing interaction within phematin. Rather, if the critical interaction dictating the stability of the heme precipitate is hydrogen bonding, the Fe-(O) interactions might be observed, but the precipitate would be soluble if such hydrogen bonding was diminished. Thus, a relatively weak or geometrically offset n-n heme-metalloporphyrin complex might allow the formation of Fe(O)propionate linkages, but prevent the formation of other stabiKzing interactions. Similarly, a tight or axial geometry between inhibitor and heme would prevent Fe-(0)propionate linkages, but not completely interfere with hydrogen bonding within a complex heterogeneous precipitate. Such an explanation is consistent with the counter-intuitive observation of Fe-(O) propionate linkages in the heterogeneous precipitate of the most efficient metalloporphyrin inhibitors, but not in the others. It is also consistent with the emerging picture of the structure of hemozoin [60]. This work is also notable in that Cole et aL performed detailed aggregation studies of the hemeiMPPIX using UV-vis and fluorescence spectroscopies to detect the formation of n-n hetero-metalloporphyrin assemblies under assay conditions. By employing UV-vis absorbance spectroscopy, the aggregation of porphyrin and metalloporphyrin systems may be examined. The in vitro assay system used for hemozoin

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formation induced acid-triggered ^-aggregation characterized by significant wavelength shifts, hypochromicity and broadening of the Soret band [110]. The deconvolution of the complex spectra expected for the hetero-metalloporphyin inhibition complexes demonstrated that aggregation processes other than continued simple self-aggregation were occurring. No doubt, the species and the associative processes involved represent extremely complicated equilibria. Nevertheless, the wavelength shifts and hypochromicity were indicative of acid-triggered aggregation processes driven by n-n interactions between hetero-metalloporphyrin assemblies. Similar behavior was observed for all of the investigated hemeiMPPIX inhibitor complexes under these experimental conditions. Such complex aggregation processes may also be examined by such methods as the analysis of the fluorescence behavior of the heteroporphyrin assembly [111], the deconvolution of powder diffraction data [50,59,60,67] and the application of X-ray absorbance spectroscopy (XAS) near-edge multiplet analysis. In the fixture, such techniques will undoubtedly provide essential details to describe the nature of the heme aggregates. Recent investigations have shown that a number of porphyrins have potential applications as anti-cancer chemotherapeutics [112,113,114] and anti-viral agents [115,116]. Similarly, structurally related compounds such as the phthalocyanines and expanded porphyrins and their metal complexes have been studied as potential MRI contrast agents [117] and photodynamic therapuetic agents [118]. Although porphyrins and their metal complexes have critical shortcomings in terms of their biotransportation across the cell membrane and localization to the digestive vacuole, they represent an outstanding scaffold on which to base important structure-function relationship studies. The emerging results from these studies are uncovering principles critical to the design of effective heme aggregation inhibitors. SUMMARY The malaria parasite, P. falciparum, obtains most of its requisite amino acids from the cataboHsm of host hemoglobin via a defined metaboHc pathway which occurs in speciahzed digestive vacuoles at low pH (4.85.2). Ironically, one molecule of hemoglobin contains four molecules of

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the heme cofactor that are poisonous to the parasite in their monomeric form. Indeed, when levels of free heme rise to moderately high levels, the hemoglobin protolytic enzymes are inhibited, lipid peroxidation increases and free radical concentrations rise. Consequently, the parasite dies. To balance the demands for amino acids against rising levels of free heme, the malaria parasite has evolved a detoxification pathway which removes the monomeric heme from solution by forming the biomineral hemozoin (malaria pigment, P-hematin). This parasite specific detoxification pathway presents an intriguing target for the development of novel antimalarial compounds. Exciting developments on a number of fronts promise to facilitate the development of new hemozoin inhibitors. On a physiologically relevant time scale, hemozoin does not form spontaneously. This suggests that biomolecules found in the digestive vacuole may mediate the formation of malaria pigment. Two possible candidates include endogeneous lipids and the histidine-rich proteins. While both classes of molecules clearly mediate the in vitro aggregation of heme into hemozoin, it remains to be seen which is of primary biological relevance. The fact that so little is understood about the process involved in the formation of this critical biomineral highlights the need for additional research in this area. Recently, the crystal structure of hemozoin was determined using Xray powder diffraction techniques. The startling new structure was not the expected extended polymeric chain of five-coordinate Fe(III)protoporphyrin IX's linked by axial propionate linkages, but rather a hydrogen bonded network of dimeric heme units linked by reciprocating iron-carboxylate bonds from one of the propionic side chains of each porphyrin. This new model of hemozoin has significant implications concerning both the biomineralization of hemozoin and currently proposed mechanisms of action for a number of antimalarial drugs. For over 300 years, the quinoline family of drugs, and chloroquine in particular, has been used as the primary treatment for malaria. Recent studies have demonstrated that this drug inhibits the aggregation of free heme into hemozoin, allowing levels of monomeric heme to rise until cell lysis occurs. Although the determined structure of hemozoin makes the polymer termination scheme proposed by Sullivan et aL unlikely, hemozoin:drug:heme interactions appear critical in the inhibition mechanism. Thus, researchers have identified the characteristics of

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planar aromaticity, peripheral Fe(III)-coordinating moieties and potential sites of propionate interaction as critical parameters for new hemozoin inhibitor drugs. The importance of these design parameters is seen in the development of the porphyrins, hydroxyxanthones and N4O2 Schiff-base coordination complexes as not only well defined platforms on which to base structure-function relationships, but as potential new antimalarial therapies. ACKNOWLEDGEMENTS I would like to thank my students who have played a crucial part in the rapid progress that we have made on the role of template-mediated synthesis of hemozoin. I would also like to thank Professor D. Scott Bohle for providing a preprint of the most recent powder diffraction model of hemozoin and for reading sections of this manuscript prior to submission. Without his assistance and cooperation, this review would have been instantly out of date. I would also like to thank the Research Corporation, the Hunkele Charitable Trust of Pittsburgh, the National Foundation of Infectious Diseases, and Duquesne University for financial support of this work. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9]

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Desjardins, R.E.; Canfield, J.; Haynes, D.; Chulay, J.D. Antimicrob. Agents Chemother., 1979, 16, 710. Egan, T.J.; Ross, D.C.; Adams, P.A. FEES Lett., 1994, 352, 54. Pandey, A.V.; Tekwani, B.L. FEES Lett., 1997, 393, 189. Ignatushchenko, M.V.; Winter, R.W.; Bachinger, H.P..; Hinrichs, DJ., Riscoe, M.K. FEES Lett., 1997,409, 67. Egan, T.J.; Hempelmann, E.; Mavuso, W.W. J. Inorg. Eiochem., 1999, 73, 101. Warhurst, D.C. Eiochem. Pharm., 1981, 30, 3323. Riddley, R.G.; Dom, A.; Vippagunta, S.R.; Vennerstrom, J.L. Ann. Trop Med ParasitoL, 1991, 91,559. Fitch, CD. Trans. Am. Clin. Climatol Assoc., 1998, 109, 97. Chou, A.C.; Fitch, CD. Eiochem. Eiophys. Res. Commun., 1993, 915, 422. Fullerton, D.S. In Textbook of Organic Medicinal and Pharmacological Chemistry 9th Ed., Delgado, J.N.; Remers, W.A., Eds., J.B. Lippencott Co.: Philadelphia, 1991, pp. 205-225. Ridley, R.G.; Hofheinz, W.; Matile, H.; Jaquet, C ; Dom, A.; Masciadri, R.; Jolidon, S.; Richter, W.F.; Guenzi, A.; Girometta, M.-A.; Urwyler, H.; Huber, W.; Thaithong, S.; Peters, W. Antimicrob. Agents Chemother., 1996, 40, 1846. Thompson, P.F.; Werbel, L.M. Antimalarial Agents: Chemistry and Pharmacology, Academic Press: New York, NY, 1972, pp. 57-158. Ridley, R.G.; Matile, H.; Jaquet, C ; Dom, A; Hofheinz, W.; Luepin, W.; Masciadri, R.; Theil, F.P.; Richter, W.F.; Girometta, M.-A.; Guenzi, A.; Urwyler, H.; Gocke, E.; Potthast, J.-M.; Csato, M.; Thomas, A.; Peters, W. Antimicrob. Agents Chemother., 1997, 41, 677. Ismail, F.M.D.; Dascombe, M.J.; Carr, P.; Merette, S.A.M.; Roulault, P. J. Pharm. Pharmocol., 1998, 50, 483. Macomber, P.B.; Sprinz, H.; Tousimis, A.J.; Nature, 1967, 214, 937. Chou, A.C; ChevH, R.; Fitch, CD. Eiochemistry, 1980., 19, 1543. Dom, A.; Vippagunta, S.R.; Matile, H.; Jaquet, C ; Vennerstrom, J.L.; Ridley, R.G. Eiochem. Pharmacol., 1998, 55, 727. Orjih, A.U.; Banyal, H.S.; Chevli, R.; Fitch, CD. Science, 1981, 214, 668. Orjih, A.U.; Ryerse, J.S.; Fitch, CD. Experientia, 1994, 50, 34. Slater, A.; Pharmacol. Ther., 1993, 57, 230. Sullivan, D.J.; Gluzman, I.Y.; Russel, D.G.; Goldberg, D.E. Proc. Natl. Acad. Set, t/5/i, 1996,93, 11865. Constantinidis, I.; Satterlee, J.D. J. Am. Chem. Soc, 1988, 110, 927. Constantinidis, I.; Satterlee, J.D. J. Am. Chem. Soc, 1988, 110, 4391. Sullivan, D.J.; Matile, H.; Ridley, R.G.; Goldberg, D.E. J. Eiol. Chem., 1998, 273,31103. Hawley, S.R.; Bray, P.G.; Mungthin, M.; Atkinson, J.D.; O'Neill, P.M.; Ward, S.A. Antimicrob. Agents Chemother., 1998, 42, 682. Vennerstrom, J.; Eaton, J. J. Med. Chem., 1988, 31, 1269. Balca, M. Meshnick, S.; Sigler, C ; Lebind, P.; HoUingdale, Am. J. Trop. Med. Hyg., 1990, 42, 532.

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101] Tsang, B.W.; Mathias, C.J.; Green, M.A. J. Med. Chem., 1994, 37, 4400. 102] Ziegler, J.; Shuerle, T.; Kelly, C ; Elamin, A.; Pasierb, L.; Cole, K.A.; Wright, D.W. Inorg. Chem. In Press. 103] Polyakov, V.R.; Sharma, V.; Crankshaw, C.L.; Piwnica-Worms, D. Inorg. Chem., 1998, 37, 4740. 104] Basilico, N.; Monti, D.; Olliaro, P.; Taramelli, D. FEBSLett., 1997, 409, 297. 105] Martiney, J.A.; Cerami, A.; Slater, A.F.G. Mol. Med., 1996, 2, 236. 106] Monti, D.; Vodopovec, B.; Basilico, N.; Olliaro, P.; TaramelH, D. Biochemistry 1999, 38, 8858. 107] Cole, K.A.; Ziegler, J.; Wright, D.W. J. Inorg. Biochem., 2000, 78, 109. 108] Atwood, J.D. Inorganic and Organometallic Reaction Mechanisms, 2nd ed., VCH: New York, 1997, pp. 82-85. 109] Adams, P.A.; Egan, T.J.; Ross, D.C.; Silver, J.; March, P.J. Biochem. J., 1996, 318,25. 110] Shelnutt, J.A. J. Phys. Chem., 1984, 88, 4988 and references therein. I l l ] Endisch, C ; Fuhthop, J.H.; Buschmann, J.; Luger, P.; Siggel, U. J. Am. Chem. 5c>c., 1996, 118,6671. 112] Ding, L.; Etemad-Moghadam, G.; Meunier, B. Biochemistry, 1990, 29, 7868. 113] Ding, L.; Casas, C ; Etemad-Moghadam, G.; Meunier, B.; Cros, S. New J. Chem., 1990,14,421. 114] Ding, L.; Casas, C ; Etemad-Moghadam, G.; Cros, S.; Auclair, C ; Meunier, B. J. Med Chem., 1991,34,900. 115] Dixon, D.W.; Marzilli, L.G.; Shinazi, R. Ann. N.Y. Acad. Sci., 1990, 616, 511. 116] Ding, L.; Balzarini, J.; Schols, D.; Meunier, B.; DeClerq, E. Biochem. Pharmacol., 1992,44, 1675.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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BIOACTIVE PEPTIDES AS SIGNAL MOLECULES IN PLANT DEFENSE, GROWTH, AND DEVELOPMENT ANDREAS SCHALLER Institute of Plant Sciences, Federal Institute of Technology ZUrich, CH-8092 ZUrichy Switzerland ABSTRACT: Until recently, intercellular communication in higher plants was thought to be mediated by the five classes of classical phytohormones i.e, auxins, cytokinins, gibberellins, abscisic acid, and ethylene. Hormone action in plants thus appeared to be fundamentally different from that in animals. This view is changing, however, since over recent years brassinosteroids and jasmonates resembling animal steroids and prostaglandins, respectively, have been added to the group of chemical messengers in plants. Furthermore, there is now compelling evidence for the existence of plant (poly)peptide hormones. The present arsenal of endogenous plant peptide signals includes just four groups of hormones involved in wound signal transduction, in cell proliferation, and in the regulation of salt/water homeostasis, i.e. systemins, phytosulfokines, enod40, and natriuretic peptides, but many more are likely to exist. Plants appear to possess the receptors for a plethora of peptide signals. These signals include both endogenous peptides as well as peptides of microbial origin. Furthermore, plant proteases have been identified likely involved in the generation of peptide signals from larger precursor proteins. This article discusses the evidence in support of a general role for bioactive peptides in plant signal transduction with emphasis on the structure and bioactivity of the peptides themselves.

INTRODUCTION The biochemical machinery necessary for peptide synthesis, secretion, and posttranslational modification is present in every living cell. An enormous structural diversity can be generated by use of this preexisting cellular machinery. Not surprisingly, peptides are commonly used as signal molecules for intercellular communication in prokaryotes, fungi, and animals. Peptide signals in animals include vast numbers of peptide hormones, growth factors and neuropeptides. Are plants any different in this respect? Until very recently they appeared to be. Plants seemed to rely on only five different classes of phytohormones comprising auxins, cytokinins, gibberellins, abscisic acid, and ethylene for the regulation of growth and development [1]. These phytohormones are small diffusible

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molecules which were thought to be much better suited to penetrate the rigid cell walls between adjacent cells as compared to large peptide hormones [2]. Research in recent years, however, has indicated that peptides may be widely used as chemical signals in plants as well. In the present work, I intend to summarize the evidence in support of this hypothesis. Endogenous bioactive peptides, Le, plant-derived peptides that evoke specific cellular responses, provide the most direct evidence for a general role of peptides in the regulation of plant growth and development. Their number, however, is still very limited. Presently, endogenous regulatory peptides in plants include systemin, phytosulfokines, and enod40 [3,4]. Furthermore, there is some indication that plants contain peptides similar in structure and function to natriuretic peptides in animals [5]. Following the initial discovery of systemin as an 18~amino-acid peptide in tomato plants, closely related peptides have been discovered in other solanaceaeous species. Systemins are mediators of the defense responses triggered by the attack of herbivorous insects. Phytosulfokines are small sulfated peptides of four or five amino acids which exhibit mitogenic activity. The enod40 group of peptides is involved in cell proliferation as well. The biosynthesis of these peptides, their biological activity, as well as the structural requirements for bioactivity and signal perception will be the first focus of the following discussion. The endogenous bioactive peptides thus far identified are likely to represent only the tip of the iceberg. Research in recent years has shown that plants have the capacity to generate and to perceive peptide signals providing indirect evidence for a general role of peptides as plant growth regulators. The perception of peptide signals requires receptor proteins for proteinaceous ligands. In higher plants, a large number of receptor-like kinases (RLKs) have been identified possessing extracellular domains which are likely to be involved in protein/protein interaction [6]. Thus, RLKs were hypothesized to be the receptors of (poly)peptide ligands. For two RLKs, involved in meristem and organ development in Arabidopsis (CLAVATAl) and in the determination of self-incompatibility in Brassica (SRK), the respective peptide ligands have been identified very recently (see below). Likewise, the generation of peptide signals in animals requires proteases that are involved in the maturation of peptide hormones from inactive precursor proteins by limited proteolysis. Proteases of the subtilisin superfamily play a predominant role in this process [7]. Proteases of this family have been identified and may serve a

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similar function in plants [8]. They provide further indirect evidence for the common use of peptides as chemical signals in plants. Plants do not only perceive endogenous peptide signals but also react specifically to a large number of exogenous peptides produced by pathogenic microorganisms. Microbial peptides act as elicitors of both general and race-specific defense responses. Numerous structurally diverse peptides from phytopathogenic fungi have been implicated in the latter. In contrast, the attenuation of general resistance responses depends on the recognition of conserved structural features of (poly)peptides common to a wide range of microorganisms. This distinction between „self' and „non-self * is a prerequisite for the development of resistance [9,10]. The nature of peptide elicitors of general and race-specific resistance responses, the structural requirements for their bioactivity, and the molecular basis for recognition by the plant cell will be discussed. ENDOGENOUS PEPTIDE SIGNALS Systemin Tomato plants respond to local injury by herbivorous insects with the induction of a systemic defense response which is characterized by the transcriptional activation of a large number of defense genes and the concomitant accumulation of the respective defense proteins (Fig. (1); [11-13]). The search for the signal molecule that allows tomato plants to respond systemically to a local stimulus (Le, wounding) led to the identification of the first plant peptide with signaling function in 1991. The 18-amino-acid peptide was isolated from leaves of tomato plants on the basis of its ability to induce the expression of defense genes, the hallmark of the wound response (Fig. (1)). To emphasize its central role and the systemic nature of the wound response the peptide was named systemin [14]. In subsequent years, it was established that systemin is both sufficient and necessary for systemic wound signal transduction. While there is clear evidence for additional signals to exist, a central role for systemin in wound signaling in tomato plants and in other members of the Solanaceae [15] is now generally accepted. Both the discovery of systemin as well as its role in wound signaling have been discussed extensively and the reader is referred to recent review articles for further information [4,13,16-19]. In this chapter, I will concentrate on unresol-

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ved questions concerning systemin perception as well as its synthesis and degradation. The careful analysis of structural aspects that are relevant for its biological activity have been instrumental in the search for the systemin receptor and will, therefore, be the starting point of the following discussion. I II III kD

Defense Proteins Serine Proteinase Inhibitor I Serine Proteinase Inhibitor II Cysteine Proteinase Inhibitor Aspartic Proteinase Inhibitor Polyphenol Oxidase

96 — 67 —

Signal Pathway-Associated Prosystemin Lipoxygenase Calmodulin Nucloetide Diphosphate Kinase AcylCoA-Binding Protein

45 —

31 —

21

Systemic Wound Response Proteins in Tomato Plants

?*S^

^fffw

f^^'d^

m Mil'

14 — 'ASs^

^^

Proteolysis-Associated Leucine Aminopeptidase Carboxypeptidase Aspartic Proteinase Cysteine Proteinase Ubiquitin-hke Protein Other Proteins Threonine Deaminase

Fig. (1). The wound response in tomato plants. Tomato plants respond to wounding with the transcriptional activation and accumulation of Systemic Wound Response Proteins including defense proteins, proteolysis-associated proteins, signaling-associated proteins, and proteins of yet unknown function in plants defense. The change in gene expression can be monitored on SDSPAGE gels. In comparison to control plants (I), treatment with systemin (II), or overexpression of the prosystemin cDNA (III) leads to the accumulation of SWRPs (arrowheads) and the downregulation of other, unidentified proteins (triangles). The figure was modified after [13].

Sequence analysis of the defense gene-inducing principle isolated from tomato leaves revealed the amino acid sequence AVQSKPPSKRDPPKMQTD as the primary structure of systemin (Fig. (2)). The most salient

371 1 MGTPSYDIKN KGDDMQEEPK VKLHHEKGGD EKEKIIEKET 41 PSQDINNKDT ISSYVLRDDT QEIPKMEHEE GGYVKEKIVE 81 KETISQYIIK lEGDDDAQEK LKVEYEEEEY EKEKIVEKET 121 PSQDINNKGD DAQEKPKVEH EEGDDKETPS QDIIKMEGEG 161 ALEITKWCE KIIVREDLAV QSKPPSKRDP PKMQTDNNKL Systemin Fig. (2). The primary structure of tomato (pro)systemin. The amino acid sequence deduced from the prosystemin cDNA is shown. The systemin precursor comprises 200 amino acids. The 18 amino acids of the systemin oligopeptide (bold, underlined) are located close to the carboxy terminus.

feature of this sequence is the palindromic structure around a central pair of basic residues with two proline doublets at positions 6 and 7, and 12 and 13 [14]. Several studies aimed at the identification of secondary and tertiary structural elements within systemin. At acidic pH, twodimensional NMR spectroscopy revealed a Z-like-p-sheet structure which is frequently found in DNA-binding proteins [20]. Based on this observation, systemin was suggested to bind to the promoter region of defense genes thereby regulating their expression [20,21]. At neutral pH in aqueous solution, however, proton NMR experiments did not provide evidence for persistent secondary or tertiary structural elements in the systemin polypeptide [22]. Circular dichroism, as a method particularly sensitive to secondary structure, revealed a poly(L-proline) II type, 3i helical structure for a substantial part of systemin in aqueous solution [23]. The 3i helicity has been observed in oligopeptide ligands of Src homology 3 (SH3) proteins [23]. Hence, the presence of this structural feature in systemin does not support the suggested interaction between systemin and DNA but would rather favor a proteinaceous receptor for systemin. Despite experiments aiming to determine the relevance of the proline residues for bioactivity, it remains unknown whether or not the 3i helicity is relevant for systemin activity in vivo. Each of the 18 amino acids of systemin was individually replaced by alanine to assess the contribution of single amino acid side chains to the biological activity of systemin. The Pro 13Ala substitution resulted in a dramatic loss of defense protein-inducing activity, second only to the Thrl7Ala derivative which was completely inactive. Pro 13 may thus be structurally important.

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Systemin derivatives with Ala substitutions for any of the other proline residues, however, retained most (>10%) of the bioactivity [24]. Progressive deletions from the N- and C-termini indicated that the 18amino-acid peptide is the minimal structure having full biological activity. Deletions from the N-terminus resulted in a progressive loss of activity, while the deletion of a single amino acid from the C-terminus completely inactivated the peptide. Interestingly, the C-terminally truncated peptide as well as the Thrl7Ala derivative of systemin acted as competitive antagonists of systemin activity [24]. Based on these observations it was proposed that systemin may bind to its receptor via the N-terminal part while the C-terminus is essential to activate downstream responses [24]. The analysis of the systemin structure/function relationship identified residues that can be modified without impairing systemin activity. In subsequent studies, some of these residues (Val2, Ser8, and Met 15) were modified to generate labeled systemin derivatives as affinity probes for the systemin receptor. A biotinylated systemin derivative (biotinylCysSSer systemin) was used in initial attempts to isolate the systemin receptor from tomato plasma membranes. The biotinylated peptide could be specifically crosslinked to a 50 kDa protein (SBP50) in plasma membrane preparations from tomato leaves [25]. Binding to SBP50, however, was slow and was saturated only at high concentrations of biotinyl-systemin. Furthermore, competitive displacement of the ligand with alanine-substituted systemin derivatives revealed a lack of correlation between the structural requirements for binding to SBP50 and the biological activity of systemin [25]. Therefore, SBP50 is not likely to be the systemin receptor. In contrast, SBP50 exhibited characteristics of proteases related to yeast kexin and was suggested to be involved in systemin processing thereby facilitating its activity or degradation. [25]. Further attempts to identify a high-affinity binding site for systemin in tomato plasma membrane preparations using either biotinylated, or radiolabeled systemin derivatives were not successful (Doares, Schaller, and Ryan; unpublished). Progress was made possible by use of a different source for membrane preparations. It had been shown by Felix and Boiler [26] that cell suspension cultures of a wild tomato species (Lycopersicon peruvianum) give a characteristic response to systemin. After addition of systemin an alkalinization of the culture medium was observed paralleled by an efflux of K^. Systemin also caused an increase in the activities of 1-

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aminocyclopropane-1-carboxylate (ACC) synthase and phenylalanine ammonia-lyase, Le, two enzymes with established functions in plant defense [26]. Medium alkalinization in response to systemin treatment provided a convenient assay for systemin activity. The alkalinization response was dose-dependent and saturated at about 1 nM systemin. The structural requirements within systemin were the same for medium alkalinization and for defense gene induction [26,27]. Therefore, the two responses appeared to be mediated by the same perception system and cultured cells of L. peruvianum were subsequently used in the search for the systemin receptor. In microsomal membrane preparations from L. peruvianum cells, Meindl et al. [28] characterized a high-affinity, saturatable binding site for a systemin derivative extended at the C-terminus by ^^^I-iodotyrosine. Using I-Tyr2,Alal5-systemin as the ligand, a similar binding site was observed by Scheer and Ryan [29] on the surface of L. peruvianum cells. The binding sites described by the two groups exhibited very similar characteristics and are likely to reside in the same protein. Both groups found antagonists of systemin activity to be able to compete for binding of the respective radioligands. Furthermore, the biological activities of alanine-substituted systemin derivatives were correlated to their ability to compete with the binding of the radioligands [29]. While the C-terminal part of systemin is essential for bioactivity, a corresponding systemin fragment was not able to displace the radioligand from its binding site. Meindl et al [28] concluded that systemin perception requires a two-step mechanism involving binding to the receptor via its N-terminal part and activation of cellular responses by the C-terminus of systemin. Thus, the systemin-binding site in L. peruvianum cells exhibited characteristics that had been predicted for the systemin receptor from the analysis of the systemin structure/activity relationship [24]. Photoaffinity labeling localized the binding site to a 160-kDa protein. This protein likely represents the functional systemin receptor in L. peruvianum cells and, hence, was named systemin receptor 160 (SRI60) [29]. The abundance of SRI60 at the cell surface was found to increase in response to treatment with methyl jasmonate [29]. Upregulation at the transcriptional level by wounding and jasmonates, which are downstream signaling molecules in the wound signal transduction pathway [30,31], has also been described for other proteins of the signaling pathway including lipoxygenase [32], allene oxide synthase [33,34], prosystemin [13,35], calmodulin [36], and the catalytic and regulatory subunits of a

374

polygalacturonase [37]. The increase in the abundance of the systemin receptor and of other components of the wound signal transduction pathway due to their de novo biosynthesis may enhance the capacity of tomato plants to respond to continuous attack by herbivores with a further increase in defense gene expression. It is expected that the characterization of SRI60 will provide profound insights into systemin perception and the transduction of the wound signal into cellular responses. Presently, this can only be inferred from the changes observed after systemin treatment of cultured cells or differentiated plants. The earliest systemin-triggered changes in tomato leaf mesophyll cells are an increase in the cytosolic free calcium concentration within 1 - 2 minutes [38] and a transient depolarization of the plasma membrane potential [39]. Depolarization of the plasma membrane in response to systemin has also been observed in cultured L. peruvianum cells [26] and was shown to depend on the influx of Ca^"^ as well as the activity of a protein kinase [40]. By use of inhibitors of the plasma membrane H"^-ATPase [40] and of various ionophores (Frasson and Schaller, unpublished) it could be shown that depolarization of the plasma membrane potential is sufficient to induce the expression of defense genes. However, when membrane depolarization was suppressed by application of fusicoccin which activates the plasma membrane H^ATPase, defense gene expression was inhibited [40]. These data indicate that the depolarization of the plasma membrane potential and the influx of Ca^"^ are necessary elements in the signal transduction pathway toward the activation of defense gene expression [4]. Either one or both of these events in concert with the activity of a protein kinase [41,42] lead to the activation of phospholipase A2 [43] and the subsequent release of linolenic acid from membrane lipids [43-45]. Linolenic acid, via the octadecanoid pathway, is converted to jasmonic acid which ultimately triggers defense gene induction [31,46]. For further details on this signaling pathway, which resembles eicosanoid signaling in the inflammatory response of animal macrophages, the reader is referred to the comprehensive review article by Ryan and Pearce [19] and to a recent paper from the same laboratory [43]. In addition to the problem of how the systemin signal is perceived and transduced in target tissues, another complex of open questions revolves around the factors governing systemin synthesis and degradation. Systemin is synthesized as 200-amino-acid precursor protein, prosystemin (Fig. (2); [35]). In analogy to animal systems, it had been assumed that

375

processing of prosystemin to release systemin is necessary for the activation of the peptide signal [27,47]. Recent data show that this may not be the case. The prosystemin polypeptide was overexpressed in both prokaryotic [48] and eukaryotic [49] hosts. When the recombinant protein was tested for biological activity, it turned out to be active in inducing medium alkalinization in L. peruvianum cell cultures as well as defense gene expression in tomato plants [49,50]. The activity was shown to reside exclusively in the C-terminal, Le. systemin, part of the prosystemin structure [50], and the perception systems used by prosystemin and by systemin are likely to be identical [49,50]. Systemin has originally been isolated as the actual defense gene-inducing factor and it has been shown to be the minimal structure that retains full biological activity [14,24]. Thus, prosystemin is processed to generate systemin in vivo, and mature systemin is not likely to be a random degradation product of prosystemin. While the data show that proteolytic processing is not necessary for the activation of systemin, it may still be required to facilitate systemin release and/or systemic translocation. The proteases involved in systemin maturation still remain to be identified, however. The expression of several proteases including exopeptidases (leucine aminopeptidase, carboxypeptidase) and endoproteinases (aspartic proteinase, cysteine proteinase) is induced systemically upon wounding of tomato or potato plants [13,51-57], and for some of these proteases a role in the regulation of systemin activity has been discussed [12,54,56]. However, whenever this was investigated, induction by wounding was found to be rather slow, resembling the induction of defense genes rather than that of signal pathway components [32,35-37,54,56]. Therefore, these proteases may rather contribute directly to deterring the insect predator [58] or else, they may be responsible for rapid protein turnover facilitating C and N salvaging in damaged leaves and providing an adequate amino acid pool for the synthesis of abundant defense proteins in systemically induced leaves [56,59]. Systemin exerts its biological activity at extremely low concentrations (fmol/plant). Therefore, in analogy to animal systems, proteases have to be postulated that inactivate systemin and clear the system from residual active hormone. Experimental data indeed support the existence of such enzymes. Felix and Boiler observed that the transient nature of systemintriggered alkalinization response in L. peruvianum cells is due to proteolytic inactivation of systemin rather than desensitization of the perception system [26]. Inactivation of systemin was, in fact, observed in

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cell-free growth medium of L. pemvianum cultures [26], and the site of proteolytic cleavage was identified as the Lysl4/Metl5-bond of systemin [27]. The alkalinization of the culture medium in response to a systemin derivative in which this peptide bond had been stabilized by A^methylation was no longer transient. Furthermore, in tomato plants, the AT-methylated systemin derivative exhibited an enhanced defense geneinducing activity as compared to native systemin [27], indicating that this processing event contributes to systemin inactivation in vivo. While the protease responsible for processing of systemin at the Lysl4/Metl5-bond has not been isolated yet, such an activity was detected in plasma membrane preparations from tomato leaves [25,27], and SBP50 which shares several characteristics with the yeast kex2 protease is a possible candidate [25]. Obviously, many questions remain open with respect to the identity of the proteolytic systems involved in systemin maturation and inactivation, as well as their tissue specific and subcellular localization and they will attract the attention of future studies. Phytosulfokines It is well known that suspension-cultured plant cells require a critical initial cell density for growth. In cultures below that threshold density the cells fail to divide. In 1969, Stuart and Street demonstrated that the growth of Acer pseudoplatanus cells in low-density culture could be restored by the addition of 'conditioned' medium [60]. Conditioned medium was derived from high-density cultures after separation of the cells by dialysis. Hence, a low-molecular-weight 'nursing' factor must be released by high-density cultures, and Stuart and Street initiated work to establish the chemical nature of this factor [60]. For decades, however, all attempts to purify this nursing factor failed, likely because a fast and sensitive assay system was not available. A highly sensitive bioassay was eventually developed by Matsubayashi and Sakagami [61]. In this assay system, conditioned medium and fractions thereof were tested for mitogenic activity on mechanically dispersed Asparagus mesophyll cells in 24-well microplates. The mitogenic activity was purified and found to reside in two factors subsequently named phytosulfokine-a and -p (PSKa and PSK-p). Amino acid sequence analysis and mass spectrometry identified the phytosulfokines as a sulfated pentapeptide (Tyr(S03H)-IleTyr(S03H)-Thr-Gln) and its C-terminally truncated tetrapeptide deriva-

377

live (Tyr(S03H)-Ile-Tyr(S03H)-Thr), respectively (Fig. (3)). Synthetic PSKs exhibited the same mitogenic activity as the purified native compounds, thus confirming the structures [61]. a)

1 MVNEGB.TAE^..LC.LLCLALLX....liGQDTHSRKL LLQEKHSHGV 41 GNGTTTTQEP SRENGGSTGS NNNGQLQFDS AKWEEFHTDY 81 lYTODVKNP

b)

SO3H SO3H

H-Tyr-Ile-Tyr-Thr-Gln-OH PSK-a

SO3H SO3H

H-Tyr-Ile-Tyr-Thr-OH PSK-P

Fig. (3). The structure of (prepro)-phytosulfokines. a) The amino acid sequence of preprophytosulfokines deduced from the sequence of the cDNA is shown. The signal peptide for secretion (dotted hne) and the position of phytosulfokines within the precursor (bold, underlined) are indicated, b) Structures of the sulfated penta- (PSK-a) and tertrapeptides (PSK-P).

The work on phytosulfokines culminated last year in the cloning of the cDNA for the PSK precursor protein from rice (Fig. (3); [62]). The rice cDNA has the capacity to encode a 89-amino-acid prepro-phytosulfokine indicating that PSKs have to be released from their precursor by limited proteolysis, a feature they share with systemin and with animal peptide hormones in general. By virtue of a 22-amino-acid signal peptide at the amino terminus, prepro-phytosulfokine was shown to be targeted to the secretory pathway of cultured rice cells resulting in the accumulation of correctly processed PSK in the culture medium [62]. The PSK precursor was found to be expressed in all analyzed tissues of rice seedlings but, consistent with its mitogenic activity in-vitro, expression was highest in root and shoot apices [62]. The rate of cell division was correlated to the expression level of prepro-phytosulfokine in transgenic rice cultures thus confirming the role of PSKs in mitosis [62]. The PSK gene was detected in Oryza sativa, Asparagus officinalis, Arabidopsis thaliana, Daucus carota, and Zinnia elegans and PSK-a was found to be present in cell culture media of all these species suggesting conservation of phytosulfokines between monocot and dicot plants [61-64].

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Matsubayashi and Sakagami also initiated work towards the identification of the phytosulfokine receptor. In order to generate labeled PSK derivatives as affinity probes for the PSK receptor, functional groups had to be identified within PSKs that can be modified without impairing their bioactivity. The structure/activity relationships of PSKs were analyzed using the same bioassay of PSK activity that had already been used during PSK purification [61,65]. PSK-a exhibited half-maximal activity at 4 nM and was shown to be more active than PSK-p. The active core was shown to reside within the N-terminal tripeptide of PSK-a, which still retained 20 % of the full PSK-a activity. In contrast, the Nterminally truncated derivative was essentially inactive. Both sulfate groups were found to be essential for biological activity. The mono(Tyrl)- and mono(Tyr3)-sulfated peptides retained 0.6 % and 4 % of PSK-a activity, respectively, while the unsulfated peptide was found to be inactive [65]. Both the isoleucine and the threonine residues of PSK-a were shown to be functionally relevant since replacement with either valine or serine, respectively, resulted in a 20-fold reduction of bioactivity [65]. A radioligand was synthesized by introducing ^^S into the tyrosine sulfate to generate [^^S]PSK-a. Specific binding sites for [^^S]PSK-a were detected on intact rice suspension cells and in plasma membraneenriched fractions [63]. The ability of PSK derivatives to displace the radioligand from its binding sites was correlated to their mitogenic activity in the bioassay [63]. Characterization of the plasma membranelocated binding site required the synthesis of a second radioligand ([^H]PSK-a) with higher specific activity by catalytic tritiation of a tetradehydroisoleucine-containing PSK-a analog [66]. Using [^H]PSK-a, saturatable, reversible binding was demonstrated to plasma membrane preparations and high (Kd of 1.4 nM)- and low (Kd of 27 nM)-affinity binding sites were characterized. The observed correlation between the bioactivity of PSK analogs and their ability to compete with the radioligand for binding, as well as the affinity of binding which corresponds to the threshold of bioactivity in the bioassay indicate that the high-affinity binding site within rice plasma membranes may represent the functional PSK receptor. Whereas systemin is a peptide signal involved in the regulation of plant defense responses [14,67], phytosulfokines appear to be the first true plant peptide growth regulators. They clearly display mitogenic

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activity which is distinct but depends on the activity of other plant hormones like auxins and cytokinins [68]. Furthermore, under certain circumstances, their activity may extend to the regulation of cell differentiation [64]. Further work in this area holds the promise of exciting new discoveries related to the perception of the phytosulfokine signal and the cellular machinery involved in signal transduction. £nod40 Soil-borne bacteria of the family Rhizobiaceae and leguminous plants form a symbiotic relationship during which a new organ, the root nodule, is developed. Within these root nodules the bacteria fix atmospheric dinitrogen and the product of nitrogen fixation, ammonia, is exported to the plant [69,70]. Root nodules develop from primordia which are established at specific sites in the root cortex shortly after Rhizobium infection. The peptide enod40 is believed to play a critical role in inducing the de-differentiation and the mitotic division of root cortical cells, i.e. the initial steps in nodule development. This however, is not entirely undisputed [3,4,69-72]. During early stages of nodule development, early nodulin (ENOD) genes are expressed in the plant root and they are postulated to mediate nodule morphogenesis. The spatial and temporal patterns of expression in the root pericycle and cortical cells prior to the onset of mitotic cell divisions suggested the ENOD40 gene to be responsible for the initiation of nodule formation [73,74]. As a matter of fact, root cortical cell division was induced in Medicago trunculata plants overexpressing ENOD40 [75] and, furthermore, the induction of ENOD40 gene expression was shown to be required for nodule development [76]. In transgenic M. trunculata overexpressing ENOD40, the extent of nodule formation was correlated to the expression level of ENOD40 [76]. While a role for the ENOD40 gene in nodule initiation is well documented, it is less obvious what the gene product is that triggers the cellular responses. The transcripts of ENOD40 genes do not contain long open reading frames (ORFs) and it was initially assumed that the mRNA itself is the active principle [77]. A sequence comparison of all cloned ENOD40 genes, however, revealed the presence of two conserved regions. The 5'-proximal conserved region contains a short ORF with the capacity to encode the enod40 peptide of 10 - 13 amino acids (Fig. (4)).

380

a)

GjnEN0D4 0 a GjnEN0D4 Ob PVENOD40 LJEN0D4 0 SrEN0D4 0

ME - LCWQTSIHGS ME-LCWLTTIHGS MK-FCWQASIHGS MR-FCWQKSIHGS MK-LCWQKSIHGS

b)

PSENOD40 ysEN0D4 0 MSEN0D4 0 MtEN0D4 0 TrEN0D4 0

MKFLCWQKSIHGS MKLLCWQKSIHGS MKLLCWQKSIHGS MKLLCWEKSIHGS MKLLCWQKSIHGS

c)

NtENOD40 0SEN0D4 0 OjbENOD4 0 ZinEN0D4 0

MW WDEAIHGS ME-DEWLEHAHGS ME-DEWLEHAHGS ME-DAWLEHLHGS

Fig. (4). The primary structures of enod40 peptides. Enod40 peptides are compared from legumes with determinate nodules (a), legumes with indeterminate nodules (b), and non-legumes (c). Gaps (-) were introduce to optimize the alignment. The invariant residues are shown in bold face. The figure was modified after [79].

The 3'-distal part of the mRNA, in spite of the conserved region, does not contain a protein-coding ORF. Stimulated by this unexpected finding, several independent methods were employed to demonstrate that the enod40 peptide is actually expressed in vivo. Using immunological methods, the peptide was shown to be present in nodules, but not in roots, of soybeans as well as in the medium of tobacco protoplasts expressing the GmENOD40 transgene but not in wild-type protoplasts [78]. Furthermore, when a translational fusion was made between the ORF of soybean enod40 and green fluorescent protein (GFP) and expressed in tobacco protoplasts, GFP fluorescence in these protoplasts was similar in intensity to the GFP fluorescence expressed with its own translational start site [78]. The same technique was used to demonstrate that the AUG start codon of the conserved ORF is the only AUG that functions as an efficient translational start site in tobacco ENOD40 [74]. These studies allow the conclusion that enod40 is the primary translation product. This is in contrast to both systemin and phytosulfokines which are synthesized as larger precursor proteins.

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When the peptide-coding part of the ENOD40 gene was transiently expressed in M. trunculata, an induction of cortical cell division was observed indicating that indeed the peptide is the inducing factor [75]. Surprisingly however, expression of the conserved 3'-untranslated region (3'UTR) was found to stimulate the identical cellular responses [75,78]. A rationale to explain this finding has been put forward according to which the 3'UTR may regulate the translation of the ORF. It is assumed that the endogenous ENOD40 mRNA is expressed but is not translatable as a result of the binding of an inhibitory protein to the 3'UTR. This protein is titrated by transient expression of extra 3'UTR, thus allowing translation of the endogenous enod40 [3]. The data strongly support a role for the enod40 peptide in nodule initiation which may reside in its mitogenic activity. Notwithstanding, there is clear evidence that ENOD40 function extends beyond the initiation of nodule formation. In alfalfa, antisense inhibition of ENOD40 expression arrested callus growth, while overexpression of ENOD40 gave rise to embryogenic tumors [77]. Furthermore, ENOD40 genes were found in non-legumes like tobacco [78] and even monocot plants like maize and rice [79]. In rice plants, ENOD40 expression was found to be confined to the parenchyma cells surrounding the protoxylem during early stages of lateral vascular bundle formation and a function in the differentiation of the vascular bundles was suggested [79]. While evidence for the enod40 peptide being an endogenous plant growth regulator is accumulating, definite proof is still missing. This is mainly due to the lack of a convenient assay system. As seen above, such an assay system was instrumental for the purification and characterization of both systemin and phytosulfokines. Furthermore, it allowed the generation of labeled peptide derivatives for the characterization of receptor sites and it will ultimately lead to the isolation of the receptor proteins. Undoubtedly, the development of a suitable assay for enod40 activity would greatly advance this field of research. Natriuretic Peptides Natriuretic peptides (NPs) are a group of peptide hormones in animals that are critically involved in salt and water homeostasis (for review see [5]). NPs include atrial NP (ANP), brain NP (BNB), C-type NP (CNP), and urodilatin all of which are synthesized as larger precursor proteins. In

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the initial processing steps, the 24-amino-acid signal peptide and the two C-terminal arginine residues of preproANP are cleaved to yield proANP. ANP, which corresponds to the 28 C-terminal amino acids of proANP is believed to be the main biologically active ANP. Active ANP is circular in structure due to the formation of an intramolecular disulfide bridge [5]. The cellular perception of ANP involves binding to two of three different receptor proteins with cytoplasmic guanylate cyclase domains, the formation of cyclic guanosine-3',5'-monophosphate (cGMP), and the subsequent regulation of cation channel activity [5]. Evidence is accumulating that a NP signaling system, surprisingly conserved in structure and function, is operating in plants as well. The first line of evidence is provided by a series of studies revealing specific effects of synthetic rat ANP (rANP) in planta. In Tradescantia, rANP induced stomatal opening in a concentration-dependent manner [80]. The effect was shown to depend on the circular secondary structure of rANP: Linearization of rANP by disulfide reduction abolished the activity of the peptide [81]. This finding was interpreted as an indication for a highly specific, receptor-mediated process. As a matter of fact, spcific binding of ^^^I-rANP to microsomal and plasma membrane preparations of Tradescantia was demonstrated in vitro [80,82], and to tissue sections in situ [82]. Further experiments indicated a possible conservation of the rANP-triggered signal transduction machinery between vertebrates and plants. The involvement of cGMP was suggested by the finding that rANP-induced stomatal opening was suppressed by inhibitors of guanylate cyclase, while a membrane-permeable cGMP analog was able to mimic the rANP effect [81]. Furthermore, rANP was found to induce radial water movements out of the xylem in Tradescantia multiflora shoots and this response was similarly dependent on cGMP [83]. Despite this evidence, NPs presently cannot be considered plant peptide hormones. The establishment as a new peptide hormone requires the identification and the structure elucidation of the plant-derived molecule as well as its functional characterization. So far, an endogenous plant NP (PNP) has not been identified. Several steps toward this goal have been taken, however. Immunological evidence was obtained for the presence of NPs throughout the plant kingdom using antibodies directed against different parts of proANP [84]. PNP has been partially purified from ivy leaves by immunoaffinity chromatography using immobilized antisera directed against human and canine ANP [85]. While the resulting

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protein preparation still contained a number of proteins, immunoreactive polypeptides were present that corresponded in mass to rANP [86]. Immunopurified ivy PNP was found to be biologically active, causing a rapid and transient increase of cGMP concentrations and cation influx in the stele tissue of maize roots [86,87]. Like rANP, ivy PNP caused stomates to open and it was found to be more active on a molar basis as compared to rANP [85]. Considering the apparent functional and structural (immunological crossreactivity) conservation between plant and vertebrate NPs, it should be possible to identify Arahidopsis sequences related to preproANP in databases upon completion of the Arabidopsis genome project which is expected within the current year. While still unpublished, such a sequence may have already been identified [5]. The molecular characterization of the cDNA-encoded polypeptide will eliminate all doubts as to whether or not NPs rank among plant peptide hormones. INDIRECT EVIDENCE FOR ENDOGENOUS PEPTIDE SIGNALS Receptor-like Protein Kinases Indirect evidence for a general role of peptide hormones for intercellular signaling in plants stems from the observation that plants possess putative receptor proteins for (poly)peptide ligands known as receptor-like protein kinases (RLKs). While in all cases the direct biochemical interaction with a putative peptide ligand remains to be shown, the existence of RLKs involved in cell differentiation [88], morphogenesis [89], embryogenesis [90], meristem development [91], self-incompatibility [92-97], pathogen infection [98-102] and hormonal responses [103-105] points to a possible involvement of peptide signals in these processes. RLKs share a Cterminal, cytoplasmic serine/threonine kinase domain, a transmembrane domain and an N-terminal extracellular domain which is thought to interact with the peptide ligand (Fig. (5); [2,106-109]. Based on the structure of their extracellular domains, RLKs can be assigned to seven different classes. Most RLKs belong to the leucine-rich repeat (LRR) and the S-domain classes, respectively [6,108,109]. Exceptions include kinases with extracellular domains resembling epidermal growth factor (EOF) repeats [110,111], tumor necrosis factor receptors (TNFRs) [88], lectins [112], pathogenesis-related proteins [100], or kinases with novel

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extracellular domains [113]; Fig. (5)). In analogy to animal systems, RLKs are expected to interact with (poly)peptide ligands. This seems to hold true for CLAVATAl (CLVl) [91] and the S-locus receptor kinase (SRK) [92], the only two plant RLKs for which the endogenous ligands have been identified. Therefore, the discussion in this paragraph will be restricted to CLVl and SRK and their respective ligands.

Fig. (5). Modular structure of plant receptor-like kinases (RLKs). Schematic diagrams are shown of (A) SRK, a Brassica S-locus receptor kinase with sequence similarity in the extracellular domain to the S-locus glycoprotein (SLG); (B) Erecta, an Arabidopsis RLK with 20 LRRs; (C) WAKl, an Arabidopsis RLK with two epidermal growth factor-like repeats; (D) lecRK, an Arabidopsis RLK, with an extracellular domain resembling lectins; (E) CRINKLY4, a maize RLK with a region similar to tumor necrosis factor receptor and seven novel repeats; (F) PR5K, a RLK the extracellular domain of which resembles the pathogenesis-related protein PR5, and (G), a novel RLK from Catharanthus roseus (references are given the text). While the putative ligandbinding domains in the apoplast are highly divergent, RLKs have a conserved cytoplasmic protein kinase domain (PKD). The figure was modified after [109].

CLAVATAl CLVl belongs to the class of LRR-RLKs. By 1998, more than forty plant kinases had been identified in this group [6]. The extracellular LRR motif

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has been described as a versatile peptide-binding domain. Hence, LRRRLKs are believed to bind (poly)peptide ligands like animal receptor kinases do. Within a scaffold of positionally conserved leucines, variable residues confer specificity for different peptide ligands to the LRR motif [107]. Hence, the sequence of the extracellular ligand-binding domain does not provide any clues as to the nature of the respective peptide ligands, and the ligands for the LRR-RLKs have remained elusive. Genetic tools may now have allowed the identification of the ligand for CLVl. All organs of the plant shoot are derived from the shoot apical meristem as meristematic cells divide continually and start to differentiate to form new organs. Meristem maintenance requires a balance between the proliferation and the differentiation of meristematic cells. In Arabidopsis thaliana, three mutants clv(clavata)l, clv2, and clv3 have been described in which this delicate balance is disturbed. In these mutants, excessive meristem proliferation results in the formation of a club (latin: c/ava)-like structure. Genetic analyses revealed that the gene products of the two unlinked loci CLVl and CLV3 act in closely associated steps, or else form a complex (e.g. a receptor/ligand complex), in the same signaling pathway [114-116]. Therefore, the identification of CLVl as a LRR-RLK suggested CLV3 as a possible ligand of CLVl. The recent cloning of two tagged alleles of CLV3 provided strong evidence in support of this hypothesis [117]. CLV3 was shown to encode an extracellular, 96-amino-acid protein. Expression of CLV3 in the uppermost cell layer of the shoot apical meristem was sufficient to control cell proliferation and differentiation across the entire meristem. Apparently, CLV3 produced in one region of the meristem acts on the CLVl RLK located in another region of the meristem, i.e. CLV3 acts in a non-cell-autonomous manner [117]. These results are consistent with CLV3 being the ligand of CLVl or, alternatively, a molecule involved in CLVl ligand formation (e.g. a precursor). While a direct biochemical interaction between CLVl and CLV3 has not been demonstrated, there is strong evidence for an interaction in vivo [118]. It was shown that CLVl exists in two protein complexes of either 185 or 450 kD. The 185 kD complex represents the inactive form of the receptor complex consisting either of a CLVl homodimer or possibly of a CLV1/CLV2 heterodimer [118,119]. In presence of functional CLV3, the 185 kD complex recruits additional protein components (a protein phosphatase and a small GTP-binding protein) and is converted into the

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450 kD form which represents the activated receptor complex. Apparently, binding of CLV3 leads to activation of CLVl, phosphorylation, and association with other signaling factors [118,120]. S'locus Protein Kinase Most higher plants have hermaphroditic flowers, containing both male (anthers) and female (stigma, style, and carpel) organs. While this arrangement facilitates the transfer of pollen from one flower to the other by pollinating insects, it also promotes self-pollination. Self-pollination, however, leads to inbreeding and, consequently, to a reduction in geneflow. Thus, plants have evolved different strategies to prevent selffertilization, including the spatial separation of male and female flower parts as well as a separation in time of the maturation of male and female sex organs. Furthermore, many plants are able to identify and reject their own pollen, a phenomenon that has been described as self-incompatibility (SI) [121,122]. SI is controlled genetically by the highly polymorphic S locus [123]. In Solanaceae, the S locus encodes an allele-specific ribonuclease expressed in female flower tissues which is thought to provide the biochemical basis for the rejection of pollen carrying the same allele [124-126]. In Brassica (cabbage), the biochemical basis of pollen rejection is not known yet, but again, SI is controlled by the S locus complex, a highly polymorphic cluster of genes [127,128]. When a pollen grain is deposited on the surface of the stigma containing the same S allele as the pollen, an incompatible reaction leading to pollen rejection is triggered by action of the S locus gene products. Apparently, the S locus gene product expressed in the stigma recognizes the S locus gene products present in the pollen. Two of the S locus genes, those for the S locus glycoprotein (SLG) [129] and the S-locus receptor kinase (SRK) [92,93,95], are required for the phenotypic expression of SI [130]. They are co-expressed in female tissues and absent from male reproductive tissues [131], and have been suggested to interact functionally as the female determinant of self-incompatibility [131]. While SLG appears to have a stabilizing function, SRK itself is viewed as the receptor for the male determinant of self-incompatibility in pollen (for review see [121]). In a recent paper, Schopfer et al. describe a secreted pollen protein called

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SCR (S locus cysteine-rich protein) and propose it to be the ligand of SRK[132]. The SCR gene was identified as a single copy gene located between SRK and SLG at the polymorphic S locus. Loss-of-function and gain-offunction studies showed that the SCR gene product is both necessary and sufficient to determine the specificity of SI [132]. SCR gene expression was found to be restricted to anthers. Three SCR genes were sequenced. They were shown to code for cysteine-rich, small (8.4 to 8.6 kD), basic (isoelectric point of 8.1 to 8.4), secreted proteins. Apart from the signal sequence at the N-terminus and 8 conserved cysteine residues, sequence conservation at the SCR amino acid level was found to be very limited. The cysteines of SCR were proposed to engage in disulfide bridges, resulting in a protein fold with highly divergent, exposed surface loops. The extensive sequence divergence and the pattern of expression are consistent with SCR being the male determinant of SI [132]. While a direct interaction of SCR and SRK resulting in the activation of the receptor and downstream signaling remains to be shown, the data strongly support the hypothesis that SCR is the ligand of SRK. Hence, SCR appears to be a new plant peptide hormone involved in the determination of self-incompatibility. Processing Proteases Plants do not only possess the tools for the perception of peptide signals, they also have enzymes potentially involved in the processing of peptide prohormones. The existence of such enzymes provides additional indirect evidence for a general role of peptide hormones in plant signal transduction processes. In animals, most (poly)peptide hormones, growth factors, and neuropeptides are generated from larger, biologically inactive precursor proteins. The close examination of precursor primary structures indicated that the active peptides are flanked by pairs of basic amino acids. Therefore, the maturation of precursors to release the active peptides was postulated to involve limited endoproteolytic processing at sites marked by pairs of basic residues followed by the exoproteolytic trimming of the peptide termini [133]. The search for proteases with the required substrate specificity resulted in the initial discovery of the yeast kex2 protease (kexin), which is necessary for the maturation of the a-mating factor

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pheromone, followed by the identification of seven related proteases in mammals called proprotein convertases (PCs). PCs are critically involved in the processing of polypeptide precursors of hormones, growth factors, neuropeptides, receptor proteins, bacterial toxins, and viral glycoproteins. The function of PCs in proprotein processing has been reviewed extensiveley [7,134-139]. Mammalian PCs, just like kexin, cleave their substrates carboxyterminal of paired basic residues and they share a conserved catalytic domain resembling that of bacterial subtilisins. The catalytically important residues Asp, His, and Ser are arranged in the catalytic triad in a way that is typical for subtilisins but distinct from the arrangement found within the (chymo)trypsin clan of serine proteases. The subtilisins and (chymo)trypsins have thus served as a prime example of convergent evolution [140,141]. Until recently, proteases of the subtilisin clan of serine proteases (subtilases) were thought to be restricted to prokaryotes. The discovery of the PCs, Le. mammalian subtilases, greatly stimulated the interest in these enzymes. As of 1997, 200 subtilases were known and their number is steadily growing [141]. They have been grouped into six distinct families, the subtilisin, thermitase, proteinase K, lantibiotic peptidase, pyrolysin, and kexin families (Fig. (6)). Four of these families are restricted to micro-organisms while only the pyrolysins and kexins are found in both micro-organisms and higher eukaryotes and these are the two families that are relevant to the following discussion of plant enzymes possibly involved in the generation of peptide signals. Subtilisin Thermitase

\

Kexin Pyrolysin

K

v^

^

Lantibiotic peptidases Proteinase K

/y

Fig. (6). Families of subtilases within the clan of subtilisin-like serine proteases. A general layout of the relationship between the six subtilase families is shown. The dendrogram is based on a sequence alignment of the catalytic domains only. The figure was modified after [141].

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Several lines of evidence point to the existence of plant proteases related to mammalian PCs in both structure and function. In transgenic plants, the existence of a kexin-like activity was demonstrated. Tobacco plants were engineered to overexpress the precursor of the KP6 killer toxin. The KP6 preprotoxin is encoded by a double-stranded RNA virus which is present in some natural isolates of Ustilago maydis, a fungal pathogen of maize. In U. maydis, the processing of the KP6 protoxin to release the active toxin requires a kexin-like protease within the secretory pathway [142]. In transgenic tobacco plants overexpressing the preprotoxin, correct processing of the precursor and secretion of the toxin was observed indicating the presence of a kexin-like activity in the secretory pathway of tobacco plants [143,144]. The protease was later shown to be a Golgi-resident enzyme and the kexin-like substrate specificity was confirmed [145]. A protein (SBP50) was identified in preparations of tomato leaf membranes that interacts specifically with the wound signal systemin. Competition experiments with a series of Ala-substituted systemin derivatives identified those amino acids within the systemin primary structure relevant for the interaction with SBP50 [25]. They constitute a sequence motif typically found in furin, a PC with an extended sequence requirement for substrate recognition [146,147]. A possible function of SBP50 as a PC-like protease was supported by the observation of an immunological relationship between SBP50 and a PC from Drosophila. Also, processing of systemin on the carboxy side of the central dibasic (Lys9-Argl0) pair indicated the presence of a PC-like activity in tomato plasma membrane preparations [25,27]. While the data suggest the existence of a kexin-like protease(s) in plants, ultimate proof remains to be provided. The situation is reminiscent of that in animals where the existence of proprotein convertases had been postulated already in the early 1960s. Nonetheless, the hunt for these proteases lasted for three decades and more. The PCs evaded all attempts of biochemical characterization and purification. The breakthrough came in 1984 with the identification of kexin by genetic complementation of a yeast mutant. The discovery of kexin paved the way for the identification of mammalian PCs by molecular biological techniques exploiting the sequence conservation within the catalytic domains [148]. The same rationale was used in an attempt to identify PCs in plants. The most highly conserved regions surrounding the catalytically important residues were identified by sequence comparison of kexin and mammalian PCs.

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Oligonucleotides were derived from these regions and employed as primers in the polymerase chain reaction (PCR) to amplify related sequences from the tomato genome. The gene family of subtilases was found to comprise a minimum of 15 members in tomato plants and its complexity thus exceeds that of mammalian PCs [8]. In contrast to mammalian PCs, however, tomato subtilases belong to the pyrolysin rather than the kexin family of subtilases. The observation that no kexins were isolated while the PCR primers used in these experiments were actually based on sequences conserved among kexins may indicate that kexins do not exist in tomato. This notion is corroborated by the fact that the Arabidopsis genome project, while being close to completion, did not yield any sequences related to kexin-like subtilases. Pyrolysins are more closely related to bacterial subtilisins than kexin and they were thought to share with subtilisins their broad substrate specificity resulting in a degradative function rather than a role as processing proteases. The recent discovery of two mammalian pyrolysins indicates that this is not necessarily so. The site-1 protease (SIP) from hamster is a pyrolysin involved in the regulation of lipid composition of animal cells [149]. It participates in the activation of a transcription factor (nuclear sterol regulatory element binding protein, nSREBP) by cleaving one (site 1) of two processing sites in the nSREBP precursor. SIP cuts between Leu and Ser of the site-1 processing site Arg-Ser-Val-Leu-Ser. Recognition requires the Arg and Leu residues while Ser and Val could be replaced with Ala without reducing cleavage efficiency [150]. A second pyrolysin called subtilisin/kexin-isozyme 1 (SKIl) was cloned from man, rat and mouse. SKIl exhibited a substrate specificity similar to that of SIP - cleaving pro-brain-derived neurotrophic factor (pBDNF) between Thr and Ser of the sequence Arg-Gly-Leu-Thr-Ser - but different from the PC specificity [151]. Apparently, processing proteases in mammals include members from both, the kexin and the pyrolysin family of subtilases. Considering the likely absence of kexins from higher plants, their respective functions would have to be carried by pyrolysins alone. As a matter of fact, there is increasing evidence for plant pyrolysins playing a role both in protein processing as well as in protein degradation. The first pyrolysin to be cloned from a higher plant was cucumisin from Cucumis melo, an extracellular protease highly abundant in melon fruit [152]. Cucumisin was shown to have a broad substrate specificity in that it cleaves a variety of small peptide substrates and eight peptide bonds within the oxidized insulin B chain [153-155]. A similar, broad

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Specificity was also observed for related subtilases from Helianthus annuus, Madura pomifera, and Taraxacum officinale [156-158]. Therefore, these proteases are likely to serve degradative functions during fruit ripening and leaf senescence, respectively. Several cDNAs encoding pyrolysins have been cloned from Alnus [159], Arahidopsis [159,160], and Lilium [161], but the respective enzymes have not been characterized and their functions remain unclear. The so far most comprehensive analysis of plant subtilases has been done in tomato. Fifteen genes were identified in the haploid tomato genome which fall into 5 distinct subfamilies including the P69, tmp, L^SBTl, L^SBT2, and LeSBT3/4 subfamilies (Fig. (7); [8]. Members of the P69 subfamily were initially identified as inducible components of the plant defense response triggered by pathogen infection [162-164]. Later, P69A and P69D were suggested to play a role in plant development, while P69B and P69C were shown to be expressed following pathogen infection [165] and treatment with salicylic acid or the fungal toxin fusicoccin [165,166]. For P69E and P69F, a highly specific expression has been described in the root tissue and in hydathodes, respectively [167]. Unfortunately, the substrate specificity of the P69 subtilases has not been characterized and - with the notable exception of LRP - a tomato cell wall protein of unknown function, none of the in vivo substrates has been identified [168]. Therefore, a function for the P69s as either degradative or processing proteases remains to be established. The tomato protease tmp is highly similar to LIM9 from Lilium logiflorum [161,169]. This protease has been identified as an extracellular protein which is differentially expressed in anthers during late stages of microsporogenesis [161]. The highly specific pattern of expression points to a very restricted role of LIM9 in pollen development, and possible functions in the remodeling of the extracellular matrix and tapetal cell apoptosis have been discussed [161]. Unfortunately, as for the P69s, the specificity in vitro and the substrates in vivo remain to be identified. The latter is also true for enzymes of the LeSBTl, L^SBT2, and LeSBT3/4 subfamilies. One of these subtilases however, L^SBTl, has been overexpressed in the baculovirus/insect cell system and the recombinant enzyme characterized biochemically [170]. L^SBTl was shown to be an extracellular protease that is secreted in form of an inactive zymogen. Zymogen activation requires the sequential processing of the signal peptide for targeting to the secretory pathway, the prodomain, and a 21amino-acid auto-inhibitory peptide at the amino terminus [170]. In

392 P69Arw

^P69B

P69E P69C

SBTl

tmp

Fig. (7). Phylogenetic relationship of tomato subtilases. An unrooted phylogenetic tree is shown based on the amino acid sequences deduced from tomato subtilase genes and cDNAs. Numbers indicate PAM distances (accepted point mutations per 100 residues) between sequences. The figure was modified after (8).

contrast to other plant subtilases, L^SBTl exhibited a narrow substrate specificity cleaving polypeptide substrates preferentially but not exclusively carboxy-terminal of Gin residues. These properties make L^SBTl a likely candidate for a proprotein processing protease potentially involved in the generation of a peptide signal, as opposed to an enzyme with merely degradative function. Further evidence for a role of plant pyrolysins in signaling processes was provided by the analysis of the sddl mutant in Arabidopsis, which is affected in the density of leaf stomata. The SDDl gene was isolated by positional cloning and found to encode a subtilase. Loss of SDDl function resulted in an increase in stomate density suggesting a role for the protease in pattern development (T. Altmann; Max Planck Institute of Molecular Plant Physiology, Golm, Germany; pers. communication). The major challenge for future work will be the identification of the in vivo substrates of SDDl and L^SBTs. These substrates may well include the precursors of endogenous bioactive peptides with signaling function in plant growth and development.

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EXOGENOUS PEPTIDE SIGNALS Plants, being sessile organisms, have to be able to adapt to the everchanging environment with appropriate biochemical responses. A prerequisite for adaptation is the ability to perceive these changes which include biotic as well as abiotic stress factors. Microbial infection is a particularly threatening form of biotic stress. Consequently, plants have evolved to sense the presence of a pathogen and to react to infection with appropriate defense responses including the development of resistance. Hence, a surveillance system must be present which - similar to the phenomenon of self-incompatibility (discussed above) - allows the distinction between "self and "non-self [9,10]. Two types of resistance are known and are referred to as general and race-specific resistance, respectively. For the induction of a general resistance response, a structure has to be recognized by the plant cell that is absent from plant cells but common to a wide range of microorganisms [9,171]. In contrast, racespecific resistance depends on the presence of a specific protein in a certain race only of a microbial species (i.e. the avirulence (avr) gene product) and a corresponding protein in the resistant plant species (i.e. the resistance gene product). This type of interaction is described by the gene-for-gene concept [172-176]. In general resistance and in race-specific resistance as well, it is a molecule produced by the microorgnism (or generated by an enzyme produced by the microorganism) that is recognized by the plant cell as being foreign, followed by the induction of a defense response. Such molecules have been referred to as elicitors [10, 177]. Elicitors can be (poly)peptides, carbohydrates, lipids, small secondary products, or a combination thereof. The structural diversity of elicitors is immense and beyond the scope and intention of this review. Thus, the following discussion will be restricted to microbial (poly)peptide elicitors of plant pathogen resistance and will focus on a few examples that are most instructive in demonstrating the underlying principles. General peptide elicitors General Peptide Elicitors in Bacteria Unlike vertebrates, plants do not have an immune system but they nevertheless are able to detect the presence of a pathogen and to respond

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with a broad set of defense responses including localized cell death {i.e. the hypersensitive response, HR) and induced resistance against a broad array of pathogens (Le. systemic acquired resistance, SAR). The first bacterial elicitor of HR and SAR to be characterized was harpin, a heatstable, glycine-rich protein from Erwinia amylovora the causal agent of fire blight in apple and pear [178]. Subsequently, several harpins have been characterized in many different phyto-pathogenic bacteria ([179,180] and references therein). Harpins are extracellular proteins secreted via the Sec-independent type-Hi secretion system [181]. General characteristics of harpins include high glycine- and low cysteine-content, heat stability, low mobility during SDS-PAGE, and the ability of fulllength and truncated polypeptides to elicit the HR ([182], and references therein). Harpins induce defense responses and resistance in a variety of non-host plants [180,182,183]. Hence, they are general elicitors in the sense of the above definition, i.e. elicitor activity does not depend on the presence in the infected plant (or cell culture) of a corresponding resistance gene. However, plants also respond to mutants lacking secreted harpins and to bacteria that lack the type-III secretion system [171,181]. Consequently, additional and more general structures must exist that are recognized by the plant cell. Very recently, the bacterial flagellum has been identified as such an elicitor-active structure [171]. The bacterial flagellum consists of a rotary motor anchored in the cell surface and a long, helical filament composed of multiple subunits of a single protein, flagellin. Within the flagellin protein, it is the most conserved region close to the N-terminus that is recognized by a specific chemo-perception system of the plant cell resulting in the activation of defense responses including an extracellular alkalinization, the oxidative burst, the HR, and the induction of pathogenesis-related gene expression [171,184]. As a convenient assay system to further characterize the response, elicitor-induced pH changes of the growth medium of tomato cell suspension cultures were monitored. In this cell culture system, flagellin was found to cause a rapid and transient alkalinization of the growth medium. A 22-amino-acid peptide (flg22) corresponding to the conserved N-terminal region retained full flagellin activity and causes half-maximal alkalinization at 30 pM. Competitive antagonists of flg22 also inhibited the response to flagellin or crude bacterial extracts from Erwinia carotovora, E. chrysanthemi, Pseudomonas syringae, P. aeruginosa, P. fluorescens and E. coli indicating that the flg22 epitope is the major if not the only determinant of recognition by the plant cell.

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Interestingly, this epitope is not conserved in the flagellins from Agrobacterium tumefaciens and Rhizobium meliloti, two plant-associated bacteria. Accordingly, peptides corresponding to the respective regions of these flagellins did not exhibit elicitor activity in both the cell culture bioassay and in Arabidopsis plants. Apparently, the modification of the respective region within the flagellin protein, while retaining full flagellin functionality, enables Agrobacteria and Rhizobia to evade the plant chemo-perception system [171,184]. Treatment of Arabidopsis seedlings with flg22 resulted in growth arrest allowing for a convenient screen for natural and mutagen-induced genetic variation in this response. Three genetic loci designated FLSl, FLS2, and FLS3 were identified and FLS2 was identified by positional cloning [184]. Recently, the interesting finding was reported that FLS2 encodes a putative LRR-RLK. FLS2 thus resembles Xa21 the rice gene conferring resistance to Xanthomonas oryzae, and CLVl (discussed above), the LRR-RLK involved in the regulation of meristem proliferation [185]). It therefore seems likely that FLS2 is a functional receptor of flagellin or fragments thereof. General Peptide Elicitors in Fungi General resistance against Phytophthora, a widespread phyto-pathogenic oomycete, is elicited by a family of small (10 kDa) secreted proteins called elicitins [10,186]. Phytophthora elicitins, while highly similar, have been grouped in acidic a-elicitins (e.g, capsicein, parasiticein, amegaspermin) and basic p-elicitins (e.g. cryptogein, cinnamonin, (3megaspermin). The p-elicitins, and cryptogein in particular, are more potent elicitors of general defense responses (including HR and SAR) than the a-elicitins [187-189]. Consequently, most studies concentrated on the highly active cryptogein. The threedimensional structure of cryptogein has been elucidated by both X-ray crystallography and by NMR in solution yielding nearly identical results. The overall structure has a novel fold comprising three disulfide bridges, six a-helices, and a beak-like motif composed of an antiparallel two-stranded p-sheet and an Q-loop [190,191]. In tobacco plasma membranes, a single class of high affinity (Kd = 2 nM) binding sites was observed for [^^^I]cryptogein [192]. A later study using [^^^I] derivatives of four different elicitins revealed that binding relies on those amino acid residues that are conserved among

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elicitins and that all elicitins bind to a common site in tobacco plasma membranes which is believed to be the elicitin receptor [188]. Binding to the receptor is thought to stimulate cellular responses including the influx of Ca^"^, extracellular alkalinization, acidification of the cytosol, depolarization of the plasma membrane potential, the oxidative burst, protein phosphorylation and changes in gene expression ([188,193-196], and references therein). While the characteristics of binding to tobacco plasma membranes were very similar for all elicitins, the activation of cellular responses correlated with the in-vivo activity of a-, and p-elicitins [188]. As discussed for bacterial elicitors of general defense responses, additional determinants of general resistance must exist in fungi, since elicitins are found in the entire genus Phytophthora and some Pythium species but are notably absent from many other phytopathogenic fungi [10]. These additional elicitors include glucans, chitin and chitosan oligosaccharides derived from the fungal cell wall, ergosterol, i.e, the main sterol in most higher fungi (reviewed in [197]), but also oligopeptide elicitors derived from fungal glycoproteins [198]. Such an elicitor and its interaction with cultured parsley cells have been studied in considerable detail. In parsley cell cultures, a surface glycoprotein from the soybean pathogen Phytophthora megasperma elicits typical non-host, general resistance responses including the influx of Ca^"^, the alkalinization of the apoplast, the depolarization of the plasma membrane potential, the oxidative burst, protein phosphorylation, defense gene activation, and phytoalexin production [198-201]. The elicitor activity was found to reside solely in the protein moiety of the 42-kDa fungal glycoprotein and could be confined to the 11-amino-acid core of a 13amino-acid peptide (Pep 13) within the C-terminal hydrophilic region of this protein. Systematic replacement with alanine identified two amino acids (Trp2 and Pro5) within Pepl3 (VWNQPVRGFKVYE) that are critical for elicitor activity [202]. Rapid, high-affinity binding of radiolabeled Pep 13 to parsley plasma membranes was demonstrated. Binding of structural Pep 13 analogues correlated well with their respective elicitor activities [202]. The binding site, which is thought to be the elictor receptor, was shown to reside in a = 100-kDa membrane protein and was partially purified from parsley microsomal membranes [203,204]. Activation of the receptor by elicitor binding stimulates a plasma

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membrane Ca^"*"-permeable ion channel resulting in increased cytosolic Ca^"*" which is necessary for subsequent cellular responses [205]. Notwithstanding, the structural diversity among peptide elicitors of general pathogen resistance and the obvious involvement of different receptor proteins, the initial cellular responses observed after receptor activation are very similar. The first responses include ion fluxes across the plasma membrane (Ca^"^/H'*"-influx, K^Cl- efflux), resulting in a depolarization of the plasma membrane potential and an alkalinization of the apoplast. Hence, the responses mediated by general peptide elicitors closely resemble those triggered by the endogenous defense signal systemin [4,26,38-40,206]. In contrast, race-specific peptide elicitors (discussed below) cause a hyperpolarization of the plasma membrane potential and the acidification of the apoplast rather than extracellular alkalinization [175,207,208]. Race-specific Peptide Elicitors As opposed to non-host resistance discussed in the previous two paragraphs, race-specific elicitors are involved in defense reactions that can be described genetically in terms of the "gene-for-gene" concept. This concept was developed in the 1940s by Flor [172,209] who observed that in the interaction of flax with the flax rust fungus Melampsori lini the development of resistance depends on the presence of two dominant genes, the avr (avirulence) gene in the fungus and the corresponding R (resistance) gene in the plant. As a biochemical basis for the gene-forgene concept, a ligand-receptor model of disease resistance was proposed. According to this model, resistance develops as a consequence of the activation of a receptor (Le. the R gene product) by interaction with its ligand, the race-specific elicitor. The elicitor may be produced by the action of an avr gene-encoded enzyme or else, the avr gene product itself has elicitor activity. In the latter case, the avr gene product qualifies as a bioactive peptide in the sense that it elicits specific cellular responses. The cloning of many avr genes and, more recently, the cloning of matching plant resistance genes provided good evidence in support of this model. It cannot be the aim of this review to cover this area of research comprehensively. This has been done in recent review articles [176,210216].

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Two examples have been chosen for discussion, Le, the interactions of tomato with Pseudomonas syringae pv. tomato and with Cladosporium fulvum, a bacterial and a fungal pathogen, respectively. These two plant pathogen interactions are among the few that have been characterized not only in genetic terms but also at the molecular and biochemical levels and they are thus well suited for a discussion of the fundamental principles. Race-specific Peptide Elicitors in Bacteria Most plant pathogens propagate in the extracellular space of the host. Hence, R proteins were expected to be cell surface receptors of extracellular signals. While this appears to be true for Xa21 (the product of the rice R gene for Xanthomonas oryzae resistance [98]), most R genes for bacterial pathogens rather encode cytoplasmic proteins [212,217]. This finding was difficult to reconcile with the receptor/ligand model of plant disease resistance. Upward of 40 avr genes have been cloned from bacteria. Most of them encode hydrophilic proteins that lack signal sequences for secretion [218,219]. Avr proteins do not induce the HR when injected into leaves of plants possessing the corresponding R gene. Only living bacteria carrying the avr gene are able to induce the resistance response. Apparently, avirulence depends on additional factors [219]. Additional genes required for avirulence are located in the hrp (hypersensitive response and pathogenicity) cluster. Several of the hrp genes code for components of the contact-dependent type III secretion system. In some mammalian pathogens {e.g, strains of Yersinia, Salmonella, Shigella), type III protein translocation complexes function in the translocation of bacterial proteins into the cytoplasm of target cells of the host [219]. Therefore, bacterial avirulence factors are now believed to be delivered directly into the host cells by the type III secretion apparatus [220-226]. The data imply that these race-specific elicitors are perceived intracellularly and they are in good agreement with the surprising finding that plant R genes for bacterial pathogens - i,e, putative receptor proteins for the avr gene products - code for cytosolic proteins as well. While this is also true for Pto, the tomato R gene that confers resistance to Pseudomonas syringae carrying the corresponding avirulence gene avrPto [216,227], Pto differs from other R genes for bacterial pathogens in that it does not encode a protein comprising a leucine rich repeat (LRR) domain and a nucleotide binding site, but rather an intracellular

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serine-threonine protein kinase lacking any obvious receptor domain [227,228]. Nevertheless, the Pto-avrPto system is the only one for which a direct interaction of the respective R and avr proteins has been demonstrated. Pto and avrPto were found to interact specifically in the yeast two-hybrid system, an assay which tests for interaction of two proteins by reconstitution of a functional transcription factor. Furthermore, mutations that disrupt the interaction resulted in a loss of avrPto recognition within the plant cell [229,230]. Thus, formation of the Pto-avrPto complex is necessary for the resistance response but it is not sufficient, since the activation of cellular responses requires Prf, a second serine-threonine protein kinase [228,231-233]. Like most other bacterial avr genes, avrPto was identified by selecting for clones in a bacterial DNA library that confer an avirulent phenotype to a Pseudomonas syringae strain that is normally virulent on tomato cultivars carrying the Pto resistance gene [234]. AvrPto encodes mRNAs of 0.7 and 0.75 kb whose translation product is a hydrophilic 164 amino acid protein of 18.3 kDa. AvrPto bears no similarity to other proteins in the GenBank and EMBL databases [235]. Likewise, the deduced protein products of other bacterial avr genes range from 18 to 100 kDa in size and lack substantial sequence similarity to proteins of known biochemical activity or motifs indicative of specific functional domains [219]. Hence, the biochemical function of these avr gene products remains unknown. It is believed that bacterial avr proteins play a role in pathogenicity in the compatible plant/pathogen interaction. Once recognized by the R protein as a component of the plant surveillance system, however, they become avirulence factors [212]. The avrBs2 gene of Xanthomonas campestris pv. vesicatoria provides an example in support of this hypothesis. Deletion of this avr gene causes a reduction in pathogenicity confirming a role as virulence factor on a compatible host [236,237]. AvrBs2 bears resemblance to Agrobacterium tumefaciens agrocinopine synthases and may play a role in the Xanthomonas adaptation to the host extracellular space, or it may provide the bacterium with a carbon and nitrogen source during colonization of the host plant [212,237]. Race-specific Peptide Elicitors in Fungi The interaction of tomato with the leaf mould fungus Cladosporium fulvum is a typical gene-for-gene relationship. The development of

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resistance depends on the presence of a matching avr gene for each Cf resistance gene [210,215,238]. Avirulence gene products are race-specific elicitors. They have been detected in the apoplastic fluid of C. fulvuminfected tomato plants [239]. AVR4 and AVR9 proteins were isolated from apoplastic fluids based on their ability to elicit a hypersensitive reaction on tomato plants carrying the corresponding Cf-4 and Cf-9 resistance genes, respectively. Both AVR4 and AVR9 are synthesized as prepro-proteins that are processed upon secretion to yield mature proteins of 86-88, and 28 amino acids, respectively [240-242]. The production of mature AVR9 involves both fungal and plant proteases [243]. AVR4 and AVR9 are characterized by 8 and 6 cysteine residues, respectively, that are engaged in the formation of disulfide bridges. Disulfide bridge formation and the rigidity of the resulting structure are crucial for the function of the proteins as avirulence factors [244], For AVR9, the threedimensional structure as well as the structural requirements for elicitor activity have been determined. The polypeptide forms a so-called cystine knot in solution in which the Cys3-Cys6 disulfide bond penetrates a ring formed by the Cysl-Cys4, and Cys2-Cys5 disulfide bonds and the intervening polypeptide chain [245,246]. This structural motif is common among inhibitory and toxic polypeptides [247]. The rigid structure is thought to provide resistance against degradation by plant and fungal proteases present in the extracellular space [238,244]. Systematic substitution with alanine and the synthesis of mutant peptides revealed the hydrophobic residues present in both solvent-exposed surface loops as relevant for elicitor activity [248,249]. The AVR9 peptide was labeled with iodine-125 at the N-terminal tyrosine residue, and ^^'^I-AVR9 was used to identify a high-affinity (Kd = 0.07 nM) binding site (HABS) in tomato plasma membrane fractions [250]. According to the receptor/ligand model of gene-for-gene interactions, one would expect the HABS to be identical with Cf-9, the resistance gene product. Surprisingly, however, the binding site was shown to be present in both resistant and susceptible tomato cultivars, as well as in other Solanaceae [250] showing that Cf-9 is not the AVR9 receptor per se. Nevertheless, the HABS appears to be required to initiate the resistance response, since a positive correlation exists between the binding affinity and the necrosis-inducing activity of mutant AVR9 peptides [251]. Possibly, Cf-9 does not recognize AVR9 itself as its ligand but rather the AVR9/HABS-complex, and the interaction of all three components may be necessary to elicit the defense reaction.

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Alternatively, the HABS may not be involved in the defense reaction but may rather represent the target of AVR9 as a virulence factor in the compatible plant/pathogen interaction. In the latter case, a second binding site for AVR9 must exist which may by formed by Cf-9 and a second signal-transducing component yet to be identified [238,251]. This model resembles the perception of CLV3 which is thought to be the endogenous peptide signal in the regulation of meristem proliferation/differentiation (cf. above, [238]). The perception of CLV3 appears to involve the formation of a heterodimeric complex including CLVl (a LRR-RLK) and CLV2. CLV2 resembles Cf-9 in that it is a transmembrane protein comprising an extracellular LRR domain and a very short cytoplasmic domain [119]. Like CLV2, Cf-9 has an extracellular LRR domain but lacks a cytoplasmic domain with any obvious signaling function [252]. A similar structure was determined for the R gene products Cf-2, Cf-4, and Cf-5 [215,253-255], and the specificity for the avr protein was shown to reside in the extracellular LRR domains of Cf proteins [256]. Therefore, the LRR appears to provide the recognition element for the race-specific elicitors but an additional factor is obviously required for the activation of cellular responses. This factor may be the HABS, or else, a CLVl-like LRR-RLK. Biochemical data will be essential to either support or reject this model of AVR9 perception. GENERAL CONCLUSIONS The examination of general and race-specific elicitors of plant defense reactions revealed that plant cells are able to perceive a large number of structurally diverse peptide signals. It seems likely that during evolution the machinery to perceive and transduce exogenous peptide signals was not generated de novo but was rather recruited from pre-existing cellular signaling components. Thus, the recognition of exogenous peptide elicitors by the plant cell may indicate the presence of endogenous peptide signals and the corresponding signal perception/transduction machinery. The structural similarity between CLVl, a LRR-RLK involved in meristem maintenance, and FLS2 or Xa21, i.e, the putative receptors of exogenous peptide elicitors, provides support for this hypothesis. Likewise, structural similarity was observed between the resistance gene product Cf-9 and CLV2. Both proteins have been suggested to be part of a receptor complex and contain LRRs likely to

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provide the ligand binding sites. Hence, LRRs appear to be versatile perception modules for extracellular and intracellular elicitors as well as for endogenous peptide signals. In some cases, structural similarity is not restricted to the perception system for exogenous and endogenous peptides but rather extends to the signals themselves. The AVR9 peptide elicitor and SCR, the male determinant of self-incompatibility in Brassica, for example, are both small cysteine-rich proteins in which disulfide bridges provide a rigid structural scaffold for exposed surface loops. A comparison of systemin, an endogenous peptide signal for plant defense, with peptide elicitors of general resistance shows that even the initial cellular responses are highly similar. These observations, in addition to the presence in planta of receptor-like kinases and processing proteases as indirect evidence for a general role of peptide signals in intercellular communication in plants, all seem to indicate that the plant peptide signals identified thus far may just be the tip of the iceberg. Obviously, we are just beginning to unravel the complexity of peptide signaling in higher plants. The years to come will likely see the identification of new peptide signals as regulators of plant growth and development and promise exciting new discoveries in the elucidation of both signal perception and cellular responses. ABBREVIATIONS avr HABS hrp LRR HR PC R RLK SAR SBT SCR SI SLG SRK SWRP

= = = = = = = = = = = = = = =

avirulence high-affinity binding site hypersensitive response and pathogenicity leucine-rich repeat hypersensitive response proprotein convertase resistance receptor-like kinase systemic acquired resistance subtilase S locus cysteine-rich protein self-incompatibility S locus glycoprotein S locus receptor-like kinase systemic wound response protein

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ACKNOWLEDGEMENTS I thank Dr. N. Amrhein for critical reading of the manuscript, helpful discussions, and support. Work in the author's laboratory was supported by grants from the ETHZ and the Swiss National Science Foundation. REFERENCES [I] [2] [3] [4] [5] [6] [7] [8] [9] [10] [II] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

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De Wit, PJ.G.M.; Spikman, G. Plant Pathol., 1982, 21, 1. Scholtens-Toma, I.M.J.; De Wit, PJ.G.M. Physiol. Mol. Plant Pathol., 1988, 33, 59. Van den Ackerveken, G.F.J.M.; Van Kan, J.A.L.; Joosten, M.H.A.J.; Muisers, J.M.; Verbakel, H.M.; De Wit, P.J.G.M. Plant J., 1992, 2, 359. Joosten, M.H.A.J.; Cozijnsen, A.J.; De Wit, P.J.G.IVI. Nature, 1994, 367, 384. Van den Ackerveken, G.F.J.M.; Vossen, P.; De Wit, P.J.G.M. Plant Physiol., 1993,103,91. Joosten, M.H.A.J.; Vogelsang, R.; Cozijnsen, T.J.; Verberne, M.C.; De Wit, P.J.G.M. Plant Cell, 1997, 9, 1. Vervoort, J.; Van den Hooven, H.W.; Berg, A.; Vossen, P.; Vogelsang, R.; Joosten, M.H.A.J.; De Wit, P.J.G.M. FEBS Lett., 1997,404, 153. Van den Hooven, H.W.; Apelman, A.W.J.; Zey, T.; De Wit, P.J.G.M.; Vervoort, J. Eur. J. Biochem., 1999, 264, 9. Pallaghy, P.K.; Nielsen, K.J.; Craick, D.J.; Norton, R.S. Protein ScL, 1994, 3, 1833. Kooman-Gersmann, M.; Vogelsang, R.; Hoogendijk, E.C.M.; De Wit, P.J.G.M. Mol. Plant Microbe Interact., 1997, 10, 821. Mahe, E.; Vossen, P.; W., V.d.H.H.; Le-Nguyen, D.; Vervoort, J.; De Wit, P.J.G.M. J. Peptide Res., 1998, 52, 482. Kooman-Gersmann, M.; Honee, G.; Bonnema, G.; De Wit, P.J.G.M. Plant Cell, 1996, 8, 929. Kooman-Gersmann, M.; Vogelsang, R.; Vossen, P.; Van den Hooven, H.W.; Mahe, E.; Honee, G.; De Wit, P.J.G.M. Plant Physiol., 1998,117, 609. Jones, D.A.; Thomas, CM.; Hammond-Kosack, K.E.; Balint-Kurti, P.J.; Jones, J.D.G. Science, 1994, 266, 789. Dixon, M.S.; Jones, D.A.; Keddie, J.S.; Thomas, C.M.; Harrison, K.; Jones, J.D.G. C^//, 1996,84,451. Dixon, M.S.; Hatzixanthis, K.; Jones, D.A.; Harrison, K.; Jones, J.D.G. Plant Cell, 1998, 10, 1915. Thomas, CM.; Jones, D.A.; Parniske, M.; Harrison, K.; Balint-Kurti, P.J.; Hatzixanthis, K.; Jones, J.D.G. Plant Cell, 1997, 9, 2209. Parniske, M.; Hammond-Kosack, K.E.; Golstein, C ; Thomas, CM.; Jones, D.A.; Harrison, K.; Wulff, B.B.H.; Jones, J.D.G. Cell, 1997, 91, 821.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

413

ENZYMES INVOLVED IN THE BIOSYNTHESIS OF BRASSINOSTEROIDS JOCHEN WINTER Max-Planck-Institutfur Zuchtungsforschungy Carl-von-Linne-Weg 10, D50829 Koln, Germany; tel: +U9'221'5062-251; fax: ++49-221-5062213; email: [email protected] ABSTRACT: Brassinosteroids are essential phytohormones with a multitude of physiological activities. Since the first report on growth-promoting "brassin", termed later "brassinolide", many new members of the brassinosteroid hormone family have been characterized. The biosynthesis of brassinosteroids has very carefully been investigated by in vivo feeding experiments of plant cell cultures and by studying the distribution of native brassinosteroids in diverse plant species. A major progress in understanding brassinosteroid biosynthesis and mode of action resulted from the discovery oidv/arf Arabidopsis mutants that proved to be brassinosteroid-sensitive or deficient. The best known dwarf mutants in Arabidopsis, tomato and pea exhibit deficiencies in genes implicated in the biosynthesis of brassinosteroids. Cytochrome P450-dependent monooxygenases and dehydrogenases were shown to play a major role in the conversion of sterol precursors to the biologically active hormone, brassinolide, by genetic, molecular biological, biochemical and metabolite feeding studies.

INTRODUCTION In 1979, a new class of plant growth-promoting substances, termed "brassins", was isolated from rape pollen {Brassica napus). The active compound of brassin proved to be a phytosterol, brassinolide, the structure of which was elucidated using X-ray analysis. The structure of brassinolide, (22/?,23/?,245)-2a,3a,22,23-tetrahydroxy-24-methyl-Bhomo-6a-oxa-5a-cholestan-6-one, resembles the insect moulting hormone 20-hydroxyecdysone [1]. Brassinolide shows a growth promoting biological effect at very low concentrations, i.e. elongation, bending, cell division [2,3]. Since the discovery of brassinolide many new brassinosteroids have been isolated. All brassinosteroids share a full steroid skeleton bearing a 5a-configuration between ring A and B, a 60X0- or 7-numbered B-ring lactone-function and a 22/?,23/?-cis-diolmoiety in the side-chain [4-7].

414

The biosynthesis of brassinosteroids has been investigated by in vivo feeding of plant cell cultures, in addition to the analysis of native brassinosteroid-patterns in diverse plant species. The proposed pathway (Fig. (1)) leads from unpolar sterol precursors to the polyhydroxylated phytohormone brassinolide, that is known to exhibit the highest biological activity. A major breakthrough in understanding the biosynthesis pathway and mode of action of brassinosteroids resulted from molecular genetic characterization of Arabidopsis dwarf mutants turned to be brassinosteroid-insensitive or -deficient. To date other dwarf mutants in Arabidopsis, pea and tomato are known to be deficient in genes coding for enzymes involved in brassinosteroid biosynthesis. Cytochrome P450dependent heme-thiolate monooxygenases and dehydrogenases seem to play a major role in regulating the conversion of sterol precursors to biologically active brassinosteroid intermediates and the end product, brassinolide [8-11]. During the initial molecular genetic studies the functions of mutated Arabidopsis genes were determined by feeding mutant plants with intermediates of brassinosteroid biosynthesis. Thus brassinosteroid precursors preceding the proposed biosynthetic blockage, led to no change in the mutant phenotypes, whereas brassinosteroids occurring after the block in the pathway bypassed the deficiency, leading to a rescue of mutant to wild type phenotype [12-16]. Some biochemical functions defined by the Arabidopsis dwarf mutants were later confirmed by heterologous expression of genes and by in vivo conversion of postulated substrates [17-20]. As part of these physiological and biochemical studies, tomato cell suspension cultures have also been established to investigate intermediates and enzymes of brassinosteroid biosynthesis and metabolism [21-23]. Enzyme activities from partially purified protein extracts were first detected in this model system [24]. This review will focus on enzymes converting sterols, but will not deal with proteins representing elements of signal transduction cascades, i.e. kinases or receptors.

415

^(^Vs^

Squalene

^,^,^k^Js^Cycloartenol

H O ^ ^ * " ^ ^ Episterol \STE1

24-MethyIdesmosterol

24-Methylenecholesterol

DWFl y 1KB /

V'vAy^

S,XN^*N^

Campestcrol DWF41

%y^N^\^

H ^

22a-Hydroxycampesterol

DWF4 j

^

5-Dehydroepisterol

DJFF-/ I

l o ^ N ^ j ^ Campestanol

HO^^-TX 6-Oxocampestano!

'I

5»H ,

^^^-^ I OH

6-Deoxocathasterone CPD \

tf"

3.Dehydro-6-deoxoteasterone

a-Oehydroteasterone

Ho^A^^fY^Typhasterol 6-DeoxotyphasteroI

DWARF 6-Deoxocastastcrone f

OH

H O ^ ' ^ H r y ^ Brassinolide

Fig. (1). Proposed pathways and genes involved in sterol and brassinosteroid biosynthesis.

416

BIOSYNTHESIS OF BRASSINOSTEROID PRECURSORS Most known brassinosteroid mutants are defective in genes that code for enzymes required for the biosynthesis of brassinosteroid precursors. This chapter will summarize the upstream part of brassinosteroid biosynthesis pathway. Desaturation of episterol: The Arahidopsis mutant dwp/stel is defective in C5-desaturation of episterol (Fig. (2)) [18], thus impaired in an enzyme function involved in a very early step of brassinosteroid precursor biosynthesis. The enzymatic block of dwp/stel was determined by feeding experiments using '"^C-labelled mevalonic acid and a subsequent analysis of endogenous sterol and brassinosteroid precursors. The mutant accumulates episterol with a simultaneous decrease of downstream intermediates (24-methylenecholesterol, campesterol, castasterone, brassinolide).

Episterol

5-Dehydroepisterol

Fig. (2). Desaturation of episterol, the proposed enzymatic reaction of STEl (DWARF7).

417

Formation of campesterol: The conversion from 24-methylenecholesterol to campesterol (Fig. (3)) is catalyzed by the product of a gene deficient in the Arabidopsis mutant dwarf 1 [25], which is allelic with dim [16] and ccbl [14], as well as with Ikb from pea (Pisum sativum) [26]. The nature of dwfl deficiency was defined by feeding experiments with brassinosteroid intermediates together with the analysis of endogenous brassinosteroid patterns. The deduced amino acid sequence of the DWFl gene showed significant similarity to membrane-bound flavin-adenine dinucleotide-dependent oxidorecuctases. The reduction of 24-methylenecholesterol to campesterol seems to be catalyzed by a two step reaction. First, 24methylenecholesterol is isomerized to 24-methyl-desmosterol which, in a second step, is reduced to campesterol.

24-Methylenecholesterol

24-Methyldesmosterol

Fig. (3). Two step formation of campesterol.

Campesterol

418

Conversion of 22a-hydroxycampesterol to (22S,24/f)-22hydroxyergost-4-en-3-one: Another dwarf mutant of Arabidopsis , saxl, defines a step upstream of DWFl in the brassinosteroid biosynthesis pathway [27]. Rescue experiments with intermediates showed that saxl is involved in the oxidation and isomerization of 3p-hydroxyl,A ' precursors to 3-oxo-A^4,5 steroids (Fig. (4)). OH

22a-Hydroxycampesterol

Fig. (4). Conversion of 22a-hydroxycampesterol.

OH

(22/?, 245)-22-Hydroxyergost-4-en-3-one

419

5a-reduction of (24/iL)-24-methylcholest-4-en-3-one: The Arabidopsis mutant detl is involved in a 5a-reduction of the A"^'^ double bond of (24/?)-24-methylcholest-4-en-3-one (Fig. (5)) [13, 17]. Exogenously applied brassinosteroids were able to rescue the mutant to wild type phenotype. Both feeding experiments and analysis of endogenous brassinosteroids supported the role of detl early in the pathway. The DET2 gene codes for a protein with high similarity to a mammalian 5a-reductase. Heterologous expression of the detl-cDNA in mammalian cells showed that the enzyme was able to metabolize several 3-oxo,A'^'^ steroids, such as progesterone, testosterone and androstenedione with NADPH as electrondonor, but failed to convert 3|3hydroxyl, A^'^ steroids including cholesterol, pregnenolone and dehydroepiandrosterone [28].

(24/?)-24-Methylcholest-4-en-3-one

(24/?>24-MethyI-5a-cholestan-3-one

Fig. (5). 5a-reduction of (24/?)-24-methylcholest-4-en-3-one.

420

C22a-hy droxy lation: The first side-chain hydroxylating step (Fig (6)) in brassinosteroid biosynthesis is catalyzed by a cytochrome P450 (CYP90B1) defective in the Arabidopsis dwarf4 mutant [15, 18]. The function of CYP90Blas a steroid side-chain 22a-hydroxylase, was revealed by feeding with 22ahydroxylated intermediates. Interestingly, not only cathasterone and its 6deoxo-derivative succeeded in rescuing dwarf4, but also the 22hydroxylated precursor 22a-hydroxycampesterol was effective.

6-Oxocampestanoi

Cathasterone

Fig. (6). Formation of cathasterone.

BIOSYNTHESIS OF BRASSINOSTEROIDS: This chapter deals with the conversion from cathasterone to brassinolide, the biologically most active brassinosteroid. Only two mutants have been found to be involved in the downstream subpathway. Nevertheless, enzyme activities from enriched protein fractions have first been detected in this part of brassinosteroid biosynthesis.

421

Cathasterone 23a-hydroxylation: The Arabidopsis mutant cpd codes for another cytochrome P450, named CYP90A1 [12]. This enzyme is responsible for hydroxylating cathasterone at side-chain C23 leading to teasterone (Fig. (7)). The proposed enzymatic block was verified by applying biosynthetic precursors and intermediates of the brassinosteroid pathway to explants. Brassinosteroid precursors preceding the blockage of biosynthesis showed no significant effect, whereas intermediates occurring after the block were able to compensate the gene defect leading to a wild type phenotype. Studies on the activity of the CPD promoter exhibited a similar phenomenon. Brassinosteroid precursors preceding the block were found to have no siginificant effect on promoter activity. All intermediates of the early and late C6 oxidation pathway and the corresponding 24-epimers bearing the side-chain c/^-diol-function showed a strong inhibition [29]. Binding spectra of the heterologously expressed cytochrome P450 could only be recorded with cathasterone as the substrate, and intermediates carrying side-chain hydroxyl groups at C22 and C23 (Winter, unpublished). In summary, biosynthetic precursors preceding cathasterone seem to have no visible effect , neither on the plants, nor on the promoter and enzyme activity.

OH

Cathasterone

Fig. (7). Cathasterone 23-hydroxylation.

Teasterone

422

Oxidoreductive epimerization of teasterone and typhasterol: The next step in the proposed pathway is the epimerization of teasterone to typhasterol producing 3-dehydro-teasterone as intermediate (Fig. 8)). No mutant is thus far described being responsible for this conversion. Nevertheless, a corresponding enzyme activity has been detected in different plants.

3-Dehydroteasterone

Typhasterol

Fig. (8). Epimerization of teasterone.

The first data confirming this oxidoreductive epimerization were obtained by measuring the enzyme activity in protein extracts from a tomato cell suspension [24]. It could be demonstrated, using a very sensitive fluorimetric detection method, that two different enzymes were involved in this subpathway. No enzyme activity could be detected with 24-^/7/-teasterone as substrate and NAD^ or NADP"^ as electronacceptors. But using the proposed intermediate 3-dehydro-24-^p/-teasterone as substrate, enzymatic conversion to 24-e/7/-teasterone was measured in a microsomal fraction of tomato cell cultures (Fig. (9)). 3-dehydro-24-e/7/teasterone-reductase showed a specific activity of 361 fkat/mg protein with NADPH as the only accepted electrondonor. 3-dehydro-24-e/7/-teasterone being used as substrate was enzymatically reduced to 24-e/?/-typhasterol. This enzyme activity was observed in the soluble lOO.OOOg supernatant showing a specific activity of 82 fkat/mg protein with NADH as only accepted electrondonor. 24^/?/-typhasterol was converted to 3-dehydro-24-^/7/-teasterone by the same protein fraction with a specific activity of 21 fkat/mg protein and NAD^ as only accepted electronacceptor .

423

24-£'/7/-Teasterone

3-Dehydro-24-e/;/-teasterone

24-^/7/-Typhasterol

Fig. (9). Conversion of 3-dehydro-24-e/7/-teasterone.

Enzyme activities were detectable only in protein extracts derived from cell cultures after induction with the corresponding substrates. Two different enzyme activities were shown to be involved in this subpathway, one located in the microsomal fraction, and one with a typical cytosolic dehydrogenase activity. Interestingly, the conversion of 3-dehydro-24-^/7/-teasterone to lA-epiteasterone follows the opposite direction of the proposed biosynthesis pathway. Feeding experiments support this observation: Exogenously applied 3-dehydro-24-^/?/-teasterone was converted to 24-^/7/-teasterone as the major metabolite (95%), and to only traces (5%) of lA-epxtyphasterol [23]. Thus, it is very likely that there is a different pathway in tomato cell cultures. In addition, preliminary studies with protein extracts of Arabidopsis cell cultures also reveal differences in enzyme distribution and specificity depending on the origin of the culture [Winter, unpublished]. In contrast, enzymatic conversion from teasterone to typhasterol could be detected in cultured cells of Marchantia polymorpha. It was demonstrated that teasterone as substrate is converted to 3dehydroteasterone by a cytosolic protein in a cell culture . 3dehydroteasterone is further reduced to typhasterol by the same protein extract with nearly the same specific enzyme activity. No exogenously applied redox equivalents were necessary for these enzyme activities. The reverse reaction from typhasterol to teasterone was not detected [30].

424

C6 oxidation of 6-deoxocastasterone: The tomato Dwarf gene also encodes a cytochrome P450, named CYP 85 [31]. The proposed function of CYP85 was confirmed by feeding experiments, the analysis of endogenous brassinosteroid patterns, and by heterologous expression of this enzyme [20], catalyzing the conversion from 6-deoxocastasterone to castasterone, in yeast (Fig. (10)). 6deoxocastasterone was exogenously applied to a yeast cell culture expressing the tomato cytochrome P450. The substrate was first hydroxylated at C6, giving 6a-hydroxycastasterone, then was subsequently converted to the corresponding 6-oxo-compound castasterone. CYP85 is shown to be a multifunctional enzyme, exhibiting a hydroxylase connected with a dehydrogenase enzyme activity.

6-Deoxocastasterone

6a-Hydroxycastasterone

Castasterone

Fig. (10). C6-oxidation of 6-deoxocastasterone, the proposed enzymatic reaction of CYP85.

425

Bayer-Villiger-oxidation of 24-^/?i-castasterone: The key step in the biosynthesis of brassinosteroids is the conversion of castasterone to brassinolide. This reaction is a lactonization of the steroidal B-ring or a Bayer-Villiger-oxidation. Tomato cell suspension cultures have been extensively studied in respect to the metabolism of 24-^/7/-castasterone and 24-ep/-brassinolide [21, 22, 32, 33]. A microsomal fraction of tomato cell cultures, induced by lA-epicastasterone, was able to convert this substrate into 24-^/7/-brassinolide (Fig. (11)). The specific enzyme activity was determined to be 230 fkat/mg protein with NADPH serving as the only accepted electrondonor [24].

OH

OH

HO/, HO^^

24-e/7/-Castasterone

Fig. (11). Bayer-Villiger-oxidation of 24-e/7/-castasterone.

24-£/7/-Brassinolide

426

CONCLUSIONS An enormous progress in investigating the brassinosteroid biosynthesis was made by mutant analysis and the use of very sensitive analytical methods, leading within few years to a logical and very attractive putative biosynthetic pathway. Nevertheless, there are many questions still open because of problems to detect enzyme activities corresponding to each step of the pathway. The model of biosynthesis pathway was put together by studying the metabolism of exogenously applied intermediates in cell cultures of various origins and combining these results with data of native brassinosteroid patterns. It is more or less accepted that there are three pathways in parallel, the early and the late C6 oxidation pathway, as well as the 24/?-epimers follow ing the same route. Some observations in the analysis of native brassinosteroid patterns suggest a possible connection between the pathways. It was shown that seeds of Arabidopsis contain castasterone and 24-^p/-brassinolide [34]. Also members of both 24-epimers, brassinolide and 24-^/7/-brassinolide were detected in tomato seeds [Winter, unpublished]. At present, only few data from studying of purified enzyme fractions are available. This could either be due to a very low level of enzyme activity, or due to the application of unsuitable substrates that are not metabolized. The future will give us the chance to characterize more enzymes in the brassinosteroid pathway, leading not only to an improved understanding, but also to the possibility of modifying internal brassinosteroid levels, thus getting a new insight in the mode of action of brassinosteroids. ACKNOWLEDGEMENT The author thanks Csaba Koncz (Cologne) for all his support. The author is financially supported by a joint research program between the MPI and Hoechst Schering AgrEvo GmbH.

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Grove, M.D.; Spencer, F.G.; Rohwedder, W.K.; Mandava, N.B.; Worley, J.F.; Warthen, J.D.; Steffens, G.L.; Flippen-Anderson, J.L.; Cook, J.C.; Nature, 1979,287,216-217 Mandava, N.B.; Ann. Rev. Plant Physiol. Plant Mol. Biol., 1988, 59, 23-52 Clouse, S.D.; Sasse, J.M.; Ann. Rev. Plant Physiol. Plant. Mol. Biol., 1998, 49, 427-451 Sakurai, A.; Fujioka, S.; Plant Growth Reg., 1993,13, 147-159 Fujioka, S.; Sakurai, A.; Natural Product Reports, 1997,14, 1-10 Yokota, T.; Trends Plant Sci., 1997, 2,137-143 Adam, G.; Schneider, B.; In Brassinosteroids: Steroidal Plant Hormones.; Sakurai, A; Yokota, T; Clouse, S.D.; Eds.; Springer-Verlag: Tokyo, 1999; p. 113-136 Clouse, S.D.; Plant J., 1996, 20, 1-8 Szekeres, M.; Koncz, C ; Plant Physiol. Biochem., 1998, 36, 145-155 Koncz, C ; Trends Plant Sci., 1998, 3, 1-2 Altmann, T.; Planta, 1999, 208, 1-11 Szekeres, M.; Nemeth, K.; Koncz-Kalman, Z.; Mathur, J.; Kauschmann, A.; Altmann, T.; Redei, G.P.; Nagy, F.; Schell, J.; Koncz, C ; Cell, 1996, 85, 171182 Li, J.; Nagpal, P.; Vitart, V.; McMorris, T.C.; Chory, J.; Science, 1996, 272, 398-401 Kauschmann, A.; Jessop, A.; Koncz, C ; Szekeres, M.; Willmitzer, L.; Altmann, T.; Plant J., 1996, 9, 701-713 Azpiroz, R.; Wu, Y.; LoCascio, J.C; Feldmann, K.A.; Plant Cell, 1998,10, 219-230 Klahre, U.; Noguchi, T.; Fujioka, S.; Takatsuto, S.; Yokota, T.; Nomura, T.; Yoshida, S.; Chua, N.-H.; Plant Cell, 1998,10, 1677-1690 Fujioka, S.: Li, J.; Choi, Y.-H.; Seto, H.; Takatsuto, S.; Noguchi, T.; Watanabe, T.; Kuriyama, H.; Yokota, T.; Chory, J.; Sakurai, A.; Plant Cell, 1997, 9,19511962 Choe, S.; Dilkes, B.P.; Fujioka, S.; Takatsuto, S.; Sakurai, A.; Feldmann, K.A.; Plant Cell, 1998,10, 231-243 Choe, S.; Noguchi, T.; Fujioka, S.; Takatsuto, S.; Tissier, C.P.; Gregory, B.D.; Ross, A.S.; Tanaka, A.; Yoshida, S.; Tax, F.E.; Feldmann, K.A.; Plant Cell, 1999,77,207-221 Bishop, GJ., Nomura, T.; Yokota, T.; Harrison, K.; Noguchi, T.; Fujioka, S.; Takatsuto, S.; Jones, J.D.; Kamiya, Y.; Proc. Natl. Acad. Sci. USA, 1999, 96, 1761-1766 Hai, T.; Schneider, B.; Adam, G.; Phytochemistry, 1995, 40, 443-448 Winter, J.; Schneider, B.; Strack, D.; Adam, G.; Phytochemistry, 1997,45, 233-237

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IMMUNOPOTENTIATING EFFECTS OF A GLYCOPROTEIN FROM CHLORELLA VULGARIS STRAIN CK AND ITS CHARACTERISTICS *KUNIAKI TANAKA, YIKIHIRO SHOYAMA, AKIRA YAMADA, KIYOSHI NOD A, FUMIKO KONISHI AND KIKUO NOMOTO K, Z, K,K, E K: Research Laboratories, Chlorella Industry Co,, Ltd., 1343 Hisatomi, Chikugo City, Fukuoka 833-0056; Y. S,: Department of Pharmacognosy, Facuhy of Pharmaceutical Sciences, Kyushu University, 3'1'1 Maidashi, Higashi-ku, Fukuoka 8 J2-0054; A. Y: Kurume University Research Center for Innovative Cancer Therapy, and Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume City 830-0011; K N.: Department of Immunology, Medical Institute ofBioregulation, Kyushu University, 3-1-I Maidashi, Higashi-ku, Fukuoka 812-0054, Japan. Abstract: Chlorella vulgaris strain CK, a unicellular green alga, has been used as a health food for the past 30 years in Japan and in otlier countries. Oral administration of C vulgaris results in several pharmacological effects, including augmenting host defenses in animal models and in human experiments. The oral administration of C vulgaris showed clear prophylactic effects in stress-induced peptic ulcer models, presumably through the "immune-brain-gut" axis, and it also suppressed a Meth A tumor growth in an antigen-specific manner. C. vulgaris in active form, known as CVS, was purified from the culture supernatant of C. vulgaris and found to be a glycoprotein with a molecular weight of 63,100 amu. CVS contains 67% carbohydrate with a p-l,6-D-galactopyranose backbone and 35% protein. A protein moiety is essential for CVS to exhibit immunopotentiating activity, and 15-mer of the partial amino acid sequence at the NH2-terminus has been determined. CVS exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis by intratumor (i.t.) injection. Prophylactic effects of CVS were observed on 5-fluorouracil-induced myelosuppression and indigenous infection by subcutaneous injections. From these results, it became evident that CVS augments antimetastatic inununity through T cell activation in lymphoid organs and accelerates recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis and/or tlie progression of residual tumors. CVS may also be beneficial for the alleviation of adverse effects of cancer chemotherapy, causing an early recovery of hematopoietic stem cells without affecting the antitumor activity of chemotherapeutic agents. 1. OVERVIEW 1-1.

From the discovery of Chlorella algae to the mid 1990s

Chlorella was one of the first algae to be isolated as a pure culture; this was accomplished by Beijerinck in the 1890s [1]. Otto Warburg introduced

430

the use of Chlorella in a study of photosynthesis in 1919. Since the latter part of the 1940s, scientific attention has been drawn towards the potential of unicellular green algae for mass cultivation as a food source because of its high protein content and highly nutritious nature. The taxonomic criteria for determination of the genus Chlorella were established by Fott and Novakova [2]. C vulgaris strain CK has been maintained by Chlorella Industry Co., Ltd., for over 30 years. The dried powder of C. vulgaris has been used as a health food for the past 30 years in Japan and in other countries. The pharmacological effects of Chlorella have been reported mainly using C vulgaris, with only a few reports listing the potential uses of other Chlorella species, including radioprotection by C kessleri [3], an anti-malignant glioma effect C pyrenoidosa [4], and anti-dopaminergic effects of C stigmatophora [5]. Various pharmacological effects of C vulgaris strain CK were studied in animal models and human experiments, such as anti-cardiovascular disease [6,7], anti-hyperlipidemia [8], immunomodulation in tumor-bearing mice [9-11], and host-mediated protection against opportunistic infections [12-15]. 1-2.

Pharmacology of Chlorella algae in the last five years

Pharmacology of oral administration ofC. vulgaris strain CK

Oral administration of the algae shows an anti-peptic ulcer effect in human experiments [16]. Peptic ulcers are formed by a destruction of the balance between ulcer-promoting factors, including gastric acid and pepsin secretion, and protective factors, including the repair of injured mucous membrane, improvement of the blood stream through epithelial tissues, and prostaglandin secretion. C vulgaris showed clear and strong prophylactic effects against ulcer formation with regard to its effect on protective factors, as observed in 16-hr-stressed Wistar rats at 23 °C, and in a cysteamine-induced duodenal ulcer model, although no anti-ulcer effect occurred in the former models [17]. The algae may prevent ulcer formation through "immune-brain-gut" axis. Oral administration of whole organisms of C. vulgaris increased the frequency of bowel movements and the total stool amount and also improved the feeling of comfort and the stool consistency in female students aged 18 to 20 [18]. The effect on the frequency of bowel movement was recognized even after stopping C vulgaris administration. Thus, constipation is improved by the intake of C vulgaris, which suggests its usefulness to persons troubled by constipation as a simple and easy diet therapy. Komaki et al studied the protein digestibility of whole organisms of C vulgaris in in vitro and in vivo experiments to evaluate the difference of

431

whole organisms of C. vulgaris with or without cell rupture processing [19]. The results suggest that C vulgaris should b^e an efficient protein source even without cell rupturing. Chemically induced tumor promotion was inhibited by glyceroglycolipids or sterols from C vulgaris strain CK according to Morimoto et al [20] and Yasukawa et al [21]. The administration of C. vulgaris should be useful in preventing gastrointestinal absorption and for promoting the excretion of dioxin already absorbed into tissue [22]. Chlorophyll-related compounds in the algae show such an effect and are useful as a new approach in the treatment of patients exposed to lipophilic xenobiotics [23]. Host-mediated activity ofglycoprotein-rich extracts ofC. vulgaris strain CK

We reported previously that a hot-water extract of C. vulgaris strain CK augmented the resistance against Escherichia coli infection via the accumulation and activation of neutrophils [12] and against murine cytomegalovirus infections via activation of natural killer (NK) cells [14]. Augmented resistance against opportunistic infections was expressed even in leukopenia induced by cyclophosphamide, an anti-cancer drug, and the effect was observed even by oral administration of the extracts [13,15]. The elimination of Listeria monocytogenes, a facultative intracellular bacterium, was impaired in mice with LP-BMS murine leukemia virus-induced murine acquired immunodeficiency syndrome (MAIDS). Oral administration of glycoprotein-rich, hot-water extracts of C. vulgaris restored or enhanced the capacity of MAIDS mice to eliminate L monocytogenes in association with improvement of the deteriorated immune response to L monocytogenes [24]. Oral administration of the extracts enhanced resistance to L monocytogenes through augmentation of Listena-specific cell-mediated immunity in normal mice and mice with MAIDS. In these mice, oral administration of the extracts augmented the expression of interferon y (IFNy) and interleukin (IL)-12 mRNA in the spleen after Listeria infection, while it reduced the expression of IL-10 mRNA [25]. The extracts may preferentially augment Thl responses against Listeria via the activation of macrophages to produce IL-12 and enhance host defense against Listeria infection both in normal and MAIDS mice. Thus glycoprotein-rich, hot-water extracts of the algae are potent biological response modifiers (BRMs), as assessed by their ability to increase host defenses against bacterial and viral infections in normal and immune-compromised hosts. Oral administration of the hot-water extracts in mice suppressed the immunoglobulin E (IgE) production against casein antigen, accompanied by increased IFNy and IL-12 mRNA expression [26]. Oral administration of the extracts enhanced Thl response to casein in the spleen of casein-immunized mice. The hot-water extracts may be useful for the pre-

432

vention of allergic diseases with a predominant Th2 response. Antitumor immuno-activity of whole organisms and glycoprotein-rich extracts ofC. vulgaris strain CK

The growth of a transplantable Meth A tumor, methylcholanthrene-induced fibrosarcoma of BALB/c origin, was significantly suppressed when whole organisms or glycoprotein-rich fractions extracted from C vulgaris were administered by not only the i.t. route but also through oral administration in an antigen-specific manner [9,10]. These effects were found to be due to enhanced host defenses rather than to any direct activity against tumor cells or targets. Polymorphonuclear leukocytes from such mice show an enhanced antigen-nonspecific antitumor activity in the Winn assay using lymphoid and inflammatory cells from mice treated with a hot-water extract [11]. CDFl ((BALB/c x DBA/2 )F1) mice that had received an diet containing whole organisms of C vulgaris strain CK for the previous 5 weeks were inoculated subcutaneously with Meth A tumor cells on day 0, and rechallenged subcutaneously (s.c.) in the right or left flank with Meth A or Meth I tumor cells, another syngeneic tumor of BALB/c origin, on day 9, respectively. Meth A tumor growth inoculated on day 0 was less affected, but the growth of the rechallenge Meth A tumors was inhibited about 50% at more than 8 days after the rechallenge tumor inoculation in mice administered the diet containing C. vulgaris. At the same time, the growth of the Meth I tumors was not inhibited. As determined by a Winn assay, lymph node leukocytes of C vw/gam-administered Meth A-inoculated mice inhibited the tumor growth significantly, about 54%, at 13 days after the inoculation of the mixture of leukocytes and Meth A tumor cells into untreated normal mice. A similar effect was observed in the lymph node cells from the glycoprotein-rich acetone extract of C. vulgaris-treated mice. Cytostatic activity against Meth A tumor cells was observed in the peritoneal cells obtained from tumor-inoculated, acetone extract-treated mice on day 12, but no cytolytic activity was observed in the spleen cells of the same mice. Glycoprotein-rich hot-water extracts of C vulgaris strain CK showed stronger antitumor immuno-activity by i.t. administration [9]. Based on these findings, we decided to purify the pharmacologically active components from the crude extract of C vulgaris strain CK. 2. DISCOVERY OF ANTITUMOR IMMUNO-ACTIVE ACIDIC GLYCOPROTEIN FROM THE CULTURE SUPERNATANT OF C VULGARIS STRAIN CK22 We found a crude antitumor active substance from the fresh culture supernatant of C vulgaris strain CK22, a substrain of CK. The higher

433

molecular weight (MW) fraction, CVS-U, was tested on Meth A, B-16, 3LL, MH134, and Sarcoma 180 tumors, and significant antitumor effects were observed in all the tumor lines following i.t, administrations [27,28]. Antitumor effects were also observed in some tumor lines after intravenous (i.v.) or intraperitoneal administrations. Table 1. The inhibition of both primary and rechallenge tumor growth by i.t injection of CVS-U into the primary tumors [27] Rechallenge l^imors

Priinary I\imors Drug, Dose,Tiimes (mg/kg)

'nimor Volume/Suppress, l^imor Voliime/Suppress./Comp.Reg. (X 10^ mm^)

A. Meth A PBS, CVS-U, 50,

(%)

(X 10^ mm^)

(%)

(%)

140.0 ± 62.2 135.9 ±46.3

3

0 0 40

3* 1*

192.1 ±43.3 175.7 ±44.2

36 6*

137.8 ±22.2^

28

46.6 ± 50.8^

67

119.8 ±38.8^

27.9 ±49.1^

80

50

10* 3^^ 3-^

102.0 ±44.5^ 174.2 ±41.6

38 57 9 -2

73 46 9

70 10

194.4 ±34.3

37.9 ±61.7^ 75.0 ± 60.9^ 127.0 ±57.4

CVS-U, 10,

3* 3*

222.2 ± 39.5 130.7 ±24.9^

41

186.1 ±42.2 59.0 ± 65.9^

68

0 50

CVS-U, 10,

10*

112.6 ±40.8^

49

37.9 ± 58.9^

80

60

3* CVS-U, 50, 10* OK432,0.5KE, 3* OK432, IKE, 5*

136.5 ± 45.9^

39

51.2 ±79.0^

91.1 ±21.9^ 175.0 ±58.4 202.0 ± 33.9

59

29.8 ±54.1^ 132.4 ±100.4 107.9 ± 98.7^

72 84

60 70

29 42

22 33

CVS-U, 50, CVS-U, 50, CVS-U, 50, CVS-U, 50, CVS-U, 50, B. Meth A PBS,

CVS-U, 50,

21 9

0

CDFl mice were inoculated s.c. with 5 x 10^ Meth A cells into the right flank on day 0 and rechallenged s.c. with the same tumor as primary inoculated tumor into the left flank on day 9. CVS, OK432 or PBS was injected i.t. into therightflank-tumor.Primary tumor sizes were measured 11 (A) or 13 (B) days, and rechallenge tmnor sizes were measured 10 (A) or 11 (B) days after either the primary and rechallenge tumor inoculation, respectively, and are expressed as the mean ± S.D. Suppress: % suppression of tumor growth was calculated as ((tumor volume of PBS-treated mice tumor volume of drug-treated mice) / tumor volume of PBS-treated mice) x 100. Comp. Reg.: % of rechallenge tumor regressed mice completely at 14 days after the tumor inoculation was calculated as (number of mice without a rechallenge tumor / number of used mice) x 100. fj: significantly different from PBS group in each experiment by students f-test. ^; drugs were injected i.t. 1, 3, 5, 6 or 10 time(s) every 2 daysfromday 2. ^J CVS-U was injected i.t. three times every 2 daysfromday 7. ": CVS-U was injected i.t. three times every 2 days from day 10.

We also tested the antitumor activity of CVS-U against rechallenge tumors using Meth A and CDFl mice. We observed a strong antitumor effect against the rechallenge Meth A tumor after three i.t. administrations

434

of 50 mg/Kg CVS-U were given once every other day for six days (Table 1). The effects of CVS-U were stronger than those of the standard dose of an already established BRM, OK-432. When the rechallenged and the primary-inoculated tumor system were compared, the former was found to be more sensitive and dose-dependent. 2-1. Methodology (1) Purification of antitumor immuno-active BRM

For the screening of antitumor immuno-active components, 5 x 1 0 ^ Meth A tumor cells in 0.2 ml phosphate buffered saline (PBS; 0.8% NaCl, 0.02% KCl, 0.29% NaHP04 and 0.02% KH2PO4) were inoculated s.c. into the right and left flank of 8-12 week-old CDFl mice on days 0 and 9, respectively, Fig.(l). Each test fraction (2.0, 10 or 50 mg dry wt/kg body wt/each time) was injected into the right flank-tumor 5 times every two days from day 2, to evaluate an antitumor activity against both tumors at 8, 10 or 12 days after the tumor inoculation. Antitumor activity was estimated as the tumor size of the longest and shortest diameters of growing ellipsoid tumors over the skin. BALB/c

V

Q

I

9

CVS, i.t

Measurement of tumor size

I

Meth A Meth A SxlO^s.c. SxlO^s.c. Fig. (1). Experimental design of screening test.

(2) Analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)

Since, the broad protein and carbohydrate band overlapped each other in the 42 - 200 kDa range of SDS-PAGE and in the pH 3.0 - 4.3 range of lEF-PAGE (data not shown), MALDI MS of ARS2 was performed as previously reported for hapten-carrier protein conjugates For more detail MW determination [29-31 ]. ARS2 (1-10 pmole) was mixed with a 100-fold molar excess of sinapinic acid in an aqueous solution containing 10% trifluoroacetic acid (TFA). The mixture was placed inside a JMS-LDI 1700 time-of-flight (TOF) MS spectrometer (JEOL, Japan) and irradiated with a nitrogen laser (337 nm, 3 ns pulse). The ions formed by each pulse were accelerated by a 30 kV potential into a 1.7 m evacuated tube.

435

(3) Formation ofpolyclonal antibodies against ARS2 and immunoassay using anti'ARS2 antibody

ARS2 dissolved in PBS was emulsified with an equal volume of Freund's complete or incomplete adjuvant. The emulsion was s.c. injected into male JW rabbits at an initial dose of 1.0 mg, followed by 0.5 mg injections every 2 weeks. Serum was collected 2 weeks after the last booster injection. The anti-ARS2 antibody level was determined by usual ELISA as described previously [27,28]. (4) Chemical characterization ofARS2

Purity was confirmed by gel-filtration using a HPLC column packed with Asahipak GS-520HQ and elution with 100 mM sodium phosphate buffer containing 300 mM sodium chloride (pH 6.7). The content of total protein, total sugars, uronic acids, sulfates, nucleic acids, phosphate or fatty acids was assayed by the BCA [32] and Lowry method [33], the phenol-sulfuric acid method [34], the Blumenkrantz method [35], nephelometry [36], absorption at 260 nm, the Bartlett method [37] and the GLC method after methyl-esterification [38], respectively. ARS2 was hydrolyzed with 2.5 M TFA at 100°C for 6 hr for neutral sugars, and with 2N HC1/2N TFA at 100°C for 7 hr for amino sugars, which were derivatized with 2-aminopyridine. Thefluorescentderivatives were analyzed by HPLC using TSKgel Sugar AXI, for sugar composition [39]. For amino acid analysis, ARS2 was hydrolyzed with 5.7 M HCl at llO^C for 24 hr, derivatized with phenylisothiocyanate, and analyzed using a TSKgel ODS-80TS column [40]. (5) NMR spectra ofARS2 and methylation analysis

1H (400 MHz) and l^c (125 MHz) NMR spectra were measured by Brucker AM-400 and Varian Unity 500p spectrometers using D2O as solvent, respectively. Deproteinized ARS2 (5.0 mg) was methylated according to Hakomori [41] and purified with a Sep-Pak CI8 cartridge [42]. The eluted fraction was hydrolyzed in 90% HCOOH at 100°C for 12 hr and then in 2 M TFA at 100°C for 6 hr. It was then reduced with NaBD4 and acetylated with AC2O. The derivatives were analyzed by GLC-MS. Ionizing voltage, 70 eV; column, 2% OV-17 on Chromosorb W (2 mm x 1.1 m); injection temp., 240''C; oven temp., programmed from 170°C at 3''C/min to 230°C; carrier gas. He, 30 ml/min.

436

(6) Enzymatic and chemical modification ofARS2

ARS2 (100 mg) was treated successively with 5.5 U Actinase E at 60^C for 2 hr, and 43 U Thermolysin at 70°C for 3 hr in 10 ml of 100 mM Tris-HCl buffer (pH 7.4) containing 100 mM CaCl2. A quantity of 50 mg ARS2 was chemically deproteinized with 2.5 ml anhydrous hydrazine at lOO^C for 10 hr, and then N-acetylated with acetic anhydride and pyridine. For proteolysis, 20 U thermolysin in 100 mM Tris-HCl buffer (pH 7.4) containing 100 mM CaCl2 at 65 ^C for 3 h, 20 U trypsin in 100 mM Tris-HCl buffer (pH 7.7) at 37 X for 17 h, or 1100 U pepsin in 20 mM HCl (pH 1.9) at 37 X for 17 h were also used. For degradation of the carbohydrate moiety of 50 mg ARS2 sample, 10 or 25 U exo-P-galactosidase were added and the mixture was incubated at 3TC for 18 hr; 10 ml of 100 mM acetate buffer (pH 4.2) containing 25 U endo-p-l,4-galactanase or 1 mg crude endo-|3-l,6-galactanase [43] were added and the mixture was incubated at 45°C for 48 hr. Fifty unit exo-a-mannosidase or 0.5 U endo-P-galactosidase in 50 mM potassium phosphate buffer (pH 6.0) at 37 ^C for 18 h was also used. The sugar chain of ARS2 was oxidized, partially cleaved with 30 mM NalO^ at 4°C in the dark, and reduced with NaBH4 at room temperature overnight. For controlled Smith degradation, the polyol derivatives of ARS2 were hydrolyzed with 100 mM H2SO4 at room temperature for 16 hr. (7) Heat, acid or alkali treatment

ARS2 (50 mg) in 10 ml PBS was heated with a dry thermo-bath at 100 ^C or autoclaved at 121 °C for 30 min with an autoclave, treated with 20 ml of 1 M HCl (below pH 0.5) for 1 h at 80 X or 2M HCl at 100 °C, or treated with 20 ml of 0.1 M NaOH (pH 12.8) for 16 h at 4 X or at 25 X . 2-2. Results C vulgaris, strain CfC22, was cultured as previously reported [27,28]. Purification steps of antitumor immuno-active components and their antitumor activities are shown in Fig (2). ARS2 showed a broad single [M + H]"^ peak at m/z 63,100, Fig. (3). ARS2 is a white powder readily soluble in water. Quantitative analyses revealed that ARS2 contained both carbohydrate (67%) and protein (35%) moieties in a molecule. The carbohydrates included D-galactose (88 mol%), mannose (8%), iV-acetylglucosamine (2%) and xylose (1%), respectively. The amino acid components were Ala (15 mol%), Gly (10%), Thr (10%), Val (10%), Leu (10%), Glx (9%), Asx (7%), Ser (6%), He (4%),

437

IC. vutgaris coirtafailng cultivation fluid Centrifneatlon (C2Mx|.Mahi.) | c vtUgmis, ligiicl

| SupernatanTl l]ItraflltraUon(MW; M.M«) CVS-U Q^Sepharose Fast Flow

I Q2

Ql

Q4

Q3

AF-Chdate TOYOPEARL 650M

I Q2C2

Q2C1

1 QOH

Q2C3

Con A-Agarose

r

A2

Al

A3

|±4j RCA 12<^At:arase

I A2R1

|A2R2 JdL-

A2R3

A3R3

S i m e r d n lOOng

I ARSl

A3R2

AJRl

ARS2

Jb±dL

ARS3

Fig. (2). Separation of glycoproteins obtained from C. vulgaris strain CK22 andtiieiranti-rechallenge tumor activity.

n I 40000 70000

I I ] I n 1 Y100000 ISOOOO 1*0000 190000 3-40000 XSOOOO UOOOO

*/* Fig. (3). Molecular weight analysis of ARS2 by MALDI-MS. 10000

Phe (3%), Lys (3%), Pro (3%), Met (3%), Arg (2%), Tyr (2%), His (2%) and Hyp (1%). Protein moiety of ARS2 was sequenced by Edman degradation, and the NH2-terminal amino acid sequence was determined as DVGEAFPTVVDALVA. No amino acid sequence homology was found in a pattern much search using GeneWorks with the SWISS-PROT data bank. Therefore, it is evident that ARS2 is a novel glycoprotein. The I H - N M R 8: 4.45 (d, gal H-1), 3.51 (dd, gal H-2), 3.65 (dd, gal H-3),

438

3.92 (t, gal H-4), 3.90 (m, gal H-5), 3.89 (m, gal H=6), and 4.01 (d, gal H.6). 13C-NMR 5: 106.1 (C-1), 73.5 (0-2), 75.3 (C-3), 71.4 (0-4), 76.6 (C-5) and 72.2 (C-6). The individual signals of the ^H NMR spectrum were confirmed by a C-H Cosy spectrum (data not shown). In view of these findings, the major part of ARS2 is composed of 6-linked p-galactopyranosyl residues. A ^^C-NMR spectrum also revealed carbonyl carbon signals of the amide bonds at 170-175 ppm, and a broadband signal at 130-135 ppm, which may be related to aromatic carbons, as well as several weak signals at 57 to 68 ppm (data not shown). It is suggested that these minor signals may depend on the protein moiety of ARS2 (data not shown). The permethylated product consisted only of 2,3,4-tri-O-methylgalactose. No 2,3,4,6-tetra-O-methylgalactose arising fi*om terminal non-reducing galactose units was found. This suggests that the carbohydrate moiety of ARS2 may consist of straight chains as confirmed by ^^C- NMR. As indicated in Fig. (4)-A, ARS2 produced two peaks following successive enzymatic digestion with Actinase E and Thermolysin. On the other hand, when ARS2 was treated with exo-a-galactosidase, exo-p-galactosidase or endo-P-l,4-galactanase, no pronounced changes were occurred in the chromatographic profile, whereas endo-P-l,6-galactanase only partially degraded ARS2. Controlled Smith degradation modified the physicochemical nature of ARS2, leading to a collapse of the peak. Fig. (4)-B. Upon digestion of ARS2 with Actinase E and Thermolysin the antitumor activity was totally abolished. Fig. (5)-A. A similar phenomenon was observed following hydrazinolysis (data not shown). Partial digestion with trypsin or pepsin gave the decrease of antitumor activity. On the other hand, the Smith-degradation product exhibited almost the same antitumor activity, as did the original ARS2, Fig. (5)-B. Gelfiltrationprofile of ARS2 collapsed after autoclaving at 121 °C for 30 min without the decrease of antitumor activity (data not shown). Even by the treatment with IM HCl at 80 °C for 1 h, the antitumor activity was stable, while the gelfiltrationpeak was expanded (data not shown). Under the more excessive condition with 2M HCl at 100 °C for 1 h, the activity was decreased. The antitumor activity was not influenced either by the alkali treatment with 0.1 M NaOH at 4 °C for 16 h, though the absorption at 280 nm was decreased (data not shown). Even by the treatment with 0. IM NaOH at 25 °C for 16 h, its activity still remained. From these results, it becomes evident that the activity of ARS2 belongs to its protein moiety. Further studies are underway regarding the nature of protein linkage to the carbohydrate moiety and the fiill amino acid sequence of the protein moiety.

439

Fig. (4). Influences of protease digestion (A) and controlled Smith degradation (B) on gel filtration profile of ARS2 [28]. Treatment with heat-inactivated proteases (A, upperfigure)or Actinase E and Thermolysin (A, Lower figure); column: Sephacryl S-300HR, eluent: 0.1 M KPB (pH 6.8), flow rate; 0.4 ml/min: Native ARS2 (B, upper figure) and ARS2 degraded by controlled Smith degradation (B, lower figure); column: GPC300 + GPC60, eluent: 0.1 M KPB (pH 6.8), flow rate; 0.8 ml/min. Protein was determined by absorbance at 280 nm (solid line); sugar content was determined by absorbance at 490 nm by the phenol-sulfiiric acid method (A, dotted line) or detection was done via refi-active index (RI) (B, dotted line). 300

B

CM

E E

CO

200 H

\_ o E ZJ

CD

clOO CD

"co IZ

o

CD

DC

oJ

8

— I —

11

day

14

— I —

11

14

day

Fig. (5). Influence of protease digestion (A) and controlled Smith degradation (B) on the antitumor activity of ARS2 [28]. The sizes of rechallenge tumors are given after i.t. injection of: protease- (A, D ) or inactivated protease- (A, A ) treated ARS2, native ARS2 (B, D ) , ARS2 degraded by controlled Smith degradation (B, A ) , or PBS (O). *: Significantiy differentfiromtiiePBS ^oup at the same measuring day (students Mest, p<0.05). •*: Significantly differentfiromthe inactivated protease-treated ARS2 ^oup at the same measuring day (p<0.05).

440

3. IMMUNOPHARMACOLOGICAL EFFECT OF ACIDIC GLYCOPROTEIN CVS OBTAINED FROM THE CULTURE SUPERNATANT OF C. VULGARIS STRAIN CK22 3-1. Anti-metastatic immunopotentiation [44] We examined the antitumor effect of CVS against spontaneous tumor metastasis after surgically removing a s.c,-inoculated primary Meth A tumor mass from the right foot pad in BALB/c mice, Fig. (6)-Protocol A. The survival rates of the Meth A tumor-inoculated mice markedly increased when CVS was injected i.t. three times every other day following the tumor inoculation, Fig. (7)-A. A similar result was obtained in the EL-4 lymphoma and C57BL/6 syngeneic system (data not shown). CVS displayed a strong antitumor effect against experimental metastasis induced by i.v. tumor rechallenging, Fig. (6)-Protocol B. BALB/c mice were inoculated s.c. with Meth A tumor cells in the rightflankon day 0 and rechallenged intravenously with the same tumor on day 9 or 58. The primary tumor was removed on day 11. A significant dose-dependent increase in survival rate was observed when CVS was injected i.t once on day Protocol A. Spontaneous metastasis BALB/c

CVS, i t

f s^abtofo^tS

^ Removalofprimarytumor

Protocol B: Rechallenge metastasis BALB/c

9 ^ (Day) f

9orSS

CVS, i t

AMh A

^1^6^ 1x10 ,s.c.

11

I XyfAtli A

I «

•

^ ^ 6 ^ Removal of primary tumor 1x10 ,i.v ^ ^

Protochol C: Rechallenge s.c. system (for histochemical study) BALB/c

<j^

(Day) 0

^ CVS, i.t. Meth A 5xlO*,s.c.

9 ^ Meth A 5xl0^s.c.

Fig. (6). Experimental design of metastasis experiments [44].

441

3, Fig. (7)-B. A more prominent effect was observed when mice received a single i.t. injection of CVS on day 3 followed by subcutaneous injections of CVS twice weekly. A similar effect was observed when the i.v. rechallenge was performed on day 58, Fig. (7)-C. The antimetastatic effect of CVS was confirmed by determining the number of metastatic foci on the lung surface after Wexler staining [45] and by measuring the lung weight (Table 2). Furthermore, the percentage of metastatic areas in lung sections supported the antimetastatic activity of CVS (data not shown). 100-o

90

120

150

Day

Fig. (7)A-C. Inhibition of metastasis by It. administration of CVS into s.c. inoculated tumor [441. Meth A cells (Panel A; 5 x 10^, Panel B,C; 1 x 10^ from BALB/c mice were inoculated s.c. into therightfootpad (Panel A) or into therightflank(Panels B,C) on day 0. The primary tumor was removed on day 8 (Panel A) or day 11 (Panels B,Q. CVS was " ' '* ' '^ ,. ^ . . (0,c)inpanel injected' ' ' ' " A ig once, and 50 week. In panel m C, CvS was given i.t. (50 mg/1cg) once (A, i) or 3 times (O, jJ. D, PBS control (groups a, d, h in panel A, B, C, respectively). Mem A cells (1 x 10"), used to rechallenge, were inoculated i.v. on day 9 (Panel B) or on day 58 (Panel C). Number of mice used in a group was nine in panel A and ten in panel B, C. All of surviving mice 60 days after the tumor inoculation (dav 60, day 69 or day 127 in panel A, B or (j, respectively) were still alive until 90 days after the rechallenge tumor inoculation, and none oi metastatic foci under the microscopic observation after staining was detected atttieperiod. Significant difference by Mann-Whitney U test: h versus I, h versus j: p<0.05; a versus c, d versus f, e versus g: p<0.01; d versus g: p<0.001. Table 2. Antimetastatic effect of CVS [44] Group

Injection Method

Metastatic Foci MI

PBS CVS CVS

i.t. once i.t. once i.t. once and s.c. 2-times/week

2.83 ± 0.41 2.00 ±0.89^ 0.50 ±0.55^'^

Long Weight (mg) 235 ± 7 3 170 ±12^ 159 ±9^

BALB/c mice were injected s.c. with 1x10" Meth A cells into therightflankon day 0 and i.v. rechallenged on day 9. The primary tumor was removed on day 11. CVS (50 mg/kg) was injected i.t. into the primary tumor on day 3. Subcutaneous injections of CVS (150 mg/kg) were started from day 5. Metastatic foci and lung weight were measured on day 23 (14 days after the rechallenge tumor inoculation). Number of metastatic foci was evaluated as metastatic index (MI: 0; no me-

442

tastasis, 1; 1-10, 2; 11-100, 3; 100>). Each value is the mean ± SD for n=7 (students ^test, ^^: significantly different from PBS group with p<0.05, p<0.001, respectively; ^: significantly different from CVS i.t. once group with p<0.01). Tumor specificity of the CVS-induced antimetastatic effect

Meth A and Meth I fibrosarcomas of BALB/c origin bear different tumor-specific antigens that are recognized by syngeneic immune cells [46,47]. When mice were inoculated with Meth A tumor cells as the primary tumor and intravenously rechallenged with Meth I tumor cells, or vice versa, CVS did not exhibit any antitumor effect against rechallenge tumor growth (data not shown). Participation ofT-cell subsets in antitumor effects

The effect of CVS is T-cell mediated, since no effect was observed in athymic nu/nu mice of BALB/c background which were hereditarily T cell deficient (data not shown). Therefore, we examined the participation of T-cell subsets in the antitumor effect of CVS following their in vivo depletion with monoclonal antibodies in the Meth A and BALB/c system. Fig. (6)-Protocol B. CVS was injected i.t. 3 times from day 2, and tumor i.v. rechallenging was performed on day 9. Anti-CD3 treatment completely inhibited the antitumor effect of CVS (data not shown). Anti-CD4 and anti-CD8 treatment partially inhibited the effect of CVS. Less than 1.0% of the CD3-, CD4-, or CD8-positive cells were detected in the spleen or lymph nodes following in vivo treatment with the corresponding antibodies byflow-cytometricanalysis using FACS (Becton Dickinson, USA). Participation of the T-cell subsets was also examined histochemically in subcutaneously rechallenge tumor sections. Fig. (6)-Protocol C. The accumulation of lymphocytes in the tumor tissues and around the tumor increased in the CVS-treated group, 2 days after the s.c. tumor rechallenging. Fig. (8)-B. Immunohistochemical staining revealed increased infiltration of mature CD3 (D), CD4 (F), and CDS (H)-positive T-cells into the tissue in the CVS group. The tumor mass disappeared in about 50 percent of the CVS-treated mice 7 days after tumor inoculation, while in all control PBS mice it increased progressively until death (data not shown). Restoration of delayed hypersensitivity to Meth A tumor by CVS

We evaluated delayed hypersensitivity to examine the participation of CVS administration on the cellular immunity to Meth A tumor antigens according to the delayed footpad reaction (DFR) [48] in the tumor i.v. rechallenge system. On day 9, 16, or 23, a quantity of 5 x 10^ mitomycin C-treated Meth A cells in 0.05 ml PBS were injected into the right hind footpad of each BALB/c mouse. PBS injected into the left hind footpad

443

PBS

CVS B

in
o :^>r'^v,;.?

o

8 :

A,B;

200 jim, C--H; 60 fun

Fig. (8). Increased infiltration of CDS, CD4 and CDS positive cells into the sx. rechallenge tumor following CVS treatment [44]. BALB/c mice were injected s.c. with 5 x 10" Meth A cells in the ri^t and left flanks on day 0 and 9, respectively. CVS (50 mg/kg) (B,D,F,H) or PBS (A,C,E,G) was injected i.t. into the primary tumor 3 times every 2 days from day 2. Rechallenge tumor sections, including subcutaneous tissues, were obtained 2 days after tumor rechallenge and stained with hematoxylin-eosin (A,B) or immunohistochemically (C-H). a: Subcutaneous muscle layer, b: interstitial space, c: tumor tissue.

444

served as the control. Footpad thickness was measured 24 h later with a dial thickness guage, and footpad swelling was expressed according to,the following formula: footpad swelling (x 0.01 mm) = (thickness of right footpad) - (thickness of left footpad). The effect of CVS on the DFR to the Meth A tumor was examined in the i.v. rechallenge system, Fig. (6)-Protocol B. Administration of CVS slightly augmented the DFR against Meth A tumor[s] on day 9, and the effect was maintained until day 23, Fig. (9), when the control mice started to die. 80 1

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Fig. (9). Restored delayed footpad reaction (DFR) against Meth A tumor antigen following CVS treatment [44]. BALB/c mice were injected s.c. with 1x10" Meth A cells in therightflank on day 0 and rechallenged i.v. on day 9. The primary tumor was removed on day 11. CVS (50 mg/kg) or PBS was injected i.t. once into the primary tumor on day 3. Mitomycin C-treated Meth A was injected into the footpad on days 9,16 or 23. Footpad swelling was measured 24 h after the elicitation. Each value is the mean ± SD for ten mice, unprimed: age-matched normal mice that were not primed the Meth A tunior. ^: Tumor unprimed mice were s.c. injected with CVS on day 3 and DFR was elicited on day 9. . significantly different (students f-test, p<0.001)fi-omthe correspondingly timed PBS group. Organ weight and leukocyte numbers in the thymus, spleen, and lymph nodes

We examined the effects of CVS on body and organ weights on day 9 at the time of tumor i.v. rechallenging. Fig. (6)-Protocol B. The weights of the right axillary and inguinal lymph nodes (regional lymph nodes of the s.c.-inoculated primary tumor) were markedly increased in the CVS group, Fig. (10). A similar tendency was observed in the left lymph nodes, with statistical significance. The number of leukocytes in the regional lymph nodes of the tumor-primed PBS and CVS groups was approximately 5.5- and 10.1-fold greater than in those of the tumor-untreated normal mice, respectively (data not shown). In the contralateral lymph nodes, the number of leukocytes

445

increased by about 2.4- and 5.2-fold in the PBS and CVS groups, respectively. Body weight was slightly reduced in the PBS controls, whereas no decrease was evident in the CVS group, Fig. (10). Thymus weight slightly decreased and spleen weight increased in the PBS controls. The decrease in thymus weight was prevented and the increase in spleen weight augmented by CVS. There were no remarkable alterations in the weights of organs such as the liver and kidney. 120 r

90 I30 h

Tumor Size

Thymus

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Fig. (10). Effects of CVS on body and organ weights on day 9 upon tumor rechallenge [44]. BALB/c mice were injected s.c. with 1 x 1 0 " Meth A cells in the ri^t flank on day 0. CVS (50 mg/kg) was injected i.t. 3 times into the tumor. Mice were sacrificed on day 9 and the organs were removed. Experimental groupsj— M age-matched untreated (no-tumor) normal mice; • tumor-primed PBS-treated control; »-* tumor-primed C VS-treated group. Each value is the mean ± SD for six samples from 12 mice (organs from two mice were pooled to make one sample). B.W.: body weight, R: right, L: left. A: axillary, I: inguinal, LN: lymph nodes. Significantly different fi"om tumor-unprimed normal mice by students r-test; a: p<0.001, b: p<0.01, c: p<0.05. Significantly different from tumor-primed PBS-injected mice; d: p<0.001, e: p<0.01, f: p<0.05.

Fig. (11). Increased accumulation of CD4 and CDS positive cells in CD3 positive population in regional lymph nodes following CVS injection on day 9 upon tumor rechallenge [44]. See legend to Fig. (10).

446

Influence of CVS on T-cell surface markers

The effects of CVS on the numbers of T-cell subsets were examined on day 9 at the time of tumor i.v. rechallenging, Fig. (6)-Protocol B. Cells were stained with fluorescein 5'-isothiocyanate (FITC)-, phycoerythrin (PE)-conjugated and/or biotinylated mAb and were additionally stained with RED-613-streptavidin for flow-cytometric three-color analysis. In the regional lymph nodes, the numbers of CD4 or CDS single positive and CD4CD8 double positive T cells increased in the PBS controls, and the increment was augmented markedly by CVS, Fig. (11). A similar tendency was observed in the numbers of these cells in the contralateral lymph nodes, but to a lesser event. ax«^

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447

The CD4-positive population in the regional lymph nodes displayed a pronounced increase in the proportion of CD 18 bright, CD44 bright, CD25, CD54, CD69 and CD71 positive cells following treatment with CVS, Fig. (12). In the spleen, the absolute numbers of CD4 or CDS single positive T cells and CD4CD8 double positive T cells increased in the tumor-bearing state. Fig. (11). The increment in CDS single positive T cells was augmented significantly by CVS administration. In the thymus, the absolute number of the double positive T cells slightly increased in the CVS-injected mice. 3-2. Prophylactic effects against myelosuppression by 5-fluorouracil [491 Whole organisms and glycoprotein-rich hot-water extracts of C vulgaris strain CK show not only an antitumor immuno-potentiation [9,10,44] but also a protective effect on bacterial [12,13,15,24,25] and viral [14] infections in murine systems. Hot-water extracts accelerate the recovery of neutrophils and restore protection against infection with E, coli in neutropenic states by a cyclophosphamide treatment [13,15]. Thus, glycoprotein-rich extracts may not only activate mature leukocytes but also stimulate hematopoietic stem cells in the bone marrow. Chemotherapeutic agents for cancer therapy are sometimes subjected to a limit of use because of the adverse effects. One of the critical adverse effects is leukopenia induced by a suppression of bone marrow hematopoiesis resulting in opportunistic infections. In this regard, an improvement of impaired hematopoiesis is required to obtain a satisfactory outcome in the cancer chemotherapy. Protective effect of CVS against 5-fluorouracil (SFUJ-induced lethality in normal CDFl mice

Most of the normal CDFl mice died between 11 and 17 days after the intraperitoneal treatment with 500 mg/kg of 5FU. The 50% lethal dose (LD50) of 5FU was 400 mg/kg, whereas the LD50 was increased to 520 mg/kg or 700 mg/kg when CVS was pre-injected with 50 mg/kg or 500 mg/kg, respectively (data not shown). Thus, CVS reduced the adverse effect of 5FU. Protective effect against indigenous infection

The protective effect of CVS against indigenous bacterial infection caused by 5FU treatment [50,51] has been examined. At 5 days after 5FU

448

treatment, no indigenous bacterial infection was detected in the liver or blood. On day 8, dissemination with bacteria became evident in the liver, and on day 11, dissemination was evident in both the liver and blood of all 5FU-treated mice (Table 3). In CVS-pre-injected, 5FU-treated mice, the indigenous bacterial infection in the systemic circulation was significantly protected in not only bacterial number infected indigenously but also in the number of mice bacteria detected in their organs. This resuh indicates that CVS is protective against indigenous infection induced by 5FU. Table 3. Protective effect of CVS against indigenous infection induced by 5FU [49] Day 11

Day 8

Treatment

Detected mice No.

Bacterial No. (LoglO)

Detected mice No.

Bacterial No. (LoglO)

Liver 5FU 5FU + CVS

8/8 3/8

8.31 ±0.47 <3.69±3.23*

8/8 7/8

9.68 ±0.35. <6.84±3.12^

Peripheral blood 5FU 5FU + CVS

5/8 3/8

<1.99±1.44 <1.29±0.57

8/8 7/8

3.20 ± 0.48 <1.78±1.00^

CDFl mice were injected with CVS (50 mg/kg, six times, s.c.) on days -14 to - 1 , and treated with 5FU (550 mg/kg, i.p.) on day 0. Bacterial counts are mean ± SD. Detected No.: number of bacteria-detected mice in their organs/number of mice used. Bacterial No.: mean ± SD of bacterial number in their organs. ^: /? < 0.01, and ": /? < 0.05 versus the group given 5FU alone by students Mest.

Table 4. Restorative effect of CVS on progenitor cells in the bone marrow of 5FU treated mice [49] 5FU

+ +

Days

4 7 9 11

IL-3

GM-CSF

CVS(-)

CVS(+)

CVS(-)

4188 ±541

NT

7378 ± 953

24±7 235 ± 45 684 ±165 3463 ± 2039

83 ± 27^ 1385 ±47^ 3565 ± 2328^ 4077 ±1940

24 ± 6 47 ± 9 259 ± 6 6953 ±4094

CVS(+) NT 50 ±16^, 785 ±266*^ 5805 ±3790^ 21455 ±10213^

CDFl mice were injected with CVS (50 mg/kg, six times, s.c.) on days -14 to -1, and were treated with 5FU (250 mg/kg, i.p.) on day 0. Results are means ± SD of viable cells responding to IL-3 or GM-CSF/fumer. NT; not tested. ^:p< 0.001, ^:p< 0.01, and ^: /? < 0.05 versus the group given 5FU alone by students Mest. Effect on 5FU-induced

myelosuppression

5FU impairs the proliferation and differentiation of hematopoietic stem cells resulting in peripheral leukopenia. The above-mentioned indigenous infections should be attributable to the leukopenia-related impairment in the host defense system.

449

The number of leukocytes after treatment with a sub-lethal dose of 5FU decreased and reached its lowest level on day 7, followed by gradual recovery (data not shown). In the CVS group, the overall kinetics were almost the same, but the levels of leukopenia on day 4 were weaker and a rapid recovery was observed. CVS administration did not influence the total leukocyte counts or the differential counts in untreated normal mice (data not shown). Accelerated restoration of the progenitor cells in the bone marrow

The influence of CVS on the kinetics of hematopoietic stem cell numbers was examined in the bone marrow of 5FU-treated mice by in vitro colony-forming assay. With 5FU treatment, the number of colony-forming cells responding to IL-3 decreased to about 1/200 of the normal level 4 days after treatment and was restored by day 11 (Table 4). In CVS-injected mice, the number of colonies responding to IL-3 was 3.4-fold higher on day 4, and a complete recovery was observed as early as on day 9. granulocyte macrophage colony-stimulating factor (GM-CSF)-responding colonies also showed an accelerated recovery following administration of CVS. A similar recovery of viable cells (cfii) was observed in the spleen (data not shown). It is assumed that CVS produces some factor(s) accelerating hematopoiesis. A significant level of CSF activity assessed by in vitro soft-agar colony formation in bone marrow cells was detected in the sera of a few hours after thefinalinjection of 50 mg/kg CVS. Thus, CVS induced some CSF, contributing to the accelerated hematopoiesis and early recovery of the number of viable colonies, which resulted in the protection of mice against indigenous infection by 5FU treatment. Protective effect against SFU-induced adverse effects in tumor-bearing mice

With these experiments it became clear that CVS is highly protective against adverse effects in 5FU-treated normal mice. We carried out similar experiments using the Meth A tumor-bearing mice. In normal mice, an intraperitoneal 250 mg/kg 5FU treatment is not lethal, but 100% mortality was observed within 2 weeks after the same dose of 5FU treatment in mice bearing Meth A tumors. The mean survival time of Meth A-inoculated, 5FU-treated mice (23.1 ± 1.2 days) is significantly shorter than that of 5FU-untreated tumor-bearing mice (29.0 ±3.5 days). In CVS-administered tumor-bearing mice, the mean survival time is 48.5 ± 8.7 or 37.6 ± 6.2 days with or without 5FU treatment, respectively. With respect to the antitumor effect as determined by tumor growth, 5FU was

450

superior to CVS, Fig. (13)B, but the combination of 5FU plus CVS appears to be better than or comparable to the eflfect of 5FU alone. Subcutaneous injections of CVS near the tumor prevented weight loss in the tumor-inoculated, 5FU-treated mice. Fig. (13)A. CVS might prevent the adverse effects of 5FU in tumor-bearing states. These results indicated that the injection of CVS is highly effective in promoting the antitumor effects of a chemotherapeutic agent and in reducing its adverse effects. A) Body weight 5D

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4. DISCUSSION Whole organisms or hot-water extracts of C. vulgaris strain CK exhibit various pharmacological effects on host defense, such as the prevention of opportunistic infections of £". coli, L monocytogenes or cytomegalovirus in normal and immune-compromised hosts, suppression of IgE production against exposure to immunogens, and antitumor immunopotentiation. Prevention ofE. coli infection by C vulgaris strain CK was due to accelerated neutrophils in number and activity. Protection from L monocytogenes and the antitumor effect by C vulgaris strain CK should be due to augmentation of cellular immunity to their antigens, mediated by activation of CD4-positive helper T cells. Thus, glycoprotein-rich hot-water extracts of C. vulgaris strain CK enhanced or restored not only nonspecific host defense but also Thl-type helper T-cell response in the Th2-dominant host exposed to allergens. Peptic ulcers are formed by a destruction of the balance between ulcer

451

promoting factors and ulcer protective factors. Oral administration of the whole organisms of C vulgaris enhances the protective factors. It was reported that endogenous ILl showed a protective effect on gastric mucosa against ulcerogenic stimuli through the "immune-brain-gut" axis, and the algae may prevent ulcer formations mainly through this axis. We found antitumor effects against Meth A tumors in glycoprotein-rich higher MW extracts obtained from the whole organisms of C vulgaris containing culture medium, especially by i.t. injections via augmentation of concomitant immunity [9,10] or mediated by hot water extract-induced polymorphonuclear leukocytes [11]. Thus, antitumor effects against Meth A tumors were obtained by not only administration of whole organisms of C vulgaris but also by glycoprotein-rich higher MW extracts obtained from the organisms containing culture medium. We established that antitumor immunopotentiating principles were secreted into the culture medium of C. vulgaris. A clear effect against the rechallenge Meth A tumor[s] was observed when three i.t. administrations of 50 mg/lcg CVS-U (crude extract) were given every other day for 6 days (Table 1). The effect was augmented as an increment of injection times. A significant anti-rechallenge effect was also observed when CVS-U was administered from 7 days after the tumor inoculation, when the primary-inoculated tumor was palpable. These reactions are advantages under clinical application, in addition to the stability of antitumor activity after various treatments such as acid, alkali or heating against the active principle. The antitumor activity was concentrated in fraction Q2C2 of the copper-charged chelate column chromatography, Fig. (2). Although we tried to achieve fiirther purification by the addition of hydroxyapatite, hydrophobic interaction, gelfiltrationand affinity chromatographies, no increase of purity was observed. Subsequently, we performed two more lectin-column chromatographies and obtained the most purified galactose-rich acidic glycoprotein, ARS2. The antitumor effect of ARS2 is the strongest among the active fractions. ARS2 exhibited a broad band on SDS-PAGE and lEF-PAGE (data not shown). The main bands of carbohydrate and protein moiety of each antitumor fraction on two kinds of electrophoreses or main peaks on chromatographies of separation procedures overlapped. Fig. (4). Moreover, all antitumor immuno-active products obtained by enzymatic or physicochemical treatments also showed overlapping of the main bands of carbohydrate and protein moieties. Judging from these findings, we conclude that the antitumor immuno-active principle might be a glycoprotein, because it consists of a protein and a galactose-rich carbohydrate. The broad bands on SDS- and lEF-PAGE might be due to the heterogeneity of carbohydrate moiety, which is in good agreement with previous reports [52,53].

452

Thus, we found an antitumor immunopotentiating principle that is a galactose-rich glycoprotein, ARS2 (Mr, 63,100), consisting of a 6-linked p-l,6-galactopyranose-rich carbohydrate (67%) and protein (35%), secreting into the culture supernatant of C. vulgaris [28]. Several BRMs, OK-432 [54], PSK [55], lentinan [56] and sizofilan [57], are now commercially available. OK-432 and PSK are prepared from the whole body oi Streptococcus pyogenes and a crude glycoproteinfractionof Coriolus versicolor, respectively. The last two BRMs are purified from Lentinus edodes Sing, and Schizophyllum commune Fries, respectively. The p-glucan moiety is the most common active site in commercially used BRMs such as PSK, lentinan and sizofilan [58]. The activity of the acidic glycoprotein of C. vulgaris was obviously sensitive against proteolysis but resistant to enzyme hydrolysis and Smith degradation of carbohydrate moiety. Fig. (5). Therefore, we suggested that the antitumor active site of acidic glycoprotein must be located in the protein moiety. Kita et al obtained an antitumor crude glycoprotein (MW 38,000 - 40,000) from Corynebacterium kutschei [59,60]. The antitumor activity of the glycoprotein disappeared with trypsin digestion just as that of ARS2 disappeared. However, two glycoproteins from C. vulgaris and C. kutschei can be easily distinguished with each other based on the following physicochemical points. The ratios of the protein content per carbohydrate content are approximately 5.2 and 0.7 in the two glycoproteins obtained from C. kutschei and C. vulgaris, respectively. While the glycoprotein from C. kutschei is inactive at pH 2 and pH 12, that from C vulgaris is stable by heating (80 T ) at a pH lower than 0.5 (1 M HCl) or at 4 ^C at pH 12.8 (0.1 M NaOH). Based on these comparisons, we speculate that glycoproteins of either bacterial or ftmgal and green algal origins are quite different in their characteristics, even though both show antitumor effects. The antitumor activity of the acidic glycoprotein from C vulgaris is heat-stable and acid- and alkali-resistant, while the protein moiety of the acidic glycoprotein from C vulgaris is an important active site. The fimction and in vivo fate of chimeric mouse-human IgGl are dramatically affected by its carbohydrate structure [61]. The thermodynamic stability of lysozyme is controlled by its glycosylation signal sequence [62,63]. Hyp and Ser glycosylation sites enhanced conformational stability and molecular recognition in hydroxyproline-rich glycoproteins of plants [64]. Thus, the carbohydrate moiety may be important for preservation of the structure and activity of the acidic glycoprotein from C vulgaris. To our knowledge, this is the first successftil finding of a galactose-rich glycoprotein having its active site in the protein moiety and a high antitumor stability. However, the active unit of the acidic glycoprotein may be smaller because antitumor activity remained after partial degradation by acid or alkaline treatment. Further scrupulous study is necessary to determine the antitumor active principle.

453

BRMs isolated from plant tissues and bacterial products show nonspecific [65,66] and T cell-mediated antitumor effects [67,68]. Administration of these BRMs improves the quality of life, without causing complete regression of the cancer in most cases. The antitumor effect of the acidic glycoprotein in the tumor rechallenge system was at least comparable to that of OK.432 (Table 1). The therapeutic effect induced by glycoprotein-rich fractions extracted from C. vulgaris might depend on a T cell-mediated mechanism in an antigen-specific manner [9,10,44]. The antigen-specific suppression of glycoprotein-rich extracts of C vulgaris on Meth A tumor growth in a syngeneic system may be mediated by cytostatic T cells rather than cytolytic T cells with macrophage participation [9,69,70]. As reported elsewhere, cytostatic T cells (presumably CD4-positive helper T cells) are responsible for delayed type hypersensitivity, and these T cells activate macrophages, which may be thefinaleffectors in suppressing the growth of target tumor cells [71-73]. Thus, antitumor T cells activated by glycoprotein-rich extracts of C. vulgaris could be CD4-positive T cells. CVS exhibited stronger antitumor activity against the rechallenge tumors than against the primary tumors, as the glycoprotein-rich extracts did. Moreover, i.t. administration of CVS inhibits spontaneous and i.v. rechallenge metastasis of syngeneic tumors, with subsequent subcutaneous injection of CVS enhancing the inhibition of metastasis by i.t. injection of CVS. Thisfindingmight have clinical application. We demonstrated that the antimetastatic effect of CVS was T cell-mediated in an antigen-specific manner, since no antimetastatic effect was observed in athymic nude mice (data not shown). Augmentation of the antimetastatic effect against the rechallenge tumors was observed only when the rechallenge tumor lines were homologous to those of the primary tumors. The underlying T cell-mediated mechanism involved in the antimetastatic effect of CVS is further supported by the following findings: (1) the accumulation or infiltration of CD3-, CD4- and CD8-positive cells around and into the s.c. rechallenge tumor was augmented by CVS, (2) the antimetastatic effect of CVS was inhibited by in vivo treatment with anti-CD3, anti-CD4 or anti-CD8 antibody, and (3) the DFR against Meth A tumor antigens was restored by CVS. The antitumor effects of CVS can be explained by the changes CVS evoked in the number of total leukocytes, as well as in the percentage of CD4, CD8 single positive or CD4CD8 double positive T-cell subsets in the regional lymph nodes and/or thymus. On day 9 (when the antitumor immune response developed and the rechallenge tumors were inoculated), a large increase in the absolute numbers of total leukocytes and of all T-cell subsets in the regional lymph nodes was observed in the CVS group. It is possible that CVS facilitated the migration of effector T cells to the regional lymph nodes. These resuks suggest that the regional lymph nodes

454

may play an important role in CVS-induced antitumor immunity. We found that the expression of activation-related surface antigens was augmented by CVS administration, especially in the CD4-positive cell population. The CD 18 (P-subunit of lymphocyte function-associated antigen-1) and the CD44 (the memory T-cell marker) antigens were highly expressed by CD4-positive cells of the regional lymph nodes following CVS administration. Expression of CD25-, CD69- and CD71-positive cells also increased in CD4-positive populations of the regional lymph nodes after CVS injection. These findings indicate that CVS activates CD4-positive helper T-cells, causing them to proliferate and resulting in the induction of CD8-positive cytotoxic T lymphocyte populations. Antimetastatic immunopotentiation of CVS was strongly exhibited by i.t. or subcutaneous injection near the tumor site, while a decrease in the total number of thymocytes was prevented by CVS. Local accumulation of effector cells could also play an important role in antitumor immunopotentiation [74]. CVS might have augmented the migration of thymocytes to the periphery without causing any decrease in thymocyte number on day 9. Our results suggest that CVS enhances the de novo generation of thymocytes in the tumor-bearing state. Alternatively, CVS might prevent apoptosis of immature thymocytes observed in tumor-bearing hosts [75,76]. It appears that CVS is a promising candidate for a novel type of anticancer drug intended to prevent tumor metastasis or recurrence after surgical resection. We observed significant antitumor activity when i.t. injection of CVS was started within 7 days of tumor inoculation. It may be that presurgical treatment with CVS at early stages of tumor growth would prevent metastasis orfiirthertumor progression. A combination of CVS or other BRMs with cytokine gene therapy or therapeutic vaccination could provide a novel approach to cancer therapy. CVS reduces the adverse effects of 5FU, such as aggravated lethality, weight loss or bacteremia, not only in normal but also in tumor-bearing mice. The indigenous infection following bacterial translocation from the gut appears to be responsible for 5FU-induced lethality [50,51]. In addition, 5FU-induced suppression of the bone marrow hematopoiesis should result in a rapid impairment of the host defense system, depending upon bone marrow-derived leukocytes. Once intestinal bacteria enter the circulation, leukopenic hosts are no longer capable to copying with the invading bacteria and bacteremia or septicemia will develop. It is generally accepted that prevention of marrow dysfunction and recovery of hematopoiesis overcome the development of a septic state after bacterial translocation [77,78]. The observations of accelerated recovery of stem cell hematopoiesis and protection against peripheral leukopenia suggested that CVS contributed to the prevention of indigenous infection mainly through these effects. Bone marrow stem cells are known to be susceptible to 5FU, but

455

pluripotent stem cells are not [79]. In our experiment, the numbers of colony-forming cells responding to IL-3 or GM-CSF were increased in the bone marrow of the 5FU-treated mice 4 days after CVS administration (Table 4). It is possible that CVS accelerated the differentiation of stem cells into the 5FU-resistant stage. CSF activity was detected in the sera of mice treated with CVS (data not shown). CVS may produce the factors contributing to accelerated hematopoiesis and induce an early recovery after treatment with 5FU. These effects of CVS were expressed in tumor-bearing mice without ahering the therapeutic efficacy of 5FU. The clinical application of CVS may soon be available to prevent recurrence or metastasis of primary tumors and to relieve the adverse effects of chemotherapeutics and support cancer immuno-chemotherapy ABBREVIATIONS CVS

— -

BRM i.t. s.c. i.v. PBS MW DFR 5FU IL GM CSF

= = = = = = = = = = =

immuno-active acidic glycoprotein obtained fi'om the culture medium of C. vulgaris biological response modifier intratumor subcutaneously intravenous phosphate buffered saline molecular weight delayed footpad reaction 5-fluorouracil interleukin granulocyte/macrophage colony stimulating factor

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 25 © 2001 Elsevier Science B.V. All rights reserved.

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HEPATOPROTECTIVE EFFECT OF PLANT COMPONENTS: INHIBITION OF TUMOR NECROSIS FACTOR-a-DEPENDENT INFLAMMATORY LIVER INJURY KOJI HASEj QUANBO XIONG,' SHIGETOSHI KADOTA *^ ^Institutefor Consumer Healthcare, Yamanouchi Pharmaceutical Co., Ltd, Tokyo 174-8612, Japan; and^Instituteof Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan ABSTRACT: There is evidence to suggest that the upregulation of serum tumor necrosis factor (TNF-a) and the emergence of hepatocyte apoptosis are involved in the pathogenesis of human alcoholic and viral hepatitis. In experimental liver injuries induced by D-Galactosamine (D-GalN)/lipopolysaccharide (LPS), D-GalN/TNF-a or Propionibacterium acneslL?S, TNF-a induces hepatocyte apoptosis which triggers an inflammatory reaction and subsequent massive hepatocyte necrosis, playing a central role in the pathological process. These models provide a promising experimental basis not only for understanding the pathophysiological mechanisms of various hepatic disorders but also for evaluating the hepatoprotective efficacy of natural products. A diverse array of plant-derived compounds including saponins, polyphenols, iridoids and alkaloids have been reported to have a hepatoprotective effect in the TNF-adependent inflammatory liver injury models. Some of these compounds impede TNF-amediated hepatocyte apoptosis and consequently block the progression of liver injury, whereas others protect against hepatocyte necrosis occurring at the final stage. The protection against apoptosis by hepatoprotective compounds can be explained by the inhibition of TNF-a production from macrophages or by attenuation of the hepatotoxic fimction of TNF-a. This review discusses the recent progress in TNF-a-dependent liver injury models and the hepatoprotective action of plant constituents in such models.

INTRODUCTION A number of medicinal plants including Glycyrrhiza glabra, Silybum marianum, Picrorrhiza kurroa and Artemisia capillaris have been traditionally used for the treatment of hepatitis. These plants contain various compounds with unique structures such as alkaloids, terpenoids and polyphenols. To study the hepatoprotective phytoconstituents of medicinal plants is of value to provide a scientific basis for the traditional use. Additionally, the active constituents could be used as pharmacological tools for the study of the mechanisms underlying liver disease or as lead compounds for the development of new drugs for hepatitis. In fact, * To whom correspondance should be addressed. Fax: 81-76-434-5059. E-mail: [email protected]

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glycyrrhizin isolated from G. glabra, silymarin from S. marianum and picroliv from P. kurrooa have been developed as hepatoprotective drugs for viral chronic hepatitis and alcoholic hepatitis in Asia and Europe. Recent progress in the study of medicinal plants for treating liver diseases has resulted in the isolation of about 170 different phytoconstituents from 55 plant families [1]. In previous evaluations of hepatoprotective activity, direct hepatotoxins such as carbon tetrachloride (CCI4), bromobenzene, acetoaminophen or D-galactosamine (D-GalN) were used to induce e)q)erimental liver injury. These chemically induced liver injuries result from plasma membrane perturbation due to the generation of cellular radicals or the impairment of membrane component synthesis [2-4]. Consequently, too many phytoconstituents with antioxidant or membrane-stabilizing activity tended to exhibit hepatoprotective activity [5-10]. However, the majority of the cause of human hepatitis are triggered by immunological responses to viral infection, endotoxin or autoantigen [11-12]. Thus, liver injury models induced by direct hepatotoxins are considered to reflect only a limited aspect of human hepatitis. The evaluation for hepatoprotective activity of phytoconstituents in such models might produce e>q)erimental results inconsistent with those in the clinical situation. Therefore, new models related to immunological reactions have been recently proposed, namely, liver injuries induced by lip op oly saccharide (LPS), tumor necrosis factor-a (TNF-a) or concanavalin A (Con A) in intact or D-GalN-sensitized mice [13-14]. Recent progress in these models has revealed that TNF-a secreted from LPS-stimulated macrophages is a strong inducer of hepatocyte apoptosis, which tri^ers an inflammatory reaction and massive hepatocyte necrosis [15-16]. The hepatic lesions induced in these models resemble those of human hepatitis, because upregulations of serum TNF- a concentration and hepatocyte apoptosis have been repeatedly reported as pathogenic symptoms in human hepatitis [17-22]. The TNF-a-dependent inflammatory liver injury models may therefore provide a more promising e)q)erimental basis for the evaluation of hepatoprotective agents. The first part of this review deals with the recent findings in the TNF-a-dependent liver injury models. The second part discusses the effect of plant-derived hepatoprotective agents on inflammatory liver injury and their pharmacological mechanisms. 1. TNF-a-dependent liver injury models TNF-a was originally recognized for its oncolytic effects on solid tumors, and subsequent investigations have demonstrated a pleiotropic effect mediating both acute and chronic inflammatory disorders. Several e>q)erimental liver injury models including D-GalN/LPS-, D-GalN/TNF-a-, Propionibacterium acnesILV?>', and concanavalin A (Con A) models are

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induced by host-immune responses including the activation of macrophages and T lymphocytes. A central role for TNF-a in these models has been reported in studies using neutralizing anti-TNF-a antibodies or knockout mice for TNF-a itself or receptors [23-27]. TNFa triggers hepatocyte apoptosis as well as necrosis, and induces chemoattractants which are essential for lymphocyte infiltration into liver [28-29]. This chapter deals with proceedings on TNF-a-dependent liver injury models. D-GaiN/LPS and D-GalN/TNF-a models: Administration of endotoxin (lipopolysaccharide; LPS) into different animals has been reported to cause multiple organ failure, resulting in lethal shock [30]. The liver is one of the main target organs for LPS toxicity. Numerous animal models have been developed to study the effects of LPS on liver injury. The simplest of these is bolus intravenous or intraperitonal injection of hi^-dose (ca. 1-40 mg/kg in rodents) of LPS [31-33]. However, this model may not be suitable for the evaluation of hepatoprotective efficacy, because LPS has a wide range of physiological activities and exerts unspecific deleterious effects on extrahepatic org^s such as the circulatory system, kidneys and lungs as well as liver. When other hepatotoxins are administered along with LPS, a synergic increase in liver injury occurs. For instance, co-injection of D-GalN (300-700 mglcg) and a subtoxic dose (1-100 /ig^g) of LPS into mice causes severe hepatitis [34]. In this model, liver injury is induced at 8 hr after intoxication without affecting other parts of the animal [35]. In contrast, a single injection of the above dose of D-GalN or LPS hardly affects mice. LPS is known as a strong stimulator that induces macrophages to secrete proinflammatory cytokines. Among the secretions, tumor necrosis factor-a (TNF-a) is thought to play predominant roles in liver injury and lethal shock induced by LPS, because administration of an anti-TNF-a antibody or a down-regulator of TNF-a production such as pentoxifylline abolish deleterious effects of LPS [23,24,30,36]. Although 10 | i ^ g of LPS is enough to induce TNF- a release, liver injury dose not occur in mice. This may be due to the fact that TNF-a triggers two distinct biochemical pathways; one that leads to apoptotic cell death in hepatocytes and another that leads to the induction of hepatoprotective proteins, probably nitric oxide synthase (NOS) and acute phase proteins [37-41]. D-GalN is converted to UDP-galactosamine (UDP-GalN) in hepatocytes, which depletes hepatocellular uridine phosphate such as UDP and UTP [42]. Depletion of uridine phosphate leads to inhibition of protein synthesis in hepatocytes, resulting in the suppression of the endogenous hepatoprotective proteins. Therefore, the susceptibility of animals to LPS hepatotoxicity increases more than 1000 times with the sensitization by D-GalN [34]. A similar sensitizing effect is seen when transcriptional inhibitors such as Actinomycin D (Act D) are administered with LPS or TNF-a [43]. The D-GalN/LPS-induced liver injury model has been

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frequently used to evaluate hepatoprotective agents [35,44,45]. Apoptotic symptoms such as DNA fragmentation and apoptotic bodies are observed in the livers of mice intoxicated with D-GalN/LPS or D-GalN/TNF-a [16]. A time course study on the pathological changes showed that hepatic fragmented DNA amount and serum ALT activity increase at 5 and 8 hr after intoxication, respectively. This means that hepatocyte apoptosis is induced at the early stage of the liver injury process and precedes hepatocyte necrosis [16]. Apoptosis has been recognized as a silent form of cell death under physiological conditions [46]. However, it appears that in some forms of liver disease, hepatocyte apoptosis actually triggers inflammation. Leist etal [16] postulated that in the D-GalN/LPS model, hepatocyte apoptosis may act as an initial step of liver damage, and subsequently, neutrophils are attracted by dying hepatocytes that are not removed swiftly enough under this pathological condition. Thus, neutrophils are thought to infiltrate liver tissue and to cause massive hepatocyte necrosis at the late stage. This concept was proved recently by a study with the caspase inhibitor Z-VAD, which allowed the selective blockage of apoptosis. Z-VAD treatment not only prevented caspase activation and apoptosis but also suppressed neutrophil transmigration and hepatocyte necrosis [15]. This indicates that a large number of hepatocytes undergoing apoptosis can represent a stimuli for primed neutrophils in sinusoids to transmigrate and activate, leading to massive hepatocyte necrosis. Therefore, prevention of apoptosis may be a new therapeutic approach to modulating inflammation and liver injury, although it remains to be elucidated how apoptosis signals inflammation [47,48]. Propionibacterium acnes/LPS model: Thehepatotoxicity of LPS and TNF-a can be enhanced by priming agents. It has been reported that severe hepatitis is induced by priming rodents with heat killed gramnegative bacteria namely Propionibacterium acnes or bacillus CalmetteGuerin (BCG) (ca. 1 mg^mouse), followed by injecting the mice with a small dose (ca. 1 /xg^mouse) of LPS after an interval of 7days [49,50]. The initial injection of BCG or P. acnes recruits mononuclear cells (MNCs) from the circulating system into the liver, which is prerequisite for the induction of LPS hepatotoxicity. Under this condition, the injection of a normally innocuous amount of LPS activates the infiltrating MNCs to release a huge amount of TNF-a, resulting in severe liver injury [27]. Plasma TNF-a activity rose sharply and reached a maximal level at 1 hr after LPS challenge, and then declined to near zero at 3 hr. Plasma ALT activity increased gradually from 4-24 hr, and more than 50% of mice died within 24 hr after LPS challenge. This liver lesion pathologically mimics fulminant hepatitis in humans, especially that associated with septic shock. This model has been one of the most popular animal models for the evaluation of hepatoprotective agents. The essential role of TNF-a in this model has been demonstrated by the observations that passive

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immunization against TNF-a or down-regulators of TNF-a such as PGE2 and dexamethasone protect mice from lethality and liver injury [27]. Liver damage in this model is histologically characterized by hepatocellular loss due to apoptosis and necrosis. Tsuji etal [51] recently reported that TNF receptor p55-deficient mice (TNFRp55"^") resist LPS-elicited hepatocyte apoptosis and liver injury as well as P. acwe^'-induced mononuclear cell infiltration, suggesting important roles forTNFRp55 in these pathological events. It should also be mentioned that Fas, which is a member of the nerve growth factor/TNF receptor superfamily, may contribute to induction of hepatocyte apoptosis in this model. The extent of P, acwe^'/LPS-induced hepatocyte apoptosis and liver injury is reported to be moderate in Fas-deficient Lpr/Lpr mice compared with wild mice. Fasseems to induce apoptosis together with TNFRp55 but through different pathways [51]. Alcohol and D-GalN models: It appears that TNF-a somewhat contributes to the pathogenesis of alcohol- and D-GalN-induced liver injury. In these two models, passive immunization of TNF-a markedly reduces liver injury [52]. Besides, ethanol or D-GalN-induced liver injury is significantly attenuated when Kupffer cells, a main source of TNF-a production, are destroyed with GdCla [53,54]. Ethanol-induced liver injury is diminished when gram negative bacteria in gut microflora are reduced by treatment with antibiotics or lactobacillus [55,56]. It is therefore hypothesized that chronic ethanol feeding or D-GalN administration increases the permeability of the gut to endogenous bacteria, resulting in the increased translocation of endotoxin into the blood, which stimulates Kupffer cells to produce TNF-a [57,58]. This hypothesis may be of clinical importance because patients with alcoholic liver disease and cirrhosis frequently have endotoxemia and hypercytokinemia [20,59,60]. In addition, serum TNF-a levels in patients with alcoholic hepatitis correlate inversely with patient survival ratio [18,19]. Pharmacological intervention with prostaglandins and glucocorticoids, which decrease TNF-a production, has been reported as an effective therapy in clinical liver diseases [61,62]. Con A model: As described above, LPS-related liver injury models are dependent on the activation of hepatic macrophages. On the other hand, a number of reports have suggested the potential involvement of T lymphocytes in chronic viral hepatitis and primary biliary cirrhosis [63]. In the 1990's, animal models of activated T cell-mediated liver injury were developed. Intravenous injection of mice with concanavalinA (Con A) at a dose of more than 1.5 mg^g leads to T-cell activation, resulting in liver injury within 8 hr of intoxication [64]. Con A is a T cell mitogenic plant lectin with high affinity for the hepatic sinus. Accumulation of Con A in the liver increases infiltration of circulating lymphocytes into the hepatic sinus and subsequent local proliferation. The activated lymphocytes in the liver release large amounts of cytokines that are essential for the pathology

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of liver injury in this model [13,65]. Pharmacological intervention with immunosuppresive drugs such as FK506 and dexamethasone abolishes the Con A-induced liver injury. Furthermore, mouse strains lacking T cells such as severe combined immunodeficient (SCID) mice and athymic nude mice show resistance to Con A-hepatotoxicity [64,65]. These results clearly revealed a central role for T cells in the development of liver injury. And, CD4'^ helper T cells are likely to be effector cells because pretreatment of mice with monoclonal antibody against CD4'^ but not CD8^ cells protected against Con A [64]. However, it is interesting to note that the activated lymphocytes start to infiltrate the liver tissue 8 hr after ConA injection, when liver damage has already begun. On the other hand, serum concentrations of most cytokines rise to a maximal level before the lymphocytes infiltrate, supporting that an early increase in cytokines is essential to induce liver injury [65,66]. Among these cytokines, interferon-y (IFN-y) is thou^t to predominantly correlate with the pathology, because passive immunization with anti-IFN-y but not with anti-IL-1 or anti-IL-6 antibody inhibits liver injury in this model [13,67, 68]. In contrast, IL-6 and IL-10 actually protect against liver injury by negatively regulating the production of IFN-y, suggesting a counter effect of some cytokines in this model [65,69]. The involvement of TNF-a in this model has been a subject of debate. Passive immunization with antiTNF-a abolished liver injury [13,65], whereas TNF-a gene-deficient mice are just as sensitive to Con A-induced liver injury as wild-type mice [70]. The reason for such a discrepancy in the involvement of TNF-a in this model is still unclear at present. Either the dose of Con A or genetic background of the mice used in each e^qperiment might affect the results. Additionally, mechanisms whereby INF-y and TNF-a induce liver injury also remain to be elucidated. Several authors suggested that similar to the D-GalN/LPS model, hepatocyte apoptosis is induced at the early stage of Con A-induced liver injury, via the TNF-receptor or Fas-lig^nd dependent pathway [13,16,70-72]. However, it is unclear why TNF-a could induce hepatocyte apoptosis without transcriptional arrest by D-GalN or Act D. Hepatocyte apoptosis may be caused by the synergistic action of TNF-a with IFN-y. On the other hand, other authors reported that the main pathological feature of the hepatic injury in the Con A model was hepatocyte necrosis, and that hepatocyte apoptosis is not found until the later stage of liver injury [73]. They reported that Con A administration caused a marked intrasinusoidal hemostasis, which consisted of erythrocyte agglutination, lymphocyte sticking to endothelial cells and platelet aggregation, resulting in congestion of the liver and consequently hepatocyte damage. Co-treatment with anti-TNF-a and anti-IFN-y monoclonal antibodies completely protected mice from hemostasis and liver injury. This suggested that IFN-y and TNF-a may be required for the induction of intrasinusoidal hemostasis [73]. Furthermore, TNF-a is well known as a strong inducer of adhesion molecules to recruit circulating

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lymphocyte in various cells including endothelial cells [31]. This effect is suggested to help the development of Con A-induced liver injury [68]. Thus, it is plausible that IFN-y and TNF- a are prerequisite for this model, and these cytokines may be related to the recruitment of lymphocytes to the hepatic sinus or the induction of intrasinusoidal hemostasis rather than direct cytotoxicity to hepatocytes. Tabid. Inflammatory liver injury models predominantly mediated byTNF-a Trigger Sensitiser Animal species D-GaIN (300-700 mg/kg, i.p.) Propionibacterium acnes (0.7-1 mg/mouse,/.v.) Pb acetate (15 mg/kg, /. v.) D-GalN (16 mg/mouse, i.p.) D-GalN (700 mg/kg, i.p.)

Effector cells M(l)

Ref

LPS(l-40mg/kg,/./?or/.v.) LPS(l-100^g/kg,/.p.) LPS(1-25 |ig/mouse, /.v.)

Mice, Rats, Others Mice, Rats, Rabbits M(j) M(}) Mice

[30,311 [34,351 [27,501

LPS(100ng/kg,/.v.)

Mice, Rats, Chicks, M(|) Baboons Mice M(t), T cells M(|), T cells Mice

[74,751

[761 SEB (50-100 pg/mouse, i.p.) [771 Anti-CD3 mAb (10 ^ig/mouse, /.v.) Md), T cells Con A (7.5-3Q__mg/kg, /.v.) Mice [Ml D-GalN, D-galactosamine; LPS, lipoplysaccharide; SEB, Staphylococcal enterotoxin B; Con A, concanavalin A; mAb, monoclonal antibody

2. Natural products with hepatoprotective activity against TNF-adependent liver injury Several crude drugs have traditionally been used for the treatment of liver diseases. Their pharmacological and biochemical actions have been evaluated scientifically using various e>q)erimental liver injury models. Following the experimental confirmation of a hepatoprotective property in some plants, numerous active compounds have been purified and identified. There are several reviews dealing with various aspects of hepatoprotective natural products [1,78,79]. We and other investigators have tried to find new hepatoprotective agents from natural resources using the inflammatory liver injury models mentioned in the first chapter. As a result, a series of plant-derived compounds including flavonoids, tetrahydroxanthone, caffeic acid derivatives, phenylethanoids, iridoids and alkaloids have been shown to have hepatoprotective activity. This chapter updates proceedings on the hepatoprotective effect and pharmacological mechanisms of phytoconstituents in TNF-a-dependent inflammatory liver injury. Glycyrrhizin (1) is a hepatoprotective saponin which consists of an aglycone glycyrrhizic acid and two molecules of glucuronic acid. Glycyrrhizin is derived from the root of licorice {Glycyrrhiza glabra L.) and allied plants (Leguminosae). There are many reports that suggested various bioactivities, namely, anti-inflammatory, anti-viral and hepatoprotective, of glycyrrhizin [80-84]. It has been used extensively as a therapeautic drug for chronic active hepatitis in Japan for decades. The

466

hepatoprotective effect of gjycyrrhizin on chemically-induced liver injuries is well established. For instance, pretreatment with glycyrrhizin (200 mg/kg,/.v. or i.p.) markedly inhibited the elevation of serum ALT and AST levels and the development of pericentral hepatocyte necrosis in CCI4- or allyl formate-induced liver injury in rats [85]. An in vitro study using primary cultured rat hepatocytes demonstrated that this compound exerts direct hepatoprotective activity against CCI4- or D-GalNcytotoxicity. This effect was much stronger in the aglycone glycyrrhizinic acid than glycyrrhizin. Additionally, we recently found that the oral or intraperitoneal administration of glycyrrhizin (100 or 200 mglcg) significantly inhibited the elevation of serum ALT activity after intoxication with D-GalN and LPS in mice [86]. Sibayama [85] reported that the mortality of rats after intoxication with LPS markedly decreased when glycyrrhizin (200 mg^g) was administered intravenously, althou^ no significant difference was seen in serum transaminase levels. These results prove the efficacy of glycyrrhizin in TNF-a-dependent inflammatory liver injury. In our data, glycyrrhizin provided 61% protection against elevation of the serum ALT activity at 8 hr after intoxication with D-GalN and LPS, whereas it inhibited neither hepatic DNA fragmentation nor apoptotic body formation, both of which are indicators of apoptosis, at the 5 hr time point [87]. This suggests that the hepatoprotective effect of glycyrrhizin in this model may be due to protection from secondary hepatocyte necrosis triggered at the fmal stage rather than inhibition of inflammation, although an anti-inflammatory effect through adrenocorticosteroid-like action of glycyrrhizin was proposed previously [88,89]. It is assumed that the membrane-stabilizing effect of glycyrrhizin is involved in the protection against necrosis [84]. COOH

GlcA-GlcA-0

,. -,

Fig. (1). Glycyrrhizin isolated from Glycyrrhiza glabra

It was recently examined whether glycyrrhizin affects T-cell- or TNFa-mediated cytotoxicity in vitro [90]. Glycyrrhizin suppressed T-cellmediated cytotoxicity against antigen-presenting cells; however, the effective concentration was as high as 200 [ig/mL. Since the serum concentration of glycyrrhizin after intravenous injection at the clinical dose is reported to be 30-60 |Lig^mL at the maximal level [91], this compound is unlikely to suppress T-cell-mediatedcytotoxicity in vivo. On the other hand, glycyrrhizin suppressed TNF-a-mediated cytotoxicity

467

against TNF-a-sensitive L929 cells. A significant inhibitory effect is observed even at a concentration of 2 jLtg^mL. However, it should be noted that the effect was very moderate: less than 50% protection even at 200 ILig^mL, the highest concentration tested. Although it was considered that such inhibition of immune-medicated cytotoxicity is one of the mechanisms whereby glycyrrhizin suppresses elevated serum transaminases levels in patients with chronic viral infection [90], there remains room for further investigation on this point. Tetrahydroswertianolin (THS; 2) is one of the main hepatoprotective constituents of SwertiajaponicaMakino, a very popular folk medicine in Japan. THS possesses an unique structure characterized by a partially-saturated xanthone frame (Fig. 1). Dreiding model analysis along with an analysis of the J-value from ^H-NMR andNOE experiments suggested that the cy clohexene ring in THS is likely to be in distorted halfchair conformation with an equatorial 5-OH and axial 8-O-glucose [92]. Additionally, using Mosher's method, its absolute configuration was determined to be 5-(i?) and 8-(iS) (Fig. 2). As to its hepatoprotective activity, pretreatment with THS (25 and 50 mglcg, s.c.) before intoxication with D-GalN and LPS in mice significantly attenuated the serum ALT elevation by 68 and 84%, respectively. A significant inhibition (80 and 91%) of the serum ALT elevation was also observed with the oral administration of THS (20 and 200 mg/kg). The hepatoprotective effect of THS was comparable to that of glycyrrhizin used as a positive reference. To find the active center of THS, the hepatoprotective activity of aglycone (tetrahydrobellidifolin; 3) and its derivative (l-hydroxy-3methoxyxanthone; 4) was investigated. The hepatoprotective activity was preserved on tetrahydrobellidifolin, whereas l-hydroxy-3metho)^^xanthone did not show any activity. These observations suggest that the cyclohexene-ring moiety may be important for the hepatoprotective activity of THS [86]. OCH3

O R O^ OH 2 : R = Glc 3 :R = H

r^j^^^N^Ov^^-'^j^iY^OCHa

O

OH

4

Fig. (2). Tetrahydroswertianolin and its derivatives

Further study demonstrated that THS significantly inhibited hepatic DNA fragmentation, the emergence of apoptotic bodies and chromatin condensation in D-GalN/LPS-induced liver injury in mice. The extent of the inhibitory effect of THS on hepatocyte apoptosis at 5 hr paralleled that on serum ALT elevation at 8 hr [87]. As mentioned above, in this model, a large number of hepatocytes undergoing apoptotic cell death can

468

represent a stimulus for primed neutrophils in sinusoids to transmigrate and activate, leading to hepatic inflammation and massive hepatocyte necrosis [15,30,48,93]. Therefore, prevention ofapoptosis will protect to some extent ag^nst hepatocyte necrosis. Our data clearly showed that THS inhibited hepatocyte apoptosis at the early stage of the development of liver injury, indicating that the suppression of hepatocyte apoptosis is one of the mechanisms by which THS protects mice from liver injury. Since it is established that the induction of hepatocyte apoptosis in the DGalN/LPS model is mediated by TNF-a, the interaction of THS with TNF-a might be related to their hepatoprotective activities. In a separate study, we found that THS significantly prevented the serum TNF-a elevation and hepatic mRNA induction that occurred as an early pathological event after D-GalN/LPS-intoxication. The inhibitory effect (65 to 78%) of THS on serum TNF-a elevation was observed at a dose range (10-200 mglcg) similar to that observed with the inhibitory effect (65% at a dose of 50 mg/kg)on hepatic apoptosis. On the other hand, pretreatment with THS did not attenuate mrTNF-a-induced hepatic apoptosis significantly in D-GalN-sensitized mice [87]. These results suggest that protection by THS against hepatic apoptosis induced by D-GalN and LPS may be due predominantly to the inhibition of TNF-a production. TNF-a is responsible for not only induction of apoptosis but also the initial inflammatory response such as e>q)ression of adherent molecules and sequestration of neutrophils in sinusoids [15,31]. The inhibition of TNF-a by THS, therefore, may contribute to the suppression of the initial inflammatory response as well. Acteoside (verbascoside; 5) is aphenylethanoid widely distributed in medical plants such as Cistanche deserticola Y. C. Ma. Phenylethanoids are a group of water-soluble natural products and were reported to have various bioactivities. Their antioxidant activity in particular is well documented [94]. There have been several reports on the hepatoprotective effect of acteoside and some other phenylethanoids on CCI4- or D-GalNinduced cytotoxicity in rat primary culture hepatocytes [95,96]. We recently found that pretreatment with acteoside (10 or 50 mg^kg, s.c.) significantly inhibited the elevation of serum ALT, fragmentation of hepatic DNA and formation of apoptotic bodies in the D-GalN/LPSinduced liver injury model. Also, this compound improved the mortality of mice after intoxication [97]. A similar effect was observed when acteoside was administered orally at a dose range of 20 to 100 mglcg. These results clearly showed that acteoside is effective against the hepatocyte apoptosis as well as necrotic liver damage. Interestingly, acteoside had no apparent effect on the elevation of serum TNF-a, but it partially prevented mouse TNF-a (100 ng^mL)-induced cytotoxicity in D-GalN (0.5 niM)-sensitized mouse hepatocytes at a concentration of 50 |LiM or more. Thus, the protective effect of acteoside on hepatocyte apoptosis and subsequent liver injury could be explained by attenuation of

469

the cytotoxic function rather than the production of TNF-a. It can be speculated that this compound may interfere with apoptotic signals such as the activation of caspase-3 protease. The fruits of Hovenia dulcis Thunb. (Rhamnaceae) is a traditional Chinese medicine used as a dotoxify ing agent for alcoholic poisoning [98]. The accelerating effect on alcoholic metabolism of the H2O extract of H, dulcis has been confirmed by e}q)eriments using rodents and human beings [99-101], although the mechanisms and active component(s) remain to be clarified. Recently, the MeOH extract of this plant was shown to possess hepatoprotective activity against CCI4- or D-GalN/LPS-induced liver injuries in rats and mice [102,103]. The MeOH extract has a good deal of dUiydroflavonoids such as (+)-ampelopsin (dihydromyricetin; 6) and its 0-methylated derivative hovenin I (7). Pretreatment with ampelopsin (100 mg/kg, p,o,) and hovenin I (25 or 50 mg1<:g, i.p.) protected significantly against the elevation of the serum transaminase activity in the CCI4- and D-GalN/LPS-induced liver injury models. The hepatoprotective efficacy of these two compounds seems comparable, suggesting that 0-methyl substitution in the B ring does not affect hepatoprotective activity. In a separate e>q)eriment, pretreatment with ampelopsin (100 mg/kg, p.o.) significantly reduced the extent of hepatic DNA fragmentation, and prevented the emergence of hepatocytes with chromatin condensation and apoptotic bodies at the early stage of D-GalN/LPS-induced liver injury in mice. The elevation of the serum TNF-a concentration was also suppressed by pretreatment with ampelopsin, which did not inhibit TNFa-induced hepatotoxicity in D-GalN-sensitized mouse hepatocytes (unpublished data of the author's group). The inhibitory effect of TNF-a production was commonly observed on other flavonoids, dismosin, hesperidin, naringin and rutin [104]. Particularly, intraperitoneal administration of naringin (8) (1 mg/mouse) was reported to inhibit LPS-induced TNF-a production by 94% in mice. Also, naringin pretreatment (1 or 3 mg/^mouse, ip.) markedly inhibited hepatocyte apoptosis after intoxication with D-GalN and LPS, and consequently protected mice from liver injury [104]. Based on these results, inhibition of hepatocyte apoptosis via suppression of TNF-a production may be of prime importance to the hepatoprotective mechanisms of flavonoids such as ampelopsin and naringin. Curcumin (9) is an ary Iheptanoid isolated from turmeric, the rhizome of Curcuma longa L. Turmeric has been used as a remedy for inflammatory in Asia for centuries. There are a number of reports supporting an anti-inflammatory effect of curcumin. Its hepatoprotective activity against inflammatory liver injury was also demonstrated in a recent study [105], where pretreatment with curcumin (50 mglcg, p.o.) significantly inhibited the elevation of serum transaminase activities after intoxication with D-GalN and LPS in mice. The hepatoprotective effect is presumably

470

mediated by inhibiting TNF-a production, because an in vitro study demonstrated that curcumin (5 |iM) markedly inhibited LPS-induced production of TNF-a and IL-1 in a human monocytic macrophage cell Hne, Mono Mac 6 [106]. A gel mobility assay indicated that the inhibition of these cytokines by curcumin is based on down-regulation of nuclear factor kappa B ( N F - K B ) , a key transcriptional factor which regulates the e>q)ression of cytokine genes through stimulation of LPS. Curcumin possesses two polyphenolic and one diketone functional group and is well known to have antioxidativeproperties. SinceNF-KB is an oxidative stress sensitive transcription factor [107], the antioxidant property of curcumin may help to inhibit N F - K B activation [106]. (-)-Epigallocatechiii gallate (EGCG; 10) is a main compound of green tea (Camellia sinensis) polyphenols and accounts for more than 40% of all polyphenols. EGCG has various bioactivities including scavenging of 0}q)ression [108,109]. Furthermore, it was recently reported that oral administration of green tea polyphenols (100 or 500 mg/kg) significantly suppressed the elevation of serum TNF-a concentration and markedly improved rat mortality after intoxication with LPS (40 mg1<:g, i,p.) [33]. An in vitro study using the macrophage cell line RAW264.7 endorsed the dir-ect inhibition by EGCG of LPS-stimulated TNF-a production. TNF-a mRNA and nuclear NF-KB-binding activity as well as protein synthesis were also suppressed when treated with EGCG. These effects were not due to cytotoxicity of EGCG. These observations suggest that the hepatoprotective effect of green tea polyphenols on the LPS-induced liver injury can be attributed to its down-regulation of TNF-a gene e>q)ression throu^ suppression of N F - K B activation [33]. Similar to curcumin, the antioxidant property of tea polyphenols is believed to play an important role in the inhibition of N F - K B . This mechanism may be consistent with the mode of action of some other polyphenolic compounds that inhibit TNF-a production, such as THS and ampelopsin. HO^^^

p

OHQ

^,0^"

Rha-Glc-O,

OH O 8

9

Fig. (3). Polyphenolic compounds with hepatoprotective acltlvity against TNF-a-dependent liver Injury

471

Gentiopicroside (11) and sweroside (12) are bitter secoiridoid glycosides widely distributed in Swertia spp, and Gentiana spp, (Gentianaceae). Thehepatoprotective effect of these compounds has been suggested in chemical liver injury models induced by CCI4 or Cd [110,111]. Besides, we found that pretreatment with gentiopicroside and sweroside (each 25 or 50 mg/kgj.p,) moderately inhibited the elevation of serum ALT activity in inflammatory liver injury induced by D-GalN and LPS in mice [86]. This finding was in line with that reported by Kondo et al. [110]; pretreatment with gentiopicroside (30 or 60 mglcg, Lp.) significantly inhibited the increase in the serum transaminase activities in a dosedependent manner after LPS challenge in BCG-primed mice. The hepatoprotective activity against inflammatory liver injury may be explained at least in part by the suppression of TNF-a production, because gentiopicroside significantly suppressed the acute increase in the serum TNF- a activity in a dose-dependent manner in the BCG/LPS model [110].

11

12

Fig. (4). Iridoids with hepatoprotective acitivity against TNF-a-dependent liver injury

The methanol extract (200 mglcg, ip,) of roots of Angelica Jurcijuga Kitagawa (Umbelliferae) was reported to protect mice from inflammatory liver injury induced by D-GalN (350 mg/kg, ip,) and LPS (10 lig^g, /./7.). By phytochemical analysis, coumarines and poly acetylenes were isolated as hepatoprote ctive compounds. Intraperitoneal administration (12.5 or 25 mg/kg) of isoepoxypteryxin (13), anomalin (14), isopteryxin (15) and falcarindiol (16) was found to significantly inhibit the increase in serum transaminase activities after intoxication with D-GalN and LPS [112]. These coumarines and poly acetylene were proposed to inhibit LPSinduced nitric oxide (NO) production by macrophages. However, it should be noted that the role of NO in the systemic inflammatory response is two-faced. On the one hand, overe)q)ression of NO is partly responsible for the vascular collapse and ensuing circulatory failure caused by LPS or TNF-a [113,114]. On the other hand, liver-derived NO is now considered an endogenous hepatoprotective factor [115]. Administration of NOS inhibitors (L-arginine-analogues) aggravates rather than inhibits hepatotoxicity and lethality induced by BCG/LPS, P. acnes/LPS, or TNFa [113,116,117]. Recent data indicated that NO prevents TNF-a-

472

induced hepatocyte apoptosis by inhibiting caspase-3 activation [118]. Inhibition of coagulation by NO is also proposed to contribute to the hepatoprotective effect [116,119]. Given these findings, it seems unreasonable to suppose that the inhibition of NO production is part of the hepatoprotective mechanism of coumarines and poly acetylene isolated from^. furcijuga. Instead, these compounds could impede the secretion of other deleterious factors like TNF-a from macrophages, resulting in hepatoprotection.

16 Fig. (5). Hepatoprotective coumarins and polyacetylene of Angelica furcijuga

Reports have suggested that some alkaloids protect against TNF-adependent liver injury. Pretreatment with an anti-malaria drug quinine (17) (230 mg/kg, up,) was shown to abolish plasma TNF-a elevation as well as subsequent hepatic DNA fragmentation and plasma transaminase and sorbitol dehydrogenase elevations in D-GalN/LPS-liver injury in mice. Also, it markedly improved mortality in mice after intoxication [120]. Activation of the K^ chaimel was reported to be important for LPSstimulated TNF-a production in human alveolar macrophages. Since quinine is a K^ channel blocker, it is plausible that this effect helps to inhibit TNF-a production both in vitro and in vivo [120,121]. Similar to quinine, intraperitoneal administration (30 or 100 m ^ g ) of sinomenine (18), an epimorphinan alkaloid from Sinomenium acutum Rehder et Wilson, was reported to significantly inhibit the elevation of serum transaminase activities in D-GalN/LPS-induced liver injury in mice in a dose-dependent manner [122]. Intraperitoneal administration (10 mg/kg)of bisbenzylisoquinoline (BBI) alkaloids, chondocurine (19), cycieanine (20), tetrandrine (21) and berbamine (22), also inhibited the elevation of serum ALT activity and improved mortality after LPS challenge in BCGprimed mice [123,124]. The hepatoprotective effect of sinomenine and BBI alkaloids is thought to result from suppression of TNF-a production.

473

because pretreatment with these alkaloids significantly reduced the increase in serum TNF-a activity after intoxication [122,124]. Colchicine (23), an alkaloid isolated from Colchicum autumnale, shows a very potent hep atop rotective effect against D-GalN/LPS-induced liver injury in mice; intravenous administration of only 0.5 mglcg colchicine suppressed the elevation of serum ALT activity to the normal level [125]. Interestingly, pretreatment with colchicine also markedly protected against liver injury and lethality induced by intravenous injection of TNF-a or LPS-treated macrophages into D-GalN-sensitized mice. On the other hand, in vitro treatment with colchicine (20 or 200 |iM) had no effect on LPS-stimulated TNF-a production in primary cultured bone marrow-derived macrophages. From these observations, the authors concluded that the hep atop rot ective effect of colchicine is due to antagonization of the hepatotoxic and lethal function of TNF-a [125]. This effect may be related to down-regulation of TNF-a receptors on target cells by colchicine as a consequence of microtubule depolymerization [126].

"N-CH3 H3C0.

23

H3CO..

H3C.,

Fig. (6). Alkaloids with hepatoprotective activity against TNF-a-dependent liver injury

474

The crude drug Saiko, the roots of Bupleurum falcatum, has been used in Oriental medicines for the treatment of hepatobiliary diseases. The effect of Sho-Saiko-To (Xiao-Chai-Hu-Tang), a preparation containing Bupleurum Radix, on chronic hepatitis is well defined by clinical trials in Japan and China [1,127]. Experimental data suggested that pretreatment with Sho-Saiko-To (500 mg^g, p.o.) blocks human TNF-a-induced lethality in D-GalN-sensitized mice. In this model, all of the control mice died within 24 hr after TNF-a challenge, whereas 80% of the Sho-SaikoTo-treated mice survived 72 hr. Decrease in rectal temperature seen at 1 hr after TNF-a challenge was also improved by Sho-Saiko-To pretreatment [128]. Based on phytochemical analysis of Bupleurum Radix, saikosaponins and other saponins have been proposed to have hepatoprotective effects. In a recent study, Bupleuroside i n (24), IV (25) and Xffl (26), and Scorzoneroside A (27), B (28) and C (29) were reported to show heap atop rot ective activity against D-GalN/LPS-induced liver injury at the dose range of 10 to 20 mg/kg[129,130].

Glc—Fuc—O^ ''CH2OH

Glc—Fuc—C ""CHgOH

24 : R = P-OH 25 : R = a-OH

26 OH OH OH

\

}

f

H

H

H

,vCOO—CHgi'-C—-C—C-'iCHgO—R2

CH2OH 'OH Ri—O' XHgOH

27: Ri = Fuc-GIc, R2 = Glc 28 : Ri = Fuc-GIc, R2 = H 29: Ri = Fuc, R2 = H

Fig. (7).Triterpene glycosides of Bupleurum scorzonerifolium

There is much interest in the pharmacological role of dietary nutrients in human health. Some nutrients have been recognized to have a hepatoprotective effect throu^ interaction with TNF-a. For instance, dietary supplementation (5%, at the e>q)ense of casein) with glycine (30), a non-essential amino acid, attenuated LPS-induced liver injury and

475

subsequent mortality in rats [131]. It has been shown that Kupffer cells have voltage-dependent Ca^^ channels, and increase of the intracellular Ca^"^ concentration ([Ca^^Jj) is essential for Kupffer cells to produce cytokines after LPS stimulation. An in vitro study using primary cultured Kupffer cells proved that a supplement of glycine (1 mM) to culture medium largely prevents the increase in [Ca^ ji due to LPS. This inhibitory effect by glycine appears to be mediated by an increase in chloride flux via activation of glycine-gated chloride channels [132]. Thus, the hepatoprotective effect of glycine is associated with a blunting of the LPS-induced elevation of [Ca ]i in Kupffer cells, thereby minimizing the production of proinflammatory cytokines includingTNF-a [131,132].

30

Fig. (8). Dietary neutrients reported to inhibit TNF-a production

Choline (31) has apparently similar biological activity to glycine. Previous studies have demonstrated that a choline-deficient diet significantly increases the mortality of rats due to endotoxin shock [133], whereas rats fed with a diet containing excess choline (0.025-0.4%) show resistance to LPS-induced liver and lung injuries, and subsequent mortality [134]. However, in contract to glycine, in vitro treatment with choline does not affect the LPS-induced [Ca^"^]i increase and TNF-a production in liver and alveolar macrophages, ahhough Ca^^ influx and TNF-a production in macrophages isolated from choline diet-fed rats is blunted by 40-60% compared with in control rats. In addition, glycine and choline synergistically protect against TNF-a production and endotoxin shock, suggesting that the protective mechanism of glycine against endotoxin shock is distinct from that of glycine [134]. It is hypothesized that choline alters the signaling cascade triggered by the binding of LPS to receptors on macrophages, possibly by increasing the ratio of membrane phosphatidylcholine to phosphatidylinositol whose turnover is essential for macrophage activation. Alternatively, choline may improve the LPSinduced decrease in membrane fluidity [134]. However, it remains to be elucidated whether the protective effect of choline against endotoxin shock is due only to inhibition of TNF-a production, because in the same report it was described that feeding choline had no effect on the increase in serum TNF-a after intoxication with LPS. Choline may affect also the susceptibility of hepatocytes and other target cells to TNF-a cytotoxicity.

476 Table 2. Natural products with hcpatoprotcctive activity against TNF-a-dependent liver injury Dose' Adm- Models Compounds Original plants Ref. (m^g) inist. Route Alkaloids: [123,1241 Berbamine Berberis vulgaris 10 X 3 i.p. BCG/LPS Colchicine Colchicum autumnal 0.5 X 2 i.v. D-GalN/LPS, 11251 D-GalN/TNF-a [123,1241 Chondocurine Chondodendron tomentosum 10 X 3 i.p. BCG/LPS Cycleanine Cissampelos imularis 10 X 3 i.p. BCG/LPS 1123,1241 Sinomenine Sinomenium acutum 30 i.p. D-GalN/LPS [1221 Tetrandrine Stephania tetrandra 10 X 3 i.p. BCG/LPS [1241 Quinine Cinchona officinalis 230 i.p. D-GalN/LPS [1201 Coumarlns: Isoepoxypterv'xin Anomalin Isoptety'xin

Angelica furcijuga Angelica furcijuga Angelica furcijuga

Iridoids: Gentiopicroside Sweroside Polyphenols (Flavonoids etc.): Acteoside (+)-Ampelopsin (Dihydromyricetin) Curcumin (-)-Epigallocatechin gallate Gomisin A Hovenin I Naringin Lithospermate B Tetrahydroswertianolin

12.5 12.5 12.5

i.p. D-GalN/LPS i.p. D-GalN/LPS i.p. D-GalN/LPS

Gentiana macrophylla

30 X 5

Swertiajaponica

25 X 2

i.p. BCG/LPS D-GalN/LPS, s.c. D-GalN/LPS

Cistanche deserticola Hovenia dulcis

10 X 2 100 X 2

P.O.

Curcuma longa 50 Camellia sinensis 100 Schizandra chinensis 100 Hovenia dulcis 25 Citrus aurantium var. daidai Umg/moiise) Salvia miltiorhiza 50 Swertiajaponica 50 X 2

Triterpenes: Bupleuroside III, IV and XlII Bupleurum scorzonerifolium Ginsenoside Re and Rgi Panax notoginseng Glycyrrhizin Glycyrrhiza glabra

D-GalN/LPS D-GalN/LPS

D-GalN/LPS LPS i.p. P. acneslLVS i.p. D-GalN/LPS i.p. D-GalN/LPS s.c. D-GalN/LPS P.O. D-GalN/LPS

P.O. P.O.

186,1101 [861 [971 [al [1051 [331 [91 [1031 [1041 [1351 [86,871

or s. c. 10 or 20 20 X 2 100 X 2 10

Scorzoneroside A, B and C

Bupleurum scorzonerifolium

Other phytoconstituents Falcarindiol Celosian

Angelica furcijuga Celosia argentea

Nutrients Choline

—

0.4%

—

5%

Glycine

s.c

[1121 [1121 [1121

12.5 10

i.p D-GalN/LPS i.p. D-GalN/LPS P.O. D-GalN/LPS or s. c. D-GalN/LPS i.p.

i.p. D-GalN/LPS s.c. D-GalN/LPS, P.acnesIL?^ Dietary LPS

[1291 [1361 [85,871 [1301 [1121 [44,1371 [1341

siippl.

Dietary LPS

[131,1321

suppl.

Minimally effective dose, [a] unpublished data of the author's group. D-GalN, D-galactosamine; LPS, lipoplysaccharide; BCG, bacillus Calmette-Guerin

CONCLUSION The TNF-a-dependent models are mostly characterized by the apoptotic cell death of hepatocyte at the early stage of liver injury. In these models, a large number of hepatocytes undergoing apoptosis can represent a Stimulus for primed neutrophils in sinusoids to transmigrate and activate, leading to hepatic inflammation and massive hepatocyte necrosis. This

477

means that prevention of apoptosis is an effective therapy for disrupting the progression of Uver injury. Indeed, some hep atop rot ective phytoconstituents protect against hepatocyte apoptosis and consequently block the progression of liver injury, althou^ others seem to prevent secondary hepatocyte necrosis tri^ered at the final stage without affecting apoptosis. Inhibition of hepatocyte apoptosis may be one important mechanism whereby phytoconstituents exert hep atop rot ective activity. Therefore, measuring apoptotic markers besides necrotic makers such as serum transaminase levels is useful to distinguish the mode of action of hepatoprotective compounds on inflammatory liver injuries. The protection against hepatocyte apoptosis and liver injury by natural products is presumably mediated by an interaction with TNF-a. The majority of the hepatoprotective compounds described here possess inhibitory activity against TNF-a production by macrophages. On the other hand, a few compounds attenuate the cytotoxic action of TNF-a. These observations clearly suggest that certain hepatoprotective phytoconstituents bear pharmacological potency that impedes the intracellular signaling pathway essential for TNF-a production in effector cells or TNF-a cytotoxicity in target cells. Elevation of the serum TNF-a level is frequently seen in patients suffering from alcoholic, viral or fulminant hepatitis, or cirrhosis, and there is an inverse correlation between the TNF-a elevation and the survival ratio of these patients. Continuing investigations on phytoconstituents with the TNF-adependent liver injury model are e>q3ected to provide new effective hepatoprotective agents. ABBREVIATIONS ActD ALT AST BCG Con A CCI4 D-GalN IFN-Y LPS MNCs NO NOS RT-PCR THS TNF-a TNF-R

=

actinomycin D alanine transaminase aspartate transaminase bacillus Calmette-Guerin concanavalin A carbon tetrachloride D-galactosamine interferon-y lipopolysaccharide mononuclear cells nitric oxide NO synthase reverse transcription-polymerase chain reaction tetrahydroswertianolin tumor necrosis factor-a TNF receptor

478

ACKNOWLEDGEMENT We would like to thank Emeritus Professor Tsuneo Namba and Dr Purusotam Basnet for their support and valuable comments on the manuscript. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11 [12 [13 [14 [15 [16: [17 [18 [19: [2o: [21 [22 [23 [24 [25 [26 [27 [28

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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INDUCTION AND REGULATION OF BIOSYNTHETIC ACTIVITY OF PHT^TOALEXIN IN CARROT CELLS FUMIYAKUROSAKI Faculty ofPharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-0194, Japan ABSTRACT: The intracellular production of 6-methoxymellein, a phytoalexin of carrot, is induced by the addition of a wide variety of substances called elicitors. Active elicitor molecules appear to be fragments of pectic substances from carrot cell walls, and are liberated by partial hydrolysis of the walls with extracellular pectinase or proteases secreted by invading fimgi. The accumulation of 6-methoxymellein is controlled primarily by the rate of transcription of the genes which encode the biosynthetic enzymes. Transduction of elicitor signals in plant cells may involve a mechanism similar to that reported in odor-sensitive animal cells. It is likely that Ca^"^ acts as a second messenger, and plays a central regulatory role in expression of the genes that encode the enzymes for 6-methoxymellein biosynthesis. The increase in the cytoplasmic Ca^^ level is mediated by activation of the phosphatidylinositol cycle, liberating inositol trisphosphate and diacyl glycerol as messenger molecules. In addition, evidence has been gathered suggesting that cyclic AMP stimulates Ca^'^-influx by gating of cyclic AMP-sensitive cation channels with no accompanying cyclic AMP-dependent protein phosphorylation. Biosynthesis of 6-methoxymellein is catalyzed by two inducible enzymes, 6hydroxymellein synthase and 6-hydroxymellein-O-methyltransferase. 6-Hydroxymellein synthase is a multifunctional polyketide biosynthetic enzyme, and is an active catalyst only in the homodimeric form. Acetyl and malonyl moieties, which are the building units of 6-hydroxymellein, bind to the transacylase domain of the synthase, and are channeled to two SH-groups at the reaction center. The reaction catalyzed by 6hydroxymellein-0-methyltransferase proceeds by a bireactant sequential mechanism, and the activity of the enzyme is strictly controlled by its products.

INTRODUCTION It is widely recognized tliat plant cells are potentially rich sources of commercially important secondary metabolites. The production of secondary metabolites could be controlled by a mechanism by which

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enzymes are expressed or repressed. It is likely that the genes encoding specific enzymes in the biosynthetic pathway of desired products are sometimes repressed, or expressed very weakly. However, the mechanism involved in 'switch on' of the genetic information on secondary metabolism is at present very poorly understood. It is known that microbial invasion in plants often triggers the accumulation of antimicrobial substances called phytoalexins. Interaction between plants and microorganisms has been studied in several hostpathogen systems, and experimental results show that signal molecules which induce the synthesis of secondary products in plant cells are produced in the early stage of microbial infection. These molecules are called elicitors, and include peptides, polysaccharides and glycoproteins derived from microbial and plant cells. The response of plant cells to these elicitors was first studied from the phytopathological point of view to elucidate the regulation mechanism of phytoalexin production. Recent investigations have indicated that the treatment of plant cells with possible elicitors occasionally results in a rapid accumulation of secondary products. Here, several questions are addressed regarding the production of secondary metabolites by elicitors. 1) How is the signal of the elicitor recognized by plant cells? 2) How is the signal transduced in the cells? 3) How does the signal trigger the expression of genes? 4) How are enzyme activities controlled to produce secondary metabolites? Effective use of elicitors in producing usefiil metabolites in plant cells requires the elucidation of these biochemical mechanisms by which external stimuli regulate the genetic information. These questions are under active consideration, although at present very little is known about the molecular mechanisms underlying the recognition and transduction of elicitor signals. STIMULATION OF 6-METHOXYMELLEIN PRODUCTION IN CARROT CELLS Liberation of elicitors during host-pathogen interaction 6-Methoxymellein, an antifungal isocoumarin[Fig. (1)], was first isolated as the metabolite that is responsible for the bitter taste in cold-stored

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carrot roots [1]. Condon and Kuc [2] have shown that this compound accumulates in

Extracellular hydrolases

Cell wall fragments as elicitors

CH3O'

"^^

^ ^

'"'t

6-Methoxymellein production Fig. (1). Interaction between invading fungi and host plants in the elicitation of phytoalexin production

carrot roots after inoculation with Ceratocystis fimbriate, which causes black rot disease in sweet potato but is not pathogenic to carrot. The resulting production of 6-methoxymellein accounted for the resistance of carrot tissue to microbial infection. This compound inhibits the growth of various fungi in the concentration range 0.05 - 0.5 mM [3]. Preliminary studies indicate that heat-stable and water-soluble substances which show elicitor activity are released during interaction of carrot cells and the fungus [4]. The elicitor lost its activity after digestion with pectinase or proteases, suggesting that oligogalacturonides and/or peptides are jointly essential in inducing activity. Also, partial hydrolysates of pectic fractions of carrot cell walls prepared with these enzymes showed strong elicitor activity. These results suggest that extracellular hydrolases secreted by fungi, including pectinase and proteases, act to liberate oligosaccharides and peptides from carrot cell walls, and the fragments of the extracellular matrix of carrot bring about 6-methoxymellein production [Fig. (1)]. This was confirmed by an experiment in which filter-sterilized pectinase and trypsin were directly added to carrot cell

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culture. Biosynthetic activity of 6-methoxymellein was induced in the carrot cells, implying that eliciting substances are released from live carrot cells by the enzymatic action of these hydrolases. Stimulation of 6methoxymellein production was also observed on exposure of carrot cells to ethylene or metal ions [5]. Effect of esterification of oligogalacturoiiides on elicitor activity As with other secondary metabolites, phytoalexin production in in vitro culture of plant cells is influenced by culture age [6]. When 6methoxymellein production was triggered by adding partial hydrolysates of cell walls, its rate of production was very low in growing cells, but high in cells in the early stationary phase [7]. The release of active elicitor depended also on the growth stage of carrot cells. Partial hydrolysates obtained from carrot cells in the late logarithmic and early stationary phases yielded highly active elicitors, while those from the early logarithmic and late stationary phases showed very low activity. Asamizu et al. [8] reported that polygalacturonides in cell walls of cultured carrot are highly esterified, while those in aged cultures, which are a poor source of elicitor, consist mostly of non-esterified uronic acids. These results suggest that the elicitor activity of pectin fragments of carrot cell walls depends largely on the degree of esterification of uronide complexes. To verify this, partial hydrolysates of the pectic fraction of carrot cell walls were further treated with pectin esterase. After hydrolysis the elicitor activity had decreased to less than 20%, suggesting that some degree of esterification of polyuronides is required for the induction of 6-methoxymellein synthesis [9]. By contrast, Jin and West [10] reported that, in the induction of casbene synthesis in castor bean seedlings, the eliciting activity of tridecagalacturonides was decreased by methylesterification of carboxyl groups. The effect of chemical modification of oligogalacturonides therefore varies according to plant species. TRANSMEMBRANE SIGNALING MECHANISMS IN STIMULATION OF PHYTOALEXIN PRODUCTION Participation of Ca^^ as a second messenger

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When elicitor-active pectic fragments were analyzed by ion exchange and gel-filtration chromatography, the activity was found to be distributed in many fractions [11], suggesting that the elicitor consists not of a single molecule but a mixture of several active substances. This result led to an examination of whether these elicitors share a common signaling mechanism. Ca^"*" is an important second messenger in many physiological processes in both animal and higher plant cells, and calmodulin (CAM), a Ca^^-binding protein, plays a central role in many of these systems [12, 13]. 6-Methoxymellein production induced by oligogalacturonide was appreciably inhibited in the presence of the Ca^"^channel blocker verapamil [14]. Trifluoperazine and W-7 [N-(6aminohexyl)-5-chloro-l-naphthalenesulfonamide], a different class of inhibitors of CAM-dependent reactions, also caused marked inhibition. In addition, it was found that appreciable 6-methoxymellein biosynthesis was induced in carrot by treatment with Ca^"^-ionophore A23187. These observations strongly suggest that the increase in cytoplasmic Ca^^ level is an essential early event in eliciting 6-methoxymellein production. In potato and soybean, phytoalexin production is also a Ca^'^-dependent process, and the elicitor-induced responses were significantly inhibited by several Ca^'^-inhibitors [15, 16]. Activation of phosphatidylinositol cycle Further support for the hypothesis that Ca plays a central role in regulating phytoalexin accumulation is provided by experiments in which the tumover of phosphatidylinositol was measured in the plasma membrane of elicitor-treated carrot cells [17]. The carrot cells were first labelled with ['^H]myo-inositol and, after the addition of elicitors, acid extracts of the cells were analyzed chromatographically for the production of inositol trisphosphate (IP3). In cells treated with elicitor, the release of radioactive IP3 increased with time and attained a maximum at 3 - 5 min after treatment. Phospholipase activity responsible for the degradation of phosphorylated phosphatidylinositol increased correspondingly. Several reports have shown that IP3 induces rapid release of Ca^"^ from intracellular stores in animal cells [18, 19]. Studies on plant cells have also demonstrated that exogenous IP3 releases Ca^"^ from microsomal preparations at micromolar concentrations, although only limited

488

information is available [20, 21]. Schumaker and Sze observed IPsinduced release of Ca^"^ from intact vacuoles of Avena seedlings [22]. Vacuoles are the most prominent organelles in plant cells, and normally contain 0.1 to 10 mM Ca^^; therefore they may serve as the principal Ca^^ store. Diacyl glycerol, another of the hydrolysates of phosphorylated phosphatidylinositol, is a known activator of protein kinase C in animal cells [23]. The present experimental results suggest that this protein kinase also participates in the expression of phytoalexin biosynthesis in carrot cells. We found that the synthetic diacylglycerol l-oleoyl-2-acetylrac-glycerol, which has been shown to be intercalated into cell membranes and to activate protein kinase C, induced 6-methoxymellein production in the absence of elicitor. A similar result was obtained for the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate, another activator of protein kinase C [17]. On the other hand the addition of H-7 [l-(5-iso-quinolinesulfonyl) -2-methyl-piperazine], a specific inhibitor for protein kinase C, resulted in suppression of phytoalexin production. These observations strongly suggest that a rapid breakdown of phosphatidylinositol in the plasma membrane of carrot cells takes place upon contact with elicitor molecules, resulting in the liberation of two types of second messengers, IP3 and diacyl glycerol. Role of cyclic AMP as a second messenger In contrast to Ca^^, the role of cyclic AMP (cAMP) as a second messenger in plant cells is still obscure, because there is no proof of the presence of cAMP-dependent protein kinase in plant cells. The existence of cAMP itself in plant cells has been confirmed [24, 25]; more recently, various works suggest that the cyclic nucleotide is involved in physiological events in plants [26]. We have found [14] that the addition of dibutyryl cAMP (Bt2cAMP) to carrot cell culture causes 6-methoxymellein production even in the absence of elicitor. Addition of several reagents which are known to change the intracellular level of cAMP, namely cholera toxin, which is an activator of adenylate cyclase, and theophylline, a phosphodiesterase (PDE) inhibitor, also led to production of 6methoxymellein, suggesting that elevation of the cAMP concentration in carrot triggers phytoalexin production in the cells. In fact, treatment of carrot cells with uronide elicitors led to a rapid but transient increase in the concentration of intracellular cAMP. Similar observations have been

489

reported by Bolwell et al. [27], who tested the effect of various modulators of signal transduction processes on the induction of phenylalanine ammonia-lyase in Phaseolus vulgaris cell cultures. They found that cholera, petussis toxins and forskolin all stimulated synthesis of the enzyme. These reagents are known to activate adenylate cyclase, either through interaction with G-protein or directly. We examined changes in the activity of protein phosphorylation in carrot cells following treatment with either Bt2cAMP, forskolin or Ca^^ionophore A23187 [28]. Addition of cAMP to cell extracts prepared from these treated cells did not cause any change in phosphorylation activity, indicating that cAMP-dependent kinase activity is absent or very low in carrot cells, as well as in most of the other plants. By contrast, the activities of Ca^^- and Ca^VCAM-dependent protein kinases increased markedly in both cytosolic and microsomal fractions after the treatment. Phosphorylation activity was stimulated not only by Ca^^-ionophore but also by Bt2cAMP and forskolin. Furthermore, although Bt2cAMP and forskolin can stimulate phytoalexin production in carrot cells, the effect was severely suppressed by diverse Ca^"^ channel blockers and CAM antagonists [28]. These observations suggest that cAMP acts as second messenger by stimulation of the Ca^'^-cascade, rather than by activating cAMP-dependent protein kinases. This view is supported by experimental results in which changes in the concentration of cytosolic Ca^^ in carrot cells were measured by a fluorescent Ca^"^-indicator (fluo-3) after treatment with the reagents [28]. The Ca^^ level in the cytoplasm of untreated carrot cells was found to be about 0.1 \iM, A marked increase in the intracellular concentration of Ca^"^ to 0.6 - 0.8 |iM was observed 3 6 min after the addition of BticAMP or forskolin. These results suggest that the increase in cytoplasmic cAMP level leads to the Ca^'^-influx into carrot cells. This conclusion was also drawn from an experiment in which the effect of cAMP on the Ca^^-flux was examined using '*^Ca^"^-loaded vesicles of plasma membrane [28]. Plasma membranes prepared by the two-phase partitioning method are generally composed of differently oriented sealed vesicles, some normal and some inside-out [29]. Incubation of these vesicles with "^^Ca^"^ in the presence of ATP results in selective placement of the radiolabelled ions into the inside-out vesicles by the plasma membrane-located Ca^^-ATPase [29]. When the ^^Ca^^loaded vesicles were incubated with cAMP a rapid release of "^^Ca "^ from the vesicles was observed; this discharge was specifically observed with

490

cAMP among the nucleotides tested. These observations are consistent with the hypothesis that the cytoplasmic level of cAMP is raised by an appropriate stimulus, and the nucleotide triggers Ca^^-influx v^ithout accompanying cAMP-dependent protein phosphorylation, probably through cAMP-sensitive ion channels. Synthesis and degradation of cyclic AMP Addition of forskolin to carrot cell culture caused an appreciable increase in adenylate cyclase activity. However, the increase was transient although the activator was present throughout the experiment [30]. In contrast to cyclase reported from other plant sources [31, 32] the forskolin-stimulated activity of the enzyme in carrot cell extracts was detected only when EGTA was included in the assay mixture, and the addition of exogenous Ca^"^ strongly inhibited the enzyme activity. The effect of various concentrations of Ca^"^ on adenylate cyclase activity was therefore studied using buffers with the concentration of free Ca^^ adjusted by the EGTA-Ca^"^ buffer system [33]. The activity of the cyclase was markedly affected by the free Ca^"^ concentration, and was maintained at a high level only when the Ca^"^ concentration was below 0.1 |LIM. This figure is close to the Ca concentration in cytoplasm in the resting state of various plant species. Constitutive activity of PDE was found in cultured carrot cells; this activity did not depend on either Ca^"^ or CAM. By contrast, a CAMdependent isoform of PDE (CAM-PDE) was induced in the cells by adding forskolin or Bt2cAMP to the culture [30]. Induction of CAMPDE activity in Bt2cAMP-treated carrot cells was markedly inhibited in the presence of verapamil, and addition of Ca^'*"-ionophore A23187 induced CAM-PDE [34]. These results suggest that increased Ca^"^, but not cAMP, in the stimulated carrot cells triggers induction of the PDE isoenzyme. Affinity of CAM-PDE to the substrate was low compared to constitutive PDE (Km values, 0.14 and 0.07 |uiM, respectively); however, V for the induced PDE was approximately 2.7 times higher than for the constitutive isoenzyme. These results suggest that synthesis and degradation of cAMP in cultured carrot cells are both controlled and switched on/off according to the concentration of Ca^^ in carrot cytoplasm. Adenylate cyclase activity is induced in the cells only in the resting state, and the enzyme activity is

491

automatically inhibited when the concentration of cytoplasmic Ca^^ increases and reaches the level of the excitatory state. The constitutive PDE, w^hich is insensitive to the cytoplasmic Ca^"*" level, is important in maintenance of the resting state of carrot cells, by keeping cellular cAMP and Ca^^ levels very low, while CAM-PDE induced in excited cells hydrolyzes the messenger nucleotide rapidly under conditions of high cAMP and Ca^"^, in vivo, as a response-decay mechanism. In animal cells, the cAMP-induced Ca'^'^'-influx through the nucleotidesensitive channels is terminated by the hydrolysis of cAMP, the ligand of the channels [35]. However, in cultured carrot cells, the cytoplasmic Ca^"^ concentration elevated by the stimulation of cAMP began decreasing even though the level of intracellular cAMP was high [28]. Furthermore, when a Ca^'^-influx was triggered by treating the cells with Bt2cAMP, the cytolasmic concentration of Ca^"^ returned to its base level after a few minutes, by which time the cAMP analogue was still present at a high concentration [28]. These results clearly indicate that, in contrast to animal cells, degradation of cAMP is not the immediate reason for the response decay of the cAMP-gated cation channel in carrot cells. We found [36] that the discharge of Ca^"^ from inside-out sealed vesicles of carrot plasma membrane was strongly inhibited when 9+

the suspension of the vesicles was supplemented with 1 ^iM free Ca , while Ca^"^ concentrations lower than 0.1 ^M did not affect Ca^'^-release. In addition, the inhibited Ca^^-flux across the plasma membrane was restored by the addition of CAM inhibitors and anti-CAM IgG [36]. These results suggest that the Ca^^-influx initiated by increases in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca^^ concentration reaches the threshold excitatory level in the cells. It is probable that CAM located in the plasma membrane plays an important role in the decay response of the cyclic nucleotide-gated cation channels. Ca^'^-dependent CAM binding to several target proteins in the plasma membrane has been reported in the pea [37]. However, this does not seem to be the case in closing the cAMP-sensitive cation channels of carrot cells, because Ca^^-loaded vesicles of plasma membrane which were repeatedly washed with EGTA-containing buffer showed similar resuks. CAM involved in this transmembrane signaling process should therefore be EGTA-stable, and probably partially embedded in the lipid bilayer as reported in the pea [37].

492

Regulation of Ca^^-ATPase activity As with other eukaryotic cells [38], maintenance of low Ca^"^ concentration in the cytoplasm of non-stimulated higher plant cells is essential. The cytoplasmic Ca^"^ concentration of plant cells in the resting state, as described above, is generally maintained at approximately 0.1 ^M by the action of Ca^"^4ransporting systems [39] which sequester the ion into internal organelles, including endoplasmic reticulum, mitochondria, and vacuoles, or mediate its efflux to the cell exterior. It is known that Ca^^-pumping ATPase at the plasma membrane plays a key role in transporting Ca ^ to apoplastic spaces [39]. Characteristics of Ca^"^-translocating ATPase have been reported from a wide range of plants [39], although some are highly variable depending on the plant species. One of the most serious controversies over properties of ATPase is the role of CAM in regulation of the enzyme; inconsistent observations on the CAM-dependence of enzyme activity have been reported from several plants [40-43]. It is not yet clear whether this discrepancy represents genuine variation across species or is an experimental artifact. However, it seems that results depend partly on the fact that plasma membrane preparations obtained from higher plant cells sometimes contain the membranes of other organelles. A highly purified plasma membrane fraction from cultured carrot cells was prepared by the aqueous two phase-partition method [29], in order to reevaluate the role of CAM in regulating Ca^'^-ATPase at the plasma membrane of the cells. The Ca^'^-translocating activity of ATPase was considerably inhibited in the presence of different classes of CAM antagonists or anti-CAM IgG [44]. This Ca^"^-pumping activity decreased significantly when the plasma membrane preparation was washed with EGTA-containing buffer; however, it was restored to almost the control level upon adding exogenous CAM. These results suggest that Ca^^-ATPase at the plasma membrane of carrot cells is regulated by CAM, and the modulator protein associates with the enzyme in a manner dependent on the Ca^^ concentration [44]. The biochemical basis of CAM-induced stimulation of Ca^^-ATPase activity in carrot cells was studied further by determining the parameters of the Ca^"*"-translocating reaction of the enzyme in the presence and absence of exogenous CAM, using EGTA-treated plasma membrane [45]. The affinity of Ca^^-ATPase for Ca^^ was considerably increased by

493

association with CAM, and Km values decreased from 11.4 |aM to 0.7 )LiM. These figures are close to those of CAM-dependent Ca^^-ATPase at the plasma membrane in animal cells [39]. Affinity of the enzyme for ATP was also increased in the presence of CAM, although the increase was low compared to that for Ca^"^ (Km values of 914 and 670 |LIM in the absence and presence of CAM). In contrast to the affinities for the substrates, the relative V values of the ATPase were similar or slightly decreased by the addition of CAM. It is well known that, in the excited plant cells having high Ca^^ concentration, CAM is activated by binding to the ion, and is able to associate with various CAM-dependent proteins [38]. The Ca^"^ concentration in resting plant cells, by contrast, is too low to activate CAM, resulting in the dissociation of the modulator from its target proteins, including Ca^"*"-ATPase [38, 39]. The Kca of the ATPase associated with CAM is similar to that of the cytoplasmic Ca^"^ level of excited plant cells (0.7 jaM), while the Kca of the ATPase without CAM increased markedly (11.4 |aM) though the cytoplasmic Ca^^ concentration in the resting cells is quite low. These observafions suggest that Ca^^ATPase at the carrot plasma membrane plays an important role in the excited cells only as an 'acute' enzyme. However, Rasi-Caldogno et aL [43] pointed out that Kca decreased from about 10 |aM to about 0.1 |iM if the level of free Ca^"^ alone is considered. This low Km value of CAMdepleted Ca -ATPase for Ca is consistent with the transport protein involved in maintaining cytoplasmic Ca^"^ concentration at the submicromolar range being a 'house keeping' enzyme in resting cells. These results strongly suggest that, on binding of CAM, the affinity of the carrot Ca^^-ATPase for Ca^"*" is markedly increased, and this is the most important biochemical change behind the CAM-induced increase in pumping activity of the enzyme. Possible scheme for transduction mechanisms of elicitor signals These studies all support the hypothesis that external stimuli of the elicitor cause an increase in the cytoplasmic Ca^^ level via the phosphatidylinositol cycle and/or the adenylate cyclase system. Although an authoritative picture of this process cannot yet be given, possible signal transduction mechanisms are summarized in Fig. (2). At present the data

494

are still fragmentary, so that it is important to leam more about the biochemical nature and function of the components involved in signal Elicitors

PLC

-CX

<^

Receptors DAG

^v AC CCAM^ /^—K; ATP

PKC

Ca2+

AMP^

Ca'*-ATPase

^

A^<^>

/

cl

Ca'7CAM PK PDE

CAM-PDE

Activationl ooff CCa -cascade

i

Gene expression Fig. (2). Schematic presentation of early events of phytoalexin production

transduction process in plants. Evidence has been accumulated suggesting that plant cells contain the major components of the phosphatidylinositol cycle, while the function of cAMP described here is unique. This class of signal transducing mechanism is rare and is seldom seen in animal and microbial cells. However, a similar gating action of cAMP has been reported in olfactory transduction in animal sensory cells [46]. In these cells the cAMP content increases in response to odoriferous substances, and this change induces an influx of Ca^^ into the cells without cAMP-dependent protein phosphorylation. Krupinski et al. [47] have suggested that the amino acid sequence of an adenylate cyclase from the bovine brain is topographically similar to ion channels such as Ca^"^ and K"*". Based on this assumption, Schultz et al. [48] tested the poreforming ability of adenylate cyclase from Paramecium in an artificial lipid layer, and suggested that the enzyme has a secondary function as a carrier of ions. Enzyme activity of the cyclase and pore-forming activity

495

were strictly interdependent. Although no information on the structure of plant adenylate cyclase is presently available, the possibility that the cyclase itself operates as a transmembrane ion channel in plant cells cannot be excluded. Stimulant

AC

CAM PDE

o

cAMP

I

CAM CAM-PDE

Fig. (3). Signal cross-talking between cAMP and Ca^^-cascade

Activation of Ca^'*'-cascade

The characterization of the functional proteins involved in cAMPinduced cellular events suggests that most components of these signal transduction processes are correlated, and regulate each other. A plausible scheme for signal cross-talking of the messenger nucleotide with the Ca^'*"-cascade in the early stages of transmembrane signaling processes is as follows [Fig. (3)]. 1) In the resting state only constitutive PDE is active, and both adenylate cyclase and cAMP-sensitive channels are inactive; therefore cAMP and Ca^^ are both maintained at low levels. 2) Upon the arrival of elicitor signals on the receptor protein located at the plasma membrane, adenylate cyclase is activated, and the increased level of cAMP associates with cAMP-sensitive channels as the ligand to open the ion gates. 3) Influx of Ca^"*" activates the Ca^'^-cascade leading to the expression of genes encoding the biosynthetic enzymes of the phytoalexin.

496

In parallel, activity of adenylate cyclase is inhibited by Ca^"^, and the ion activates the membrane-embedded CAM to close the cAMP-dependent channels. In addition, CAM-PDE is induced to hydrolyze the messenger molecules rapidly. 4) Finally, Ca^^ activates the cytoplasmic CAM to enhance the activity of Ca^^-translocating ATPase, causing the cells to return to the resting state. Contribution of inward K^ channels A family of new genes encoding inward K"^ channels has recently been isolated from Arabidopsis thaliana, and it has been shown that two distinct modules, a cAMP-binding domain and an ankyrin repeat motif, comprise the carboxyl-terminal half of the structures of these genes [4952]. These findings support the idea above that cAMP acts as a second messenger by activating cAMP-sensitive ion channels as the initial event in a series of signal transduction reactions in higher plant cells. We therefore tested the possibility that the inward K'^-channels regulated by cAMP contribute to signal cross-talking of the cyclic nucleotide with the Ca^'^-cascade in cultured carrot cells. The correlation was studied between the cAMP level in carrot cells and the K^-influx caused by activation of a particular K^-channel, and also links between the cAMPinduced inward K"^ current and the Ca^^-influx in the cells. Treatment of carrot cells with Bt2cAMP resulted in a transient decrease in extracellular K^, which was restored to the original level within 1 min. The Bt2cAMPinduced decrease in the K"^ concentration outside the carrot cell was almost totally inhibited in the presence of the K^-channel blockers CsCl and TEA. Similar results were obtained when the cell culture was treated with forskolin. These observations suggest that the increased level of cAMP in cultured carrot cells induces K^-influx across the plasma membrane; it is very likely that this process is mediated by a particular inward K^-channel. Next, the effect of the K'*'-channel blockers on the discharge of "^^Ca^^ was tested, employing the inside-out vesicles of carrot plasma membranes. The cAMP-induced discharge of "^^Ca^^ was inhibited appreciably in the presence of CsCl when the blocker was sealed inside the vesicles; however, the blocker mixed in the buffer outside the vesicles did not show inhibitory activity. By contrast, TEA, another K^-channel blocker, exhibited marked inhibitory effects on the discharge of "^^Ca^^ in both the interior and exterior spaces of the vesicles.

497

It is well known that TEA is able to inhibit K"^-channel mediated ion transport at both sides of plasma membranes [53, 54], and the present observations are consistent with this unique characteristic. These observations, together with the results described above, suggest strongly that elevation of the cytoplasmic concentration of cAMP in cultured carrot cells, in response to appropriate external stimuli, results in the activation of inward K'^-channels or related structures at the plasma membrane; such structures might be members of the inward K"^-channel family that include the cAMP-binding domain [49-52]. Also, this inward K"^ current across the membrane leads to Ca^^-influx into carrot cells. A likely mechanism for the relation between these two ion currents is as follows: cAMP-induced influx of K"^ resuhs in depolarization of the plasma membrane in the cultured carrot, activating voltage-dependent Ca^^-channels [55] at the membrane to allow Ca^^ entry into the cells. It has been assumed [49, 52, 56] that inward K^-channels at plasma membrane of higher plant cells act to smooth the current flow of ions in the inner and outer spaces of the membrane and also provide a structure for uptake of the ions as a nutrient. However, our experimental results strongly suggest that some inward K'^-channels located at the plant plasma membrane are regulated by cytoplasmic concentration of cAMP, and play an important role in signal transduction processes of higher plant cells through the signal cross-talking mechanism. It has been confirmed [57] that an appreciable discharge of Ca ^, following stimulation with cAMP, is observed even if K^-salts are omitted from the buffers of both sides of the membrane and are replaced by Na -salts in equivalent concentrations. This observation clearly indicates that multiple mechanisms are involved in cAMP-induced entry of Ca^"^ into cultured carrot cells, and that the activation of cAMP-sensitive inward K^channels is not a sole initial event in Ca -influx elicited by the nucleotide. It is well known that inward K'^-channels at the plasma membrane are organized as tetramers, with the assembly of the four identical subunits making up a single pore for the permeation of K^ [58]. By contrast many other cation channels are composed of single polypeptides in which a set of domains corresponding to the subunit of the inward K'^-channels is repeated four times to form the channel structures [58, 59]. It is therefore reasonable to suppose that cAMP-sensitive ion channels for the permeation of ions other than K"^ exist and function in plant plasma membranes. The activation of these unidentified channels in response to

498

elevation of cytoplasmic cAMP would induce depolarization or hyperpolarization of the membrane, followed by gating of the voltagedependent Ca^^-channels. It is also possible that, as in animal cells, elevated levels of cAMP directly open the cyclic nucleotide-sensitive Ca^^-channels[60,61]. BIOSYNTHETIC ENZYMES INVOLVED IN 6-METHOXYMELLEIN PRODUCTION Biosynthetic sequence of 6-methoxymellein Based on ^^C-NMR analyses, it has been assumed [62] that 6methoxymellein, a polyketide compound, is synthesized by head-to-tail condensation of one acetyl-CoA and four malonyl-CoA to form 3,4dehydro-6-hydroxymellein as an intermediate. This isocoumarin derivative is then reduced to its dihydro-form, 6-hydroxymellein, which then accepts a methyl-unit via 0-methyltransferase. We have shown [63] that cell extracts prepared from elicitor-treated carrot root disks catalyzes Malonyl-CoA

-.COA _V^

Acetyl+ onyl-CoA Malonyl-

O

Malonyl-CoA

XA^

^^^_k^

NAHPH

AAS.K„X

Or^

O

Malonyl-CoA -^

O

_ O

^AJl^s-EnzA^^Y^ 0<^^^^

O

O^^^-^*^-^

O

Malonyl-CoA

AAS.,„A^

A-

\ „ HO'^

OH O

g^j^ g^jj

OH O

A^O i ^ A ^ O HO-^-^^^^-^^^

^30^^^^^^'

6-HydroxyineIIein

6-Methoxyinellein

Fig. (4). Biosynthetic sequence of 6-methoxymellein

the synthesis of the dihydroisocoumarin skeleton from acetyl-CoA and malonyl-CoA in the presence of NADPH. In the absence of the reducing co-factor, triacetic acid lactone was produced as an unwanted product, but another possible intermediate, 3,4-dehydro-6-hydroxymellein, was not

499

observed [5, 64]. These results suggest that dehydro-6-hydroxymellein is not involved in the biosynthetic processes of 6-methoxymellein, but that keto-reduction of the enzyme-bound intermediate takes place during elongation of the polyketomethylene chain. It is likely that the NADPHdependent reduction occurs at the triketide stage, and condensation of the reduced pentaketomethylene chain results in the formation of the reduced isocoumarin 6-hydroxymellein. 0-methylation of the compound by a specific O-methyltransferase [65] should then generate 6-methoxymellein [Fig. (4)]. Organization of 6-hydroxymellein synthase protein Polyketide biosynthetic enzymes have been assumed to share many common properties with fatty acid synthases, on the basis that most of the partial reactions involved are similar [66, 67]. However, unlike fatty acid synthases, biochemical properties of polyketide synthetic enzymes have not been well characterized, since these enzymes are usually very unstable. Higher plants and bacterial fatty acid synthases are readily separated into individual catalytic units (type II), while animal and yeast fatty acid synthases exist as multifunctional enzymes (type I). The latter are further divided into two subclasses, lA (consisting of two identical subunits) and IB (in higher states of aggregation) [68]. The catalytic activity of 6hydroxymellein synthetic enzyme remained after chromatographic fractioning of the enzyme preparation. Comparison of the apparent molecular masses of the enzyme, estimated by gel-filtration chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting, suggests that 6hydroxymellein synthase is a multifunctional enzyme which resembles type I fatty acid synthases [69]. More recently, it has been shown [70] that 6-hydroxymellein synthase is organized as homodimers, and the two subunits are reversibly dissociated to monomers with loss of synthase activity under conditions of high ionic strength. This fact in tum implies that functional structures in the synthase subunit are not destroyed by dissociation of the native dimer, and suggests that the monomer merely loses some of the partial activities essential for 6-hydroxymellein biosynthesis. When the monomeric synthase was incubated with the radiolabelled substrates acetyl-CoA and malonyl-CoA, appreciable radioactivity was associated with the enzyme proteins, suggesting that the

500 Ketoreduction

chain elongation

SHSH

JH_JH 6-HydroxymelIein synthase

Animal fatty acid synthase Fig. (5). Organization of active homodimer of 6-hydroxymellein synthase

synthase does not lose its ability to bind the substrates even after dissociating into monomers [71]. The monomeric synthase liberated triacetic acid lactone instead of 6-hydroxymellein, suggesting that the monomer enzyme retains the capability for acyl-CoA condensation but is lacking in NADPH-dependent keto-reducing activity. These results indicate that keto-reduction of the triketomethylene chain takes place only in the dimeric form of the synthase. It is likely that the catalytic sites of the two partial reactions of 6-hydroxymellein synthase, namely chain elongation and keto-reduction, associate with each other in catalytically functional form only in the homodimeric structure. It therefore seems that the catalytic domain of ketomethylene chain elongation associates with the domain of keto-reduction belonging to another subunit in the homodimer structure. This hypothesis also implies that two reaction centers are organized in each molecule of the active enzyme; this model of the synthase resembles type lA fatty acid synthases of animal cells [72, 73], in which two multifunctional subunits are aligned in an antiparallel

501

direction to form two complete reaction centers. However, in contrast to 6-hydroxymellein synthase, the monomer of type lA fatty acid synthase lacks the acyl-CoA condensation reaction but retains keto-reduction, according to assays of the partial activities in the presence of the appropriate substrates [74]. It is widely accepted [72, 74-78] that one of the two SH-groups at the reaction center of type lA fatty acid synthase belongs to the other subunit of the homodimer. This SH-group is derived from 4'-phosphopantetheine attached to acyl carrier protein (ACP), and is located near the keto-reduction domain. By contrast, in 6-hydroxymellein synthase, the SH-group from ACP [69] would be involved in the reaction center of the acyl-CoA condensation, even in the dissociated form. These facts clearly indicate that the contribution and arrangement of the catalytic domains in the reaction center of 6-hydroxymellein synthase are different from those of type lA fatty acid synthases [Fig. (5)]. Interaction between catalytic domains of 6-hydroxymellein synthase When the catalytic reaction of 6-hydroxymellein synthase is carried out in the absence of NADPH or with monomeric enzyme, keto-reduction of the carbonyl group of the triketomethylene chain does not take place, and the synthase liberates triacetic acid lactone instead of 6hydroxymellein [64, 71]. However, the efficiencies of product formation are markedly lower than for the normal reaction. Two mechanisms could account for the low efficiency of triacetic acid lactone formation observed in the monomeric and the NADPH-depleted dimeric forms of 6-hydroxymellein synthase. These are: 1) Reduced affinity of the cosubstrates acetyl-CoA and/or malonyl-CoA for the enzyme protein with the incomplete reaction centers; 2) Reduced rate of reaction of acyl-CoA condensation and/or product liberation. To examine these possibilities, kinetic parameters of the two triacetic acid lactone-forming reactions were compared with those of the normal reaction which produces 6hydroxymellein. The Km value of 6-hydroxymellein synthase for acetylCoA in the normal reaction was estimated to be 22 |LIM, while in both the NADPH-depleted dimer and the monomer reactions the affinity of 6hydroxymellein synthase protein for acetyl-CoA was markedly lower at 284 and 318 \xM respectively. By contrast the Km values for malonylCoA in the normal and the two abnormal reactions were essentially the same (40 - 43 |LIM), indicating that the affinity of 6-hydroxymellein

502

synthase to the extender unit is not altered even if the synthase is lacking the reducing co-factor or is dissociated to monomer subunits [79]. The difference in V between the normal and abnormal reactions is much less than that in the Km values observed for acetyl-CoA. It is therefore likely that reduced affinity for acetyl-CoA is the most important difference in the incomplete reaction systems, causing low efficiencies in product formation in these reactions. Only the NADPH-associated keto-reducing domain is capable of enhancing the affinity of acetyl-CoA for 6hydroxymellein synthase, and neither the domain without NADPH nor without NADPH molecule is able to increase in affinity. It can therefore be hypothesized that the structural perturbation of the reducing domain caused by association with the NADPH molecule alters the microenvironment around the primary binding site of acetyl-CoA in the homodimer structure of 6-hydroxymellein synthase, causing an increase in the affinity of the binding site for acyl-CoA. As shown in Fig. (4), the keto-reducing domain of 6-hydroxymellein synthase contributes the biosynthetic processes of 6-hydroxymellein in the middle stage of the catalytic cycle. According to our proposal this domain is also important in the entry of the starter unit into the multifunctional enzyme, which is the earliest step in a series of partial reactions; we also gain new insight into functional correlations between catalytic domains of various classes of multifunctional enzymes in homodimeric and oligomeric forms. The unique co-operative interaction between the catalytic domains observed in 6-hydroxymellein synthase opens a novel class of 'subunit communication' of multifunctional proteins. 6-Hydroxymellein production by the synthase was also observed when NADH was employed instead of NADPH. However, the effect of NADPH on enzyme activity was not fully replicated by NADH, and the activity of NADH-dependent 6-hydroxymellein production of the synthase was usually 50 - 70% of that for the corresponding NADPHmediated reaction [80]. To clarify the biochemistry of the lower yield using NADH, the kinetic parameters of the reactions were determined under various reaction conditions. The Km value of 6-hydroxymellein synthase for NADPH was estimated to be 70 |iM, while for NADH it was 10 |j,M, so that the affinity of the enzyme protein for NADPH is appreciably lower than for NADH. It follows that the difference in affinities of NADPH and NADH for the enzyme protein is not responsible for the low efficiency of NADH-mediated product formation.

503

Relative values of V for the NADPH- and NADH-mediated reactions were essentially identical, suggesting that the difference in efficiencies of 6-hydroxymellein formation is not caused by differing reaction rates. Further experiments were therefore undertaken to test whether the NADPH-induced enhancement of acetyl-CoA entry into the enzyme protein is observed even when NADPH is replaced by NADH. The Km value of 6-hydroxymellein synthase for acetyl-CoA in the NADHmediated reaction was found to be much higher (224 |LIM) than for NADPH (22 |iM), and was close to the figure of 284 jiM obtained for the synthase from which these reducing co-factors were omitted (as described above). Therefore it is assumed that, in contrast to NADPH, NADHassociated 6-hydroxymellein synthase protein has no effect or only a very small one on the interaction between ketoreductase and transacylase domains which increases the affinity to acetyl-CoA. These results suggest that the phosphate residue attached to the 2'-position of the adenosyl part of the NADPH molecule (2'-P) plays an important role in the co-operative interaction between the NADPH-associated ketoreducing domain and the transacylase structure, enhancing the affinity of the enzyme for acetyl-CoA. In sharp contrast to acetyl-CoA, it was found that the affinity of another substrate of 6-hydroxymellein synthase, malonyl-CoA, does not depend on the reducing co-factor associated with the ketoreducing domain. It is now clear that the alternation of the enzyme structure evoked by 2'-P in NADPH influences the affinity for acetyl-CoA but not for malonyl-CoA. It has also been suggested that 2'-P affects the affinity of NADPH for the enzyme protein. The Km value of the synthase for NADPH is 7 times higher than that for NADH. Therefore, it is very likely that 2'-P in the NADPH molecule exhibits both positive and negative effects on the catalytic reaction of 6HM synthase: increase in the affinity of the enzyme protein for acetylCoA, and decrease in the affinity of NADPH itself The low efficiency of NADH in the 6-hydroxymellein -forming reaction results from the balance of these effects caused by the phosphate residue. Initial processes of 6-hydroxymellein synthase-catalyzing reactions It has been shown that the reaction center of multifunctional fatty acid synthases of yeast and animal cells comprises two SH-groups: Cys-SH of the condensation enzyme, and pantetheine-SH attached to ACP. The

504

acetyl and malonyl units are transferred from the CoA esters to these SH groups prior to initiation of the condensation reaction, and Ser-OH of the transacylase domain is regarded as important in the proper entry and channeling of the co-substrates [72, 73]. Recent genetic and biochemical approaches have shown that ACP-like and transacylase-like domains also occur in polyketide biosynthetic enzymes in fungi and bacteria [66, 67]. In sharp contrast to these microbial enzymes, ACP is not involved in the structures of chalcone and stilbene synthases, which are well characterized polyketide synthases of higher plants [81, 82], and no transacylase domain is evident. Therefore an attempt was made to identify the primary binding site(s) of the acyl-CoAs, and to elucidate the charmeling pathways of the co-substrates to the two SH groups at the reaction center of 6-hydroxymellein synthase. Accordingly, several chemically modified enzymes were prepared in which the functional groups of amino acid residues were blocked, and the binding abilities of these partially masked enzymes toward the acyl components were determined [83]. 6-Hydroxymellein synthase lost all binding ability toward its co-substrates acetyl- and malonyl-CoAs following treatment with blocking reagents for serine-OH. However, this enzyme retained its binding ability even when the two SH groups at the reaction center were blocked, and one substrate bound to the SH-blocked enzyme was readily replaced by the other. These results suggest that a transacylase-like domain in the structure of 6-hydroxymellein synthase is involved as a common primary binding site of its co-substrates; however, this domain is not able to recognize the acetyl and malonyl co-substrate components. It is therefore likely that the loading of substrates is random, and both acetyl and malonyl groups load onto Ser-OH at the transacylase structure at any stage of substrate entry and chain elongation. Introduction of the substrates into the reaction center of the synthase in the correct order should therefore depend on the other structures and/or the mechanisms of the multifunctional protein. To study the entry of 6-hydroxymelein synthase to the substrate, three functional groups at the reaction center of the enzyme, Ser-OH, Cys-SH and ACP-SH, were alkylated appropriately, and the binding abilities of the modified proteins toward the substrates were examined [83]. It appeared that cysteine-SH accepted only the acetyl group, while cysteamine-SH of ACP was preferentially malonylated in the presence of both substrates. A plausible explanation for the initiation of the synthase

505

ACP-malonylated form

Cys-acetylated form

Ac-CoA

t

Fig. (6). Mechanism of substrate channeling of 6-hydroxymellein synthase

Ma-CoA

t

506

catalyzing reactions is as follows [Fig. (6)]. Acetyl-and malonyl-CoAs first acylate Ser-OH in the transacylase domain of the enzyme, and the malonyl group is channeled from the Ser-0-ester to ACP-SH while the acetyl group is directly channeled to Cys-SH. When one of the two SH groups is acylated, the Ser residue of the transacylase domain exchanges acyl groups by rapid replacement. In ACP-malonylated enzyme, acetylated Ser channels this to free Cys-SH, while malonylated Ser exchanges groups to acetyl. In Cys-acetylated enzyme, Ser-bound malonyl group is transferred to ACP-SH, while Ser-(9-acetyl exchanges the acyl group to malonyl before acetyl transfer to ACP-SH occurs. It seems that a combination of rapid exchange of the acyl groups at Ser-OH, and limited or preferential acylation abilities of Ser-0-acetyl and Ser-Omalonyl toward the two SH groups, are essential for channeling of the two SH groups onto the proper thiols. This scheme has recently been confirmed by an experiment in which free CoA generated spontaneously in the enzyme reaction was taken up [84]. If rapid exchange of the acyl groups at Ser-OH of the transacylase structure is essential for proper channeling of the two acyl groups, deacylated free CoA should accept the undesired acyl group attached at the Ser residue. When ATPxitrate-lyase, a CoA-scavenging enzyme, was coupled with 6-hydroxymellein synthase reaction processes, the activity of the synthase was appreciably inhibited. However, synthase activity was restored to the control level upon adding free CoA or pantetheine, indicating that free CoA acts as an acyl acceptor in substrate entry to the reaction center of the enzyme. Physiological significance of 6-hydroxymelIein-(?-methyl-transferase 6-Hydroxymellein is the direct precursor of 6-methoxymellein, and Omethylation catalyzed by 6-hydroxymellein-O-methyltransferase, with 5adenosyl-Z-methionine (SAM) as the methyl donor, generates 6methoxymellein [Fig. (4)]. Also, the rate of accumulation of 6methoxymellein in elicitor-treated carrot root tissues was close to that of the 6-hydroxymellein-(9-methyltransferase catalyzing reaction in the cell homogenates [85]. It follows that 0-methyltransfer is the rate-limiting step, controlling the overall rate of biosynthesis of 6-methoxymellein. It has also been found [86] that 6-methoxymellein exhibits a considerable toxic effect on its host plant carrot cells, as well as toxicity to invading microorganisms [3]; however, the cytotoxicity of 6-hydroxymellein is

507

low compared to 6-methoxymellein [87]. These observations suggest that the methyltransfer step in 6-methoxymellein biosynthesis is a physiologically important process, since it converts a less toxic precursor to a highly toxic final product in a rate-determining step. It is therefore likely that direct mechanism(s) are involved in the regulation of 6hydroxymellein-0-methyltransferase activity in 6-methoxymellein biosynthesis, controlling the cellular concentration of this compound to avoid harmful effects caused by overproduction. Regulation of 6-hydroxymelIein-(7-methyltransferase Since preliminary studies showed that 6-hydroxymellein-O-methyltransferase activity was appreciably inhibited in the presence of the reaction products, the mode of product inhibition of the enzyme was studied in detail in order to understand the regulatory mechanism of in vivo methyltransfer. It is well known that iS-adenosyl-L-homocysteine (SAH), which is a common product of many 0-methyltransferases that use SAM as methyl donor, is usually a potent inhibitor of such enzymes. In the 6-hydroxymellein-O-methyltransferase catalyzing reaction another product of this enzyme, 6-methoxymellein, has pronounced inhibitory activity, in addition to SAH. Since the 'specific' product of the transferase reaction, 6-methoxymellein, is capable of inhibiting transferase activity [88], this observation suggests that activity of the transferase is specifically regulated in response to increases in cellular concentrations of its reaction products in carrot cells. It has been also found that 6methoxymellein inhibits transferase activity with respect not only to 6hydroxymellein but also to SAM, competitively. This competitive inhibition was also found in SAH as a function of the co-substrates of the enzyme [89]. It follows that the reaction catalyzed by 6-hydroxymellein0-methyltransferase proceeds by a sequential bireactant mechanism in which the entry of the co-substrates to form the enzyme-substrate complexes and the release of the co-products to generate free enzyme take place in random order [Fig. (7)]. This result also implies that 6methoxymellein and SAH have to associate with the free transferase protein to exhibit their inhibitory activities, and cannot work as the inhibitors after the enzyme forms complexes with the the substrate. If, therefore, 6-hydroxymellein-O-methyltransferase activity is controlled in vivo by its specific product 6-methoxymellein, this compound should

508

block the entry of both co-substrates, 6-hydroxymellein and SAM, prior to formation of the primary enzyme-substrate complexes, enzyme-6hydroxymellein and enzyme-SAM. The Ki values of 6-methoxymellein are close to those of the co-substrates SAM and 6-hydroxymellein (47 and 37 |aM), suggesting that 6-methoxymellein appreciably prevents binding of both co-substrates to 6-hydroxymellein-O-methyltransferase protein, and inhibits the formation of the enzyme-substrate complexes. S AH also exhibited comparable Ki values to these substrates (27 |aM for SAM and 26 |LiM for 6-hydroxymellein), and it is likely that, as with 6methoxymellein, this compound has a comparable capacity to inhibit entry of the two co-substrates. These observations suggest that both of the products of 6-hydroxymellein-O-methyltransferase are able to inhibit Entry of substrates 6-HydroxyinelIein SAM

Release of products 6-Methoxymellein SAH

E-S complexes

SAM

6-Hydroxymellein

t

SAH

t

6-Methoxyiiiellein

Fig. (7). Mechanisms of substrate entry and product release of 6-hydroxymellein-Omethyltransferase

the formation of enzyme-substrate complexes within the range of physiological concentrations found in vivo [89]. The results support the idea that 6-hydroxymellein-O-methyltransferase activity is strictly controlled by its own product, 6-methoxymellein, as well as SAH, so as to maintain the cytoplasmic concentration of 6-methoxymellein at appropriate levels that avoid damage to the host carrot cells themselves that might otherwise take place by overproduction of this toxic secondary metabolite.

509

CONCLUSION The last 20 years have seen a number of observations on the chemical properties of elicitors, and on biochemical changes in plant cells following the addition of elicitors. Recent studies indicate that elicitors stimulate the synthesis of various secondary metabolites other than phytoalexin in plant cell cultures [90-92]. The production of anthraquinones in cultured rhubarb tissues is stimulated by treatment of the cells with the ethylene-generating reagent 2-chloroethylphosphonic acid, or with Ca^^-ionophore A23187 [93]. Use of elicitors therefore offers a new strategy for activation of cellular synthesis of secondary metabolites and high yields of desired products. Many important problems are yet to be solved, however. For example, some of the elicitor-induced secondary metabolites are expected to share precursors with other enzymes of constitutive metabolism, as common substrates. Mechanisms regulating the balance between constitutive and induced metabolism in elicitor-treated plant cells should be studied. Although considerable further work is necessary to clarify many aspects of the action of elicitors, such studies should also shed light on the relation between the receptor and the intracellular signal transduction system, and on the molecular mechanisms regulating gene expression in plant cells. Study of the triggering of phytoalexin synthesis promises new insights into the regulatory mechanisms of plant metabolism. ABBREVIATIONS CAM = Calmodulin IP3 = Inositol trisphosphate cAMP = Cyclic AMP PDE = Phosphodiesterase CAM-PDE = Calmodulin-dependent phosphodiesterase BticAMP = Dibutyryl cyclic AMP ACP = Acyl carrier protein SAM = S'-Adenosyl-Z-methionine SAH = S-Adenosyl-i-homocysteine

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol 25 © 2001 Elsevier Science B.V. All rights reserved.

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INHIBITION OF EUKARYOTE SIGNAL TRANSDUCTION COMPONENTS BY PLANT DEFENSIVE SECONDARY METABOLITES GIDEON M. POLYA Department of Biochemistry, La Trobe University, Bundoora, Melbourne, Victoria 3083, Australia ABSTRACT: Plants are sessile and in addition to physical barriers elaborate a variety of chemical defenses against mobile organisms that consume them such as bacteria, fungi and animals. Such chemical defences include bioactive proteins and secondary metabolites that can be either constitutive or inducible as a result of wounding by herbivores or invasion by pathogens. It is important to establish biochemical sites of action of such compounds in order to define mechanisms of action, to develop better screening systems for detection of bioactive natural products and for design of targetspecific synthetic analogues of potential pharmaceutical utility. Significant differences exist between the signal transduction mechanisms of plants and plant-consuming eukaryotes. Thus plants do not have a neurotransmission system and a variety of plant bioactive metabolites (notably alkaloids) are antagonists or agonists of specific animal neurotransmitter receptors. A generality that has emerged in the last decade is that representatives of many major classes of plant defensive metabolites are inhibitors of signal-regulated protein kinases operating downstream from hormone or neurotransmitter receptors in animal and fungal signalling pathways. In particular, while 3',5'-cyclic AMP (cAMP)-dependent protein kinase (PKA) is apparently absent from plants, it is present in all other eukaryotes and cAMP acts as a "hunger signal" in all non-plant organisms that consume plants. Other signal transduction targets for plant defensive compounds include adenylyl cyclase, guanylyl cyclase, ion gradient generating ATPases, ligand- or voltagegated ion channels, neurotransmitter converting or transporting proteins, receptor tyrosine kinases and cyclic nucleotide phosphodiesterases.

INTRODUCTION METABOLITES

-

PLANT

DEFENSIVE

SECONDARY

There is a huge variety of plant defensive secondary metabolites that has been the subject of major phytochemical [1-6] or pharmacological and toxicological [7-12] compilations. This structural complexity is very briefly reviewed below before considering those plant bioactives with signal transduction targets. The major groups are the phenolics, the terpenoids and the alkaloids as well as bioactives structurally related to

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major groups of metabolites such as carbohydrates, amino acids and fatty acids. Since a very large number of plant defensive secondary metabolites are considered in this broad-ranging review, the reader is referred to some very comprehensive works for the structures of most of these compounds [1,5,6]. The phenolics include anthocyanins, anthraquinones, benzofurans, chromones, chromenes, coumarins, flavonoids, isoflavonoids, lignans, phenolic acids, phenylpropanoids, quinones, stilbenes and xanthones. Some phenolics can be very complex in structure through additional substitution or polymerization of simpler entities. Thus xanthones can be prenylated and flavonoids, lignans and other phenolics can be glycosylated. Condensed tannins involve the polymerization of procyaninidin or prodelphinidin monomers and hydrolysable tannins involve gallic acid residues esterified with monosaccharides. As detailed in this review, representatives of some major classes of plant-derived phenolics are potent protein kinase inhibitors. Terpenoids are structurally based on the isoprenoid (C5) unit and include monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids, steroids and carotenoids. These compounds can be further modified to generate greater structural complexity. Thus the saponins are surface active amphiphiles deriving from the glycosylation of steroid (C27) or triterpenoid (C30) entities. Plant triterpenoids with very specific biochemical effects include those that mimic the effects of mammalian steroid hormones or of insect developmental hormones. Alkaloids are basic plant natural products that typically have a nitrogen atom as part of a heterocyclic ring system and indeed are classified on this basis. Thus major classes of alkaloids are based on indole, isoquinoline, pyrrolidine, piperidine, pyrrolizidine, quinoline, tropane, quinolizidine or other heterocyclic ring systems. Other alkaloids are basic monoterpenoid, sesquiterpenoid, diterpenoid, steroid, purine, pyrimidine or peptide entities. Some of these compounds are exceptionally toxic [1, 6,7-12]. Finally, there are a variety of plant secondary metabolites that are fatty acid, amino acid, organic acid and carbohydrate derivatives. The bioactive carbohydrates include cyanogenic glycosides (that generate highly toxic cytochrome oxidase-inhibiting cyanide), glucosinolates (that variously generate chemically reactive isothiocyanate, thiocyanate and nitrile entities) and sweet-tasting monosaccharides and disaccharides.

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There is clearly a large structural variety of plant defensive secondary metabolites and the structural complexity can be further increased through polymerization, glycosylation, prenylation and esterification. This added complexity can have two major consequences. Firstly, covalent modification may quantitatively and qualitatively alter target interactions, yielding a modified entity with altered affinity for the biochemical targets of the precursor or indeed capable of interacting with new biochemical targets. Secondly, if such covalent modification is reversible (e.g. by deglycosylation of glycosylated natural products) there is the possibility of generation of inactive forms of a defensive secondary metabolite (e.g. through glycosylation) that can be converted to active forms after wounding or invasion of the plant (e.g. active aglycone generation through the action of wounding- or pathogen-induced glycosidases). There may be as many as 100,000 secondary metabolites (SMs) generated in the plant world and of the order of 100,000 gene products as potential targets in a higher organism. There is accordingly a potential interaction matrix for any one higher organism of 10^^ elements. The problem of identification of the key sites of action of plant SMs is massive. Thus of the order of 10,000 secondary metabolites have been isolated and structurally characterized so far but sites of biochemical interaction have been identified for only about 1,000 of such compounds. It is clearly important to establish such sites in order to define mechanisms of action and to better detect bioactive natural products as potential lead compounds for pharmaceutical development. A key problem is whether a biochemical site of interaction of a secondary metabolite established in vitro is the critical target in relation to plant defense. This problem simplifies in some instances in which interaction with the biochemical target is rapidly lethal for clearly defined reasons. Thus a variety of cyanogenic glycosides are non-toxic in themselves (and, critically, non-toxic to the producer plant) but decompose to generate cyanide after ingestion. Cyanide causes death through inhibition of cytochrome oxidase and hence blockage of aerobic ATP production by oxidative phosphorylation coupled to mitochondrial oxygen-terminated electron transport. Similarly, a variety of glucosinolates and related compounds can generate toxic isothiocyanates, thiocyanates and nitriles [1,2]. Interference with signal transduction pathways can also be rapidly lethal. Thus further examples of rapidly toxic plant SMs for which the key

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targets have been established are those SMs acting as potent neurotransmitter antagonists and which can accordingly lethally interfere with neuromuscular mechanisms regulating critical processes such as respiration by the lungs and blood circulation by the heart. However there are many examples of biochemical targets that have been found for plant SMs that may well contribute to the biological effects of these compounds in eukaryotes but which are not necessarily the only or primary sites of action. Many such interactions involve elements of signal transduction pathways that enable eukaryote organisms to respond to external physical or chemical signals. Since such pathways are intrinsically regulatory, interference with specific components may not necessarily be lethal but merely decrease survivability of the organism by altering cognitive processes or other stimulus-response mechanisms. Obvious exceptions to this are compounds interfering with neurotransmission, and in particular neuromuscular transmission, that have immediate drastic effects of cognitive shutdown or immobilization that will prove to be rapidly lethal [1,2,6]. All higher organisms have fundamentally similar mechanisms of catabolic energy extraction, basic metabolite conversions and of genetic information flow from genes to proteins. Accordingly, while such systems can be targets for plant defense, the plant must ensure that its own metabolic and macromolecular synthetic processes are protected from autotoxicity. While sessile plants have stimulus-response mechanisms, the precise mechanisms involved can differ from those in other eukaryotes. A picture is now emerging that plant defenses can exploit such differences and many plant defensive SMs are directed against the signal transduction components of fungal pathogens and animal herbivores. Before reviewing such specific interactions, an overview of eukaryote signal transduction systems is appropriate. EUKARYOTE SIGNAL TRANSDUCTION PATHWAYS Eukaryote organisms primarily respond to external signals by an initial signal perception by receptors. In general, such receptors can be either cytosolic or located on the plasma membrane [13-15]. The former mechanism applies to thyroid hormones (triiodothyronine and tetraiodothyronine or thyroxine), retinoids (e.g. retinoic acid), the insect developmental hormones such as ecdysone, steroid hormones (such as

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progesterone, testosterone and oestrogen), the glucocorticoids Cortisol and cortisone and the mineralocorticoid aldosterone. Signalling via such cytosolic receptors typically involves the following type of pathway: hormone -> hormone-cytosolic receptor complex -> dissociation of an inhibitory protein complex from a DNA-binding domain of the receptor —> receptor conformational change -^ translocation of the hormonereceptor complex to the nucleus and binding to specific promoter elements -> specific activation of transcription by a transcription activation domain -> specific gene expression —> cellular biochemical and ultimately developmental and physiological responses [13-16]. Other hormones (Hs) and neurotransmitters (NTs) act via plasma membrane (PM) located receptors (Rs) and such signalling molecules include proteins, small peptides, prostaglandins, other prostaglandinrelated eicosanoids and biogenic amines including neurotransmitter catecholamines (dopamine, epinephrine and norepinephrine), glutamate, glycine, serotonin, histamine, acetylcholine (AcCh) and y-aminobutyric acid (GABA). The action of such molecules involves binding to a specific cell surface receptor and a consequent conformational change of the receptor. Depending upon the type of receptor, receptor conformational change results in changes in membrane permeability to particular ions (and consequent changes in transmembrane potential) or activation of particular enzymes. Either of these general mechanisms results in transient changes in the cytosolic concentration of so-called "second messengers" that can in turn activate specific "second messenger"-regulated enzymes, most notably protein kinases that catalyze the reversible phosphorylation and functional modification of target proteins. Such "second messengers" include Ca^"^, 3',5'-cyclic adenosine monophosphate (cAMP), 3',5'-cyclic guanosine monophosphate (cGMP), cyclic adenosine-5'diphosphateribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), inositol-1,4,5-triphosphate (IP3), inositol-1,3,4,5tetraphosphate (IP4), diacylglycerol (DAG), phosphatidylinositol-3,4,5triphosphate (PI345P3) and phosphatidylinositol-3,4,-bisphosphate (PI34P2) [13-15]. The levels of these "second messengers" rise transiently in response to the primary signalling molecules (Hs or NTs) binding to their specific receptors. Thus Ca^"^ is pumped out of the cell by plasma membrane Ca^"*"ATPases or by Na"^/Ca^"^ antiporters which utilize the Na^ gradient

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generated by the Na"*", K'*"-ATPase. Ca^"*" is also sequestered in the endoplasmic reticulum via Ca^'^'-ATPases. The other "second messengers" are degraded by specific hydrolytic enzymes. Thus cAMP and cGMP are hydrolyzed to 5'-AMP and 5'-GMP, respectively, by cyclic nucleotide phosphodiesterases (PDEs). When "second messengers" are elevated, a variety of "second messenger"-dependent enzymes are switched on. Thus cAMP activates cAMP-dependent protein kinase (PKA) and also opens cAMP-gated Na"^ channels (thereby causing a transmembrane potential depolarization and Ca^^ elevation through the activation of voltage-gated Ca^"*" channels). Cyclic GMP activates cGMP-dependent protein kinase (PKG) and also opens cGMP-gated Na"^ channels. Ca^^ can activate Ca^"^dependent enzymes, notably particular protein kinases such as those of the PKC family and a variety of protein kinases that are activated by the Ca^"*"binding regulator protein calmodulin (CaM). DAG can activate PKC isozymes and IP3 opens IPs-gated Ca^"*" channels in the endoplasmic reticulum (ER), causing an increase in cytosolic Ca^"^ with the consequences outlined above. IP4 can also promote Ca^"*" elevation in the cytosol. Cyclic ADPR opens the ER ryanodine receptor Ca^"^ channel in a Ca^^-CaM-dependent process with resultant elevation of cytosolic Ca^^. NAADP and particular sphingolipids similarly open other specific ER Ca^"" channels [17]. PI345P3 and PI34P2 activate phosphatidylinositolphosphate-activated protein kinases (PDKl and a hypothesized PDK2) and bind to the insulin-responsive protein kinase PKB which is phosphorylated at two sites by the phosphatidylinositolphosphate-activated protein kinases. As outlined above, protein phosphorylation is a key process involved in many signal transduction pathways and reversal of this process is catalyzed by a multiplicity of phosphoprotein phosphatases (PPs). Major PPs catalyzing dephosphorylation of phosphoserine or phosphothreonine residues on proteins include PPl (inhibited by phosphorylated inhibitor protein I-l and by okadaic acid and microsystins), PP2 (also inhibited by okadaic acid and microcystins), PP2B or calcineurin (CaM-activated and having a CaM-like regulatory subunit) and PP2C (Mg^"^-dependent) [18]. These PPs have been found in all eukaryotes so far examined [18, 19]. In addition, a variety of protein phosphotyrosine phosphatases can reverse the consequences of RTK or JAK/STAT receptor activation [20]. The overall hormone (H) or neurotransmitter (NT) signalling involved here can be succinctly summarized as follows: H -> H-R —> membrane

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potential change (via the opening of an ion-specific channel in the receptor in response to voltage changes or to H/NT binding) or intracellular enzyme activation (e.g. through interactions involving hormone-activated receptor tyrosine kinases (RTKs) and related systems or hormone-receptor complex-activated G protein Ga subunit-GTP complexes) ~> second messenger elevation -> activation of second messenger-dependent enzymes (e.g. protein kinases) -^ reversible protein modification (e.g. protein phosphorylation) -^ protein functional alteration (e.g. change in catalytic activity of a key metabolic enzyme or change in activity of a transcription factor) —> change in cellular processes (e.g. change in metabolism or specific gene expression) -^ reversal (e.g. through dephosphorylation of proteins by phosphoprotein phosphatases (PPs) and second messenger removal by transport or hydrolysis) —> return to the unexcited state [13-15]. Before considering specific signalling pathways in greater detail, it is useful to consider some key components involved in the initial hormone reception and signal transduction processes at the level of the plasma membrane. Plasma membrane hormone receptors are specific for particular hormones or neurotransmitters, there may be a multiplicity of receptors for any signalling molecule and, as detailed below, there may be different signal transduction mechanisms involved after the primary binding of the signalling molecule to the receptor. Thus plasma membrane-located signal receptors include ligand-activated receptor tyrosine kinases, protein phosphotyrosine phosphatases, guanylyl cyclases, ion channels, receptor serine/threonine protein kinases, JAK/STAT-linked receptors and G protein-linked receptors. To this list we can add voltageregulated ion channels and the light receptor rhodopsin (a G proteinlinked receptor-like protein) [13-15]. A number of distinct signalling pathways have been elucidated that involve plasma membrane (PM) located receptors (Rs) and these are briefly outlined below. a. Hormone (H)-activated receptor tyrosine kinases (RTKs) include receptors for growth regulatory hormones such as insulin, insulin-like growth factor, epidermal growth factor and platelet-derived growth factor. The pathways involved can be summarized as follows: H —> H-R —> H-Rs interact and the tyrosine kinase (TK) on the cytoplasmic domain of the RTK is activated —> autophosphorylation of RTKs on tyrosine residues in

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cytoplasmic domains --> effector proteins activated by tyrosine phosphorylation and/or interaction with phosphotyrosine residues via SH2 (type 2 Src homology) domains —> downstream consequences [21-26]. b. Receptors that activate dimeric protein tyrosine kinases (JAKs) which in turn phosphorylate STATs (signal transducers and activators of transcription), represent variants of the RTK mode that initiate the JAK/STAT pathway by binding cytokines such as interleukins and interferons. This pathway can be summarized thus: H —> binds to a dimeric R (a and p subunits) -> the tyrosine kinases JAKl and JAK2 bind and are activated and reciprocally tyrosine phosphorylated -> the receptor (3 subunit is tyrosine phosphorylated ~> STAT-a and STAT-b bind to R "-> both are tyrosine-phosphorylated by JAKs -^ tyrosinephosphorylated, activated STAT-a/STAT-b dimers translocate into the nucleus —> activated dimer binds to specific promoter elements —> specific gene expression [27, 28]. c. Ligand-activated protein tyrosine phosphatases (PTPases) couple ligand binding to phosphotyrosine protein dephosphorylation thus: ligand —> ligand-PTP -> ligand-PTP activated --> dephosphorylation of tyrosinephosphorylated-proteins [20]. d. Hormone-activated guanylate cyclase (GC) is activated by atrial natriuretic factor (ANF) to generate cGMP and thence promote vascular dilation and improved cardiac function. Thus, heart stress —> ANF -> ANF-GC -> ANF-GC activated -> increased cGMP -> increased natriuresis, diuresis, vascular dilation and cardiac function [29]. e. Hormone- or neurotransmitter-gated ion channels include such receptors for acetylcholine (AcCh) (nicotinic receptors), glutamate (Nmethyl-D-aspartate (NMDA) and non-NMDA receptors), glycine, serotonin and y-amino butyric acid (GABA). However it should be noted that some of these signalling molecules (e.g. AcCh, glutamate, serotonin and GABA) can also act through G-linked receptors). The signalling sequence involves the following: ligand (L) -> L-R ~> change in conformation of the L-R complex —> ion channel (for Na"*", K"*", CI" or Ca^^) opens -^ change in transmembrane potential (A\|/m) -> downstream

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effects (notably changes in cell excitability or cytosolic free Ca^"^ levels) [13-15]. f. Voltage-gated (V-gated) ion channels are protein pores specific for particular ions (e.g. Na"*" , K^ and Ca^^ channels) which open or close in response to changes in transmembrane potential (v|/m). Thus signallinginduced A\|/m —> V-gated channels open or close -4 change in permeability (AP) for specific ions —> A\|/m -> downstream effects as outlined in (e) above [13-15, 30]. g. Hormone-activated serine/threonine-specific receptor protein kinases (S/T-RPKs) include receptors for hormones of the transforming growth factor p (TGFp) family (that suppress cell proliferation), developmentally important activins (involved in mesoderm induction) and bone morphogenetic proteins (involved in bone formation). The pathway involved is as follows: H -> H-R -^ S/T-RPK activated -^ target protein serine/threonine phosphorylation -> downstream consequences (e.g. specific gene expression as a result of specific transcription factor phosphorylation and activation) [31]. h. Hormone receptors linked to heterotrimeric G proteins mediate signalling for ATP, adenosine, many peptide hormones, catecholamines (such as dopamine, epinephrine and norepinephrine), eicosanoids and histamine. In addition, glutamate, serotonin, GABA and acetylcholine (depolarizing and hence excitatory hormones/neurotransmitters that can act via ion channel receptors) can also act via G protein-linked receptors. The G proteins are heterotrimers composed of a, P and y subunits. Interaction with H-R complexes results in dissociation of a Ga subunit (as a complex with GTP) from a Gp-Gy complex. The Ga-GTP complex can interact with various effector proteins which are then activated and produce a variety of downstream effects. Reversibility of the system is achieved through the GTPase hydrolytic activity of the Ga subunit which generates Ga-GDP which then recombines to form the inactive Ga-GDPGp-Gy complex. Specificity in such pathways is achieved through specific hormone receptors and specific types of Ga subunits. This sequence can be summarized as follows: H ^ H-R -> H-R-Ga-GDP-Gp-Gy -^ H-R +

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Ga-GTP + Cp-Gy -> Ga-GTP activates effector proteins depending on what specific Ga subunit is involved -^ downstream effects [32-35]. A variety of Ga subunits interact with specific effector proteins and can be classified into various families, namely (for simplicity), Gas, Gaolf, Gai, Gao, Gaq and Gat. Gas-GTP (and Gaolf-GTP involved in olfaction) activate adenylate cyclase (AC) (thereby increasing cAMP levels and activating PKA and cAMP-gated Na"*" channels) and can also open Ca^"*" channels. Vibrio cholerae (cholera) toxin ADP ribosylates and inhibits the Gas and Gaolf GTPase, thus causing persistent activation of adenylate cyclase. In contrast, Gai-GTP inhibits adenylate cyclase, closes Ca^"^ channels and opens K^ channels. Gai is ADP-ribosylated and thus functionally inhibited by a Bordetella pertussis (hooping cough) toxin. Gao-GTP and Gaq-GTP are also variously ADP ribosylated by pertussis toxin and can activate phospholipase C (PLC) which catalyzes the formation from phosphatidylinositol-4,5-bisphosphate (PI45P2) of diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). IP3 in turn increases cytosolic Ca^"*" by opening IPs-gated Ca^"^ channels in the endoplasmic reticulum (ER). Gat-GTP (transducin, the effector of lightactivated rhodopsin), activates cGMP phosphodiesterase, lowering cGMP levels in retinal rod cells and thereby closing cGMP-gated Na"^ channels as a critical step in visual perception. Gat is ADP ribosylated by both the cholera and pertussis toxins [32, 34]. EUKARYOTE SIGNAL-REGULATED PROTEIN KINASES All of the PM receptor-mediated signal transduction mechanisms described above ultimately act through the phosphorylationdephosphorylation of proteins through the action of signal-regulated protein kinases (PKs) [36-43] and phosphoprotein phosphatases (PPs) [18] as summarized in Fig. (1). Such reversible covalent modification is involved in metabolic regulation and in developmental regulation through control of cell division and specific gene expression. Indeed protein phosphorylation-dephosphorylation itself is regulated by the phosphorylation-dephosphorylation of protein kinases, PP inhibitors and PK- or PP-targeting proteins. Such phosphorylation-dephosphorylation processes can be the targets of particular plant-derived secondary metabolites. It has been estimated

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that there may be as many as 1000 PKs and hundreds of PPs encoded by a typical higher organism genome. Many signal-regulated PKs have been described and some of the better studied systems are briefly outlined below. Fig. (1) summarizes the signalling pathways leading to the activation of some of these major types of signal-regulated PKs. OUT

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Fig. (1). Signal transduction in eukaryotes involving plasma membrane-located receptors. PM, plasma membrane; OUT, outside the cell; IN, inside the cell; Ll-Lll, ligands 1-11; AC, adenylyl cyclase, cAMP, 3',5'-cyclic AMP; CaM, calmodulin; CaM PK, Ca^'^-CaM-dependent protein kinase; CDPK, Ca^'^-dependent protein kinase; cGMP, 3',5'-cyclic GMP; G, G protein; GC, guanylyl cyclase; IP3, inositol-1,4,5-triphosphate; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MLCK, myosin light chain kinase; P, permeability of PM to specific ions; PDK, phosphatidylinositolphosphate-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; PIP2, phosphatidylinositol-3,4-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PKA, cAMP-dependent protein kinase; PKB, insulin signalling-activated protein kinase; PKC, Ca^"^- and phospholipid-dependent protein kinase; PKG, cGMP-dependent protein kinase; PLC, phospholipase C; PLCy, phospholipase C-y; P-Pr, phosphoprotein; P-STAT, tyrosine phosphorylated STAT dimer (signal transducers and activators of transcription); PTPase, protein phosphotyrosine phosphatase; R, receptor; Ras, small G protein; Raf, MAPK kinase kinase; RSTK, receptor serine/threonine protein kinase; RTK, receptor tyrosine kinase; RPTP, receptor protein phosphotyrosine phosphatase; AP, change in permeability of PM to specific ions; A^', change in transmembrane potential.

a. Cyclic AMP-dependent protein kinase (PKA) is inactive as the holoenzyme (R2C2) which is activated by the binding of cAMP to the regulatory ( R ) subunits releasing the active catalytic ( C ) subunits: R2C2 (inactive) + 4 cAMP -> (R-cAMP2)2 + 2 C (active). PKA phosphorylates and activates phosphorylase b kinase, PP inhibitor-1 protein, site 1 on the

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glycogen targeting protein (thereby activating PPl) and triglyceride lipase. PKA phosphorylates and functionally inhibits CaM-dependent myosin light chain kinase (MLCK), glycogen synthase, site 2 on the glycogen targeting protein (thereby inhibiting PPl) and liver pyruvate kinase. The three dimensional structure of the PKA catalytic subunit has been determined and is very similar to the structure of the catalytic domains of a variety of other protein kinases [36]. Cyclic AMP levels are elevated by hormone receptors acting via Gas-GTP and are decreased by cAMP PDEs. A PKA inhibitor protein (the Walsh-Krebs inhibitor protein) inhibits free catalytic subunit, C. While cAMP is a "hunger" signal in eukaryotes and prokaryotes (signalling catabolite deficiency), fructose2,6-bisphosphate is a "plenty" signal in eukaryotes. Thus in animals cAMP promotes gluconeogenesis and inhibits glycolysis in the liver. PKA phosphorylates the liver dual fructose-6-phosphate-2-kinase / fructose-2, 6-bisphosphatase to inhibit the kinase and activate the phosphatase activity, thereby decreasing fructose-2,6-bisphosphate levels (a converse effect obtains with the muscle enzyme consonant with liver being a "glucose provider" and muscle a "glucose user") [43]. Finally, PKA phosphorylates CREB (cAMP response element binding) protein transcription factors that induce the synthesis of, for example, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase [44, 45]. Other targets of cAMP in eukaryotes are cAMP-gated Na^ channels (e.g. those involved in odor perception via Gaolf-GTP activation of adenylate cyclase, cell depolarization through the opening of cAMP-gated Na"^ channels and signal transmission to the central nervous system) [46], noting that Ca^^ and cGMP are also involved as second messengers in odor perception [47]. The Dictyostelium discoideum (slime mould) cAMP receptor is a 7-transmembrane a-helix-type receptor located on the PM and is involved in amoeboid form aggregation and differentiation to a spore-generating fruiting body) [48,49]. b. Cyclic GMP-dependent protein kinase (PKG) is a homodimer that is homologous to PKA and is activated by cGMP thus: E2 + 4 cGMP <—> (E-CGMP2 )2 (active) [50, 51]. Cyclic GMP is generated by ANF-activated membrane-bound guanylate cyclase (GC) [29] or by soluble NO-activated GC, NO being generated by NO synthase that can be Ca^'^-CaM-activated [52, 53]. Cyclic GMP is hydrolyzed to 5'-GMP by cGMP PDEs. Activation of PKG leads to phosphorylation of specific proteins and, for

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example, to vascular dilation. However cGMP can also act via cGMPgated Na^ channels (e.g. this being critical in vision) [13-15]. c. The Ca^^- and phospholipid-dependent protein kinase (PKC) family consists of a variety of PKC isozymes that are variously activated by Ca^^, DAG and phospholipids [38, 50]. Cytosolic Ca^"^ is increased by hormones acting via RTKs (and subsequent PLC-y activation) or via Gao-GTP and Gaq-GTP (and subsequent PLC activation), both types of pathways yielding DAG and IP3, the latter mobilizing Ca^"*" from the ER. Cytosolic Ca^"^ is also increased by transmembrane potential depolarizing neurotransmitters and opening of voltage-gated Ca^^ channels, the latter events being directly communicated to the ER ryanodine receptors (RyRs) in skeletal muscle (or indirectly via cADPR synthase and Ca^'*"-CaMactivated cADPR interaction with the ryanodine receptor in other muscle cells). Ca^"*" is removed from the cytosol across the plasma membrane by the NaVCa^"*" antiporter (driven by a Na^ gradient generated by the Na"^, K^-ATPase) and into the ER and out of the cell by Ca^^-ATPases. PKC isozymes are variously involved in growth control and transcriptional activation by phosphorylating specific transcription factors that interact with TREs (tetradecanoylphorbolacetate (TPA) response elements) (TPA and other phorbol esters being plant-derived or semi-synthetic phorbolbased PKC activators). PKC also regulates growth via phosphorylation of key components such as histone HI, the epidermal growth factor receptor (a RTK) and phosphorylation and activation of Raf (a MAPKKK) [38, 42] (see (f) below). d. Ca^^-calmodulin (CaM)-dependent protein kinases are activated by the Ca^"*'-dependent regulator protein calmodulin (CaM) [55] and include CaM-dependent PKs I-IV [55] and myosin light chain kinase (MLCK) [56]. CaMKn is involved in specific gene expression, neuronal transmission and memory consolidation. MLCK has a key regulatory role in muscle contraction and in smooth muscle phosphorylates myosin light chains, thus permitting actin-myosin interaction and muscle contraction [13-15]. e. 5'-AMP-dependent protein kinase (AMPK) is a heterotrimer that is activated by 5'-AMP and requires activating phosphorylation by 5'-AMPactivated AMP kinase kinase (AMPKK). AMPK couples nutrient stress-

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or exercise-induced elevation of 5'-AMP to requisite metabolic and other responses. Activated phospho-AMPK phosphorylates and thus inhibits various proteins with the following effects: acetyl CoA carboxylase (decreasing malonylcoenzyme A and thereby inhibiting fatty acid synthesis and stimulating fatty acyl carnitine synthetase and fatty acid oxidation), creatine kinase (inhibiting the enzyme) and hydroxymethylglutarylcoenzyme A synthetase (thereby inhibiting cholesterol synthesis). AMPK phosphorylates and activates epithelial NO synthase (elevated NO activating soluble guanylate cyclase leading to cGMP-induced vascular dilation) and AMPK is also evidently involved in promoting glucose transport through mobilizing glucose transporters and in inhibiting apoptosis and transcription of particular genes [57]. f. PKB is a key protein kinase that is activated as result of insulin binding to the insulin receptor (a RTK) as summarized in the following scheme: insulin-insulin RTK -^ RTKs dimerise and are activated —> autophosphorylation on cytoplasmic tyrosines —> insulin R substrates IRS1 and IRS-2 are phosphorylated on tyrosine residues ~> phosphorylated RTKs and IRSs interact with effector proteins via phosphotyrosinebinding SH2 domains and, in particular, wortmannin-inhibited phosphatidylinositol-3-kinase (PI3K) is activated -^ PI45P2 to PI345P3 -^ PI34P2 (via PI345P3 5'-phosphohydrolase) -> the second messengers PI345P3 and PI34P2 activate PDKl (and a supposed second protein kinase, PDK2) by binding to pleckstrin homology (PH) domains on these protein kinases —> N-terminal region PH domains on PKB also bind PI34P2 and PI345P3 —> PKB is threonine- and serine-phosphorylated by PDKl (and PDK2) -^ PICB activated. Activated PKB phosphorylates various proteins including phospho-Bad (resulting in phosphoBad sequestration by 14.3.3 proteins and inhibition of apoptosis), p70S6K (causing ribsomal S6 protein phosphorylation and translational activation) and glycogen synthase kinase 3 (GSK3) (the inactivated phosphoGSK3 being unable to phosphorylate glycogen synthase, the active dephosphoglycogen synthase catalysing glycogen synthesis). PKB also contributes to GLUT4 activation (resulting in its translocation to the PM and glucose transport), activation by phosphorylation of the glycogen targeting subunit of glycogen-bound PPl (site 1 phosphorylation causing PPl activation and dephosphorylation and activation of phospho-glycogen

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synthase) -> glycogen synthase active -^ glycogen synthesis) and phosphorylation of eIF4E binding protein (thereby abolishing inhibition of eIF4E-dependent mRNA translation) [39, 58]. g. Mitogen-activated protein kinases (MAPKs) (or ERKs, extracellular signal regulated kinases) are activated by signalling cascades initiated by various signals including hormones such as insulin binding to specific RTKs. Subsequently a variety of effector proteins bind to the activated and tyrosine-phosphorylated RTKs or to each other by phosphotyrosine-binding SH2 domains or by SH3 domains that recognize proline-rich protein regions. This type of signalling pathway can be illustrated by the following example: insulin binds to its RTK receptor —> RTK aggregation, activation and autophosphorylation —> IRS-1 and IRS-2 tyrosine phosphorylation -> SHC (an adaptor protein) binds -> GRB2 (a further adaptor protein) binds -^ Sos (guanyl nucleotide exchange factor = GEF) binds and is activated —> Ras activation to yield active Ras-GTP ~> activation (by Ras-GTP) and phosphorylation (by PKC) of Raf (a MARK kinase kinase or MAPKKK) -> serine phosphorylation of MAPK kinase (yielding activated phosphoMAPKK) --> threonine and tyrosine phosphorylation of MAPK (involving a threonine-glutamate-tyrosine sequence phosphorylation) -> transcription factor phosphorylation -> specific gene expression) [59]. h. Cell division protein kinases (CDKs) of eukaryotes critically regulate passage of cells through the cell cycle that involves successive Gl, S (DNA synthesis), G2 and M (mitosis) stages. Cycle stage-specific CDKs are activated by specific threonine and tyrosine dephosphorylation, a particular threonine phosphorylation and by interaction with stage-specific cyclin proteins. The overall process is regulated exquisitely by hormoneinduced signalling, the synthesis and degradation of specific cyclins, the activation or de-activation of specific serine/threonine or tyrosine-specific protein kinases and PPs and by other regulatory components [60]. i. Histidine kinases (present in yeast, slime moulds and plants) autophosphorylate on histidine and are involved in subsequent aspartate phosphorylation on target proteins and protein kinase cascade regulation [42, 61].

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Of course plants also have signal transduction pathways but some major differences are apparent. Thus while possible elements of a cyclic nucleotide regulatory system have been found in plants including cAMP, cGMP, adenylyl cyclase, guanylyl cyclase, cyclic nucleotide-hydrolyzing phosphodiesterases and a number of cyclic nucleotide-binding proteins, functional equivalents of PKA or PKG have not been found in plants [6265]. Nevertheless there is evidence for the involvement of cGMP in stomatal opening [66] and in plant responses mediated by phytochrome proteins [64]. Further, proteins homologous to the cyclic nucleotide-gated ion channels of other eukaryotes are present in higher plants and an Arabidopsis gene (GCRl) encodes a putative seven transmembrane element receptor-like protein with similarity to the Dictyostelium cAMP receptor protein [64, 65]. Microinjection experiments have provided evidence for cADPR, NO and cGMP in induction of plant defense responses [67]. Finally, elements of Ca -mediated signalling, namely Ca^"^ pumps and channels, CaM, CaM-dependent protein kinases and Ca^'*'-dependent protein kinases (CaM-domain protein kinases) (CDPKs) have been resolved from higher plants [68, 69]. It is notable that a homologue of plant CDPK is found in the malaria-causing protozoan Plasmodium falciparum but not in other non-plant eukaryotes, suggesting that inhibitors of this CDPK may have therapeutic potential [70]. With this sketch of major eukaryote signalling pathways in mind, we can now consider the interaction of particular plant defensive secondary metabolites with particular signal transduction components of non-plant eukaryotes such as fungi and animals that consume plants. The reader is referred to some major compilations for the structures of most of the plant defensive compounds mentioned [1, 5, 6]. SIGNAL TRANSDUCTION TARGETS FOR PLANT DEFENSIVE SECONDARY METABOLITES I. Ligand-gated ion channel neurotransmitter receptors a. Acetylcholine receptors (nicotinic) bind the piperidine alkaloid nicotine and are excitatory (depolarizing), presynaptic and postsynaptic, acetylcholine-gated NaVK"*" channels involved in neurotransmission, including neuromuscular transmission [71-74]. Major plant-derived

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agonists of this kind of receptor include the quinolizidine alkaloid cytisine and the piperidine alkaloids nicotine, anabasine, nornicotine, (-)-lobeline and (+)-coniine [75, 76]. (+)-Coniine is from hemlock, Conium maculatum, (the agent of Socrates' judicial death) and related piperidine alkaloids from this source include pseudoconhydrine, y-coniceine and (+)N-methylconiine [1]. A variety of isoquinoline alkaloids are nicotinic receptor antagonists and consequently neuromuscular transmission blockers, notably the highly toxic bisbenzylisoquinoline alkaloid (+)tubocurarine, an important component of South American Indian curare. Other curare-like isoquinoline alkaloids include further bisbenzylisoquinoline alkaloids (berbamine, dauricine, isochondrodendrine (isobebeerine), macoline and rodiasine), Erythrina isoquinoline alkaloids (erysinine, erysotrine, erythratidine, a-erythroidine, p-erythroidine and dehydro-p-erythroidine), isococculidine, berberine and magnoflorine [1]. A variety of toxic diterpenoid alkaloids have curare-like properties including avadharidine, condelphine, delcoline, delcorine, elatine, karacoline, lappaconitine, N-desacetyllappaconitine and methyllycaconitine [1, 77]. A variety of toxic indole alkaloids also have curare-like activity including calebassine, caracurine, C-curarine, sapargine and toxiferine I [1]. It is notable that the snake 8 kDa polypeptide toxin a-bungarotoxin is also a nicotinic receptor antagonist that causes neuromuscular blockage and skeletal muscle paralysis [72]. b. lonotropic GABA (Y-aminobutyric acid) receptors (GABA class A and C receptors) are inhibitory (hyperpolarizing) GABA-gated CY channels that have sequence homology with nicotinic acetylcholine and glycine receptors. GABA is the main inhibitory neurotransmitter of the mammalian central nervous system (CNS) and GABA agonists have potential as anxiolytics and anticonvulsants [78-81]. The class A receptors are hetero-oligomeric, are modulated by steroids, barbiturates and benzodiazepines and are blocked by the phthalideisoquinoline alkaloids (+)-bicuculline and N-methylbicuculline and by the alkaloids (-)securinine and (+)-tubocurarine [82, 83]. GABA A receptor natural product agonists include GABA itself, the Amanita mushroom oxazole alkaloids muscimol and dihydromuscimol and the piperidine alkaloid isoguvacine [83]. The homo-oligomeric GABA C receptors are insensitive to bicuculline, steroids, barbiturates and benzodiazepines but are activated by GABA, and muscimol and bind isoguvacine [78, 79, 84].

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c. lonotropic glutamate receptors include N-methyl-D-aspartate (NMDA)-binding glutamate receptors (excitatory (depolarizing) NaVK"*" and Ca^"^ channels whose subunit composition can be regulated by the type of innervating neuron). Non-NMDA binding glutamate receptors are excitatory (depolarizing) receptors that are Na"*"/K^ channels, the opening of which and the consequent partial depolarization permitting NMDA receptors to open in response to glutamate. Activation of GABA A receptors can also facilitate NMDA receptor activation [85-90]. NMDA (as well as non-NMDA receptors) participate in long-term potentiation (LTP) of synaptic transmission that is involved in memory consolidation [90]. Non-NMDA glutamate receptors include those binding a-amino-3hydroxy-5-methyl-4-isoxazoleprionic acid (AMPA) or kainic acid. The neurotoxins kainic acid and domoic acid are agonists of the non-NMDA glutamate receptors. The activity of the non-NMDA glutamate receptors is enhanced by PKA-catalyzed phosphorylation as a result of Dl dopamine receptor-mediated increase in cAMP concentration. Kynurenic acid is a non-selective ionotropic glutamate receptor antagonist and glycine is involved as a co-agonist in activation of NMDA receptors (Dserine also acting as a co-agonist of NMDA receptors). p-N-oxalylaminoL-alanine (L-BOAA) (from Lathyrus sativus), the causal agent of neurolathyrism in humans, has an excitatory effect on AMPA receptors and binds to NMDA receptors [91]. p-N-methylamino-L-alanine (LBMAA) (present in Cycas circinalis and implicated in a type of dementia in Guam) is also an NMDA agonist as well as a non-NMDA receptor agonist, albeit at much higher concentrations [91, 92]. The peptide derivative S-(4-hydroxybenzyl)glutathione from Gastrodia elata binds to kainic acid-binding glutamate receptors [93]. Bioactive polyamines such as spermine are present in animals and plants [1], have antifungal activity and can modulate NMDA receptors [94]. d. lonotropic serotonin (5-hydroxytryptamine) receptors (5HT3 class receptors) are excitatory (depolarizing) Na'**/K"^ channels that are involved in emesis and anxiety [95]. Plant compounds binding to the 5HT3 receptor include serotonin (5-hydroxytryptamine) itself and related bioactive and psychotomimetic indole alkaloids [1]. Ethanol and other alcohols may act via effects on the 5HT3 receptor [96].

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e. Glycine receptors are inhibitory (hyperpolarizing) glycine-gated CI" channels involved in neurotransmission and a well-known antagonist is the highly toxic indole alkaloid strychnine [1]. The benzylisoquinoline alkaloid (-)-laudanidine can produce strychnine-like effects of paralysis [1]. II. G-protein-linked receptors a. Acetylcholine receptors (muscarinic) are G protein-linked receptors (Ml, M2 and M3 variants), the cardiac receptor activating a G protein complex with resultant opening of a K"*" channel by the dissociated G protein py^^^pl^^' hyperpolarization and diminution of contraction [97]. A similar mechanism can be involved with some other G protein-linked receptors including somatostatin, serotonin, o2 adrenergic, dopamine (D2), opioid (|Li and 6) and GAB A (B) receptors [97]. Plant agonists include L(+)-muscarine, pilocarpine, pilosine, norarecoline and arecoline [1, 97]. Muscarinic receptor antagonists include the tropane alkaloids hyoscamine, atropine (the hyoscamine racemate) and hyoscine (scopolamine) [1, 97-99], the benzylisoquinoline liriodenine [100] and the steroidal alkalord ebeinone [101]. There is current interest in muscarinic agonists for treatment of some people with Alzheimer's Disease [102]. b. Adrenergic receptors for epinephrine and norepinephrine are G protein-linked receptors initiating various signalling pathways depending upon whether they are a l (elevating Ca^^ through phospholipase C activation through Gaq), a2 (mostly inhibiting adenylyl cyclase through Gai and depolarizing) and p adrenergic receptors (pl-AR, P2-AR and p3-AR) (elevating cAMP) [103-107]. The indole alkaloid yohimbine and the aporphine isoquinoline alkaloid xylopinine are a receptor blockers [108] and the aporphine isoquinoline alkaloid isocorydine and the bisbenzylisoquinoline alkaloid oxyacanthine are also adrenergic antagonists [1]. The legume-derived bioactive agmatine (l-amino-4guanidinobutane) binds to a2-adrenergic receptors and to the nonadrenergic imidazoline receptors that bind the hypotensive drug clonidine [109]. L-norepinephrine and dopamine are present in some plants and act as agonists for both a- and p-adrenergic receptors [1]. Further plant padrenergic receptor agonists include the phenylpropanoid alkaloids L-

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ephedrine, pseudoephedrine, D-cathinone and D-cathine [110]. The adrenergic receptors have a critical function in cardiac function, blood pressure [111], metabolism [13-15] and immune responsiveness [112]. c. Dopamine receptors are excitatory receptors involved in neurotransmission. Dopamine Dl receptors act via Gs, adenylyl cyclase activation, cAMP and PKA-phosphorylated, voltage-gated K"*" channel closure. D2 receptors act via Gao/Gai proteins causing adenylyl cyclase inhibition and ion channel modulation [113]. Dopamine is elaborated by some plants and the isoquinoline alkaloid (-)-salsolinol is a dopamine antagonist [1]. The Aconitum alkaloid sangorine is a D2 agonist [115]. Dopamine signalling and interacting elements are important targets for treatment of Parkinson's disease and schizophrenia [116]. d. Serotonin receptors (5HT1, 5HT2 and 5HT4 receptors) are excitatory (depolarizing) G protein-linked receptors acting via cAMP and PKA-phosphorylated, voltage-gated K"*" channel closure [117]. Plant agonists include serotonin (5-hydroxytryptamine) itself and various indole alkaloids including bufotenin (5-hydroxy-N,N-dimethyl tryptamine), Omethylbufotenine, gramine, hordenine, ergotamine, N,N-dimethyl tryptamine, mescaline, norharmane, harmine, ergine and harmaline [1, 118-120]. The indole hallucinogens psilocin (from the mushroom Psilocybe mexicana) and the synthetic lysergic acid diethylamide (LSD) also act as serotonin agonists [119]. The xanthone y-m^i^gostin is a serotonin antagonist [121] and the alkaloids annonaine, nornuciferine and asimilobine bind to 5-HTl receptors [122]. Serotonin receptors and signalling are targets for treatment of depression and migraine. e. Metabotropic glutamate receptors (as opposed to the ionotropic glutamate receptors) are 7 transmembrane helix domain receptors that act via G proteins to modulate K"*" and Ca^"^ channels [123]. The mushroom compound ibotenate (the precursor of the hallucinogen muscimol) is an agonist of some metabotropic glutamate receptors [123]. f. GABA receptors (B-class receptors) are 7-TM helix, G protein-linked receptors that are like metabotropic glutamate receptors and activate Ca^"*" and K"*" channels [124]. GABA is present in a wide variety of Leguminosae seeds [1].

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g. Histamine receptors (HI, H2 and H3 receptors) are G protein-linked receptors. Allergic responses such as inflammation and bronchoconstriction involve HI receptors, heart rate increase and gastric secretion involve H2 receptors [125] and vascular H3 receptors are involved in hypertension [126]. Histamine itself occurs in some plants and the quinoline alkaloid vasicinal (7-hydroxypeganine) and the steroidal alkaloid tomatine (lycopericin) have some histamine antagonist activity [1]. h. Purine receptors bind adenosine and adenine nucleotides, with adenosine binding more tightly than ATP to PI (Al and A2) receptors and ATP binding more tightly than adenosine to P2 receptors [127-130]. Adenosine and dopamine receptors can interact. Thus adenosine Al receptors and dopamine D2 receptors act via Gai to inhibit adenylyl cyclase and adenosine A2A and dopamine Dl receptors act via Gas and Gaolf to activate adenylyl cyclase. Adenosine binding to Al and A2A receptors decreases the affinity of dopamine for Dl and D2 receptors, respectively. Thus adenosine binding to A2A receptors inhibits dopamineD2-mediated increase in Ca^"^ and inhibition of adenylyl cyclase. Adenosine binding to Al receptors inhibits dopamine-Dl-induced activation of adenylyl cyclase [130]. Thus adenosine can modulate dopamine responses in the CNS [130] and can have sedative and anticonvulsant effects in addition to having vasodilatory, bronchoconstricting and metabolic effects [127, 131]. The methylxanthines theophylline and caffeine are adenosine Al and A2 receptor antagonists but chronic administration can upregulate adenosine receptors [127]. The G protein-linked P2 receptors act via Gai or Gaq but it should be noted that P2x receptors are ionotropic and affect NaVCa^"^ channel opening [129]. i. Cannabinoid receptors include the CBl receptors (high in the CNS and coupled via G proteins to inhibit adenylyl cyclase, close Ca^^ channels and open K^ channels) and CB2 receptors (present in immune cells and acting via Gai proteins to inhibit adenylyl cyclase). CBl and CB2 receptors bind A^-tetrahydrocannabinol (from marijuana. Cannabis sativa) as well as the endogenous ligand anandamide (arachidonylethanolamide)

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[132]. However A^-tetrahydrocannabinol anatagonizes the peripheral CB2 receptor while acting as an agonist for the CNS CBl receptor [133]. Cannabinoid receptors mediate the appetite stimulant and psychoactive effects of cannabinoids which have therapeutic potential for relief from nausea and pain [132]. j . Opiate receptors mediate analgesic and anti-tussive effects and a number of well-known plant natural product narcotics and analgesics are agonists of such receptors, mimicking the effects of the endogenous peptide endorphin and enkephalin ligands [134-137]. Such plant products include the morphinan isoquinoline alkaloids morphine (the diacetate being heroin), codeine (3-0-methylmorphine), neopine (p-codeine) and thebaine [1, 138]. 0-demethylation of thebaine yields oripavine that can have both agonist and antagonist effects on opioid receptors [139]. Other isoquinoline alkaloids that have anti-tussive (but not analgesic) effects and which bind at receptors distinct from codeine and other opiates are the benzylisoquinoline papaverine, narceine and the phthalide isoquinoline alkaloids a-narcotine and narcotoline [1]. The isoquinolines (-)-salsolinol and tetrahydropapaveroline bind to an opiate receptor with affinities comparable to those of enkephalins and their analgesic effects are overcome by the opiate receptor antagonist naloxone [140]. The indole alkaloids ibogaine, ibogamine, coronaridine and tabernanthine bind to opiate receptors [141]. Plant-derived opiates and related compounds are of major importance medically because of their pain-relieving and narcotic effects [137]. k. Receptors for a variety of other peptide and non-peptide hormones acting via G proteins are potential targets for plant defensive compounds, including peptides. Thus a variety of plant secondary metabolites are uterotonic including the cyclic peptide kalata which has oxytocin-like activity in stimulating uterine contraction [142]. However these uterotonic agents are not necessarily acting as oxytocin receptor agonists and other uterotonic mechanisms are possible. IIL Ion channels and ion pumps a. Voltage-gated Na"*" channels are critical for neurotransmission and cell excitability [143]. Inactivation of the channel is blocked by the steroidal

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alkaloid veratridine [143] and by the highly toxic diterpenoid alkaloid aconitine from Aconitum [144]. A variety of related diterpenoid alkaloids having aconitine-like effects include aconifine, bikhaconitine, delphinine, falaconitine, indaconitine, jesaconitine, mesaconitine and pseudoaconitine [1]. The diterpenoid alkaloids lappaconitine, N-deacetyllappaconitine and ajacine block the Na"^ channel and thus can act as antagonists of aconitine [145]. b. K"*" channels are similarly critical to transmembrane potential- and Ca^'^-mediated signalling. Some K"*" channels are voltage-gated, others are modulated by G proteins (that are in turn regulated by particular hormones such as dopamine or adenosine) [146] and others are subject to Ca^"*"dependent activation [147]. A Ca^"^-dependent K"*" channel is opened by the lignan nordihydroguaiaretic acid (NDGA) [148]. Voltage-regulated K"*" channels involved in action potentials are blocked by the quinolizidine alkaloid sparteine (lupinidine) as well as by synthetic aminopyridine drugs [149]. ATP-sensitive K"*" channels (KATP channels) are blocked by ATP and have various roles including modulation of muscle, synaptic and endocrine functions [150, 151]. KATP channels control insulin secretion from pancreatic p-cells, are inhibited by synthetic sulphonylurea drugs used in treating non-insulin-dependent diabetes melittus (NIDDM) [150, 151] and are also inhibited by the legume-derived quinolizidine alkaloid sparteine [152]. c. Intracellular Ca^^ channels and PM voltage-gated Ca^"*^ channels are involved in signalling [153-159] and are targets for various plant defensive compounds. PM-located voltage-gated Ca^^ channels of various kinds (L, N, P, Q, R and T classes) have been resolved of which L-type Ca^^ channels are the best studied [153-156]. The L-type Ca^"*" channels are blocked by various synthetic drugs including phenylalkylamines (notably verapamil), benzazepines, benzthioazepines (notably diltiazem) and dihydropyridines [153]. Intracellular, ER-located, ligand-gated Ca^^ channels include the inositol-1,4,5-triphosphate (IP3) receptor, the ryanodine receptor (RyR), the NAADP receptor and the sphingolipid receptor [157-159]. The IP3 receptor Ca^^ channel is opened by IP3 generated as a result of G protein(or RTK)-mediated PLC activation [159].

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Ryanodine receptor Ca^* channels are opened by cADPR in a Ca^^CaM-dependent fashion and Ca^^, the diterpenoid alkaloid ryanodine and methylxanthines such as caffeine promote opening of the channel [157, 160]. Ryanodine can also negatively modulate the receptor [157]. Ryanodine receptors are involved in excitation-contraction coupling in skeletal muscle through direct interactions with V-gated Ca^^ channels [161, 162]. Both cADPR and NAADP are synthesized by ADP-ribosyl cyclase from NAD and NADP, respectively [163, 164] and cADPR may act as a second messenger (in addition to IP3) for chemokines such as interleukin-2 [165]. However there appears to be no direct involvement of the ADP-ribosyl cyclase activity of the ectoprotein CDS8 on Tlymphocytes and intracellular Ca^"*" signalling [166]. NAADP receptor Ca^^ channels are opened by very low NAADP concentrations but is blocked at higher NAADP concentrations [157]. Sphingolipid receptor Ca^^ channels may be opened by sphingosine-1phosphate or sphingosyl-phosphorylcholine [157]. d. The Na"*", K'*"-ATPase generates the Na"*" and K^ gradient across the PM that is required for cell excitability, action potential transmission and for Na^ gradient-dependent transport of metabolites and indeed of signalling molecules [14]. A variety of C23 and C24 triterpenoid-derived cardenolide and bufadienolide steroid glycosides are highly toxic but have cardiotonic activity and are potent inhibitors of the Na"^, K'^-ATPase, including the cardenolides digoxin (digoxigenin 3-O-tridigitoxoside) from Digitalis lanata and ouabain (ouabagenin 3-O-L-rhamnoside) from Strophanthus gratus and the bufadienolide scillaren A (scillarenin 3-0glucosylrhamnoside) from Scilla maritima. The diterpenoid alkaloid cassaine is also a potent inhibitor of the Na"**, K^-ATPase and the structurally related diterpenoid alkaloids cassaidine and erythrophleguine have similar digitalis-like effects [1, 167]. Digitalis, the dried leaves of foxglove {Digitalis purpurea), contains the Na"^, K^-ATPase inhibitor digitoxin and has been used as a cardiotonic for centuries [1]. Inhibition of the Na"*", K'*'-ATPase increases intracellular Na"*" and hence decreases Ca^'^/Na'*' antiport activity. Consequent elevated cytosolic Ca^"^ concentration increases heart contractility [14]. Various flavonoids inhibit the Na"", K^-ATPase [168, 169].

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e. Ca^"*" ATPases pump Ca^^ out of the cytosol across the PM or into the ER and can be Ca^'*'-CaM-activated. Thapsigargin is a sesquiterpenoid cell activator and secondary tumor promoter from Thapsia species. Thapsigargin is an inhibitor of the Ca^"*"-ATPase and consequently elevates cytosolic Ca^"^ concentration [170]. The prenylated xanthone a-mangostin is also an inhibitor of the Ca^'^-ATPase [171]. IV. Neurotransmitter converters and transporters A key element in any signalling is reversibility and neurotransmission across synaptic junctions requires neurotransmitter conversion to inactive entities or neuronal neurotransmitter uptake. Various plant defensive compounds interfere with these processes. Synaptic neurotransmitter transporters include glutamate transporters (that couple glutamate translocation to Na^ and K^ movement) [41] and transporters for GAB A, glycine, taurine, norepinephrine, dopamine and serotonin (that are coupled to Na^ and CI" movement) [42-44]. In addition there are vesicular monoamine transporters of the chromaffin granules of the adrenal medulla, stomach oxyntic cells and neurons that are variously involved in transport of epinephrine, norepinephrine, histamine, serotonin and dopamine into secretory vesicles, these transport processes being coupled to H^ movement [41, 45, 46]. A vesicle glutamate transporter in glutaminergic nerve endings similarly concentrates glutamate into vesicles in a process driven by a H^ gradient generated by a H^-ATPase [41]. a. Acetylcholinesterase (AcChE) hydrolyses acetylcholine to acetate and choline [178]. Plant derived acetylcholinesterase inhibitors include the indole alkaloids eseramine, eseridine, physostigmine (eserine) and physovenine, the sesquiterpenoid alkaloid N-(phydroxyphenthyl)actinidine, the steroidal alkaloid P-demissidine, the Amarillidaceae alkaloid galanthamine (galantamine) and the quinazoline alkaloid vasicinol (7-hydroxy peganine) [1, 179]. All of these alkaloids, like acetylcholine, have a critical protonatable N recognized by the enzyme. Physostigmine, galantamine and other AcChE inhibitors are of current interest for Alzheimer's Disease therapy [179]. b. Monoamine oxidases (MAO-A and MAO-B) catalyze the oxidative deamination of monoamine neurotransmitters, notably serotonin (5-

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hydroxytryptamine) [108]. The plant indole alkaloids harmaline and harmine [180], the chalcone isoliquiritogenin [1] and various isoquinoline alkaloids [108] are inhibitors of monoamine oxidase. c. Synaptic glutamate transporters are PM-located Na^-dependent transporters that remove glutamate (the major CNS excitatory neurotransmitter and a potential neurotoxin) from the extracellular space. In some cases K"*" is countertransported. A number of these transporters have been cloned [172, 173, 181-183] and inhibitors include analogues of glutamate and aspartate [173, 183]. d. NaVCr-linked monoamine neurotransmitter transporters are PMlocated and specific for various neurotransmitters including GABA, norepinephrine, serotonin, dopamine, glycine and taurine [172] and are targeted by some antidepressants, stimulants and antihypertensives [174]. GABA transporters (GAT1-GAT4) recover GABA after its synaptic release [172] and are variously inhibited by the phthalideisoquinoline alkaloids bicucuUine and norbicuculline and by the piperidine alkaloid guvacine, 3-aminopropionic acid (p-alanine) and the synthetic nipecotic acid (3-piperidinecarboxylic acid) [184, 185]. Some GABA transporter antagonists have anticonvulsant effects as a consequence of elevation of synaptic GABA [186]. Synaptic glycine transporters (GlyTl and GIyT2) are responsible for Na'^^/Cr -linked glycine transport and are the major means of removing synaptic glycine [187, 188]. GlyTl is inhibited by sarcosine (Nmethylglycine) [187]. Plant phorbol esters downregulate GlyTl through activation of PKC and oleoylacetylglycerol mimics this effect which is blocked by PKC inhibition [188]. Synaptic dopamine transporters are inhibited by various psychotropic alkaloids including the tropane alkaloid cocaine [189, 190], the indole alkaloid ibogaine [191] and by amphetamine (methylphenethylamine) and related compounds [192, 193]. Synaptic serotonin (5-hydroxytryptamine) transporters are inhibited by amphetamines, the tropane alkaloids cocaine and ecgonine [194] and by the indole alkaloid ibogaine (12-methoxyibogamine) and its demethylation product ibogamine [191, 195]. Hyperforin is a major antidepressant constituent of St. John's Wort (Hypericum perforatum) and inhibits serotonin uptake by elevating cytosolic Na"^ [196]. The additional

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major component hypericin does not appear to affect monoamine uptake but rather binds to apomorphine-binding sigma receptors [197]. Selective serotonin transport inhibitors are potential antidepressants [198]. e. Vesicular monoamine transporters (VMATl and VMAT2) of neuroendocrine cells transport various monoamine neurotransmitters (dopamine, epinephrine, norepinephrine, serotonin and histamine) from the cytosol into vesicles prior to exocytotic release from the cell surface. VMATl is present in central, peripheral and enteric neurones and VMAT2 is present in chromaffin cells of the adrenals and in histaminestoring enterochromaffin-like cells of the stomach oxyntic mucosa [ITSIT?]. The VMATs are inhibited by the indole alkaloid reserpine (that is employed as a tranquillizer and antihypertensive agent) and also by phenylethylamine, amphetamine and amphetamine-related compounds [199, 200]. f. Vesicular glutamate transporters located in glutaminergic nerve endings accumulate glutamate into synaptic vesicles prior to release. This process is coupled to a H"*" gradient generated by a H"^-ATPase [201]. LGlutamate is ingested by herbivores and is neurotoxic in excess [1]. V. Cyclic nucleotide signalling targets of plant bioactive compounds Cyclic AMP acts as a hunger signal in non-plant eukaryotes that consume plants and its metabolic effects are mediated by PKA as outlined above [13-15]. This provides a rationale for the inhibition of PKA by representatives of a number of major classes of plant defensive secondary metabolites. PKA is required for pathogenicity towards plants of a variety of fungal pathogens [65, 202-206] and accordingly inhibition of PKA would be expected to interfere with fungal invasion. PKA is also involved in specific gene expression and development in higher organisms and is involved in cognitive processes of animals. Accordingly, inhibition of PKA might be expected to interfere with pathogenic fungal growth and perturb cognitive processes of herbivores such as insects [65]. Conversely, agents that elevate cAMP in animal cells can be toxic as evidenced by the toxic cAMP-elevating effect of cholera toxin in animal intestinal cells that results in excessive Na"*" and H2O loss [14, 207]. Similarly, Bordetella pertussis elaborates an invasive, Ca^^-CaM-activated adenylyl cyclase and

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hence elevates cAMP in the target animal cells [208]. This toxic effect of cAMP elevation provides a rationale for the defensive utility of adenylyl cyclase activation by the plant diterpenoid forskolin [209, 210] and of cAMP phosphodiesterase inhibition by plant-derived methyl xanthines such as theophylline and caffeine [211]. a. Adenylyl cyclase catalyzes the formation of cAMP from ATP and is indirectly modulated by a variety of plant natural products that interact with G protein-linked receptors as outlined above. The plant diterpene forskolin binds to the catalytic core of adenylyl cyclase and activates the enzyme [209, 210]. b. Cyclic AMP phosphodiesterases (cAMP PDEs) catalyze the hydrolysis of cAMP to 5'-AMP and cAMP PDE inhibition consequently elevates cellular cAMP concentration. There is a multiplicity of animal cyclic nucleotide PDEs [211-214] and these are variously inhibited by plant metabolites including methyl xanthines (such as caffeine, theophylline and theobromine) [211-217], a number of saponins [218], the chalcone isoliquiritigenin [219], the protopine alkaloid allocryptopine (phomochelidone or a-fagarine) [220], the benzylisoquinoline alkaloid papaverine [221], biflavones (amentoflavone, bilobetin, sequoiaflavone and ginkgetin) [222], the isoquinoline alkaloid atherosperminine [223], phenylpropanoid glycosides [224], dihydropyranocoumarins [225], dihydrofuranocoumarins [225] and a range of flavonoids [226]. c. Membrane-associated or soluble guanylyl cyclases catalyze the formation of cGMP from GTP [29, 227]. Atrial natriuretic factor (ANF) activates membrane-associated guanylyl cyclase (29, 227). A peptide having ANF-like activity in stimulating cGMP synthesis has been resolved from plants [228]. Soluble guanylyl cyclase is activated by NO which in turn is synthesized by NO synthase (NOS) [229-232]. Soluble guanylyl cyclase can be activated by CO generated by the heme oxygenase system [233]. NO is synthesized in plants [67] and the plant-derived vasorelaxant chalcone isoliquiritigenin activates soluble guanylyl cyclase [234]. Particular NOSs are activated by Ca^'^-CaM or regulated by phosphorylation by signal-regulated protein kinases [231] and plants elaborate a variety of secondary metabolites that are protein kinase inhibitors [235, 236].

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d. Cyclic GMP phosphodiesterases (cGMP PDEs) catalyze the hydrolysis of cGMP to 5'-GMP. A number of cGMP PDEs have been resolved from animal tissues and these enzymes are variously inhibited by some plant natural products including methyl xanthines and papaverine [211-217] . Cyclic GMP acting via PKG (or through cGMP-gated ion channels) has a vasodilatory effect of importance in relief of angina and of erectile disfunction, the synthetic specific cGMP PDE inhibitor viagra (sildenafil) being of importance for treatment of impotence [213]. e. Cyclic AMP-dependent protein kinase (PKA) mediates catabolite deficiency or hunger signalling resulting in specific gene transcription and metabolic alteration toward glycogenolysis, gluconeogenesis and fatty acid oxidation [13-15]. Representatives of some major classes of plant defensive secondary metabolites are inhibitors of PKA [235, 236]. Such plant-derived PKA inhibitors include flavonoids (3-hydroxyflavone, 5hydroxyflavone, 5,4'-dihydroxyflavone, 5,7-dihydroxyflavone, 3',4'dihydroxyflavone, 7,8-dihydroxyflavone, 3,3',4'-trihydroxyflavone, galangin, apigenin, 7-0-methyl-apigenin, 2,3-dihydroapigenin, naringin, luteolin, 2,3-dihydroluteolin, 3-0-methyl-2,3-dihydroluteolin, kaempferol, 4'-0-methyl-kaempferol, 7,8,3',4'-tetrahydroxyflavone, fisetin, 2,3dihydrofisetin, 2,3-dihydrofisetin, quercetin, 2,3-dihydroquercetin, 3-0rhamnosyl-quercetin, morin, tricetin, myricetin, quercetagenin, 3',4',5'-tri0-methyl-tricetin and hesperidin) [237], flavanols ((+)-catechin and (-)epicatechin) [237], a phenolic dimeric sesquiterpenoid (gossypol) [237], anthraquinones (alizarin, quinizarin, anthrarufin, chrysazine, anthraflavic acid, chrysophanic acid, emodin, purpurin and quinalizarin) [238], prenylated xanthones (y-mangostin and a-mangostin) [239], hydrolyzable tannins ( a range of such compounds with differing numbers of phenolic substitutents) [240], condensed tannins (a range of such catechin-based flavans with different degrees of complexity e.g. procyanidin dimer, trinmer and tetramer entities) [241, 242], amphiphilic triterpenoids (18-aglycyrrhetinic acid, 18-p-glycyrrhetinic acid, ursolic acid, oleanolic acid, betulin and asiatic acid) [243], a phenylpropanoid (curcumin) [244], prenylated isoflavones (warangolone, 8-y,Y-dimethylallylwighteone, 3'Y,Y-dimethylallylwighteone and nallanin) [245], an aporphine alkaloid (apomorphine) [246], a benzophenanthridine alkaloid (sanguinarine)

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[246], a stilbenoid (piceatannol) [247] and the phenolic ellagic acid [247]. A common feature of these compounds is a planar or quasi-planar hydrophobic nucleus linked to a hydrophilic element, a motif also found with a variety of potent PKA inhibitors that are synthetic [248] or microbe-derived [248, 249] as well as with PKA inhibitors that are synthetic phenanthrene-based [250], acridine-based [251, 252] or isoquinoline-based compounds [253], oxazine alkaloids (darrow red, nile blue A and oxazine 170) [246] and semi-synthetic prenylated xanthone derivatives [254]. This pattern suggests a possible binding site at the hydrophobic cleft of the PKA catalytic subunit that accomodates the purine ring of ATP [36]. However a variation from this pattern is found with some hydrophobic anti-inflammatory triterpenoid esters that are PKA inhibitors [256]. The naturally occurring PKA inhibitors described above are not necessarily specific for PKA. Thus various anti-inflammatory triterpenoid PKA inhibitors are also protease inhibitors [243, 255, 256]. Further, a variety of flavonoids that inhibit PKA also inhibit other protein kinases [235, 236] as well as inhibiting cyclic nucleotide PDE [226] and Na"',K''-ATPase[168, 169]. VI, Ca^^-mediated signalling targets The transient, signal-induced elevation of cytsolic free Ca^"*" concentration switches on a variety of Ca^^- and Ca^"*"-calmodulin (CaM)-dependent enzymes, notably protein kinases. Plants contain CaM and CaM-regulated enzymes [68] and accordingly elaboration of CaM antagonists would be damaging unless the CaM antagonists were stored in an inactive form or in a location remote from cytosolic CaM (e.g. in seeds or plant cell walls). Similarly CDPK rather than PKC represents a major means of mediating Ca^"*" signals in plants. As outlined below, plants elaborate a variety of PKC inhibitors and activators. a. The PKC family of protein kinases contains a variety of isozymes that are variously activated by Ca^"*", diacylglycerol (DAG) and phospholipids. PKC is activated by plant phorbol esters (tigliane diterpenoids) that act at the DAG-binding domain. The phorbol esters such as phorbol-12,13-dibutyrate (PDB) and 12-0-tetradecanoyl-phorbol13-acetate (TPA) are highly inflammatory compounds and secondary tumour promoters [38]. A variety of other PKC activators are elaborated

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by plants including other tigliane diterpenoid esters (e.g. those based on 4deoxyphorbol, 12-deoxyphorbol and 4,20-dideoxy-5-hydroxyphorbol nuclei), daphnane diterpenoids (daphnoretin, gnidimacrin) [257, 258], pyranocoumarins (decursin and decursin angelate) [259], a spiroiridal triterpenoid (28-deacetylbelamcandal) [260] and ingenol diterpenoids (17hydroxyingenol-20-hexadecanoate, ingenol-20-hexadecanoate, ent16a,17-dihydroxyatisan-3-one and ent-3p, 16a, 17-trihydroxyatisane) [261]. A glycosylated hydroxy fatty acid, namely glycosylated (IIS)hydroxyhexadecanoic acid (tricolorin A), displaces TPA from PKC [262]. The secondary tumour promoting (co-carcinogen) activity of phorbol esters can be rationalized in terms of an increase in levels of signalling pathway phosphoproteins phosphorylated by PKC and predisposing cells to transformation. The same rationalization can be applied to the secondary tumour promoting activity of thapsigargin (Ca^'^'-ATPase 9-4-

inhibition leading to increased Ca concentration and hence PKC activation) and of the PPl and PP2A inhibitors okadaic acid (from dinoflagellates) and the Microcystis microcystins (inhibition of dephosphorylation of the PKC-phosphorylated phosphoprotein) [18]. A variety of plant natural products inhibit PKC including flavonoids ( fisetin, quercetin, luteolin, hirsutenone, oregonin and hirsutanonol) [263], a sesquiterpenoid (gossypol) [264], anthraquinones (alizarin, quinizarin, anthrarufin, chrysazine, anthraflavic acid, chrysophanic acid, emodin, purpurin and quinalizarin) [238], hydrolyzable tannins [240], condensed tannins [241, 242], a phenylpropanoid (curcumin) [244], an aporphine isoquinoline alkaloid (apomorphine) [246], a stilbenoid (piceatannol) [247], the phenolic ellagic acid [247], diarylheptanoids (glycosylated (3R)-l,7-bis(3,4-dihydroxyphenyl)heptan-3-ol and the aglycone) [265], phenylethanoid glycosides (calceolareoside A, calceolsareoside B, forsythiaside, plantainoside D, leucosceptoside, acteoside and poliumoside) [266], corosolic acid [267], coumaryl glycosides (vanicoside A and vanicoside B) [268] and a xanthone (norathyriol) [269], noting that some semi-synthetic prenylated xanthones also inhibit PKC [254]. The stilbenoid magnolol inhibits phorbol ester activation of PKC [270]. PKC is involved in mitogenic signalling and inflammatory responses and some PKC inhibitors accordingly have anti-inflammatory effects [38]. b. The Ca^^-binding regulator protein calmodulin (CaM) activates a variety of enzymes including CaM-dependent kinases such as myosin

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light chain kinase (MLCK). Inhibition of CaM-dependent protein kinases could occur via inhibitor binding to CaM and affecting its function or through the inhibitor binding to the protein kinase. Thus the flavonoid quercetin and the sesquiterpenoid gosspol are inhibitors of CaMdependent MLCK and also interact with CaM as detected through 94-

inhibition of Ca -dependent enhancement of dansyl-CaM fluorescence [237, 271]. Further plant-derived MLCK inhibitors include other flavonoids (5,4'-dihydroxyflavone, 5,7-dihydroxyflavone, 3,3',4'trihydroxyflavone, galangin, apigenin, naringin, luteolin, kaempferol, 4'0-methyl-kaempferol, fisetin, morin, tricetin and myricetin) [271, 272], a phenolic dimeric sesquiterpenoid (gossypol) [271], anthraquinones (alizarin, quinizarin, chrysazine, anthraflavic acid, emodin, purpurin and quinalizarin) [238], condensed tannins [241, 242], an isoflavone (genistein) [245], aporphine alkaloids (apomorphine and boldine) [246] and a stilbenoid (piceatannol) [247]. c. Ca^^-dependent protein kinase or calmodulin domain-containing protein kinase (CDPK) is a major protein kinase in higher plants that is switched on by micromolar free Ca^"*" concentration [68, 69]. This type of protein kinase is not found in other eukaryotes except for Plasmodium, the protozoan causing malaria. Accordingly there is interest in Plasmodium CDPK as a potential target for selective chemotherapy [70]. Not surprisingly, plant CDPK is not inhibited by a wide range of plant-derived flavonoids (with the exception of 3,3',4'-trihydroxyflavone, 7,8,3',4'tetrahydroxyflavone, quercetin, tricetin and myricetin, noting that glycosylation of quercetin or methylation of tricetin abolishes inhibitory effectiveness) [271]. Of a wide range of anthraquinone-based PKA and PKC inhibitors tested, only purpurin is a good inhibitor of CDPK [238] and a variety of amphiphilic triterpenoid inhibitors of PKA do not inhibit plant CDPK [243]. The prenylated xanthones y-^^angostin and amangostin are good inhibitors of plant CDPK but these defensive components are located in the hull of the mangosteen fruit and hence away from the CDPK-containing cytosol [239]. Similarly, a variety of bark- and fruit-derived procyanidin and prodelphinidin-based condensed tannins are inhibitors of CDPK [241, 242]. CDPK is also inhibited by genistein [245] and the stilbenoid protein kinase inhibitor piceatannol [247].

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d. A large number of other serine/threonine protein kinases are encoded by higher organism genomes thus far examined [42, 50] and are likely to interact with plant defensive secondary metabolites. Serine/threonine-specific protein kinases that are involved in signalling cascades initiated by receptor tyrosine kinases (RTKs) are potential targets for the design of drugs against cancer and metabolic disease. Thus PKB is a serine/threonine protein kinase that is switched on by a pathway initiated by an insulin-insulin receptor tyrosine kinase interaction and PKB phosphorylates and reversibly inactivates a glycogen synthase kinase (GSK3). GSK3 is an enzyme that in turn phosphorylates and reversibly inactivates glycogen synthase. Accordingly, a synthetic compound or natural product that inhibits GSK3 might conceivably mimic insulinpromotion of glycogen synthesis [58]. However it must be appreciated that the catalytic domains of protein kinases are similar [36, 273-276] and, as is apparent from the above survey, there can be substantial overlaps of specificity of protein kinase inhibitors that interact with the catalytic domains of serine/threonine-specific (and indeed of tyrosine-specific) protein kinases. Thus casein kinase 11 is inhibited by the anthraquinone emodin [276] as are PKA and PKC [238]. Further examples of such protein kinase inhibition overlaps are given below. VII. Tyrosine kinase-mediated signalling Receptor tyrosine kinase-mediated signalling can be subject to interference by plant natural products at various levels. Thus plant inhibitory agents could act as agonists or antagonists with respect to particular receptors or inhibit downstream components such as RTK tyrosine kinase activity, Ras, Raf, MAPK, Src (a tyrosine kinase) or phospholipase C-y (PLCy). Thus PLCy is inhibited by the flavonoid amentoflavone [277] and a variety of flavonoids and other phenolic plant bioactives inhibit Src [278]. A further complexity is introduced if one considers "cross-talk" between pathways. Thus PKC can phosphorylate particular RTKs [22] and alter their function and is also involved in the activation by phosphorylation of the serine/threonine protein kinase Raf (a MAPKKK) [279, 280]. As outlined above, a variety of plant bioactives inhibit PKC. Genes encoding RTK-mediated pathway components (protooncogenes) can undergo mutation to oncogenes whose expression can contribute to oncogenic transformation. Accordingly there is much

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interest in synthetic or naturally-occurring substances that can inhibit the function of such oncogene products and hence inhibit or reverse cancerous transformation [281, 282]. RTKs bind the activating hormone and then phosphorylate target proteins or bind target adaptor and effector proteins through phosphotyrosine-SH2 domain interactions. A variety of plant natural products have been found which directly inhibit the tyrosine kinase activity of RTKs and in many cases also inhibit cell proliferation. Such plant-derived RTK inhibitors include polyphenols (purpurogallin [283], (-)-epigallocatechin gallate [284] and butein [285]), anthraquinones (emodin [286] and damnacanthal [287]), isoflavonoids (kievitone, genistein and genistin [288]), hydroxystilbenes (resveratol [289] and piceatannol [290]), flavonoids (desmal [291], kaempferol and quercetin [292]), the phenolic ellagic acid and synthetic compounds related to ellagic acid [293]. While piceatannol and genistein have been applied as selective tyrosine kinase inhibitors, piceatannol also inhibits PKA, PKC, MLCK and plant CDPK [247] and genistein also inhibits MLCK and (albeit poorly) PKA [245]. Ellagic acid is also an inhibitor of PKA and PKC [247], as is emodin [238]. VIII. Steroid hormone, taste and smell receptors Finally, brief mention must be made of plant compounds that mimic steroid hormones, notably the phytoestrogens (including particular isoflavones, coumestans and lignans) that bind to estrogen receptors [294], compounds that mimic insect developmental hormones such as juvenile hormone (including the sesquiterpenoids dehydrojuvabione, juvabione and juvenile hormone HI) [1] and compounds mimicking insect pheromones (such as the sesquiterpenoids a- and P-famesene) [1]. Taste receptors variously bind bitter and sweet entities, the signalling pathways involving changes in membrane conductance after ligand binding [295]. A variety of monosaccharides and disaccharides bind to sweet receptors and a notable bitter receptor ligand is quinine [1]. Smell signal transduction involves adenylyl cyclase, cAMP and cAMP-gated Na^ channels [46] and can also involve Ca^"^ and cGMP as second messengers [47]. Indoles such as skatole and indole itself are well known examples of

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"bad smelling" ligands that can act as insect attractants and "nice smelling" ligands include monoterpenes such as camphol [1]. FUTURE DIRECTIONS As indicated at the outset of this broad-based review, although of the order of ten thousand plant defensive compounds have been isolated and structurally characterized, only of the order of a thousand have so far been shown to interact with specific proteins and most of these biochemical sites of interaction are signal transduction components. The elucidation of such targets now permits rapid biochemical screening of complex ecosystems for plant-derived ligands for such proteins. Incisive technologies, including plasmon resonance-based analysis, are now available for rapidly quantitating such protein-ligand interactions. The recent major advances in genome sequencing and X-ray crystallographic structure determination of members of major protein families (most notably of protein kinases and hormone receptors, including receptor tyrosine kinases) will permit a more exacting progress towards filling in the plant defensive metabolite-eukaryote target protein matrix. ABBREVIATIONS AC AcCh AMPA AMPK AMPKK ANF cADPR cAMP CaM CaMPK CDPK CNS

= Adenylyl Cyclase = Acetylcholine = a-Amino-3-hydroxy-5-methyl-4-isoxazoleprionic Acid = 5'-AMP-dependent Protein Kinase = 5'-AMP-dependent Protein Kinase Kinase = Atrial Natriuretic Factor = Cyclic Adenosine-5'-diphosphateribose = 3',5'-Cyclic AMP = Calmodulin = Ca^^-CaM-dependent Protein Kinase = Ca^^'-dependent Protein ICinase = Central Nervous System

548

CREB protein = cGMP = DAG = = ER = G = GABA = GC GEF = = GSK3 = H = IP3 = IP4 = JAK = L = L-BMAA = L-BOAA = LSD = MAPK = MAPKK = MAPKKK = MLCK = NAADP = NDGA = NIDDM = NMDA = NOS = NT = P = PDE = PDK = PI3K = PI34P2 = PI45P2 = PI345P3 = PK = PKA = PKB = PKC

cAMP Response Element Binding Protein 3',5'-Cyclic GMP Diacylglycerol Endoplasmic Reticulum G Protein y-Aminobutyric Acid Guanylyl Cyclase Guanyl Nucleotide Exchange Factor Glycogen Synthase Kinase 3 Hormone Inositol-1,4,5-triphosphate Inositol-1,3.4,5-triphosphate Janus Kinase Ligand P-N-Methylamino-L-alanine P-N-Oxazylamino-L-alanine Lysergic Acid Diethylamide Mitogen-activated Protein Kinase Mitogen-activated Protein Kinase Kinase Mitogen-activated Protein Kinase Kinase Kinase Myosin Light Chain Kinase Nicotinic Acid Adenine Dinucleotide Phosphate Nordihydroguaiaretic acid Non-Insulin-dependent Diabetes Melittus N-Methyl-D-aspartate Nitric Oxide Synthase Neurotransmitter Permeability of membrane to specific solutes Phosphodiesterase Phosphatidylinositol lipid-dependent Protein Kinase Phosphatidylinositol-3-kinase Phosphatidylinositol-3,4-bisphosphate Phosphatidylinositol-4,5-bisphosphate Phosphatidylinositol-3,4,5-triphosphate Protein Kinase cAMP-dependent Protein Kinase Insulin signalling-activated Protein Kinase Ca^^- and Phospholipid-dependent Protein Kinase

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PKG PLC PLCy PM PP P-Pr P-STAT PTPase R Ras Raf SM S/T-RPK RPTP RTK STAT TGFp TPA TRE AP A^

cGMP-dependent Protein Kinase Phospholipase C Phospholipase C-y Plasma Membrane Phosphoprotein Phosphatase Phosphoprotein Tyrosine Phosphorylated STAT Protein Phosphotyrosine Phosphatase Receptor Small G protein a MAPK Kinase Kinase Secondary Metabolite Receptor Serine/Threonine Receptor Protein Kinase Receptor Protein Phosphotyrosine Phosphatase Receptor Tyrosine Kinase Signal Transducer and Activator of Transcription Transforming Growth Factor p Tetradecanoylphorbolacetate Tetradecanoylphorbolacetate Response Element Change in Permeability of PM to specific solutes Change in Transmembrane Potential.

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[289] Jayatilake, G.S.; Jayasuriya, H.; Lee, E.S.; Koonchanok, N.M.; Geahlen, R.L.; Ashendel, C.L.; McLaughlin, J.L.; Chang, C.J.; /. Nat. Prod., 1993,56,1805-1810. [290] Geahlen, R.L.; McLaughlin, J.L.; Biochem. Biophys. Res. Commun., 1989,165,241-245. [291] Kakeya, H., Imoto, M.; Tabata, Y.; Iwami, J.; Matsumoto, H.; Nakamura, K.; Koyano, T.; Tadano, K.; Umezawa, K.; FEBS Lett., 1993,320,169-172. [292] Abou-Sher, M.; Ma, G.E., Li, X.H.; Koonchanok, N.M.; Geahlen, R.L.; Chang, C.J.; /. Nat. Prod., 1993,56,967-969. [293] Dow, R.L.; Chou, T.T.; Bechle, B.M.; Goddard, C; Larson, E.R.; /. Med. Chem., 1994,37,2224-2231. [294] Kurzer, M.S.; Xu, X.; Annu. Rev. Nutr., 1997,17, 353-381. [295] Kinnamon, S.C; Cummings, T.A.; Annu. Rev. Physiol, 1992, 54, 715-731.

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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FLAVONOIDS AND CARDIOVASCULAR DISEASES JUAN DUARTE; FRANCISCO PEREZ-VIZCAINO^ J0S6 JIMENEZ; JUAN TAMARGO^; ANTONIO ZARZUELO University of Granada, Department of Pharmacology, School of Pharmacy, Granada, Spain; and ^ University Complutense ofMadrid, Department of Pharmacology, School of Medicine, Madrid, Spain. ABSTRACT: Several epidemiological studies have found an inverse correlation between the dietary flavonoid intake and a reduced mortality from coronary heart disease and the incidence of stroke. We will focus our review on several mechanisms which have been suggested to explain these protective effects. a) Antiatherogenic eiffects. Flavonoids together with others antioxidants constitute two lines of defense in protecting cells against injury owing to oxidation of LDL: 1) at the LDL level, by inhibiting LDL oxidation due to their free radical scavenger activity, and 2) at the cellular level, by protecting the cells directly, i.e., by increasing their resistance against the cytotoxic effect of oxidised LDL. Additionally, recent studies indicate that flavonoids can prevent the expression of adhesion and chemoattractant molecules. b) Antiaggregant effects. Flavonoids prevent platelet aggregation induced by several proaggregant stimuli although relatively high doses are required. Inhibition of platelet phosphodiesterases, inhibition of arachidonic acid metabolism and antioxidant effects have been suggested as possible mechanisms of action. c) Direct effects on vascular smooth muscle. The vasodilator effects of flavonoids in vitro is mainly endothelium-independent. The main mechanism of action seems to be related to their inhibitory effects on protein kinases. Some flavonoids, however, can produce endothelium-dependent contractile responses due to increased TXA2 production. d) Antihypertensive effects. Little information about the effects offlavonoidson blood pressure is available. However, recently, the chronic oral administration of quercetin has been shown to exert potent antihypertensive effects.

INTRODUCTION Flavonoids coiiq)rise a large group of secondary metabolites occurring widely throughout the plant kingdom. These low molecular weight polyphenolic compounds are foimd practically in all parts of the plants including fruit, vegetables, nuts, seed, flowers, and bark and are integral

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part of the human diet. The term flavonoid as currently used refers to a large group of chemical compounds share the common skeleton of diphenylpyrans (C6-C3-C6). This basic structure allows a multitude of substitution patterns and variations leading several flavonoid classes, including flavonols, flavones, flavanones, catechins (or flavanols), anthocyanidins, isoflavones, dihydroflavonols, and chalcones Fig. (1). Over 4000 different flavonoids have been described, and the number is still increasing [1-3]. Until recently, the estimations most frequently cited pointed to a daily himianflavonoidintake of more than 500 mg/day (total flavonoids) [4,5]. However, more recent and accurate estimations gave an average of 23 mg/day ia of selected flavonols plus flavones [6] but this value varied among countries ranging from 6 mg/day in Finland to 64 mg/day in Japan [7]. The main sources offlavonoidsin the human diet are onions, apples, grapes, wine, tea, berries, herbs and spices. Among dietary flavonoids, quercetin is by far the most abimdant. A very wide range of biological actions offlavonoidshave been reported including antibacterial, antiviral, anti-inflammatory, antiallergic, antihepatotoxic, antiulcer, analgesic, hypoglycaemic, estrogenic, and antidiarrheic (for a review see [8-11]). In recent years, after the publication of several epidemiological studies showing an inverse association between the consumption of fruit and vegetables and cancer risk and mortality from coronary heart diseases, particular attention has been paid to their antitnutagenic, anticarcinogenic and cardioprotective activities (reviewed in ref [3,12]). In the present review, we analyse the cardiovascular effects of flavonoids in both in vitro and in vivo models and also the possible mechanisms inq)licated in the cardioprotective effects of these compounds. We will focus our review on the current status of research in these main topics: a) Their antiatherogenic effects and its relationship with their antioxidant properties (inhibition of low density lipoprotein [LDL] oxidation), b) the inhibition of platelet aggregation, c) the mechanisms involved in their vascular effects and their antihypertensive properties. EPIDEMIOLOGICAL STUDIES Cardiovascular diseases remain the main cause of death in developed countries. Studies relating the intake of dietary flavonoids to risk of cardiovascular disease (mortalityfromcoronary heart disease, incidence of

567

FLAVANONES

CATECHINS

^^X ANTHOCYANIDINS

CHALCONES

Fig. (1). Generic structure of flavonoids

ISOFLAVONES

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myocardial inferction and stroke) have been observational in nature. Several studies have shown thatflavonoidintake was inversely related with mortality from coronary heart disease (table 1). Epidemiological studies in The Netherlands [6,13], Finland [14] and USA [15] as well as a crosscultural study performed in 16 cohorts from seven countries [7], have shown a significant inverse association between dietary flavonoids (mainly quercetin) and long term mortality from coronary heart disease and to a lesser extent with incidence of first myocardial inferction [6]. A study carried out in male USA health professionals found a modest non significant inverse association regarding long term mortality from coronary heart disease in men with previous history of ischaemic heart disease [16]. In contrast to the above studies, in the Caerphilly study (in which the major source of flavonoids was tea with milk) found a non significant increased mortality from ischaemic heart disease and a significant increase in total mortality in all quartiles of high flavonoid intake compared to the lowest quartile [17]. They hypothesised that this result may have been due to binding offlavonoidin tea with protein from milk, thus reducing flavonoid absorption. To summarise, a significant protective role for flavonoids in coronary heart disease was found in 3 out of 5 prospective studies, in addition to one cross-cultural study. One study showed a weak inverse and another a weak positive association between flavonol consumption and mortalityfromischaemic heart disease (Table 1). Additionally, men in the highest quartile of flavonol and flavone intake showed a reduced incidence of stroke in a Dutch cohort [18], while a prospective study in postmenopausal women foimd no association between totalflavonoidintake and stroke mortality [15]. The potential limitations, common in all epidemiological studies of diet and disease, are: a) mis-classification of dietary exposures, and b) lack of measure of changes in diet that occurred during the follow-up period, as their analyses were based on information from a single food frequency questionnaire at the beginning of the studies. The daily consumption of flavonoids is difficult to determine because the dietary supply is strongly dependent on feeding habits and, in this field, exhaustive tables on food composition are not always available. So, given the limitations of the diet assessment, the epidemiological evidence points to a protective effect of flavonols in cardiovascular disease but it is not conclusive.

Table 1. Summary of epidemiological prospective studies on flavonoid intake and coronary heart disease (CHD), myocardial infarction (MI) and stroke risk.

7

Zotpben Elderly Zotphen Finland (Tbe Netherlaods) (Tbe Netberlaoda)

Gender

Male

Male

Age (y) Outcome

65-84 (I) CHD mortality (2) MI incidence

50-69 Stroke incidence

Follow-up (y) Population size Relative risk (95% C.I.)

5 15 805 552 (I) 0.32 (0.15-0.71) 0.27 (0.1 14.70) (2) 0.52 (0.22-1 2.3)

CHD mortality

Scvcn Coootries

Female 49-59

CHD mortality

-

Japan) 2.6 (West

intake (mglday) (high vs. low) women Refaenw

1 113)

1 [IS]

[I 41

[I 61

[I 7l

[I 51

* Relative risk of highest versus lowest flavonoi 1 intake group, adjusted for agc diet and other risk factors for coronary heart disease. (C.I. = confidence interval)

i

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Furthermore, polyphenolics present in wine, of which flavonoids are important components, have been suggested to be responsible of the so called French paradox, that is, the unexpectedly low rate of mortality from coronary heart disease in French population despite an unfavourable e>qposure to known cardiovascular risk factors such as high saturated fat consumption [19-21]. Epidemiological studies in USA [22] and Denmark [23] reported that moderate red wine drinkers had a lower risk of coronary artery disease than participants with no alcoholic beverage preference. However, controversial results about the antioxidant capacity of human serum after red wine consuniption have been reported [24-27]. It is therefore uncertain whether wine constituents other than alcohol add to the cardioprotective effects of red wine. Tea is another important dietary source forflavonoidSpInfeet,about half of the flavonoid intake in western populations is derived from black tea. Tea was the major source of flavonoids in the Dutch [6,13] and Welsh studies [17]. Only a small number of studies investigated the association between tea consumption and cardiovascular disease risk. No association between tea consimiption and cardiovascular disease risk were reported in Scottish men and women [28] and in U.S. men in the Health Professionals foUow-up study [29]. However, in a Norwegian population an inverse association was reported between tea intake, serum cholesterol, and mortality from coronary heart disease [30]. Several studies reported that tea consumption did not affect plasma antioxidant activity [31] and hemostatic factors [32]. However, a recent prospective study (the Rotterdam study) of 3,454 men and women 55 years and older followed for 2 to 3 years, showed a significant, inverse association of tea intake with severe (> 5 cm the length of the calcified area) aortic atherosclerosis. Odds ratios decreased approximately 70 % for drinking more than 500 mL/day (4 cups per day). The associations were stronger in women than in men. However, the risk reductions for moderate and mild atherosclerosis were only weak or absent [33]. The protective role of dietaryflavonoidsagainst cardiovascular diseases in the above mentioned studies has been attributed to their inhibitory effects on LDL oxidation, their inhibitory effects on platelet aggregation. PHARMACOKINETICS A limitation for the understanding and relevance of these epidemiological

571

Studies was the scarce and conflicting data on the pharmacokinetics of flavonoids (for a review see [5,12]. Absorption from the diet is a prerequisite for a causal relation between flavonols and coronary heart disease prevention. In addition, the metabolism of flavonoids after absorption should not substantially inhibit their biological activities. Absorption of flavonoids from the diet was long considered to be negligible, as they are mostly bound to sugars as p-glycosides (with exception of catechins). Onlyfreeflavonoidswithout a sugar molecule, the so-called aglycones, were considered to be able to pass the gut wall, and no enzymes that can split these predominantly p-glycosidic bonds were thought to be secreted into the gut or present in the intestinal wall. The large intestine microflora, especially anaerobic species Bacteroides distasonis, B, uniformis and B. ovatus possess glycosidases liable to hydrolyse glycosides with a high degree of specificity in the distal ileum and in the caecum. Thus, only a marginal absorption of dietary flavonoids would be expected. However, several recent studies in rats and humans have revealed that flavonoids are absorbed in appreciable amounts, both as aglycones and as glycosides, in the small intestine [34]. Some but not all common dietary flavonoid glycosides can be deglycosilated by a hepatic beta-glucosidase [35]. In rats and humans, the absorption of orally administered quercetin aglycone was about 20 %, while isoflavone absorption was 10-20% [3637]. In ileostomy patients, absorption of quercetin glycosides from onions was 52%, 24% for quercetin aglycone and 20% for quercetin rutoside [38]. In rats after in situ perfiision of jejimum plus ileimi, quercetin (66,7% of perfiised) enters the enterocytes by a still unidentified mechanism. In these cells, quercetin is readily glucuronidated, methoxylated and sulfated [39]. The resulting metabolites can subsequently leave the intestinal cell possibly via a facilitated transport system, either across the apical pole (secretion of the conjugated forms in the lumen, 52,4%) or across the basolateral side for their fiirther transfer into the portal vein (14,3%). The liver fiuther metabolises the conjugated compoxmds arising from intestine, leading to an increase of their degree of glucuronidation, sulfation, and methoxylation [39]. The main plasma metabolites after quercetin administration are conjugated derivatives of quercetin and isorhamnetin, a 3'-0-methylated form of quercetin [40-42]. The conjugated metabolites show a long lasting

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presence in plasma (elimination half life of about 25 h) implying that repeated dietary intake would lead to an accumulation in plasma [43]. Excretion in bile of glucoronides and sulphates seems to be important. Bacteria in the colon hydrolyse conjugates and glycosides which is supposed to enable absorption of the liberated aglycones. Thus, conjugates can be reabsorbed and enter an enterohepatic cycle. However, these microorganisms also substantially degrade the flavonoid moiety by cleavage of the heterocyclic ring. Three main types of ring scission, depending on the ring structure, each leading to different phenolic acids or their lactones, have been postulated. These primarily produced phenolic acids are prone to secondary reactions such as p-oxidation, demethylation and dehydroxylation. The phenolic acids are absorbed and excreted with urine [5]. In conclusion, the effects of dietary flavonoids are expected to be dependent on the absolute content and the specificflavonoidspresent in the diet. Flavonoids are highly metabolised both at the intestinal level and in the liver. The pattem of glycosilation and the presence of other food components may also influence its bioavalability. Therefore, the results of epidemiological studies probably reflect the effects of long lasting metabolites whose pharmacology has been poorly analysed. At present, only the antioxidant capacities of the circulating metabolites of quercetin (glucurono-sufo conjugates of isorhamnetin and quercetin) were tested, showing that they also exhibit antioxidant properties [44]. ANTIATHEROGENIC EFFECTS Oxidised LDL are key factors ki the pathogenesis of atherosclerosis, the underlying cause of coronary heart disease, stroke, and peripheral arterial disease [45-47]. Endothelial cells and macrophages accelerate LDL oxidation, a process that is catalysed by heavy metals as copper and iron. Oxidised LDL are chemotactic for macrophages promoting their residence in the intima, cytotoxic to the endothelium, chemoattractant for monocytes, rapidly absorbed by macrophages leading to foam cell formation, appearance of fatty streak lesions and ultimately to growth of atherosclerotic plaques [48]. There is experimental evidence that certain antioxidants inhibit the formation of foam cells and slow the progression of atherosclerosis. Flavonoids can impair the generation of and neutralise

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superoxide anion [49], hydroxyl radical and lipid peroxide radicals [50]. These antioxidant properties of flavonoids, especially toward LDL, have attracted quite a lot of attention in recent years as a mechanism to explain the possible protective effect of these compounds against coronary heart disease. Antioxidant properties Flavonoids have the ability to act as antioxidants by a free radical scavenging mechanism with the formation of less reactive flavonoid phenoxyl radicals [Eq. (1) and (2)]. On the other hand, through their known chelating ability these compounds may inactivate transition metals ions (iron, copper), thereby suppressing the superoxide-driven Fenton Reaction, Eqs. (3) and (4), which is currently believed to be the most important route to activate oxygen species [51]. ROO- + Fl-OH -^ ROOH + Fl-OHO- -H Fl-OH -> H2O + Fl-0O2" + Fe (III) -> O2 -H Fe (II) Fe (II) + H2O2 -> Fe (III) + HO- + HO"

(1) (2) (3) (4)

There is much discussion in the literature regarding the relative contributions of these two mechanisms [52]. It is widely believed that the antioxidant ability of flavonoids reside mainly in their ability to donate hydrogen atoms and thereby scavenge the free radicals generated during lipid peroxidation. Despite the early realisation that the structures of these flavonoids allow them to form heavy metal complexes, metal chelation has generally been regarded to play a minor role in the antioxidant activity of these compounds and, hence, has not been intensively studied [53]. In fact, flavonoids inhibited LDL oxidation induced by ultraviolet light in the absence of iron and copper [54]. In general, optimal antioxidant activity offlavonoidis associated with the presence of multiple phenolic groups (hydroxyl groups increase the antioxidant activity, whereas methoxy groups suppress it), a carbonyl group at C-4, and free C3 and C5 hydroxyl groups. All these structural features are summarised in Fig. (2).

574

OH

O

B

o

P

^- H

Fig. (2). Structural criteria for effective free radical scavenger activity.

Substitution patterns on the B-rings appear to be the most important contributors. The 3*,4'-orthodihydroxy structure in the B rings confers the highest ability to the aryloxyl radical formed and participates in electron delocalization. A hydroxyl group at C-3 position is also beneficial to the

575

ability of flavonoids to inhibit lipid peroxidation [51]. The maximimi radical scavenging potential can be achieved by the combination of the 5-hydroxyl group in the A ring with the 3-OH group, the 2,3 double bond and the 40X0 function in the C ring. The 2,3-double bound (in conjunction with the 4-0X0 function) and the 3-hydroxyl group in the C ring are responsible for delocalization from B ring [50]. Therefore, quercetki, the most common flavonoid which has all these molecular characteristics, is one of the most potent antioxidant flavonoids. However, a number of studies have found prooxidant effects for many of these flavonoids. Because of their ubiquitous nature, the most widely studied have been quercetin and myricetin. Some of their prooxidant properties have been attributed to the fact that they can undergo autooxidation when dissolved in aqueous buffer. The criteria for prooxidant activity of flavonoids remain unclear. Recently, it has been shown in a cell-free system, that prooxidant activities offlavonoidsincrease upon: a) the presence of easily autoxidizable catechol group in B-ring, b) an increase in the total number of hydroxy groups, and c) the conjugation between the A- and B-rings, i.e., the existence of flavone or flavonol structure [55,56]. However, the extracellular production of active oxygen species (superoxide, hydrogen peroxide, and hydroxyl radical) by dietary flavonols is not likely to occur in vivo [57]. Moreover, it has been found that naringenin, taxifoUn and kaempferol offer certain protection against quercetin toxicity, so that it is possible that the prooxidant effects can be minimised by the intake offlavonoidmixtures [58]. Mechanisms of inhibition of LDL oxidation Several mechanisms have been implicated in the protective effect of flavonoids in LDL oxidation. Fig. (3): A) Preservation of endogenous antioxidants. LDL particles contain antioxidants including tocopherols, p-carotene, lycopene, and retinyl stearate. LDL oxidation in vitro exhibits a lag phase corresponding to the time required for the endogenous antioxidants in LDL to be consumed [59]. The water soluble flavonoids (conjugated metabolites) located near the surface of phospholipid bilayers could prevent LDL oxidation by taking up the water soluble free radicals generated by copper through the Fenton reaction, decreasing the consumption of the LDL antioxidants contained ki

576

the lipid-water interface [44,60]. In fact, flavonoids decrease the consumption of endogenous a-tocopherol, thus delaying the increase in LDL uptake and oxidation by macrophages [60]. When flavonoids were added to the LDL solution, once the oxidation process was initiated, their antioxidant effect was related to the presence of vitamin E. When vitamin E was still present in the LDL,flavonoidsprolonged the lag phase for the formation of conjugated dienes, delaying the consumption of vitamin E and, therefore, the whole oxidation process. However, when flavonoids were added later on during the oxidation process, when vitamin E was already consumed, their addition neither regenerated the vitamin nor decreased or stopped the oxidation process. So, the flavonoids decrease LDL oxidation only if they are present in the early stages of the oxidation process, when endogenous antioxidants are still present [60]. B) Flavonoids reduce the formation or release of free radicals. LDL is oxidised by free radicals generated from endothelial cells, monocytederived macrophages, and smooth muscle cells [61]. Several flavonoids inhibit the release of reactive o>Q^gen species by stimulated human leukocytes or neutrophils [62,63] and attenuate this cell-mediated oxidation of LDL when added to the extracellular medium. Under these conditions, it is not clear whether the antioxidant effects of flavonoids were exerted within the lipoprotein particle, in the extracellular medium, or at the cellular level. Macrophage-mediated oxidation of LDL is considered to be of major importance during early atherogenesis, and it depends on the oxidative state of LDL and of the macrophages [61]. The LDL oxidative state is elevated when the ratio poly/mono unsaturated fatty acids is increased, and it is reduced by elevation of LDL-associated antioxidants. Flavonoids inhibit the oxidation of LDL by macrophages with concentrations producing 50% inhibition (IC50) ranging from 1-2 |aM for quercetin, fisetin, morin, and gossypetin to 15-20 |iM for galagin and chrysin [64]. Macrophage-mediated oxidation of LDL is affected by the balance between cellular oxygenases and antioxidants, such as the glutathione system. Flavonoids are known to inhibit cyclo-oxygenase and lipoxygenases. However, it appears that free radicals produced as a byproduct of these enzymes are not essential for LDL oxidation by macrophages [65]. In contrast, the activation of the macrophage NADPH oxidase, the enzyme responsible for the reduction of O2 to 02", can lead to a substantial LDL oxidation [66]. Flavonoids inhibit NADPH-oxidase activity in vitro, leading

577

to a decrease of superoxide anion and hydrogen peroxide production [67], AntiQM
Endothelial cell

'

Oiidant \( Antioxidant

FLAVONOIDN

IL-1,MCP-1

\ \ \

@

^

• ^X-LDK^

Smooth muscle cell Fig. (3). Mechanisms implicated in the protective efifect of flavonoids in LDL oxidation. OXLDL: oxidized LDL; CE: cholesteryl ester; UC: unesterified cholesterol; GSH: glutathione; SOD: superoxide dismutase; ROS: radical oxygen species. Dashed lines represent inhibition.

Inhibition of LDL oxidation by flavonoids has also been observed in in vivo experiments. Consumption of quercetin and catechin by atherosclerotic apoliproprotein E-deficient (Eo) mice resulted in a significant reduction in the development of atherosclerotic lesions along with a reduction in LDL oxidation [68]. Recently, glabridin (20 |ag/mouse/day for a period of 6 week), a lipophilic isoflavan isolated fi^om liquorice root, has been reported to reduce by 50% the atherosclerotic lesion area in apolipoproteiti E-deficient mice [69]. This inhibition of atherosclerosis was also related to reduced macrophage-mediated oxidation of LDL. Glabridin may possess this property not only because of its binding to LDL but also by its accumulation in macrophages, where it reduces cellular oxidative stress by inhibiting protein kinase C (PKC) and, hence.

578

the translocation of P-47, a cytosolic component of NADPH oxidase, reducing NADPH oxidase activation. Glabridin, such as others flavonoids that inhibited PKC activity, were found to be competitive inhibitors with respect to adenosine triphosphate (ATP) binding. These effects of glabridin were found to be specific to the glabridin hydroxyl groups on the isoflavan B ring, since 2',4'-o-dimethylglabridin was inactive [69], In endothelial cells, the main source of Oa*" (at least in cultured bovine coronary endothelium) is the membrane-associated NADH-oxidoreductase [70], that is also inhibited byflavonoids[71]. Therefore, at least in vitro, the protective effects offlavonoidsagainst LDL against oxidation depend on their structural properties in terms of the response of the particular flavonoid to copper and iron ions, whether chelation or oxidation, their partitioning abilities between the aqueous compartment and the lipophilic environment within the LDL particle, their hydrogen-donating antioxidant properties and their capacity to reduce the formation or release offireeradicals by the cells (probably related to the inhibition of NAD(P)H-oxidoreductase and/or PKC activity). Inhibition of LDL oxidation by tea and wine flavonoids In China and Japan, rates of cigarette smoking are very high and LDL samplesfi-omcigarette smokers have been shown to be more susceptible to oxidative modification than samples from non-smokers. Nonetheless, mortality ratesfi-omcoronary artery disease are much lower in China and Japan than in the West (Far East paradox). The relative low average plasma LDL concentrations and ratios of LDL to high-density lipoprotein (HDL) in these populations may accoimt, in part, for the low coronary artery disease mortality rates. Furthermore, most people in China and Japan drink large quantities of green tea. Teaflavonoids,of which catechin and theaflavins are the two major groups, may play a significant role in this socalled Far East paradox. Ishikawa and co-workers [72] reported, in an in vitro study, that teaflavonoids,associated on the surface of or within LDL particles, inhibited both copper-catalysed and mouse peritoneal macrophages oxidation of LDL in the following order: quercetin > theaflavin > myricetin > a-tocopherol. In the same study, serum lipids and vitamin E concentrations did not change in 14 healthy volunteers consuming 750 mL black tea/day for 4 weeks as compared with 8 control

579

volunteers. However, LDL oxidation was significantly delayed after the subjects had consumed tea for 4 weeks. As mentioned above, in the Rotterdam study, teaflavonoidshave been implicated in the reduction of severe aortic atherosclerosis [33]. It was postulated that the inhibition of LDL oxidation by red wine may help to e5q)lain the French paradox. Since Frankel and co-workers [73] showed that red wine inhibit the oxidation of LDL, several in vitro studies have confirmed thisfinding[26,74,75]. There is controversy over whether or not the consumption of red wine by humans reduces the oxidation of LDL ex vivo. While several studies found resistance of LDL oxidation after 2 weeks of red wine consimiption in healthy subjects [75,76], others found no effect on the oxidation of LDL ex vivo [26,77]. Recently, Stein and coworkers [78] showed that short-term ingestion of purple grape juice (7.7 ± 1.2 mL/Kg/day for 14 days) reduced the susceptibility to oxidation in coronary artery disease patients and that this is a potential mechanism by which flavonoids in purple grape products may prevent cardiovascular events, independent of alcohol content. Protection from LDL-induced toxicity. As mentioned above, oxidised LDL is cytotoxic for the cells implicated in atherosclerotic process. Besides of the effects offlavonoidsto inhibit LDL oxidation, these compoimds were also able to prevent directly the cytotoxic effect of oxidised LDL at the cellular level [79]. In fact, incubating human endothelial cells in the absence or presence of genistein, a tyrosine kinase inhibitor derived fi-om a soy diet, and challenging the cells with already oxidised lipoprotein revealed that, in addition to its antioxidative potential during LDL oxidating processes, genistein effectively protected the vascular cells fi'om damage by oxidised lipoproteins. Genistein was foimd to block upregulation of two tyrosine-phosphorylated proteins of 132 and 69 kDa in endothelial cells induced by oxidised LDL. However, parallel experiments with the inactive analogue to inhibit protein tyrosin kinase activity daidzein, showed that the cytoprotective effect of the isoflavones seems not to be dependent on tyrosine phosphorylation [80]. High concentrations of myricetin and gossypetin (> 100 pM), can modify LDL increasing its uptake by macrophages by a non oxidative mechanism (non depletion of a-tocopherol in presence of myricetin was

580

observed) due to the aggregation of LDL particles caused by the covalent crosslinking of apo B-lOO molecules [81], The question arises as to whether or not theseflavonoidsmay modify LDL in vivo. This possibility seems unlikely as theseflavonoids,which are not the mainflavonoidsin the diet, probably do not reach plasma concentrations high enough to exert their effects. In addition, the modification of LDL by myricetin is prevented by serum, possibly because serum proteins, rather than the apo B-lOO of LDL, bind the bulk of myricetin. Thus, ifflavonoidsdo interact with LDL in vivo, they are likely to act by inhibiting its oxidation rather than modifying the LDL by themselves [64]. In conclusion, flavonoids together with others antioxidants constitute two lines of defense in protecting cells against injury owing to oxidation of LDL: a) at the LDL level, by inhibiting the LDL oxidation and the subsequent cytotoxicity, and b) at the cellular level, by protecting the cells directly, i.e., by increasing their resistance against the cytotoxic effect of oxidised LDL. Effects on adhesion and chemoattractant molecules Recent evidence suggests that atherosclerosis is a chronic inflammatory process. The recruitment of mononuclear leukocytes and formation of intimal macrophage-rich lesions at specific sites of the arterial tree are key events in atherogenesis. Alterations of chemotactic and adhesive properties of the endothelium play an important role in this process [82]. Quercetin has been reported to inhibit the expression in glomerular cells of monocyte chemoattractant protein-1 (MCP-1) [83] a potent chemoattractant for circulating monocytes. Red wine reduced MCP-1 mRNA and protein expression in abdominal aorta of cholesterol fed rabbits after balloon injury and this effect was associated with a reduced neointimal hyperplasia [84]. The antioxidant-mediated inhibition of nuclear factor K B (NFKB) and the subsequent non selective reduction of cytokine transcription have been suggested to be responsible for these effects [85], Additionally, quercetin downregulated both phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis fector-a (TNFa)-induced intercellular adhesion molecule-1 (ICAM-l) expression in himian endothelial cells [86].

581

EFFECTS OF FLAVONOIDS ON PLATELET AGGREGATION Platelet-blood vessel interactions are implicated in the development of thrombosis and atherosclerosis [87]. Since Gryglewski and co-workers [88] showed that quercetin bound to platelets membranes, dispersed platelet thrombi adhering to rabbit aortic endothelium and prevented platelet adhesion and aggregation, several others researchers have established that particular flavonoids are effective inhibitors of platelet adhesion, aggregation and serotonin secretion (for a review see [2,5,8]). The degree of inhibition is dependent on the type of inducer and on the structure of the flavonoid. At 30 |LIM, fisetin, kaempferol or quercetin inhibit the platelet aggregation induced by arachidonic acid, whereas morin and myricetin are effective only at concentrations greater than 150 |nM [89]. Aggregations induced by adenosine diphosphate (ADP) and especially by platelet activating factor (PAF) are less affected by flavonoids, except by myricetin. Quercetin, fisetin and myricetin show a more pronounced inhibitory effect against collagen-induced aggregation. The flavonoid component of grape products, including red wine and purple grape juice inhibited collagen-mediated platelet aggregation [90]. This variety of flavonoids effects against different inducers influencing several pathways involved in platelet function, strongly suggests that the antiaggregatory effects of flavonoids cannot be attributed to a single biochemical mechanism [89]. Several mechanisms have been implicated on the inhibition of platelet aggregation induced by flavonoids: A) Inhibition of platelet phosphodiesterases (PDEs) [91], Quercetin and myricetin potentiated the anti-aggregatory action of prostacyclin (PGI2), a potent stimulator of platelet adenylate cyclase synthesised by the vascular endotheliimi, on ADP-induced platelet aggregation in washed himian platelets, and the elevation of platelet cyclic adenosine monophosphate (cAMP) elicited by PGI2 [89,92,93]. These effects are probably due to an inhibition of PDEs. As suggested by Ferrell and co-workers [92], this inhibition arisesfromthe similarity between the pyranone ring of flavonoids and the pyrimidine ring of adenine. B) Inhibition of arachidonic acid metabolisnL Most flavonoids tested (flavone, chrysin, phloretin,flavanone,apigenin and kaempferol) inhibited

582

cyclooxygenase activity although with marked differences ki potency. Myricetin and quercetin, however blocked both cyclooxygenase and lipoxygenase pathways at high concentrations (50 |LIM). At low concentrations (10 ^M) lipoxygenase was the primary target of inhibition [89]. Tzeng and co-workers [94], determined that flavonoids (iBsetin, kaempferol, morin and quercetin) were antiaggregant because they inhibited thromboxane B2 (TXB2) formation and also antagonised platelet aggregation induced by U46619, a thromboxane A2 (TXA2) mimetic receptor agonist. C) The antioxidant actions of flavonoids appear to participate in their antithrombotic action. Flavonoids bind to platelet membranes and scavenge platelet-generated lipid peroxides and free radicals, restoring the biosynthesis and the action of endothelial prostacyclin and nitric oxide (NO), respectively [49,88]. D) Other mechanisms. Genistein apparently can affect platelet function in ways possibly unrelated to protein tyrosine phosphorylation [95]. For example, platelet tyrosine phosphorylation stimulated by thrombin was only weakly ^ected by genistein but it inhibited platelet aggregation and serotonin secretion. On the other hand, this isoflavone suppressed platelet aggregation, serotonin secretion and protein phosphorylation triggered by collagen and U46619. Moreover, daidzein, an inactive analogue, is capable. Eke genistein, of inhibiting the binding of U46619 to platelets with an associated reduction in collagen- or U46619-induced platelet responses. An inhibition of the intracellular mobilisation of Ca^"^ and of its influx across the plasma membrane could also play a role [96,97]. Inhibition of platelet aggregation by dietary flavonoid has been suggested to play a role in the protective effects of flavonoid-containing foods on ischaemic diseases [6]. As described above, the most abundant dietary flavonoid, quercetin, inhibited platelet aggregation in vitro (13-150 jiM) [94,98,99] and, in laboratory animals (5-50 mg kg"^) [100,101]. However, the effects of quercetin supplementation on human platelet aggregation have been poorly evaluated. Conquer and co-workers [102], investigated in a double-blind study with healthy volunteers, the influence of four capsules daily of a quercetin-containing supplement (1 g quercetin/day) or rice flour placebo for 28 days on plasma quercetin status and risk factors for heart disease. Quercetin intakes were - 50-fold greater than the dietary intakes associated with lower coronary heart disease

583

mortality on the basis of epidemiological studies. Subjects consuming quercetin-containing capsules had plasma quercetin concentrations ^ 23fold higher than those of subjects consimiing the control capsules. However, they did not observe any inhibition of either platelet aggregation or platelet release of TXA2. This may be due to the fact that the concentration of quercetin in the plasma of supplemented subjects (1.5 |j.M) did not reach high enough levels to exert an inhibitory effect on platelet activity. So, inhibition of platelet aggregation appears to require a minimum of 10 |LIM quercetia Similar results have been reported by Janssen and co-workers [103] in subjects consuming 220 g onions daily (containing 114 mg quercetin) or 5 g dried parsley daily (containing 84 mg of apigenin). These results suggests that the protective effect of food containing quercetin may be mediated via effects on risk factors other than inhibition of platelet aggregation and/or to fectors other than quercetin contained in those foods. VASCULAR EFFECTS OF FLAVONOIDS Another potential mechanism by which flavonoids may be protective in cardiovascular diseases is by their direct effects on vascular smooth muscle cells either as vasodilators or as inhibitors of proliferation. However, vasoconstrictor effects have also been reported for some flavonoids. Various natural, chemically modified and mixtures of flavonoids are widely used therapeutically as venous protective or venotonic drugs in chronic venous insufficiency and haemorrhoidal attacks. Flavonoids have been found to inhibit increased vessel wall permeability,fluidchanges in the capillary bed and diffusion of plasma proteins. In addition, they may exert a protective effect on the perivascular tissues due to their antihyaluronidase effect and the inhibition of lysine oxidase (producing crosslinks in collagen and elastin) and lysosomal hydrolases (degrade glycosamines). All these effects may account for the venotonic effects of these drugs [5]. However, the venous effects offlavonoidsare out of the scope of the present review. Vasodilator effects Several studies foimd that flavonoids and other polyphenolic compounds

584

present in food exhibit vasodilator effects in different isolated vascular preparations [104-117]. The vasorelaxant response evoked byflavonoidsis considered mainly endothelinm-independent [108-111,115-117]. However, others investigators have found that some polyphenolic compounds [104106] produce endothelium-dependent vasorelaxation, Endothelium-dependent relaxation

The vascular endothelium lies at the interface between the circulating blood cells and the vascular smooth muscle cells and plays a crucial role in regulating blood flow and vascular tone [118]. In addition, vascular endothelium can synthesise and release different relaxant &ctors such as NO, prostanoid derivatives and the so called endothelium-derived hyperpolarizing fector [119] and also endothelium-derived contracting fectors (endothelins, vasoconstrictor prostanoids and superoxide anions) [120]. In many vascular pathologies, such as hypertension, diabetes and atherosclerosis, endothelium-dependent vasorelaxation to different vasodilator agonists is reduced. One of the mechanisms accounting for this endothelial dysfunction is a decreased release of NO [121]. Certain wines, grape juice, grape skin extracts [111], red wine polyphenol conqx)unds (RWPCs) [104-106] and defined polyphenols contained in wine, such as, leucocyanidol [104], delphinidin and oligomeric condensed tannins [105] cause endothelium-dependent vasorelaxation in vitro. The imderlying mechanisms involved in this vasodilator effect are far fi-om clear. Fitzpatrick and co-workers [111] foimd that certain wines, grape juice and grape skin extracts, which are known to contain polyphenols, can induce endothelixmi-dependent vasorelaxation of rat aorta probably via NO release, enhanced biological activity of NO or protection against breakdown by superoxide anions. As described above, flavonoids scavenge superoxide anions protecting the inactivation of NO induced by this fi'ee radical [50]. In feet, the synthetic flavonoid 6, 7-dimethoxy-8-methyl-3', 4', 5-trihydroxyflavone protects endothelium-dependent relaxation in rabbit ear and basilar arteries fi'om high levels of superoxide anion possibly by scavenging superoxide anion [113]. However, very recently, the group of Stoclet showed that a red wine extract enriched in RWPCs induced endothelium-dependent relaxation in rat aorta via an enhancement of endothelial NO synthesis, rather than enhanced biological activity of NO or

585

protection against breakdown by superoxide anions [104,106]. This effect was aboUshed after removal of extracellular calcium or in the presence of

endothelial cell

Flavonoid

NO

vascular smooth muscle cell

<3jAD(P)H

Flavonoid Relaxation

Fig. (4). Vasodilatory mechanisms of flavonoids. RWPC: red wine polyphenolic compounds; NO; nitric oxide; NOSe: nitric oxide synthase endothelial; O2': superoxide anions; OONO' peroxynitrites; PKC: protein kinase C; AC: adenylate cyclase; GC: guanylate cyclase; PDE phosphodiesterase.

586

the Ca-entry blocker lanthanum, indicating an extracellular calciumdependent mechanism. This effect was inhibited by N-ethylmaleimide, a sulphydryl alkylating agent, that is thought to interfere with plasma membrane-located guanosine triphosphate (GTP)-binding proteins, suggesting that such coupling proteins may be involved in the signal transduction of RWPCs-induced endothelial NO production. However, neither pertussis toxin or cholera toxin-sensitive G proteins, the phospholipase C or phospholipase A2 (PLA2) pathways, PKC, or tyrosine kinases were implicated in the effects of RWPCs [106], Fig. (4). Recently, ingestion of purple juice for 14 days has been shown to improve endothelial function, as measured byflow-inducedvasodilation, in patients with coronary artery disease [78]. The authors attributed this effect to theflavonoidiccontent of the purple juice. These benefits were observed despite use of antioxidant vitamins, lipid lowering medications and small increases in total cholesterol and triglycerides levels. Endothelium-independent relaxation

Structure-activity relationship. The endothelium-independent vasodilator effects showed by flavonoids are related to the structure of the compound tested. Structure- activity relationships have been studied to flavonoids selectedfi-omfivegroups:flavonols,flavones,flavanones,isoflavones, and flavanols in rat isolated aorta on the contractions induced by noradrenaline, KCl and the phorbol ester derivative PMA, as well as the interactions of theseflavonoidswith isoprenaline and sodiimi nitroprusside. Table (2). From these results, several structural features are involved in the vasodilator activity [108,114]: A) The lower activity erfiibited by flavanols against the contractions induced by any of the agonists tested, suggests that the 4-carbonyl group is required for the vasodilator activity. B) The pattem of hydroxylation in B-rings is very important for the activity, (i) The absence or the methylation of the hydro^g^l group in position 3' accoimts for a lower vasodilator potency. In contrast, the lack of the 3'-hidroxyl group is related with a potentiation of the effects in the presence of sodium nitroprusside suggesting a reduced inhibitory effect on cyclic guanosine monophosphate (cGMP) PDE activity, (ii) Morin, with a

587

Table 2. ICsa values (^M) of differentflavonoidsto inhibit the contractions induced by 80 mM KCl, 10'^ M PMA or 10'^ M noradrenaline (NA) in the absence or in the presence of 10'^ M isoprenaline (NA+ISO) or 10*^ M sodium nitroprusside (NA+SNP)« Data was taken from [108,114]

1 FLAVANOLS 1 Quercetin

NA

NA + ISO

NA + SNP

KO

PMA

10±2

1011

1613

3415*

1112

15±2

811**

911**

3511*

183112

18114

120113*

118110*

309125

473136**

405185

468165*

3318* 4417** 313137

Luteolin

4815

2114*

3413

64110 *

3313*

Apigenin

3913

2713

1812*

4316

94113*

1

2113**

3.310.4**

1813**

3015**

1

1 Kaempfbrol Myricetin

1 1 1

IFLAVANES

Chiysin 1 FLAVANONES Flavanone

6.610.7 115113

63110*

3514**

7619

108110

Naringenin

95114

230165*

59113*

9617

4617*

1

Hesperetin

96110

128148

10613

138123

88112

1

1ISOFLAVONES Genistein

5412

3212

42113

2613

2115

1 CATECHINS (+)-Cathechm

5901100

4701150

620170

>1000

> 1000

1 (->Epicatfiechm

5301100

5401120

380190

>1000

>1000

1

*P < 0.05 and P < 0.01 compared to NA

2',4' substitution pattern, is less potent than quercetin, with a 3 \ 4' orientation. Thus, the ortho orientation on the B ring is important for the vasodilator effect, (iii) The presence of three contiguous hydroxyls in the B ring (myricetin) potentiates, at low concentrations, the contractions induced by the agonists tested. In fact, potentiation of the contractile response exerted byflavonoidspossessing three contiguous hydroxyls on either A (5,6,7-trihydroxy; baicalein, scuteUarein, and quercetagetin) or B (3\4\ S'-trihydroxy; myricetin) rings has been observed in rat tail and femoral artery to both transmural nerve stimulation-evoked and exogenous agonist-evoked contractile response [122]. C) The lower relaxant activity exhibited by flavanones against the contractions induced by any of the agonists tested, as compared with the others flavonoids, suggests that the presence of a double linkage C2-C3

588

that gives a coplanar conformation of the benzopyran ring system (i.e., flavonol, flavone, and isoflavonoid) is required for the vasodilator activity. Moreover,flavanonesare more potent agents for induction of relaxation in tissues contracted by these agonist than are flavanols (catechin or epicatechin, IC50 > 500 |LIM) , which confirms that the presence of a 4carbonyl group is an essential structural feature for the vasodilator effect of flavonoids. D) The isoflavonoid genistein also inhibited the contractions induced by noradrenaline, KCl or PMA with similar IC50 values than that of flavonok and flavones, which suggests that position 2 or 3 of the phenyl ring is not essential for the vasodilator activity. Mechanism of action. Several mechanisms have been proposed for the vasodilator eflfects of flavonoids including inhibition of A) PKC, B) Ca^^ entry, C) cyclic nucleotide PDEs and D) tyrosin kinases. A) Inhibition of PKC. PKC has been proposed to play a key role in the maintenance of tonic contractions of vascular smooth muscle [123]. Phorbol esters (i.e., PMA) activate PKC and induce a slowly developing sustained contraction in rat aorta without changing intracellular Ca^^ concentration, possibly by increasing the Ca^^ sensitivity of contractile proteins [110,124]. PKCfromrat brain is inhibited by plant flavonoids in a concentration-dependent manner depending on the flavonoids structure, being competitive inhibitors with respect to ATP binding, and noncompetitive with respect to protein substrate, and the inhibition was independent of Ca^ phosphoUpid and enzyme activator [125]. The minimal essential features required for PKC inhibition includes a planar flavone structure with free hydroxil substituents at positions 3* , 4* and 7, i.e. similar structural determinants as those for smooth muscle relaxation as described above. Furthermore, Fig (5) shows that there is a good correlation between the potency of flavonoids to relax previously contracted aortae, particularly when activated by PMA [108,114] and their potency to inhibit PKC from rat brain reported by Ferriola and co-workers [125]. However, these drugs are rather unspecific since they also inhibit a number of protein kinases such cAMP-dependent protein kinase, myosin Ught kinase and phosphatidylinositol-4-phosphatase kinase [126,127]. As previously described for other PKC inhibitors in rat aortae [128,129], most flavonoids inhibited the tonic contraction induced by PKC activator, PMA,

589

to a similar extent to the contractions evoked by noradrenaline or KCl. In contrast, in rat aorta, drugs acting through other mechanisms (i.e. activators of guanylate and adenylate cyclase, PDE inhibitors, K^ channel openers) were more potent on contractions induced by a-adrenoceptor activation than by phorbol esters [130]. Quercetin inhibited to a similar extent the contractile response induced by PMA in the presence or in the absence of extracellular Ca^^ and after depletion of noradrenaline-sensitive intracellular stores, suggesting that the main vasodilator effects of quercetin may be related to the inhibition of PKC [109]. Unfortunately, there is no information concerning the effects of quercetin on the various PKC isoenzymes in vascular tissues. 100 LutMlin Q. C 0

2

60

Qu«ro«tin

S5

0 eo 100 % R e l a x a t i o n o n the P M A - i n d u c e d contractions

Fig. (5). Correlation between the potency of flavonoids to relax aortae previously contracted with phorbol 12-myristate 13-acetate (PMA) and their potency to inhibit PKC from rat brain. Data taken [108, 114, 125].

Quercetin also inhibited rat aortic contractions induced by noradrenaline, S-hydroxytryptamine, and cajBFeine in Ca^^ -free media [110]. PMA enhanced this transient contraction elicited by noradrenaline, an effect that was abolished by quercetin. Furthermore, quercetin or the PKC inhibitor staurosporine, had no effect on '^^Ca^^ efQux vmder resting conditions or when stimulated by noradrenaline. So, the inhibitory effects of quercetin on phasic contractile response induced by receptor agonists in

590

Ca^^ -free media do not seem to be related to changes in cellular Ca^^ regulation but to an inhibitory effect on the regulation of contractile proteins, an effect probably related to the decreased sensitivity of contractile elements to Ca^^ that apparently resulted from the inhibitory effects of quercetin on protein kinases [110]. B) Inhibition of Ca^^ entry. KCl-induced contractions are the result of an increased Ca^^ influx through voltage-stimulated type-L Ca^"^ channels, and are specifically inhibited by Ca^"^ antagonists [131]. Thus, the inhibitory effects of flavonoids on the contractile responses induced by high KCl or by increases in extracellular Ca^^ in high KCl Ca^^-free solution [108, 114, 116] could be attributed, at least partly, to a blockade of Ca^^ entry through voltage-stimulated L-type Ca^^ channels. Moreover, quercetin suppressed the spontaneous myogenic contractions recorded in portal veins [110] and apigenin reduced the high KCl- and noradrenaline-stimulated "^^Ca^^ influx in rat aorta [115]. Therefore, flavonoids may reduce Ca^^entry into smooth muscle cells through both voltage- and receptor operated Ca^^ channels. C) Inhibition of cyclic nucleotide PDEs. Elevation in cellular cyclic nucleotides induces vascular smooth muscle relaxation [132]. The cellular accumulation of cAMP and cGMP depends upon the rate of their synthesis and their breakdown. The latter is achieved by cyclic nucleotide PDEs that have been classified into seven femilies [133]. Some flavonoids (apigenin, kaempferol, fisetin and quercetin) produce an inhibitory action on cyclic nucleotide PDEs [134,135] which may collaborate in the inhibitory effect on platelet aggregation [93] and vascular smooth muscle relaxation [107,108,114]. The ability of isoprenaline or sodium nitroprusside to potentiate the relaxant effects of flavonoids has been used as a fimctional test to investigate the inhibitory effects of these drugs on cAMP or cGMP PDEs, respectively [136]. Pretreatment with isoprenaline produced a mild potentiation of the vasodilator effects of kaempferol, luteolin and flavanone, which suggests a possible role of cAMP on their inhibitory effects on the noradrenaline-induced contractions. Only the vasodilator effects of flavonoids without 3'-hydroxyl (kaen^)ferol, morin, pentamethylquercetin, apigenin, chrysin, flavanone, naringenin) were potentiated by pretreatment with sodium nitroprusside, suggesting that their relaxant effects could be partly related to increased levels of cGMP [108,114]. However, the potentiation by sodium nitroprusside did not

591

parallel potentiation by isoprenaline, which suggests that these later drugs are probably inhibiting the GMP-dependent iisoforms of PDEs (PDEl and/or PDES). D) Inhibition of tyrosin kinases. The isoflavonoid genistein inhibits tyrosine kinases [137] and this eflfect has been suggested to contribute to its vasodilator effect [138]. However, the relaxant action of genistein on KC1-, noradrenaline-, or PMA-induced contraction was not likely to be due to an inhibitory action of tyrosine kinase because daidzein, the inactive analogue, was slightly less potent than genistein to inhibit noradrenaline and KCl induced contractions in rat mesenteric resistance arteries [139]. Several other mechanisms for genistein induced vasodilator effects have been proposed: inhibition of Ca^^ entry or Ca^^ release induced by agonists [140] a direct effect upon the contractile proteins [96] and inhibition of other kinases, such as PKC [114,141], On the other hand, genistein, at concentration that inhibits tyrosine kinase activity (3 ^M), did not affect baseline diameter of the basilar artery in vivo but inhibited vasodilatation in response to acetylcholine and bradykinin without affecting vasodilatation produced by sodium nitroprusside. Because the vasorelaxant effect produced by these agonists is mediated primarily by NO, activation of tyrosine kinase may have an important role in NO production in the basilar artery in vivo [142]. Thus, it was postulated thatflavonoidswhich inhibit tyrosine kinase activity may interfere with the endothelium-dependent vasodilator response induced by agonists by inhibition of endothelial cells NO production. In conclusion, flavonoids exert endotheliimi-independent vasodilator effects in isolated vascular smooth muscles that are related to the structure of the compound tested. The main vasodilator mechanism of flavonoids seems to be related to the inhibition of PKC, although an inhibitory effect on cyclic nucleotide PDEs and Ca^^ uptake and other protein kinases may also contribute to these actions. Fig. (4). Vasoconstrictor effects Some flavonoids (baicalein, myricetin, quercetagenin and scutellarein) under certain conditions may exert vasoconstrictor rather than vasodilator effects [122]. As described above, myricetin has been shown to exhibit a biphasic response in pre-contracted rat thoracic aorta. At low

592

concentrations (< 50 |LIM), it potentiates the response to different vasoconstrictor agents in rat isolated aorta, whereas at higher concentrations, it exerts a vasorelaxant eflFects on precontracted vessels [114]. A potentiation of the contractile response induced by transmural stimulation and by different adrenergic agonists has been reported for other flavonoids possessing three contiguous hydroxyls. Recently, we have characterised the contractile effect induced by myricetin in rat thoracic rings and the underlying mechanisms [143]. Myricetin endothelial cell

Contraction Fig. (6). Vasoconstrictor mechanism of myricetin. PLA2: phospholipase A2; PL: phospholipids; AA: arachidonic acid; COX: cyclooxygenase; PGG2: prostaglandin G2; PGES: prostaglandin endoperoxide synthase; PGH2: prostaglandin H2; TXS: Thromboxane synthase; TXA2: Thromboxane A2; Tp: Thromboxane receptor; PKC: protein kinase C.

593

Myricetin induced endothelium-dependent contractile response which developed slowly, reached a peak within 6 min and then declined progressively. Myricetin induced contractions were almost abolished by the phospholipase A2 inhibitor, quinacrine, the cyclo-oxygenase inhibitor, indomethacin, the thromboxane synthase inhibitor, dazoxiben, and the putative TXAi/prostaglandin endoperoxide receptor antagonist, ifetroban. These contractions were also abolished in Ca^^-free medium. In cultured bovine aortic endothelial cells (BAEC), myricetin produced an increase in cytosolic free calcium concentration which was abolished after removal of extracellular Ca^^ in the mediimL Myricetin also increased TXB2 production both in aorta with and without endothelium and in BAEC, and this effect was abolished in Ca^^ -free media and by indomethacin. Taken together, these results suggests. Fig. (6), that myricetin stimulates Ca^"^ influx and subsequently triggers the activation of the phospholipase A2 and cyclooxygenase pathways releasing TXA2 from the endothelium to contract rat aortic rings. The latter response occurs via the activation of Tp receptors on vascular smooth muscle cells. Even when there is no information concerning the bioavalability and plasma levels of myricetin in humans, myricetin is present in the diet in smaller amounts than the more common flavonoid quercetin [3]. Thus, it is unlikely that in subjects on a normal diet, plasma concentrations of myricetin reach levels high enough to exert a vasoconstrictor effect. However, it cannot be excluded that these levels might be reached after selected meals (e.g. broad beans and red wine) with a high content of myricetin. Effects on vascular cell proliferation The greater potential for growth of vascular smooth muscle cells is one of the key abnormalities in the development of atherosclerosis. However, few studies have analysed the effects of flavonoids on vascular cell proliferation. Huang and co-workers [144], have shown that baicalein inhibits the proliferation of smooth vascular myocytes induced by fetal calf serum or by platelet-derived growth factor (PDGF). Additionally, the isoflavonoid genistein has been reported to inhibit endothelial cell proliferation and in vitro angiogenesis at concentrations giving half maximal inhibition of 5 and 150 |iM, respectively [145]. These effects may partly account for the anticarcinogenic effects of thisflavonoidby inhibiting

594

neovascularization of solid tumours. CARDIAC EFFECTS The effects of some flavonoids have been analysed in isolated cardiac preparations. Quercetin and 3-methylquercetin exerted a positive chronotropic effects in isolated guinea-pig atria and enhanced the positive chronotropic effects of isoproterenol which could be related to the inhibition of cAMP PDE 3 [146]. Luteolin also increased the amplitude and rate of contractions in spontaneously beating atria and in isolated guineapig hearts [147]. Apigenin also produced a positive chronotropic response in isolated rat atria probably as a result of a reduction in noradrenaline uptake as well as in monoamineoxidase activity [148]. Occhiuto and coworkers [149] compared 16 flavones in guinea pig and rat heart on myocardial ischaemia and in several arrhythmia models (pytressin-induced coronary spasm, coronary artery ligation-reperfiision and hyperkinetic ventricular arrhythmias induced by reperfusion). Most of these compounds exerted an antiarrhythmic effect and significantly reduced the area of ischaemia and the aglycones (apigenin, luteolin) being less effective than the corresponding glycosides (rhofolin, vitexin, and orientin). ANTIHYPERTENSIVE EFFECTS OF FLAVONOIDS Hypertension is an important risk factor for coronary heart disease and stroke. Since ancient times, hypertensive patients have been treated orally with plant extracts based on folk medicine. However, and despite their in vitro vasodilator effects, Uttle information about the protective effects of flavonoids on hypertension is available in the literature. Flavonoids have been considered as active principles of several antihypertensive plant extracts (e.g. rhamnoglycoside of Umocitrin isolatedfi'omCitrus limonum^ kaempferol 4'-0-glucose and hyperin fi*om Euphorbia maddeni, moracenins ifrom Morus alba, procyanidin glycoside fi-om Rhamnus lycioides) [150,151]. In all cases, only the acute antihypertensive effects after i.v. administration in anaesthetised normotensive and/or hypertensive animals have been described. In addition to the direct vasodilator effects discussed above, the inhibition of angiotensin-converting enzyme reported

595

for some flavonoids [151,152] could be involved in these antihypertensive properties. Rutin had no acute effect on arterial blood pressure of guinea pigs and did not modify the hypertensive effect of noradrenaline or the vasodilator effect of acetylcholine [153]. In normotensive anaesthetised dogs, rhoifolin, apiin and vitexin decreased mean arterial aortic and pulmonary pressures [154]. In anaesthetised guinea-pigs Luteolin (0.3 mg/kg iv) caused a brief increase in systolic and diastolic blood pressure that lasted 4-5 min, while at higher concentration produced a dose-dependent increase in blood pressure [147], This biphasic effect was attributed by the opposite effects of luteolin on the vascular smooth muscle and the heart, at low doses predominates its cardiac stimulatory effect while at high dose its vascular effect would fevour the decrease in blood pressure. Recently, Takizawa and co-workers [155] found that the treatment with the flavonoid baicalein (60 mg Kg'^ s.c.) did not affect the blood pressure in normotensive rats but decreased the blood pressure in rats with angiotensin Il-induced hypertension. The antihypertensive effect of baicalein was prevented by pretreatment with indomethacin and partially reversed by the administration of 5,6-dihydro-PGl2 antiserum which binds PGI2 and block its vasodilator action. Treatment of the hypertensive rats with baicalein also caused selective increases in the rate of conversion of exogenous prostaglandin H2 (PGH2) to PGI2 by aortic rings, release of 6-keto-PGF2a from aortic rings, concentration of 6-keto-PGF2a in blood, and renal excretion of 6-ketoPGF2a. These results suggest a contribution of PGI2 to the acute antihypertensive effect of baicalein in rats with angiotensin Il-induced hypertension. Baicalein may promote PGI2 formation by interfering with the production of some inhibitors of PGI2 synthase, including lipoxygenasederived fatty acid hydroperoxides and reactive oxygen species. We have recently analysed the effects of an oral dose of quercetin, the most abundant dietary flavonoid, on the hypertension, oxidant status and renal, cardiac and vascular alterations induced in rats by chronic inhibition of NO synthesis with N-nitro L-arginine methylester (L-NAME). Administration of this NO synthase inhibitor to rats for six weeks induced a progressive increase in systoUc blood pressure, but concomitant administration of an oral daily dose of quercetin (10 mg Kg"^) inhibited the development of hypertension induced by L-NAME (Duarte et al., 1999, Meth. Find. Exp. Clin. Pharmacol. 21 (suppl. A), 40). Moreover, when

596

hypertension was established after four weeks of L-NAME administration, quercetin (50 mg Kg"^) induced a progressive reduction in systolic arterial pressure which was evident even at the &st week of treatment. Fig. (7). Also, quercetin reduced proteinuria, the increased endothelium-dependent contractions, and mortality in this model of chronic NO deficient rats.

^

180 n

X

E E o* 160 H 3

m

s I.

o

140

**

CO

120 i

Weeks of treatment

F>S* CO' Effects of quercetin (50 mg Kg per day) in the established phase of N-nitro L-arginine methylester (L-NAME)-induced hypertension in rats. L-NAME groups (D); L-NAME plus quercetin ( • ) .

Although the acute vasodilator effects, as shown in in vitro studies (see above), may participate in the antihypertensive effects, the reduced blood pressure persisted even 42-48 h after the last administration of quercetin, when the plasma quercetin concentration and its metabolites fall bellow 25% of the peak post-administration levels [43]. Furthermore, the antihypertensive effects of quercetin did not appear to be related to its antioxidant properties since quercetin did not lower the urinary isoprostane F2a excretion, a prostaglandin-like compound produced in a non enzymatic reaction of arachidonic acid in membrane lipids and superoxide, which is currently used as a reliable marker of oxidative stress. The mechanisms involved in the antihypertensive effects and protection from organ damage

597

produced by quercetin in this model of hypertension remain unclear even when its vasodilator, platelet antiaggregant and antioxidant effects may partly account for. The molecular targets of quercetin include multiple enzymes involved in signal transduction such as protein kinases (PKC or tyrosine kinases), enzymes involved in arachidonic acid metabolism (cyclooxygenase, lipooxygenases, and citochrome P450 monooxygenases), PDEs and Ca^^ ATPases [8]. Furthermore, the chronic results obtained with a single daily oral dose of quercetin probably reflect the effects of its long lasting metabolites (3'- and 4'-methylquercetin, with a half life of approximately 25 h) whose pharmacology has been poorly analysed. Thus, quercetin, and flavonoid in general, are extremely non-selective agents and its preventive effects on cardiovascular diseases may not be attributed to a single mechanism. The supplementation with an oral dose of 1 g of quercetin for 1 month to healthy normotensive subjects has been recently compared to placebo, showing no differences in selected cardiovascular risk fiictors including blood pressure [102]. However, these data do not exclude an antihypertensive effect of quercetin in patients with essential hypertension. In fact, a diet rich in finit and vegetables (and presumably rich in flavonoids) lowered blood pressure in hypertensive but not in normotensive subjects [156,157]. SUMMARY AND CONCLUSIONS Flavonoids constitute a large group of plant metabolites with a broad spectrum of pharmacological effects. Both their pharmacodynamic and pharmacokinetic properties are heterogeneous, depending on their aglycone structure and its pattem of glycosilation. The degree of metabolisation within the intestine and the liver is likely to be important for the in vivo pharmacology of flavonoids. The large amount of flavonoids (and particularly of quercetin) present in the normal human diet make them an important part of the recently coined term nutraceuticals (pharmacologically active compounds more or less ubiquitously found in food). The epidemiological evidence suggests that dietary flavonoids may exert protective effects in cardiovascular diseases such as ischaemic heart disease or stroke. However, prospective randomised clinical trials must be performed to confirm it before supplements containing flavonoids can be

598

used therapeutically in the fotxire. Meanwhile, several mechanisms which coxild account for their possible protective actions have been proposed including antiatherogenic, antioxidant, antiaggregant, vasodilator and antihypertensive properties. Given the lack of selectivity offlavonoids,their actions probably resultfroma combination of all these mechanisms.

ADP ATP cAMP cGMP CHD BAEC GTP HDL IC50 ICAM-1 LDL L-NAME MCP-1

MI NFKB

NO PAF PDGF PDE PGF2a PGH2 PGI2 PKC PLA2 PMA RWPCs TNFa TXA2

= = = = = = = = = = = s=s

= = = = = = = = = = = = = = = =

Adenosine diphosphate Adenosine triphospate cyclic adenosine monophosphate cyclic guanosine monophosphate Coronary Heart Disease Bovine Aortic Endothelial Cells Guanosine triphosphate High Density Lipoprotein Concentration producing 50% inhibition Intercellular Adhesion Molecule 1 Low Density Lipoprotein N-nitro-L-arginine methylester Monocyte Chemoattractant protein 1 Myocardial Infarction Nuclear factor K B Nitric oxide Platelet Activating Factor Platelet Derived Growth Factor Phosphodiesterase Prostaglandin F2 a Prostaglandin H2 Prostacyclin Protein kinase C Phospholipase A2 Phorbol 12-n:tyristate 13-acetate Red Wine Polyphenol Compounds Tumour necrosis factor a Thromboxane A2

599

TXB2

=

Thromboxane B2

ACKNOWLEDGEMENTS The authors are supported by CYCIT (SAF 98-0160, SAF 99-0069 and SAF 98-0157) and CAM (08.4/0019/1998) Cirants. REFERENCES [I] [2] [3] [4] [5] [6] [7]

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Ferrell, J.E.; Chang Sing, P.G.D.; Loew, G.; King, R.; Mansour, J.M.; Mansour, T.E.; MoL Pharmacol, 1979,16, 556-568. Beretz, A.; Stierle, A.; Anton, R.; Cazenave, J.P.; Biochem. Pharmacol, 1981, 31,3597-3600. Tzeng, S.H.; Ko, W.C; Ko, F.N.; Teng, CM.; Thromb, Res., 1991, 64, 91100. Nakashima, S.; Koike, T.; Nozawa, Y.; Mol Pharmacol, 1991, 39,475-480. Ozaki, Y.; Yatomi, Y.; Jinnai, Y.; Kume, S.; Biochem, Pharmacol, 1993, 46, 395-403. Murphy, C.T.; Kellie, S.; Westvsdck, J.; Eur J. Biochem., 1993,216, 639-651. Chung, M.-I.; Gan, K.-H.; Lm, C.-N.; J, Nat. Prod., 1993, 56, 929-934. Xiao, D.; Zhen-Lun, G.; Bai, J.-P.; Wank, Z.; Acta Pharmacol Sinica, 1995, 16,223-226. Xiao, D.;Gu, Z.-L.; Quian, Z.N.; Acta Pharmacol Sinica, 1993,14, 505-508. Nuraliev, I.; Avezov, G.A.; Eksp. Klin. Farmakol, 1992, 55,42-44. Conquer, J.A.; Maiani, G.; Azzini, E.; Raguzzini, A.; Holub, B.J.; J. Nutr., 1998, 128, 593-597. Janssen, P.L.T.M.K.; Mensink, R.P.; Cox, F.J.J.; Harryvan, J.L.; Hovenier, R.; Holhnan, P.C.H.; Katan, M.B.; Am J. Clin. Nutr., 1998,67,255-262. Andriambeloson, E.; Kleschyov, A.L.; MuUer, B.; Beretz, A.; Stoclet, J.C; Andriantsitohama, R.; Br. J. Pharmacol, 1997,120,1053-1058. Andriambeloson, E.; Magnier, C ; Haan-Archipoff, G.; Lobstem, A.; Anton, R.; Beretz, A.; Stoclet, J.C; Andriantsitohaina, R.; J. Nutr., 1998, 128, 23242333. Andriambeloson, E.; Stoclet, J.C; Andriantsitohaina, R.; J. Cardiovasc. Pharmacol, 1999,33,248-254. Beretz, A.; Stoclet, J.C; Anton, R.; Planta Med, 1980, 39,236-237. Duarte, J.: Perez-Vizcaino, F.; Utrilla, M.P.; Jhn6nez, J.; Tamargo, J.; Zarzuelo, A.; Gen. Pharmac, 1993,24, 857-862. Duarte, J.; P6rez-Vizcaino, F.; Zarzuelo, A.; Jimenez, J.; Tamargo, J.; Eur. J. Pharmacol, 1993,239, 1-7. Duarte, J.; P^rez-Vizcaino, F.; Zarzuelo, A.; Jimenez, J.; Tamargo, J.; Eur. J. Pharmacol, 1994,262,149-156. Fitzpatrick, D.F.; Hirschfield, S.L.; Coffey, R.G.; Am. J. Physiol, 1993, 265, H774-H778. Fitzpatrick, D.F.; Hirschfield, S.L.; Ricci, T.; Jantzen, P.; Coflfey, R.G.; J. Cardiovasc. Pharmacol, 1995,26,90-95. Girard, P.; Sercombe, R.; Sercombe, C; Le Lem, G.; Seylaz, J.; Potier, P.; Biochem. Pharmacol, 1995,49,1533-1539. Herrera, M.D.; Zarzuelo, A.; Jim^iez, J.; Marhuenda, E.; Duarte, J.; Gen. Pharmac, 1996,27,273-277. Ko, F.-N.; Huang, T.-F.; Teng, C.-M.; Biochim. Byophys. Acta, 1991, 1115, 69-74. Sanchez de Rojas, V.R.; Somoza, B.; Ortega, T.; Villar, A.M.; Tejerina, T.;

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R.; Schweigerer, L.; Proc. Natl Acad. Sci. USA, 1993,90,2690-2694. Laekeman, G.M.; Cla^^s, M.; Rwangabo, P.C; Herman, A.G.; Vlietinck, AJ.; PlantaMed, 1986, 52,433-437. Abdalla, S.; Zarga, M.A.; Sabri, S.; Phytother. Res., 1994, 8,265-270. Lorenzo, P.S.; Rubio, M.C.; Medina J.H.; Adler-Graschinsky, E.; Eur. J. Pharmacol, 1996; 312, 203-207. Occhiuto, F.; Busa, G.; Ragusa, S.; De Pascuale, A.; Phytother. Res., 1991, 5, 9-14. Villar, A.; Paya, M.; Terencio, M.C.; Fitoterapia, 1986, LVII, 131-145. Terencio, M.C.; Sanz, M.J.; Paya, M.; J. Ethnopharmacol, 1991, 31,109-114. Kameda, K.; Takaku, T.; Okuda, H.; Kimura, Y.; Okuda, T.; Hatano, T.; Agata, I.; Arichi, S.; J. Nat. Prod., 1987, 50,680-683. Yildizoglu-An, N.; Allan, V.M.; Altinkurt, O.; Phytother. Res. 1991, 5,19-23. Occhiuto, F.; Limardi, F.; Phytother Res., 1994, 8, 153-156. Takizawa, H.; DelliPizzi, A.; Nasjletti, A.; Hypertension, 1998, 31, 866-871. Appel, L.J.; Moore, T.J.; Obarzanek, E.; Vollmer, W.M.; Svetkey, L.P.; Sacks, F.M.; Bray, G.A.; Vogt, T.M.; Cutler, J.A.; Windhauser, M.M.; Lin, P.-H.; Karanja, N.A.; New Engl J. Med, 1997, 336, 1117-1124. Moore, T.J.; Vollmer, W.M.; Appel, L.J.; Sacks, F.M.; Svetkey, L.P.; Vogt, T.M.; Conlin, P.R.; Simons-Morton, D.G.; Carter-Edwards, L.; Harsha, D.W.; Hypertension, 1999, 34,472-477.

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EFFECTS OF FLAVONOIDS ON GASTROINTESTINAL DISORDERS J. GALVEZ, F. SANCHEZ DE MEDINA, J. JIMENEZ, A, ZARZUELO Department of Pharmacology, School ofPharmacy, University of Granada, Campus de Cartuja s/n, 18071 Granada (Spain) ABSTRACT: This diapter reviews the curroit litCTature about the effects of flavonoids on diseases of the gastrointestinal tract, namely gastric idcer, diarrhoea, chronic inflammation and cancer. The activity-structure relationships and putative mechanisms of action are detailed whenever possible. Flavonoids exert a protective effect on gastric mucosa against experimental ulceration in which a number of mechanisms may participate, including blockade of acid secreticMi, a direct prostaglandin E2-dependent cytoprotective effect, an antioxidative action and a bactericidal effect on Helycobacter pylori, Flavonoids may amelicwate acute and chronic diarrhoea by inhibition of intestinal motility and secreticMi and may also be helpful in reducing chrcMiic inflammatory injury in the gut by protecting it from oxidative stress and preserving mucosal function through an as yet unidentified mechamism. Despite initial reports on their carcinogenic potaitial, it appears that flavonoids behave predominantly as antimutagens/anticancerous agents. However, epidemiological demonstration of this effect in humans is not without controversy and further studies are mudi warranted. The mechanism of action is unclear but seems to involve induction of apoptosis and specific antiproliferative activities rather than an unspecific toxic effect.

INTRODUCTION Flavonoids comprise a large group of polyphenolic compounds that occur naturally in almost all higher plants. Over 4000 different flavonoids have been chemically described and nev^ structures are still being reported. The coreflavonoidstructure consists of two aromatic rings connected by a C3 unit, which usually constitutes a heterocyclic ring with diferent degrees of oxidatioa Flavonoids are classified according to their chemical structure in several major classes, which include flavonols, flavones, flavanones, catechins (orflavanols),isoflavones, anthocyanidins, dihydroflavonols and chalcones. The different chemical modifications that occur in each of these classes, such as hydrogenation, hydroxylation, sulphation, methylation, acylation, and glycosylation, give rise to the enormous diversity of flavonoids found in nature. Manyflavonoidsoccur naturally as glycosides.

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carbohydrate substitutions including D-glucose, L-rhamnose, glucorhamnose, galactose, lignin and arabinose [1]. The ubiquitous presence of flavonoids in fruits, vegetables, nuts, seeds, flowers and barks as well as in beverages such as tea and red wine implies their regular intake as part of the normal human diet [1,2,3]. However, daily flavonoid intake is very variable depending on the dietary habits of a given population. One of the first studies which attempted to estimate the flavonoid content of the human diet was performed by Kiihnau [1] in 1976, who established that the totalflavonoidintake in the USA was around 1 g per day expressed as glycosides, the current form offlavonoidsin nature, or 650 mg per day when expressed as aglycones. More recent studies, which encompass more specific food analyses, have pointed out that this estimation may be too high and that flavonoid intake is actually much lower than that originally estunated by Kiihnau. Hertog et al [4] showed that the average dietary intake of flavonols and flavones in 16 cohorts participating in a crosscultural study, the Seven Countries Study (including Finland, USA, Serbia/Croatia, Greece, Italy, The Netherlands and Japan), was between 6 and 64 mg per day. Subsequent studies performed in other European countries, Denmark and Germany, also revealed values in daily flavonoid intake in that range: 56 and 11 mg per day respectively [5, 6]. Recently, an elegant study by Noroozi et al [7] stimated a mean intake offlavonolsof 35 mg/day, 91% of which was quercetin. In fact, quercitrin and rutin are the most common flavonoid glycosides in the diet, their common aglycone (sugar-free flavonoid) being quercetin, the predominant flavonoid found in foodstuff [3]. In addition to their presence in foods, flavonoids constitute the active principles of medicinal plants, representing one of the most interesting groups of biologically active compounds. In fact, numerous phytomedicines containingflavonoidsare marketed in different countries for the treatment of several pathologies in different body systems: digestive, urinary, cardiovascular, nervous and skin [8]. Furthermore, flavonoids have been reported to exhibit a wide range of biological effects, including antibacterial [9], antiviral [10, 11], anti-inflammatory [12, 13], spasmolytic [14, 15], vasodilatory [16, 17], inhibition of platelet aggregation [18, 19] and cytotoxic [20, 21] activities, among others. Many of the alleged effects flavonoids at pharmacological doses are related to their well-known properties as strong antioxidants and/orfree-radicalscavengers [22, 23], as well as to their ability to interfere with the activity of several enzyme systems [24]. Quercetin has been the subject of most studies investigating

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the biological effects offlavonoids,since it is the predominant flavonoid in food and medicinal plants [3]. It is evident that flavonoid ingestion, either through the diet or as pure compounds or active principles in phytomedicines, results in the presence of these biologically active compounds in the gastrointestinal tract during a certain period of time. Thus, this system can be considered as the first putative target of flavonoid effects, especially if we consider the results obtained from the different studies which deal with the bioavailability of flavonoids, (extensively reviewed by Hollman and Katan [25]). Despite the potentially significant health effects of flavonoids, information about the absorption, metabolism and excretion of individual flavonoids in humans is scarce [26]. Initially, it was stated that flavonoids present in food cannot be absorbed from the intestine because they are usually bound to sugars as glycosides [1], and only the aglycones were considered to be able to cross the gut wall. Although mammalian species, including man, lack the enzymes necessary to cleave the 6-glycosidic bonds offlavonoidglycosides, the aglycones may be released within the intestinal lumen byfi-glycosidasesfromintestinal bacteria, which are predominantly located in the large intestine [1,26,27]. Different studies have supported this assumption; in particular, Griffiths and Barrow [28] showed that germ-free rats excreted large amounts of unchanged glycosides in the faeces when glycosidicflavonoidswere orally administered, whereas only small amounts of glycosides were found in the faeces of rats with a normal microflora. It should be noted however that intestinal lactase-phlorizin hydrolase has been recently shown to hydrolyseflavonoidsand isoflavones in vitro^ although the significance of thisfindingis uncertain [29]. Once the aglycone is released in the colonic lumen, it can be either absorbed, eliminated with the faecal contents or degraded fiuther by ring cleavage, a process dependent also on the presence of intestinal microflora [25, 30], However, the requisite of hydrolysis for the intestinal absorption offlavonoidshas been questioned by Hollman et al [31], who studied the small intestine absorption of glycosylated and unglycosylated flavonoids in ileostomy patients. This approach has the added advantage of avoidingflavonoidlosses as a result of degradation by colonic bacteria. Their results showed that different quercetin glycosides derived from onions were absorbed in the small intestine. Furthermore, the absorption rate was found to be higher than that of rutin and quercetin, administered as pure compounds, suggesting that other components in food may influenceflavonoidabsorption. Subsequent studies have added even more controversy to this unsolved issue. Thus, Manach et

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al [32] showed that quercetin, but not rutin, was absorbed from the small intestine. This discrepancy with the observations made by Hollman et al [31] was attributed to species differences or to modifications of the digestion process in ileostomized patients, such as bacterial colonizatioa However, in a recent follow-up study Holhnan et al [33] reported further evidence of absorption of flavonoid glycosides in the small intestine by normal subjects. Clearly, more research on this important issue is much warranted In summary, from all of the above it can be concluded that flavonoid administration results in the presence of these biologically active compounds, either as glycosides or as aglycones after hydrolisis of the sugar moiety, in the gastrointestinal system, where they can exert their potential beneficial effects. In this regard, the present chapter will review the current status of research on the effects of flavonoids on several gastrointestinal disorders, namely gastric ulcer, diarrhoea, inflammatory bowel disease, and cancer. It is important to note that much, if not all the available information about the effects of flavonoids on gastrointestinal conditions has been obtained in both in vitro and in vivo experimental models, and for this reason critical analysis of published data is essential in order to design pharmacological and/or dietary approaches to the use of flavonoids in the treatment of the aforementioned conditions in humans. ANTIULCER ACTIVITY From ancient tunes, there have been many crude drugs which have been used in folk medicine for the treatment of gastric complaints, including ulcer disease. Many efforts have been made in order to validate the traditional use of these remedies by using several experimental models of gastric lesions. As a result of these investigations, it has been possible to correlate the antiulcer activity of some of these crude drugs in several experimental models with the presence offlavonoidsas active principles responsible for their beneficial effects (Table 1). These experimental models generally involve the administration of a gastric damaging substance (/.e. absolute ethanol, nonsteroidal antiinflammatory drugs -NSAIDS-, serotonin, reserpine, etc.), keeping the animal in a stressed enviroment (/.e cold, restraint, water-immersed) or a surgical manouvre (/.e. ligature of the pylorus or ischaemia-reperfiision). The proposed gastroprotective flavonoid or flavonoid containing extract is usually administered prior to induction of injury. All these experimental models have been developed in order to

611

reproduce in laboratory animals the gastroduodenal imbalance of acid secretory mechanisms, so-called aggresive factors, and mucosal protective factors that has been postulated to occur in peptic ulcer [34], In addition, these models also give information about the possible mechanism of action involved in the beneficial effects exerted by flavonoids in this conditioa histamine M

H,^

1.- Blockade of acid secrehon 2.' Bactericidal on H. pylori 3.- Reduced ROM formatioii 4.- Potentiation of POEj-dependent mucosal protectioii

/ FLAVONOID

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ATPWtH*/K*

0^® \\*—•—1— 1 ^ {proUfenition I

1 •

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Fig. 1. Schematic diagram showing the different mechanisms of action proposed for the antiulcer action of flavonoids, 1, Blockade of acid secretion by decreasing histamine iM'oduction or inhibiting the proton pimip. 2. Bactericidal effect on H. pylori, 3. Antioxickitive activity by scavenging free radicals and j^eventing ROM formation, 4. Potentiation of the mucosal protective fectors. PAF: platelet activating fector; ROM: reactive oxygen metabolites; H2: histamine receptor 2; M: muscarinic receptor; G: gastrin receptor.

Different studies have revealed that flavonoids or crude drug extracts containing flavonoid-like compounds are able to limit the deleterious effect of excessive gastric acid secretion, one of the aggresive factors involved in peptic ulcer. This is the basis of two widely used e^qperimental models: the pylorus-ligated assay in rats or mice and the cold-restraint stress ulcer model [35], Vela et al [36], in an attempt of validate the use of Stachytarpheta cayenmmis in folk medicine for the treatment of gastric disorders, including gastric ulcer, showed that an aqueous extract protected mice against ulcers induced by restraint in cold and reduced gastric acid secretion both in basal

612

eonditions and when induced by histamine or bethanecol in the pylorusligated assay in mice. After a guidedfractionationof the aqueous extract, the authors ascribed these effects to the presence of flavonoids in the crude drug. The purified flavonoid fractions obtained from Genista rumelica [37] and Erica andevalemis [38] have been also reported to reduce the gastric lesions induced after pylorus ligation, the latter two being active also in the cold restraint model. In addition, different pureflavonoids,such as vexibinol [39], sophoradin [40], hypolaetin-8-glucoside [41], catechin [42], silymarin [43] aiKl rutin [44] have shown beneficial effects in these assays. One of the mechanisms that can account for the effectiveness of flavonoids in the inhibition of gastric acid secretion/experimental ulcers may be the inhibition of histidine descarboxylase [45, 46, 47], an enzyme involved in the formation of histamine from histidine, resulting in diminished histamine release and histamine-evoked gastric acid secretion via H2 receptors [48]. In feet, H2 receptor antagonists such as cimetidine, ranitidine and famotidine have played a major role in the treatment of peptic ulcer disease. It has been also hypothesized that the gastroprotective effect exerted by flavonoids could be related to an inhibition of gastric lf",K^ATPase, also known as the proton pump. This enzyme plays a pivotal role m the final step of gastric acid secretion, catalyzing Yt transport at the expense of ATP hydrolisis [49]. Therefore, the inhibition of this enzyme leads to the reduction of acid secretion. Selective Hr,K^-ATPase inhibitors, known as proton pump inhibitors (PPIs), like omeprazol and lansoprazole show a powerfiil antisecretory activity and are clinically used for the therapy of peptic ulcers. Experimental data initially supported this hypothesis, since several naturally occurring flavonoids and derivatives, including flavonols, flavones,fliavanones,chalcones, isoflavones and catechins [50-54] were able to inhibit this enzyme in vitro, when activated with ATP. The kinetic analysis suggested a competitive mechanism between most flavonoids assayed and ATP, since inhibition increased when ATP concentration was lowered. The first attempt to establish the structure-activity relationship of proton pump inhibition by flavonoid compounds was carried out by Murakami et al [53] on catechin derivatives. The authors proposed that the intensity of the inhibitory activity depended on the number of hydroxyl groups in the molecule. Thus, the more hydroxylated catechins such as (-)epigallocatechin gallate and (-)-epicatechin gallate, with eight and seven hydroxyl groups respectively, had a stronger inhibitory activity than (+)catechin and its esteroisomer, (-)-epicatechin, both possesing only five hydroxyl groups. A more extensive structure-activity study performed by the

613

same group with over 80 flavonoids allowed them to further characterize the structural requirements of flavonoids to act as inhibitors of H^,K^-ATPase [54], They concluded that increasing the number of hydroxyl groups in the molecule resulted in a higher potency only up to four hydroxyl groups; no additional enhancement was observed above this limit. In addition, glycosylation or methylation of hydroxyl groups resulted in a decrease in potency. They authors also proposed that, in addition to the number of hydroxyl groups, the hydroxylation pattem is determinant to obtain a high potency of inhibition, and they established that two (catechol type) or three (pyrogallol type) adjacent hydroxyl groups or hydroxylation at C-3, C-5 and C-7 are a minimum requirement. Finally, they showed that a ketone at C-4 is not essential for enzyme inhibitioa However, these relationships are not in accordance with a previous study performed by Beil et al [50], who showed that flavanone, which has no hydroxyl groups, presented a lower IC50 towards Kr,K^-ATPase activity than quercetin, a flavonol with five hydroxyl groups. Finally, they concluded that, although proton pump inhibition may participate in the gastroprotective effect of flavonoids in vitro, it is unlikely to play a key role, because when the inhibitory activity of flavonoids on acid secretion was studied in isolated parietal cells [39, 50], it greatly depended on the agent used to stimulate acid secretion (i.e. histamine, pentagastrin, carbachol, 2-desoxy-D-glucose), while agents like PPIs, which interfere with the final step of acid secretion, block acid production in response to all stimuli [55]. In consequence, other mechanisms must participate in the gastroprotective activity attributed to flavonoids. In this regard, there has been an increased interest about the ability of flavonoids to promote the gastric protective factors that counteract the deleterious effect exerted by acid gastric secretion on gastroduodenal mucosa This effect has been studied in experimental animal models of gastric damage induced by the administration of necrotizing agents like absolute ethanol (p.o.) [56] or NSAIDs like indomethacin [57] or aspirin [58]. Several crude drug extracts containing flavonoids have shown gastroprotective activity in these experimental models, such as an aqueous extract of Stachytarpheta cayennensis [36] or an ether extract of Bidem avrea [59] or a flavonoidenriched ether extract from Dittrichia viscosa [60], as well as a number of isolated flavonoids: quercetin [61], naringenin [62], naringin [63], tematin [44], rutin [44], genistein [37], liypolaetin-6-glucoside [41] and the flavonoid cyanidin IdB 1027 [64]. Different mechanisms have been proposed to participate in this effect, considering the pathophysiology of these lesions.

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Initial exposure to ethanol produces rapid necrosis of mucosal cells, which leads to the release of vasoactive mediators, followed by venoconstriction and arteriolar dilation, resulting in hyperemia, edema and hemorrhage [65]. It has been proposed that reactive oxygen metabolites (ROMs) may fimction a cytotoxic agents which could trigger this sequence of events and play a key role in ethanol-induced injury [66]. If so, it is plausible that the antioxidant and/or free radical scavenging properties of flavonoids may contribute to their gastroprotective activity. Surprisingly, no direct evidence about this mechanism has been collected in the different experimental studies dealing with the antiulcer activity of flavonoids. However, some authors seem to point in this direction [60, 61]. Thus, Alarcon de la Lastra et ah [67] demonstrated the gastroprotection exerted by silymarin in the ischaemiareperfusion model gastric of mucosal injury, whose pathogenesis has been ascribed to the generation of oxygenfreeradicals which promote neutrophil recruitment via activation of adhesion molecules that in turn generate more ROMs resulting in a lipoperoxidation process in the gastric mucosa [68]. These authors propose that the gastroprotective effect of sylimarin can be attributed, at least partially, to the reported antioxidant properties of this flavonoid [69, 70]. In addition, other studies have reported the ability of silymarin to increase the glutathione content in different tissues, including the liver, the stomach and the intestine [71], an effect that could contribute to its protection against oxidative stress, given that glutathione is considered one of the most important endogenous antioxidant mechanisms in tissues [72]. In fact, this effect of sylimarin may protect the cellsfromthe toxicity of glutathione depletors, like ethanol [69] or acetaminophen [73], which is frequently associated to an increase in lipid peroxidation. Therefore, this mechanism may contribute to the therapeutic effects of the flavonoid in many hepatic conditions and digestive diseases such as gastric ulcer. The fact thatflavonoidsare able to counteract glutathione depletion has been also proposed as a mechanism involved in the gastroprotective activity of quercetin [74] and of a flavonoid fraction from Bidens aurea agakist different necrotizing agents, including absolute ethanol [59]. Nevertheless, it is difficult to establish a correlation between the antioxidant properties of flavonoids and their gastroprotective effect in the absolute ethanol nK)del, sinceflavones,which have been show to possess a low potency as inhibitors of Upid peroxidation compared toflavonolslike quercetin and rutin [22], did show a higher potency as gastroprotective agents, requiring ten fold lower doses to obtain a more pronounced effect [75] thanflavonols[61, 62]. This could be the reason why these studies have not focused on the antioxidant

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properties of flavonoids. However, the participation of an antioxidative mechanism in the gastroprotection exerted byflavonoidscannot be ruled out at the moment. Platelet activating factor (PAF) is another mediator that might be involved in gastric mucosal damage in rats treated with ethanol, since gastric PAF levels have been shown to be increased after ethanol administration [76] and PAF antagonists are able to reduce the gastric lesions induced by this necrotizing agent [77]. In fact, PAF is one of the most potent ulcerogens known and its role in the digestive tract has been extensively reviewed by Izzo [78]. PAF induces changes in microvascular circulation with stasis, which is thought to contribute to its ulcerogenic action [79], as it has been proposed to occur in the absolute ethanol experimental model of gastric lesions. The antiulcerogenic activity of several flavonoids, including flavone, quercetin, naringin, rutin and kaempferol was investigated with regard to the production of PAF in a rat model of gastric damage induced by acidified ethanol [76]. The results obtained revealed that intraperitoneal administration of quercetin, rutin, kaempferol (25-50 mg/kg) and naringin (200-400 mg/kg) resulted in a reduction in gastric damage and this effect was correlated with a decrease in gastric PAF formatioa However, Yanoshita et al [80] studied the inhibition of lysoPAF acetyltransferase activity, the enzyme involved in the synthesis of PAF, by flavonoids in vitrOy and concluded that, of the flavonoids tested, only luteolin and quercetin exhibited significant inhibitory effects. Furthermore, only luteolin was considered to act as a specific inhibitor, since it did not suppress the activity of other related enzymes such as choline acetyltransferase. In consequence, it is unlikely that inhibition in PAF synthesis plays a key role in flavonoid gastroprotection, and the reduction in PAF levels observed by Izzo et al [76] must be the result of inhibition at a distal step in the signalling cascade. The gastroprotective effect offlavonoidshas been linked to an interaction with mucosal eicosanoid production, sinceflavonoidshave been described as inhibitors of the main eicosanoid generating enzymes. In fact,flavonoidscan act as weak phospholipase A2 inhibitors [81, 82], and have been reported to possess inhibitory activity on cyclooxygenase/lipojg^genase [83, 84, 85], showing a higher potency against the latter. This may be relevant for the beneficial effects offlavonoidsin gastric ulcers. The ability offlavonoidsto inhibit lipoxygenase activity, and thus to decrease leukotriene B4 production, has been proposed to collaborate in their antiulcer activity, since leukotrienes are considered as important aggressive mediators which are released in response to necrotizing agents such as absolute ethanol and HCl [86, 87],

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acting as an important chemoatractant compound for neutrophils. However, there is no direct evidence supporting this hypothesis. On the other hand, the mhibitory activity of flavonoids on cyclooxygenase seems paradoxical in view of their reputed gastroprotective effect. Theoretically, a compound intended to act as gastroprotective should facilitate prostaglandin synthesis and release, since endogenous prostaglandins, mainly those derivedfromthe E series, which resultfromthe action of cyclooxygenase on arachidonic acid, are considered one of the most important cytoprotective factors in gastric mucosa, playing a key role in the maintenance of gastroduodenal integrity [88]. Although it has been described that prostaglandins are able to directly inhibit gastric acid secretion [89], their cytoprotective effect is obtained through the combination of different mechanisms, such as preservation of microvascular fimction, which has been considered to be altered in these gastric conditions, or an increase in gastric mucus production, which protects the gastroduodenal mucosa from agressive secretions such as HCl and pepsin, orfecilitatingmucosal cell tumover, to replace damaged cells [90]. In fact, it has been shown that exogenous prostaglandins protect the gastrointestinal mucosa from damage induced by a wide range of irritants [91], and prostaglandin analogues, like misoprostol, are used as antiulcer agents [92]. The effects of flavonoids on gastric prostaglandin production have been controversial. Thus, Beil et al [50] showed that flavone and flavanone were able to increase basal prostaglandin E2 (PGE2) release in gastric mucosal cells in vitro, but not when estimulated with arachidonic acid. In contrast, quercetin did not increase PGE2 release, confirming previous results obtained by Alcaraz and Hoult [93] in fragments of rat caecum. Additional evidence of the participation of prostaglandins in the gastroprotective effects of flavonoids comes from studies in which their beneficial effect was partially reversed after treatment with an inhibitor of prostaglandin synthesis like indomethacin [61, 62], although no definitive conclusion was reached. However, different studies have shown that flavonoids such as quercetin, naringemn and hypolaetin-8-glucoside [41, 61, 62] or a flavonoid fraction from Bidem aurea [59] are able to increase gastric mucus production, whereas the anthocyanidin IdB 1027 increased gastric bicarbonate secretion [94], effects that have been postulated to be related to prostaglandin productioa Subsequent studies with IdB 1027 demonstrated that this compound induced a marked increase in gastric mucosal release of PGE2 after oral administration in ten healthy subjects [95].

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From all of the above, it is evident that flavonoids have antiulcer activity in several models of experimentally induced gastric lesions, and this beneficial effect is probably achieved through the combination of several mechanisms. However, most of the eixperimental studies have dealt with acute treatments, which do not resemble the typical scenario which takes place in gastroduodenal ulcers in humans. Very few reports describe the healing effect of flavonoids in chronic treatment, perhaps due to the lack of appropiate experimental models. One of the proposed methods consists in the injection of acetic acid (5 % or 20 %) in the antral areas of the rat stomach, resulting in gastric lesions which share many of the characteristics of the human peptic ulcer, both macroscopically and histologically [96, 97]. It has been stown that pure flavonoids, like naringenin, quercetin [98] and IdB 1027 [64], as well as a flavonic fraction of Bidens aurea containing mainly aurones and chalcones [99], exert a healing effect in this chronic e:?q)erimental model, together with a stimulation of gastric vessel proliferation, an effect which may be attributed to a greater release of endogenous prostaglandins inducing a cytoprotective action, thereby facilitating gastric mucus secretioa In addition, an impairment in neutrophil function was proposed to collaborate in the healing effect obtained with the flavonicfractionof Bidens aurea, since these leukocytes have been involved in the pathogenesis of acetic acid-induced chronic ulcers [100]. Thus, flavonoids would block the activation of these cells by inflammatory mediators like leukotriene B4, PAF, free radicals, etc., and the subsequent release of tissue damaging factors. As a consequence, all the aforementioned mechanisms supossedly exerted byflavonoidsin the gastroprotection against acute experimental ulcers may also account for the beneficial effect in this chronic model, Le, lypoxygenase inhbition, prostaglandin production and even antioxidant activity. Finally, it is universally accepted at present that Helicobacter pylori infection has a definitive ethiological role in peptic ulcer disease, and that erradication therapy is warranted in these clinical scenarios. The majority of therapeutic trials have included the application of triple therapy with proton pump inhibitors or ranitidine bismuth citrate, clarithromycin and either amoxycillin or metronidazol and is to date the treatment of choice. However, recent studies have reported antibiotic resistance which can be one reason for failure of treatment of Helicobacter pylori infection [101-103], and new treatment strategies are therefore wellcome. Flavonoids, in addition to their gastroprotective activity previously commented, have been also shown to inhibit Helicobacter pylori growth in vitro. In this way, Beil et al [50]

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reported thatflavone,flavanoneand quercetin exerted bactericidal action on tliis microorganism, flavone being slightly more potent, with an activity similar to that reported for colloidal bismuth subcitrate, a drug usually employed in the erradication therapy. More recently, Bae et al [104] have investigated the inhibitory effect of someflavonoidson K pylorU including different glycosides (hesperidin, poncirin, naringin and diosmin) and their corresponding aglycones (hesperetin, ponciretin, naringenin and diosmetin), as well as that of phenolic acids, which could be generated from flavonoids by human intestinal microflora They observed that the unglycosylated flavonoids did inhibit Helicobacter pylori growth, whereas the glycosides or the phenolic acids showed a much weaker effect. The structure-activity relationship established by these authors revealed that the highest activity was shown by thoseflavonoidsposssesing a flavanone skeleton, and this activity was further increased when a methoxy group in the para position was present. In fact,flavonolsandflavonesshowed a much weaker activity. This is not in accordance with the previous observations made by Beil et al [50], who described thatflavonewas abnost as potent asflavanone.In any case, this reported activity opens up a newfieldin the study offlavonoidsfor therapeutical use in the treatment of gastroduodenal ulcers, particularly if flavonoids are considered as chemical structures susceptible to undergo structural modifications to obtain new compounds with a higher and more selective activity against Helicobacter pylori. The reported antiulcer activity of flavonoids has led to the synthesis of fiavonoid derivates with the purpose of increasing their efectiveness, meciadanol [105], solon [106], and the sophoradin derivative SU-840 [107].. The protective effects of these compounds have been described to be related with a cytoprotective action, mediated mainly through a rise in the mucosal generation of PGE2, a mechanism similar to that proposed for different naturalflavonoids.Similarly, Ares et al [108] established as a goal of their research the synthesis offlavoneanalogs that would be gastroprotective but have a minimal impact on drug metabolizing enzymes, an undesirable effect of flavonoids [109] that may be especially relevant if they are to be administered simultaneously with other drugs like NSAIDs to prevent the gastric side effects of the latter, as previously proposed [110]. The authors observed that, with regard to flavones, substitution of the 3-, 6- or 8positions in theflavonoidcore led to reduction of gastroprotective activity in the rat ethanol damage model. However, a methoxy substitution in the 5- or 7-positions maintained the activity of the parent flavone, showing an even lower gastroprotective ED50 than that of flavone. They also described the

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core ring changes that resulted in a marked loss of biological activity, e.g. moving the phenyl ring from the 2- to the 3-position, reducing the C2-C3 double boimd,fragmentingthe heterocycle ring, or replacing the oxygen in position 1 with nitrogea On the other hand, when the phenyl ring in position 2 was replaced with alkyl or heteroaryl groups, the gastroprotective activity was retained. In conclusion, different experimental studies have shown the ability of flavonoids to prevent ulcer formation in several experimental models, an effect that can be ascribed to several mechanisms of action. Fig. (1). It is probable that the activity at different levels, rather than at a single one, can better justify the beneficial effect of flavonids in these gastric conditions. ANTIDIARRHOEIC ACTIVITY Many crude drugs with antidiarrhoeic activity have been widely used in folk medicine. Despite the widespread occurrence of flavonoids in plants, most studies have empirically attributed the reputed antidiarrhoeic activity to other polyphenolic compounds like tannins rather than to flavonoids. However, different studies have revealed that, in addition to tannins, flavonoids can account for the antidiarrhoeic activity of some of these traditional crude drugs. One of the first studies to suggest this possibility was performed by Lutterodt [111], who proposed that the presence of quercetin in Psidium guajava leaf extracts, both as aglycone or as different glycosides, could justify its use in the treatment of acute diarrheal diseases for many centuries. In a subsequent study, the same author demonstrated the antidiarrhoeic activity of Psidium guajava extracts in experimentally induced diarrhea in rats [112]. However, it was two years later when Lozoya et al effectively reported that the antidiarrhoeal activity of this crude drug could be ascribed to the presence of several quercetin glycosides, such as guajavarin, isoquercitrin, hyperin, quercitrin and quercetin 3-0-gentobioside [113]. Other studies have corroborated that different flavonoid containing extracts are able to exert an antidiarrheic effect. Thus, we demonstrated the antidiarrhoeic activity of Euphorbia hirta whole plant decoction in several e}q)erimental models of diarrhoea (castor oil, arachidonic acid and prostaglandin E2) and isolated quercitrin as theflavonoidresponsible for the antidiarrheic activity of the crude drug [114]. Similarly, Rao et al [44] investigated the antidiarrheal properties of tematin, a tetramethoxyflavone from Egletes viscosa^ a brazilian herb commonly used in popular medicine

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for the treatment of intestinal colic. Other examples include 7-0methyleriodictyol, a flavanone isolated from Artemisia monosperma^ or cirsimaritin, a flavone isolated from Artemisia judaica, identified as the agents responsible for the antidiarrhoeic activity of these plants, which have been used in folk medicine for this purpose [115,116]. Despite the lack of studies correlating the antidiarrheic activity of crude drugs with the presence offlavonoids,these have been also studied as pure compoimds in this regard. The most used assay of antidiarrhoeic activity is the castor oil test, in which diarrhea is induced by the oral administration of castor oil to mice. Different flavonoids have been shown to possess antidiarrheal activity in this test: quercetin, kaempferol, morin, myricetin, rutin (Lp.) [117, 118], quercitrin (p.o.) [114, 119], and tematin (i.p.) [44], all showing a dose-dependent activity in the range between 25 and 100 mg/kg. However, flavonoids are not only able to exert a preventive antidiarrheal effect in this acute model of ejq^erimentally-induced diarrhea, but also in chronic models. Thus quercitrin showed beneficial effects in a model of lactose-induced chronic diarrhea in rats, since it reduced the diarrheal output and facilitated colonic mucosal repair in lactose fed [120], Given that diarrhea, considered as the abnormally frequent expulsion of faeces of low consistency, is due to a disturbance in the hydroelectrolytic transport across the intestinal mucosa and/or to abnormal intestinal motility, the study of the mechanisms by which flavonoids can exert their antidiarrheic effect has naturally focused on both intestinalfimctions,which of course are intimately connected to one another. In fact, several in vivo and in vitro studies have demonstrated thatflavonoidsinhibit intestinal motility and secretion, delaying small and large intestinal transit in mice and rats [44, 117, 119, 121], and counteracting the intestinal accumulation of fluid and electrolytes elicited by different secretagogue compounds like castor oil [117], prostaglandin E2 or sodium picosulphate [119]. Di Carlo et al described that these effects were greatly influenced by the structure of the molecule [117], In fact, flavonols (quercetin, kaempferol, morin, rutin and myricetin) were the most potent inhibitors in vivo [117, 121], and modifications in theflavonolstructure {i.e. absence of the 3-hydroxyl group and/or saturation of the C2-C3 double bond, lack of the C4 carbonyl group or opening of the B ring) resulted in a decrease or total abolition of the activity. Interestingly, it was also observed that glycosylation increased the biological activity of these compounds. This is in accordance with the results obtained by our group [119], showing that orally administered quercitrin delayed small intestinal transit when stimulated with castor oil, whereas the

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oral administration of the aglycone, quercetin, was devoid of any effect on intestinal transit. In vitro experiments have yielded additional information about the ability of flavonoids to affect intestinal motility and hydroelectrolytic transport. Thus, flavonoids inhibit isolated guinea pig ileum contractions induced either electrically [14, 112, 122] or chemically with different contracting agents [15,44,122-127]. It is unportant to note that this inhibitory effect was reversible upon removal of theflavonoidfromthe tissue bath [116,123,124, 126], suggesting that mostflavonoidsdo not impair energy production in smooth muscle. Bambhir and Banerjee [125], on the basis of the different spasmolytic activity offlavonoidsagainst histamine and acetylcholine in the guinea-pig ileum in vitro, tried to establish the structure-activity relationship. They observed that the polyhydroxylated flavonol quercetin, with five hydroxyl groups, showed the highest potency, and that a reduction in the number of hydroxyl groups resulted in a progressive decrease in activity, fromfisetin(four hydroxyl groups) toflavonol(one hydroxyl group), which were one and two orders of magnitude less potent than quercetin, respectively. On the other hand,flavoneshowed a similar potency than that of fisetin, and mono- or di-hydroxylation in the flavone structure reduced its activity, whereas polyhydroxylation tended to restore it. These structural requirements confirm previous reports of flavonoid spasmolytic activity against acetylcholine and prostaglandin E2 [123] or prostaglandin E2 and leukotriene D4 [15] in isolated guinea pig ileum. Furthermore, these observations are in agreement with the higher activity showed by flavonols as inhibitors of small intestine transit in vivo [117]. However, glycosylation of the flavonoid structure results in a marked loss of activity in vitro, as opposed to the results obtained in vivo. This suggests that, although the aglycone is the active moiety of the glycosidicflavonoid,glycosylation may make it more accesible to the target organ than when the aglycone is administered as such in vivo. This is m accordance with the observed effects of quercetin and its glycoside quercitrin on netfluidtransfer in isolated loops of rat colon in situ [120]. In this assay quercitrin showed no effect on colonic hydroelectrolytic transport when estimulated by prostaglandin E2 or sodium picosulphate, whereas quercetin inhbited water secretion. However, if the colonic lumen was not rinsed before instillation of a quercitrin containing solution, the glycoside exerted a similar activity to that of the aglycone, indicating an influence of bacterial metabolism. Subsequent studies performed with the Ussing chamber technique have confirmed these observations, since the quercetin glycoside rutin, in contrast to the aglycone.

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had no effect on intestinal electrolyte transport in rat colon preparations [128]. Different in vitro studies have dealt with the mechanisms of action involved in the effects of flavonoids on intestinalfiinctions,i.e. motility and secretion; however it is important to note that most of these studies have focused on quercetin, the most abundant and also the most active flavonoid on intestinal functions. The fact that flavonoids inhibit both pharmacologically (acetylcholine, prostaglandin E2, histamine) and electrically induced guinea pig ileum contractions suggests a nonspecific nature of the antispasmodic activity, not related to a direct interaction with a particular receptor. In fact, quercetin inhibition of carbachol induced guinea pig ileum contractions has been shown to be noncompetitive, i.e. concentration-response curves were shifted downwards in the presence of different concentrations of the flavonoid without modification of the ED50 values of the agonist [126]. In consequence, it is likely that flavonoids act at a distal stepfi-omreceptor activation, common to the response to all stimuli. Most of these studies pointed to an interference with calcium availability for the contractile machinery in the intestinal smooth muscle cell. Thus the spasmolytic activity of severalflavonoidsis augmented by lowering extracellular calcium or by adding the calcium channel blockers verapamil in vivo [15, 117, 118] or nifedipine in vitro [123]. The smooth muscle of the guinea-pig ileum exhibits a biphasic mechanical response when exposed to a bathing medium containing acetylcholine [129], which corresponds to an initial mobilization of calciumfi*omintracellular sources [130] followed by calcium influx across the membrane from the extracellular medium [131]. It has been shown that pretreatment with quercetin inhibits both components of the biphasic response [126]. Thus calcium influx through receptor-operated cdcium channels seems to be blocked by this flavonoid, since: first, quercetin shows a similar behaviour to that of the reputed calcium antagonist verapamil [126]; second, the inhibitory effect of quercetin is reverted by addition of the calcium ionophore A23187 to the organ bath [124]; and third, quercetin causes inhibition of the concentration-response curves to calcium chloride in a high-potassium depolarizing medium [126, 132]. On the other hand, quercetin is also able to inhibit smooth muscle contraction induced by acetylcholine or histamine in a calcium-free solution containing ethylenediamine tetraacetic acid or ethylene glycol-bis(B-aminoethyl ether) N,N,N',N'-tetraacetic acid [124, 126], suggesting that it also affects the release of calcium from intracellular stores or that it prevents released calcium from binding to the corresponding target proteins (i.e. calmodulin.

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myosin light chain kinase, etc.), which are thoxight to mediate exclusively the smooth muscle contractory response in these conditions [133]. In addition to its effects on calciimi pathways in the smooth muscle cell, other mechanisms can contribute to the spasmolytic effect of quercetia For instance, quercetin inhibits cAMP phosphodiesterase, giving rise to elevations of intracellular cAMP levels, as shown in other tissues [134, 135, 136]. Because smooth muscle relaxation is known to resultfromthe activation of phosphatases as a consequence of increased intracellular cAMP [137], this mechanism may play a role in the effect of quercetin. Other mechanism that can be involved is protein kinase C inhibition [138], which has been postulated to contribute in the relaxant effects of flavonoids in other tissues, such as the vascular smooth muscle [16]. On the other hand, most of the studies dealing v^th the antisecretory mechanisms of action offlavonoids(mainly quercetin) have been performed in vitro using sheets of native rat intestinal tissues or human epithelial cell line monolayers, such as T84 cells, mounted in Ussing chambers [139], a system that allows the continuous monitorization of electrolyte transport across epithelial cells. Although it has been shown that quercetin has no effect on basal colonic absorption in vivo [119], different in vitro studies have revealed a secretory effect of this flavonoid [128, 140, 141]. On the basis of its known properties as a phosphodiesterase inhibitor, which results in increased intracellular cAMP levels [135], this effect was initially ascribed to an activation of the cAMP-mediated signalling pathway [140], a mechanism similar to that exerted by colonic secretagogue compounds like the adenylate cyclase activator forskolin. However, subsequent studies [128] suggested that the enhancement of electrogenic chloride secretion elicited by quercetin in the rat small and large intestine is cAMP independent, as well as different from that shown by the isoflavone genistein, a protein tyrosine kinase inhibitor that has been also shown to induce chloride secretion in preparations of rat colon [142] and in T84 cells [143, 144], via direct stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel [145, 146]. Otherflavonoids,like tangeritin or nobiletin, have been also shown to stimulate electrogenic chloride secretion in T84 cells [147]. Similarly to other in vitro assays with flavonoids, glycosylation resuked in loss of activity [128, 140, 147]. The secretory activity of quercetin seems paradoxical in view of its aforementioned antidiarrheal effects [118, 119, 121]. Interestingly, it has been reported that quercetin is able to inhibit the secretory response to carbachol both in T84 cells [141] and in the rat colon [128] when mounted in Ussing chambers, although the

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response to vasoactive intestinal peptide (VIP) was not altered in T84 cells [141]. Similarly, a previous study reported the ability of quercetin to reduce carbachol-evoked chloride secretion augmented by Trichinella spiralis antigen in guinea pig submucosa-mucosa colonic preparations obtained from immune animals challenged with Trichinella spiralis [148], This inhibitory effect on carbachol secretion suggests that quercetin may interfere with the calcium mediated signalling pathway by reducing calcium availability within the enterocyte, in parallel with the proposed mechanism for the spasmolytic activity of quercetin. However, other mechanisms cannot be ruled out, since quercetin also inhibits chloride secretion induced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) [141], an effect that may be related to a direct inhibitory action of quercetin on protein kinase C, in view of its known ability to downregulate this enzyme [138]. Furthermore, quercetin has been also shown to inhibit the secretory response induced by other secretagogues like prostaglandin E2 in the T84 cell line [141], confirming similar results obtained in vivo [119]. No mechanism has been proposed to explain this effect. In conclusion, the effects offlavonoidson intestinal motility and secretion can justify their antidiarrheal properties, although more research in this field is required in order to fiiUy understand the mechanims of action involved. INTESTINAL ANTIINFLAMMATORY ACTIVITY The term inflammatory bowel disease (IBD) refers to two related diseases with overlapping features, namely ulcerative colitis and Crohn's disease. Although their etiology is at present unknown, it has been proposed that either overactivation or inadequate stimulation of gut-associated lymphoid tissue, which in tum generates a disturbed intestinal immune response, is a trigger event in the development of the disease. These intestinal conditions are characterized by intestinal inflammation resulting from the local release of proinflammatory mediators (cytokines, eicosanoids and reactive oxygen and nitrogen metabolites), increased vascular permeability and recruitment of inflammatory cells, ultimately giving rise to mucosal ulceration Although these chronic inflammatory diseases of the gut have received a great deal of attention by researchers in the last few years, therapy remains paliative rather than curative today, often exhibiting distressing side effects. As a consequence there is a clear demand of new agents for the treatment of patients with IBD which improve their quality of life by alleviating or

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reducing their symptoms with minimal toxicity. In addition to their demonstrated low toxicity [149, 150], flavonoids show different features that make them potential antiinflammatory drugs applicable to IBD. First, flavonoids are inhibitors of several enzymes which are activated in inflammation [8]; second, a number of cells of the immune system are downregulated by certain flavonoids in vitro [24]; and third, most flavonoids show potent antioxidative/radical scavenging effects in vitro [22] and some of them have been shown, when administered orally to normal rats, to aflFect the oxidative status of the rat intestine in vivo, by increasing the intestinal glutathione content, thus protecting this tissue from lipoperoxidative insult [71,151]. The first study that pointed out the potential beneficial effect of flavonoids in IBD was performed by Galsanov et al, who reported that quercitrin, at doses between 25 and 100 mg/kg, was beneficial in an allergic rat model of intestinal inflammation [152]. Subsequent studies have confirmed these preliminary observations using two well established models of colonic inflammation, the acetic acid and the trinitrobenzene sulphonic acid (TNBS) models of experimental colitis, which bear some resemblance to human intestinal inflammation [153]. A number of flavonoids have been shown to exert intestinal antiinflammatory activity in these models, including glycosides like quercitrin [154], rutin [155, 156], diosmin [157] and hesperidin [157], and aglycones like morin [158]. These beneficial effects have been achieved in both the acute and chronic phases of experimental colitis. Thus flavonoids prevent acute colonic damage when administered orally prior to the colonic instillation of the phlogogen agent {le, acetic acid or TNBS) [154-158], but also facilitate colonic healing in the chronic phase of the experimental model of colitis in postreatment protocols [154,156]. It is difficult to establish a structure-activity relationship, because only a few flavonoids have been studied until now. However, different flavonoid classes, including flavonols (glycosides like quercitrin or rutin and aglycones like morin), a flavone glycoside (diosmin) and a flavanone glycoside (hesperidin) have been demonstrated to be effective. Of all the flavonoids assayed, quercitrin has been shown to be the most potent, exerting its protective/curative activity at the doses of 1 and 5 mg/kg [154], whereas the rest of the con^unds manifested their beneficial effects in a dose range between 10 and 25 mg/kg in the case of the glycosides [156,157] and between 10 and 200 mg/kg when the aglycone morin was tested [158]. Several mechanisms have been postulated to participate in the intestinal antiinflammatory activity of flavonoids, one of them being their

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antioxidant/antiradicalary properties. In fact, all flavonoids ameliorate colonic oxidative stress associated to e>q)erimental colitis [154-159], since they are able to reduce colonic glutathione depletion or to decrease colonic malonyldialdehyde (MDA) production, two biochemical markers of induced lipoperoxidative insult. This effect can be considered of great value because free radicals, including reactive oxygen and/or nitrogen metabolites, have been proposed to play a key role in the pathophysiology of human IBD [160, 161]. Moreover, in the last few years special attention has been paid about the dual role of nitric oxide (NO) m the pathogenesis of IBD [162]. It has been shown that physiological levels of NO, generated by the constitutive isoforms of nitric oxide synthase (cNOS), exert a direct protective effect in the acute intestinal inflammatory states, mainly through inhibition of leukocyte-endothelium interaction and of the increase in epithelial permeability, two key events in the early stages of intestinal inflammation [163]. On the contrary, NO synthesis is markedly augmented in states of chronic intestinal inflammation, largely due to the upregulation in the inducible isoform of NOS (iNOS), which results in several indirect deleterious/proinflammatory effects via generation of reactive nitrogen oxide species derivedfromthe interaction of NO and O2", such as nitrogen dioxide, dinitrogen trioxide and peroxynitrite, that promote oxidative stress and tissue injury [164]. It is plausible that the antioxidant and/or scavenging properties of flavonoids can interfere with NO metabolism; first, flavonoids may preserve the beneficial effects of NO by directly scavenging superoxide anions [165], which are thought to inactivate the physiological NO that is generated within endothelial cells and protects the gut from inflammatory insult [163]; second, it has been shown thatflavonoidsinhibit iNOS [166] and act as potent scavengers of peroxynitrite [167], and thus they could ako prevent the indirect deleterious effects of NO on the intestinal system. However, the antioxidant activity offlavonoidscannot be considered the only mechanism involved in their beneficial effects. Another mechanism that can participate is their ability to inhibit lipoxygenase activity, decreasing leukotriene B4 (LTB4) production [24]. LTB4 has been long thought to be a key proinflammatory mediator in IBD. In fact, blockade of LTB4 synthesis [168] or of the LTB4 receptor [169] has proven beneficial in experimental colitis. Diflferent flavonoids have been shown to decrease colonic LTB4 production in colitic rats [154-159], but a cause-effect relationship has not been consistently found [154, 158]. It is plausible that both mechanisms, antioxidative activity and inhibition of LTB4 synthesis, may cooperate in their beneficial effects, since LTB4 has been shown to potently promote

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neutrophil chemotaxis, adherence and degranulation in the TNBS treated colon [169], whereas free radical overproduction results in a multitude of deleterious effects in the gastrointestinal tract, including direct cytotoxicity, which promotes further release of proinflammatory mediators [161, 170]. In fact, flavonoid treatment resulted in most of the studies in a significant reduction of colonic myeloperoxidase activity, an enzyme predominantly found in the azurophilic granules of neutrophils which is considered as a sensitive marker of neutrophil infiltration, and is extensively used for this purpose [171], Moreover, it has been demonstrated that flavonoids, mainly as aglycones, are inhibitors of neutrophil fimction and MPO activity [24], which can also contribute to the intestinal antiinflamatory activity. In addition to the above mentioned mechanisms, a preservation of colonic absorptive function, which is profoundly altered in intestinal inflammation, can account for the beneficial effects of flavonoids in these intestinal conditions. In fact, it has been proposed that abnormal intestinal permeability is an underlying factor in the pathogenesis of human IBD [172], because a leaky epithelium may allow the entry into the lamina propria of bacterial o dietary antigens that are poorly cleared by the mucosal immune system or of a bacterial product that initiates an uncontrolled inflammatory response [173], It has been shown in different e}q)erimental models of rat colitis that altered in vivo colonic fluid absorption can be considered a sensitive measure of injury to the intestinal mucosa, resulting from mucosal leakiness and extensive epithelial necrosis in the acute stages of colitis and from unclear mechanisms in the chronic phase [154, 155,157,158,173,174], Moreover, it has been described that colonic hydroelectrolytic transport is one of the last parameters to recoverfrominflammation [154], being altered even when the inflammatory status has been essentially resolved [175], Different flavonoids, like quercitrin [154], rutin [155], hesperidin [157] and morin [158], have been reported to improve colonic fluid absorption in colitic animals. However, only quercitrin was able to conqjletely restore normal colonic fluid transport in colitic rats, acconq)anied with a reduction in the incidence of diarrhoea compared to non-treated animals, one of the symptoms that characterize intestinal inflammatioa Alternatively amelioration of colonic transport can be viewed as a consequence, rather than a cause, of a quicker recovery of mucosal barrier function and/or mucosal protectionfrominflammation. Nevertheless, a role for flavonoids in the regulation of hydroelectrolytic transport has been envisaged, as previously discussed. Thus a possible inhibitory effect of quercitrin on the secretory response to inflammatory mediators, including PGE2 [161, 176,

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177], cannot be excluded, given the antisecretagogue properties of this flavonoid as well as those of its aglycone quercetin [119, 141], which is supposed to be released after hydrolysis of the glycoside by bacterial enzymes in the colon [27]. It is important to note however that this response is tipically downregulated in the inflamed intestine, so that the relevance of this mechanism may be less than previously thought. Data obtained from a nmnber of in vitro assays suggest other possible lines of actuation of flavonoids, albeit not substantiated as yet by in vivo experiments. In particular, flavonoids may interfere with the effect of several cytokines, like tumor necrosis factor-a (TNF-a), which is considered of central pathogenic importance in intestinal inflammation [172]. In fact, a monoclonal antibody against TNF-a has been found to be a promising new therapeutic approach in IBD, providing long-term remission at least in Crohn's disease [178, 179]. In this regard, Habtemariam [180] studied the ability of seventeen flavonoids as modulators of the cytotoxicity of TNF-a in a tumor cell line, and reported that all flavonols tested, including quercetin, morin and rutin, protected these cells from TNF-induced cell death. Quercetin was the most potentflavonol,being sbc and and twelve times more potent than morin and its glycoside derivate rutin, respectively. The mechanism whereby flavonoids inhibit the cytotoxicity is yet to be established, but it may be associated with inhibition of the enzyme activities upregulated following receptor activation or with their antioxidant properties. On the other hand, it has been shown that severalflavonoidsare able to inhibit the expression of adhesion molecules stimulated by TNF and other cytokines at the transcription level [181], an early process in the cascade of events leading to derangement of intestinal mucosal homeostasis in IBD [172]. Considering that the TNBS model of rat colitis has shown to have an upregulated proinflammatory cytokine pattern, it is probable that the effects of flavonoids on cytokine production contribute to their intestinal In conclusion, the beneficial effects offlavonoidsin experimental IBD are mostly related with a preservation of intestinal fimction and/or with their ability to interfere simultaneously with different steps in the inflammatory cascade; i,e. eicosanoid generation, oxygen and nitrogen reactive metabolites production and proinflammatory cytokine release. The interest of flavonoids as potential drugs in the treatment of IBD is growing, and recently the intestinal antiinflammatory effects of the derivate flavonoid DA-6034 (7carboxymethyloxy-3',4*,5-trimethoxy flavone) has been reported in three

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experimental models of rat colitis [182], suggesting that it could be a promising drug in IBD therapy. ANTICANCER ACTIVITY Epidemiological evidence collected in the past decade has consistently established a link between the dietary intake of vegetables and tea and a reduced risk of certain diseases including cancer. Flavonoids have been proposed as one of the phytochemical groups responsible for the cancer preventive effect of foodstuflFs of vegetal origin. In particular, the low incidence of breast and prostate cancer in Asian countries has been attributed to the high consumption of tea and soy beans, which are rich sources of catechins and isoflavonoids, respectively [183, 184]. It is important to note that there is a great deal of controversy on this subject, since while some epidemiological studies have confirmed the protective effect of flavonoids against gastrointestinal and other types of cancer [185-187], others seem to rule out this hypothesis [4,188]. Nevertheless, there is sufficient evidence to warrant further studies offlavonoidsas antineoplastics in the gastrointestinal tract. Although there are preliminary data supporting the antitumoral activity of quercetin, the most commonflavonoid,in humans in the course of a Phase I clinical trial [189], direct evidence of the anticancer effect offlavonoidsis derived almost exclusively from studies performed in animal models as well as studies performed on cultured cell lines. Fig. (2). Most animal studies on gastrointestinal cancer have focused on colon cancer using the azoxymethane (AOM) model in rats or mice [190-197]. There are also available reports on models of cancer of the stomach (induced by benzo[a]pyrene [198] or N-methyl-N*-nitro-N-nitrosoguanidine [199]), oesophagus (N-methyl-N-amykiitrosamine [200]), and the tongue/oral cavity (methyl-(acetoxymethyl)-nitrosamine [198], 7,12-dimethylbenz[a]anthracene -DMBA- [201] and 4-NQO [202,203]). The anticancer effect of soy, the only relevant dietary source of isoflavones and the main candidate foodstuff responsible for the cancer protection conferred by Asian diets, has been tested in a number of animal studies. Messina et al [204] reviewed 26 such studies published up to 1994 (gastrointestinal and non gastrointestinal). Seventeen of them (65%) reported a protective effect and none found a potentiation of carcinogenesis. One study has examined the effect of feeding a soy protein diet in a genetic

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model of intestinal cancer, the Apc(Min) mouse, which is heterozigons for a nonsense mutation in the Ape (Adenomatous Polyposis Coli) gene [205]. No differences in the incidence of spontaneous adenomas were observed. Soybeans are rich in isoflavonoids, namely genistein and daidzein, a fact that may account for their putative cancer preventive effect [197]. In fact genistein, administered as a pure compound, has been reported to reduced AOM-induced colonic foci of aberrant crypts in rats, an early marker of neoplasia [191, 193, 197]. On the other hand. Booth et al [206] found genistein (administered subcutaneously) to have no effect on small intestinal homeostasis. Genistein may not be the only factor involved in cancer prevention by soy diets. Thus Hawrylewicz et al [207] found that dietary methionine supplementation reversed the growth inhibiting effect of soy protein diets in non gastrointestinal rat tumours, suggesting that the low content in methionine in soy is also involved. Genistein has been shown to significantly reduce the incidence of experimentally induced gastric cancer [199] in rats. A number of studies carried out with other flavonoids. Table (1), have shown their effect on oral [198, 201-203], oesophageal [200], gastric [198,208] and colonic cancer [190,192,209,210]. Little information about the mechanism of action of flavonoids is anticipated from in vivo studies. The mechanism of catechin and morin seems to be related to an increase of the activity of detoxifying enzymes like glutathione-S-transferase and NADPH:quinone reductase [198, 211], Similarly, EGCG effect at the colonic level is associated to an increase in tissue superoxide dismutase levels, suggesting that it may act through a potentiation of the antioxidative defense [210]. Another experimental approach is the use of cultured cell lines of gastrointestinal origin to test the antiproliferative or pro-apoptotic activity of flavonoids. The main drawback of this model is that cell lines are generally cancerous in nature, making it critical to distinguish an unspecific toxic effect from true antitumoral activity. This is of special relevance if we consider the vast array of biochemical processes that may be potentially modulated by flavonoids [24]. Few investigators have compared the effects of these compoxmds on both cancer and 'normar cell lines, and even in these cases one has to take into account that the latter display a transformed phenotype rather than the normal, physiological scenario. In this regard, Kawaii et al [212] recently evaluated the antiproliferative effect of 27 Citrus flavonoids against several tumor and normal cell lines; seven of them (in decreasing order of potency: luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3*,4*,5,6,7,8-heptamethoxyflavone) showed

631

specific activity against the tumor cell lines while exerting a weak antiproliferative effect on normal cells. The deducted structure-activity relationship included an 0-catechol moiety in ring B together with a C2-C3 double bond and a 3-hydroxyl group for maximum activity. In another recent study. Booth et al [213] examined the antiproliferative activity of genistein and other isoflavonoids on both normal (IEC6, IEC18) and cancerous (SW620, HT29) intestinal epithelial cell lines and compared it with that of estradiol, tamoxifen (a estrogen receptor antagonist), and tyrphostin (a tyrosine kinase inhibitor). They concluded that all isoflavonoids, but mainly genistein, inhibited proliferation and stimulated apoptosis similarly in all four cell lines and that this effect was associated to tyrosine kinase inhibition. Musk et al [214] compared the antiproliferatice effect of quercetin on naive and detransformed/differentiated HT29 cells and found that the flavonoid had a more pronounced effect on the former. Chen et al [215] reported a modest selectivity of EGCG towards cancer cell lines (12-fold). Conversely, in a study utilising normal, dysplastic or cancerous oral cell lines, Khafif e/ al [216] found that EGCG showed a weak antiproliferative effect which was lower in cancer cells than in the normal or dysplastic counterparts. Along the same path is the report of Kuo [217], who found no specificity of quercetin or genistein against cancerous (HT29, Caco-2) as oppossed to normal (IEC6) cell lines of intestinal origin. Taken together, these studies suggest that flavonoid selectivity for cancer cells is modest at best. PROCARCINOGENIC ri-% 4I ^^''© O '

Citochrome l

P450

» ^

^ © T P-glycoprotdii CARCINOGENIC — - _ _ » Detoxication or Extrusion GST ROM ^ © - ^

©

DNA INTERACTION

1

MUTAGENIC

• Apoptosis

Neoplastic cell

Fig. 2. Schematic diagram delineating some of the multiple stages of mutagenesis and the interference by flavonoids. 1. Flavonoids induce apoptosis and enhance mutagen detoxification and extrusion from the cell. 2. Flavonoids interfere with the metabolic activation of mutagens and protect DNA by means of their antioxidative action. GST: glutathi(me-S-transferase; ROM: reactive oxygen metabolites.

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On the other hand, there is strong evidence that the effects of flavonoids on cultured cells are not merely toxic but via induction of apoptosis or inhibition of proliferatioa Thus signs of apoptosis, such as induction of caspase-3, fragmentation of DNA and chromatin condensation, are frequently detected upon cell exposure to flavonoids [213, 217-221]. In addition, the effects of flavonoids are generally reversible upon removal or addition of serum [222-224]. Kunz et al [221] attempted to establish a structure-activity relationship for the antineoplastic potency of flavonoids in gastrointestinal cell lines, examining more than 30 flavonoids for antiproliferative activity against Caco-2 and HT29 cells. In contrast with the findings reported by Kawaii et al [212], the authors found no apparent relationship between activity and either 'core' subclasses {le.flavones,flavonols,flavanones,isoflavones) or substituents. However, most studies point to quercetin, genistein and EGCG as the most potent compounds overadl. In this regard, it is important to note that although some flavonoids are active at low concentrations in vitro, like genistein at 6 |iM [225] or quercetin at 10 nM-10 |iM [222], the anticancer effect is morefrequentlyreported at relatively high concentrations, i.e. in the 1-100 ^M range [218,221,224,226]. Different mechanisms have been postulated to contribute to the antitumorigenic activity of flavonoids at the gastrointestinal tract, taking into consideration evidence collectedfromexperiments with both gastrointestinal and nongastrointestinal cell lines. A brief review of these activities with a special emphasis on their relevance in cancer is provided betow. a) Antioxidant/antimutagenic activity Flavonoids have well characterized antioxidant and free radical scavenger properties. This is relevant in view of thefeetthat oxidative stress may cause oxidative modifications in DNA and originate mutations. Many investigators have demonstrated the antimutagenic/anticlastogenic activity of flavonoids on both prokaryotic models of spontaneous and induced mutagenesis [227229] and eukaryotic cells [230-235]. The antimutagenic effect of flavonoids may be also achieved via other mechanisms: first, some flavonoids inhibit the metabolic activating process of dietary carcinogens such as benzo[a]pyrene, aflatoxin Bl, 2-amino-3-methylimidazo[4,5-f|quinoline (IQ), MelQ, Trp-P-1, etc. [227, 233, 236]. In particular, flavonoids have been shown to inhibit the citochrome P450-monooxygenase system, which is

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involved in the oxidative activation of mutagens such as benzo[a]pyrene [236, 237]. Second, flavonoids may confer protection through activation of transporters that mediate the extrusion of mutagens from the cell, like Pglycoprotein [238] and the multidrug resistant protein, MDRP [239], or detoxifying enzymes like NADPH:quinone reductase [240] or glutathione-Stransferase [241]. Third, reactive nitrogen metabolites have also been involved in mutagenesis, particularly ui the context of chronic inflammation; flavonoids inhibit inducible nitric oxide synthase [166], the main source of NO and subsequent nitrogen reactive substances in inflammation, and are capable of scavenging these reactive molecules [167]. And fourth, myricetin has been shown to stimulate DNA repair via activation of DNA polymerase p [234] in hepatocytes, an action that may contribute to its antimutagenic effect. The fact that flavonoids behave predominantly as antimutagenic/antioxidants in vivo must be reconciled [241] with their well documented properties as pro-oxidants and mutagenic agents in their own right in vitro [242-253]. In this regard, it is possible tiiat environmental conditions, e.g. the low concentration of metal ions in vivo [232] or the interaction with other dietary polyphenols, like curcumin [198, 216] or even carcinogens [227, 233, 236], are determinant. Zhu et al [254] have also suggested that rapid metabolization of flavonoids may prevent their toxic effects. The dose may be another important factor; thus quercetin has been reported to increase the multiplicity of AOM-induced colonic adenocarcinomas in one study [194], in which the doses were quite elevated (16.8 and 33.6 g/kg chow, i.e, about a hundredfold tlK)se exerting antiinflammatory effects in rat colitis in our laboratory [154]. This carcinogenic behaviour of quercetin may be related to the knoAvn mutagenic properties of manyflavonoids.Genistein has also been reported to increase non-invasive and total adenocarcinoma multiplicity, without affecting incidence, in this model [196]. In addition, some flavonoids may actually favor mutagen activation [236]. However, the predominant effect of flavonoids in vivo seems to be antimutagenic/anticarcinogenic. b) Inhibition of enzymes involved in signal transduction Flavonoids exert a modulatory activity on a nimiber of enzymes that participate in signal transduction, like protein kinase C, phosphatidylinositol 3-kinase, and tyrosine protein kinases (see [24] for review). Many of these

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enzymes are upregulated in neoplastic cells, evidencing a state of activation, and therefore inhibition byflavonoidsmay play a role in their anticancer effects [255, 256]. In particular, some tumors are highly dependent on an increased activity of receptor tyrosine kinases like the epidermal growth factor receptor, and may especially benefit from this approach [225, 257, 258]. Quercetin has been slK)wn to downregulate the e5q)ression of K-, Hand N-ras in both colon cancer cell lines and primary colorectal tumors [259]. c) Modulation of DNA-related enzymes and cell cycle factors Someflavonoids,including quercetin, inhibit eukaryotic topoisomerase I at the religation step [260]. Topoisomerases are involved in DNA replication, transcription and recombination and play therefore a key role in cell proliferation. Genistein inhibits topoisomerase n [261, 262], suggesting that it may be useful to treat rapidly proliferating carcinomas expressing high levels of this enzyme. Constantinou et al [261] also reported inhibition of both topoisomerase I and n by myricetin, quercetin, fisetin and morin. On the other hand, EGCG has been shown to inhibit telomerase, an enzyme necessary to preserve the chromosome tips of proliferating cancer cells, in HT29 cells [263]. Flavonoids seem to induce the expression of the antioncogene/755 [264]. In support of this hypothesis, quercetin is ineffective in reducing txmK)rigenesis mp5S -A knockout mice [265] and in arresting cell cycle in p53 knockout cells [264]. However, one study carried out in a lung cancer cell line found no evidence ofp53 induction by flavone [266]. Instead, the authors reported the induction of p21, an inhibitor of cyclin-dependent kinases, as well as dephosphorylation of RB protein, leading to cell cycle arrest in Gl. Genistein has been also shown to induce the expression of p21 as well as to downregulate cyclin Bl specifically [262]. Finally, Heruth et al [267] showed that genistein could downregidate the oncogene c-myc in colonic cancer cell lines as a consequence of tyrosine kinase inhibitioa It is noteworthy that EGCG, quercetin andflavonols/flavonesseem to block the cell cycle at the Gl or Gl/S phase [216, 222-224, 268], whereas genistein, an isoflavonoid, causes blockade at G2/M [223]. Only one study offers conflicting results, showing cell cycle blockade at G2/M for quercetin, apigenin and luteolin [264].

Table 1. Summary of the experimental studies showing the effect of flavonoids on animal models of gastrointestinal cancer. The model of cancer, the animal species and the main effects observed are shown.

rutin liquiritin

oesophageal colonic colonic

MNAN AOM AOM

rat CFI mice F344 rat

hesperidin 12031 & incidence of squamous cell carcinoma. 4multiplicity of carcinoma, papilloma and preneoplasia [200] -1hyperproliferationand focal areas of dysplasia [I901 & hyperproliferative markers [192]

. OI

01 u

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d) Antiestrogenic activity Flavonoids exhibit weak estrogenic/antiestrogenic properties [269]. In particular, flavonoids have been shown to bind type II but not type I estrogenic binding sites in the cytosol and nucleus [270]. Type 11 estrogen binding sites (EBSII) were initially described as receptors distinct from the 'classical* ones with a lower affinity (--10-20 nM) and which were highly correlated with hypertrophy and hyperplasia of the rat uterus, an effect which is selectively blocked by flavonoids [270]. Subsequently flavonoids, particularly quercetin and genistein, were shown to have antiproliferative effects on the human breast cancer model cell line MCF-7 and this activity was correlated to the blockade of EBSII [270, 271]. Furthermore, quercetin was found to induce EBSII in these cells [272]. The expression of EBSII is not limited to breast or ovarian cancer cells, since Ranelletti et al [222] and Di Domenico et al [273] described their occurrence in several cell lines of colonic origin and in human colorectal cancer biopsies. Moreover, the antiproliferative activity offlavonoidsagainst these cell lines was correlated to the number of EBSII. In Caco-2 cells estradiol but not other estrogens stimulates cell proliferation and activates c-src, c-yes, erk-1 and erk-2 [273]. Inhibition of cell proUferation by genistein was associated to downregulation of erk-2. Thus antagonism of EBSII seems to be involved in the anticancer activity offlavonoids.Nevertheless, this is clearly not absolutely required sinceflavonoidsare active also on cell lines that do not express estrogen receptors [274-276]. Some studies have proposed additional antiestrogenic actions of flavonoids, such as inhibition of aromatase [277] and of 17phidroxysteroid [278]. e) Others The flavonoids fisetin and genistein exhibit antiangiogenic activity as evaluated in in vivo studies [279]. Flavonoids may also exert a limiting effect on tumor metastasis by way of their inhibitory activity on proteolytic enzymes such as trypsin, leucine aminopeptidase and other methalloproteinases [280, 281]. Bergan et al [282] examined the effect of genistein, a putative antimetastatic agent, on cell adhesioa They showed that the isoflavonoid caused cell flattening, associated to the specific accumulation of focal adhesion kinase in contact areas and the formation of a

637

complex with Pi-integrin. Finally, flavonoids have been proposed to act as antineoplastic drugs sensibilizing agents via inhibition of the heat shock activating cascade, since heat shock proteins are thought to confer resistance to chemotherapy [283,284]. It is important to bear in mind that many of these activities have not been studied in gastrointestinal cells and that they do not apply to all flavonoids. The only purpose of the above compilation is to serve as a reference for the possible mechanisms of action of flavonoids as antineoplastics in the gastrointestinal tract. Further investigation is needed to clarify the respective contributions of the many activities proposed. Finally, we would like to mention two synthetic flavonoid derivatives with gastrointestinal anticancer activity for the sake of completeness: flavone acetic acid andflavopiridol.Flavone acetic acid (FAA, LM975) is the active principle of a parent esterified compound (LM985) which emerged from a series offlavonoidssynthesized by Lyonnaise Industrielle Pharmaceutique and screened by the National Cancer Institute [285]. FAA showed dramatic activity against solid tumors in mice including colonic transplantable tumors* Its mechanism of action was found to be complex, including antiangiogenic and TNFa releasing properties, as well as activation of natural killer cells. Unfortunately it showed no effect in humans for unclear reasons. On the other hand,flavopiridol(L86-8275) is a potent inhibitor of cdc-2 kinase (at least 250-fold more potent than quercetin or genistein), an enzyme involved in cell cycle regulation, as well as of protein kinase C [286]. Flavopiridol is capable of promoting mitomycin C-induced apoptosis in gastric cancer cells [287] and induces cell cycle arrest and apoptosis in oesophageal cell [288]. It has undergone Phase I clinical trial. In summary, the evidence pointing to the possible use offlavonoidsin the treatment or prevention of gastrointestinal cancer, as derived from epidemiological, in vivo and in vitro studies, is still promising but inconclusive. Further investigation about the effects of these compounds on gastrointestinal cancer is needed, particularly with regard to their mechanism of action. ABBREVIATIONS 4-NQO = 4-nitroquinoline 1-oxide AOM = azoxymethane cAMP = cyclic adenosine monophosphate

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CFTR = Cystic Fibrosis Transmembrane conductance Regulator cNOS = constitutive Nitric Oxide Synthase DMBA = 7,12-dimethylbenz[a]anthracene DMH: 1,2-dimethylhydrazine EBSn = Estrogen Binding Site 11 ED50 = Eflfective Dose 50 EGCG = (->Epigallocatechin Gallate GST = Glutatliione S-Transferase IBD = Inflammatory Bowel Disease iNOS = inducible Nitric Oxide Synthase IQ = 2-amino-3-methylimidazo[4,5-fjquinoline LTB4 = leukotriene B4 MAN: methyl-(acetoxymethyl)-nitrosamine MDA = malondialdehyde MDRP = Multidrug Resistance Protein MelQ = 2-amino-3,4-dimethylimidazo[4,5-f|quinoline MNAN: N-methyl-N-amylnitrosamine MNNG: N-methyl-N'-nitro-N-nitrosoguanidine MPO = myeloperoxidase NADPH = Nicotine Adenosine Dinucleotide Phosphate, reduced form NS AIDs = Nonsteroidal Antiinflammatory Drugs PAF = Platelet Activating Factor PGE2 = prostaglandin E2 PMA = Phorbol 12-Myristate 13-Acetate PPIs = Proton Pump Inhibitors ROM = Reactive Oxygen Metabolites TNBS = Trinitrobenzene Sulphonic Acid TNF-a = Tumor Necrosis Factor a Trp-P-1 = 3-amino-l,4-dimethyl-5H-pyrido[3,4-b]indole VIP = Vasoactive Intestinal Peptide ACKNOWLEDGEMENTS The authors acknowledge funding by the Spanish Ministry of Education and Culture (SAF98-0157 and SAF98-0160).

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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BIOACTIVITY OF THE PHENOLIC COMPOUNDS IN HIGHER PLANTS JUAN M. RUIZ and LUIS ROMERO Departamento de Biologia VegetalFacultad de Ciencias, Universidad de Granada, 1807 l-Granada,Espana. ABSTRACT: Phenylpropanoid compounds encompass a wide range of stmctural classes and biological functions. Among plant polyphenols, of which several thousand have now been described, flavonoids form the largest group. However, phenolic quinones, lignans, xanthones, coumarins and other groups exist in considerable numbers and there are also many simple monocyclic phenolics. In this review, we examine the principal biosynthetic pathway of phenolic compounds in higher plants, and we define the different enzymes responsible for the regulation of phenolic metabolism. The central focus, however, is on the different factors, both biotic and abiotic, which influence the synthesis or degradation of phenolic compounds (bioactivity), analysing the physiological and ecological implications of these compounds in tlie adaptation by plants to adverse conditions. Finally, we related bioactivity of phenolic compounds with tlie appearance of physiological disorders in different crops.

GENERAL PHENYLPROPANOID PATHWAY Higher plants use amino acids not only as protein building blocks but also, and in even greater quantities, as precursors for a large number of secondary metabolites [1]. Phenylpropanoid compounds are among the most influential and widely distributed secondary products in the plant kingdom. These compounds serve a range of important functions in plants, providing structural components (such as lignin), protection against biotic and abiotic stresses (anti-pathogenic phytoalexins, antioxidants and UV-absorbing compounds), pigments (particularly the anthocyanins), and signalling molecules (e.g. flavonoid nodulation factors) [2]. Limiting this discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds that are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant [3,4].

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Biosynthesis of phenylpropanoid compounds The general phenylpropanoid pathway links the shikimate pathway to the lignin branch pathway. The latter pathway leads to the formation of a series of hydroxycinnamic acids and hydroxycinnamoyl-CoA esters varying in their degrees of hydroxylation and methylation [5]. There are diffent pathways by which all phenolic compounds are synthesized [6,7]. The shikimate/arogenate pathway leads, through phenylalanine, to the majority of plant phenolics, and therefore we shall centre the present revision on the detailed description of this pathway. The acetate/malonate pathway leads to some plant quinones but also to various side-chain-elongated phenylpropanoids (e.g. the group of flavonoids). Finally, the acetate/mevalonate pathway leads by dehydrogenation reactions to some aromatic terpenoids. The shikimate/arogenate pathway leads to the formation of three aromatic amino acids: L-phenylalanine, L-tyrosine, and L-tryptophane. This amino acids are precursors of certain homones (auxins) and of several secondary compounds, including phenolics [6,7]. One shikimate/arogenate is thought to be located in chloroplasts in which the aromatic amino acids are produced mainly for protein biosynthesis, whereas the second is probably membrane associated in the cytosol, in which L-phenylalanine is also produced for the formation of the phenylpropanoids [7]. Once Lphenylalanine has been synthesized, the pathway called phenylalanine/hydroxycinnamate begins, this being defined as "general phenylpropanoid metabolism" [7]. The general phenylpropanoid pathway begins with the deamination of Lphenylalanine to cinnamic acid catalyzed by phenylalanine ammonia lyase (PAL), Fig. (1), the branch-point enzyme between primary (shikimate pathway) and secondary (phenylpropanoid) metabolism [5-7]. Due to the position of PAL at the entry point of phenylpropanoid metabolism, this enzyme has the potential to play a regulatory role in phenolic-compound production. The importance of this is illustrated by the high degree of regulation both during development as well as in response to environmental stimuli. The enzyme PAL was first detected by Koukol and Comm [8], and is found in most higher plants as well as many microorganisms. This enzyme has an optimal pH of 8.8 and does not require a cofactor for its activity. Some preparations of PAL show activity towards the tyrosine although PAL

653

is especially specific towards the substrate from which it draws its name [6,7].

Phony lalan mo

SaJioylic acid (SA)

CiMnam»c acid

\^^

4-coumaric a o d

4 -coumaroyl-CoA

Ftnuiic acio n3C3{[]> sinapic ac:d Feruloyl-CoA

Sinapyl-alcohol

Guaiacyl.siibunils

Syringytsubunits

Lignin

Coumartns Isoflavonoids (via C H R and CHS)

Naringenm chalcoru)

c:K::n<>

Naringomn ^UF3H "^ OH 0«hydrokaomp(erol

ILX^^^^i

Au

r^;^t{]> Phlobapher c-s 1L_^ FlavonoH

C3c=)t{> Flavonois

^.

Loucopoiargonidin

•'' Anthocyanins Tannins

Fig. (1). Schematic view of some branches of phenylpropanoid metaboHsm. Solid arrows indicate enzymatic reactions with the respective enzyme indicated on the right. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; CHS, chalcone synthase; CFI, chalcone flavavone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol reductase; CHR, chalcone reductase. Broken arrows indicate metabolic branches towards several classes of phenylpropanoids, or several subsequent enzymatic steps. In some cases the enzymes indicated are also involved in other reactions, not shown. •Taken from Weisshaar and Jenkins, 1998, [2].

654

All phenylpropanoids are derived from cinnamic acid, which is formed from L-phenylalanine by the action of PAL. Several simple phenylpropanoids (with the basic C6-C3 carbon skeleton of phenylalanine) are produced from cinnamate via a series of hydroxylation, methylation, and dehydration reactions, including /7-coumaric, caffeic, ferulic, and sinapic acid and simple coumarins, Fig. (1). The free acids rarely accumulate to high levels inside plant cells, but instead are usually conjugated to sugars (e.g. salicylate-glucose conjugates), cell-wall carbohydrates (e.g. ferulate esters), or organic acids (e.g. chlorogenic acid) [4]. Salicylic, benzoic, and /7-hydroxybenzoic acids, although not strictly phenylpropanoids themselves because they lack the three-carbon side chain, originate from the phenylpropanoids cinnamate and /7-coumarate, Fig. (1) [9]. Lignin and suberin, complex polymers formed from a mixture of simple phenylpropanoids, Fig. (1), vary in composition from species to species [5,10]. A large number of stress-induced phenylpropanoids are derived from the CI5 flavonoid skeleton, which is synthesized via the chalcone synthase (CHS)-catalyzed condensation of/?-coumaroyl-CoA and three molecules of malonyl-CoA, Fig. (1) [6,7]. In most plant families, the initial product of CHS is a tetrahydroxychalcone, which is fiither converted to other flavonoid classes, such as flavones, flavonones, flavonols, and anthocyanins [11]. In a number of species (e.g. pine, grapevine and peanut) the condensation ofpcoumaroyl-CoA or cinnamoyl-CoA with three malonyl-CoA molecules can also give rise to stilbenes by action of stilbene synthase (SS), Fig. (1) [12]. In legumes, isoflavone synthase (IFS) rearranges the flavonoid carbon skeleton, leading to the accumulation of a wide range of simple isoflavonoids, coumestrans, pterocarpans, and isoflavans. Structural diversity among the phenylpropanoids is brought about by a variety of modifications, including regro-specific hydroxylation, glycosylation, acylation, prenylation, sulfatron, and methylation [4]. Oxidation of phenolic compounds The metabolism of phenolics is regulated by the activity of various enzymes. As indicated above, the main and determining enzyme of phenolic synthesis is PAL, while in oxidation processes, the enzymes involved are peroxidase (POD) and primarily polyphenoloxidase (PPO).

655

PPO (known also as catecol oxidase, phenolase or diphenol-oxygen oxidereductase) and POD catalyse the oxidation of o-diphenols to odiquinones, as in the hydroxylation of monophenols [13]. PPO is located exclusively in the plastids of healthy tissues, while most phenolic compounds are localized in the vacuoles, the two compounds thus being physically separated [13]. PPO apparently, however, exists free in the cytoplasm in degenerating or senescent tissues such as ripening fruit [14]. Reports of PPO in other organelles may be artifactual due to a POD activity mistakenly identified as PPO, solubilization of the enzyme during organelle isolation, or fragmentation of plastid parts containing PPO into other organelle [13]. Three lines of experimental evidence are used to support the idea that PPO is solely a plastid enzyme: fractionation studies, histochemistry, and chemical or genetic modifications of the plastid [13]. Futher confirmation of a plastid localization was reported by Henry et al. [15] after coupling fractionation techniques with PPO cytochemistry. The only structures with cytochemically detectable PPO activity were recognized as plastid parts. Because PPO can readily convert monophenols to o-diphenols in vitro [16], some investigators have assumed that the same process is involved in phenolic compounds synthesis in vivo. There are several arguments against this assumption. As discussed above, PPO is located exclusively in the plastids of healthy tissues and is apparently not even activated until it crosses the plastid envolope. The vast majority of phenolic compounds in higher-plant cells are located in the vacuole. One might argue, however, that the plastid location of PPO is necessary to provide a strong reducing environment in order to prevent fiirther oxidation of o-diphenols to odiquinones by PPO [13]. Although some investigators have found relationships between phenolic compound content and extractable PPO activity from cells or tissues, the relations may be due to POD activity, given that several works have demonstrated close relationships between POD activity and the oxidation of phenolics [17]. Moreover, there is considerable evidence that PPO is not active as a phenol oxidase in chloroplasts, but is limited as a phenol oxidase by latency or lack of substrate [18]. The latent form of the enzyme can be activated by a wide variety of treatments, including detergents [19], fatty acids [20] trypsin [21] and Ca^"" [22]. The results of Tolbert [21] indicated that light could activate latent PPO because polyphenols can be oxidized photochemically by chloroplast membranes in the absence of other

656

activators. The only clear roles that PPO has in phenolic metabolism are in those cases in which plastid and vacuole contents are mixed. These cases can be divided into two categories: 1) senescence and 2) injury. In the first case, PPO has been invoked to explain the development of pigmentation in black olives, and other dark brown or black, usually dead, plant tissues. The increase in extractable PPO activity often seen during senescence is due to activation of previously synthesized enzyme [23]. Many researchers believe, however, that there is a fiinctional significance to the rapid production of quinones caused by injury [16]. Whether due to mechanical injury or to cellular disruption from disease, the quinones produced by the resulting PPO-phenolic compounds interaction are very reactive, making them good candidates for involvement in protection from other organisms. Thus, disruption of the plastid results in activation of latent PPO, which reacts with phenolics released from the vacuole. Finally, recent research suggests that different types of stress caused either by biotic or abiotic factors activate PPO and POD, thereby stimulating phenolic boactivity [24-27]. PHYSIOLOGICAL IMPLICATIONS: COMPOUNDS VS. PLANT HORMONES At present, it is still not known definitively whether phenolics play a physiological role in the growth and metabolism of plants. Many phenolic compounds are capable of exerting significant effects on growth and developmental processes in plants, when these compounds are applied to tissues at physiological concentrations [6]. With so much variation in structure, it is unlikely that phenolics as a group of substances have one particular universal role in regulating growth and development. Rather, it is possible that individual classes or individual subtances may carry out significant activities in certain physiological processes. Many of the observed physiological interactions may be incidental to the wider fiinction of phenolics as protective agents in plants [6]. Even though most phenolics are not hormones themselves, they may affect plant growth by interaction with one or other of the major kinds of plant hormones, such as the auxins. Physiological studies have suggested

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that several phenolic compounds regulate auxin, indole-3-acetic acid (lAA), and others affect polar transport of auxins. Hydroxyphenolic, chlorogenic acid, was shown to be a protector of auxin against oxidation by lAA oxidase in sunflower leaves, thereby stimulating plant growth [28]. Dihydroxyphenolic compounds have been considered inhibitors of lAA oxidation, whereas monohydroxyphenolic derivatives stimulated lAA oxidation and thus have a potentially inhibiting effect on growth [29]. Certain flavonols, such a quercetin, were found to inhibit polar transport of lAA [30]. Changes in the levels of lAA as well as abscisic acid had been measured during growth and ripening in the pericarp tissue of two cultivars of tomato fruit [31]. Recently, Buta and Spaulding [32] found that the decline of chlorogenic acid and rutin levels during fixiit ripening paralleled the decline in lAA levels measured previously in the pericarp tissues of two varieties of tomato fhiit during maturatio (Lycopersicon esculentum fruit var. Ailsa Craig and Pik-Red). These phenolics are among the ones that have been suggested as regulators of auxin metabolism. Another hormone that can be affected by phenolics is ethylene. Coumaric acid is necessary as a cofactor for the biosynthesis of ethylene [33]. On the other hand, caffeic acid inhibits an enzyme (peroxidase type) for which the cofactor is p-coumaric acid. Therefore, the balance between /7-coumaric and caffeic acid can in theory influence the regulation of ethylene biosynthesis [6]. Phenolics can react with other hormones by synergism or inhibition, and both situations can be observed in the case of gibberellin. There is evidence that dihydroxyconiferyl-alcohol gives rise to synergetic effects over gibberellin, stimulating hypocotyl elongation [34]. On the contrary, the replacement of hydroxyconiferyl-alcohol for one or several hydroxycinnamic acids results in the inhibition of effects caused by gibberellin [34]. Finally, in view of the above examples, phenolics appear to interact specifically with certain plant hormones which effect growth. ECOLOGICAL IMPLICATIONS AND FUNCTIONS OF PHENOLIC COMPOUNDS

PHYSIOLOGICAL

There is increasing evidence that the functional aspects of phenolics must be considered in making any distinction between primary and secondary

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compounds. For instance, it is known that many secondary compounds are valuable storage vehicles and indispensable elements in anatomical and morphological structures. However, there is increasing knowledge that they are also of prime ecological importance for the improvement in plant survival [7]. First of all, phenolics are of great importance as cellular support materials. They form an integral part of cell wall structures, mainly in polymeric materials such as lignins and suberins. Lignin is the second most abundant plant polymer after cellulose and forms a major component of terrestrial biomass [35]. Lignin is the term given to a group of complex phenolic polymers that provide important strengthening and waterproofing properties to plant cell walls. It is not suprising, therefore, to find lignin playing fiindamental roles in mechanical support, solute conductance, and disease resistance in higher plants [5]. The prospect of manipulating the quantity and composition of lignin in plants has several attractions, particularly in the modification of the digestibility of plant material and in the development of resistance to microbial pathogens. Lignification is ultimately dependent on phenylpropanoid metabolism for the supply of the basic building blocks of lignin and this dependency has led to the targeting of phenylpropanoid metabolism as a suitable point at which to manipulate lignin biosynthesis. Phenolics are of great ecological importance along with various toxic nitrogen-containing compounds as well as attractant or repellent terpenoides. The most significant fimction of the phenolic flavonoids, especially the anthocyanins, together with flavones and flavonols as copigments, is their contribution to flower and finit colours [36]. This is important for attracting animals to the plant for pollination and seed dispersal [6]. On the other hand, phenolics may protect plants against predators. Herbivores react sensitively to the phenolic content in plants. The rise in cumestrol and cumarin levels can be toxic for herbivores, causing anti-coagulation and estrogenic effects [37]. Phenolics may influence competition among plants, a phenomenon called allelopathy. A series of experiments in both the field and the laboratory have indicated a role for a number of phenolic derivatives (hydroquinone, hydroxybenzoates and hydroxycinnamates) as allelopathic agents. These are chemicals excreted by the plant, which may be autotoxic or affect the growth of other plants in the environment [6,7]. A role in recognition and signalling has been determined for a variety of

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phenolics. Thus, phenolics induce the germination and development of haustorium in parasitic plants [38], the expression of v/> genes responsible for T-DNA transfer from Agrobacterium to the plant cell [39], and regulate the expression of "nod" genes during the formation of the legumeRhizobium symbiosis [40]. Li the last case,flavonoidsand isoflavonoids from seed and/or root exudates or extracted from roots, were identified as regulatory molecules [40]. The processes of isoflavonoid exudation and accumulation are partly regulated by environmental factors. The isoflavonoid exudation and accumulation is inhibited by phosphate [41] and various N forms with nitrates being a stronger inhibitor than ammonium or urea [40]. However, most of the literature available relates phenolic compounds to resistance against the following types of stress: (i) pathogen attack, (ii) woimds, (iii) ultraviolet radiation, (iv) environmental pollution (specially ozone), and finally (v) thermal stress. Due to the importance and incidence of these types of stress for the survival and adaptation of plants, this topic will be explored in more detail in the forthcoming sections. Bioactivity of phenolics in resistance to pathogen attack Three groups of phenolic compounds are involved in defense responses: (1) the signal molecule salicylic acid [42], (2) phenylpropanoid compounds (phenolics and phytoalexins) [43], and (3) lignin and related polyphenolics [44,45]. Plants react to pathogen attack through a variety of active and passive defense mechanisms. At the site of infection, a hypersensitive response is often initiated in resistant plants, which isfrequentlymanifested as necrotic lesions resultingfromhost cell death. In addition, the distal uninfected parts of the plant can develop systemic acquired resistance, which results in enhanced long-lasting defense against the same or even unrelated pathogens [45]. Both the hypersensitive response and systemic acquired resistance are associated with increased expression of a large number of defence or defence-related genes [46]. Examples of the defence reactions include the lignification and suberization of the plant cell wall, deposition of callose, de novo synthesis of pathogenesis-related proteins, production of active oxygen species, and biosynthesis of secondary metabolites (phytoalexins) [47-50]. Much evidence suggests that the increases in salicylic acid levels are

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essential for the induction of systemic acquired resistance [42]. Pathogen-induced necrosis on the leaves of many plants results in the production of a mobile signal in the phloem that triggers systemic resistance to subsequent pathogen [51]. The development of systemic acquired resistance depends on the rate at which the pathogen causes necrosis in the infected leaf Pathogen-induced necrosis in the inoculated leaf is accompanied by the accumulation of salicylic acid at the site of inoculation, in phloem fluids, and in healthy, uninoculated leaves [52]. Several reports suggest that PAL is a key regulatory enzyme in the synthesis of salicylic acid. Fig. (2), and the establishment of systemic acquired resistance. Mauch and Slusarenko [53] showed that in Arabidopsis, PAL activity was essential for the accumulation of salicylic acid and expression of the hypersensitive response. Recently, it was also reported that tobacco plants epigenetically suppressed in PAL activity were unable to express systemic acquired resistance [54].

Fig. (2). Proposed pathways of SA and 4HBA biosynthesis. Enzymatic steps for which the enzymes have been identified include PAL, CA4H (cinnamic acid 4-hydroxylase), and BA2H (benzoic acid 2hydroxylase). *TakenfromSmith-Becker et al., 1998, [55].

Smith-Becker et al. [55] have shown that the first measurable effect of the mobile signal for systemic acquired resistance in cucumber inoculated

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with P. syringae is the transient stimulation of PAL activity in the petiole of the inoculated leaf and in the stem above the inoculated leaf The transient increase in PAL activity precedes a transient increase in salicylic acid and 4hydroxybenzoic acid in phloem fluids, and suggests that the two compounds are produced de novo in stems and petioles, perhaps in vascular tissues.

Host Defense Activation by H.O, Elevation

Fig. (3). Hypothetical representation of host defense activation by extracelluiarly produced H2O2 in GO-transgenic plant. Salicylic acid biosynthesis is elevated through stimulation of benzoic acid 2hydroxylase activity. The expression of defense-related proteins is induced by increased salicylic acid and perhaps also by H2O2 itself via a separate pathway. •TakenfromWu et al, 1997, [42].

A metabolic engineering approach has now provided direct evidence for the role of salicylic acid in systemic acquired resistance. Transgenic tobacco plants were produced expressing the "nah G" gene from Pseudomonas putida, which encodes a salicylate hydroxylase that converts salicylic acid

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to catechol [56]. These plants had greatly reduced salicylic acid levels and were unable to establish systemic acquired resistance. Moreover, not only did they fail to exhibit resistance to virulent challenges following inoculation with avirulent pathogens, but also they were no longer able to express hypersensitive resistance against the primary avirulent challenge [56], reflecting an important role for salicylic acid in the expression of local resitance. Fig. (3). A range of defence response genes, including those encoding the so-called pathogenesis-related proteins, are activated in systemically protected leaves and in response to exogenously applied salicylic acid [51]. Plants have envolved a wide array of chemical defences against pathogens. These include secondary metabolites with anti-microbial properties [57]. Some secondary metabolites are constitutively present in normally developed healthy plants, whereas others are induced by pathogen invasion [58]. The latter include phytoalexins, which are synthesized de novo after a pathogen attack [43], and phytoanticipins [59]. The first direct demonstration of the potential significance of phytoalexins in plant defence was provided by the introduction of a grapevine SS gene into tobacco plants [60]. The foreign gene product was able to divert a portion of the substrates of chalcone synthase to the synthesis of the stilbene phytoalexin resvaratrol, resulting in plants with increased resistance to thefiingalpathogen Botrytis cinerea. Generally, the increase in phenolic content is the response associated with plant disease resistance. Diibeler et al. [61] found a direct relationship between, on the one hand, the accumulation and qualitative composition of phenolic compounds in Fagus sylvatica and resistance against Cryptococcus fagisuga, Li addition, Valette et al. [62] carrying out histochemical and cytochemical investigations of phenolic compounds in roots of banana infected by the burrowing nematode Radopholus similis showed the importance of phenolic accumulation to differentiate plants which were resistant from tiiose sensitive to infection by this nematode. Using Neu*s reagent, these researchers indicated the presence of flavonoids and caffeic esters in parenchyma and vascular cells and of ferulic acid in walls of parenchyma cells in the resistant cultivar, while these compounds were not detected in susceptible roots. Finally, Valette et al. [62] concluded that, in resistant banana, phenols incluidingflavonoids,caffeic esters, and dopamine may limit root penetration by the nematode, and the high level of vascular lignification and suberization of endodermal cells restricts xylem

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invasion by, and prevents multiplication of, the pathogen in the vascular tissues. As indicated above, PAL, the first enzyme of phenylpropanoid metabolism, plays a significant role in the regulation of phenol biosynthesis in plants as a response to pathogen infection [55,63]. The phenylpropanoid skeletons serving as building blocks for lignin induce lignification, which has been proposed as a mechanism of disease resistance in plants against the invasion of fungal pathogens [64]. Lignin precursors and free radicals are known to inhibit fungal enzymes, and lignin accumulation may act as a barrier and check to the translocation of host nutrients into the pathogen. Nagarathna et al. [63] suggested that an increased amount of PAL increases lignin synthesis, leading to a hypersensitive response at the site of infection in the incompatible interations and thereby restricting fungal growth. Bioactivity of phenolics against wounds Woxmds may be inflicted by severe weather, insects, large herbivores, the activities of man, or even during the normal development processes of the plant, such as abscission or growth cracks [65]. In commerce, even minimal processing of fresh fruit and vegetables involves many mechanical processes (e.g. abrading, cutting and peeling), which injure the tissue [66]. Such wounding induces alterations in many physiological processes, which often make the processed item more perishable than the unprocessed fresh product and diminish the shelf life of thefinalminimally processed product [67]. Wounding also elicits several physiological responses associated with wound healing [68]. Foremost among these reactions are alterations in phenolic metabolism and the concomitant increase in the propensity of the wounded tissue to brown [66]. The activity of PAL and the concentration of phenolic compounds (e.g. chlorogenic acid, dicaffeoyl tartaric acid, and isochlorogenic acid) increase in excised iceberg lettuce midrib segments after wounding [69]. In addition, Thypyapong et al. [25] provide evidence that wound-responsive expression of potato PPO is regulated at the levels of transcriptional activity or mRNA stability. Similarly, wounding increases the steady-state PPO mRNA levels in apple [70]. Phenylpropanoid biosynthesis gives rise to caffeic acid, lignins.

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flavonoids, and salicylic acid. The coordinated regulation of PPO and phenylpropanoid biosynthesis underscores the importance of PPO in wound responses. Wound induction of the phenylpropanoid pathway leads to intracellular accumulation of phytoalexins and the extracellular polymerization of phenolics, e.g. lignification, providing barriers to pathogen ingress [4]. Once pathogens overcome these barriers and disrupt plant cells, PPO activity diverts phenolics to quinone production, causing cell death and providing additional polymerized phenolic barriers to sencondary infection [71]. PPO catalyses the dependent oxidation of phenolics to quinones. The secondary reactions of quinones lead to the formation of polymeric brown or black pigments, which are responsible for significant post-harvest losses offiiiitsand vegetables [72]. Finally, induced PPO activity consists of both systemic and localized components. Systemic induction of PPO in tissues in response to all types of injuries may represent a broad, defensive role for PPO in protection of juvenile tissues from subsequent attack by a broad spectrum of pathogens and pests [71]. Bioactivity of phenolics in resistance to ultraviolet rays In recent years, there has been considerable concem over reductions in stratospheric ozone concentrations resulting from human activities. Since stratospheric ozone is the primary screen of solar ultraviolet radiation, ozone reduction would increase ultraviolet radiation (UV-B, wavelengths between 280 and 320 nm) reaching the earth's surface. UV-B is biologically significant, since it is potentially mutagenic and its influnce is increasing due to ozone depletion [73]. Among the most important physiological and biochemical processes affected by UV-B exposure is photosynthesis. UV-B radiation generally results in reductions in net photosynthesis, coinciding with ultrastructural damage to the chloroplast. UV-B radiation also affects stomatal resistance, chlorophyll concentration, soluble leaf proteins, lipids, and carbohydrate pools [74]. Finally, exposure to UV-B give rise to variations in anatomical and morphological plant characteristics. Commonly observed changes include plant stunting, reductions in leaf area and total biomass, and alterations in the pattern of biomass partitioning into various plant organs. In sensitive plants, evidence of cell and tissue damage often appears on the upper leaf

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epidermis as bronzing, glazing and chlorosis [74]. Interception of UV-B by epidermal flavonoids is often proposed as an adaptive mechanism preventing UV-B from reaching the mesophyll and affecting photosynthesis [75], Flavonoids have high absorption capacity for UV-B and they may be present in mature leaves at levels exceeding 50 |imol g"* f w. [76]. When higher plants are subjected to supplemental UV they tend to synthesize flavonoids and anthocyanins [77], and some fimgi also react by forming such pigments [78]. Liu et al. [79], analysing the effect of UV-B radiation on phenolic metabolism, growth and photosynthesis in barley primary leaves, indicate that UV-B significantly slows rates of leaf elongation with a simultaneous, possibly related, increase in wall-bound ferulic acid esters in the epidermis. UV-B also markedly increases flavonoid accumulation in both the lower epidermis and underlying tissues. On the other hand, since UV-B had no significant effect on photosynthesis rates, photosynthetic pigments, fresh weight or dry weight, this authors conjecture thatflavonoidsin the barley primary leaf provide considerable constitutive, as well as inducible, resistance to damage from this type of radiation. Finally, Liu et al. [80] showed that UV-B sharply boosted PAL activity while reducing POD activity; this would explain the synthesis and accumulation of flavonoids and ferulic acid in barley primary leaves. Bioactivity of phenols against ozone exposure Phenolic compounds take part in protection, regeneration and degradation processes caused by toxic pollutants [81]. An increase in the phenolic level is commonly observed after exposure to hydrogen fluoride [82], sulphur dioxide [83] and especially to ozone [84]. Ozone in recognized as one of the most damaging secondary gaseous air pollutants essentially generated from nitrogen oxides produced by anthropogenic activities [85]. Among its numerous impacts on the environment, it has been shown to affect crop yield significantly [86]. Currently, to indicate phytotoxic environmental concentrations of ozone, the assessment of visible ozone injury (necrotic spots and bronze-coloured symptoms that follow a chlorotic fecking of leaves) of more or less ozone sensitive plant varieties is becoming widely used [85]. Tobacco, Nicotiana tabacum [87], and clover, Trifolium sp., [88] are the most important ozone

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bioindicators. From a physiological standpoint, molecular and biochemical studies have suggested that ozone stimulates phenolic metabolism and the biosynthesis of lignin or other substances partly derived from coniferyl alcohol [89]. Lignification of mesophyll cell walls might confer some protection against oxidation, and thus be a defence response against ozone [90]. Studies have shown that phenylpropanoid metabolism can be stimulated by ozone. The activity of PAL increased in soybean [91], Scots pine {Pinus sylvestris L.) [92], and parsley {Petroselinum crispum L.) [93] soon after treatment with 150-200 nmol O3 mor\ Rapid increases in transcript levels for PAL in response to ozone have been observed in parsley [93], Arabidopsis thaliana L. Heynhold [94] and tobacco {Nicotiana tabacum L.) [95]. Transcript levels for 4-coumarate:CoA ligase (4CL), the last enzyme in the general phenylpropanoid pathway, increased commensurately with PAL transcripts in ozone-treated parsley seedlings [93]. Phenolic compunds reported to accumulate in leaf tissue in response to ozone include hydroxycinnamic acids, salicylic acid, stilbenes, flavonoids, furanocoumarins, acetophenones, and proanthocyanidins [85, 92, 93, 96, 97]. A rise in transcript levels and activity of cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in lignin biosynthesis, were observed in Norway spruce {Picea abies L.) needles [84] and parsley leaves [93] after treatment with ozone. Increased lignin in sugar maple {Acer saccharum Marshall) foliage following exposure to ozone has also been reported. However, higher levels of lignin were not detected in the foliage of several ozone-treated conifers and other hardwood species [96]. Booker and Miller [98], studying the phenylpropanoid metabolism and phenolic composition of soybean leaves following exposure to ozone, found that the activities of general phenylpropanoid pathway enzymes (PAL and 4CL) were stimulated by ozone 6h after their application, whereas the activity of the key enzyme in lignin synthesis, CAD, was stimulated 27 h after ozone exposure. In addition, these authors report that the levels of cellwall-bound total phenolics, acid-insoluble lignin and lignothioglycolic acid extracted from leaf tissue from ozone-treated plants increased on the average by 65%. However, histochemistry, UV and IR spectra, radiolabelling and nitrobenzene oxidation assays all indicated that lignin and suberin did not increase with ozone treatment. Finally, taking into

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account these results, Booker and Miller [98] concluded that ozone-induced incrases in phenolic metabolism, resemble certain elicited defense response, and thus occur in concert with effects characteristic of the browning reaction and wound responses. Bioactivity of phenolics in resistance against thermal stress Most plants suffer damage, both physiological and biochemical by exposure to temperatures higher or lower than optimal for growth [99]. The results of these injuries, which are reflected in most metabolic processes may be a reduced growth capacity of the crops and therefore lower commercial yield [100]. It has been demontrated that thermal stress induces the production of phenolic compounds [3,4,101], Phenolic compounds may be involved in plant responses to cold stress and in plant acclimation to low temperature. Acclimation of apple trees to cold climates was found to be associated with a seasonal accumulation of chlorogenic acid [102]. Strengthened frost tolerance in a variety of plants were attributed to thicker cell-wall lignification or suberization [102]. Thickening of cell walls and increased production of suberin-type lipids were observed in cold-acclimated winter rye leaves [103]. The presence of suberin in cell walls may favour membrane cell-wall adhesion, a major factor in the resistance of plant cells tofreezing[104]. Stimulated of PAL activity as a response to chilling treatment was observed in chilling-sensitive tissues such as potatoes and sweet potato tubers, as well as in apple fruits [105]. Solecka and Kacperska [106] studying in the leaves of winter oilseed rape plants grown for 3 weeks at 2^C and then exposed to a brief freezing and thawing, found pronounced changes in PAL activity. These changes included: a) a marked boost in the total and specific activities of the PAL, noted during thefirst2 days of plant treatment with chilling temperature, b) a sharp, swift and transient rise in PAL activities noted directly after thefreezingand thawing treatment. Recently, working in our laboratory with tomato and watermelon plants exposed to different temperature, we found that the optimal growth temperatures were 25 and 35 °C respectively, while over 35 ^C and below 15 °C, respectively, caused thermal shock . The results of this experiment indicate that in plants the response against thermal stress was similar regardless of the temperature. With regard to phenolic metabolism, thermal stress in tomato and watermelon plants is characterized by

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increased PAL activity and foliar accumulation of phenolic compounds, and also by a sharp drop in the activity of enzymes governing phenolic oxidation (PPO and POD)^ These results, together with those of the other works described above indicate that phenylpropanoid metabolism may play an important role in the development of plant acclimation to thermal stress. ABIOTIC FACTORS COMPOUNDS

AND

BIOACTIVITY

OF

PHENOLIC

Given the scope of the processes in which phenolic compounds are involved (e.g. interactions with plant hormones, resistance to pathogen attack, wounds, UV rays, ozone and thermal stress, implications for agriculture), knowledge of the factors that regulate the metabolism of these compounds could enable the manipulation of their synthesis or degradation, depending on the conditions chosen or the results desired. Among the abiotic factors most widely used to manipulate and vary the metabolism of phenolics, are biocides, primarily herbicides and fungicides, and different nutrients. Biocides Herbicides are the biocides most likely to affect the metabolism of plants, including secondary metabolism [107]. The synthesis of hydroxyphenolics and anthocyanin in plants can be influenced by a variety of environmental and chemical stimuli. Some herbicides were found to raise the levels of these compounds in plants [108] whilst others had the opposite effect [109]. The products of secondary metabolism are controlled by enzymes, including PAL and chalcone isomerase (CI), and several herbicides appear to intensify the activities of those enzymes involved in the accumulation of hydroxyphenolic compounds and anthocyanin biosynthesis in several plant species [109-111] whereas others depress this activity [112]. For example. Rivero, R.M.; Ruiz, J.M.; Garcia, P.C.; L6pez-Lefebre, L.R.; S^chez, P.C.; Romero, L. Resistance to thermal stress: accumulation of phenolic compounds in tomato and watermelon plants (in consideration).

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PAL activity in soybean seedlings was increased by several herbicides such as DPX-4189, glyphosate and acifluorfen [112,113]. Also, the activities of PAL and tyrosine ammonia lyase (TAL) in both maize and soybean seedlings were also increased by alachlor [110] and metolachlor [111]. Depending upon the type and concentration of the herbicides, a range of activity from little impact on plant growth to severe symptoms of toxicity were found [114] and these varied effects proved to be associated with marked changes in secondary metabolism. Nemat Alia and Younis [115] studing the effects of different herbicides (trifluralin, fluometuron, atrazine, alachlor, and rimsulfuron) on phenolic metabolism in maize {Zea mays L.) and soybean {Glycine max L.) seedlings, detected various modes of action on this metabolic process, depending on the herbicide applied. The results of that work showed that the activities of PAL and CI were greatly enhanced in both species by alachlor and rimsufuron, but diminished by trifuralin. Moreover, dydroxyphenolic compounds were increased in both species by alachlor and rimsulfuron and decreased by trifuralin and atrazine. Similarly, anthocyanin content was augmented in both seedlings by alachlor and rimsulfuron, but reduced by trifluralin and fluometuron, whereas atrazine decreased the anthocyanin content in maize only. As indicated above, although less data is available that for herbicides, fungicides also have been implicated in the appearance of variations in phenoUc metabolism. Molina et al. [116] have demostrated that the systemic acquired resistance signal transduction pathway, a salicylic aciddependent plant-defence mechanism, mediates fungicide action in the plant. Ruiz et al. [27] found that the application of the fungicide carbendazim (bencimidazol 2-il methyl carbamete, C9H9N3O2) in tobacco plants not infected by pathogens caused significant variations in the bioactivity of phenolics with respect to control plants. The foliar application of 2.6 mM carbendazim boosted phenol biosynthesis and accumulation, since PAL activity was stimulated and, in addition, the oxidative enzymes PPO and POD were inhibited. According to these researchers, this may imply an increased resistance of plants to infection by pathogens, given the essential role of phenolic compounds in the lignification and suberization of the plant cell wall.

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Nutrients Different works show that the nutritional status of certain nutrients, such as boron (B), calcium (Ca), nitrogen (N), phosphorus (P) and iron (Fe) can trigger changes in phenolic metabolism. Of these nutrients, B is attributed with a clear and significant effect on the metabolism of these secondary compounds. As we shall discuss below, the relationship between B metabolism and phenolics is complex and depends largely on the sensitivity of the plant to B deficiency or toxicity. Boric acid has the particular ability to form stable complexes with compounds that present cis-hydroxyl groups (cis-diol groups) [117]. Several compounds, such as sugars and their derivatives and some phenolics (o-diphenols) have these cis-diol groups and therefore can form stable complexes with B [117]. The cell wall is a structure rich in compounds with a cis-diol configuration. On the other hand, it has been found that B is a predominant element in this structure, principally under deficiency conditions [118]. It has been estimated that the B present in the cell wall represents roughly 96% of the total of cellular B in carrots (Dancus carOta) [119]. In tobacco cells, the B in the cell wall reportedly makes up 90% of the total B in the cell with deficient levels and 60% with normal (or adequate) levels of this micronutrient [119]. Increases in the phenolic concentration under conditions of B deficiency are observed particularly in plants with high requirements of this element, such as sunflower {Helianthus annuus) [120]. The formation of cis-diol complexes with B, in some sugars and phenolic compounds, play a decisive role in the accumulation of phenolics in the B-deficient tissues. This element forms complexes with 6-phosphogluconic acid, inhibiting the activity of 6-phosphogluconic dehydrogenase activity, which augments the concentration of 6-phosphogluconic acid in the tissues deficient in B [121]. Consequently, under conditions of B deficiency the substrate is moved from glycolysis to the penthose phosphase shunt, which increases the synthesis of the phenolics [121]. The formation of B-phenolic complexes can also affect the quantity of these compounds in the tissues. As indicated by Pilbeam and Kirkby [122], the bonding of B and caffeic acid blocks the formation of quinones, and therefore facilitates the synthesis and accumulation of phenolics. The accumulation of phenolics in B-deficient tissues is a critical step in

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the start of different cell responses, since a characteristic of these is their phytotoxicity, even at low concentrations (l|iM) [123]. In B-deficient plants, caffeic, chlorogenic, ferulic and vanillic acids can accumulate [124], this phenomenon being most pronounced with stronger light intensity [125]. The stimulation of phenolic synthesis by intensifying luminosity has also been demonstrated, and is attributed to inducing PAL by light [126]. However, several authors have corroborated that PAL activity, regardless of light intensity, is increased by B deficiency [120]. Phenolic accumulation in B-deficient tissues can activate a group of enzymes that use these compounds as substrates. In B-deficient sunflower leaves, PPO activity progressively augments as symptoms characteristic of this deficiency appear. Plants with sensitivity to B deficiency differ in PPO activity. The accumulation of phenolics and PPO activity are reportedly greater in more sensitive plants, such as sunflower, than in less deficiency-sensitive plants, such as com {Zea mays) [125]. Phenolic oxidation by PPO and POD in B-deficient leaves leads to the production of quinones. These compounds are known for their high toxicity, and for being responsible for the production of oxygenated radicals such as: H2O2 and O2". The accumulation of quinones in plants that act as indicators of the deficiency has been considered as the prime cause of cell damage and of growth reduction [127]. In most cases, B deficiency is associated with brown coloration in foliar tissues. In sunflower plants, Cakmak et al. [128] demonstrated that the foliar pigmentation intensified in B-deficient plants exposed to intense light. This pigmentation may be caused in B-deficient tissue by a stronger phenolic concentration and their subsequent oxidation by PPO. The quinones produced by PPO activity are consecutively polymerized, giving rise to this pigmentation. Also, in fruits, the brown colour during ripening or after harvest is associated with high phenolic content and strong PPO activity [128]. As commented above, most works that relate B to phenol metabolism centre on the effect of B deficiency. Nevertheless, other works show the effect of growing dosages of the micronutrient on the metabolism of these secondary compounds. For example, Fawzia et al. [120] found that as the B dosage increased to 500 ppm, the phenolic concentration declined, as did the PAL and PPO activity, while the contrary effect was evident in POD activity. The high POD activity, together with the low PAL activity perhaps decreased the phenolic content. These same authors found

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completely opposite responses under conditions of B deficiency. In addition, Ruiz et al. [26] reported a foliar accumulation of phenolics under the lowest B dosages (0.5 jiM) and the highest (20 |iM), while intermediate dosages (5 and 10 |iM) resulted in oxidation of these compounds. The explanation of these results may be based on different forms in which B is found in the leaves, either forming complexes with the phenols or in free form. According to these authors, the lowest and highest dosages reflect, respectively, deficiency and high levels of B. Under these conditions, the proportion of free B would be minimum, while the proportion of B forming complexes with phenolics could be high. Different works indicate that the application of B at deficient or excessive dosages causes the formation of complexes between B and compounds such as pectins and phenols (more than 90% of the total B) [118,130]. Therefore, the lowest proportion of free or metabolic B in these treatments would account for the high PAL activity, the low availability of phenolics for oxidation due to their bonding with B, the low PPO and POD activities, and the highest concentrations of phenolics [26]. On the contrary, the intermediate B dosages (5 and 10 (iM) gave rise to adequate levels of this micronutrient, possibly raising the levels of free or metabolic B. These may inhibit PAL and increase phenol oxidation, due primarily to two causes: i) a greater availability of uncomplexed phenolics with B, and ii) an effect of free B boosting the activities of PPO and POD. Finally, Ruiz et al. [27] observed that the foliar application of fiingicide carbendazim B dosage of 8 mM strengthens synthesis (PAL) and subsequent oxidation of phenols (PPO and POD). The oxidation of phenols under this treatment could generate active oxygen species, which perform multiple important fiinctions in early defence responses of the plant. In short, these authors conclude that joint application of carbendazim with certain dosages of B could imply an increase in the resistance of plants to infection by pathogens, and suggest that the application of carbendazim-B could reduce the recommended applications of fimgicide without decreasing effectiveness. As opposed to the case of B, very few works investigate the direct effect of Ca on phenolic metabolism. Castaneda and Perez [131], working with lemon trees, observed that the application of 10 juM of CaCb increased PAL activity one hour earlier than control only if the trees were treated with cell walls isolated from the fungus Alternaria alternata, or

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when the trees were inflicted with injury. Therefore, the authors suggested that the external application of Ca favours the transduction signals produced by the inductors or by the wound, for two responses: one related to plant defence and the other to injury repair. According to the results of the above work, Ca appears to act as a second messenger. However, as the authors indicate, it is difficult to determine whether the increase in cytosol Ca, which would act as a second messenger, is due to the ingress of extracellular Ca or to the exit of Ca from other cell organelles. To address this latter doubt, the authors administered seedlings with 50 |iM of Varopimil, a compound well known for its effective blockage of Ca channels. The result was a sharp drop in Ca absorption, and also a decline in PAL activity. On the other hand, the increase in cytoplasmic Ca from the uptake of the culture medium would involve the appearance of a series of transduction signals which would cause the rapid and efficient initiation of response against mechanical damage or pathogen attack. Finally, these researchers indicate that different CaCk concentration added to the reaction medium of PAL did not alter the activity of this enzyme, suggesting that the Ca participates in the cell response and not directly in the activity of the enzyme [131]. Another number of works [22,69,132] showed the effect of Ca on the enzymes responsible for the oxidation of phenolics. This subject is surrounded by controversy, since, as we shall discuss below, some researchers claim a positive effect of Ca on PPO and POD activities, while others maintain the contrary: i) Soderhall [22] indicated that the presence of CaCb stimulated PPO activity. Taking into account that this enzyme is normally found in latent form, this researcher explained the increased PPO activity on adding 6 mM of CaCb by the conformational alteration of the active site of the PPO to make itself accessible to the substrate. ii) Lastly, other workers have reported that an increase in the Ca concentration depresses the PPO and POD [69,132]. Another of the nutrients which has been related to the bioactivity of phenolics has been N. In present-day agriculture, the main types of stress commonly generated as a consequence of the heavy use of inorganic fertilizers are related to the nutritional status of certain nutrients, primarily N, given its extensive use. In general, although only scant literature is available on the relationship between N and phenol metabolism, it appears

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that the relationship between the N availability and the accumulation of phenolic compounds is usually positive [133, 134]. Recently, we found that the toxicity of N (27 mM) in green bean plants is characterized by the inhibition of the oxidative enzymes PPO, POD and catalase (CAT), and by the stimulation of PAL, thus resulting in a foliar accumulation of phenolic compounds . On the other hand, and studying the N deficiency in green bean plants in relation to phenolic metabolism, we found that N deficiency (1.35 mM) is characterized by a stimulation of the oxidative enzymes PPO, POD and CAT, which inhibited PAL activity, resulting in the lowest foliar accumulation of phenolic compounds . Finally, several works have also implicated the nutrients P and Fe as possible inductors of changes in phenolic metabolism. However, studies of these relationships have been scarce. With regard to the former nutrient, P deficiency has been observed to raise the level of anthocyanins, but the reason for this rise remains unclear [4]. Meanwhile, low levels of Fe can increase the release of phenolic acids, presumably to help solubilize metals and thereby facilitate their uptake [135]. IMPORTANCE OF BIOACTIVITY PRESENT-DAY AGRICULTURE

OF

PHENOLICS

IN

The accumulation or oxidation of phenolic compounds principally in stored agricultural products normally gives rise to the physiological disorders that result in a major loss of commercial value of the products. Among the most common of these disorders in which phenol metabolism is involved is fruit browning and the russet spotting (RS) in harvested lettuce. Due to the importance of visual appearance as a parameter of produce cosmetic quality, tissue browning has long gained the attention of horticultural researchers [136]. Fruit browning, as a consequence of bruising, is due to phenolic oxidation [136,137]. The destruction of fruit Sdnchez, E.; Ruiz, J.M.; Garcia, P.C.; L6pez-Lefebre, L.R.; Rivero, R.M.; Romero, L. Response to the bioactivity of phenolic compounds and oxidative metabolism in green bean plants undergoing nitrogen toxicity (in consideration). ^ Sanchez, E.; Ruiz, J.M.; Garcia, P.C.; L6pez-Lefebre, L.R.; Rivero, R.M.; Romero, L. Behaviour of phenolic and oxidative metabolism as bioindicators of nitrogen deficiency in green bean plants (Phaseolus vulgaris L. Cv. Strike). Plant Biol., 2000 (in press).

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cellular compartmentation allows the phenolic substrates to be accessible to PPOs which catalyze phenolic oxidation [137]. The concentration and composition of phenolic compounds and/or activity of PPOs are often the major factors governing the development and intensity of tissue browning [137]. Infruitsof apples [138] and avocado [139] positively correlated with the tendency of the fruit to tum brown was total phenol content and PPO activity. Cheng and Crisosto [24] found that browning in the buffer extracts of peach and nectarine skin tissue depends on the presence of PPO activity and chlorogenic acid, which are major contributors to enzymatic browning. RS is a physiological disorder in iceberg lettuce that manifests itself as oval, brownish spots or lesions, mainly on the achlrophyllous midribs, although in advanced stages may spread over the entire leaf blade [140]. Several studies have described a relationship between the activity of PAL in iceberg lettuce leaf tissue and the development of RS symptoms [141-146]. Hyodo et al. [141] observed that an ethylene-induced increase in PAL activity parallelled the appearance of RS symptoms. These researchers also measured an increase in total phenolic compounds. It has been proposed that ethylene induces PAL activity and the resulting accumulation of phenolic compounds in cells leads to their discoloration and eventual death [141,143,145]. Taking into account the essential role of PAL activity in the appearance of RS, Peiser et al. [146] studied whether the application of various inhibitors of PAL activity could prevent the development of RS lesions in lettuce midribs. The use of the inhibitor 2-aminoindan-2-phosphonic acid (AIP) sharply diminished the formation of phenolic compounds, although this did not reduce the number of lesions associated with RS [146]. In short, according to the results of Peiser et al. [146], the early development of RS lesions is independent of the increase in PAL activity and phenolic compounds, rather than resulting from these increases as previously suggested. However, the accumulation of phenolic compounds does contribute to the subsequent browning symptoms indicative of RS. FUTURE As indicated throughout this review, phenolic compounds play an essential part in different physiological processes, in the adaptation of plants to adverse conditions, as well as in the commercial quality of many

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agricultural products. Therefore, future research should centre on the different stages that define the metabolism of these phenolics, in addition to studying the regulation of this metabolic process in order to control synthesis or oxidation of phenolics, depending on the conditions chosen or the result desired. With regard to the first point, most of the final enzymes of phenolic metabolism, that is, those that give rise to the formation of lignin, have not been characterized nor are their biochemical properties known, and therefore in the future it would be useful to elucidate these stages of phenylpropanoid metabolism. Also, in studying the regulation of phenolics, there are two options. Firstly, through genetic engineering, the genes that determine the synthesis or oxidation of phenolics can be strengthened or inhibited. However, at present, the use of transgenic plants best adapted or lease sensitive to some type of stress would be quite difficult due to the social controversy surrounding the commercialization of agricultural products derived from these types of plants. An altemative, which is possibly more rapid and useful in the short term, and which is currently generating a great number of studies, is to explore and define a series of abiotic factors which serve primarily activate or inhibit some of the stages of synthesis or oxidation of phenylpropanoid metabolism. ABBREVIATIONS PAL CHS SS IFS POD PPO lAA 4CL CAD CI TAL CAT RS AIP

= = = = = = = = = = = = = =

phenylalanine ammonia lyase chalcone synthase stilbene synthase isoflavone synthase peroxidase polyphenoloxidase indole-3-acetic acid 4-coumarate:CoA ligase cinnamyl alcohol dehydrogenase chalcone isomerase tyrosine ammonia lyase catalase russet spotting 2-aminoindan-2-phosphonic acid

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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BIOACTIVE NATURAL PRODUCTS FROM MARINE SOURCES M J . ABAD* AND P. BERMEJO Department of Pharmacology, Faculty of Pharmacy, University Complutense, 28040Madrid, SpainTel: ^34-l'3941871; Fax: +34-1394J 764; E-mail: mjahaddeticmos, sim. vcm. es ABSTRACT: Natural products from plants and microorganisms have traditionally provided the pharmaceutical industry with one of its most important sources of "lead" compounds in the search for new drugs and medicines. Although twenty thousand plant species are used in traditional medicines, most species have not been thoroughly examined chemically or pharmacologically. Natural product research is increasingly tiu^ning to marine animals, plants and microbes as source organisms. The oceans with their millions of species are a rich source of marine plants and animals. In recent years, a number of potential therapeutic agents have been isolated from marine flora and fauna. Several marine natural products are currently in preclinical and clinical evaluation, others show promising biological activities in vitro and in vivo assays, and others are making significant contributions to our understanding of cellular processes at the biochemical level. Although only initiated in the late 1970s, natural drug discovery^ from thev world's oceans has been accelerated by the chemical uniqueness of marine organisms, and by the need to develop drugs for contemporary, difficult to cure, diseases. The isolation, structure, biological activities, chemical properties and synthesis of compounds from marine soiwces, have attracted the attention of chemists, biologists and phannacists. Current research activities have generated convincing evidence that marine drug discovery has an exceedingly bright future. This article deals principally with bioactive constituents characterized in tha past decade from marine sources in order to obtain a better understanding of the biological significance of marine flora and fauna. The structural diversity of the medicinal constituents is discussed.

INTRODUCTION It is well knovv^n that plants are an exceptional source of biologically active products w^hich may serve as commercially significant entities in themselves, and v^hich may provide lead structures for development of modified derivatives possessing enhanced activity and reduced toxicity. It is likely that many compounds still await discovery. However, in the last decade the source of natural drugs has expanded to include lower plants, microorganisms and animals as well as marine organisms.

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The oceans cover more than 70% of the earth's surface, which represents over 95% of the biosphere. The oceans are therefore an unexplored area of opportunity for the discovery of pharmacologically active compounds. Although it has been one of man's principal sources of food for thousands of years, the sea was not considered as a supply of biologically active substances until forty years ago. In the last two decades, the search for marine-derived natural products has been extended to all oceans of the world. The enormous potential of the sea as a source of energy, food and chemicals has led to its being the subject of intense research. The results of this search had been reported in numerous reviews [1-8], Marine organisms have been shown to be a very rich source of unique and biologically active secondary metabolites that have attracted the interest of both chemists and pharmacologist. Plant and animal marine life forms have been studied with a view to obtaining such products. In particular, in pharmacognosy, the centre of interest is biologically active substances with therapeutical possibilities. Marine natural products represent a vast potential source of new drugs with diverse and often unique stmctures, many associated with interesting biological properties. Among the properties which have been reported for different marine natural products are very diverse: toxicity, antiviral, antibacterial, antimalarial, antifungal activity, antitumor, anti-inflammatory, analgesic, hypocholesterolemic and hypolipidemic activity. Success in these areas is demonstrated by the agents now in pre- or clinical evaluation. Biologically active natural products, or secondary metabolites, have become fine tools for pharmacologists and biochemists. Several marine natural products have entered pharmaceutical development, and others are making significant contributions to our understanding of cellular processes at the biochemical level [9,10]. As ligands for cellular receptors, they are used to explore fundamental processes that elicit behavioral responses in living systems, both in homeostasis and in disease states. Biological investigations of marine secondary metabolites have already yielded promising candidates for fliture drugs, e.g., didemnin B and bryostatin in the area of cancer chemotherapy [11], or have proven, useful as biological probes in studying cellular events, e.g., saxitoxin as a sodium channel blocker [12]. The marine environment, comprising approximately half of the total global biodiversity, offers a rich diversity of species, which is in many ways

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comparable to that of tropical rain forests. This environment also contains a great number of organisms for which there are no terrestrial counterparts, and which offer an enormous source of novel and biologically active compounds. Biological and chemical investigations of marine ecosystems have provided insights into a wonderful and complex underwater world. As a direct result of these investigations, the structural classes which can be obtained from certain taxonomic groups is to some extent becoming predictable, and some emphasis is now being placed on the biological properties of extracts, fractions and isolated pure metabolites. Ecological pressures on marine organisms, which include competition for space, maintenance of an unfouled surface, deterrence of predation, and the ability to reproduce successfully, may have led to the evolution of unique secondary metabolites, which are responsible for the chemical components of these actions and interactions. The oceans support a stable and thriving community of sponges, corals, echinoderms and many other invertebrates, that have adapted to the freezing temperatures, low nutrient levels and periodic low light levels. It has been suggested that marine habitats lack sufficient predation pressure to drive sessile invertebrates to produce defensive metabolites. Sessile marine organisms possess various defense systems against predators, larvae of other sessile organisms and pathogenic microorganisms. Since marine invertebrates do not produce antibodies, their defense mechanisms are based primarily on phagoc>1:osis by leukocytes, aided by producing and exuding secondary metabolites. The presence of endogenous secondary metabolites is believed to endow marine organisms with a chemical means of defense. Both chemists and biologists have been intrigued for many years as to the role of secondary metabolites of terrestrial and marine origins. Dudley Williams (1989) [13] proposed that "secondary metabolites are a measure of the fitnees of the organism to survive by repelling or entrapping other organisms". The accumulation of biologically active substances in marine invertebrates has been observed as a general phenomenon which reflects the defensive strategy of these often sedentary filter-feeding organisms. Although only initiated in the late 1970s, natural drug discovery from the world's oceans has been accelerated by the chemical uniqueness of marine organisms, which have the highest probability of yielding natural products with unprecedented carbon skeletons and interesting biological activity. Marine organisms, specially sponges, have attracted the attention

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of natural product chemists because of their versatility in using different metabolic pathways, that have no counterpart in the terrestrial world. Because life began in the ocean, it is hardly surprising that marine organisms have not only adapted to the high salt concentrations in the ocean, but have incorporated halogens into their chemical constitutions [14]. While ocean water is universally known for its chloride ion content, it is also an abundant source of bromide, and to a lesser extent iodide. An important consequence of halogen ion availability has been the extensive utilization of halogenation reactions by various marine organisms in their evolutionary biosyntheses of defensive and other necessary constituents. Additionally, during the past decade interest in the secondary metabolites of marine microorganisms and fungi has been increasing at a slow but definitive pace. Marine microorganisms have become recognized as an important and untapped resource for novel bioactive compounds [15-17]. The vast area of research into marine microorganisms, comprising marine bacteria, ranging from archaebacteria to glinding bacteria, fungi, and a whole range of microalgae, e.g., dinoflagellates, diatoms and protozoa, is just emerging and already showing immense potential. Microbiological investigations of marine environments have yielded a number of new biologically active microorganisms [18-22]. Moreover, symbiotic marine organisms, e.g., sponges and algae and/or microorganisms are common in all marine environments, and are believed to be of great importance in the biosynthesis of biologically active natural products within these organisms. Cases where secondary metabolites have been intimated as products of the microorganisms-containing symbionts of a sponge or algae have attracted much attention. However, it is difficult to rigorously sort out which compounds are metabolites elaborated by the marine organisms or by the symbiont, and most of the suggestions on this point are based on circumstantial rather than hard experimental evidence [23]. In this review, data have been presented to illustrate the diversity of organisms living in the sea and the plethora of chemical compounds that have been discovered from them. Since the late 1970s, there has been a veritable explosion of activity and many new marine metabolites have been isolated and identified. Therefore, the present review will cover only recent development in this area. The information was obtained from a review of the scientific literature, and the sources are referenced at the end of the chapter. Bioactive compounds found in marine environments include

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terpenoids and steroids, alkaloids, peptides and proteins, phospholipids, poliketides, carbohydrates, macrolides and toxins. TERPENOroS The diverse, widespread and exceedingly numerous class of natural products that are derived from a common biosynthetic pathway based on mevalonate as parent, are synonymously named terpenoids, terpenes or isoprenoids, with the important subgroup of steroids, sometimes singled out as a class in its own right. Monoterpenes, sesquiterpenes, diterpenes and triterpenes are ubiquitous in terrestrial organisms and play an essential role in life, as we know it. Although the study of terrestrial terpenes dates back to the last century, marine terpenes were not discovered until 1955. Sponges remain the primary target in the search for "drugs from the sea". It is known that sponges produce the greatest variety of secondary metabolites of any animal group. Diterpenes are one of the most abundant non-steroidal secondary metabolites isolated from marine sponges, with a wide range of biological properties. Structurally, the diterpenes from sponges possess polycarbocyclic skeletons, which are sometimes very degraded with loss of one or two carbon atoms to give nor- or bis-nor-derivatives.

,0H H3C CH3 .CH3

CN3

m

CH3

Fig. (1). Structure of agelasimines

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From the Thai marine sponge belonging to the genus Mycale, two labdanes diterpenes, mycaperoxides A and B were isolated. Both compounds exhibited antibacterial and antiviral activity, and showed significant cytotoxicity against tumor cell lines [24]. The marine orange sponge Agelas maiiritiana and others of the same genus, yielded two novel derivatives of a biclycic diterpene, agelasimine A and B, Fig. (1), both exliibiting a wide range of biological activity [25]. Cytotoxicity was reported from both compounds, and both caused relaxation in smooth muscle of rabbit gut and bovine coronary artery. Recently, these marine diterpenes have been reproduced by chemical synthesis using sigmatropic rearrangement and Ritter reaction [26]. Novel diterpenoids, including nakamurol A with unique thelepogane skeleton, were isolated from another Agelas sponge species, Agelas nakamnrai [27]. A Philippine marine sponge of the genus Strongylophora yielded new meroditerpenoids with antimicrobial and antiflmgal activity [28], while sponges of the genus Diacarnus yielded epidioxy-substituted nor-diterpenes with antimalarial properties [29]. Additionally, marine organisms have provided a large number of compounds of mixed biogenesis, originating partly from mevalonate and partly from a benzenoid precursor. A number of linear or cyclic prenylhydroquinones have been described with a terpenoid portion from one to eight isoprene units. Many sponges belonging to the familiy Spongiidae are chemically characterized by a series of terpenoids containig 21 carbons and displaying two (J-substituted fliran moities at the end of the molecule [30,31].These unusual compounds are probably biogenetically derived from higher terpenoids. These marine sponges are a well known source of novel furanoterpenes. Hippospongia sp., from the southern Australian sea, produced hippospongins A-F, with antibiotic activity [32]. The sponges Spongia officinalis and Fasciospongia cavernosa yielded fliranoditerpenes, including the novel ambliofuran 2 [33]. Ircinia sp. yielded bioactive fliranoterpene sulfates, which specifically inhibited the neuropeptide Y receptor in vitro, and also showed cytotoxicity against KB cells [34]. The fliranoterpene ircinin has been shown to inhibit phospholipase A2 (PLA2) activity and to affect human neutrophil functions like superoxide generation and degranulation [35]. Recently, this marine natural product has been synthesised [36]. Sponges of the genus Acanthella have previously been shown to be rich sources of terpenes having various nitrogen-containing groupings, with

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antimicrobial and antifungal activities [37,38]. From the Okinawan sponge Acanihella cavernosa, novel kalihinane diterpenoids. Fig. (2) were isolated, with potential antimalarial activity [39,40]. Recently, 15 diterpenes which contain isonitrile, isothyocianate and isocyanate groupings were also reported from the tropical marine sponge Cymbasiela hooperi. The majority of them demonstrate significant and selective in vitro antimalarial activity [41,42].

HO.,

Fig. (2). Structure of kalihinol A

Besides sponges, other marine organisms such as corals and algae are begining to receive attention from natural product chemists. Soft coral are symbiotic associations of coral animals with their algal partners. They are a rich source of terpenoids, notably cembranoid diterpenes with cytotoxic and antifungal activity [43,44]. Their abundant production and accumulation of diterpenoids is intriguing, as it seems unlikely that these compounds act solely as repellents against predators. Recently, new bioactive cembrane-type diterpenoids have been isolated from octocorallia [45,46].

HO

1

OH

Fig. (3). Structure of pseudopterosin E

Pseudopterosins are a series of tricyclic diterpene glycosides from the

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Caribbean coral Pseudopterogorgia elisabethae discovered by Fenical et al [47]. Pseudopterosin E, Fig. (3) is the one with the best pharmacological profile, combining low toxicity and potent antiinflammatory activity. In human neutrophils, pseudopterosin E inhibits degranulation and formation of leukotrienes [2]. In 1991, phase I clinical trials were initiated with pseudopterosin E as a topical anti-inflammatory agent. Recently, novel anti-inflammatory natural products have been isolated from this Caribbean soft coral [48,49]. Japanese researchers reported the isolation of a group of compounds designated helioporins AE, which are related to the pseudopterosins [1]. Several other soft: corals have been investigated in recent years. Eleiitherobia aurea yielded two novel diterpenoid glycosides, elenthosides A and B [50]. Seo et al. [51] isolated three pigments of the guaiazulene class from the gorgonian Calicogorgia granulosa. From the Japanese soft coral Similaria imnolobata^ new amphilectane-type diterpenoids with cytotoxicity activity were isolated [52], while a sample species of Sinularia genus from the Indian Ocean yielded aromadendrane diterpenoids with larvicidal activity [53]. Another Sinularia species also showed interesting biological properties, such as antispasmodic activity from Sinularia flexibilis [54]. New cytotoxic and antitumor diterpenes were isolated from the Caribbean gorgonian Eunicea toiirneforti [55], the Formosan gorgonian coral Briareum excavatum [56,57], the Okinawan soft coral of the genus Xenia [58], and the European Eunicella cavolinii [59]. Brown algae of the family Dictyotaceae yielded diterpenes of the dolabellane, xenicane, crenulide as well as extended germacrane and hydroazulenoid types. Some of these compounds were identified as capable of demonstrating appreciable selectivity as antimalarial agents [60], and are being synthetised in the laboratory [61]. The brown alga Dilophus ligulatus yielded diterpenoids with cytotoxic activity [62]. The novel xenicane diterpenoid dilopholide, was also obtained in this study. New secospatane diterpenes were recently isolated from another Dilophus alga, Dilophus okamurai [63]. From the marine alga Stypopodium flabelliforme^ several diterpenoids with interesting biological properties were isolated. The diterpenoid epitaondiol exhibited a potent anti-inflammatory activity related to inhibition of human PLA2 activity and leukocyte accumulation [64]. Additionally, epitaondiol has been shown as a potent calcium antagonist in

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a study of the cardiovascular system [65]. The diterpenoid 14-ketostypodiol diacetate, isolated from this alga, inhibited the proliferation of human prostate cells [66]. This compound and several derivatives are being synthetised in the laboratory as the racemate in a stereoselective manner [67]. More recently, new terpenoid compounds have been reported from another Stypopodium species, Stypopodium zonale, as tyrosine kinase inhibitors [68]. From the blue-green alga Tolypothrix nodosa, an anti-inflammatory diterpenoid, tolypodiol, was isolated [69]. Tolypodiol showed strong anti-inflammatory activity in the mouse ear edema assay. Another anti-inflammatory diterpene, pheophytin, was isolated from the edible green-alga Enteromorpha prolifera [70]. Bioactive diterpenoids were also isolated from marine microorganisms, such as phomactin derivatives. Fig. (4) reported from the marine fimgus Phoma sp. as platelet activating factor antagonists [71]. OHO

Fig. (4). Structures of phomactin derivatives

Marine organisms have also been intensively examined for their sesquiterpene content. Dysidea herbacea is a sponge species which has yielded new metabolites for more than 20 years, and no doubt further collectionsfromdifferent locations will continue to reveal new chemistry.

Fig. (5). Structure of herbadysidolide

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The terpenoid metabolites reported from Dysidea sp. are predominantly sesquiterpenes [72]. They possess a spiro moiety as in herbadysidolide, Fig. (5), herbasolide. Fig (6) and spirodysin. Fig. (7), or they are fUranosesquiterpenes such as forodysinin. Fig. (8). However, Dysidea herbaceafromtwo collection sites on the Great Barrier Reef less than 120 km apart also yielded enantiomericfiiranosesquiterpenes[73].

.O Q

Fig. (6). Sructure of herbasolide

These results suggest that samples of this sponge differ in their enzymatic capabilities concerning the cyclization of geranyl-geranylpyrophosphate.

Fig. (7), Structure of spirodysin

More recently, two new isonakafuran-type sesquiterpenes were isolated from this sponge species [74]. These types of compounds possess interesting antitumor and antifungal activity, and attempts to synthesize them are being conducted [75]. Other bioactive metabolites, such as antifouling sesquiterpenes, have also been recently isolatedfromDysidea herbacea [76].

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Fig. (8). Structure of furodysinin

From another Dysidea sponge species, Dysidea avara, the sesquiterpenes avarol. Fig. (9) and avarone. Fig. (10), which show a wide variety of biological activities, were first isolated. Both compounds are potent antileukemic agents in vitro and in vivo. They were determined to be neither direct mutagens nor premutagens, and they displayed antimutagenic activity

Fig. (9). Structure of avarol

Both avarol and avarone inhibit replication of the etiological agent of acquired immuno-deficiency syndrome (AIDS) [77]. Additionally, avarol and avarone effectively control acute inflammation in experimental models after either oral or topical administration. Their anti-inflammatory activity may result from inhibition of eicosanoid release and depression of superoxide generation in leukocytes [78]. Several studies reviewed the structures and bioactivity of compounds related to avarone as an antihuman immuno-deficiency vims (HIV), antitopoisomerase II activity and as proteinkinase C (PKC) inhibitors [3, 79].

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Fig. (10). Structure of avarone

From two sponge samples, Luffariella sp. and Accmthella klethra , and from red algae of the genus Laurencia, several sesquiterpenes with an isonitrile or isothiocyanatefixnctionalitywere obtained [80]. Some of them exhibited detectable cytotoxic activity against cultured tumor cells [81,82], as well as antibacterial [83] and antimalarial activity [84]. More recently, these types of compounds with antimalarial and antifouling activities were also isolated from marine sponges of the g^mxs Axinyssa [85,86]. Additionally, sesquiterpene-substituted quinones and related compounds constitute an important class of cytotoxic natural products of marine origin. Natural products of mixed sesquiterpene and quinol biosynthesis are common to marine algae and sponges. For example, several sesquiterpenoid/quinols have been isolated from a deep water collection of the marine sponge Siphonodictyon coralliphagum [87]; cyclorenierins from the sponge Haliclona sp. [88] and a Philippine sponge of the genus Xestospongia [89]; dactyltronic acids from the sponge Dactylospongia elegans [90]; two sesquiterpene hydroquinones from Polyfibrospongia australis [91]; vinylfurans from Euryspongia de lieu lata [92]; and several sesquiterpene quinones and hydroquinones from Thorecta choanoides, a marine sponge from the southern Australian sea [93], and from Perithalia caudata, an Australian marine brown alga [94]. In many cases, these compounds showed other interesting biological properties, such as antibiotic [95], anti-inflammatory [96,97], antiviral activity, e.g., peyssonols A, Fig. (11) and B, two anti-HIV sesquiterpenes hydroquinones isolated from the Red Sea alga Peyssonelia sp. [98], and cardioactive properties, e.g., halenaquinol, recently isolated from the sponge Petrosia seriata [99].

695 JCMO

Fig. (11). Structure of peyssonol A

From the marine sponge Haliclona sp. (also known as Adocia sp.), a family of hexaprenoid hydroquinones called adociasulfates, have been recently reported as inhibitors of kinesin motors [100,101]. These types of compounds were also found in several soft corals, such as Lemnalia africana [102], Okinawan soft coral of Nephthea sp. [103], and the gorgonian Alertogorgia sp., which yielded the cytotoxic tricyclic sesquiterpene, suberosenone [104]. Marine organisms, specially sponges, have also provided a large number of biologically active sesterterpenoids. The sesterterpenes are the smallest class of terpenoid compounds and consist of alcohol, aldehyde and ketone derivatives of terpene hydrocarbons. The occurrence of sesterterpenes in nature is somewhat uncommon, but for the last two decades an increasing number of examples have been reported. Interestingly, many of the recent additions have been isolatedfi*ommarine sponges of the order Dictyoceratida. These metabolites may be listed in two main groups: linear sesterterpene molecules terminated by a fiiran ring at one end and by a tetranoic acid or lactone ring at the other end, and tetra-or pentacyclic-sesterterpenes which are analogues of the scalarane skeleton. The scalaradial group of marine metabolites exhibit potent biological activity, mainly anti-inflammatory properties [2,105]. Scalaradial, Fig. (12) and other scalaranes were found to completely inactivate the enzyme PLA2 from bee venom directly and irreversibly. Marine sponges are a wellknown source of bioactive scalaradial sesterterpenes. Phyllospongia sp., collected in the South China Sea, yielded two new scalarane-type sesterterpenes, phyllactone H and I [106]. Scalarolide and scalarin were reported from Cacospongia and Ircinia sponges, besides other scalarane

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sesterterpenes [107,108].

Aco . cno f

CHO

Fig. (12). Structure of scalaradial

These types of compounds also showed other interesting properties, such as antineoplasic and cytotoxic activity reported from scalarane-type sesterterpenes of the Indian Ocean sponge Hyrtios erecia [109,110], and antituberculosis properties [111]. In recent years, many marine sesterterpenes which are promising candidates for new drugs have been discovered. The sesterterpenoid manoalide. Fig. (13), obtained from the sponge Luffariella variabilis^ was detected in a program searching for new anti-inflammatory compounds. Manoalide proved to be a potent inhibitor of PLA2 and has become a usefiil biochemical tool. Inhibition of phospholipase C and the ability of manoalide to function as a calcium channel blocking agent allows this compound to be used in the study of the role of calcium mobilisation in inflammatory processes, and in a more general sense, in signal transduction pathways [105,112]. A significant number of manoalide derivatives has been isolated and evaluated for their biological activity [113,114]. A total synthesis of manoalide employs an organometallic coupling strategy [115]. Clinical trials are currently underway with some of these and synthesised derivatives, and it is probable that a manoalide-inspired derivative will reach the market [116].

Fig. (13). Structure of manoalide

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New and interesting anti-inflammatory sesterterpenes have been reported in recent years from marine sponges. Petrosaspongiolides, Fig. (14), isolated from the Caledonian marine sponge Petrosaspoiigia nigra [117], were found to potently inhibit PLA2 on acute and chronic inflammation [118]. In a similar manner, cacospongiolide B, Fig. (15), a sesterterpene isolated from Fasciospongia cavernosa [119,120], was shown to be a potent inhibitor of human synovial PLA2 [121].

'

GMaOAc

Fig. (14). Structure of petrosaspongiolides

Fig.(15). Structure of cacospongiolide B

Several other marine sponges have been investigated in the last decade, in the search for novel bioactive sesterterpene molecules. A sample of the sponge Dysidea herbacea from the Red Sea is unique in that it contains cytotoxic sesterterpenes with a scalarin skeleton, e.g., scalardysin. Fig. (16) and the C2i-furanoterpene flirospongolide. Fig. (17).

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Fig. (16). Structure of scalardysin

This compound also showed antispasmodic activity [122]. A variety of cytotoxic compounds was isolated, including a bishomosesterterpene and dysidiolide from another Dysidea sp., a sulfated sesterterpene hydroquinone from a Hippospongia sp., and two new sesterterpenes, lintenolides F and G from the Caribbean sponge Cacospongia linteiformis [123,124].

Fig. (17). Structure of furospongolide

From the Maldives' Black marine sponge Hyrtios erecta^ several cytotoxic sesterterpenes were isolated, such as the penytacyclic sesterterpenes designated sesterstatins [125-127] and puupehenone. Fig. (18) with a quinone-methide system [128]. Three novel cytotoxic norsesterterpenes, rhopaloic acid A, Fig. (19), B and C, were recently isolated from the sponge Rhopaloeides sp. These compounds also inhibited the gastrulation of the starfish (Asterina pectmifera) embryo [129,130]. Both racemic and enantiomeric forms of rophaloic acid A have been synthesised by very diflFerent strategies [131,132].

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Fig. (18). Structure of puupehenone

Additionally, during the search for biologically active sponge metabolites belonging to the sesterterpenoid class, a sulfated sesterterpene hydroquinone, halisulphate. Fig, (20), was isolated from the dark brown sponge Halichondriidae sp.

OOOH

Fig. (19). Structure of rophaloic acid A

It demonstrated in vitro antimicrobial, antifungal and anti-HIV activities [24,133]. Recently, a halisulphate derivative with antithrombin and antitrypsin activity was isolated from the marine sponge Coscinoderma mathewsi [134], The absolute configuration of halisulphate has been determined by application of the chiral amide method coupled with chemical degradation procedures [135]. NaOaSO'

Fig. (20). Structure of halisulphate

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Fig. (21). Structures of malabaricane triterpenes

Triterpenoids are a minor group of sponge metabolites; of these malabaricane/isomalabaricane triterpenoids. Fig. (21) are known from Jaspis and Stelletta sponge species. All of this group shows cytotoxicity and anti-HIV activity [136-139].

Fig. (22). Structure of sodwanones

Other cytotoxic triterpenes, designated as sodwanones A-M, Fig. (22), were recently reported in Axinella weltneri^ a marine sponge from the Indian Ocean [140]. The investigations of small samples of the Mediterranean sponge Raspaciona actileata revealed the presence of raspacionins. Fig. (23), triterpenoids containing two perhydrobenzoxepine systems [141]. Besides sponges, other marine organisms have been reported to produce bioactive triterpenes, including algae from Lanrencia genus [142], and the \\o\o\h\xn2inPsolusfabricci [143].

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OCOCHa

Fig. (23). Structure of raspacionins

As we indicated in the introduction section, halometabolites frequently occur in marine organisms and are known to have basic functions related to the survival of the living creatures producing them. Bromine is by far the halogen most frequently found in these metabolites. Halogenated marine terpenes were first isolated only in 1963. Since most marine organisms have been found to contain halogenated compounds, there are certainly thousands of different, new organohalogenated terpenes in marine organisms awaiting discovery. Among marine organisms, red algae, particularly species oiLaurencia and Plocamium, have provided a rich and diverse collection of halogenated terpenes over the past 25 years [14]. Red algae of the genus Plocamium have been shown to be a rich source of acyclic and cyclic halogenated monoterpenes that vary for a given species depending on collection location and season. These algae can be found in many locations ranging from Antarctica to tropical waters. Numerous chemical studies of these species show the presence of bioactive halogenated monoterpenes, whose structure and yield vary greatly [4]. Ahhough the red algae Plocamium have been investigated for its chemical content for many years, several new bioactive compounds have been identified recently from these species. For example, plocamadiene A is a polyhalogenated monoterpene which causes histamine release from mast cells of the guinea-pig and rat in vitro [144]. The species Plocamium cartilagineum found on the Portuguese coast, produced acycUc polyhalogenated monoterpenes [145]. More recently, new halogenated monoterpenes were isolated from Plocamitmi costatimi. These compounds have been shown to deter settlement of barnacle larvae, suggesting a potential ecological role [146]. An array of new and unusual halogenated terpenes have been isolated and characterized from Laurencia red algae [14]. This genus is well known as a source of halogenated sesquiterpenes. New chamigrane-type derivatives were isolated from Laurencia species, some from Laurencia

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nidifica [147] and some from Laurencia nipponica growing in Japan [148]. Examples of iodinated terpenes, which are quite rare and interesting, were found in Laurencia majuscula collected in the South China Sea [149]. More recently, Rovirosa et al [150], reported the isolation of new halogenated sesquiterpenes from Laurencia claviformis, a species endemic to Easter Island.

Fig. (24). Structure of halomon

Several other red algae have been investigated in recent years. Among them, it is interesting to point out the halogenated monoterpene halomon. Fig. (24) and related compounds, isolated from the red alga Portieria hornemannii^ which exerts potent antitumor activity in vitro and in vivo [151-153]. Species of sea hares, a marine moUusk, have also provided a rich source of halogenated terpenes [14]. In many cases, these compounds are derived from the sea hare's algal diet. New chamigrene-type halogenated sesquiterpenes were isolated from Aplysia dactylomela [154], and from the Spanish sea hare Aplysia punctata^ which exert potent cytotoxic activity [155]. STEROIDS Since the start of the twentieth century, steroids have continued to be the focus of the research activities of natural product chemists, synthetic chemists, biochemists and clinicians. The reasons are several-fold and related to the fascination of the chemical complexity of sterols and their biochemical functions in living organisms. Sterols and steroids are excellent compounds for the organic chemists to practise their skills upon in the development of new reactions and synthetic procedures. The biological functions of sterols, for example as an essential constituent of membranes, have proved thought-provoking to lipid biochemists. Marine organisms have been found to be storehouses of sterols.

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particularly in terms of unique side-chain structures and unusual fiinctionalization. For example, marine sponges are a rich source of steroids with highly functionalized nuclei and modified side chains. Numerous new sterols have been isolated from marine sponges. Although most have side chains that are polyoxygenated or alkylated, other occurring sterols are known to contain a methylether. Extensive studies on sterols from marine sponges during the past decades have resulted in the identification of a plethora of unusual forms with interesting biological activities [156]. For example, topsentinols A-J, Fig. (25), new sterols with unusual polyalkylated side chains, were isolated from the Okinawan marine sponge Topsentia sp. [157], while sponges of the genus Ircinia yielded new epoxy sterols [158]. Sterol composition has also been reported from the sponge Faciospongia cavernosa growing in the Adriatic, Aregean and Tyrrhenian Seas [159]. Recently, a new sterol containing an unprecedented seven-membered cyclic enol-ether has been isolated from the Australian Euryspongia arenaria [160]. Most of these compounds showed interesting biological properties, such as antiplasmodial and cytotoxic activity of the steroids from Agelas oroides, a Maltese marine sponge [161]; novel cytotoxic steroids from sponges of the genus Xestospongia sp. [162,163], Biemna sp. [164] and Scleritoderma sp. [165]; an antifouling epidioxy sterol from Lendenfeldia chondrodes, a Palauan marine sponge [166], and antiviral sterols from the marine sponge Petrosia weinbergi [167]. It is interesting to point out the cytotoxic activity of camptothecin and related compounds, with which clinical developments have recently been initiated [168-170].

Fig. (25). Structure of topsentinols

Additionally, sulfated sterols have been described from a wide variety of marine organisms, particularly sponges and echinoderms, and several of these steroidal sulfates have exhibited a broad range of activities. Halistanol sulfates are a group of sulfated polyhydroxysteroids from

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Sponges, which are very attractive because of their biological activity. Halistanol disulfate B, isolated from the marine sponge Pachastrella sp., was shown as a potent inhibitor of endothelium converting enzyme [171], while halistanol trisulfate, a sulfated steroid derivative isolated from the marine sponges of the genus Topsentia, inhibits protein tyrosine kinase activity [172,173]. New trisulfated trihydroxysteroids were also isolated from two different collections of the sponges Trachyopsis halichondroides and Cymbastela coralliophila [174], while tamosterone sulfates, new polyhydroxylated steroid sulfates, have been reported from a new oceanapiid sponge genus [175]. Acanthosterol sulfates A-J, isolated from the Western Japan sponge Acafithodendrilla sp., exhibited antifungal activity [176]. More recently, some cytotoxic bis-steroid sulfates called crellastatin, were isolated from the Vanatua marine sponge Crella sp. [177,178]. Fom the marine sponge Jaspis sp., several steroidal sulfates have been reported as inducers of larval metamorphosis and inhibitors hatching enzyme activity in the ascidian Halocynthia roretzi [179]. Additional unusual steroid derivatives have been isolated from marine sponges, e.g., polymastiamides, steroid/aminoacid conjugates isolated from Polymastia holetiformis, a Norwegian marine sponge [180], and an aminoimidazolium sah of steroid trisulfate from Topsentia sp. [181]. Some of these marine sterols are being reproduced by chemical synthesis in the laboratory [182]. Besides sponges, other marine organisms have been investigated in recent years for their steroid content, such as octocorallia [183]. Sarcoaldesterols A and B, two new polyhydroxylated sterols together with novel epoxy steroids were isolated from the soft coral Sarcophytum sp. [184,185], Gorgonian of the genus Mtaicella sp. from Jaejn Island of Korea, yielded calicoferols. Fig. (26), new secosteroids with significant cytotoxicity and inhibitory activity against PLA2 [186,187]. Recently, chemical examination of the soft corals Cimndaria viridis, Nephthea chahroli and Simdaria disseda yielded novel polyhydroxy steroids [188190]. Red algae are known to be important sources of cholesterol and desmosterol in the marine environment. Some of these compounds, e,g., oxygenated desmosterols and clerosterols isolated from the red algae Codinm arabicum and Galaxaiira marginata, showed interesting cytotoxic properties [191,192]. More recently, a new sterol amide with antimicrobial activity, boophiline, was isolated from the cattle tick Boophihis micropliis [ 193 ].

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Fig. (26). Structure of calicoferols

ALKALOIDS AND RELATED COMPOUNDS Alkaloids are extremely difficult to define because they do not represent a homogeneous group of compounds from either the chemical, biochemical or physiological viewpoint. All do occur in plants, but some are found in animals, and practically all have been reproduced in the laboratory by chemical synthesis. Most possess basic properties due to the presence of an amino nitrogen, and many, specially thoses pertinent to pharmacy and medicine, possess marked physiological activity. Marine organisms are known to be a rich source of alkaloids with unique chemical features and pronounced chemical activities, which suggest potential value as lead structures for the development of new pharmaceuticals [194], Extensive studies on alkaloids from marine organisms during the past decades have resuhed in the identification of a plethora of compounds, sometimes with interesting biological activities. For example, indole alkaloids isolated from marine sponges such as Raphisiapallida [195], Ircinia sp., a Okinawan marine sponge [196], and Hamacantha sp., with antifungal activity [197]. Imidazole alkaloids such as leucettamine A and related compounds isolated from the marine sponge Leucetta microrciphis^ have been shown as potent antagonists of leukotriene B4 receptor [198], and antitumor agents [199]. Recently, new imidazole alkaloids were reported from an Australian marine sponge Axinella sp., with interesting bactericidal activity [200,201]. Stellettamide A and B, Fig. (27), indolizidine alkaloids isolated from sponges of the genus Stelletta have been reported as inhibitors of

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calmodulin [202,203]. The absolute configuration of these molecules have been established by synthesis of their enantiomers [204]. From this sponge species, new alkaloids named stellettazoles B and C, which exhibit antibacterial activity have recently been reported [205]. From the Caribbean marine sponge Agelas dispar, novel betaines alkaloids which also exert antibacterial activity have recently been isolated [206], Cytotoxic guanidine-alkaloids have been reported from different samples of marine sponges [207-210].

Fig. (27). Structure of stellettamides hAeO

PR^

MeO Fig. (28). Structure of lamellarins

Another cytotoxic compound, a sulfur-containing alkaloid, was isolated from the ascidian Polycarpa aurata [211]. Recently, another series of ascidian alkaloids, the lamellarins. Fig. (28), have been shown as selective inhibitors of HIV virus replication in cell culture [212], together with new indolocarbazole and ergoline alkaloids isolated from the ascidians Eudistoma toealensis and Botryllns leachi, which showed moderate cytotoxic activity [213-215]. From a Philippine marine sponge, Oceanapia sp., an unusual sesquiterpene alkaloid, oceanapamine, was isolated [216], while marine sponges of the genus Corticium sp. yielded unusual steroidal

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alkaloids [217]. Additional cytotoxic alkaloids were reported from other marine sponges, e.g., peptide alkaloids from Lissoclinum sp. [218], and tryptophan-derived alkaloidsfromthe Okimy^^n Aplysina sp. [219]. From the Southern Australian sponge Spongosorites sp., a new class of marine alkaloids, dragmacidins, have been reported as potent inhibitors of protein phosphatases [220]. Some of these cytotoxic marine alkaloids are promising candidates for new drugs. For example, ecteinascidins. Fig. (29) are a family of tetrahydroisoquinolone alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata, which have been selected for clinical development. These compounds are presently in pre-clinical and clinical trials for human cancers [221-225]. A series of totally synthetic molecules that are structurally related to the ecteinascidins is currently being prepared and evaluated as antitumor agents [226]. 0CH3 H>,CO.

H3CO

Fig. (29). Structure of ecteinascidins

However, pyrroloquinolines and pyridoacridines are the alkaloids of major interest as metabolites in sponges and ascidians [227]. Many of these compounds have generated interest both as challenging problems for structure elucidation and synthesis as well as for their cytotoxicities [228230]. A family of alkaloids characterized by a pyrroloquinone skeleton has been isolated in recent years from several sponges. Included in this family are the batzellines, isobatzellines, damirones, makaluvamines, discorhabdins, prianosins and wayakin. These alkaloids have shown a

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variety of biological activities including cytotoxicity against human tumor cell lines, in vivo tumor inhibition and inhibition of topoisomerase I and II. Among these, the makaluvamines. Fig. (30) are the most potent inhibitors of topoisomerase II, suggesting their efficacy as anticancer agents. The principal structural feature of these alkaloids is the core of a planar iminoquinone moiety which can intercalate into DNA and cleave the DNA double helix, or inhibit the action of topoisomerase II [231]. This family of makaluvamines alkaloids was mainly isolated from the Philippine marine sponge Zyzzya fidiginosa [232-234]. Recently, these alkaloids are being reproduced in the laboratory by chemical synthesis employing a strategy based upon intramolecular nucleophilic substitution reactions [235-237].

Fig. (30). Structure of makaluvamines

Discorhabdin alkaloids. Fig. (31), in contrast, are of high cytotoxicity, but they exhibit no inhibition of topoisomerase II. They were isolated from the Anthartic sponge Latnmculia apicalis [238], and more recently from a deep-water marine sponge of the genus Batzella sp. [239]. The new discorhabdin derivative isolated from this sponge showed in vitro cytotoxicity against tumor cell lines.

Fig. (31). Structure of discorhabdin

Marine sponges of these genus Batzella sp. also yielded novel

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pyrroloquinolines alkaloids, batzelladines. Fig. (32), with interesting biological properties [240,241]. Many of these types of alkaloids were also isolated from other marine sponges, e.g., Agelas sp. [242], and tsitsikammamine A and B reported from a South African marine sponge, which exhibited antimicrobial activity [243]. As representative of the derivatives of pyridoacridine, eilatin, a marine alkaloid inhibits in vitro cell proliferation in chronic myeloid leukemia patients [244]. Other members of the pyridoacridines, such as alkaloids isolated from a Cystodytes sp. ascidian, inhibit topoisomerase II [245]. Additionally, analogues derivatives of these type of alkaloids showed interesting anti-HIV activity [246].

Fig. (32). Structure of batzelladine A

Fused tetracyclic and pentacyclic alkaloids constitute a relatively new class of natural products isolated mostly from ascidians and sponges. Cytotoxic, antimicrobial and antiviral activities have been reported for many of these compounds. The manzamine alkaloids. Fig. (33) are characterized by a complex pentacyclic diamine linked to C-1 of Pcarboline moiety. Manzamine have been isolated mainly from six different genera of marine sponges: Haliclona, Pellina^ Xestospongia, Ircinia, Pachypellin and Amphimedon.

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Fig. (33). Structure of manzamines

Haliclonacyclamines, manzamine alkaloids with pronounced cytotoxic activity, were isolated from Haliclona sp., a tropical marine sponge [247249]. Other manzamine-type alkaloids with cytotoxic and antibacterial activity were isolated from the Philippine marine sponge Xestospongia ashmorica [250]. Some of these compounds are very attractive because of their biological activity, e.g., manzamine alkaloids isolated from another Xestospongia sp., also reported in the marine sponge Agelas novaecaledoniae^ which are potent somatostatin and vasoactive intestinal peptide inhibitors [251]. These compounds could be promising agents in the research on compounds for therapeutical interventions in cystic fibrosis, Alzheimer's disease and some tumors.

Fig. (34). Structure of norzoanthamine

Manzamine-type alkaloids were also reported in another samples of marine sponges, e.g., the Okinawan Amphimedon sp. [252-254], Pachypellina sp. [255], and a novel alkaloid called hyrtiomanzamine from Hyrtios erecta, with interesting immunosuppressive activity [256]. From

711

the colonial zoanthid Zoanthus sp., a zoanthamine-type alkaloid. Fig. (34) has been reported as a good candidate for an osteoporotic drug [257], while the marine bacteria Agrobacterium sp. yielded agrochelin, a new cytotoxic thiazole alkaloid [258]. Recently, investigations have been conducted to reproduce these type of compounds by chemical synthesis in the laboratory [259-261].

X = Br{9S/^R = 6:4) X = H (9S^f^ = 1 1)

Fig. (35). Structure of tauroacidins

Although very few terrestrial plant alkaloids contain halogen, brominated alkaloids have been reported from the marine environment. From the Okinawan marine sponge Hymeniacidon sp., several bromopyrrole alkaloids have been described, e.g., tauroacidins A and B, Fig. (35) [262], konbuacidin A, Fig. (36) [263] and spongiacidins A-D [264]. Several species of sponges contain hymenialdisine. Fig. (37), which has been shown as a potent inhibitor of nuclear factor kappa B and interleukin-8 production in vitro [265,266].

H;^.

.N

Bf Fig. (36). Structure of konbuacidin A

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New bromopyrrole alkaloids were also isolated from different species of Agelas sp., such as the Caribbean Agelas dispar [267], Agelas nakamurai^ si Papua New Guinean marine sponge [268,269] and Agelas wiedenmayeri [270]. Two samples of the marine sponge Stylissa carteri collected in Indonesia, yielded two new bromopyrrole alkaloids [271]. Brominated indole alkaloids have been reported from the Caledonian marine sponge Orina sp. [272], while bromotyrosine alkaloids with cytotoxic and antitumor activity have been isolated from several marine sponges, such as Aplysina aerophoha [273], the Okinawan Psammaplysilla purea [274] and Psendoceratina verrucosa [275].

Fig. (37). Structure of hymenialdisine

Additionally, marine organisms have proven to be a rich source for a wide variety of modified nucleosides considered worthy for clinical application. For example, arabinoside-nucleosides, constituents of the Caribbean sponge Cryptotethya crypta^ have led researchers to synthesise analogues with improved antiviral and anticancer activity [4].

Fig. (38). Structure of tubercidin

Other bioactive nucleosides have been reported from the marine

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environment, e.g., pyrrolo-pyrimidine nucleosides such as tubercidin. Fig. (38) and analogues derivatives from the ascidian Didemmim voeltzkowi [276]; caissarone. Fig. (39), a sea anemone iminopurine with adenosine receptor antagonist activity [277]; phenethylguanidine analogues from Petrosia contignata, a Indo-Pacific marine sponge [278]; and bioactive bisguanidines from Stylotella miraritium, with potent cytotoxic, antibiotic and immunosuppressive activity [279]. More recently, nucleosides have also been reported in the Australian marine sponge Carteriospongia sp. [280].

II

H

0:Vo Me

M«

Fig. (39). Structure of caissarone

PEPTIDES AND PROTEINS The "term" peptide includes a wide range of compounds varying from low to very high molecular weights, and showing marked differences in physical, chemical and pharmacological properties. The lowest members are derived from only two molecules of aminoacids, but higher members have many aminoacid units and form either peptides, simple proteins or more complex proteins, conjugated proteins, for example, lipoproteins in which proteins are combined with lipids. Marine organisms are a well-established source of unique and biologically active peptides. Complex cyclic peptides and depsipeptides have emerged as an important new class of metabolites present in extracts of marine organisms. Many of these peptides have been found to be extremely potent cytotoxic and /or enzyme inhibitors.

714

Fig. (40). Structure of didemnin B

Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. Didemnin B, Fig. (40), one member of this family obtained from the tunicate Trididemnum solidum^ has antiviral, immunosuppressive and potent cytotoxic properties [281-283]. The compound is too toxic to be useful as an antiviral or immunosuppressive agent, but has been in phase I clinical trials as an anticancer agent, and phase II clinical trials are currently underway [284]. Arenastatin A, Fig. (41) is another potent cytotoxic depsipeptide isolated from the marine sponge Dysidea arenaria, which shows selective toxicity against tumor cells [285]. This compound have been reproduced by chemical synthesis in the laboratory [286]. In a similar manner, hapalosin and aplidine, marine cyclic depsipeptides with inhibitory activity against human tumor cell lines, have been obtained by chemical synthesis by a route involving a macrolactamization as an important ring-forming step [287-289]. Other cytotoxic and antiproliferative depsipeptides were recently isolated from the Vanatua marine sponges Axinella carteri [290], Jaspis splendcms [291], Geodia sp. [292], and the Papua New Guinea sponge Cymhastela sp. [293]. Marine depsipeptides also showed other interesting biological properties, such as antiviral [294], antifimgal [295,296] and hemolytic activity [297]. It is interesting to point out the biological activity of papuamides A-D, new cyclic depsipeptides isolated from the Papua New Guinea sponges Theonella sp., which showed interesting anti-HIV and cytotoxic activity [298].

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A ? ^-^=^M Fig. (41). Structure of arenastatin A

Marine organisms, specially sponges, have provided a large number of other biologically active peptides in the last decades. Cyclotheonamides, Fig. (42), a family of cyclic pentapeptides isolated from marine sponges of the above mentioned genus Theonella sp., have been shown as potent thrombin, trypsin and other serine proteases inhibitors [299-302]. From the marine sponge Theonella swinhoei a highly cytotoxic peptide, polytheonamide B, was recently isolated [303].

O

^N O^

^

NHCOMe

H^ ^N. -. ^ .

Fig. (42). Structure of cyclotheonamide B

Phakellistatin, Fig. (43) is a series of cyclic hepta and octopeptides isolated from the Indian Ocean marine sponge Plakellia sp., with interesting antineoplasic activity [304,305]. These compounds have recently been reproduced in the laboratory by chemical synthesis using a combination of stepwise coupling and segment condesation [306]. New cytostatic heptapeptides, isolated from marine sponges, were also chemically synthetized using a new synthetic method to elaborate peptide bond [307309]. Additional biologically active peptides of marine sponge origin include dipuupehedione, a cytotoxic compound from the New Caledonian

716

Hyrtios sp. [310,311], and the antifungal peptides halicylindramides from Halicoridria sp. [312].

Fig. (43). Structure of phakellistatins

Besides sponges, other marine organisms have been reported to produce bioactive peptides, which are promising candidates for new drugs. For dolastatins, e.g. dolastatin 10, Fig, (44), potent antineoplasic peptides isolated from the Indian Ocean molusk Dolahella amictilaria, clinical trials are pending [313]. Recently, a structural derivative of dolastatin called auristatin, has been evaluated in human tumor cell lines and has undergone clinical trials [314].

iHgCO H

O

Fig. (44). Structure of dolastatin 10

Microcolins, Fig. (45) are lipopeptides isolated from a strain of the blue-green alga Lyngbya majtiscula^ which revealed interesting cytotoxic and immunosuppressive activity [315]. Several synthetic derivatives are also being evaluated [316]. These compounds resemble majusculamides, which were isolated from another chemovariant of the same species and from marine sponges [317]. Dendroamides, new cyclic hexapeptides, were isolated from another blue-green alga [318].

717

b

d^

o Fig. (45). Structure of microcolin A

Marine microorganisms have also been reported to produce bioactive peptides, such as marinostatin from the marine bacterium Alteromonas sp. [319], pentapeptides from the cydinohdiCttnum Anahaena cylindrica [320], and new anti-inflammatory cyclic peptides from the marine Strepiomyces sp. [321]. From the marine fungus Hypoxylon oceariicum, several lipodepsipeptides with antifungal activity have recently been reported [322,323]. Besides peptides, marine organisms have been reported to produce biologically active proteins, which are probably involved in the protection of organisms against physiological and stress conditions. Recently, these molecules have been cloned from sponges [324] and marine microorganisms [325]. The marine protein variabilin. Fig. (46) has been shown as a potent dual inhibitor of human secretory and cytosolic PLA2 with anti-inflammatory activity [326]. An interleukin-6 cytokine family antagonist protein was reported from the marine sponge Callyspongia sp. [327]. From the marine sponge Pachymatismajohnstonii, a, cytotoxic glycoprotein, pachymatismin was isolated [328,329]. Another active glycoprotein, niphatevirin, isolated from the marine sponge Niphates erecta was reported as an HIVinhibitory agent [330], together with cambrescidins, proteins isolated from marine invertebrates which also exert antiviral activity [331]. The activities of the purple fluid of the sea hare Aplysia dactylomela, such as toxic, antimicrobial and hemagglutinating properties, have been attributed to a substance of protein nature [332]. Proteoglycans and adhesive glycoproteins present in the extracellular matrix of vertebrates, have also been reported in sponges. These molecules are probably involved in the cell adhesion systems of sponges [333]. Recently, novel marine proteins have been reported, such as silicatein from sponge biosilica [334], and a metallothionein protein from the marine alga Fiicus vesiculosus [335]. Metallothioneins have also been isolated from Arctic

718

bivalves as possible indicators of the availability of trace metals in the Arctic [336]. Additionally, proteins were also isolated from other species in the marine environment, e.g., from the Kuruma prawm Penaeus japonicus [337,338], and the shore crab Carcirms maenas [339].

OH Fig. (46). Structure of variabilin

Enzymes are also colloidal in nature and consist of protein or contain proteins as an essential part. Several enzymes have been reported from marine organisms, specially sponges and algae, e.g., exopolyphosphatases from the marine sponge Tethya lyncuhum [340], tauropine dehydrogenases from the Demospongia Halichondria japonica [341], and more recently phenylalanine hydrolases from the sponge Geodia cydonium [342]. From this marine sponge Geodia cydonium, oligoadenylate synthetases were also isolated which may be useful as biomarkers for environmental monitoring [343,344]. Additionally, these sponge species contain high levels of telomerase activity, suggesting that they possess a high proliferation capacity [345]. A protease hydrolyzing casein with proteolytic activity has been reported from the Papua New Guinea sponge Callyspongia schulzi [346], while the glutathione-S-transferase activity of the sponge Suberites domuncula has been used as marker of thermal stress [347]. This enzyme was also reported in a marine fish, Pleuronectes platessa [348]. Additionally, isomerases were reported from the marine alga Ptilota filicina [349], while the cyanobacterium Synechocystis sp. yielded pcarotene hydroxylases [350]. Cyanobacteria also yielded oligomeric forms of dehydrogenases [351]. Besides sponges and algae, enzymes were also isolated from marine organisms and microorganisms. For example, polymerases and proteases from marine Vibrio sp. [352], marine bacterium such as Alcaligenes faecalis [353], and from archaeons, such as the psychrophilic Cenarchaeum symbiosiim [354], and the hyperthermophile archaeons Pyrococciisfuriosus [355], Sulfolobus solfataricus [356], and Aeropyrum pernix [357]; transferases from marine bacterium such as Vibrio vulnificus

719

[358], and Photobacterium damsela [359,360]; dehydrogenases from different strains of Nocardioides sp. [361]; novel alginate lyases from marine bacterium Alteromonas sp. [362], and phenoloxidases from the colonial ascidian Botryllus schlosseri [363], and the marine bacterium Marinomonas mediterranea [364]. More recently, enzymes of the lysozyme family were purified from marine bivalves and conchs [365,366]. Additionally, the marine sponge SpirastreUa sp., in symbiotic associations with marine fungi and bacteria, produces enzymatic activities, e.g., serine-type acetylcholinesterase with the marine bacterium Arthrobacter ilicis [367]; urethanase activity with Micrococcus species [368]; and asparaginase and amylase activity produced by the ftingus Mucor sp. associated with this sponge [369,370]. Aminoacids occur in plants and animals, both in the free state and as the basic units of proteins and other metabolites. Aminoacid derivatives have been reported in marine environment, such as from marine sponges of the genus Jaspis sp. [371,372], fi-om Suberea creba^ a Coral Sea marine sponge [373], and the marine ascidian Leptoclinides dubius [374]. Some of these compounds have been shown to possess interesting biological properties, e.g., cytostatic activity exhibited by axinastatin-4, an aminoacid derivative isolated fi'om a marine sponge [375]. However, tyrosine-derived halometabolites frequently occur in marine organisms. Marine sponges of the order Verongida are of much current biological and chemical interest. An unusual secondary metabolites containing up to four bromotyrosine residues has been isolated from sponges belonging to this order which includes, among others, the genera Aplysinia^ lanthella^ Psammaplysilla^ Pseiidoceratina and Verongida. Bastadins, Fig. (47) are a family of bromotyrosine-derived metabolites isolated from different samples of the marine sponge lanthella basta, which exhibit a wide range of biological activity, such as antineoplasic [376], antimicrobial [377], and inhibitory activity of the endothelin A receptor [378]. These types of compounds have recently been reported from another marine sponge, Psammaplysilla purpurea, together with two new dibromotyrosine-derived metabolites [379]. This sponge also afforded brominated benzenoacetonitriles, unusual dibromo-tyrosine derivatives [380]. From the marine sponge Verongida gigantea, a bromotyrosinederived metabolite, verongamine, has been reported as a potent histamine receptor antagonist. This compound and new acetylenic derivatives are being developed by chemical synthesis. [381].

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Fig. (47). Structure of bastadin 8

Other biologically active bromotyrosine-derived metabolites of marine origin include aeroplysinin. Fig, (48) as cytotoxic and tyrosine kinase inhibitor [382,383], fistularin isolated from Aplysina archeri which exhibited antiviral activity [384], and ceratinamides A and B, antifouling metabolites iromPseiidoceratinapurpurea [385].

Fig. (48). Structure of aeroplysinin

PHOSPHOLIPIDS Lipids are esters of long-chain fatty acids and alcohols or of closely related derivatives. The chief difference between these substances is the type of alcohol; in fixed oils and fats, glycerol combines with the fatty acids; in waxes, the alcohol has a higher molecular weight, e.g., cetyl alcohols. Several monounsaturated phospholipid fatty acids exist in nature, but few cases are known of very long-chain monounsaturated acids longer than 22 carbons. However, marine sponges are unusual in that they have very long-chain fatty acids in their phospholipids. Sponges have provided the most interesting examples of long-chain phospholipid fatty acids since

721

acids with chain-lengths between 24 and 30 carbons have been reported. This unusual ability of these marine invertebrates to biosynthesize very long-chain fatty acids has been responsible for the many interesting structures which have been reported without counterpart in the terrestrial world. Sponges have long been recognized as a rich source of structurally novel lipids including unique fatty acids, phospholipids and triglycerides. The fatty acids of sponges have attracted considerable interest because of their unique characteristics, such as increased chain length, branching and unusual unsaturation patterns, and because of the implications the structural variations may have, when present in phospholipids, for membrane fiinction. Common phospholipid fatty acids from marine sponges include 5,9 hexacosadienoic, which occurs in most know^n sponges, 5,9 heptacosadienoic and 5,9 octacosadienoic. For example, marine sponges of the class Demospongiae contain high levels of characteristic C24-C30 fatty acids and are unique in that they seem to be able to biosynthesize these compounds with amazing ease. Studies have shown that many of these "demospongic acids" possess unusual unsaturation and/or methyl branching not found in the fatty acids of other more common organisms. Many of these compounds have been shown to possess interesting biological properties, e.g., antifungal [386], amidolytic [387], inhibitory activity of PKC and anti-inflammatory activity [388,389] and topoisomerase I [390], and inhibitory activity of HIV reverse transcriptase reported from taurospongin A, Fig. (49), a fatty acid derivative isolated from the Okinawan marine sponge Hippospongia sp. [77,391].

o Fig. (49). Structure of taurospongin A

Branched fatty acids of longer than unusual chain-length have also recently countered in several other sponges. Sphingosine derivatives, such as plakoside A and B, Fig. (50), two unique prenylated glycosphingolipids isolated from Plakortis simplex , have been reported as potent immunosuppressive agents [392]. The Okinawan marine sponge Age las

722

mauritianus yielded a family of new glycosphingolipids named agelasphins, which have been shown as strong antitumor compounds [393]. These lipids have been recently reproduced by chemical synthesis in an efficient manner [394]. Glycosyl ceramides are a family of agelasphin derivatives also with antitumor and immunomodulating activity, which have been reported fi*om different Age las sp. such as the above Agelas mauritianus [395] and Agelas dispar [396]. These compounds were also isolated from other marine sponges, e.g., Haliclona koremella, as an antifouling substance against macroalgae [397], and Spirastrella abata as inhibitors of cholesterol biosynthesis [398]. Recently, these compounds have also been reproduced by chemical synthesis [399]. From marine sponges of the genus Peirosia sp., several glicerol derivatives have recently been isolated, showing interesting biological properties, such as cytotoxicity against human tumor cell lines [400], inhibitory DNA replication [401], and inhibitory activity of HIV reverse transcriptase [77]. New glycerol derivatives were also isolated from the sponge-associated hdiCiQrmm Micrococcus hiteiis [402].

C10H21

R « -<^^4^

v^^*^**^

^C4aH2i

Fig. (50). Structure of plakosides

Besides sponges, other marine organisms and microorganisms have been reported to produce bioactive fatty acids and phospholipids [403]. These compounds. Fig. (51) have been isolated from green algae, such as the Southern Australian Dictyosphaeria sericea [404], red algae such as Pachymeniopsis lanceolata [405], the cyanobacterium Lynghya majuscula [406], and more recently from the brown algae Laminaria sp. [407,408], and Dictyota ciliolata, which yielded a sulfonoglycolipid with nitric oxide synthetase activity [409]. From a symbiotic marine alga, Symbiodinitim sp., a long-chain product, zooanthellatoxin B, was also isolated and caused

723

rabbit platelet aggregation [410] and possess potent vasoconstrictory activity [411]. Exophilin A , a new antibiotic compound, was isolated from the marine microorganisms Exophiala pisciphila [412]. Unusual fatty acids were also reported from the Atlantic salmon, Salmo salar [413], and more recently from the turbot, Scophthalmus maximus [414], the Antartic lamellibranch Laternula elliptica [415], and marine bivlaves [416].

Fig. (51).

In the marine environment, several polyacetylenic compounds biogenetically related with the fatty acids have been reported in the last decade. These types of molecules, with interesting cytotoxic properties, have been isolated mainly from marine sponges of the genus Petrosia sp. [417-419]. However, other samples of marine sponges also yielded bioactive acetylenic compounds, such as the Okinawan Adocia sp. [420,421], and the marine sponges Pellina sp. [422], Reinera sp. [423] and Callyspongia truncata, which yielded polyacetylene derivatives that inhibit fertilization of starfish gametes and showed potent antifouling activity against larvae [424,425]. From the marine sponge Siliquariaspongia japonica, new polyacetylenic derivatives with antitumor and antifungal activity have been recently isolated [426,427]. These types of compounds have also been reported in corals [428], and more recently in marine ascidians, such as the Australian Synoicum prunum [429]. POLYKEXroES Many of the unusual compounds that indicate the exciting chemistry to be discovered in marine natural products are polyketides. Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. They are produced primarily by microorganisms through a specialized metabolism that is a variation of fatty acid biosynthesis [430]. Polyketides fall into two structural classes: aromatic and complex. Polyketides are formed by enzyme complexes

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consisting of 4 to 7 monoflinctional proteins in which the P-carbonyl groups of the intermediates resulting from the condensation of acetate residues are largely not reduced, and cyclization of the intermediates typically produces aromatic compounds. Complex polyketides are composed of acetates, propionates or butyrates, and the extent of pcarbonyl reduction varies from one cycle to the next. However, a strong sequence and mechanistic similarity among many of the fatty acid and polyketide synthase enzymes has led to paradigms for explaining polyketide biochemistry.

Fig. (52). Structure of callystatin A

In the marine environment, several polyketides with important medicinal value have been reported in recent years. They have been isolated mainly from marine sponges of the genus Callyspongia and Plakortis^ which yielded potent cytotoxic polyketides, e.g., callystatin A, Fig. (52) isolated from Callyspongia truncata [285,431], and several cyclic polyketides named plakortides. Fig. (53), isolated from Plakortis lita [432] and Plakortis simplex [433].

Q ^ ,^^C00CH3 Fig. (53). Structure of plakortide

These polyketides, plakortides, have also been shown as potent activators of cardiac calcium-pumping ATPase [434]. New cyclic polyketides were recently isolated from the Red Sea marine sponge Acarnus bergquistae [435], while cytotoxic polyketides have also been reported from sea hares of the genus Aplysia and Dolabella [436], and the marine sponge Theonella swinhei [437]. Marine microorganisms also produced a variety of polyketides wich

725

sometimes incorporate aminoacids, such as marine myxobacteria and cyanobacteria [438-440]. The saltwater culture of marine ftmgi isolated from sponges yielded new polyketides, e,g., the cultured fungus Aspergillus sp. and Paecilomyces sp. separated from the Indo-Pacific sponge Jaspis coriacea [441,442], and Trichoderma longibrachiattim from Haliclona sp. [443]. Some of these marine fiingi producing bioactive polyketides were also isolated from marine tunicates, such as the fungus Phitomyces sp. separated from the Indo-Pacific tunicate Oxycorynia fascicnlaris [444]. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases to generate natural and novel polyketides [445,446]. Additionally, manipulation of the biosynthetic pathways of microbial polyketides through engineering permits the biosynthesis of bioactive polyketides not generated naturally [447,448]. CARBOHYDRATES Carbohydrates are aldehyde or ketone alcohols containing carbon, hydrogen and oxygen in which the hydrogen and oxygen are generally in the same ratio as in water. In the marine environment, it is known that algae produce the greatest variety of carbohydrates, specially polysaccharides, which exhibit a wide range of biological activity. For example, polysaccharides have been reported in the Clorophyta Ulva sp. [449], in the marine alga Fucus vesiculosus, which yielded anti-HIV polysaccharides [450], and in the green marine alga Codium dwarkense with anticoagulant activity [451]. However, it is interesting to point out the biological activity of calcium spirulan, a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis [452]. Extensive studies on calcium spirulan during the past decade have resulted in the identification of a wide variety of biological activity, suggesting it as a usefial candidate for a new drug. Calcium spirulan exhibit anti-herpes and anti-HIV activities, related to the inhibition of enveloped virus replication [453]. Additionally, this compound has been shown as a potent antithrombin agent, and more recently as an inducer of plasminogen activator in fibroblast and as inibitor of metastasis and tumor invasion [454,455]. Recently, polysaccharides have also been reported from marine

726

prokaryotes, both bacteria and archaea, which oflFer a number of novel material properties and commercial opportunities, ranging from emulsifiers to adhesives [456]. MACROLroES Macrolides are a group of compounds containing a macrocyclic lactone ring and up to 9 conjugated trans double bands. In recent years, new macrolides have been isolated from marine organisms, some of them reported as promising candidates for future drugs.

Fig. (54). Structure of bryostatin 1

Bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa [457]. They were first discovered in the bryozoan Bugula neritina, but problems with supply of sufficient quantities of this natural product hampered the study of this interesting group of marine metabolites for many years. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for PKC isoenzymes, compete for the phorbol ester binding site on PKC and stimulate kinase activity in vivo and in vitro. Bryostatin 1, Fig. (54), one member of this family, is a PKC modulator in a variety of tumor systems [458,459]. Bryostatin 1 is currently in phase II

727

clinical trial in Europe and USA sponsored by the National Cancer Institute as an anticancer chemotherapeutic agent [460]. A significant number of bryostatin derivatives have been reproduced by chemical synthesis in a computer model, and it is probable that a bryostatin-inspired derivative will eventually reach clinical phase trials [461-463].

Fig. (55). Structure of jaspisamide A

Additionally, other macrolides constitute an important class of cytotoxic natural products of marine origin, speciallyfi*ommarine sponges. For example, mycalolides, a family of cytotoxic trisoxazole-containig macrolides isolated fi*om marine sponges of the genus Mycale sp. [464,465]; superstolide B isolated from the New Caledonian sponge Neosiphonia superstes [466]; jaspisamides. Fig. (55) from the Okinawan marine sponge Jaspis sp. [467]; and altohyrtin A, a macrolide isolated from Hyrtios ahum [285].

f

Fig. (56). Structure of halichondrin B

H

H 1 H

728

Marine sponges of the genus Haliclona contain a diverse array of active secondary metabolites, including highly potent cytotoxic macrolides, e.g., halichondrin and related compounds. Fig. (56) [468], and salicylihalamides A and B, Fig. (57) [469]. New macrolides chemically related to salicylihalamides, apicularens A and B, were recently isolated from the myxobacteria Chondromyces sp. [470]. From marine bacteria, other cytotoxic macrolides have been isolated, such as octalactin A, Fig. (58) and B, which have been shown as a cell cycle-specific anticancer drug [471], and swinholide. Fig. (59), isolated fi*om symbiotic cyanobacteria with the marine sponge Theonella swinhoei [472].

OH

O

OH

O

Fig. (57). Stnictiireofsalicyhalamides

HO,

Fig. (58). Structure of octalactin A

Many macrolides of marine origin have been reported to show other interesting biological properties, such as immunosuppressive [473,474], antifungal [323,475,476], anti-actin [477], and anti-inflammatory activity

729

reported from lobophorins A and B, which are two new macrolides recently obtained from a marine bacterium isolated from the Caribbean brown alga Lobophora variegata [478]. Recently, efforts have been conducted in order to design the chemical synthesis of these marine natural products based on a macrolactamisation strategy [479-484].

Fig. (59). Structure of swinholide

TOXINS Toxins are bacterial waste products which are considered poisonous for the animal body. These compounds are released by marine organisms in both fresh-water and marine environments and, when ingested by man and other animals, can cause detrimental or even lethal effects [485,486]. Recent reports have shown that marine toxins are the causative agents of seafood poisoning [487], e.g., pectenotoxin 2 isolated from the European alga Dinophysis fortii [488], manauealides. Fig. (60) from the red alga Gracilaria coronopifoUa in Hawai [489], and domoic acid, neurotoxin isolated from the diatom Pseudo-nitzschia miiltiseries [490,491]. Several chromatographic and fluorimetric methods are being developed for the detection of these paralytic poisoning toxins [492-495].

730 HO J

R^==^Cf; R 2 = H

R< = H; R2r:Ac

Fig. (60). Structure of manauealides

Biological investigation of marine toxins, which has had ramifications in many areas of biomedical sciences, has reported a wide range of pharmacological properties, and other research has yielded usefiil candidates as biological probes in studying cellular events [12]. For example, saxitoxins, brevetoxins and more recently, ciguatoxins and maitotoxins involved with ciguatera poisoning, which have been employed in the study of sodium channel action. Saxitoxins are produced by various dinoflagellates and are linked with paralytic shellfish poisonings [496]. This toxin blocks neuronal transmission by binding to the voltage-gated sodium channel [497]. The brevetoxins are "red-tide" toxins of the temperate zones that also cause so-called shellfish poisoning [498,499]. In contrast to these well-understood poisoning, ciguatoxins. Fig. (61) was a problem that was apparently associated with tropical coral reefs [500]. These compounds, originally produced by dinoflagellates, are being sequestered by herbivorous fish and reach high concentrations in carnivorous, predatory species. The related neurotoxin maitotoxin has been shown to induce necrotic cell death in cerebrocortical and breast cancer cell cultures, and calcium-dependent excitatory effects on excitatory membranes such as skeletal, smooth or cardiac muscle [501,502]. This toxin, obtained from the dinoflagellate Gambierdiscus toxicus as a putative calcium-channel activator, has been recently reported as a potent hemolytic and ichthyotoxic agent [503], and to cause shape change followed by aggregation in platelets [504].

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Fig. (61). Structure of ciguatoxin

Additionally, a number of marine toxins with medical and toxicological importance have been isolated from marine flora and fauna. Okadaic acid. Fig. (62) is the main toxin produced by dinoflagellates, which can accumulate in the hepatopancreas of mussels and caused diarrhetic shellfish poisoning in consumers [505,506]. However, this toxin is also a tumor promoter and a specific potent inhibitor of protein phosphatases which may provokes mitotic arrest and apoptosis of leukemia cells [507509]. These types of compounds have been reported in shellfish and phytoplankton, and more recently, in Spanish mussels [510], Portuguese bivalves [511], and the diatom Thalassiosira weissflogii [512]. Following the first report of tumor promotion by okadaic acid, additional tumor promoters of the okadaic acid activity class have been identified, e.g., microcystin [513,514], and calyculin derivatives. Fig. (63) reported in marine sponges such as Discodermia calyx [515] and Theonella swinhoei [516] as potent inhibitors of tumor cell proliferation . A two-sponge association, Poecillastra sp. and Jaspis sp., yielded cytotoxic toxins which exhibited selective activity against several tumoral cell lines [517].

Fig. (62). Structure of okadaic acid

732

227

Fig. (63). Stmcture of calyculin J

Other biological marine toxins have been recently isolated from sea anemones [518,519], the venomous gastropod Conns [520], and marine sponges, e.g., Haliclona exigua and Niphates sp. which yielded xestospongins, neurotoxins that produce depolarizing effects in nerve fibers and inhibit nitric oxide synthase activity [521,522], and a Red Sea sponge which affords latrunculin A, Fig. (64), a potent inhibitor of immunological phagocytosis by macrophages [523]. A related compound, latrunculin B, was recently isolated from the East African nudibranch Chromodoris hamHtoni [524]. Paralytic shellfish toxins were also isolated from blue-green algae, such as Cylindrospermopsis raciborskii from Brazil [525], and the tropical cyanobacterium Lyngbya mqjtiscula [526]. From this alga, curacin A, Fig. (65), was isolated a potent brine shrimp toxin, which has shown promise as an antiproliferative agent due to its inhibition of tubulin polymerization, a mechanisms of proven value in the treatment of neoplasic disorders.

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Fig. (64). Structure of latrunculin A

Additional biological properties have been reported from toxins of marine origin, such as actin-inhibitory activity [527,528], and inactivation of a serotonin-gated ion channel [529]. More recently, marine toxins have also been identified from sea cucumbers [530], and coral reef animals [531]. These toxins, which have been detected in zoanthid species of the genus Palythoa, also occur in various marine organisms living in close association with zoanthid colonies, e.g., sponges, soft corals, mussels and crustaceans.

Fig. (65). Structure of curacin A

Some of these marine toxins have been shown as promising candidates for new drugs. For example, mycalamides, potent antitumor and antiviral compounds isolated fi-om a New Zealand marine sponge, which are undergoing to preclinical evaluation, A significant number of mycalamide derivatives have been synthetised and evaluated for their biological activity [532,533]. Efforts are currently being conducted to design the chemical synthesis of this group of marine natural products [534-536]. To finalize this review, we have to consider that the accumulation of toxic and persistent substances in the marine environment continuously

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increases owing to antropogenic activities [537-539]. Particular attention is being paid to the presence of heavy metals, because of their irreversible effects on man. In fact such elements tend to concentrate in all marine environment matrices, and for this reason they are present in the aquatic food chain, becoming dangerous for humans too, as a consequence of the consumption of marine products [540-542]. In recent years, a number of reports have suggested analytical procedures for the determination of heavy metals in marine organisms, showing that a correct purging procedure considerably reduces the metal content in these matrices [543545]. ABBREVIATIONS PLA2 = Phospholipase A2 AIDS = Acquired immuno-deficiency syndrome HIV = Immuno-deficiency virus PKC = Proteinkinase C ACKNOWLEDGEMENTS The technical acknowledged.

assistance

of

Ms.

Brooke-Turner

is

gratefully

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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BIOLOGICAL ACTIVITIES OF NATURAL HALOGEN COMPOUNDS GERHARD LAUS Immodal Pharmaka GmbH, Bundesstrasse 44, A-6111 Voiders, Austria ABSTRACT: Algae, bacteria, fiingi, lichens, sponges, plants, and mammals produce a wide variety of halogen-containing secondary metabolites. Biological activities also cover a wide range. Many natural halogen compounds exhibit antibiotic activity, for example the altematamides, the pyralomycins, the longamides, clathramides, celenamide, axinellamides, and the macrolide flurithromycin. Cytotoxicity is displayed by the polychlorinated phenolethers russuphelins, the macrolide phorboxazoles, the cyclopropane derivatives grenadadiene and callipeltosides, the aurantosides, the aurisides, the rubrosides, the didemnolines, the spiro isoxazole derivates purealidins, and the cyclic peptides geodiamolides. The chloropyrrol-containing hexapeptide cyclocinamide A, and the nostocyclophanes, natural cyclophanes, also belong to this categoiy. Spongistatins are among the most cancer cell growth inhibitory compounds yet discovered. Some natural halogen compounds display a gallery of activities, like the cytotoxic cyclodepsipeptide jaspamide which is also antifungal and insecticidal. The mycorrhizin-related chloroepoxide lachnumone combines antifungal, nematicidal and antimicrobial activities. The chlorosteroid glycoside blatellastanoside is a pheromone of cockroaches. Polyhalogenated monoterpens are insecticides, some deter the settlement of larvae. The volutamides, barbamide, and parguerol show antifeedant properties. Kalihinol A inhibits the settlement of barnacle larvae. Some natural halogen compounds have emerged as potent lead structures for the development of new synthetic drugs, like the analgesic chloropyridine epibatidine, or pyrrolnitrin and dioxapyrrolomycin which have lead to powerful fungicides and insecticides. Even polychlorinated dibenzodioxins have been found in nature. In some cases, the halogen is essential for activity like the antifungal chloroorcinols, ichthyotoxic malyngamides, and cytotoxic makaluvamines. The biological activities of these and related compounds are reviewed, and 173 references are given.

INTRODUCTION Algae, bacteria, fungi, lichens, sponges, plants, and mammals produce a wide variety of halogen-containing secondary metabolites. Natural halogen compounds have long been considered as chemical freaks. The antibiotics aureomycin and chloramphenicol, and the antimycotic griseofulvin are classic examples. Today there are more than 3000 examples known. Most of them are of marine origin. Ocean water is approximately 0.5 M in chloride, 1 mM in bromide, and 1 |aM in iodide. Given this high halogen content, it is not surprising that marine organisms have developed means to incorporate halogens into their metabolites

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which are sometimes passed on to symbionts mid possibly modified for defensive purposes. The origin of marine metabolites can be obscured by these symbiotic associations. Recent reviews concern origin and occurrence of natural organobromine compounds [1], organochlorine compounds [2,3], the diversity of natural organohalogen compounds in living organisms [4,5], and natural chemistry of chlorine in the environment [6]. Marine sponges continue to be a rich source of secondary metabolites with novel structures and desirable biological activities [7]. Various aspects of this field, e.g. marine haloperoxidases [8], ascidian metabolites [9], marine invertebrate chemical defenses [10], bioactive metabolites of symbiotic marine microorganisms [11], marine bacteria [12], biosynthesis of marine natural products [13], microalgal metabolites [14], bioactive sponge peptides [15], marine pyridoacridine alkaloids [16], and marine toxins have been reviewed earlier [17], The present review is divided into major areas of activity although some overlapping does occur due to the many and diverse bioactivities of some natural halogen compounds. It emphasizes the newer literature and refers to older publications only when they are of special relevance. A discussion of the more striking structural features is given where appropriate. BIOSYNTHESIS OF HALOGENATED ORGANIC COMPOUNDS Haloperoxidases are enzymes that catalyze the oxidation of a halide by hydrogen peroxide, a process which results in tlie concommitant halogenation of organic substrates. The nature of the oxidized halogen intemiediate has been shown to depend on the nature of the organic substrate [18]. Haloperoxidases have been isolated from all classes of marine algae and many other marine organisms. Two general types of marine haloperoxidases have been identified: vanadium haloperoxidase and Fe-heme haloperoxidase. A remarkably thermostable iodoperoxidase has been isolated fi-om the sea weed Saccorhiza polyschides [19]. In addition, other terrestrial haloperoxidases are known. Mechanistic considerations of the vanadium haloperoxidases have been reviewed recently [20]. CYTOTOXICITY Marine tunicates are a rich source of intriguing structures and interesting biological activities. Eudistomidin A (1) fi"om the Okinawan tunicate Eiidistoma glqucus possesses powerfiil calmodulin antagonistic activity (IC50 3 X 10'^^ M). Eudistomidins B-D (2-4) show potent cytotoxicity

759

against murine leukemia L1210 (IC50 3.4, 0.36, and 2.4 |ig/ml) and L5178Y (ICsq 3.1, 0.42, and 1.8 jig/ml). In addition, eudistomidm B activates rabbit heart muscle actomvosin ATPase by 93% at 3 x lO'*' M, while eudistomidin D induces Ca'^^ release from the sarcoplasmic reticulum, about 10 times more potent than caffeine [21].

3 (eudistomidin C)

4 (eudistomidin D)

The remarkably antineoplastic spongistatins have been obtained from a Maldivian Spongia sp. Spongistatin 1 (5) possesses two spiro ketals and is extremely potent against highly chemoresistant tumor types (GI50 typically 3 x 10"^^ M) [22]. Spongistatins 4 and 5 have been obtained from the African sponge Spirastrella spinispirulifera. Mean growth inhibition (GI50) values of 10'^ M are found in the National Cancer Institute's (NCI) 60 tumor cell lines. Human breast cancer cell lines are even more sensitive (GI50 down to 10" M) [23,24]. The names altohyrtin and cinachyrolide A have also been used for spongistatins 1 and 4, respectively [25]. Thus, cinachyrolide A from a sponge of the genus Cinachyra is highly cytotoxic against L1210 murine leukemia cells with an IC50 of <0.6 ng/ml [26]. Spongistatins 1 and 9 have been found to potently inhibit the glutamate-induced polymerization of tubulin with IC50 values of 3.6 and 4.2 |LIM. They appear to be the most cancer cell growth inhibitory antimitotic substances discovered to date [27]. Two antifungal and cytotoxic macrolides, phorboxazoles A (6) and B (7), have been isolated from the Indian Ocean sponge Phorbas sp. They are active against Candida albicans and Saccharomyces carlsbergensis at 0.1 |ig/disk. Testing in 60 tumor cell lines shows exceptional inhibition of cell growth. Both compounds have mean GI50 values of <7.9 x 10"'" M; however, most cell lines are still inhibited at this concentration. Thus, the phorboxazoles are among the most potent cytostatic agents yet discovered [28].

760

OCH3 5 (spongistatin 1)

6 R = ^ ^ ^ (phorboxazoleA) 7 R = />^ (phorboxazole B)

Discorhabdins A (8), B (9) and C (10) have been isolated from several sponge species of the genus Latrunculia from New Zealand. They are powerfiil cytotoxins with IC50 values against the murine leukemia P-388 cell line in the range 0.03 - 0.01 |ag/ml. Discorhabdin A has also been isolated from a Japanese sponge of the genus Prianos [29]. Discorhabdin P (11) from a Bahamas sponge of the genus Batzella exhibits cytotoxicity against P-388 cells and human lung carcinoma A549 cells with IC50 values of 0.025 and 0.41 ^g/ml, respectively. It also inhibits calcineurin (IC50 0.55 |ig/ml) [30]. Discorhabdin Q (12) has been isolated from the Australian sponges Latrunculia purpurea, Zyzza massalis and Zyzza fuliginosa. It exhibits moderate, generalized

761

cytotoxicity in the NCI's 60 cell line antitumor screen (mean panel GI50 0.5 |ig/ml). It is a hypothesis that full aromatization of the pyrroloiminoquinone system confers reduced cytotoxicity relative to the saturated members of the family [31].

8 (discorhabdine A) O

10 R = H (discorhabdine C) 11 R = CH3 (discorhabdine P)

12 (discorhabdine Q)

Grenadadiene (13), a unique cyclopropane-containing metabolite from Lyngbya majusciila, shows an interesting profile of cytotoxicity in the NCI's 60 tumor cell lines [32]. o

13 (grenadadiene)

Cyclocinamide A (14), an unusual cytotoxic halogenated hexapeptide from the marine sponge Psammocinia whose tetrapeptide core, consisting of a 14-membered ring, along with the dipeptide side chain terminating in the proline derived iV-methyl chloropyrrole has no parallel among other natural products. This compound exhibits striking in vitro selective activity against Colon-38 tumor cells [33].

762

HN

O^

^O

NH.

14 (cyclocinamide A)

A new bromopyrrole alkaloid 15 along with racemic 16 was isolated from the Japanese marine sponge Homaxinella sp. They exhibit weak cytotoxic activity against P-388 lymphocytic leukemia cells with ED50 values of 21.5 |Lig/ml and 30 |Lig/ml, respectively [34].

'^iXf .OCH,

•

^

H3C0^''^0

O

16

15

The macrolide glycosides, aurisides A (17) and B (18), from the Japanese sea hare Dolabella auricularia show cytotoxicity against HeLa S3 cells with IC50 values of 0.17 and 1.2 |ig/ml, respectively [35]. OH H3(X>,., ^X^

H^ca,

^0CH3

17 (auriside A) O

R=

A,

H^N'^

O

18 (auriside B)

763

The bromotriterpene aurilol (19) also was isolated from the sea hare Dolabella auricularia, Aurilol exhibits cytotoxicity against HeLa S3 cells with an IC50 of 4.3 |Lig/ml [36]. H

19 (aurilol)

20 (discodermindol)

Discodermindol (20), a brominated aminoimidazolinylindole, from the sponge Discodermia polydiscus shows IC50 values of 1.8 |ig/ml against P-388 (murine leukemia), 4.6 \iglm\ against A-549 (human lung), and 12 |ig/ml against HT-29 (human colon) [37]. Pigments from the marine sponge Theonella swinhoei, aurantosides A (21) and B (22), are cytotoxic against P-388 (IC50 1.8 and 3.2 |ig/ml, respectively) and LI210 leukemia cells (IC50 3.4 and 3.3|ig/ml) [38]. A recent reinvestigation has led to a revision of the geometry of the terminal double bond. Aurantoside F (23) exhibits the highest cytotoxicity against P-388 (IC50 0.05 ^ig/ml) [39].

IHO

OH

RO" 21 R = CH3 (aurantoside A) 22 R = H (aurantoside B)

Eight tetramic acid glycosides named rubrosides A-H have been isolated from the marine sponge Siliquariaspongia japonica, along with aurantosides A and B. Rubrosides are closely related to aurantosides in having the common tetramic acid and the polyene, but aurantosides terminate in olefines, whereas rubrosides terminate in a tetrahydrofiiran ring. The rubrosides induce morphological changes in 3Y1 rat fibroblasts, namely numerous large vacuoles and spindle-shaped cells. In particular, rubroside A (24) is cytotoxic against P-388 murine leukemia cells (IC50

764

0.05 |ig/ml) and moderately antifiingal against Candida albicans and Aspergillus fumigatus [40]. CI

CI

OH

O

V

HO

OH

^'... ^ O RCO'" 23 (aiirantoside F)

OH

O

24 (rubroside A)

H3CO"

Meridianins, brominated indole alkaloids from the tunicate Aplidiiim meridianum, show cytotoxicity against LMM3 (murine mamarian adenocarcinoma cell line) with IC50 values of 11.4 \xiA for meridianin B (25), 9.3 |iM for meridianin C (26), 33.9 \xM for meridianin D (27), and 11.1 laM for meridianin E (28) [41].

25 26 27 28

Ri Ri Ri Ri

= OH R2 = R4 = H R3 = Br (meridianin B) = R3 = R4 = H R2 = Br (meridianin C) = R2 = R4 = H R3 = Br (meridianin D) = OH R2 = R3 = H R4 = Br (meridianin E)

765

From the New Zealand ascidian Cnemidocarpa bicornnta, the brominated phenylethylamine 29 has been isolated. It exhibits mild cytotoxicity to P-388 cells (IC50 46 \M) [42]. The tribrominated bisindole alkaloid dragmacidin (30) from the marine sponge Dragmacidon sp. has antitumor activity against P-388 cells (ICso 15 |ig/ml), A-549 (human lung), HCT-8 (human colon), and MDAMB (human mammary) cancer cell lines (IC50 1-10 |ig/ml) [43].

^^v*

OH

NH. 30 (dragmacidin)

29

Didemnolines A (31) and C (32) from the marine ascidian Didemnum sp. are cytotoxic to human epidermoid carcinoma KB cells (IC50 values of 6.1 and 0.28 i^ig/ml, respectively) and antimicrobial tov^ard Staphylococcus aureus. Bacillus subtilis, and Escherichia coli [44,45].

H3C\,

32 (didemnoline C)

31 (didemnoline A) CI

C!

33 Ri = R2 = CH3 (russuphelin A)

OCH3

34 Rj = CH3 R2 = H (russuphelin B) 35 R] = R2 = H (russuphelin C)

36 (russuphelin D)

Several chlorinated hydroquinone derivatives with cytotoxic activity have been isolated from the toxic mushroom Russula subnigricans, russuphelins A-D (33-36) and the tetramer russuphelol (37), the latter

766

being optically active due to the bulkiness of the vicinal bisphenoxy system. In vitro IC50 values against P-388 are 4.64, 15.4, 0.94, 12.1, and 7.19 |ig/ml, respectively [46,47,48]. HO

a. ci

o.

H3C0 37 (russuphelol)

A series of bromotyrosine alkaloids has been isolated from the Okinawan marine sponge Psammaplysilla purea. Purealidins N (38), P (39) and Q (40) exhibit cytotoxicity against murine lymphoma L1210 cells (IC50 values: 0.07, 2.8, and 0.95 i^ig/ml, respectively) and human epidermoid carcinoma KB cells (IC50: 0.074, 7.6, and 1.2 |Lig/ml, respectively) in vitro. Purealidins J (41), K (42), P, and Q show inhibitory activity against epidermal growth factor (EGF) receptor kinase (IC50: 23, 14, 18, and 11 |ig/ml, respectively) [49]. Recently, a novel bromotyrosine derivative 43 has been isolated from the Caribbean sponge Aplysina cauliformis. It exhibits toxicity against HeLa cells (IC50 50 |Lig/ml) [50]. OCH

38 (purealidin N)

OCH,

39 (purealidin P)

767 OCH3

Br

Br^ HO"^

\

/

H

0

"V^r"

VK Br

40 (purealidin Q)

OCR

!

"

^

>

42 (purealidin K)

41 (purealidin J)

OCH,

43

Natural polychlorinated dioxins and furans have been identified in peat bogs. A single tetrachlorodibenzofuran isomer (2,4,6,8-TCDF 44) predominates over all other isomers, whereas two isomers of tetrachlorodibenzodioxin are prominent (1,3,6,8-TCDD 45 and 1,3 J,9-TCDD 46). The pattem is replicated by in vitro oxidative coupling of 2,4dichlorophenol using chloroperoxidase. Significant incorporation of CF occurs in peat. Autoclaving decreases incorporation while adding casein hydrolyzate increases it. Thus, the local origin of the organochlorines is suggested [51].

768

44 (2,4.6,8-TCDF)

45(1,3,6,8-TCDD)

46(l,3J,9-TCDD)

Two new jaspamide derivatives 47 and 48 along with jaspamide (49) have been isolated from the marine sponge Jaspis splendcms collected in Vanuatu. They inliibit the growth of the human NSCLC-N6 cancer cell line with IC50 values of 3.3, 1.1, and 0.36 |ig/ml, respectively. Jaspamide has been shown to possess remarkable biological properties such as antifungal, anthelminthic, antimicrobial, insecticidal (LC50 of 4 ppm against He Hot his verescens), and ichthyotoxic activities [52]. The closely related geodiamolides have been isolated from a Caribbean species of Geodia and from a Papua New Guinea sponge Cymbastela sp. These cyclodepsipeptides contain a common 12-carbon polypropionate unit and differ in the type of halogen in their tyrosine fragment. Geodiamolides A-F (50-55) show cytotoxicity against L1210 cells with EC50 values of 3.2, 2.6, 2.5, 39, 14, and 6 ng/ml [53,54]. Eight new geodiamolides have been reported recently. It has been suggested that these compounds are actually of microbial origin [55].

47R = 0 48R = OH

49 (jaspamide)

769

50 X=I R=CH3 (geodiamolide A) 51 X=Br R=CH3 (geodiamolide B) 52 X=C1 R=CH3 (geodiamolide C) 53 X=l R=H (geodiamolide D) 54 X=Br R=H (geodiamolide E) 55 X=C1 R=H (geodiamolide F)

Dichlorolissoclimide (56), a naturally occurring succinimide from the New Caledonia ascidian Lissoclinum voeltzkowi, is cytotoxic to KB cells (IC50 14 ng/ml) and P-388 cells (IC50 1 ng/ml) [56]. The polyhalogenated monoterpenes 57-59 from the Spanish sea hare Aplysia punctata show identical cytotoxic properties against P-388 mice lymphoma and HT-29 human colon carcinoma (ED50 2.5 |Lig/ml), A-549 human lung carcinoma and MEL-28 human melanoma cell lines (EDso 1.5^g/ml)[57].

a

.o

o

CI 56 (dichlorolissoclimide)

58

57

59

Halomon (60) and related antitumor monoterpenes 61-63 from the Philippinian red alga Portieha hornemannii display a unique differential cytotoxicity profile against the NCI's panel of 60 human tumor cell lines,

770

with comparable panel-averaged potency (GI50 values of 0.676, 1.32, 0.741, and 0.691 jiM). The cyclic compound 64 show^s comparable potency (GI50 1.15 |iM) but little differential response [58]. a

a Br

-Br

Br

a

61

60 (halomon)

62

63

Makaluvamine N (65) from the Philippine sponge ZyzTya fuliginosa shows cytotoxicity against the human colon tumor cell line HCT-116 with an LC50 of 0.6 fxg/ml [59].

65 (makaluvamine N)

66 (dtbromophakellstatin)

Dibromophakellstatin (66) from the Indian Ocean sponge Phakellia mauritiana shows GIso-level growth inhibition of one-third of the NCFs 60 cell line human tumor panel at submicromolar concentrations [60]. Palauamine (67) from the Pacific sponge Stylotella agminata is quite active against P-388 (IC50 0.1 pg/ml) and A549 (IC50 0.2 |ig/ml) and possesses antibiotic activity against Staphylococcus aureus and Bacillus subtilis and also antifungal activity against Penicillium notatum. In the mixed lymphocyte reaction palauamine shows an IC50 ^ 0.018 jxg/ml, while the cytotoxicity assay against murine lymphocytes shows an IC50 of 1.5 |uig/ml. It is reasonably non-toxic (i.p. LD50 in mice 13 mg/kg) [61],

771

H2N

a

-NH2

67 (palauamiiie)

68 Ri = H R2 = CI (manauealide A) 69 Ri = H R2 = Br (manauealide A) 70 Ri = Br R2 = H (aplysiatoxin)

The macrolides manauealide A (68) and B (69) have been identified along with aplysiatoxin (70) in a toxic specimen of red alga Gracilaria coronopifolia which caused food poisoning in Hawaii. A 1 |ag injection of either 68 or 69 causes diarrhea in mice. It is possible that the causative agents originate from epiphytes, blue-green algae, on the algae and not from the Gracilaria itself [62]. Steroids from the Okinawan sponge Xestospongia sp., aragusteroketal C (71) and aragusterol C (72), exhibit potent cytotoxic activities against KB cells with ICso values of 0.004 and 0.02 i^ig/ml, respectively [63].

H.CO H3CO 71 (aragusteroketal C)

72 (aragusterol C)

Marine sponges of the genus Theonella often afford bioactive cyclic peptides containing unusual amino acid residues, e.g. theonellamide, cyclotheonamide, and theonellapeptolide. Orbiculamide A (73) from Theonella sp, contains 2-bromo-5-hydroxytryptophan and is cytotoxic against P-388 murine leukemia cells (IC50 4.7 ^ig/ml) [64]. The keramamides B-D, also from a Theonella sp,, inhibit the superoxide generation of stimulated human neutrophils at 5 x 10"^ M [65].

772

y.^^„ 73 (orbiculamide A)

Keramamide E (74) exhibits cytotoxicity against L1210 murine leukemia cells and KB human epidermoid carcinoma cells with IC50 values of 1.60 and 1.55 |ag/ml, respectively [66]. The bicyclic peptides, theonellamides A-E, from a marine sponge Theonella sp. are cytotoxic to P-388 murine leukemia cells with IC50 values of 5.0, 1.7, 2.5, 1.7, and 0.9 ^ig/nl, respectively [67]. Theonellamide F (75) inhibits growth of various pathogenic fiingi {Candida spp., Trychophyton spp., and Aspergillus spp.) at concentrations of 3-12 |ig/ml. It is also cytotoxic against L1210 and P388 cells with IC50 of 3.2 and 2.7 |ig/ml, respectively [68]. The theonellamides contain bromophenylalanine and, four of them, bromophenyl-octadienoic amino acid.

74 (keramamide E)

773

OH

/—OH HN—/ ( V-NH

HN

(

p

NH2

HN—/

O


HO—/

\

^

O^ ' "W

V-NH

.-^^^^Br HN

HO

V-NH OH

75 (theonellainide F)

Recently, bromodeoxytopsentin (76) and isobromodeoxytopsentin (77) have been isolated from the Korean sponge Topsentia genitrix. They exhibit cytotoxicity against the human leukemic cell line K-562 (LC50 0.6 and 2.1 |ig/ml, respectively) [69]. Nortopsentins A-C (78-80) with a unique imidazolediylbisindole skeleton from the Caribbean sponge Spongosorites ruetzleri exhibit cytotoxicity against P-388 cells (IC50 7.6, 7.8, and 1.7 |ig/ml) and antifiingal activity against Candida albicans (MIC 3.1, 6.2, and 12.5 |ig/ml). Trimethyl and tetramethyl derivatives of nortopsentine B show significant improvement in P-388 activity (IC50 0.90 and 0.34 |J.g/ml) compared with the parent compound [70]. Nortopsentin D (81) from the axinellid sponge Dragmacidon sp. is inactive on KB tumoral cells in vitro. However, permethylation leads to high cytotoxicity on KB cell lines (IC50 0.014 \xglm\) [71].

76 Rj = Br R2 = H (bromodeoxytopsentin) 77 Rj = H R2 = Br (isobromodeoxytopsentin)

78 Rj = R2 = Br (nortopsentinA) 79 R] = H R2 = Br (nortopsentin B) 80 Ri = Br R2 = H (nortopsentin C)

774

81 (nortopsentin D)

Hiburipyranone (82), a brominated isocoumarine from the sponge Mycale adhaerens collected off Hiburi Island, Japan, is cytotoxic against P-388 cells with an IC50 value of 0.19 jig/ml [72]. Secobatzelline A (83) from a sponge of the genus Batzella is active against P-388 (IC50 0.06 jig/ml) and human lung carcinoma A-549 cells (IC5o0.04^g/ml)[73]. HN-

H2N 82 (hiburipyranone)

W ;^

NH

OH OH

83 (secobatzelline A)

Psammaplysin E (84), ceratinamine (85), and molokaiamine (86) from the sponge Pseudoceratina purpurea exhibit potent cytotoxicity against P-388 murine leukemia cells with IC50 values of 2.1, 3.4, and 2.1 |Lig/ml, respectively [74]. Recently, the related waianaeamines have been isolated from an undescribed verongid sponge from Molokai Island [75]. H3CD,

84 (psammaplysin E)

775

NHo

HO 85R = COCN (ceratinamine) 86 R = H (molokaiamine)

OH

87 (trachycJadine)

Trachycladine A (87) from the Australian axinellid sponge Trachycladiis laevispirulifer possesses a branched 5-deoxy-2-C-methyl sugar. It shows cytotoxicity against several human cell lines including leukemia CCRF-CEM (IC50 0.4 ng/ml), colon tumor HCT-116 (IC50 0.9 |ig/ml), breast tumors MCF-7 (IC50 0.2 |ig/ml), MDA-MB-435 (IC50 0.25 |Lig/ml), and MDA-N (IC50 0.1 ^g/ml). It also exhibits moderate toxicity against brine shrimp (LD50 0.26 |ig/ml) [76]. The same structure was proposed for kumusine from an Indonesian sponge Theonella sp. which shows cytotoxicity against P-388 cells with an IC50 of 5.0 |ag/ml [77]. Callipeltoside A (88) from the New Caledonian sponge Callipelta sp. is a cytotoxic glycoside macrolide featuring a dienyne cyclopropane side chain. It has a moderate activity v^th IC50 values against the NSCLC-N6 human bronchopulmonary non-small-cell-lung carcinoma and P-388 of 11.26 and 15.26 |ig/ml, respectively [78]. Callipeltosides B and C, minor metabolites differing in the saccharide moieties, have IC50 values of 15.1 and 30.0 ^ig/ml against NSCLC-N6 cells [79].

H,CO'

88 (callipeltoside A)

776

ANTIBIOTIC AND ANTIFUNGAL ACTIVITY The first marine bacterial metabolite to be reported was pentabromopseudiline (89) which is composed of more than 70% bromine by weight. It shows antibiotic properties against Gram-positive bacteria with minimum inhibitory concentrations from 0.0063 to 0.2 ^ig/ml [80]

89 (pentabromopseudiline)

90

91

Polybrominated diphenyl ethers from the Indonesian marine sponge Dysidea herbacea are active against the Gram-positive bacteria Bacillus subtilis (MIC 0.20 |ig/ml) and the phytopathogenic fungus Cladosporium cucumerinum. Compounds 90 and 91 are also active in the brine shrimp lethality test (LC50 0.96 and 0.94 |ig/ml) [81]. Alternatamides A-C (92-94), bromotryptamine peptides from the Atlantic bryozoan Amathia alternata, show modest antibacterial activities against Staphylococcus aureus^ Staphylococcus epidennidis. Staphylococcus haemolyticus. Bacillus subtilis, Enterococcus faecalis, Enterococcus faecium, and Streptococcus pyogenes with MIC values ranging between 4 and 32 |ig/ml [82]. NHCH,

92 Rj = Br R2 = CH3 (altematamide A) 93 Ri = Br R2 = H (altematamide B) 94 Ri = H R2 = H (altematamide C)

The antimicrobial pyralomycins have been isolated from a culture of Actinomadura spiralis strain. Pyralomycins la-Id (95-98) have a cyclohexene ring connected to the benzopyranopyrrole chromophor.

777

Another series , pyralomycins 2a-2c (99-101), have a tetrahydropyran ring in place of the cyclohexene ring. They inhibit the growth of Micrococcus luteus at 0.2-25 |Lig/ml [83]. OH

95 Ri = H R2 = CI R3 = CH3 R4 = CH3 9 6 R i = H R 2 = CH3R3 = ClR4 = CH3 97 Ri = H R2 = CI R3 = CH3 R4 = H 98Ri =C1R2 = CIR3=CH3R4 = H

99 R1 = H R2 = CI R3 = CH3 R4 = CH3 100 Ri = H R2 = CH3 R3 = CI R4 = CH3 1 0 1 R i = H R 2 = ClR3=CH3R4 = H

A polycyclic xanthone 102, produced by a culture of an Actinoplanes sp., exhibits very potent antifungal activity against a variety of fungal pathogens including yeasts, dermatophytes, dind Aspergillus [84]. Chlorochimaphilin (103) from the British Columbian medicinal plant Moneses uniflora shows good activity against a range of bacteria and pathogenic fungi [85]. Investigation of the Caribbean marine sponge Agelas dispar for biologically active constituents has led to the isolation of bromopyrrole alkaloids. Longamide B (104) and keramadine (105) show moderate activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus with MIC values of about 50 |ig/ml. Clathramides C (106) and D (107) show antifungal activity against Aspergillus niger [86].

OCH.

a

102

o

103 (chlorochimaphilin)

778

NH

Br

NHo

105 (keramadine)

106 Ri 107 Ri

H R2 = COOH COOH R2 = H

Celenamide E (108), a tripeptide alkaloid from the Patagonian sponge Cliona chilensis, shows antibiotic activity against Bacillus subtilis. Staphylococcus aureus^ Micrococcus luteus, and Enterococcus faecalis at 50 ^ig/disk. An unusual feature is the presence of a N-tenninal dehydroamino acid [87]. Axinellamines, e.g. axinellamine B (109), from the Australian marine sponge Axinella sp. with a unique heterocyclic skeleton have bactericidal activity against Helicobacter pylori, the bacterium associated with pepticular and gastric cancer, at 1 mM [88].

108 (celenamide E)

779 HO HN "•

HN

a

I 'OH H N ^ ^ O

HN

Br

HN-^^

109 (axinellamine B)

From strains of Sorangium cellulosum a chlorodivinylether, maracen (110), was isolated. It is active against Mycobacterium tuberculosis (IC99 < 12.5 |ig/ml) and only slightly toxic against murine fibroblasts L929 (> 24 |ig/ml) [89]. .0^

<^^^^=^

..i>s.

^^

^O

110 (maracen)

Bromopyrroles are characteristic antibacterial and antiviral metabolites of sponges of the genus Agelas. Sceptrin (111), ageliferin (112), and bromoageliferin (113) are active against Bacillus subtilis and Escherichia coli at 10 |uig/disk. They are also active against Herpes simplex (20 |Lig/disk) and Vesicular stomatitis virus (100|ig/disk), along with dibromosceptrin (114) and dibromoageliferin (115) [90]. Br

n

H,N

P

NH,

H NH7

Br

R

\

/ \^

NH2

H 111 R = H (sceptrin) 114 R = Br (dibromosceptrin)

1 1 2 R i = R 2 = H (ageliferin) 113 Rj = Br R2 = H (bromoageliferin) 115 R] = R2 = Br (dibromoageliferin)

Ageliferins may provide useful chemical tools for the study of the molecular mechanisms of actin-myosin contractile systems, since they

780

elevate the ATPase activity of myofibrils fi'om rabbit skeletal muscle to 150, 190, and 200% of control in concentrations of 3 x 10" (ageliferin), lO'^(bromoageliferin), and 10"^ M (dibromoageliferin), respectively [91]. 5-Bromopyrrole-2-carbamide (116) was isolated from the Papua New^ Guinean sponge Agelas nakamurai. It exhibits antimicrobial activity against Staphylococcus aureus (IC50 0.78 |Lig/ml) and other bacteria and fungi [92]. Welwitindolinone A isonitrile (117) from the blue-green alga Hapalosiphon welwntschii is active against Aspergillus oryzae, Penicillhim notatum, Saccharomyces cerevisiae, and Trichophyton mentagrophytes [93].

Br'

n N' H

NH. O

116

117 (welwitindolinone A isonitrile)

Aurantoside E exhibits potent antifungal activity against Aspergillus fumigatus and Candida albicans (MIC 0.16 and 0.04 |ig/ml, respectively) [39]. From a marine isolate of the fungus Emericella unguis collected in Venezuelan waters, an antibacterial depside, guisinol (118), has been isolated. It is active against Staphylococcus aureus [94]. OH

118 (guisinol)

119(tnarinone)

Marinone (119), a sesquiterpenoid naphthochinone from a marine actinomycete, shows significant activity against Gram-positive bacteria [95]. Flurithromycin (120), a fluorinated erythromycin macrolide, has been isolated from a mutant strain of Streptomyces erythraeus. It shows antibacterial activity against Streptococcus pneumoniae (MIC 0.0015-

781

0.006 |ig/ml), Haemophilus influenzae (MIC 0.012-0.4 |ig/ml), and Staphylococcus aureus (MIC 0.1-3.1 |ig/ml) [96].

120 (fliirithromycin)

PHYTOTOXICITY 5'-O-Sulfamoyl-2-chloroadenosine (121) from a strain of Streptomyces albiis possesses potent herbicidal activity on broad leaf weeds in postemergent application (80 percent control on Sinapis alba, Stellaha media, Veronica persica, and Xanthium spinosum at <24 g/acre). Unfortunately, this compound also exhibits strong mammalian toxicity (LCso in mouse fibroblasts 0.013 |ig/ml). Nucleocidin glucoside (122), a fluoronucleoside from a Streptomyces strain, is a weak herbicide [97]. The germination of the monocotyle Setaria italica and the dicotyle Lepidium sativum is inhibited by the mycorrhizins, e.g. 123, lachnumon (124), and halogenated isocoumarin derivatives, like 125, which have been isolated from the ascomycete Lachnum papyraceum [98].

NH.

N

^a HO HO

^

O

OH

OH 122 (nucleocidin glucoside)

782

CI

OH

O

O

H3CO'

125

123 (mycorrhizin A)

ANTIVIRAL ACTIVITY A number of antiviral brominated p-carbolines, eudistomins, have been extracted from the Caribbean tunicate Eudistoma olivaceum. The eudistomins can be subdivided into five classes: the simple p-carbolines (eudistomin D, J, N, and O), the pyrrolyl-p-carbolines (A and M), the pyrrolinyl-p-carbolines (G, H, I, P, and Q), the 2-phenylacetyl-Pcarbolines (R, S, and T), and the tetrahydro-P-carbolines which contain an oxathiazepine ring (C, E, F, K, and L). In particular, eudistomins C (126) and E (127) are active in the Herpes simplex virus, type 1 (HSV-1), assay at 50 ng/disk, whereas the halogen-free eudistomin I shows only questionable inhibition. Eudistomin K inhibits HSV-1 growth at 250 ng/disk and eudistomin L at 100 ng/disk [99]. Some eudistomins also are active against Bacillus subtilis or Saccharomyces cerevisiae. A mixture of eudistomins N and O displays a remarkable degree of synergism [100]. It is interesting to note that also Ritterella sigillinoides, Lissoclimim fragile, and Pseudodistoma aureum are species known to produce eudistomins [101].

H.N 126 Ri = Br R2 = H (eudistomin C) 127 R] = H R2 = Br (eudistomin E)

128 (amathaspiramide E)

Amathaspiramide E (128) from the New Zealand bryozoan Amathia wilsoni exhibits strong activity in the antiviral assay against Polio virus [102].

783

ANTIFOULING ACTIVITY Fouling in the marine environment is an economic burden. Sessile marine organisms, such as mussels and bamacles, often cause serious problems by settling on ships' hulls, on cooling systems for power plants, and on fishing nets. Organotin compounds have been used as antifouling agents. Because of environmental concerns, antifouling substances with reduced toxicity are needed. The kalihinanes, terpenoids from the Japanese marine sponge Acanthella cavernosa, completely inhibit metamorphosis of cyprid larvae of the barnacle Balanus amphitrite at a concentration of 5 |ig/ml. In particular, the isocyanate 129 and isothiocyanate derivatives are highly antifouling (EC50 ca. 0.05 ^ig/ml) [103]. Kalihipyran B (130) from the Japanese sponge Acanthella cavernosa inhibits settlement and metamorphosis of the barnacle Balanus amphitrite with an IC50 of 0.85 |ig/ml. Kalihinol A (131) is even more potent with an IC50 of 0.087 lag/nil [104]. Ecological studies indicate that the Australian sponge Stylotella aiirantium inhibits the settlement of ascidian larvae. A dichloroimine metabolite, stylotellane B (132), was isolated which displayed weak P388 activity [105]. NHCHO

129 Ri = NCO R2 = NHCHO 131 Ri = NC R2 = NC (kalihinol A)

130 (kalihipyran B)

a a CI 132 (stylotellane B)

133

1,8,8-Tribromo-3,4,7-trichloro-3,7-dimethyl-1,5-octadiene (133) from the Australian alga Plocamium costatum deters the settlement of Balanus amphitrite larvae at 10 jig/ml [106]. Elatol (134) from the red alga Laurencia rigida completely inhibits the settlement of Balanus amphitrite larvae at 0.1 |ag/cm [107].

784

Styloguaiiidins, e.g. 135, chitinase inhibitors from the marine sponge Stylotella aurantium, inhibit the moulting of cyprid larvae of barnacles at a concentration of 10 ppm [108]. They are closely related to palauamine (67). Br.

Br

sJC™ HO^ Bn.

N-i^N^^O

H.N'

HC'

NH2 134 (elatol)

135 (dibromostyloguanidine)

The bromotyramine derivatives ceratinamide A (136) and psammaplysin A (137) from the sponge Pseudoceratina purpurea inhibit the settlement and metamorphosis of cyprid larvae of the barnacle Balanus amphitrite (ED50 0.10 and 0.27 |ag/ml). Interestingly, psammaplysin A induces larval metamorphosis of the ascidian Halocynthia roretzi (ED 100 1-2 |ig/ml) [74].

136R = CHO (ceratinamide A) 137 R = H (psammaplysin A)

Oroidine (138) and its dimer mauritiamine (139) from the sponge Agelas mauritianci inhibit larval metamorphosis at ED50 values of 19 and 15 f^ig/ml, whereas 4,5-dibromopyrrole-2-carbamide promotes larval metamorphosis of the ascidian Ciona savignyi at a concentration of 2.5 |.ig/ml [109].

785

138 (oroidine)

I39 (mauritiamine)

The related pseudoceratidine (140) inhibits Balanus amphitrite with anEC5oofl5^g/ml[110].

o 140 (pseudoceratidine)

CHEMICAL DEFENSE Many halogenated metabolites are thought to be involved in chemical defense roles to keep predators away from a particular organism. Some of these compounds may also be of pharmacological interest. Fungal secondary metabolites might play an important role in the interactions between fungi and nematodes living in the same enviromnent. Nematicidal (against Caenorhabditis elegans) compounds produced by the ascomycete Lachnum papyraceum include mycorrhizin A (123), chloromycorrhizin A, lachnumon (124), and laclinumol. Mycorrhizin A shows the highest nematicidal, antimicrobial and cytotoxic activities [111]. The marine cyanobacterium Lyngbia majuscula is toxic to the mollusc Biomphalaria glabratcL This activity (LCioo 10 ^g/ml) is due to barbamide (141), which contains a trichloromethyl group and the methyl enol ether of a p-keto amide [112]. One of the antifeedants isolated from the marine moUusk Aplysia brasiliana, which is distasteful to fish and rejected by sharks, is the bromoallene panacene (142) [113].

786

141 (barbamide)

142 (panacene)

Volutamides A-E (143-147), halogenated alkaloids of amino acid origin, have been isolated from the Atlantic bryozoan Amathia convoluta. Several of the volutamides deter feeding by potential predators and are toxic toward larvae of the co-occurring hydroid Eudendrium carneum, suggesting that these metabolites form the basis of an effective chemical defense. [114].

I

"

Y

143 (volutamide A)

xc

H3CO.

144 R = H (volutamide B) 145R = CH3 (volutamide C)

OCH3

147 (volutamide E)

OCH,

787

A dibromotyrosin-derived metabolite present at 1.0% sponge dry weight, aerothionin (148), from Aplysina fistularis completely inhibits fish feeding when assayed in a 1.0% food pellet [115]. A series of bipyrrole compounds, the tambjamines, e.g. 149-150, act as chemical defenses for several nudibranch species. They deter feeding of the spotted kelpfish, Gibbonsia elegans, at concentrations from 1-10 |ig/mg food pellet [116]. OCH.

NHR, 149 R] = Br R2 = R3 = H (tambjamine B) 150 Ri = H R2 = Br R3 = i-Bu (tambjamine OCH3 Brv.

^^

0CH3 ^Br

Br^

J^

^Br

148 (aerothionin)

Elatol (133) and isolaurenterol (151), algal metabolites, deter feeding of herbivorous fishes [117]. The Caribbean sacoglossan Costasiella ocellifera feeds on the green alga Avrainvillea longicaulis and sequesters the metabolite avrainvilleol (152) in its tissues. It deters feeding of the wrasse fish Thalassoma bifasciatum [118]. The bubble snail Haminoea cymbalum from Guam exudes kumepaloxane (153) when the animal is disturbed, which deters feeding of reef fishes [119].

.•••'

""Ill

OH 151 (isolaurenterol)

O ^

K-^^^^^^^^*

OH 152 (avrainvilleol)

153 (kumepaloxane)

788

OAc AcO,,.

154 Ri = OAc R2 = Ac (parguerol triacetate) 155 Ri = R2 = H (deoxyparguerol acetate)

Parguerol triacetate (154) and deoxyparguerol acetate (155), diterpenoids from the red alga Laurencia saitoi, show potent feedingdeterrent activity against the herbivorous abalone Haliotis discus hannai and the sea urchin Strongylocentrotus nudus [120]. HALOGEN ESSENTIAL FOR ACTIVITY Chlorinated orcinol derivatives have been isolated from diseased bulbs of the edible lily Lilium maximowiczii. The antifungal activity of these compounds was tested against Bipolaris leersiae. Inhibition of the conidial germination depends on the number of chlorine atoms in the compounds. The trichlorinated orcinol 156 shows the highest activity. Dichlorinated orcinols, e.g. 157, are next, and the monochlorinated ones, e.g. 158, are still less active. The nonchlorinated orcinol shows lowest activity [121].

OCH,

156

OCH,

157

158

Chlorinated malyngamides K (159) and L (160) from the tropical cyanobacterium Lyngbya majusctila are more toxic to brine shrimp and fish than the halogen-free malyngamide J (LC50 brine shrimp 6, 8, and 18 |Lig/ml, respectively; LC50 gold fish 7, 15, and 40 |ag/ml, respectively) [122]. Malyngamide N (161) shows cytotoxicity to mouse neuroblastoma (NB) cells with an IC50 value of 4.9 ^g/ml [123].

789

OCH3

O

159 (malyngamide K)

OCH3

O

OH

160 (malyngamide L)

161 (malyngamide N)

Twenty-five cryptophycins, e.g. cryptophycin 1 (162), have been isolated from a Nostoc sp. strain They exhibit various degrees of cytotoxicity against three tumor cell lines, namely KB (human nasopharyngeal), LoVo (human colorectal adenocarcenoma), and SK0V3 (human ovarian carcinoma) Removal of the chlorine atom leads to a 10-fold reduction in cytotoxicity [124]. The antitumor activity of a number of cryptophycins from Nostoc sp. has been evaluated [125].

OCH,

162 (cryptophycin 1)

Rubrolides A, B, and C (163-165) from the Northeastern Pacific tunicate Ritterella rubra are active against Staphylococcus aureus (MIC 9, 2 and 11 |Lig/disk) and Bacillus subtilis (MIC 9, 2 and 11 |ig/disk). The most active compound, rubrolide B, contains an additional chlorine atom [126].

790

Cytotoxic and antifungal pyrroloquinoline alkaloids have been isolated from the Caribbean deep water sponge Batzella sp, Isobatzelline A (166) is considerably more active than the halogen-free isobatzelline B (P-388 leukemia: IC50 0.42 and 2.6 jxg/ml, Candida albicans: IC50 3.1 and 25 |ig/ml, respectively) [127]. SCH3

H,N 163 Ri = Br R2 = H (rubrolid A) 164 Ri = Br R2 = CI (rubrolid B) 165 Ri = H R2 = H (rubrolid C)

166 (isobatzelline A)

The cytotoxicity of makaluvamine F (167), a pyrroloiminoquinone from the Fijian sponge Zyzzya cf. marsailis, against the human colon tumor cell line HCT-116 (IC50 0.17 |LIM) and Chinese hamster ovary cell line xrs-6 (IC50 0.08 |iM) is one to two orders of magnitude higher than the activity of the halogen-free makaluvamines A-E [128]. The chlorinated cylindrol Bi (168) inhibits bovine famesyl-proteine transferase two-fold more potently (IC50 2.8 jiM) than the corresponding halogen-free analogue [129].

168 (cylindrol Bi)

Dolabellin (169), a bisthiazole metabolite from the Japanese sea hare Dolahella auricularia containing 7,7-dichloro-octanoic acid, shows cytotoxicity against HeLa S3 cells (IC50 6.1 ^ig/ml). The dechloro analogue has only one third of the toxicity [130]. The chlorocyclohexene derivative from the marine microorganism Periconia byssoides, pericosine A (170), is 33 times more cytotoxic in the P-388 lymphocytic leukemia test (IC50 0.12 ng/ml) than the corresponding methoxy compound [131].

791

OH

HO, H,CO

OCH,

HO'

a

o

170 (pericosiii A)

169 (dolabellin)

Hymenialdisine (171) from the Indonesian sponge Stylissa carteri is cytotoxic to human monocytic leukemia cells (MONO-MAC 6) with an IC50 value of 0.2 |ig/ml, whereas debromohymenialdisine is an order of magnitude less active (IC50 2.4 jig/ml) [132]. Structure-activity relationship studies of vancomycin-type glycopeptide antibiotics (e.g. vancomycin 172) have shown that removal of chlorine reduces the activity by ten-fold [133].

171 (hymenialdisine)

172 (vancomycin)

MISCELLANEOUS An alkaloid, epibatidin (173), has been isolated from skin of the Ecuadorian frog Epipedobates tricolor which exhibited a 200-fold potency compared with morphine as an analgesic in the Straub-tail test. Extracts from 750 frogs provided only 1 mg of the Straub-tail alkaloid. The researchers had to wait for a decade until more sensitive instruments

792

had been developed to allow the spectroscopical elucidation of the structure. Epibatidin contains a chloropyridine group, and synthetic analogues with promising activities have been developed. The history of this discovery has been reviewed recently by the discoverer himself [134].

173 (epibatidin)

Ambigol A (174), a polychlorinated biphenyl from a strain of the terrestrial cyanophyte Fischerella ambigua, exhibits a strong inhibition of cyclooxygenase and HIV-1 reverse transcriptase as well as potent antibacterial activity against Bacillus subtilis and strong moUuscicidal activity toward Biomphalaria glabrata, Ambigol B (175), a polychlorinated phenyl ether, shows only moderate activity [135].

174 (ambigol A)

175 (ambigol B)

6-Bromo-5-hydroxyindole (176) has been isolated from the midintestinal gland of the muricid gastropod Drupella fragum from Japan, a predator on corals. It exhibits antioxidative activity, higher than that of a-tocopherol and almost equal to that of BHT [136]. Agelastatin A (177) from the Indian Ocean sponge Cymbastela sp. with a unique tetracyclic pyrrole skeleton exhibits potent activity against brine shrimp (LC50 1.7 ppm) in addition to insecticidal activity against larvae of beet army worm, Spodoptera exigua, and tobacco budworm, Diabrotica undecimpunctata [137]. Agelastatin A has been isolated earlier from the New Caledonian axinellid sponge Agelas dendromorpha and shows cytotoxicity against KB cells (EC50 between 0.1 and 0.5 |ig/ml) and inhibits LPS-induced proliferation of murine spleen cells [138].

793

176

177 (agelastatin A)

Aurantoside C from the Philippinian sponge Homophymia conferta differs from aurantoside A (21) and B (22) in polyene chain length and in the trisaccharide structure and is mildly toxic to brine shrimp (LCso 50 ^g/ml)[139]. The Tasmanian ascidian Clavelina cylindrica has yielded two interconvertible tricyclic alkaloids, cylindricine A (178) and B (179), which are toxic to brine shrimp [140].

178 (cylindricine A)

r ^

179 (cylindricine B)

HO'' \

HC

180 (puwainaphycin C)

794

The blue-green alga Anabaena sp. produces a chlorine-containing cyclic decapeptide, puwainaphycin C (180), which elicits a strong inotropic effect in isolated mouse atria (ED50 0.2 ppm) without a concommitant chronotropic response [141]. Rumbrin (181) from the fungus Anxarthron umbrinum exhibits cytoprotective activity against cell death caused by calcium overload in 3T3-Swiss albino cells. It also shows inhibitory activity against lipid peroxidation in rat brain homogenate and low intraperitoneal toxicity in mice [142].

181 (rumbrin)

Dioxapyrrolomycin (182), a member of the well-known group of pyrrolomycin antibiotics, exhibits also appreciable anthelminthic activity against Haemonchus contortus in jirds and lambs [143]. Nostocyclophane D (183) was obtained as the major toxin from the cyanophj^e Nostoc linckia [144].

H^ca,

H^C 182 (dioxapyrrolomycin)

183 (nostocyclophane D)

The red tide alga Prymnesium parvum poses a serious threat to fish farming. Prymnesin-2 (184) (C98H138CI3NO36), the major toxin of the phytoflagellate, shows potent hemolytic and ichthyotoxic properties. The minimum concentration to cause hemolysis of a 1% mouse blood cell suspension and to kill fresh water fish, Tanichthys albonubes, is 3 nM [145].

795

184 (prymnesin-2)

2,6-Dichlorophenol (185) is the sex pheromone of the Lone Star tick Amblyomma americanum, whereas the chlorinated steroid glucoside blatellastanosid A (186) is used by the German cockroach Blatella germanica as an aggregation pheromone [146]. The first chlorinated steroids of marine origin, e.g. kiheisterone C (187), have been found in the sponge Strongylacidon sp [147].

°iir° 185

187 (kiheisterone C)

796

BIOCHEMICAL ASPECTS In recent years a number of bioactive natural products has been discovered by employing mechanism-based screening approaches involving biochemical assays. Some examples are presented in the following. The enediyne antitumor antibiotic calicheamicin y (188) from Micromonospora echinospora cleaves DNA selectively. The unusual NO bond organizes the two halves of the molecule into a shape that complements the shape of the minor groove [148]. The mechanism of the DNA scission has been proposed to involve reductive cleavage of the allylic methyl trisulfide, cyclization of the resulting thiol to form dihydrothiophene, cyclization at the diyne moiety to generate a benzene 1,4-diyl that, when bound in the minor groove of DNA, abstracts a hydrogen from the deoxyribose backbone to give C-centered radicals which initiate a cascade of reactions leading to the observed cleavages. Noncovalent interaction of the aryl iodide with guanosine is suspected to confer a thermodynamic advantage for the selective binding [149]. IC50 values of natural calicheamicin y against a series of tumor cell lines are typically 10'^ M, whereas a modified calicheamicin 0, with a thioacetyl group instead of the methyl trisulfide, exhibits IC50 values of 10' M [150]. \

s-^^\ HO

HO

^ 0

rrJ/=^^--ci£r \

188 (calicheamicin)

Another DNA-damaging antitumor agent is kedarcidin from an actinomycete which consists of an apoprotein and an enediyne chromophore (189) with a chloropyridine moiety [151,152].

797

189 (kedarcidin chroinophor)

Isochromophilones VII (190) and VIII (191) from a PeniciUium sp. inhibit diacylglycerol acytransferase activity with IC50 values of 20.0 and 127 |iM and acyl-CoA:cholesteroi acyltransferase activity with IC50 values of 24.5 and 47.0, respectively. They also show moderate antimicrobial activity [153]. CI

a Q .0

O II OH

Y 0 o

190 (isochromophiloii Vll)

HO

V I

o

191 (isochromophiloii Vlff)

The antimicrobial napyradiomycins A (192) and Bj (193) produced by a Streptomyces strain have been shown to be estrogen-receptor antagonists [154]. Tauroacidins A (194) and B (195) from an Okinawan sponge Hymeniacidon sp. exhibit inhibitory activity against EGF receptor kinase and c-erbB'l kinase (IC50 20 [ig/ml each) [155].

798 O

OH O

OH

193 (napyradiomycin B])

192 (napyradiomycin A)

NH

H

O

194 R = Br (tauroacidin A) 195R = H(tauroacidinB)

HN..^

"SO3H

196 (konbuacidin)

Another bromopyrrol from Hymeniacidon, konbuacidin A (196), inhibits cyclin dependent kinase 4 (cdk4) with an IC50 value of 20 |ig/ml but is not cytotoxic to L1210 and KB ceils [156].

197 R = Br (spongiacidin A) 198 R = H (spongiacidin B)

H,N 199 (dragmacidin E)

799

Spongiacidins A (197) and B (198), azepine-type bromopyrrole alkaloids from Hymeniacidon which are geometrical isomers of hymenialdisine (171), inhibit c-erbB-l kinase (IC50 9.0 and 8.5 |ig/ml) and cyclin dependent kinase 4 (IC50 32 and 12 |ig/ml) [157]. Dragmacidins D and E (199) from the Australian sponge Spongosorites sp. inhibit serine-threonine phosphatases [158]. The pentapeptides microginin 299-A (200) and B (201) from the cyanobacterium Microcystis aeruginosa inhibit leucine aminopeptidase with IC50 of 4.6 and 6.5 |ig/ml, respectively [159]. The glycopeptides aeruginosin 205 A (202) and B from Oscillatoria agardhii both inhibit trypsin with an IC50 of 0.07 |ig/ml and also inhibit thrombin with IC50 values of 1.5 and 0.17 |ig/ml, respectively [160].

200 R = CI (microginin 299-A) 201 R = CI2 (microginin 299-B)

The quinolizidine alkaloid halichlorine (203), isolated from the marine sponge Halichondria okadai, inhibits the induction of vascular cell adhesion molecule-1 (VCAM-1) at IC50 7 |ig/ml, which may be useful for treating atherosclerosis, coronary artery diseases, angina and noncardiovascular inflammatory diseases [161].

HO. OH 202 (aeniginosin 205 A)

a

OH 203 (halichlorine)

800

Destruxin-A4 chlorohydrin (204), a depsipeptide from a Mycelium sterilium strain, induces erythropoietin gene expression 5-fold at 0.2 |LIM [162].

204 (destruxin-A4 chlorohydrin)

Compound MC-033 (205), structurally similar to chlorothricin, has been isolated from a cultured broth of Streptomyces sp., and inhibits the biosynthesis of cholesterol from mevalonate with an ICso value of 1.05 x 10" M [163]. CXX)H

205 (MC-033)

NEW METABOLITES Some very interesting natural halogen compounds have been discovered only recently. They are included here due to their unusual structural

801

features, although data on biological activity are not yet available but possibly will come up in the near future. Eighteen polychlorinated diketopiperazines, the dysamides, have been isolated from the Micronesian sponge Dysidia chlorea. The presence of dichloromethyl and trichloromethyl groups is a rare structural feature in natural products. The structure of dysamide K (206) is shown as an example [164].

CO,

206 (dysamide K)

The sponge Dysidea herbacea from the Great Barrier Reef contains the novel metabolite herbacic acid (207) as the major trichloroleucine metabolite [165]. C€I, ^3^...

207 (herbacic acid)

Pantoneurins A (208) and B (209) have been isolated from the Antarctic red alga Pcmtoneura plocamioides. They contain a very rare dibromomethyl group [166]. Pantopyranoids A, B, and C (210-212), and pantoisofuranoids A, B, and C (213-215) have also been isolated from this species [167]. Ci

Br

208 (pantoneurin A)

210 R = Br (pantopyranoid A) 212 R = CI (pantopyranoid C)

211 (pantopyranoid B)

802

OH

HO-^

OH

^ o X ^ ^ B r

213 (pantoisofuranoid A)

HoAj

OH

^o^^^C^Br

H O ^

214 (pantoisofuranoid B)

^Q^^^^^BT

215 (pantoisofuranoid C)

An examination of the digestive gland extracts from the South African sea hare Aplyski dactylomela has yielded new halogenated sesquiterpenes, e.g. algoane (216) and ibhayinol (217) [168]. AcO

216 (algoane)

217 (ibhayinol)

Three new chlorinated welwitindolinone alkaloids, e.g. 218, have been isolated from the terrestrial cyanophytes Fischerella muscicola and Fischerella major [ 169]. Didemnimides B (219) and D (220) have been isolated from the Caribbean mangrove ascidian Didemnum conchyliatum [170]. a

H N

N-^

218

219 R = H (didemnimide B) 220R = CH3 (dideninimide D)

Brominated fatty acids are rare in nature. They have been found in sponges and other marine animals. Recently, the presence of (5£,17£)18-bromo-octadeca-5,17-diene-15-ynoic acid (221) and 18-bromooctadeca-5,7,17-triynoic acid (222) has been described in a halophilic (present in hypersaline environments) terrestrial organism, the Central Asian lichQn Acorospora gobiemis [171].

803

222

Two brominated C15 nonterpenoid compounds, japonenyne A (223) and B (224), with a novel 2,7-dioxabicyclo[4.3.0]nonane skeleton, have been isolated trom the red alga Laurencia japonensis [172].

^..•' 223 (japonenyne A )

224 (japonenyne B)

Recently, seven new compounds with a unique carbon skeleton have been isolated from the poecilosclerid sponge Hamigera tarangaemis from New Zealand, e.g. hamigeran B (225) which shows interesting antiviral activity [173].

225 (hamigeran B)

CONCLUSION Natural products represent an unparalleled source of molecular diversity for iimovative drug discovery. In the pharmaceutical industry, there is an urgent need to identify novel lead structures for effective drug development in many therapeutic areas. It is likely that natural halogen compounds will continue to contribute leads to future research. ACKNOWLEDGEMENT The author wishes to express his sincere gratitude to Professor H.Schottenberger, University of Innsbruck, Austria, for his generous support of literature research.

804

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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MARINE SULFUR-CONTAINING NATURAL PRODUCTS CARLOS JIMENEZ Departamento de Quimica Fundamental Universidade de A Coruna, 15071A Coruna, Spain, ABSTRACT: This chapter provides a comprehensive overview of the sulfur-containing natural products that are non-sulfated and have been isolated from marine organisms. The overview covers the published literature from 1985 to 1999. A total of 482 compounds and 371 references are recorded. These secondary metabolites are organized in sections according to structural classifications by sulfur functional groups and by structural families of compounds. Comments on structural characterization, biogenesis, and biological activity have also been included.

INTRODUCTION One of the most promising marine natural products to be commercialized as an anticancer drug, ecteinascidin 743 (1), and the most active antineoplastic compound known to date, dolastatin 10 (2), are sulfurcontaining marine metabolites [1]. These compounds represent the most outstanding examples of the biological activity present in this type of compounds. The wide distribution of sulfur-containing compounds in marine organisms can be explained by the fact that sulfur is the fourth most common element in sea water, after chlorine, sodium, and magnesium [2]. Many comprehensive reviews dealing with marine natural products have been published to date and these focus on structures [3], biosynthesis [4, 5], and biological activity [6]. However, reviews that deal specifically with naturally occurring sulfur compounds from marine organisms are very rare. The first survey to review this type of compound (up to mid1985) was written by C. Christophersen and U. Antoni and was entitled "Organic sulfur compounds from marine organisms"[7]. The survey deals with about 90 naturally occurring marine sulfur compounds and also includes a survey of selected sulfiir-containing primary metabolites. C. Christophersen reported a much smaller selection of biologically active sulfiir compounds from marine organisms in 1989 [8]. More recently, a

812

very extensive review dealing with more than 500 sulfated compounds arranged according to the phyla was reported [2],

\

\

^

"

:

.1.

I

A

:J

OM^O

\ / ^

^ 2, dolastatin 10

l,ecteinascidin743

In the present chapter a survey of non-sulfated sulfur-containing marine natural products is outlined. This survey covers the published literature from 1985 to mid-1999. In reviewing an area as nonhomogeneous as this, it is a difficult task to find the optimum method of classification. Due to the interest of many readers in structural discussions, these secondary metabolites are organized in sections according to structural classifications by sulfur functional groups. In order to lend more coherence to this classification, the compounds are also organized by structural families in which the predominant sulfur functional group determines the section in which it is located. The methods used in the characterization of these compounds are described in most cases, including the confirmation of their structures by total synthesis where applicable. In some cases, diagnostic spectroscopic signals are mentioned when they clearly show the presence of a particular structural family of compounds. Comments on biogenesis and the real origin of these metabolites are also included. Special attention is paid to the description of their biological activity, including a number of discussions, when available, regarding the relationship between structure and pharmacological activity. More than 460 compounds are included. It is worth noting that while sulfated marine metabolites are mostly found in the phylum Echinodermata (350 out of 500), the non-sulfated systems are more widely distributed among all the phyla.

813

THIOL METABOLITES AND THEIR RELATED DERIVATIVES The thiol functionality is only rarely encountered in marine natural products and most of these examples correspond to amino acid derivatives. The histidine-derived compounds comprise the widely distributed 2-thiolhistidines (e.g. ergothioneine 3) and the less abundant 5thiolhistidine derivatives, which were extensively described in a previous review on organic sulfur compounds [7]. The thiol-containing amino acids, ovothiols A-C (4-6), represent new examples of 5-thiolhistidine derivatives. Ovothiol C (6) was found to be present at high concentrations in the eggs of the sea urchin Strogylocentrotus purpuratus, while ovothiol A (4) and B (5) were obtained from the ovarian tissue from the starfish Evasaterias troschelii and from the scallop Chlamys hastata, respectively [9]. Their structures were determined by ^H NMR and that of 4 was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. Ovothiol A (4) and C (6) confer NAD (P)-02 oxidoreductase activity on ovoperoxidase. '^^2H =(

NH2

SH SH 3, ergothioneine

C02V y

+N- ^1 j^g

4, ovothiol A Ri = R2 = H 5, ovothiol B Rj = H, R2 = Me 6, ovothiol C Ri = R2 = Me

The original structures assigned to the l-methyl-5-thiolhistidine and its corresponding symmetrical disulfide, both detected in the unfertilized eggs of certain echinoderms [10], were revised after their subsequent unambiguous synthesis to be 4 and 7 [11]. Caledonin (8) is a modified peptide isolated from the tunicate Didemnum rodriguesi and bears an (5)3-amino-5-mercaptopentanoic acid as a new sulfur-containing p-amino acid. The structure of caledonin was proposed by analysis of spectroscopic data and chemical degradations. The absolute stereochemistry of the primary amino group was determined as S by derivatization with {R)- and (iS)-methoxyphenylacetic acid and NMR analysis. Compound 8 behaves as a natural bolaphile, binding strongly to Zn(II) and Cu(II) ions [12].

814

q

Me N

NO

NH2

NH2

SH

S~)2

7, l-methyl-5-thioI-L-histidine disulfide

8, caledonin

The thiol group can also be found in heterocyclic compounds such as 2mercaptobenzothiazole (9) and echinoclathrine C (10). Compound 9 was isolated from the symbiont bacterium Micrococcus sp., which was obtained from the sponge Tedania ignis [13]. The pyridine alkaloid echinoclathrine C (10) and its S-acetylated derivative, echinoclathrine B (11), were isolated from the Okinawan sponge Echinoclathria sp. [14]. The position of the hydroxyl and acylamino group on the phenyl ring in echinoclathrines (11 and 10) was recently corrected [15]. Compound 11 showed weak immunosuppressive activity in the mixed lymphocyte reaction assay with an IC50 of 9.7 ^ig/ml [14].

^YVsH

N^MV^S-R

10, echinoclathrine C R = H

12, T-cadinthiol

11, echinoclathrine B R = Ac

Marine sesquiterpene metabolites bearing a thiol group are represented by T-cadinthiol (12) and thiofurodysinin (13). T-cadinthiol (12) was isolated from the tropical sponge Cymbastela hooperi and exhibits a weak antimalarial activity toward cultured Plasmodium falciparum (IC50 = 3.6 fig/ml) [16]. Thiofurodysinin (13) belongs to a series of sulfur-containing furanosesquiterpenes related to dysinin, which were obtained from sponges belonging to Dysidea genus. Thiofurodysinin acetate [(+)-14] and its regioisomer, thiofurodysin acetate (19), isolated from an unidentified Australian species of that sponge, were the first thiol acetates obtained from natural sources [17]. Thiofurodysinin (13) was later isolated as its

815

free thiol from Dysidea avara [18]. Subsequently, (methylthio)furodysinin (15) and dithiofurodysinin disulfide (16) were obtained from the nudibranch Ceratosoma brevicaudatum [19] and this led to suggestions that this mollusc feeds on Dysidea sponges. A Palau sponge Dysidea sp. yielded furodysinin lactone thioacetate (17). The structure of 17 was determined on the basis of spectral data and on its semi-synthesis by photo-oxidation of 14 [20]. Finally, the antipode form of thiofurodysinin acetate, (--)-14, along with methoxythiofurodysinin acetate lactone (18) were obtained from a Fijian sponge Dysidea herbacea [21]. In relation to their biological activities, furodysinin lactone thioacetate (17) caused intracellular Ca^^ mobilization that was blocked by leukotriene B4 (LTB4) receptor antagonists [20]. Compound 17 binds LTB4 receptors with high affinity and activates the receptor-mediated signal transduction processes related to LTB4 [22]. On the other hand, (~)-thiofurodysinin acetate, (-)14, shows anthelminthic in vitro activity against Nippostrongylus brasiliensis but was inactive in in vivo screening against a mixed helminth infection oiNippostrongylus dubias and Hypselodoris nana [21]. R~S

13, thiofurodysinin R = H 14, thiofurodysinin acetate R = Ac

17, furodysinin lactone thioacetate R = H

15, (methyhhio)furodysinin R = Me

18, methoxythiofurodysinin acetate lactone R = Me

16, dithiofurodysinin disulfide

19, thiofurodysin acetate

816

SULFIDES Most of the marine metabolites bearing a sulfide group correspond to well-defined families of alkaloid compounds and they have mainly been obtained from tunicates and sponges. To a lesser extent they have also been found in bryozoans, molluscs, and algae. Sulfur-containing P-Carboline Marine Alkaloids The sulfur-containing P-carboline marine alkaloids isolated from tunicates belong to three main chemical families: the eudistomins, the eudistomidins, and the didemnolines. Eudistomins The sulfur-containing eudistomins consist of a family of tetrahydro-pcarbolines that contain an oxathiazepine ring. Eudistomins C, E, F, K, and L (20-24) were first isolated and characterized by Rinehart et al from the Caribbean tunicate Eudistoma olivaceum, which was the most active antiviral species assayed during the Alpha Helix Caribbean Expedition in 1978 [23, 24]. Subsequent NMR spectroscopic analysis of eudistomin K (23), reisolated from the New Zealand tunicate Ritterella sigillinoides [25], allowed the stereochemistry of the N-O bond to be revised (as 2a instead of 2p). X-ray analysis of the ;?-bromobenzoyl derivative of eudistomin K confirmed this revision [26] and led to the suggestion that eudistomins C, E, F, and L probably had the same stereochemistry, a fact that was confirmed by the total synthesis of these compounds [27]. The tunicate Ritterella sigillinoides was also the source of the additional oxathiazepine derivatives debromoeudistomin K (25) [28] and eudistomin K sulfoxide (26) [29]. The structure of sulfoxide 26 was deduced by spectroscopic analysis and then confirmed by its semisynthesis from eudistomin K (23). Furthermore, the stereochemistry of the sulfur-oxygen bond in 26 was assigned as a by using the model compound studies by Buchanan and Durst and by comparing the ^^C-NMR shifts of C-10, C-11, and C-13 between the acetyl derivatives of 23 and 26 [29]. From a biogenetic point of view these compounds were considered to be biosyntheticaly derived from tryptophan and cysteine [23].

817 Eudistomins

20, eudistomin C R = R3 = H, Ri = OH, R2 = Br 21, eudistomin E R = Br, Ri = OH, R2 = R3 = H 22, eudistomin F R = H, Ri = OH, R2 = Br, R3 = C02Me 23, eudistomin K R = Ri = R3 = H, R2 = Br 24, eudistomin L R = R2 = R3 = H, Ri = Br 25, debromoeudistomin K R = Ri = R2 = R3 = H

26, eudistomin K sulfoxide

The oxathiazepine eudistomins exhibited very interesting biological activities. In in vitro assays against Herpex simplex Type I virus exhibited antiviral activity: eudistomins C (20) and E (21) showed very strong activity at 50 ng/disk > eudistomin L (24) at 100 ng/disk > eudistomin K sulfoxide (26) at 200 ng/disk > eudistomin K (23) at 250 ng/disk > debromoeudistomin K (25) 400 ng/disk [23, 28]. Similar activity levels and trends were found in in vitro assays against Polio vaccine Type I virus for eudistomins C (20) and K (23), debromoeudistomin K (25), and eudistomin K sulfoxide (26) [28]. In relation to the cytotoxic activity, eudistomin C (20) was toxic to L1210 (IC50 0.36 |ig/ml) and L5178Y (IC50 0.42 |ig/ml) cells [30] while eudistomin K (23) was patented as an effective inhibitor of the growth of L1210, P388, A549, and HCT-8 cells at varying concentrations [31]. Eudistomidins The p-carboline alkaloid eudistomidins were isolated from the Okinawan tunicate Eudistoma glaucus, A methyl sulfide group is present in eudistomidins C (27) [30] and E (28) [32] while a methyl sulfoxide occurs in eudistomidin F (29) [32]. Their structures were elucidated on the basis of spectroscopic data. The S configuration at C-10 of eudistomidin C (27) was established by synthesis of 10-(i?)-O-methyleudistomidin C [30]. The

818

same configuration was suggested for eudistomidins E and F (28-29), since they are biogenetically related to eudistomidin C (27) and they were isolated from the same tunicate. Compound 27 showed potent cytotoxicity activity against L1210 (IC50 of 0.36 |ag/ml) and L5178Y (IC50 0.42 |ag/ml), and exhibited calmodulin antagonistic activity (IC50 3 x 10"^ M) [30]. Eudistomidins

27, eudistomidin C

28, eudistomidin E R = SMe 29, eudistomidin F R = S-Me O

Didemnolines The didemnolines A-D (30-33) isolated from the tunicate Didemnum sp. represent an additional family of P-carboline metabolites [33]. They differ from the other derived p-carboline compounds in that they are substituted at the N9 position rather than at the C-1 position. All of these compounds showed moderate cytotoxic activity against KB cells, with the sulfoxidecontaining compound, didemnoline C (32), being the most active with an IC50 value of 0.28 jig/ml. Compounds 30 and 32 also exhibited antimicrobial activity. Didemnolines 30, didemnoline A Rj = Br, R2 = SMe 31, didemnoline B Rj = H, R2 = SMe 32, didemnoline C Ri = Br, R2 = S-Me O 33, didemnoline D Ri = Br, Ro = S-Me O

819

Other Sulfur-containing jS-Carboline Marine Alkaloids The New Zealand bryozoan Cribicellina cribraria was the source of several p-carboline alkaloids. One of these examples, compound 34, has a unique methylsulfone substituent. Spectroscopic studies identified 34 as 1ethyl-4-methylsulfone-P-carboline [34]. Hyrtiomanzamine (35) represents the first example of a 6-OH-P-carboline ring associated with a betaine unit to be obtained from a natural source. This compound was isolated from the Red Sea sponge Hyrtios erecta and its structure was determined on the basis of its spectral data [35]. Compound 35 displayed immunosuppressive activity with an ED50 of 2 mg/ml in the B lymphocytes reaction assay. The lack of cytotoxicity against KB cells indicated that the immunosuppressive activity of 35 is specific and not due to a general cytotoxic effect.

Me

^^

Me

35, hyrtiomanzamine

Sulfide Pyridoacridines Since the discovery of amphimedine by Schmitz and co-workers in 1983 [36], the polycyclic alkaloids based on the pyrido[A:,/]acridine skeleton have emerged as a well-defined class of marine metabolites, with significant biological activities, isolated from sponges and tunicates [37]. The less common group of sulfide pyridoacridines were only obtained from tunicates and they include the shermilamines, the varamineslissoclins-diplamine group, and tintamine, another polycyclic alkaloid closely related to them. Shermilamines The shermilamines constitute a group of alkaloids characterized by a thiazinone ring attached to the pyridoacridine ring system. Shermilamine A (36) was the first known compound of this series and was isolated by Scheuer et al. from the tunicate Trididemnum sp. [38]. The complete

820

elucidation of 36 had to be deduced through X-ray analysis since the presence of the ^^Br peak obscured the ^^S in its HREIMS. The isolation of its debromo derivative, namely shermilamine B (37), was reported at the same time from the tunicates Trididemnum sp. [39] and Eudistoma sp. [40]. The structure of 37 was elucidated in both cases by spectroscopic data by comparison to those of shermilamine A. Additional shermilamines were isolated from tunicates belonging to the Cystodytes genus. Thus, shermilamine C (38) was obtained from a Fijian Cystodytes sp. [41] while shermilamines D (39) and E (40), along with tintamine (41), an alkaloid possessing a new unprecedented tropono-l,2-dihydro-3,6-phenanthroline ring system, were isolated from the Madagascar Cystodytes violatinctus [42]. Shermilamines B (37) and C (38) (and also diplamine 46) showed dosedependent inhibition of proliferation in HCT cells in vitro and inhibited the topoisomerase (TOPO) Il-mediated decatenation of kinetoplast DNA (kDNA) in a dose-dependent manner [41]. These results suggest a possible cytotoxicity mechanism for these compounds. Furthermore, shermilamine B also displayed cytotoxicity against KB cells [43] and was reported as a potent regulator of cellular growth and differentiation, affecting cAMPmediated processes [44].

OH

'Nv^^S 36, shermilamine A Ri = Br, R2 = NHCOMe 37, shermilamine B Ri = H, R2 = NHCOMe 38, shermilamine C Ri = H, R2 = NHC0CH=CMe2 39, shermilamine D Rj = H, R2 = NMe2 40, shermilamine E Rj = H, R2 = N(0)Me2

41, tintamine

821

Varamines, Diplamine, and Lissoclins A methyl sulfide group linked to the pyridoacridine ring system is present in the varamine-lissoclin-diplamine group. Varamines A (42) and B (43) were isolated from the Fijian tunicate Lissoclinum vareau [45]. The structure elucidation of 42 was carried out on its trifluoroacetate salt derivative because this form displayed the best dispersion in the ^H-NMR spectrum in methanol-d4. Lissoclins A (44) and B (45) were isolated from the Great Barrier Reef tunicate Lissoclinum sp. [46] while diplamine (46) was obtained from the Fijian tunicate Diplosoma sp. [47]. The iminoquinones 44-46 are structurally related to the cystodytins [48], which possess the same heteroaromatic carbon skeleton but lack the thiomethyl group. Diplamine (46) can be easily obtained from the oxidative demethylation of varamine B (43) with eerie amonium nitrate [47]. The total synthesis of 46 by Heathcook et al confirmed the proposed structure [49]. The spectoscopic data of diplamine were used in the structural elucidation of its analogs, lissoclins A (44) and B (45) [46]. Varamines A (42) and B (43), and diplamine (46) were shown to be cytotoxic towards L1210 cells with IC50 of 0.03, 0.05, and 0.02 |ig/ml, respectively [45,47]. The importance of the thiomethyl group in the cytotoxicity of these compounds was suggested since they are about 1 order of magnitude more toxic than the related cystodytins. Diplamine exhibited antimicrobial activity against E, coli and S, aureus [47].

YrV •<%-. ^

OMe 'SMe

R

42, varamine A R = Me

44, lissoclin A R =

43, varamine B R = H 45, lissoclin B R = 46, diplamine R = Me

822

Sulfide Pyrroloquinolines The sulfide marine metabolites having a pyrroloquinoline skeleton can be divided into three groups: the batzellines-isobatzellines, the prianosinsdiscorhabdins, and the makaluvamines. All of these types of compounds have been isolated from sponges. Batzellines and Isobatzellines These compounds are 1,3,4,5-tetrahydropyrrolo[4,3,2-Je]quinolinecontaining marine alkaloids isolated from the Caribbean sponge Batzella sp. Most of them are characterized by the presence of a methyl sulfide group at C-2. The spectral data of batzelline A (47), with a structure confirmed by X-ray analysis [50], were used for the structural determination of batzelline B (48) [50] and those of isobatzellines A, B, and D (49-51) [51]. The proposed structures were confirmed by the total synthesis of compounds 47-50 [52, 53]. The isobatzellines A and B, but not the batzellines, were found to exhibit in vitro cytotoxicity against P388 (IC50 0.42 and 2.6 |ag/ml, respectively) and moderate antifungal activity against Candida albicans [51].

47, batzelline A R = Me

49, isobatzelline A R = CI

48, batzelline BR = H

50, isobatzellineBR = H 51, isobatzelline D R = CI, A^

PrianosinS'Discorhabdins These complex sulfide-containing pyrroloiminoquinone alkaloids consist of the prianosins A-D (52, 53, 56, 57), isolated from the Okinawan Prianos melanos [54, 55], the discorhabdins A and B (52, 54), obtained from three different species of the New Zealand Latrunculia [56], discorhabdin D (57) from Latrunculia brevis and Prianos sp. [57], and discorhabdin Q (55), from Latrunculia purpurea, and several species of

823

Zyzzya genus [58]. The structure of prianosin A (= discorhabdin A) (52), including its absolute configuration, was unequivocally defined by X-ray analysis [54], while those of discorhabdins B (54) and D (57) were based on spectral data. The previous structures proposed for prianosins C and D [55] were revised to 2-hydroxydiscorhabdin D (56) and discorhabdin D (57), respectively [59]. A plausible biosynthetic pathway for these compounds suggests the involvement of a-amino acids tyrosine (C-l-^N9) and tryptophan (C-10'-C-21) [55]. All of the prianosins were cytotoxic against LI210, L5178Y, and KB cells in vitro with respective IC50 values as follows: 52 (0.037, 0.014, and 0.073 |ig/ml), 53 (2.0, 1.8, and >5.0 fig/ml), 56 (0.15, 0.024, and 0.57 |ag/ml), and 57 (0.18, 0.048, and 0.46 )ag/ml) [55]. The discorhabdins were also highly active in in vitro P388 assays with the following receptive ED50 (in ^ig/ml) values: 52 (0.05), 54 (0.1), and 57 (6). However, in the in vivo P388 model, 52 and 54 were found to be inactive (T/C < 120%) while 57 was considered to have significant in vivo activity (T/C 132% at 20 mg/Kg) [57]. Discorhabdin Q (55) exhibited a moderate and generalized cytotoxicity in the NCI 60 cell line antitumor screen [58]. In addition, compounds 52 and 57 induced Ca^"^ release from sarcoplasmic reticulum, which is 10 times more potent than caffeine in this assay [54, 55], and compounds 52 and 54 showed antiviral and antimicrobial activities [56]. Finally, a symbiotic origin was suggested for these metabolites since the pyrroloquinoline skeleton has been found in marine natural products obtained from different sources, mainly tunicates and sponges [60].

52, prianosin A (discorhabdin A)

55^ prianosin C (2-hydroxydiscorhabdin D) R = OH

53, prianosin B (A^^)

^^^ ^^^^^^^ ^ (discorhabdin D) R = H

54, discorhabdin B (A^) 55, discorhabdin Q(A^'*^

824

Makaluvamines Makaluvamines represent another group of pyrroloiminoquinones isolated from the sponge Zyzzya of. marsailis [61]. One such compound, makaluvamine F (58), contains a benzoindole moiety. The structure deduced for 58 was recently confirmed by total synthesis [62]. Makaluvamine F (58) and discorhabdin A (52) (also isolated from the same sponge) were found to be cytotoxic against HCT-116 cells (IC50 0.17 and 0.08 |ig/ml, respectively). Both compounds showed differential toxicity toward the topoisomerase Il-sensitive (CHO) cell line xrs-6 (IC50 0.08 and 0.02 jxg/ml, respectively), and displayed topoisomerase II inhibition in vitro. This activity may be mediated by intercalation into DNA and single-stranded breakage [61].

58, makaluvamine F

Sulfide Isoquinolines Simple Isoquinolines Imbricatine (59) represents the first example of a benzyltetrahydroisoquinoline alkaloid from a marine organism and it is also characterized by the unusual thioether linkage to a histidine substituent. The isolation of 59 from the starfish Dermasterias imbricata in 1986 [63] was followed by the determination of its absolute configuration through a partial synthesis in 1991 [64] and total synthesis in 1999 [65]. D-3-(3,5-Dihydrophenyl)alanine is proposed instead of Ldopa as the precursor of the isoquinoline fragment [64]. Compound 59 was responsible for eliciting the unusual anemone "swimming" behavior and displayed significant antineoplastic activity {in vitro against L1210 with an ED50 < 1 |ig/ml and in vivo against P388 with T/C 139 at 0.5 mg/Kg) [64, 66].

825

More simple sulfiir-containing isoquinolines were isolated from two bryozoan: perfragilins A and B (60 and 61) from Membranipora perfragilis [67] and 2-methyl-6-methylisoquinoline-3,5,8-(2//)-trione (62) from Biflustra perfragilis [68]. The structures of perfragilins A and B were established by X-ray analysis [69] and confirmed by synthesis of perfragilin B [70]. Compounds 60 and 61 showed cytotoxicity against P388 with ED50 values of 0.8 and 0.07 |ig/ml, respectively, but they lack antiviral activity [67]. Compound 61 was active in the brine shrimp bioassay and was also able to inhibit the growth of cultured marine bacteria [68]. Their structural relationship to mimosamycin, isolated from the terrestrial actinomycete Streptomyces lavendulae [71], prompted to the authors to speculate a bacterial origin.

O 60, perfragilin A 59, imbricatine

61, perfragilin B R = SMe 62, R=H mimosamycin R = Me

Ecteinascidins The ecteinascidins constitute a group of very complex alkaloid tris(tetrahydroisoquinolines) with potent in vivo antitumor activity and were isolated from the colonian Caribbean tunicate Ecteinascidia turbinata. The compounds are abbreviated as Et followed by a number that represents the value of the highest mass ion observed in the (+)FABMS. Reports of the potent in vivo activity of extracts of that tunicate date back to 1969, when it was reported that such extracts gave T/C values of up to 272 vs P388, with four of six cures in one experiment, and they were also found to be powerful immunomodulators [72]. After a decade of effort, two research groups reported at the same time the structures of four [Et 743 (1), Et 729 (63), Et 745 (64), and Et 770 (66)] [72] and two [Et 743 (1), Et 729(63)] [73] ecteinascidins, respectively. The development of an isolation process that was efficient on a large scale allowed Rinehart's group to obtain additional ecteinascidins, Et 743 A^^^-oxide (67), Et 722

826

(68), and Et 736 (69), in 1992 [74], and subsequently the putative biosynthetic precursors Et's 597 (70), 583 (71), 594 (72), and 596 (73) in 1996 [75]. This class of compounds can be divided into three types: the Et-743 type (1, 63-67) with a tris(tetrahydroisoquinoline) skeleton, the Et-736 type (68 and 69) in which a tetrahydroisoquinoline is substituted by a tetrahydrocarboline unit, and finally the Et's 597, 583, 594, and 596 (7073), which are characterized by the presence of just two tetrahydroisoquinoline units. The structures of these compounds were assigned primarily by spectroscopic methods. The recrystalization of the natural Et N^^-oxidc (67) and the 21-0-methyl-//^^-formyl derivative of compound 63 gave single crystals and allowed X-ray analysis of these systems [74]. The absolute stereochemistry of 70 was determined by chiral GC of the L-Cys unit and by ROESY spectrum of its acetyl derivative [75]. The structures of Et's are related to the microbially derived safracins and saframycins antitumor agents first isolated from cultured Streptomyces species [76] as well as to the sponge metabolites renieramycin and xestomycin [77]. Et's 743 (1), 729 (63), 736 (69), and 722 (68) showed promising efficacy in vivo, including activity against P388, B16 melanona, M5076 ovarian sarcoma, Lewis lung carcicoma, and several humor xenograft models in nude mice. The most abundant analogue, but not the most active, is ecteinascidin Et 743 (1). This compound has been licensed by the Spanish company Pharmamar and is now in phase II clinical trials against small cell lung cancer, melanoma and breast cancer in several European countries and in the United States [75]. It was found that 1 acted as an antimitotic agent, not binding to tubulin, but by disorganizing the microtubule network in some fashion. In addition, it is a DNA minor groove guanine-specific alkylating agent [1]. The Et's showed potent inhibition of DNA and RNA synthesis and of RNA polymerase activity, but its inhibition of DNA polymerase activity is much less marked [75]. The potent activity of Et's was attributed, at least in part, to the unit C since the related saframycin A lacks this unit and has lower efficacy than Et 729 in comparable tumor models [74, 75]. More recent structural information on Et 743-DNA adduct was obtained by NMR spectroscopy [78]. An enantioselective total synthesis of Et 743 has been achieved by Corey et al. [79].

827 Bunit

unit

Ecteinascidin 743-type

WV»^NAA/S/VSA^VS'WW*^>»

XY = -O-CH2-O. l,Et743R, = Me,R2 = OH 63,Et729Ri = H,R2 = OH

Ecteinascidin 736-type

64,Et745Ri = Me,R2 = H

XY = .O-CH2-O-

65, Et 759B Ri = Me, R2 = OH, S-oxide

68, Et 722 Ri = H, R2 = OH

66,Et770Ri = Me,R2 = CN Me 67, Et743N*^-oxideR2 =0H, Ri = <;^

69,Et736Ri=Me,R2 = OH

W W W W ^ y / V I VS/WV>i^

oV

H NH2

70, Et597Ri = Me,R2 = OH X - O M e , Y = OH 71,Et583Ri = H,R2 = OH X = OMe,Y = OH

o 72, Et 594 Ri = Me, R2 = 0H X Y = -O-CH2-O73,Et596 Ri = Me, R2=0H X = OMe, Y = OH

828

In relation to their biosynthesis, it was proposed that A-B units of Et's are most likely formed by condensation of two Dopa-derived building blocks and that the tetrahydroisoquinoline ring in unit B is closed by condensation (Pictet-Spengler) with a serine(or glycine)-derived aldehyde, as in the case of the related saframycins. SAdenosylmethinonine is the likely source of the methyl groups. A plausible route for the formation of unit C was proposed later [75]. This was partially demonstrated by incorporation of radiolabeled tyrosine and cysteine by Kerr and Miranda [80] and by incorporation of labeled methionine, glycine and tryptophan by Morales and Rinehart [81]. Sulfide Diketopiperazines Several sulfide diketopiperazines were isolated from marine bacteria. Gliovictin (74) composed 27% of media extract of cultured broths of the marine deuteromycete Asteromyces cruciatus [82]. Compound 74 was previously found in terrestrial fungi of the genera Helminthosporium and Penicillium [83]. Maremycins A and B (75 and 76) were additional diketopiperazines obtained from the cultured broth of the marine Streptomyces sp. and the structures of these compounds were elucidated by spectral methods and chemical means [84].

r

Me

T

IN

O

SMe

75, maremycin A R= ••••••OH 76, maremycin B R= —OH

The Structure of cyc/o-(L-Pro-L-Met) (77), obtained from a bacteria strain of Pseudomonas aeriginosa associated with the bioactive Antarctic sponge Isodictya setifera, was determined by spectroscopic methods and confirmed by synthesis [85]. Although the sulfur-containing diketopiperazine cyc/o-(L-Pro-L-thioPro) (78) was isolated from the Bermudian sponge Tedania ignis, a bacterium origin of this metabolite was suggested. The structure of 78, including the 5,5 absolute stereochemistry, was elucidated through spectral analyses and confirmed by synthesis from commercially available L-amino acids [86].

829

NH SMe

o 77, cvc/o-(L-Pro-L-Met)

78, cvc/o-(L-Pro-L-thioPro)

Miscellaneous Sulflde-containing Marine Metabolites Several structurally diverse marine alkaloids bearing a sulfide flinctionality were isolated from sponges and nudibranch. The sulfurcontaining phloeodictines (79-81), isolated from the New Caledonian sponge Phloeodycton sp., belong to a group of alkaloids characterized by the presence of a unique 6-hydroxy-1,2,3,4-tetrahydropyrrolo[ 1,2ajpyrimidinium skeleton. The structures of these systems were determined by extensive spectroscopic analysis and they exhibited in vitro antibacterial activity against Gram-positive and Gram-negative bacteria as well as being moderately cytotoxic against KB tumor cells [87, 88].

79, phloeodictine CI n = 2 80, phloeodictine C2 n = 1 81, phloeodictine B n = 1, A

The Structure of corallistine (82), a new polynitrogen compound from the New Caledonian sponge Corallistes fluvodesmus, was determined by X-ray single crystal analysis of its 6-isobutyloxycarbonyl derivative [89]. Compound 83 {9-[5*-deoxy-5'-(methylthio)-p-D-xylofuranosyl])adenine} was reported as the first naturally occurring purine carrying a xylosederived substituent and is an analog of methylthioadenosine (MTA), the ubiquitous biological natural methyl donor. This compound, along with its epimer at C-3' (84), were isolated from the nudibranch Doris verrucosa and their structures were deduced by spectroscopic methods [90]. Radiolabeled experiments identified adenine and methionine as the

830

biological precursors of nucleoside 83 and demonstrated that is produced by oxidation-reduction of its epimer [91, 92]. p

NH2 N

N N"^N

MeS

NH2

MeS

83, Ri =0H,R2 = H 84,R, =H, R2 = 0H

82, corallistine R2 OH

A methyl sulfide group is present in the cyclic heptapeptides phakellistatin 5 (85), obtained from the Micronesian sponge Phakellia costada [93], and hymenamide F (86), which was isolated along with its S-oxide form [Mso] (87) from the Okinawan sponge Hymeniacidon sp. [94]. Their structures were determined by spectral and chemical methods. The structure elucidation of hymenamide F (86) was carried out mainly on the basis of the spectroscopic data of [Mso] hymenamide F (87), which appeared to have been generated from hymenamide F through autoxidation of the methyl sulfide residue during purification. Compound 85 exhibited an unusual pattern of selective growth inhibition against the US NCI human cancer cell line panel [93]. NH2

MeS

HN^NH O^s^N

85, phakellistatin

86, hymenamide F R = SMe 87, [Mso] hymenamide F R= S—Me O

831

The macrocyclic lactones thiomycalolides A and B (88 and 89), which are characterized by a glutathione adduct, were isolated from a Japanese sponge Mycale sp. [95]. Their structures were determined by interpretation of spectral data and chemical transformations. Compounds 88 and 89 exhibited cytotoxic activity against P388 cells with an IC50 value of 18 ng/ml each. It is not known whether thiomycalolides are produced by an enzymatic reaction in the sponge or are simple adducts of co-occurring mycalolides and glutathione. H O'^ N AcO

R

Meb

6 ^ / 0 OMe

V.

,

^" O

N-\

OH

^

Jl y\ ^^y^

, O

88, thiomycalolide A R = O 89, thiomycalolide B

N/ OMe

H OMe

Umbraculumin C (90), characterized by the presence of a trans'3(methylthio)-acrylic acyl residue, is a diacylglycerol obtained from the skin of the opisthobranch mollusc Umbraculum mediterraneum [96]. Taylor and co-workers determined the absolute configuration of 90 by total synthesis [97] while Sodano et al. reached the same conclusion by derivatisation of the natural compound [98]. Compound 90 displayed ichthyotoxic activity against the mosquito fish at 0.1 |ag/ml and should represent the deterrent of this organism against predators [96].

OH O 90, umbraculumin C

91,R = CH20H 92, R = COjMe

The fouling bryozoan Dakaira subovoidea has been shown to contain two thiophene compounds, 1 -hydroxymethyl-6-oxo-6//-anthra[ 1,96c]thiophene (91) and its 1-methoxycarbonyl derivative 92 [99]. Their structures were determined by spectral and crystallographic analyses. The

832

acetylated derivative of 91 displayed antioxidant activity, showing 99.5% inhibition of lipid peroxide formation in rat liver microsomes at 10 |ig/ml. An unprecedented sulfur-containing yellow pigment, namely benzylthiocrellidone (93), was isolated from the Australian sponge Crella spinulata [100]. This compound represents the first recorded example of a natural product containing a dimedone unit. The structure of this compound, determined by spectroscopic methods in conjunction with an X-ray crystal study, was confirmed by total synthesis [101]. The first example of a naturally occurring 5-thiosugar, 5-thio-D-mannose (94), was reported from the Australian sponge Clathria pyramida [102]. This compound had to be isolated as its peracetylated derivative, after treatment of the crude material with Ac20/pyridine, and then be deacetylated with methanolic ammonia.

HO^ OH OH 00

94, 5-thio-D-mannose

93, benzylthiocrellidone

Additional pharmacological studies on the cytotoxic compound acanthifolicin (95), the 9,10-episulfide derivative of okadaic acid obtained from sponge Pandaros acanthifolium [103], showed it to be an inhibitor of phosphatases 1 and 2a (PPl-1 and PP-2a) with similar potencies to okadaic acid [104].

" 95, acanthifolicin 9,10^^^^^''" okadaic acid 9, 10

C=C

" 6 H Me

833

DISULFIDES AND POLYSULFIDES Tunicates are by far the most important source of marine metabolites containing disulfides and polysulfides, followed in importance by marine microorganisms and, to a lesser extent, sponges and algae. Dopamine-derived Polysulfides A series of sulfur-containing dopamine-derived metabolites has been isolated from tunicates, predominantly belonging to the genus Lissoclinum. Varacin (96), the first reported naturally occurring benzopentathiepin, was isolated from Lissoclinum vareau [105]. The evidence for a benzopentathiepin system was provided by tandem MS in the (-) FAB mode on the A^-trifluoroacetyl derivative. The structure of 96 has been confirmed by several syntheses [106]. A closely related compound, lissoclinotoxin A (97), which was obtained from the tunicate Lissoclinum perforatum, was originally reported to have a cyclic benzotrithiane structure [107] but it was subsequently shown to be a benzopentathiepin [108]. The synthesis of lissoclinotoxin A and its regioisomer 98 (referred to as isolissoclinotoxin A) led to a second revision of its structure [109]. Varacin (96) and lissoclinotoxin A (97) were found to be chiral, displaying unusual stereoisomerism due to restricted inversion about the benzopentathiepin ring. However, their optical activity may be lost during isolation [110, 46]. Two tunicates belonging to Lissoclinum genus have been shown to contain additional members of this series. The benzopentathiepin lissoclinotoxin B (105) was isolated as a minor component of L. perforatum [108] while the dithiomethyl lissoclinotoxin C (106) was obtained, along with the dimeric lissoclinotoxin D (107), from Lissoclinum sp. [46]. The energetically more favorable dimeric "head-totail" situation for 107 was proposed for lissoclinotoxin D but the alternative "head-to-head" cannot be excluded. Faulkner, Carte, and coworkers described the isolation of five additional varacin-lissoclinotoxin derivatives and used extensive spectral analysis in their structural determination [111]. These compounds include A^,A^-dimethyl-5(methylthio) varacin (99) and 3,4-dimethoxy-6-(2'-iV,A^-dimethylaminoethyl)-5-(methylthio)benzotrithiane (102) from L. japonicum, an

834

inseparable 2:3 mixture of 5-(methylthio)varacin (100) and the corresponding trithiane (103) from a different unidentified Lissoclinum species, and 3,4-desmethylvaracin (101) from Eudistoma sp. The tunicate Polycitor sp., another source of this type of compounds, yielded a benzotrithiepin, varacin A (104), and two benzotrithiepin S-oxides, varacins B (108) and C (109) [112]. ^H-NMR and MS studies of varacin A (104) and varacin (96) (also isolated from this tunicate) revealed that they readily equilibrate to give a mixture of both compounds. As a matter of fact, equilibrium reactions between the benzopentathiepin 96 and the benzotrithiepin 104 + Sg were observed to occur in CHCI3, MeOH, and pyridine. For example, 96 generated 104 when it was dissolved in MeOHd4. More recently, lissoclin disulfoxide (110) was obtained from an unidentified species of Lissoclinum [113]. It was proposed that these natural products derived from an overlapping manifold of tyramine biosynthesis coupled with hypovalent sulfur metabolism [46]. Benzopentath iepin 96, varacin Ri = R2 = Me, R3 = R4 = H

'sylp^N^^^ RsO-^Y^Rj

97, lissoclinotoxin A Rj = R3 = R4 = H, R2 = Me 98, isolissoclinotoxin A Ri = Me, R2 = R3 = R4 = H 99, N, N-dimethyl-5-(methylthio) varacin Ri = R2 = Me, R3 = SMe, R4 = Me

OR,

100,5-(methylthio)varacin Ri = R2 = Me, R3 = SMe, R4 = H 101,3,4-desmethylvaracin Ri = R2 = R3 = R4 = H

Benzotrithiepin S'^S

Sv JV^.,X-V^N; ^

II

^

102, Rj = R2 = R4 = Me,

R3 =

SMe

103, benzotrithiepin 5-(methylthio)varacin Rj = R2 = R4 = Me, R,= H 104, varacin A Rj = R2 = Me, R3 = R4 = H

835

MeO SMe

MeO

OH

S-S'^ y MeO NH2

106, lissoclinotoxin C

OH

NH2 107, lissoclinotoxin D MeO O OMe MeO^ . A . ^S-Y'^Y'^^®

105, lissoclinotoxin B

-S^y^SMe

MeS 109, varacin C

NH2

NH2

110, lissoclin disulfoxide

The polysulfides showed a wide range of biological 108, dopamine-derived varacinB activities: antimicrobial (96, 97, 99, 102, 105, acetates of 104, 108, and 109), antifungal (96, 97, 107, acetates of 104, 108, and 109), cytotoxic (96, 97), and antimalarial (97) [105-113]. Furthemiore, a differential toxicity of 1.5 for varacin (96) toward the CHO cell lines EM9 (chlorodeoxyuridine-sensitive) versus BRl (BCNU-resistant) indicated that varacin's mechanism of action involves the formation of single stranded DNA breakage [114]. Lissoclin disulfoxide (110) was found to be a potent inhibitor against both IL-8Ra and IL-8RP receptors with IC50 values of 0.6 and 0.82 \xM, respectively, and also had activity against PKC (IC50 1.54 |ag/ml) [113]. Furthermore, compounds 99, 100, 102, and 103 showed selectivity for inhibition of PKC in comparison to PKA [111]. A number of structure-activity relationships were deduced. The importance of the presence of a benzopentathiepin/benzotrithiepin ring in the activity of these compounds was suggested since lissoclinotoxin C (106) [46] and other tris(methylthio) derivatives [111], which do not contain such a system, were inactive. On the other hand, the activity of these compounds seems not to depend upon the presence of a free amino group in their side chains since varacin (96) and its iV-acetylated derivative displayed similar

836

activities [112]. Finally, the structural relation of this system to dopamine was also found to be related to the potent activity. Polycarpine and Polycarpamines Tunicates belonging to the Polycarpa genus are another source of disulfide metabolites. The isolation of the dimeric disulfide alkaloid polycarpine (111) from P. aurata was reported by Schmitz et al [115] and from P. clavata by Kang and Fenical [116] at the same time. Compound 111, as its dihydrochloride salt, was converted into the free base by silica gel chromatography, a process that also gave several degradation products (e.g. I l i a and 111b) that appear to be artifacts of the isolation process. Spectral studies, chemical transformations, and X-ray crystallography allowed the structural elucidation of these compounds. The synthesis of 111 in three steps from /7-methoxyphenacyl bromide confirmed its structure [117]. Polycarpine inhibited the enzyme inosine monophosphate dehydrogenase, but this inhibition could be reversed by addition of excess dithiothreitol, suggesting that 111 reacts with sulfhydryl groups on the enzyme [115]. Furthermore, 111 showed cytotoxicity against HCT-116 cells (IC50 0.9 |ig/ml) [116] and significant antitumor activity in vivo (white mice) against P388, L1210, carcinoma Ehrlich cells, and high inhibitory activity against reverse transcriptases from Raus sarcoma and avian myeloblastosis viruses in vitro (IC50 = 3.5 x 10"^ M) and Na^, K^ATPase isolated from rat brainof (IC50 = 5.0 x 10"^ M) [117]. The tunicate P. auzata has also been shown to contain the polycarpamines A-E (112116). These are benzenoid compounds with uncommon sulfiir functionalities and were characterized through interpretation of their NMR data and MS fragmentation patterns. Only the first collection of this organism yielded polycarpamines, suggesting that the tunicate may not be the true source of these compounds. Polycarpamine B (113) exhibited significant antifungal activity in vitro [118]. s

ixy^y^ ^-
MeO Ilia

S

NH2 111, polycarmine

Me

O

,^A>w-

MeO-

111b

837 NMe2

MeS^"

"

112, polycarpamine A Rj = H, R2 = OMe

MeO 116, polycarpamine E

113, polycarpamine B Rj = R2 = O 114, polycarpamine C Rj = R2 = S 115, polycarpamine D Rj = COMe, R2 = OMe

Other Disulfldes/Polysulfldes from Tunicates A number of other disulfides and polysulfides have been obtained from tunicates. Citorellamine (117) was the first indole disulfide dihydrochloride isolated from a marine organism - the tunicate Polycitorella mariae. The previous structure proposed for citorellamine [119] was revised and confirmed by total synthesis [120]. Compound 117 possesses both cytotoxic (against L1210 cells with an IC50 value of 3.7 |Lig/ml) and potent antimicrobial activity [119]. An unidentified New Zealand tunicate species of the Aplidium genus was the source of the trithiane 118 [121]. Spectroscopic methods and chemical degradations established the structure of 118 as c/5'-5-hydroxy-4-(4'-hydroxy-3*methoxyphenyl)-4-(2"-imidazoyl)-1,2,3-trithiane. Thus, in neutral or slightly basic solution this compound interconverted to give the trans isomer 119 and 2-vanilloyl imidazole 118a, which could arise from thione 118b. Furthermore, conformational analysis of the trans isomer showed a chair conformation for the trithiane ring with the hydroxyl functionality equatorial and the imidazole ring axial. Compound 118 displayed antimicrobial, antifungal, and modest cytotoxicity (P388, IC5o= 13 and 12 l^g/ml for 118 and 119, respectively) activities [121]. Namenamicin (120) was the first sulfur-containing enediyne compound structure to be isolated from a tunicate {Polysyncraton lithostrotum) [122]. The structure of 120 was deduced from its spectral data and by comparison to those of the

838

known enediyne antitumor antibiotics calicheamicins and esperamicins [123, 124]. Compound 120 exhibited potent in vitro cytotoxicity with a mean IC50 of 3.5 ng/ml and in vivo antitumor activity in a P388 leukemia model in mice (ILS 40% at 3 |ag/Kg). Namenamicin (120) also showed potent antimicrobial activity and cleaved DNA with a slightly different recognition pattem than calicheamicin yi^ [122]. The fact that all of the enediyne antitumor antibiotics previously isolated were products of actinomycites, as well as namenamicin's extremely low and variable yield from the tunicate, lend support to the hypothesis of a microbial origin for this natural product. HO,

HO, rH>

0.020/0NaOH ^^^.

. MeO J - ^ " " N^ N

orCD30D MeO

119

118

118a X = 0 118b X = S

OMe

3,XX^"

-''^Xx'S-)2

117, citorellamine

Me

MeS

^S

«=s-zi^o;^!>l,o/o' "°

HO

9

I MeO 120, namenamicin

Disulfides/Polysulfides from Marine Microorganisms Marine microorganisms have been an important source of disulfide and polysulfide marine metabolites. Among these compounds are the leptosins and the thiomarinols, which form a well-defined group of natural products.

839

Leptosins The polysulfide leptosins (121-135) belong to a series of epipolythiodioxopiperazines produced by a fungal strain Leptosphaeria sp. OUPS-4, isolated from the marine alga Sargassum tortile [125, 126, 127, 128]. HOv.^

\ ^

oOH

^ ^

O^^^Me

..OH

I

O ^ ^ M

121, leptosin A x = 4, y = 2

128, leptosin K n = 2

122, leptosin B x = 3, y = 2

129, leptosin Ki n = 3

123, leptosin C x = 2, y = 2

^^O, leptosin K2 n = 4

124, leptosin G x = 4, y = 3 125, leptosin Gi x = 3, y = 3 126, leptosin G2 x = 2, y = 3 127, leptosin H x = 2, y = 4

1 "^.-^^^ ^"'^

a

—k—f

133, leptosin D x = 2

131, leptosin I Rj = CH2OH, R2 = H

134^ leptosin E x = 3

132, leptosin J Rj = H, R2 = CH2OH

135^ ^^^^^^^^ p ^ ^

840

The structures of these systems have been elucidated by spectroscopic analysis, some chemical transformations, and by X-ray crystallography in the cases of 128-130 [127]. These compounds can be structurally divided into four main groups: the dimeric systems, including leptosin A type (121-127), leptosin K type (128-130), leptosin I and J (131 and 132), and the monomeric systems 133-135. All of these compounds showed potent cytotoxicity against P388 cells that range from ED50 values (in |ag/ml x 10"^) of 1.13 for leptosin I (131) to 8.60 for leptosin D (133). The dimeric leptosins displayed more potent activity than the monomeric ones and the number of sulfur atoms in the dioxopiperazine rings was not found to influence the activity [125-128]. Furthermore, leptosins A (121) and C (123) showed antitumor activity against the Sarcoma-180 ascites tumor in mice with a ratio of (T/C%) of 260 and 293 and at doses of 0.5 and 0.25 mg/Kg, respectively [125] Thiomarinols Thiomarinols A-G (abbreviated as TMA-G) (136-142) are a group of antibiotics isolated from the cultured broth of marine bacterium Alteromonas rava sp. no. SANK 73390 [129, 130, 131, 132]. Their structures were elucidated by NMR spectral analysis, chemical degradation, and by X-ray analysis in the case of 141 [131].

136, thiomarinol A Rj = R2 = OH, R3 = H, n = 5 137, thiomarinol C Rj = H, R2 = OH, R3 = H, n =5 138, thiomarinol D Rj = OH, R2 == OH, R3 = Me, n = 5 139, thiomarinol E Rj = OH, R2 = OH, R3 = H, n = 7 140, thiomarinol F Ri = OH, R2 = O, R3 = H, n = 5

841

All of these compounds showed excellent in vitro antimicrobial activity, with 136, 139, and 141 being the most active. Furthermore, compounds 136, 138, and 141 were found to inhibit the isoleucyl-transfer RNA synthetase in bacteria [132].

HO

OH

141,thiomarinolB

Me

Me^ ^

^^Y^Yy^^'

.^r>^ ^

^O

Me

O

OH 142, thiomarinol G

Additional disulfide metabolites have been isolated from marine bacteria. The unique pyridone-oxazole B-90063 (143) was isolated from the culture supernatant of Blastobacter sp. SANK 71894 [133]. The structure of 143 was determined to be bis[6-formyl-4-hydroxy-2-(2'-Ai-pentyloxazol-4'-yl)4-pyridon-3bis[6-formyl-4-hydroxy-2-(2*-«-pentyl-oxazol-4*-yl)-4pyridon-3-yl]disulfide on the basis of spectral data and chemical reactions. Compound 143 inhibited human and rat endothelin-converting enzyme (ECE) with an IC50 of 1.0 and 3.2 |aM, respectively. B-90063 also inhibited the binding of endothelin-1 to rat endotheliuA and bovine endothelin receptors, although its antagonist activities were weak. These activities led to the proposal that 143 might abolish the physiological actions of endothelin through the ECE inhibitory and receptor antagonistic mechanism [133]. The thiodepsipeptide thiocoraline (144), isolated from the mycelium Micromonospora sp. L-13-ACM2-092, was found to be a potent antibiotic and cytotoxic compound. Its structure was deduced on the basis of spectroscopic methods [134].

842

O

Hi,

C5H,,

?\

C5H1

Y

O ^

Me-N

SMe

r

s-s oo 143, B-90063

0=<

N-Me

N

yOg

MeS.

144, thiocoraline

The hyperthermophilic archaea of the genus Thermococcus, isolated from marine hydrothermal systems, has been found to be a rich source of cyclic methylene-sulfur [135]. The prokaryotic archaea, established as "the third domain of life" in addition to eukaryotes and bacteria, grow under extreme conditions such as the absence of oxygen, temperatures of 100 °C and saturated salt solutions. Thus, 23 cyclic polysulfides (145-167) could be isolated from the intact cells of Thermococcus tadjuricus (strain Ob9) and T mococcus acidaminovorans (strain Vc6bk) by using chemical screening methods. The structures of nine of these compounds were determined by spectroscopic methods, while those of the 14 remaining materials were established by GC-MS only. The compounds can be classified into four types (A to D): the 1,2,4-trithiolanes (145-154), 1,2,4,5-tetrathianes (155157), 1,2,3,5,6-pentathiepanes (158-163), which are all generally quite stable compounds, and the monoalkyl-substituted polysulfides (164-167), which tend to disproportionate into sulfur and disubstituted cyclic polysulfides. These compounds are structurally related to the cyclic polysulfide compounds reported from red alga Chondria californica in 1976 [136] and to compound 168 (3-hexyl-4,5-dithiacycloheptanone) reported from the brown algae of the Dictyopteris genus in 1971 [137]. The latter compound has more recently been found to be a potent inhibitor of bee venom-derived phospholipase A2 (PLA2) [138].

843 A: 1,2,4-Trithiolanes

C: 1,2,3, 5, 6-Pentathiepanes

s-s s-s ^S 145, Ri =R2 = Me

158, Ri = »Bu,R2 = H

146,Ri = Et,R2=Me

159, Ri = ^Bu,R2 = Me

147, Ri =*Bu,R2 = H

160, Ri = 'Bu,R2 = *Pr

148, Ri = ^Bu, R2 = Me

161, Ri ==R2 = 'Bu

149, Ri = Et, R2 = *Bu

162, Ri = Benzyl, R2 =''Bu

150, Ri = 'Bu, R2 = *Pr

163, RI = IndMe, R2 = iBu

151, Ri =R2 ='Bu 152, Ri = Benzyl, R2 = Me

IndMe=

^

153, Ri = Benzyl, R2 = *Bu 154, R , = IndMe, R2 = *Bu

D: Monoalkyl-substituted polysulfides

s-s

B: 1,2,4, 5-Tetrathianes

164,Ri ='Bu,n=l

R,—(

VR2

S-S

155,Ri = 'Bu,R2 = Me

165, Ri = Me, n = 2 166,Ri = ^Bu,n = 2 167,Ri = 'Bu,n = 3

156, Ri = R2 = *Bu 157,R, = Bn,R2='Bu

S-S

168

844

Bromotyrosine and Cysteine-derived Metabolites There are very few examples of disulfides isolated from sponges. Some bromotyrosine and cysteine-derived metabolites with a disulfide functionality were obtained from sponges belonging to the Verongidae family. The first brominated tyrosine metabolite containing a disulfide linkage, compound 169, was reported by three research groups at the same time. Two isomeric forms (169 and 170) were isolated from an unidentified sponge by Schmitz et al. [139]. Since the E,Z isomer (170) isomerizes to the EyE isomer (169), the authors postulated that the E,Z (or the Z,Z isomer) must be the natural metabolite. The {E,E) isomer (169) was reported, under the name of psammaplin A, by Crews et al. from Psammaplysilla sp. [140] and by Scheur et al from a sponge tentatively identified as Thorectopsamma xana [141]. The tetrameric bisaprasin (171) was also isolated from the latter sponge (171) [141]. The isolation of psammaplins A~D (169,172-174) along with prepsammaplin A (175), the only non-brominated component, from Psammaplysilla purpurea led to the suggestion of a biogenetic scheme for this type of compounds [142]. Psammaplins B (173) and C (174) represent the only thiocyanate bromotyrosine derivative and sulfanamide metabolite, respectively, isolated from marine organisms. Their structures, including the stereochemistry of the oxime groups, were determined from NMR data and some chemical transformations. Compound 169 showed cytotoxicity against P388 cells with an IC50 of 0.3 |ig/ml [140], while compound 174 displayed a mild in vitro activity against protein tyrosine kinase (3200 jaM) [142]. Antimicrobial activity was found in compounds 169, 171, and 174 while compounds 172 and 173, which lack the disulfide linkage, were inactive [141, 142].

169, {6EfiE) psammaplin A 170, (6£,6'Z) isomer

845

172, psammaplin B X = -SCN x-\^S~S

173, psammaplin C X = -SO2NH2 174, psammaplin DX==--s>. / \ ^ N v y ^ ^ ^

S

171, bisaprasin

O

MeO

u

if

O

O

N'

u

N'^OMe

175, prepsammaplin A

SULFONIUM COMPOUNDS Sulfonium compounds occur in a wide range of unicellular algae as well as red algae. They have been shown to be the major source of atmospheric sulfur compounds such as dimethyl sulfide and from methanethiol from the ocean, which has a central role ir lie global sulfur cycle [143]. The unicellular algae Gonyaulax polyedra contains a large amount of the cyclopropane gonyauline (176) (about 10 mg/lg wet cells), which causes the period-shortening of bioluminescent circadian rhythmicity in this photosynthetic dinoflagelate [144]. The absolute configuration of 176 was established as \R,2R by synthesis of both enantiomers from (+)-2methylthio-l,2-rra«5-cyclopropanedicarboxylic acid [145]. Gonyol (177) was first found accumulated in the cells of G. polyedra when methionine was added to the cultured medium at a high concentration [146]. It was subsequently found distributed in several dinoflagellates [147]. The structure of 177 was elucidated by spectroscopic methods and confirmed by synthesis from methyl-5-methylthio-3-oxopentanoate. Studies on the biogenesis of 177 using labeled sodium acetate in the presence of methionine showed it to be biogenetically derived from methionine and

846

acetate through dimethyl-p-propiothein or its analogous intermediates [146]. Further biosynthesis studies of 177 with ^^C-labeled methionine suggested that this compound is directly derived from L-methionine by a sequence of reactions [147]. Compounds 176 and 177 may act as osmoprotectants and as methyl donors like 3dimethylsulfoniumpropionate (DMSP) [147]. The role of these sulfonium compounds as methyl donors was also proposed by Garson [148]. Two sulfonium compounds, namely 5- dimethylsulfonio-4-hydroxy-2aminovalerate (178) and (•f)-(i?)-3-dimethylsulfonio-2-methoxypropanoate (179), have been identified from the alga Lophocladia lallemandi [149] and Diginea symplex [150], respectively.

Me I

Me^\^

CO

HV H 176, gonyauline

HO 178

NH2

177, gonyol

Me

OMe 179

ISOTHIOCYANATES The marine isothiocyanates, with more than 80 compounds isolated so far, form the largest group of naturally occurring isothiocyanates. This wellestablished group of marine natural products is constituted mainly by terpene metabolites present as sesquiterpene and diterpene derivatives. The non-terpene isothiocyanate compounds include two cylindricine alkaloids and a series of long-chain aliphatic metabolites. Marine sponges constitute the main source of these compounds, although they are also found in nudibranches and tunicates.

847

Terpene Isothiocyanates Most of the terpene isothiocyanates were isolated from three orders of sponges: Axinellida {Acanthella and Axinella genus), Halichondrida [Cymbastela, Phakellia, Axinyssa (-Trachyopsis), and Halichondria genus], and Lithistida (Theonella). In the few cases where isothiocyanates were found in nudibranches, it was postulated that these compounds were obtained by the nudibranch from sponges that make up their diet and, furthermore, that accumulation of these ichthyotoxic substances afforded the nudibranch a degree of chemical defense against would-be predators. It was proposed that these metabolites are sequestered in its non-mucous skin glands and are secreted when the nudibranch is molested [151]. The co-occurrence in some cases of thiocyanate and isothiocyanate compounds in the same extract indicates that care must be taken to distinguish the two types of functionalities. The isothiocyanate group is characterized by a strong IR band in the 2180-2070 cm"^ range, in contrast to the sharp IR stretching band of medium intensity at 2150 cm~^ for the thiocyanate group. A ^^C-NMR chemical shift ranging from 125 to 131 ppm (bs) is diagnostic of the isothiocyanate carbon, but sometimes the resonance for this functional group is not observed. In the UV spectrum these compounds show a band in a 250-245 nm range and they can be characterized by the facile loss of HNCS, HS or SCN from the molecular ion in the MS. The isolation of terpene isothiocyanate compounds associated with the corresponding isocyanates proved to be very useful in their structural characterization. Thus, adding elemental sulfur at 120 °C to the corresponding isocyanates not only verified unambiguously their presence [152, 153, 154, 155] but was also used to identify their structures by comparison to the corresponding isocyanate [152, 153]. Reduction of the isothiocyanate with LAH to give a methylamino derivative was very useful in other cases to confirm the existence of the isothiocyanate [156]. The methylamino derivative obtained can be acylated with /?bromobenzoyl chloride to give a mono-p-bromobenzamide [157] or, alternatively, submitted to Hoffman degradation in order to determine the configuration of the NCS group [156]. In other cases, the characterization was performed by the reaction of the isothiocyanate with MeNH2 in chloroform at room temperature to yield the NH-CS-NHMe derivative [158]. A direct route for the preparation of isothiocyanate terpenes

848

involving the addition of isothiocyanic acid (prepared "in situ") to terminal double bonds was reported [159]. There was considerable speculation about the biosynthetic origin of the isothiocyanate group. The isolation of isothiocyanates from marine sponges has been generally accompanied by the corresponding isocyanates and formamides (called isocyanate-isothiocyanate-formamide series), and this indicated the strict biogenetic relationship between them. The possible involvement of isothiocyanate, thiocyanate or other equivalent ions that quench the intermediate carbonium ions to form isothiocyanate and thiocyanate terpenes has been invoked [157, 160]. More recently, Garson et al, demonstrated the use of both cyanide and thiocyanate by Acanthella cavernosa in the biosynthesis of isocyanate and isothiocyanates, suggesting that these inorganic precursors may be interconverted by the sponges [161]. In the former review on sulfur compounds from marine organisms [7] were described nine terpene isothiocyanates that have also been included in this survey. Sesquiterpene Isothiocyanates These compounds range through monocyclic (with a bisabolane framework), bicyclic (with an eudesmane, amorphane, axane, guaiane, isodaucane, or gorgonane framework), tricyclic (with an aromadendrane, cubenane, maaliane, pupukeanane, or trachyopsane framework), and spiro[4,5]decane carbon skeletons. Only two monocyclic sesquiterpene isothiocyanates have been isolated to date: 7-isothiocyanato-7,8-dihydro-a-bisabolene (180) from the sponge Halichondria sp. [162] and theonellin isothiocyanate (181) from the sponge Theonella cf swinhoei [163]. The bicyclic and tricyclic sesquiterpene isothiocyanates (compounds 182-203 and 204-216, respectively) constitute the major group and they are listed in Tables 1 and 2. The sesquiterpene isothiocyanates with a spiro[4,5]decane framework are represented by axisothiocyanate-3 (217), which was first isolated from the sponge Axinella cannabina [7] and more recently from Acanthela klethra [164], 2-isothiocyanato-6-axene (218), obtained from the sponge Axinyssa {=Trachyopsis) aplysinoides [165], and 219 from Acanthella acuta [155]. Capon and MacLeod noted that most of the sesquiterpene isothiocyanates (and also the accompanying isocyanates) posses the same molecular formula, C15H25X (X = NCS or NC), which means that they have three units of unsaturation in the carbon framework [166].

849 Bisabolane skeleton

SCN

180

Amorphane skeleton NCS

HLNCS

xg SCN =

184 NCS

Eudesmane skeleton

850 Axane skeleton

197

Gogonane skeleton

Ncs

r.^.r\ SCN \

SCN SCN 199

198

^

Guaiane skeleton

Isodaucane skeleton <, NCS

Aromadendrane skeleton NCS

SCN

205

207

^

208

851 Maaliane skeleton SCN

SCN

SCN 210

211

Cubehane skeleton

Pupukeanane skeleton

212

Trachyopsane skeleton NCS

XX) R»^

213

NCS 214, Ri=H,R2 = NCS 215,Ri=NCS,R2 = H

Spiro[4,5]decane skeleton

SCN-r^^^ \==^

SCN 217

216

852

Table 1. iNo. 182

Bicyclic Sesquiterpene Isothiocyanates Isolated from Sponges Name

(-H15,6/?,7/?.10/?)-10isothiocyanato-4-amorphene

183

Origin

Ref.

Halichondria sp.

[167]

Axinyssafenestratus

[168]

184

isothiocyanato-4-amorphene (-).184

Axinella cannabina

[169]

185

(+)-185

Acanthella pulcherrima

[166]

186

(+)-4-isothiocyanato-9-amorphene

Axinyssafenestratus

[168]

187

(+)-10-isothiocyanato-4,6amorphadiene

Axinyssa fenestratus

[168]

188

10-isothiocyanato-5-amorphen-4-ol Axinyssafenestratus

[168]

189

(-)-halipanicine

Observations 1

Anthelmintic activity

1 1

Antimicrobial activity Anthelmintic activity Anthelmintic activity

1 1 1 1 1 1

Synthesis [170]

1

Halichondria panicea

[157]

Axinella cannabina

[153]

Axinella cannabina

[171] [164]

X-ray analysis

1

[164]

X-ray analysis

1

194

(+)-(!/?, 5/?, 6/?. 85)-dcc[4.4.0]ane- Acanthella klethra 1,5-dimethyl-8-( 1 *-methy!ethenyl)5-isothiocyanate (-»-)-(1/?, 5/?, 6/?, 8/?)-dec[4.4.0]ane- Acanthella klethra 1,5-dimethyl-8-( 1 *-methylethenyl)5-isothiocyanate Axinella cannabina (+)-194

195

(-)-acanthene B

Acanthella sp.

[154]

196

Axinella cannabina/ Acanthella acvta Axinella cannabina

[152]

197

(+)-6a-isothiocyano-5a//, 7a//, 10a-eudesm-4( 14)ene axisothiocyanate-1

[172]

Synthesis [173]

198

axisothiocyanate-4

Axinella cannabina

[172]

Synthesis [174]

199

(-)-lO-isothiocyanato-l 1-axene

Acanthella cavernosa

[175]

(-)-4a-isothiocyanatogorgon-11 -ene Phyllidia pustulosa (nudibranch) (-)-10-isothiocyanatoguaia-6-ene Axinyssa aplysinoides

[176]

Antifouling activity

1202

-

Unidentified sponge

[178]

1203

(+)-203

Acanthella acuta

[155]

190 191 192 193

poo 201

-5-ene (-)-191

[171]

[165]

Synthesis [177]

1

853

Table 2. INO.

Tricyclic Sesquiterpene Isothiocyanates Isolated from Sponges Name

Origin

Ref.

204

(+)-axisothiocyanate-2

Axinella cannabina

[179]

205

(+)-epipoIasin-B

Epipolasis kushimotoensis

[156]

206

(+)-1 -isothiocyanatoaromadendrane Axinyssa aplysinoides

207

(-)-1 Oa-isothiocyanoalloaromadendrane (-)-208

Observations

Synthesis [180]

[160]

Axinella cannabina

[153]

Acanthella acuta

[155]

Acanthella cavernosa

[175]

210

(+)-10/?-isothiocyanatalloaromadendrane (-)-210

Axinella cannabina

[181]

211

(-•-)-epipolasin-A

Epipolasis kushimotoensis

[156]

212

(-)-epipolasin-A

Acanthella pulcherrima

Antifeedant and 1 antimalarial [183]

213

(-)-l 3-isothiocyanatocubebane

Axinyssa aplysinoides

[151, 166] [160]

214

(+)-5-isothiocyanatopupukeanane

Axinyssa sp.

[182]

X-ray analysis

1

215

(-)-9-isothiocyanatopupukeanane

Axinyssa sp.

[183]

1

216

(+)-2-isothiocyanatotrachyopsane

Axinyssa aplysinoides

[165]

Antimalarial activity X-ray analysis

208 209

Antifouling activity

1

1

Diterpene Isothiocyanates Diterpene isothiocyanates fall into three distinct structural categories regarding their carbon skeletons: the acyclic tetraenes (220 and 221), the biflorane framework (including the kalihinol family) (222-238), and the amphilectane framework (including the cyclo- and isocycloamphilectanes) (239-241). Like the sesquiterpene isothiocyanates, most of these compounds were isolated along with the corresponding isocyanate and formamide compounds.

854

Two acyclic diterpene tetraene isothiocyanates have been isolated from sponges so far. Famesyl isothiocyanate (220) was obtained from Pseudaxinyssa pitys [184] while compound (+)-221 was isolated from Halichondria sp. [185]. Their structures were elucidated by interpretation of their spectral data.

'NCS

220

NCS 221

Diterpene isothiocyanates having a biflorane framework comprise the kalihinol metabolites (222-236) and two diastereoisomers of 10isothiocyanatobiflora-4,15-diene (237 and 238). Most of the diterpene isothiocyanates belong to the kalihinol family and are listed in Table 3. This type of compound shows a broad spectrum of biological activity including antibiotic, anthelmintic, cytotoxic, and antifouling. The kalihinols can be divided into three structurally different groups: those with a trans-d^QdXin attached to a tetrahydropyranyl group (222-226), those with /ra/i5'-decalin attached to a tetrahydrofiiranyl group (227-233), and those with cw-decalin attached to a tetrahydrofiiranyl group (234236). A proposed biogenesis of these types of terpenes was described by Crews et al. [186]. Several diagnostic spectroscopic features can be used to discern between them: tetrahydrofiiranyl groups are indicated by lower field ^"^C-NMR shifts for their ether carbons (6 82-87 ppm) compared to the corresponding shifts in the tetrahydropyranyl analogues (5 75-77 ppm). The relative stereochemical relationship of H-1, H-6, H-5, and H-7, as found in those systems having a trans-AQOdXin skeleton, can often be confirmed from the coupling pattern of H-6 (one small and three large J values). In cases where the H-6 signal is not resolved, the coupling patterns of H-5, H-7, and H-1 can provide the stereochemical information [187].

855 Trans decalin-tetrahydropyranyl

kalihinols

NCS

jj'. NHCHO

H V.NNCS

HO..

222, R = NCS

226

225

223, R = NHCHO 224, R = NC Trans decalin-tetrahydrofuranyl

kalihinols

NCS

>iCS

HO/

HO/ A = O .UNC CN ^ ^ ^ - ^ ^ ^

NCS

HO' = H SCN "

227

SCN

h^-^""

229

228

230, Ri = NCS, R2 = R3 = CH2 jjQ, ,

J. J "4^^ R2 H=^^\oo(--R3

231, Ri = NC, R2 = NCS, R3 = Me 232, Ri = NCS,R2 = NC,R3 = Me

CN

233, Ri = R2 = NCS, R3 = Me Cis decalin-tetrahydrofuranyl

kalihinol

OHCHH

H iNC

NCS

CN

HO 235

NC

"A!)

236

|N( NCS

856

Table 3.

Diterpene Isothiocyanates Belonging to the Kalihinol Family Isolated from Sponges Name

INO!

Origin

Ref.

Biological activity

222

Kalihinol I

Acanthella cavernosa

[168]

223

Kalihinol J

Acanthella cavernosa

[168]

Anthelmintic

224

(-)-kalihinol X

Acanthella spp.

[189]

Antimicrobial

225

Acanthella cavernosa

[175]

Antifouling

1

226

(+)-lOP-formamido-5 Pisothiocyanatokalihinol-A (-)-10-ep/-kalihinolI

Acanthella sp.

[188]

Antimalarial

1

227

(-)-lO-epi-kalihinolH

Phakellia pulcherrima

[187]

228

(-)-5,10-bisisothiocyanokalihinol G

Acanthella sp.

[188]

Antimalarial

1

229

(-)-kalihinol L

Phakellia pulcherrima

[187]

230

(-)-lO-isothiocyanatokalihinol C

Phakellia pulcherrima

[187]

231

(-)-kalihinol G

Acanthella spp.

[189]

Antimicrobial

1

232

(+)-kalihinol H

Acanthella spp.

[189]

Antimicrobial

1

233

(-)-lO-isothiocyanatokalihinol G

Phakellia pulcherrima

[187]

234

Acanthella cavernosa

[186]

235

6-hydroxy-10-formamido15-isothiocyano-kalihinene (-)-lO-epi-isokalihinol H

Acanthella cavernosa

[190]

236

(-)-15-isothiocyanato-1 -ep/-kalihinene

Acanthella cavernosa

[190]

1

Compounds 237 and 238 are the only isothiocyanate diterpenes with a biflorane carbon skeleton that do not belong to the kalihinol family. They are diastereosiomers of 10-isothiocyanatobiflora-4,15-diene. A planar structure was reported for compound 237, which was obtained from an unidentified sponge of the Family Adocidae [191]. A (15*,6/?*,7i?*,105*,l li?*) configuration was proposed for 238, which was isolated from the sponge Cymbastela hooperi [192]. From the latter sponge, three diterpene isothiocyanates that have an amphilectane framework (239-241) have been identified. Extensive spectroscopic experiments led to their structures being proposed as (15,35,4i?,75,85,115,125,135,15/?,20/?)-20-isocyano-7-isothiocyanatoisocycloamphilectane for 239, (15*,3S*,4/?*,75*, 85*,125*,135*)-7isocyano-15-isothiocyanatoamphilect-11 (20)-ene for 240, and

857

(IR *,35*,4i? *,75*,85*, 127? *, 13R *)-12-hydroxy-7-isothiocyanatoamphilecta-ll(20),14-diene for 241. Compounds 239-241 show significant and selective in vitro antimalarial activity [192].

NCS 237 (planar structure)

239

NCS

238

Non Terpene Isothiocyanates The only non-terpene isothiocyanate compounds reported from marine organisms are two quinoline alkaloids, namely cylindrines I (269) and J (270) [193] (they will be discussed in the thiocyanate section) and the long chain aliphatic isothiocyanate metabolites. Eighteen long chain a,cobisisothiocyanates [eight diolefinic (242-249) and ten monoolefinic (250259) and three a-isothiocyanate-co-formyl (260-262) compounds have been reported from the Fijian sponge Pseudaxinyssa sp. [194]. The absence of the corresponding isocyanate analogs suggests a different biogenesis for these compounds.

SCN 242,n=14 243, n = 8 244, n = 9 245, n = 1 0

NCS 246, 247, 248, 249,

n=ll n = 12 n=l3 n=14

SCH

'l^r^^NCS n

250,n=16 251, n = 9 252,n=10 253, n =11 254,n=12

255,n=l3 256,n=14 257,n=15 258,n=17 259,n=18

SCN^^

CHO ^ '

260,n=15 261,n = 9 262,n=16

858

THIOCYANATES Although marine organisms produce a large number of isocyanates, isothiocyanates, and formamides, the corresponding thiocyanates have rarely been encountered. Indeed, the thiocyanate functionality has only been found in six sesquiterpenes (263-268), in four tricyclic quinoline alkaloids (271-274), and in psamaplin B (172) (included in the bromotyrosine derivatives discussed in the disulfide/polysulfide section). They have been found in marine sponges, as well as in nudibranches and tunicates. The presence of a thiocyanate group can be very easily deduced from the IR spectrum, which contains a sharp, but weaker in intensity, IR band around 2150 cm~^ and an NMR carbon resonance between 112 and 115 ppm. In the case of psamaplin B (172), ^"^C-NMR additivity effects applied to the methylene carbon linked to the thiocyanate were used to distinguish between a thio- and isothiocyanate group [142]. Chemical proof can be provided by LAH reduction of the thiocyanate, which gives a thiol rather than a methylamine group as the reaction product of the reduction of the isothiocyanate [195]. Sesquiterpene Thiocyanates In 1989, the research groups of Faulkner and Clardy reported compound 263, the first sesquiterpene thiocyanate to be isolated from a marine organism - the sponge Axinyssa (= Trachyopsis) aplysinoides [165]. The structure of this compound was determined by X-ray analysis to be (15'*,45*,6iS'*,7i?*)-4-thiocyanato-9-cadinene. In 1991 the research groups of Scheur and Higa reported the isolation of two isomeric sesquiterpene thiocyanates, 2- and 4-thiocyanatoneopupukeanane (264 and 265) from two sponges, the Okinawan Phycopsis terpnis and an unidentified species collected in Pohnpei, respectively [195]. The structures of 264 and 265 were determined by spectroscopic methods and chemical transformations. A recent enantiospecific synthesis of (-)-4-thiocyanatoweopupukeanane from (i?)-carvone allowed the determination of the absolute configuration of 265 [196]. Subsequently in 1992, Fusetani et al reported the isolation of cavemothiocyanate (267) from the sponge Acanthella cf. cavernosa and its nudibranch predator, Phyllidia ocellata [197]. In the same year, Faulkner et al published the isolation of (15*,25*,3i?*,6i2*,75*,9/?*)-2thiocyanatoweopupukeanane (266) and (l/?*,2/?*,3/?*,5/?*,65*,75*)-2-

859

thiocyanatopupukeanane (268) from a Pohnpei specimen of the sponge Axinyssa aplysinoides, elucidating their structures through extensive spectroscopic studies [160]. Compound 264 displayed very modest cytoxicity against KB at 0.01 mg/ml [195]. In relation to the biogenesis of this type of compound, Garson et al. demonstrated the biosynthetic origin of the thiocyanate carbon in 2-thiocyanatOAzeopupukeanane (264) by incorporation of sodium [^^C] cyanide and [^"^C] thiocyanate into Axinyssa n.sp.[198].

A R,

r^^4^ ''

H =

^\

/^

cb r^"S--v

KySCN

1H

-SCN 263

264,Ri = SCN,R2 = R3 = H 267

268

265, R3 = SCN, Ri = R2 = H 266, R2 = SCN, Ri = R3 = H

Perhydroquinolines These non-terpene thiocyanates have been isolated from tunicates. The cylindricines constitute a series of reduced pyrrolo[2,l-y]quinolines and pyrido[2,l-y]quinolines isolated from the Tasmanian tunicate Clavelina cylindrica [193, 199]. Cylindricines I and J (269 and 270) are isothiocyanates while cylindricines F, G, and H (271-273) are thiocyanates. They represent the first examples of the isolation of iso- and thiocyanates from tunicates. Compounds 269-273 were identified by a combination of spectroscopic methods and their relative stereochemistries were assigned by using molecular modeling. A closely related compound, fasicularin (274), was isolated from the Micronesian tunicate Nephteis fasicularis [200]. Compound 274, whose structure was elucidated primarily by interpretation of its spectral data, was found to be active in a DNA-damaging assay and cytotoxic against to Vero cells with an IC50 of 14 |ig/mL

860

oOAc SCN

"SCN

SCN-^ 269, cylindrine I

270, cylindrine J

274, fasicularin

271, cylindrine F Rj = R2 = O, n = 3 272, cylindrine G Rj = R2 = O, n = 1

SCN

» ; \

273 cylindrine H Rj = OAc, R2 = H, n = 1

SULFONES, SULFOXIDES, SULFONIC ACIDS AND THEIR DERIVATIVES In this section a very structurally diverse group of compounds bearing oxygenated sulfur functionalities is covered. Sulfones Most of the reported sulfone-containing marine metabolites have been obtained from sponges and bryozoa. The hypotaurocyamine-containing agelasidines, some adociaquinone derivatives, and the euthyroideones constitute small groups of compounds bearing a sulfone group. Agelasidines Agelasidines A~D (275-279) are a series of terpene derivatives of hypotaurocyamine and were isolated from sponges of the genus Agelas. Agelasidine B (276) and (+)-€ (277) were obtained from the Okinawan A. nakamurai [201] while agelasidines (-)-C (278) and D (279) were isolated from the Caribbean A, clathrodes [202]. The structures of these compounds were elucidated by interpretation of spectral data and by

861

chemical degradation experiments. The structure of agelasidine A (275), the first reported marine natural product containing a sulfone unit [203], was confirmed by an efficient three-step synthesis based on a biomimetic approach from famesol [204]. Compounds 275-277 showed antimicrobial and antipasmodic activity, inhibitory effects on contractile responses of smooth muscle, and a potent inhibition of brain Na"^- and K'^-ATPase [201, 205]. NH

NH ''. -S—^

NH2

\S-^

276, (-) agelasidine B

275, (+) agelasidine A NH d\

NH2

.OH - ^ - ^ N

d'^0 ^

277, (-I-) agelasidine C

NH2

T

NH2

NH

279, (-) agelasidine D

278, (-) agelasidine C

Adociaquinones A series of sulfur-containing quinone sesquiterpenes, structurally related to xestoquinones and halenaquinones [206], have been isolated from sponges belonging to the Adocia and Xestospongia genus. Adociaquinones A and B (280 and 281), along with 3-ketoadociaquinone A (282), were obtained from a Pacific Adocia sp. and are characterized by the presence of a l,l-dioxo-l,4-thiazine ring [207]. Their structures were determined by comparison of their spectral data to those of xestoquinone and halenaquinone, and by chemical correlations. A subsequent total synthesis of 280 and 281 confirmed the proposed structures and determined the XAhS absolute stereochemistry for these compounds [208]. Xestoquinolide B (283), a metabolite that is structurally closely related, was isolated from the Indo-Pacific sponge Xestospongia cf carbonaria. Unfortunately, the taurine annulation regiochemistry could not be established in this case [209]. Secoadociaquinones A and B (284 and 285), isolated from the Philippine sponge Xestospongia sp., bear a taurine moiety instead of the l,l-dioxo-l,4-thiazine ring present in the closely structurally related compounds 280 and 281 [210]. Compounds 280, 281, 284, and 285

862

showed inhibition of topoisomerase II in catalytic DNA unwinding and/or decatanation assays. Furthermore, compound 281 showed activity in a KSDS assay, suggesting that 281 inhibits the enzyme by freezing the enzyme-DNA cleavable complex [210]. Compound 281 also displayed mildly cytotoxic activity [207].

O 280, adociaquinone A

O

281, adociaquinone B

282,3-ketoadociaquinone A

HO3S HO3S' O 283, xestoquinolide B

O

284, secoadociaquinone A

O

O

285, secoadociaquinone B

X=NH, Y = S 0 2 or X = S 0 2 , Y=NH

Euthyroideones and other sulfones A sulfone group incorporated within a l,l-dioxo-l,4-thiazine ring is also present in 6-(p-hydroxypheny l)-2i/-3,4-dihydro-1,1 -dioxo-1,4-thiazine (286), a compound obtained from the sponge Anchinoe tenacior [211], and in euthyroideones A-C (287-289), which are brominated quinone methides isolated from the New Zealand bryozoan Euthyroides episcopalis (order Cheilostomatida, suborder Ascophotina, family Euthyroididae) [212]. The spectral data of euthyroideone A (287), secured by X-ray analysis, was used for the structural determination of euthyroideones B (288) and C (289). Compound 288 showed a weak cytotoxic activity towards the BSC-1 cell line. Very simple sulfones have also been isolated from marine organisms. The isolation of sulfolane (290) from the sponge/tunicate composite Batzella spJLissoclinum sp. was the first report, either terrestrial or marine, of this compound as a natural product [213]. The pyrogallol Phenol B (291), obtained from the red alga Grateloupia filicina, was

863

reported as the first example of a sulfone derivative from marine algae. Its structure was deduced by spectroscopic means and confirmed by synthesis. Phenol B exhibited antibacterial activity against Bacillus subtilis at a concentration of 1 mg/disk [214]. Compound 34 is another sulfone-containing metabolite isolated from a bryozoan and has already been cited along with the sulfide P-carbolines. MeO

0

^ . N ^ J L Br II

"AN

0

OH

Q

'1

0 I

0

jlft

HO^y^OH OH

0

1

Me

Q

290, sulfolane

291, phenol B

287, euthyroideone A 288, euthyroideone B , A 2 289, euthyroideone C,A^

Sulfoxides The sulfoxide group is present in very few marine natural products. We have already described eudistomin K sulfoxide (26), eudistomidin F (29), didemnolines C (32) and D (33), [Mso] hymenamide F (87), and lissoclin disulfoxide (110). Additional examples have also been reported. The red alga Laurencia brongniartii was found in 1978 to be a rich source of polybrominated methylthioindoles [7]. Two additional methylthioindoles and two methylsulfinylindoles (292-295) were isolated in a subsequent study of an Okinawan specimen of this algae [215, 216]. The structures of itomanindole A (294) and the methylthio bisindole 293 were determined by X-ray analysis. The X-ray analysis not only showed the location of the sulfoxide group at C-2 in itomanindole A (294) but also revealed that a racemic pair of molecules are joined centrosymmetrically by two strong hydrogen bonds [216]. Br SMe Br 292

SMe N

293

Y

Br % - M e

SMe

Br^Ns^N

Me

294, itomanindole A

rVVsMe 295, itomanindole B

864

Two sulfur-containing brevetoxin analogs were isolated from two New Zealand bivalves. Brevetoxin Bi (BTXBl, 296), characterized by the presence of a taurine moiety, was obtained from the toxicated shellfish Austrovenus stutchburyi [217], while brevetoxin B2 (BTXB2, 297), characterized by the presence of a cysteine sulfoxide unit, was isolated from the hepatopancreas of greenshell mussels, Perna canaliculus [218]. The structures of both compounds were elucidated by comparison of their spectral data with those of brevetoxin B (BTXB), the major toxin produced by the Florida red tide organism, the dinoflagellate Gymnodinium breve, whose structure was secured by X-ray crystallography in 1980 [219]. Compound 297 seemed to be a diastereoisomeric mixture having two chiral centers at C-41 and the sulfoxide. Attempts to separate the diastereoisomers by HPLC, after acetylating the amino and hydroxyl groups or by methylating the carboxyl groups, were unsuccessful. A plausible biosynthetic route to Brevetoxin B2 from BTXBl was proposed [218]. Compound 296 (BTXBl) has a minimum lethal dose in mice of 0.05 mg/Kg (i.p.), producing very similar symptoms to those caused by other brevetoxins [217]. On the other hand, compound 297 (BTXB2) displayed a similar potency to BTXB in activating Na"^ channels [218],

-v:

ff

NH2

N"X^S03Na

296, brevetoxin B1 (BTXB 1)

297, brevetoxin B2 (BTXB2)

Sulfonic Acids and their Derivatives Marine natural products bearing a sulfonic acid/sulfonate group are very abundant, with a huge diverse array of structures known. In most cases, this functionality is present in the discodermin-halicylindramide depsipeptides, in sulfonolipids, as well as in assorted metabolites.

865

Depsipeptides bearing a cysteic acid residue Polydiscamide A (309), discodermins A-H (298-305) and halicylindramides A~E (306-308, 310 and 311) form a series of depsipeptides composed of 13 or 14 amino acids and bear a sulfonic acid group in a cysteic acid residue, with Cys(03H), with the //-terminus blocked by a formyl group. Their total structures, including absolute stereochemistries, were determined in most cases by a combination of spectral and chemical methods.

298, discodermin A Ri = R2 = H, R3 = R4 = Me, R5 = X, R6 = Tr,R7 = Me, 299, discodermin B Ri= R2 = R3 = H, R4 = Me, R5 = X, R^ = *Pr, R7 = Me 300, discodermin C Ri = R2 = R4 = H, R3 = Me, R5 = X, R^ = *Pr, R7 = Me 301, discodermin D Ri= R2 = R3 = R4= H, R5 = X, R^ == 'Pr, R7 = Me 302, discodermin E Ri = R2 = H, R3 = R4 = Me, R5 = Y, R^ = 'Pr, R7 = Me 303, discodermin F Rj = R2 =H, R3 = Me, R4 - '^., R5 = X, R^ = 'Pr, R7 = Mc 304, discodermin G Ri = R3 = R4 = Me, R2 = H, R5 - X, R^ = *Pr, R7 = Me 305, discodermin H Rj = H, R2 = OH, R3 = R4 = Me, R5 = X, R^ = *Pr, R7 = Me 306, halicylindramide A R| = R3 = R4 = H, R2 = Br, R5 = X, R6 = Ph, R7 = Me 307 halicylindramide B R,= R3= H, R2 = Br, R4 = Me, R5 = X, R^ = Ph, R7 = H 308, halicylindramide C Ri = R3 = H, R2 = Br, R4 = Me, R5 = X, R^ = Ph, R7 = Me

O

866 O^^NH2

O

=

Br

0

0 N^

^

^

/SO3-

O ^=

O \

"

\

N'H X+

Me,^^^

N^ ^^

O ^ ^ Y ^ O^ O

309, polydiscamide A

HN

0<5^NH2 /O3-

^ /

O^A^

^

O

310,halicylindramideD

V

Me^^^Q

^A^

O A

HN

NH

^^^

^

0,^5^ NH2

O

=

0

0

^^

O ^-

O

/03'.

Me,__^^^^ N y CONH,

Br

311, halicylindramide E

H z N ^ NH2

867

The tridecapeptide polydiscamide A (309) was obtained from an unidentified Caribbean species of Discodermia [220], while discodermins A~H (298-305) were isolated from D. kiiensis. The structures deduced previously for discodermins A-E [221, 222] were later revised on the basis of NMR data and protein sequence analysis [223]. A number of closely related compounds, the tetradecapeptides halicylindramides A-C (306-308) [224], the tridecapeptide halicylindramide D (310) [225], and the truncated linear peptide halicylindramide E [225] (311) were obtained from the sponge Halichondria cylindrata. A microbial origin for these compounds was suggested [226]. This group of compunds displayed a huge array of biological activities: cytotoxic (298-310; discodermin E and halicylindramide A with a IC50 values of 0.02 and 0.54 |Lig/ml against P388 were the most active), antimicrobial (298- 305), antifungal (compounds 303-308, 310), inhibition of the development of starfish {Asteria pectinifera) embryos (298-301), potent inhibition of phospholipase A2 (298-301), antiinflammatory activity in the mouse ear pretreated with okadaic acid and also inhibited tumor promotion by okadaic acid (298). The macrocyclic structure is essential for their cytotoxic and antifugal activities since the truncated linear peptide halicylindramide E and a degradation product of halicylindramide B, in which the cyclic structure was opened, were both cytotoxic inactive [220-226]. Arsenic-Containing Ribofuranosides (ACRs). Marine algae accumulate substantial amounts of arsenic from ambient seawater. Several authors have shown that marine algae absorb oceanic arsenate and transform it into a number of arsenic-containing ribofuranosides (ACRs) that account for 50-80% of the arsenic in the alga extract [227, 228]. It is not known if these compounds serve any function within algae. Compounds 312 and 313 are two ACRs that bear a sulfonic acid/sulfonate group and were isolated from several brown alga [229]. The structures were determined mainly by NMR spectroscopy and were assigned as 3-[5-deoxy-5-(dimethylarsinoyl)-P-D-ribofuranosyloxy]-2hydroxypropene-1-sulfonic acid (312) and 2-amino-3-[5-deoxy-5(dimethylarsinoyl)-P-D-ribofuranosyloxy]propene-1 -sulfonic acid (313). The 25 configuration was determined for a diastereoisomer of 313 that was isolated from Sargassum lacerifolium [228].

868 O

Me I

HO OH

OH O-^

312,R = OH

314R = C,5H3i

313,R = NH2

315,R = Ci3H27

AcO

O-^

O

O

316

Sulfonolipids: Sulfonoglycolipids and Sulfonoceramides Sulfonic acid-containing lipids are commonly referred to as sulfoquinovosyl diacylglycerols (SQDG). These sulfonoglycolipids are structural components of chloroplast membranes and occur widely in photosynthetic membranes of higher plants, algae and cyanobacteria (Lyngbya lagerheimii and Phormidium tenue). Such systems were also reported to be present in a sea urchin {Anthocidarias crassispind) and sponges {Phyllospongia foliascens) [230]. Sulfonoglycolipids of marine algae have received considerable attention in recent years because of their important biological functions as well as their interesting biological activities, which include anti-HIV activity [230, 231]. Illustrative examples of the sulfonoglycolipids include the 96:4 mixture of T-Opalmitoyl-3*-0-(6-sulfo-a-D-quinovopyranosyl)glycerol and its myristoyl counterpart (314 and 315) from the sea urchin Anthocidarias crassispina [232], 2',3*,4*-triacetyl-l,2-dipalmitoyl-3-0-(6*-sulfo-a-D-quinovopyranosyl)glycerol (316), obtained after acetylation of the hexane-soluble fraction from the red alga Laminaria pedicularioides [233], and, more recently, 6-sulfo-a-D-quinovopyranosyl-( 1 ->3*)- r.2*-diacylglycerol, namely KM043 (317), from the red alga Gigartina tenella, which was found to be a potent inhibitor of eukaryotic DNA polymerases and HIVre verse transcriptase type 1 [234]. Illustrative examples of sulfonoceramides are the flavocristamides A and B (318 and 319), obtained from the marine bacterium Flavobacterium sp. that was isolated from the marine bivalve Cristaria plicata. The structures 318 and 319, including absolute configurations, were determined by spectroscopic data and chemical means and both compounds were found to be DNA polymerase a inhibitors [235].

869

318,flavocristamideA A^ 319, flavocristamide B

Marine Sulfur metabolites bearing a Taurine Residue Taurine is present in a wide range of marine organisms [7]. The taurine residue (HO3S-CH2-CH2-NH-), whether joined directly to a compound or through an amide link, has been found as a portion of a very wide range of marine metabolites and is not specific to any particular phylum. The taurine residue can be easily identified by the NMR diagnostic signals at 3.0-3.9 ppm (t, y-- 6 Hz)/35-39 ppm (t) for the methylene attached to the sulfur and 2.5-3.0 ppm (t, 7 --^ 6 Hz)/48-52 ppm for the methylene attached to the nitrogen. In many cases it is possible to observe the m/z peak corresponding to [M - CH2CH2SO3H] fragment in the MS. Melemeleones A and B (320 and 321) possess a 4,9-friedodrimane sesquiterpene array linked to a quinone bearing a taurine and they are structurally related to avarol. Compounds 320 and 321 were isolated from the sponge Dysidea sp. and identified by analysis of their spectroscopic data. Compound 321 showed a moderate inhibitory activity against the pp60^"^'^^ protein Tyrosine Kinase with an IC50 of--28 |aM [236].

xvs^SOjH x^/SOjH

320, melemeleone A

321, melemeleone B

870

Several bromopyrrole alkaloids of the oroidin family, containing a taurine residue, have been isolated from the same sponges that biosynthesize this type of alkaloid. Mauritamide A (322), the first member of the oroidin alkaloid class having a taurine moiety, was isolated from Agelas mauritiana and characterized by spectroscopic methods [237]. Tauroacidins A and B (323 and 324), the taurine derivatives of the antihistaminic dispacamides C and D [238], were isolated as racemic compounds at C-9 {9SI9R = 6:4 and 9SI9R = 1 : 1 , respectively) from Hymeniacidon sp. Their structures, including the absolute configuration at C-9, were elucidated on the basis of spectral data and by chemical means. Compounds 323 and 324 exhibited inhibitory activity against EGF receptor kinase and c-erbB-l kinase (IC50 of 20 |ig/ml, each) [239]. Compound 325 is the taurine derivative of clathridine (a very stable zinc complex obtained from Clathrina clathrus) [240] and was isolated from the sponge Leucetta microrhaphis. Compound 325 is (9£)clathridine 9-A^-(2-sulfoethyl)imine and its structure was deduced by spectroscopic and chemical analysis, and confirmed by single crystal Xray analysis. The taurine residue appears to sterically inhibit the formation of the zinc complex [241].

r\

Br

N. Br" N Me O

V^

NN"^NH

/ MeOaS

HO HN

NH

O 323, tauroacidin A R = H

322, mauritamide A

324, tauroacidin B R= Br Me

{tXi>V.

"t.N

325

HO3S

•Me

S(

871

Several fatty acid derivatives bearing a taurine residue have been isolated from diverse sources. Tauropinnaic acid (326) is the taurine derivative of the mono-fatty acid containing a 6-azaspiro[4.5]decane unit and, along with pinnaic acid itself, was isolated from the Okinawan bivalve Pinna muricata and characterized by extensive 2D-NMR spectroscopic analysis. Pinnaic acid and tauropinnaic acid (326) inhibited CPLA2 activity in vitro with IC50 values of 0.2 and 0.09 mM, respectively [242]. Taurospongin A (327) is an acetylene-containing marine metabolite consisting of a taurine and two fatty acid residues and was isolated from the Okinawan sponge Hippospongia sp. [243]. Its structure was deduced by spectral data and chemical means while its absolute configuration was established by applying the Mosher methodology to a degradation product. Enantioselective total synthesis of taurospongin A confirmed the proposed structure [244]. Compound 327 showed weak inhibitory activity against cerbB-2 kinase but exhibited potent inhibitory activity against DNA polymerase p and HIV reverse transcriptase, with IC50 values of 7.0 and 6.5 |iM (Ki values of 1.7 and 1.3 jiM), respectively [243]. This inhibitory activity has been frequently associated with the sulfonic acid function [235].

CI OH 326, tauropinnaic acid HO3S C15H31

327, taurospongin A

872

The taurine residue can also be found as an amide derivative of the 26carboxylic acid function in the 3p,5a,6p,15a-polyhydroxylated steroids 328 and 329, which were obtained from the starfish Myxoderma platyacanthum [245]. The structures of both compounds were determined from spectral data and chemical correlations. The bile of the sunfish Mola mola has been shown to contain a new bile acid conjugated with taurine (330) together with sodium taurocholate. Compound 330 was identified as sodium 2-[3a,7a, 11 a-trihydroxy-24-oxo-5p-cholan-24-yl]amino]ethanesulfonate on the basis of its physicochemical data and chemical transformations [246]. SOsNa

.^\^S03Na

328, R = H 329,R = Me, A'22

Closely related to these compounds are the carolisterols A~C (331333), which were isolated from deep-water starfish Styracaster caroli [247]. Carolisterols A-C are characterized by a polyhydroxycholanic acid moiety in which the 24-carboxylic acid function is found as an amide derivative of i)-cysteinolic acid. D-cysteinolic acid has been found in fish, alga, and other marine organisms [248].

SOjNa

331, carolisterol A Ri = R3 = OH, R2 = H 332, carolisterol B Rj = R2 = O, R3= OH 333, carolisterol C Ri = R3 = H, R2 = OH

873

Several types of microbial lipids containing a taurine residue have been described in the literarure. One example to illustrate this type of compound is the glycolipid l,2-diacyl-3a-D-glucuronopyranosyl-5«glycerol taurineamide (334), obtained from the seawater bacterium Hyphomonas jannaschiana [249]. The structure of this glycolipid was determined using a combination of chromatographic, spectroscopic, enzymatic- and chemical-degradation methods. Mycosporine-like amino acids (MAAs) are a family of compounds characterized by a cyclohexenone or cyclohexenimine chromophore conjugated with the nitrogen substituent of the amino acid. These systems have been found in taxonomically varied symbiotic and non-symbiotic marine invertebrates. Several studies have demonstrated the protective function of these compounds against the damaging effects of UV radiation, a mechanism that involves filtering harmful wavelengths. An MAA bearing a taurine group, designated mycosporine-taurine (335), has been isolated as the major MAA component of the sea anemone Anthopleura elegantissima. Taurine constitutes more than 90% of the amino acid pool in this organism and, by incorporating taurine into a UV-absorbing compound, the anemone may exploit the ready availability of the most concentrated component of its free amino acid pool [250]. The presence of a taurinelike moiety has also been found in the portion linked to the pyridinium nitrogen in pyridinebetaine B (336), obtained from the Caribbean sponge Agelas dispar [251 ]. o

^^-v^soaH O

H03S^ ^^ HO3S' ^

o={

.Ac

•O"^*'*^'^^ OH -^ o==(^ O ^ ^ A ^ O H HO3S

334

JL^OMe HO'H^^^'^^-^N ^^

^SOjH

o

335, mycosporine-taurine

'—^^^3 336, pyridinebetaine B

874

Finally, the sulfonic acid group can also be found into an isoethionic acid portion in didemnaketal C (337), isolated from the Palauan tunicate Didemnum sp. [252]. The authors suggested that the previously reported metabolites isolated from this source are actually artifacts of 337 resulting from prolonged storage in methanol at 4 ^C. o \^o

\ o'^^

MeOOC

NaOaS^^^^

337, didemnaketal

THIAZOLIDINONE-CONTAINING METABOLITES The latrunculins (abbreviated as Lat) form a well-defined group of marine natural products characterized by the presence of a 2-thiazolidinone unit. After the isolation of Lats A (338) and B (339) in 1980 from the Red Sea sponge Latrunculia magnifica [253, 254], additional members of this group were discovered later on. Lats C (340) and D (341) [255], 6, 7epoxy-Lat A (342), and Lat M (343) [256] were obtained from subsequent studies of the same sponge, while Lat S (344) was isolated from the Okinawan sponge Fasciospongia rimosa [257]. The total synthesis of (4-)Lat A [258] and (+)-Lat B [259] confirmed the proposed structures for these compounds. Furthermore, a single crystal of Lat A (338) suitable for X-ray analysis confirmed its absolute configuration [260]. Compounds 338 and 339 have been shown to disrupt microfilament organization and exert profound effects on the morphology of nonmuscle cells without affecting the organization of the microtubular system [256]. Compound 338 was found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 complex with G-actin. This phenomenon affected different components of the actin-based cytoskeleton [256]. Comparison to cytochalasin showed 338 to be an order

875

of magnitude more potent and represented the only alternative to cytochalasins in pharmacological studies of both actin polymerization in vitro and actin organization and function in living cells [261]. Compound 338 was also found to inhibit macrophage phagocj^osis without interfering with cell viability, strengthening the case for participation of microfilaments in the mechanism of phagocytosis [262]. All these results illustrate the important contribution of this compound to the study of microfilament- microfilament-mediated process. Furthermore, 338 showed excellent in vitro anthelminthic activity at 50 jag/ml against A^. brasiliensis [263] and cytotoxic activity towards HEP-2 and MA-104 cells at 0.072 and 0.23 |ag/ml [264].

338, Lat A

O 339, Lat B

340, Lat C

'-

342,6,7-epoxy Lat A

-

OMe

343, Lat M

876

THIAZOLINE- AND THIAZOLE-CONTAINING METABOLITES Marine bacteria are known to produce thiazoline-containing metabolites: the antimitotic curacins and the metal chelators anguibactin and agrochelin. Thiazoline-containing Metabolites Curacins Curacins (345-348) are a small family of bioactive compounds with a unique thiazoline-containing lipid bearing a cyclopropane unit. From the cyanobacterium Lingbya majuscula, Gerwick reported the isolation of curacin A (345) in 1994 [265], curacins B (346) and C (347) one year later [266] and, finally, curacin D (348) in 1998 [267]. The structures of these compounds were determined by detailed spectroscopic analysis. The previous absolute configuration proposed for 345 by chemical degradation [268] was confirmed by several total syntheses [269, 270]. The absolute configuration of 346 was deduced by its thermally induced interconversion with 345 [266]. Compound 345 was found to be cytotoxic against several tumor cells and inhibited tubulin assembly by binding with high affinity to the colchicine site, which is one of the two distinct drug-binding locations on tubulin [271]. This result is intriguing because curacin A did not show any structural homology to known natural and synthetic colchicine-site Hgands. A later report by Hamel et al indicated that 345 affected the morphology of the microtubules that managed to form in its presence [272]. Studies on structure-activity relationships showed that the cyclopropane-thiazoline moiety appears to be necessary, but not sufficient on its own, for repressing tubulin polymerization [273]. A 95:5 mixture of 346:347 displayed a similar cytotoxic activity to 345, indicating that the geometrical isomerization of the C-7 and C-9 olefins in curacin A has little effect on their biological activities [266]. Compound 348 was comparable to 345 as a potent inhibitor of colchicine binding but it was 7 times less active in its ability to inhibit tubulin polymerization [267].

877

R

345, curacin A

OMe OMe

346, curacin B

R= X

OMe

.

.H

347. curacin C

348, curacin D OMe

Metal chelators Anguibactin (349) is a siderophore (microbial iron transport compound) that has been isolated along with 350 from the iron-deficient cultures of a fish pathogenic bacterium, Vibrio anguillarum 775 (pJMl) [274]. The structure determination of 349 was based on extensive spectroscopic data, chemical degradations, and single crystal X-ray diffraction studies of its anhydro thiazole derivative. For this reason it was not possible to determine the stereochemistry of the side chain on the thiazoline ring. Compound 349 was shown to be a very important factor for virulence, as demonstrated by its ability to cross-feed a siderophore-deficient receptorproficient mutant of V, anguillarum, which allows the establishment of this organism in the host vertebrate. Moreover, the anguibactin receptor system has been shown to be highly specific for 349 and inert toward a range of bacterial, fimgal, and synthetic iron chelators [274].

HO

OH

O OMe

349, anguibactin

350

878

Another thiazoline compound, agrochelin (351) has been isolated from the fermentation broth of a marine unicellular bacterium belonging to genus Agrobacterium. Compound 351 showed cytotoxic activity against mouse (P-388) and human (A-549, HT-29, MEL-28) tumor cell lines from 0.05 to 0.2 |ag/ml, and chelating properties to Zn^^ JQ^S [275].

OH •

HH^'^M^^'^*^ N-

351, agrochelin

Thiazole/Thiazoline-containing Cyclic Peptides The thiazoline and thiazole rings are present in many cyclic peptides isolated from marine organisms. Most of these types of compound have been isolated from tunicates belonging to the Lissoclinum and Didemnum genus, from sponges of the genus Theonella, and from the sea hare mollusc Dolabella auricularia. The isolation of closely related compounds from cyanobacteria pointed out the symbiont origin of these metabolites. Lissoclinum Cyclopeptides Tunicate Lissoclinum species, mainly L. patella^ produces a prodigious range of novel and unusual cyclic peptides that show considerable promise as potential antineoplastic agents. They are distinguished from other natural cyclic peptides by the presence of thiazole, thiazoline, and oxazoline rings in the macrocyclic skeleton. Thirty members of this series have been isolated so far and they belong to four general families: the octapeptide patellamide (352-366), the heptapeptide lissoclinamide (367377), and the hexapeptide bistratamide (378-381). These compounds are listed in Tables 4-6.

879

Table 4. Thiazole-containing Octapeptides Isolated from the Tunicate Lissoclinium patella.

^ s X ^ N ^ J ^ ^ Thr2 R3

1 No.

O

^

Ref. 1

Name

X

Y

'352" Pateliamide A

H

Me

D-Val

L-Ile

D-Val

L-Ile

[277]

Ri

R2

R3

R4

353

Patellamide B

Me

Me

D-Ala

L-Leu

D-Phe

L-Ile

[278]

354

Pateliamide C

Me

Me

D-Ala

L-Val

D-Phe

L-Ile

[278]

356

Patellamide E

Me

Me

D-Val

L-Val

D-Phe

L-Ile

[280]

357

Patellamide F

Me

H

D-Val

L-Val

D-Phe

L-Val

[281]

358

Patellamide G ( l )

Me

Me

D-Ala

L-Ile

D-Phe

L-Leu

[282]

359

Ascidiacyclamide (3)

Me

Me

D-Val

L-Ile

D-Val

L-Ile

[283, 284]

360

Me

Me

D-Ala

L-Leu

D-Phe

L-Ile

[285] 1

361

Prepatellamide B formate (2) Ulithiacyclamide

Me

Me

D-Leu

L-1/2 Cys

D-Leu

L.l/2Cys

[286]

362

Ulithiacyclamide B

Me

Me

D-Phe

L-l/2Cys

D-Leu

L-1/2 Cys

[287]

363

Ulithiacyclamide E (1,2) Ulithiacyclamide F (1) Ulithiacyclamide G (1) Preulithiacyclamide (1,2)

Me

Me

D-Leu

L-1/2 Cys

D-Phe

L-l/2 Cys

[282]

Me

Me

D-Leu

L-1/2 Cys

D-Phe

L-1/2 Cys

[282]

Me

Me

D-Leu

L-1/2 Cys

D-Phe

L-1/2 Cys

[282]

Me

Me

L-Leu

L-1/2 Cys

L-Leu

L-1/2 Cys

[288]

364 365 366

(1) Thfi has not cyclized to an oxazoline; (2) Thr2 has not cyclized to an oxazoline; (3) Ascidiacyclamide has been isolated from an unidentified tunicate species

880

Table 5. Thiazole/Thiazoline-containing Heptapeptides Isolated from the Tunicate Lissoclinium patella Q

Thri

Ri

'^^^\^

No!

Name

X

Y

R2

Ri

ReE

367

Lissoclinamide 1

thiazole

thiazole

L-Val

D-Ile

[289]

368

Lissoclinamide 2

thiazoline

thiazole

D-Ile

D-Ala

[289]

369

Lissoclinamide 3

thiazoline

thiazole

D-Ile

L-Ala

[289]

370

Lissoclinamide 4

thiazoline

thiazole

L-Val

D-Phe

[290]

371

Lissoclinamide 5

thiazole

thiazole

L-Val

D-Phe

[291]

372

Lissoclinamide 6

thiazoline

thiazole

D-Val

D-Phe

[279]

373

Lissoclinamide 7

thiazoline

thiazoline

Val

D-Phe

[292,293]

374

Lissoclinamide 8

thiazole

thiazoline

Val

Phe

[292]

375

Ulicyclamide

thiazole

thiazole

L-Ile

D-Ala

[294]

376

Prelissoclinamide 2(1)

thiazoline

thiazole

D-Ile

D-Ala

[285]

377

Preulicyclamide(l)

thiazole

Thiazole

L-Ile

D-Ala

[285]

(l)Thri iias not cyclized to anI oxazoline

881

Table 6. Thiazole/Thiazoline Bistratamide Hexapeptides Isolated from the Tunicate Lissoclinium bistratum O

[TJo!

Name

Ri

Ri

383

Bistratamide A

Ala

Phe

384

Bistratamide B

Ala

Phe

385

Bistratamide C

L-Ala

L-Val

R,

A ring oxazoline R3 = Me oxazoline R3 = Me oxazol

Cring

thiazoline X = S

thiazoline

'[295]i

thiazoline X = S

thiazole

[295]

thiazole X = S

thiazole

oxazol X = 0

thiazole

[296, [297] [296]

R3 = H

386

Bistratamide D

L-Val

L-Val

oxazoline R3 = Me

Ref. 1

B ring

The identification and absolute configurations of the amino acids, except for the thiazole residue, were established in most cases by GC retention time correlation of the methyl ester trifluoroacetyl (ME-TFA) derivatives on a chiral column. The problem of racemization of thiazole amino acids during acid hydrolysis can be solved by treatment of the peptide with singlet oxygen prior hydrolysis and, by doing so, the stereochemistry of the side chain can be preserved. Total synthesis has played a dominant role in establishing the structures of these compounds, thus allowing the revision of some previously proposed ones. Most Lissoclinum cyclopeptides displayed moderate to high levels of cytotoxicity. Ulithiacyclamide (361) is the most potent cytotoxic molecule among the related peptides from this tunicate and is followed by lissoclinamide 7 (373). Structure-activity relationship studies have revealed that the oxazoline ring is vital for the cytotoxic activity of these compounds and that the bridging disulfide unit (or the corresponding reduced dithiol moiety) greatly enhances cytotoxicity. One possible determinant of the cytotoxic activity could be the combined effects of the molecular conformation (enforced by the disulfide link in ulithiacyclamide) and the chemical reactivity of the side chains

882

(nonbonding interactions of the disulfide group). For this reason, molecular conformation studies of some Lissoclinum cyclic peptides were performed in order to obtain additional information for the structureactivity relationship. The capacity of these cyclic peptides for metal complexation has also been studied. In relation to the true origin of these metabolites, Prochloron algal symbionts associated with L. patella have been implicated in the transfer of amino acids to their tunicate [276]. The patellins 1-6 (382-387) and trunkamide A (388) [298, 299] along with tawicyclamides A (389) and B (390) [300] represent another variation of the cyclic peptides produced by Lissoclinum species. All of these compounds lack the characteristic oxazoline ring present in most Lissoclinum cyclic peptides. Compounds 382-386 and 388 were inactive in a series of in vitro cytotoxicity assays while 387, 389, and 390 showed modest cytotoxicity, supporting the importance of the presence of the oxazoline ring for cytotoxic activity [298, 300].

382, patellin 1

383, patellin 2

384, patellin 3 Rj = L-Leu, R2 = L-Leu 385, patellin 4 Rj = Val, R2 = Leu 386, patellin 5 Rj = L-Phe, R2 = L-Val 387, patellin 6 Rj = L-Val, R2 = L-Phe

883

388, trunkamide A

389, tawicyclamide A R = L-Phe 390, tawicyclamide B R = L-Leu

Other Thiazole/Thiazoline-containing Cyclic Peptides Another tunicate, Didemnum molle, has been shown to contain a novel series of cyclic peptides. They include the hexapeptides comoramides A (391) and B (392) [301] and the heptapeptides, moUamide (393) [302], cyclodidemnamide (394) [303], and mayotamides A (395) and B (396) [301]. The structures of these compounds were elucidated from spectral data, degradation experiments and, in the case of moUamide (393), by Xray analysis. The total synthesis of cyclodidemnamide led to the revision of its structure [304]. All of them exhibited cytotoxic activity and 393 also inhibited RNA synthesis [302].

)^N

J-'-'

rVv^o

391, comoramide A

o\

N-^""" ^ ^ J !

i-v-v^o

392, comoramide B

N-HT^

^ w '~~r\ 393, moUamide

884

O^yy

9

1

394, cyclodidemamide

395^ mayotamide R = H 396, mayotamide R = Me

Sponges of the genus Theonella are another source of thiazolecontaining cyclic peptides, which to date include: keramamides F, G, H, J, and K (397-401) [305, 306, 307] from a specimen collected on Okinawa, and oriamide 402 [308] from another specimen collected in Sodwana Bay. Ise O

i,ej O S ^

Dpr Ala O I

I

Trp 397, keramamide F Rj = R2 = R3 = H (135. A "'^ ) 398, keramamide G Rj = R2 = R3 = H (13/?, A "'^) 399, keramamide H Ri = OH, R2 = Br, R3 = H (135) 400, keramamide J Ri = R2 = R3 = H (135) 401, keramamide K Rj = R2 = H, R3 = Me (135)

885

The keramamides have unique structural features containing some unusual amino acids such as isoserine, A-Triptophan, and (Omethylseryl)thiazole. Their structures, including in most cases the absolute stereochemistry, were elucidated by spectroscopic methods and degradation experiments similar to those used for the patellamides or lissoclinamides. A recent total synthesis of the simplest member of this series, keramamide J (400), indicated that its structure should be revised [309]. Compounds 397 and 401 exhibited cytotoxicity against L1210 and KB [305, 307]. Ircinia dendroides is another sponge that has been shown to contain a thiazole-containing cyclic peptide, in this case waiakeamide (403) [310].

OH

OH O

O ,J

O Q-J*k^O

402, oriamide

403, waiakeamide

Thiazole-containing Dolastatins The sea hare Dolabella auricularia has been the source of the powerful cytotastic and antineoplastic constituents designated as dolastatins [311]. Some of these compounds are thiazole-containing cyclic peptides. They were found in very small quantities in the animal (ca. 1 mg each from 100 Kg), making the isolation and structural elucidation of these peptides exceptionally challenging. A review on the dolastatins, written by G. R. Pettit in 1997, covers the reported literature regarding their isolation, characterization, biological activity, and synthesis [312]. Pettit and coworkers reported the thiazole-containing dolastatins: dolastatin 3 (404) [313], 10 (2) [314], and 18 (407) [315]. The most important dolastatin and the most potent antineoplastic and tubulin-inhibitory substance known to date is the unique linear pentapeptide dolastatin 10 (2).

886 y

.

/—CONH2 / ^^^^"2

R

•-^ V Oy-^^^J^/^

\

I

2, dolastatin 10 R = H 405, symplostatin 1 R = Me

o

404, dolastatin 3

O^N

O

406, symplostatin 2

o X) 407, dolastatin 18

Compound 2 inhibited murine and human bone marrow cell colony fomiation with and ID50 of 0.1-1 pg/ml, with complete inhibition occurring at 10-100 pg/ml. It was found to be more potent than vinblastine or taxol, with an ICsoof 0.23 nM against human ovarian cancer and colon cancer cell lines. Furthermore, dolastatin 10 was shown to be powerfully effective at binding to tubulin, inhibiting polymerization and it also non-competitively inhibits the binding of vinca alkaloids to tubulin,

887

and most resembles phomopsin-A in its activity [1]. Dolastatin 10 has been reported to be in advanced preclinical development as an anticancer agent [316]. Japanese specimens of Dolabella auricularia afforded additional thiazole-containing metabolites: the cyclic hexapeptides dolastatin E (408) [317] and dolastatin I (409) [318], and the linear bisthiazole dolabellin (410) [319]. These compounds all exhibited moderate cytotoxicity activity. More recently, the isolation of symplostatin 1 (405), a dolastatin 10 analogue [320], and symplostatin 2 (406) [321], a dolastatin 13 analogue, from the marine cyanobacterium Symploca hydnoides supported the proposal of the cyanobacterial origin of the dolastatins. Symplostatin 1 (405) exhibited a cytotoxic effect against KB cells with an IC50 value of 0.3 ng/ml as opposed to < 0.1 ng/ml for dolastatin 10 (2). Since 405 induced 80% microtubule loss at 1 ng/ml when tested on A-10 cells, its mechanism of action must be similar, if not identical, to that of 2, The isolation of 405 from a cultivable source is significant, as this potentially allows the study of its biosynthesis and the isolation of further quantities for more rigorous biological evaluation [320]. A thiazoline-containing cyclic hexapeptide, keenamide A (411), was also isolated from a mollusc, the notaspidean Pleurobranchus forskalii [322]. The structure of 411 was deduced by spectral methods and chiral amino acid analysis and it showed weak cytotoxic activity against several tumor cells.

N ^O

408, dolastatin E

409, dolastatin I

Y MeO'

y^ LV

410, dolabellin

411,keenamide A

Thiazole-containing Linear Peptides Virenamides Thiazole-containing linear peptides have also been isolated from tunicates. Virenamides A-E (412-416) were obtained from the didemnid tunicate Diplosoma virens, which contains symbiotic prokaryotic algae in its cloacal cavity. The structures of virenamides A~C were determined by HPLC analysis using Marfey's procedure [323] while the absolute stereochemistry of virenamide E (416) was proven by its synthesis from virenamide A (412) [324]. Compounds 412-416 showed modest cytotoxicity toward a panel of cultured cells and 412 also exhibited topoisomerase II inhibitory activity.

412, virenamide A R = CH2CH=CMe2 414, virenamide B R = *Pr 413, virenamide D R = H

415, virenamide C R = CH2Ph

889

Thiazole-containing Polychlorinated Peptides The sponge Dysidea herbacea and cyanobacteria Oscillatoria spongeliae (a symbiont of that sponge) and Lyngbya majuscula have been shown to contain a series of linear polychlorinated peptides bearing a thiazole residue. They can be divided into three groups: the dysidenin, the isodysidenin, and the dysideathiazole series. Most dysidenin (e.g. 419) and isodysidenin (e.g. 417) metabolites were reported from D, herbacea before 1985 [7], and only one new member of the isodysidenin family has been published since that date and this is 9,ll-didechloro-13demethylisodysidenin (418), which was isolated from the same source [325]. Metabolites of the dysidenin and isodysidenin series can be distinguished by the chemical shift values of the H-6 and H-7 protons: the isodysidenin 5R series is characterized by chemical shift values close to 5 2.9 and 1.5 for H-6, and 2.7 for the H-7 signal [325]. Furthermore, it was proposed that the stereochemistry at H-5 strongly influences the optical rotation of the metabolite; thus compounds with positive [OJD values are ascribed to the isodysidenin series (e.g. 5R) whereas the negative {a\xy values are ascribed to the dysidenin series (e.g. 5S). All known natural dysidenins and isodysidenins have 25 and 75 configurations [326].

417, 13-demethylisodysidenin X = CI

419, dysidenin

418,9,11-didechloro-13-demethylisodysidenin X = H

The dysideathiazoles (420-424) were also reported from the same sponge and their structures were determined by spectroscopic methods and X-ray analyses [326]. More recently, new variations of this type of unique marine metabolite were obtained. Herbamide A (425) [327] was isolated from a collection of A herbacea, presumed to be rich in cyanobacteria because of the rich presence of chrorophyll, and barbamide (426) [328] was obtained from the cyanobacterium Lyngbya majuscula. Faulkner and

890

Unson [329] have demonstrated by flow cytometry that the polychlorinated metabolite 13-demethylisodysidenin (417) is localized in the cells of the filamentous cyanobacterium Oscillatoria spongeliae, which is the major prokaryotic symbiont of the sponge Dysidea herbacea. This result provided additional evidence regarding the symbiont origin of these compounds. Several reports on the biosynthesis of barbamide have recently been published. Feeding experiments with differently ^^C-labeled L-leucines have found the origin of the trichloromethyl group [330] while the use of [2-^'^C/^N] glycine demonstrated that cysteine is the origin of the thiazole ring in barbamide [331]. R K^ O N^S

"

420, dysideathiazole Rj = H, X = Y = CI 421, A^methyldysideathiazole Ri = Me, X = Y = CI 422,10-dechloro-//-methyldysideathiazole Rj = Me, X = CI. Y = H 423, 10-dechlorodysideathiazole Rj = H, X = CI. Y = H 424,9,10-didechloro-iV-metyldysideathiazole Ri = Me, X = Y = H

cai XJ

Me

425, herbamide A

OMe CCI3

426, barbamide

Dysidenin (419) has been shown to have inhibition activity of the iodide transfer in thyroid cells through a different mechanism than ouabain [332]. Dysideathiazoles (42(M24) were strongly deterrent in fish-feeding experiments [326] and barbamide (426) has shown moUuscicidal activity [328].

891

More recently, another cyanobacterium belonging to Lyngbya genus, L bouillonii, has been shown to contain a new tetrapeptide, lyngbyapeptin A (427), which contains the rare 3-methoxy-2-butenoyl moiety and a thiazole ring [333]. OMe

OMe

427, lyngbyapeptin A

Thiazole-containing Macrolides A series of macrolides bearing a thiazole moiety have been isolated from tunicates and sponges. The tunicate Lissoclinum patella has afforded the patellazoles A-C (428-430), which were found to be potent cytotoxins in the NCI human cell line protocol with mean ICso values of lO'^-lO"^ |ig/ml as well as exhibiting antifungal activity [334, 335]. Furthermore, patellazole B (429) exhibited very potent antiviral activity against Herpes symplex viruses [334].

428, patellazole A Rj = R2 = H 429, patellazole B R^ = H, R2 = OH 430, patellazole C Rj = R2 = OH

892

The theonezolides A-C (431-433) were isolated from the sponge Theonella sp. They are 37-membered macrocyclic compounds consisting of two principal fatty acid chains with various functionalities such as a sulfate ester, an oxazole, and a thiazole [336, 337]. Compounds 431-433 displayed cytotoxic activity against L1210 and KB cells and were found to have a unique bioactivity in terms of induction of rabbit platelet shape change and aggregation [338].

431,theonezolide A n = 3 432, theonezolide B n = 1 433, theoiiezolide C n = 5

A thiazole-containing macrolide with a rare dilactone functionality, pateamine (434), was isolated from a New Zealand sponge Mycale sp. [339]. Its relative and absolute stereochemistry was confirmed by total synthesis [340]. Pateamine was shown to be very potent and selective against P388 cell lines (although inactive in vivo) and displayed in vitro antifungal activity [339]. Furthermore, compound 434 displayed potent immunosuppressive properties (mixed lymphocyte reaction) with low cytotoxicity through inhibition of T cell receptor-mediated ILtranscription, indicating that its mode of action is similar to those of FK506 and CSA but distinct from that of rapamycin [340].

893

HoN'

434, pateamine

Thiazole-containingPyridoacridines Thiazole-containing pyridoacridines are a group of fused ring alkaloids having a pyrido[4,3,2-m,n]thiazolo[3,2-Z>]acridinium skeleton related to shermilamines, varamines and diplamine, which were discussed in the sulfide section [37]. The pyridoacridines have a characteristic UV absorption pattern [A.max(MeOH) 245, 307, and 361 nm], which is highly sensitive to the pH of the medium. Dercitin (435) was the first compound of this series to be discovered and it was obtained from an unidentifed deep-water sponge belonging to the genus Dercitus [341]. Its structure was tentatively deduced by a combination of long-range ^H-^^C and ^'^C-^'^C NMR correlations on the parent compound and its tetrahydro derivative but the regiochemistry of the thiazole moiety was assigned incorrectly. Subsequently, the same regiochemistry was proposed for cyclodercitin (436), a minor metabolite of the sponge Dercitus sp., and for nordercitin, dercitamide, and dercitamine (437-439), all of which were obtained from another deepwater sponge, Stelleta sp. The same regiochemistry was put forward because the proposed structures were based on long-range ^H-^^C correlation information and spectral comparison to dercitin [342]. The regiochemistry of the thiazole moiety was correctly assigned by interpretation of the HMBC experiment and the VH-C-N-C values in the kuanoniamines A-D (444, 440, 438, and 441), which represent the following compounds of this series and were isolated from an unidentified tunicate and its mollusc predator Chelynotus semperi [343]. Single crystal X-ray diffraction together with long range ^H-^ C coupling constants of stellettamine (445), obtained from a tunicate tentatively identified as a

894

species of Cystodytes, allowed th*^ reported regiochemistry of the thiazole moiety of dercitin and the other four related alkaloids to be corrected [344]. Compound 446 was also isolated in this study. The total synthesis of dercitin and other related compounds confirmed their structures definitively [345]. Additional members of this series were isolated from sponges belonging to the genus Oceanapia: sagitol (447), the first pyridoacridine alkaloid in which the aromatic system has been disrupted, was obtained from O. sagittaria [346] while the iV-deacyl derivative of the kuanoniamines, compound 442, was isolated from Oceanapia sp. [347]. Furthermore, compound 443 was obtained from the Fijian tunicate Cystodytes sp. [41]. The presence of similar polyaromatic alkaloids possessing a common tetracyclic ring system (a pyrido[4,3,2-/w,«]acridine skeleton) in unrelated phyla led to a symbiont origin being proposed for these compounds [343].

NMe2

435, dercitin

436, cyclodercitin

437, nordercitin R = NMe2 438, dercitamide (kuanoniamine C) R = NHCOEt 439, dercitamine R = NHMe 440, kuanoniamine B R = NHCOCH2CHMe2 441, kuanoniamine D R = NHCOMe 442, iV-deacylkuanoniamine R = NH2 443,R = NHCOCH<:Me2

895

NM2

444, kuanoniamine A

445, stellettamine

446 447, sagitol

Most of these compounds displayed very potent cytotoxic activity against several tumor cells. Dercitin (435) was also active in vivo, prolonging the life of P388-bearing mice with a T/C of 170 at 5 mg/Kg. Compound 435 inhibited both DNA and RNA synthesis, DNA polymerase and I/DNase nick translation, bound to calf thymus DNA, and relaxed supercoiled (|)X174DNA [348]. The compounds also showed immunosuppressant (435, 437, 438, and 439), antiviral (435), and insecticidal (438 and 441) activities. Furthermore, some of these compounds displayed moderate affinity to benzodiazepine binding sites of GAB A A receptors (438, 441, and 442) and affinity to A r and A2Aadenosine receptors (441). The metal binding properties of kuanoniamine D (441) with divalent metal ions such as Co^^, Cu^^, and Zn^"^ was also investigated using both ^H-NMR and fluorescence spectroscopy [341347]. Miscellanous Thiazole-containing Metabolites Other miscellanous thiazole-containing metabolites were obtained from a variety of marine organisms. Compounds 448 and 449 were isolated from the tunicate Aplidium pliciferum and their structures were established from spectral data and confirmed by synthesis [349]. Very few secondary metabolites have been reported from hydroids. The thiazole tridentatol C

896

(450) and the methylsulfides tridentatols A and B (451 and 452) are phenolic metabolites isolated from the hydroid Tridentata marginata. Interpretation of spectral data and a single crystal X-ray diffraction study of 450 allowed the determination of their structures. These metabolites may function as protection for 7. marginata from damaging solar UV radiation. Furthermore, tridentatol A (451) deters feeding by a common hydroid predator [350]. SMe SMe

N^SMe

N ^SMe

OH

448, R = 0 449, R = <

OH

450, tridentatol C

OH 451, tridentatol B

OH 452, tridentatol A

Benzothiazoles rarely occur as natural products. Four benzothiazoles (the already cited thiol 9 and compounds 453-455) were isolated from the fermentation culture extracts of Micrococcus sp., a marine bacterium obtained from tissues of the sponge Tedania ignis. The use of such benzothiazoles as starting materials in the industrial synthesis of aldose reductase inhibitors and carbonic anhydrase inhibitors was proposed as a biotechnological application. [13]. More recently, a new benzothiazole, S1319 (456), has been reported from the sponge Dysidea sp. and its structure characterized as 4-hydroxy-7-[( 1 -hydroxy-2methylamino)ethyl]-1,3-benzothiazole-2(3//)-one. This compound has been reported as the first example of a sponge-derived P2-adrenoceptor agonist. The potential of S1319 as a strong bronchodilator has been suggested since it seemed to be more potent than epinephrine and isoproterenol in the relaxation of the tracheabronchial muscle and 1000 times more active, but with similar selectivity, than salbutamol as a P2adrenoceptor [351]. Finally, the unique structure of mycothiazole (457), a disubstituted thiazole isolated from the sponge Spongia mycofijiensis, was established by extensive NMR analysis and exhaustive interpretation of its HREIMS spectrum. This compound was completely active at 50 |ag/ml in an

897

antihelminthic in vitro assay against Nippostrongylus braziliensis. A biosynthetic relationship between latruculin B (338) and mycothiazole was proposed [352].

ah N

453, R = Me

Me

XXVo 455

456

454, R = OH

457, mycothiazole

MISCELLANEOUS SULFUR-CONTAINING MARINE NATURAL PRODUCTS A group of miscellaneous sulfur-containing marine natural products are discussed in this section. Neamphine (458) is a sulfur-cont?^ming aromatic heterof^ycle that was the first example, either natural or synthetic, of the imidazo[4,5-e]-l,2thiazine ring system and it was isolated from the sponge Neamphius huxleyi [353]. Its structure has been solved by crystal X-ray diffraction analysis and bears an interesting biogenetic resemblance to ovothiol A (4). Neamphine exhibited in vitro cytotoxicity against L1210 with ED50 < 10|ig/ml. The peptide guanidine derivatives minalemines D-F (459-461), isolated from the New Caledonian tunicate Didemnum rodriguesi, were the first marine metabolites containing a sulfamic acid functional group [354]. Their structures were elucidated through their spectral data and by chemical transformations.

898

1T'^ ]f

Y

"^3?

or-

NH

"^^Y''^^-'''^^"^''rV^N\^---^N'^NH, NH

OR

458, neamphine

O

459, minalemine D R = C7H15 460, minalemine E R = CgHjy 461, minalemine F R = C9Hi9

The first examples of marine sterols with the sulfamate ester fimctionality were haplosamates A and B (462 and 463), which were obtained from two sponges from the Philippines, a Xestospongia sp. (Haplosclerida, Petrosiidae) and an unidentified haplosclerid sponge [355]. The structures of haplosamates A and B were determined by interpretation of spectral data and the compounds were found to inhibit HIV-1 integrase with IC50 values of 50 |ig/ml and 15 |ig/ml, respectively. A symmetrical ethylene bis(alkylxanthate) 464 has been isolated from the algae, Dictyosphaeriafavulosa, The structure of 464 was elucidated as carbonodithioic acid 5,5-1,2-ethendiyl 0,0-diisobutyl ester by spectroscopic analysis and confirmed by X-ray diffraction [356]. Two thiourea derivatives (465 and 466) were found in the more polar fractions of the sponge Epipolasis kushimotoensis. These derivatives are characterized by the absorption band at 1500 cm"^ due to C=S. They also showed moderate activities against L1210 tumor cells (EDso's of 4.1 and 3.7 |ig/ml, respectively) [156]. The C=S functionality can also be found in compound 467, obtained from the sponge Zyzzya massalis [357].

HO OH 462, haplosamate A Rj = SOaNa, R2 = H 463, haplosamate B Ri = R2 = S03Na

464

899

Although the unusual dimerized phosphorohydrazide thioate 468 was isolated from the marine fungus Lignincola laevis, it was postulated that this compound is a biotransfomiation product of an insecticide that was present in the seawater. However, attempts to locate a comparable structure through the chemical registry have been unsuccessful. This compound showed cytotoxicity against L1210 cell line at a level of 0.25 |ig/ml level [358].

467

465

466 468

DISTRIBUTION ACCORDING TO THE DIFFERENT PHYLA It is interesting to compare the number of the non-sulfated sulfurcontaining marine natural products reported from the different phyla of organisms. Of the 468 compounds described here, the approximate breakdown by source phylum is as follows: sponges, 207 compounds; tunicates, 133; marine microorganisms, 72; molluscs, 17; algae, 13; echinoderms, 12; bryozoans, 9; hydroids, 3; anemone, 1; fish, 1 (see Fig. (1). Indeed, sponges and tunicates are the richest source of this type of compounds. This result diverges to the distribution of the sulfated compounds from marine organisms where more than 350 sulfated derivatives out of 500 are known from the phylum Echinodermata [2]. It is worth to mention that the most abundant sulfur-containing metabolites isolated from sponges are terpene isothiocyanates (82 out of 207) while in

900

tunicates are the thiazole/thiazoline-containing cyclic peptides (50 out of 133). E3 sponges ^ • \

1

^

1%

3%-^\ \ Jj

n tunicates D microorganisms

/

/

1

^^:Xn^M'i'i*!'i'i'i'!'i

^Si'i'i'i'i'i'i'i* ilil

• molluscs

••III'!'!'!'!'!'!'!'!

L..iiiiiiilllllllllil.'•'.'•'•'.'.'•

2B%^^™**^

(Q algae • echinoderms Bbryozoans • others

Fig. (1). Phyletic distribution of non-sulfated marine sulfur-containing natural products.

ADDENDUM Since the submission of this manuscript for publication, some new references have appeared in the literature related to this issue. A new methylsulfinyl-containing theonellapeptolide-related cyclic depsipeptide has been isolated from an Okinawan marine sponge Theonella sp. The structure of 469 was elucidated on the basis of the 2D NMR data, amino acid analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS), and chemical means. Compound 469 exhibited antimicrobial activity [359]. A new isothiocyanate having an amphilectene framework, compound 470, has been isolated from the Caribbean sponge Cribochalina sp. [360]. The Australian sponge Echinodictyum sp. has been shown to contain four novel sulfur-containing bisindol compounds, the echinosulfonic acids A to C (471-473), which incorporate a very rare tertiary sulfonic acid, along with a new sulfone, echinosulfone A (474). Compounds 471-474 showed antibacterial activity [361].

901 ,^0

V, NCS

O 470

o

/^.^^.\fyl

471, R = H

474

472,R = Me 473, R = Et

A new diketopiperazine (475) and two new epidithiodioxopiperazines (476 and 477), whose structures are very similar to leptosins (e.g. 121) isolated from the fungal strain Leptosphaeria sp. [125-128], were obtained from a marine isolate of the fungus Penicillium and they exhibited cytotoxic activity [362].

476, R = H

MeS 475

^Rr° ysi'i.

477, R = OH

902

An additional sulfur-contaming p-carboline belonging to the eudistomin family, A^(10)-methyleudistomin E (478) was obtained from the Caribbean tunicate Eudistoma olivaceum [363].

.N-o MeHN''^v^^S 478, A^(10)-methyleudistomin E

New thiazole-containing cyclicpeptides were reported very recently. Homodolastatin 3 (479) and kororamide (480) were isolated from a Palau collection of the macroscopic cyanophyte Lingbya majuscula. The structures of 479 and 480 were determined by interpretation of their spectroscopic data and chemical degradation. This work constitutes an additional support of the cyanobacterial origin of the dolastatin peptides [364]. y

A-CONH2

o,V~H5 CONH2

%

^OH 479, homodolastatin 3

430. kororamide

The red alga Ceratodictyon spongiosum containing the symbiotic sponge Sigmadocia symbiotica yielded two isomers of a new and bioactive cyclic heptapeptide, cis, c/^-ceratospongamide (481) and trans, transceratospongamide (482). Their structures were established through extensive NMR spectroscopy, chemical degradation, chiral analysis, and molecular modeling. The trans, /ra/i^-isomer, compound 482, exhibited potent inhibition of SPLA2 expression in a cell-based model for

903

antiinflammation (ED50 32 nM), whereas the cis, c/^-isomer 481 was inactive. Compound 482 was also shown to inhibit the expression of a human-sPLAi promoter-based reporter by 90% [365].

QM-<<^ 481,cw, c/s-ceratospongamide

Cr^>tVO

01

'

N ^

482, trans, /m«5-ceratospongamide

During this time, the total synthesis of several marine sulfur-containing natural products cited in this review has been reported and they confirmed the suggested structures. This is the case of the synthesis of some sulfonoceramides (e.g. flavocristamide A (318) discussed in the sulfonic acid and their derivatives section [366], and the synthesis of the thiazolecontaining compounds bistratamide D (381) [367], trunkamide (388) [368], moUamide (393) [369], dolastatin I (409) [370], and virenamide B (414) [ 371]. ABBREVIATIONS ED5o(= IC50) T/C P388 L1210 B16 HCT8 KB PLA2

= Dose which inhibits cell growth to 50% of the control growth = Treated/Control Mouse lymphocytic leukemia cell cultures = Lymphoid leukemia cell cultures Melanoma cell cultures Human Colon Adenocarcicoma cell cultures = Mouth cell cultures = = Phospholipase A2

ACKNOWLEDGMENTS This work was financially supported by grants from CICYT (MAR990287) and Xunta de Galicia (XUGA-10301A97).

904

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

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ABSORPTION, METABOLISM AND BIOLOGICAL ACTIVITIES OF CHLOROGENIC ACIDS AND RELATED COMPOUNDS HIDEKO MORISHITA AND MOTOYO OHNISHI* Faculty ofEducation, Wakayama University, 930 Sakaedani Wakayama 640-8510, Japan; "^Department ofPharmaceutical Science, Institute of Medical Science, Kansai Shinkyu Medical College, J-I I, 2 Wakaba Kumatori Sennan Osaka 590-0482, Japan ABSTRACT: Chlorogenic acids are polyphenolic compounds that occur ubiquitously in foods of plant origin. They are quinic acid esters of hydroxycinnamic acid. Recently, naturally occurring plant phenolics have attracted considerable attention in relation to their physiological potential. Depending upon the conditions, phenolic compounds can be either beneficial or detrimental to biological processes. We comprehensively summarized chlorogenic acids and related compounds in absorption, metabolism and biological activity. Chlorogenic, caffeic and quinic acids are well absorbed in humans and rats. Metabolic transformations of chlorogenic acids in the human system may be crucial for their biological effect. The antioxidant activities of chlorogenic acids are preserved by inhibiting the formation of reactive oxygen species or by scavenging them. As a result, chlorogenic acids may play beneficial role in the prevention of certain oxidative diseases. The observed pharmacological activities of medicinal plants relate to the chlorogenic acids constituents of them. Despite the abundance of biological data demonstrating the antioxidant activities of chlorogenic acids, it remains controversial whether these compounds are potent antioxidants or pro-oxidants. Chlorogenic and caffeic acids switch from anti- to pro-oxidant activity, depending on their concentration, on the presence of free transition metal ions, or on their redox status.

INTRODUCTION The term "chlorogenic acid" was introduced in 1846 [1] to describe a coffee bean component that was later characterized as 5-(9-caffeoylquinic acid [2], which is an ester of caffeic acid with quinic acid. Such esters are known as depsides, and the term "chlorogenic acids" is now often used to refer to the range of depsides that have been found in plants and it is in this sense that this term will be employed throughout this paper. Chlorogenic acids occur ubiquitously in plants. They are esters of hydroxycinnamic acids with quinic acid. The structures of chlorogenic

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acids are shown in Fig. (1). Hydroxycinnamic acid compounds are almost exclusively derived from caffeic, ferulic and p-coumaric acids, while sinapic acid is comparatively rare. Hydroxycinnamic acids and their derivatives are capable of existing in Z (cis) and E (tram) forms, and while there is evidence that the natural forms are all E (tram) [3], isomerization inevitably occurs during extraction, and mixtures of isomers are frequently isolated [4]. Hydroxycinnamic acids

CH "^CH—COOH

CafiFeic acid p-Coumaric acid Ferulic acid Sinapic acid

Ri H H H OCH3

R2 OH OH OH OH

R3

OH H OCH3 OCH3

Chlorogenic acids

,COOH OR4

Quinic acid CA : caffeoyl Co : coumaroyl FA : feruloyl

3-O-Caffeoylquinic acid 4-O-Caffeoylquinic acid 5-O-Caffeoylquinic acid (Chlorogenic acid) 3,4-Di-O-caffeoylquinic acid 3,5-Di-O-caffeoylquinic acid 4,5-Di-O-caffeoylquinic acid 1,3-Di-O-caffeoylquinic acid l,5-Di-(9-caffeoylquinic acid 3,4,5-Tri-O-caffeoylquinic acid 3-O-Coumaroylquinic acid 4-O-Coumaroylquinic acid 5-O-Coumaroylquinic acid 3-(9-Feruloylquinic acid 4-(9-Feruloylquinic acid 5-O-Feruloylquinic acid 3-(9-Cafreoyl-4-<9-feruloylquinic acid 3-0-Feruloyl-4-C>-caffeoylquinic acid

^

R7

R4

R5

H H H

CA H H

H CA H

H H CA

H H H CA CA H H H H H H H H H

CA CA H CA H CA Co H H FA H H CA FA

CA H CA H H CA H Co H H FA H FA CA

H CA CA H CA CA H H Co H H FA H H

Fig. (1) Chemical structure of chlorogenic acids

Since the new lUPAC recommendations [5], the nomenclature of quinic acid isomers is very confusing in the literature. Therefore, the latest lUPAC nomenclature is used throughout this paper instead of the older, but still useful, nomenclature. In the lUPAC nomenclature quinic acid is now treated as cyclitol. In the preferred configuration, the carboxy group and the C-4 and C-5 hydroxy groups are equatorial, with the C-1 and C-3 hydroxy groups are axial. In the lUPAC system, the former 3-0-acylquinic acids are now renamed 5-0-compounds, and the

921

former 5-(9-acylquinic acids are called 3-0-compounds. Chlorogenic acid [2] (previously 3-0-caffeoylquinic acid) is now the 5-ester and neochlorogenic acid [6] (the former 5-0-caffeoylquinic acid) is now the 3-ester, while cryptochlorogenic acid [7] remains the 4-ester. Isochlorogenic acid was later identified as a mixture of several di-0caffeoylquinic acids [8]. The major dietary sources of chlorogenic acids are vegetables, fruits, and beverages. The levels of chlorogenic acids of fruits, vegetables, and spices have been described in detail by Herrmann [9]. A review of chlorogenic acids in coffee by Clifford contains tables of contents [10]. Recently, many polyphenolic compounds belonging to the tannin family have been isolated from many medical plants and characterized. Mixtures of caffeoylquinic acids, called caffeetannins, have been isolated from some species of Compositae [11]. Rosmarinic acid, which has been called labiataetannin, and its analogues and derivatives have been found in some species of Labiatae and several other families [12]. Recently, naturally occurring plant phenolics have attracted considerable attention in relation to their physiological potential. Depending upon the conditions, phenolic compounds can be either beneficial or detrimental to biological processes. The potential biological effect predicted from in vitro studies may be modulated in vivo due to metabolism after ingestion of the parent compound. Metabolic transformation of chlorogenic acids in the human system may be crucial for their biological effect. We review comprehensively chlorogenic acids and related compounds on absorption, metabolism and biological activities. ABSORPTION Although the major questions here are to what extent are chlorogenic acids absorbed from the gastrointestinal tract and what factors affect their absorption, there are few, if any, specific reports on absorption of chlorogenic acids. This subject evidently needs to be studied further. Intestinal microorganisms readily hydrolyze chlorogenic acid to quinic and caffeic acids [13]. Adamson et al [14] have investigated the fate of [^"^C] labeled quinic acid in rats and monkeys. Total radioactivity has been measured in the urine, feces and expired air of rats. As a consequence, the excreted radioactivity is contained in the intact quinic

922

acid and its metabolites. In rats, about 90% of the administered ^"^C radioactivity can be accounted for, some 33% being excreted in the urine and 22% in the feces, while 32% appeared in the expired air as CO2. In rhesus monkeys, only the urine has been examined. Urinary excretion accounts for 21 to 46% of the administered ^^C radioactivity. Booth et al [15] have reported that the ingestion of chlorogenic acid, caffeic acid, and coffee by humans and rats leads to the excretion of more than ten phenolic acids in the urine. The relative amounts of various urinary metabolites of caffeic acid are estimated by comparing the size and color intensity of the spots on paper chromatogram produced from known quantities of the pure compounds with those of appropriate dilutions of the urine. The following amounts have been excreted in human urine when 1 g of caffeic acid is ingested: vanilloylglycine 150 mg, m-hydroxybenzoic acid (m-HBA) 30 to 35 mg, ferulic acid 40 mg, feruloylglycine 35 mg, dihydroferulic acid 15 to 20 mg, and vanillic acid 10 to 15 mg. However, when rat was given 150 mg of caffeic acid by stomach tube, the total output of the various urinary metabolites were as follows: Tw-hydroxyphenylpropionic acid (m-HPPA) 18 mg, ferulic acid 20 to 25 mg and vanillic acid 5 mg. Conclusion The evidence described above suggests that chlorogenic, caffeic and quinic acids are well absorbed in humans and rats. We need further study of the mechanism of chlorogenic acids absorption across the intestinal membrane itself METABOLISM The metabolism of chlorogenic acids is relevant because a major portion of administered chlorogenic acids is excreted in the urine after more or less extensive modification in the body. Thus, a potential biological effect predicted from in vitro studies may be modulated in vivo due to metabolism after ingestion of chlorogenic acids. In the metabolism of chlorogenic acids, two compartments are important. The first consists of tissues in the body, such as the liver, where biotransformation enzymes act upon absorbed chlorogenic acids and their absorbed colonic

923

metabolites. The second metabolically active compartment is the colon, where microorganisms degrade unabsorbed chlorogenic acids. When human urine has been examined after the ingestion of chlorogenic acid [15], the most prominent metabolites are caffeic acid, m-hydroxyhippuric acid (/w-HHA), the glucuronide of m-coumaric acid, and dihydroferulic acid. It is indicated that the first stage in the metabolism of chlorogenic acid is hydrolysis to caffeic and quinic acids and both products are further metabolized. Enzymatic Transformations of Caffeic Acid in Body Tissues Within the body tissues the changes of caffeic acid are methylation of the hydroxyl group in the 3 position, p-oxidation of side chain and conjugation with glycine or glucuronic acid. In the 1970s, research on the metabolism of caffeic acid was advanced by Peppercorn and Goldman [16]. When caffeic acid was added to the diet of germ-free rats, caffeic and ferulic acids were detected in the urine, showing that o-methylation occurs. Subsequently, when dihydrocaffeic acid was fed to germ-free rats under similar circumstances, only dihydrocaffeic and dihydroferulic acids were excreted. The methylation of caffeic and dihydrocaffeic acids can be attributed to the action of catechol o-methyltransferase [17], an enzyme found in rat liver and other mammalian tissues. After oral administration of caffeic acid to rats, small amounts of vanillic acid and vanilloylglycine are excreted. The conversion of phydroxycinnamic acid into /7-hydroxybenzoic acid is found in rat liver mitochondria [18]. Studies with/7-hydroxy[U-^^C]cinnamic acid have showed that ^'*C02 is released during reaction, indicating that reaction probably followed the ^-oxidation type reactions, the two carbon being first removed as acetyl-CoA, and then oxidized to CO2. It is assumed that conversion of ferulic acid formed by methylation of caffeic acid into vanillic acid occurs in rat liver mitochondria. Vanilloylglycine, feruloylglycine and /w-coumaric acid glucuronide are identified after oral administration of caffeic acid to humans [15]. Intraperitoneal injection of the sodium salt of caffeic acid to rats also produces these conjugates. Thus, conjugation with glycine or glucuronic acid in body tissues is demonstrated.

924 Conclusion

Caffeic acid is metabolized by liver enzymes to give ferulic, vanillic acids and their glycine conjugates, which may be excreted into urine. In addition, dihydroferulic acid is produced by catechol o-methyltransferase in the liver. Because of the specificity of this enzyme, only ortho hydroxy-methoxy metabolites may be formed. These reactions may occur in rats as well in humans [15]. Fig. (2) shows the metabolic reactions of caffeic acid in body tissues.

OH

Vanillic acid

OH

Dihydrocaffeic acid

Fig. (2)

OH

Dihydroferulic acid

Metabolic reactions of caffeic acid in body tissues

Metabolism of Caffeic Acid by Microorganisms In the 1950s, it has been reported that chlorogenic acid is invariably found in cider apple juices [19]. In some varieties, it may constitute more than 0.25% (w/v) of the juice. Lactobacillus hydrolyzes chlorogenic acid to caffeic acid and quinic acid during cider fermentation [19]. Both of these acids are then further metabolized by Lactobacillus,

925

A pure culture of the organism was inoculated into a basal medium with the addition of 0.025% caffeic acid. After 7 days incubation at 25°C under conditions of reduced oxygen tension, the caffeic acid was completely metabolized. Metabolites of caffeic acid are identified as dihydrocaffeic acid and ethyl catechol, respectively. In the 1960s, it has been reported that a constitutive enzyme present in strains oiAerobacter decarboxylates caffeic acid to 4-vinylcatechol nonoxidatively [20]. Several cinnamic acids have been tested and the decarboxylation product from /?-coumaric acid has been identified as 4-vinylphenol. Thus, the bacterial enzyme activity requires a relatively unhindered 4-hydroxy group on the aromatic ring and an acrylic acid side chain. Booth et al [15] have reported on the metabolic transformation of caffeic and chlorogenic acids by the animal body. The ingestion of caffeic acid by humans leads to the appearance of a large number of metabolites in the urine, including ferulic acid, feruloylglycine, dihydrocaffeic acid, dihydroferulic acid, m-coumaric acid, m-HPPA, mHHA, m-coumaric acid glucuronide, vanillic acid, and vanilloylglycine. When rats receive caffeic acid, a species difference is noted in that mHPPA is the major metabolite, although some ferulic acid, dihydroferulic acid, and traces of 1-carbon side chain compounds may be detected in the urine. Urinary metabolites that appear after ingestion of caffeic acid indicate that these transformations are also the basis of caffeic acid metabolism in humans and experimental animals. Presumably, the intestinal micro flora of the host is responsible for these reactions, since these metabolites are diminished in response to the concurrent administration of neomycin [21, 22]. A number of different organisms have been isolated from the human gastrointestinal tract and tested for their ability to transform caffeic acid [23]. Caffeic acid is transformed by human intestinal microflora (Fig. (3)). Peptostreptococciis sp. and one of the two strains of Clostridium perfringens isolated from human feces are capable of reducing caffeic acid to dihydrocaffeic acid (reaction A). The dehydroxylation of dihydrocaffeic acid to w-HPPA requires a mixed culture o^ Escherichia coli (E. coU) and Streptococcus fecalis var, liqiiifaciens\ no reaction is detectable in an incubation with either organism alone (reaction B). Otherwise, Streptococcus fecitim decarboxylates caffeic acid to yield 4-vinylcatechol (reaction C). The microorganism(s) responsible for the reduction of 4-vinylcatechol have

926 CH.CHXOOH

/w-HPPA

4-Vinylcatechol Fig. (3)

4-Ethylcatechol

Metabolic reactions of caffeic acid by microorganisms Caffeic acid is reduced to dihydrocaffeic acid, which is dehydroxylated to m-HPPA. In another pathway, caffeic acid can be decarboxylation to yield 4-vinylcatechol which is reduced to 4-ethylcatechoI [16,23].

not been identified. They are present in successive transfers into a liquid medium containing an inoculum human feces (reaction D). None of the organisms used in this study is capable of carrying out more than one reaction of the overall pathway of caffeic acid metabolism. These results suggest that caffeic acid metabolism is the result of the interactions of different bacteria within the gastrointestinal tract and raises the question of whether the distribution of caffeic acid metabolites as they appear in urine may be correlated with the nature of the intestinal mi crofl ora of the host. Information related to this question is obtained by comparing the metabolism of caffeic acid in germ-free and various gnotobiotic rats [16, 17]. When caffeic acid has been added to the diet of conventional rats, large quantities of m-HPPA and smaller quantities of caffeic, ferulic, dihydrocaffeic, dihydroferulic acids, and ethylcatechol are detected in the urine, while germ-free rats excrete only caffeic and ferulic acids when caffeic acid has been ingested. When dihydrocaffeic acid has been fed to germ-free rats under similar circumstances, only dihydrocaffeic acid and dihydroferulic acid are excreted. Vinylcatechol is recovered

927

unchanged in the urine after being fed to germ-free rats. Thus, germfree rats do not exhibit any change in caffeic acid metabolism by intestinal flora. This observation is consistent with the role of indigenous microflora in caffeic acid metabolism in conventional rats. The methylation of caffeic and dihydrocaffeic acids, which occurs in germ-free rats, may be attributed to the action of catechol omethyltransferase, an enzyme found in rat liver and other mammalian tissues [17]. Gnotobiotic rats harboring a strain o^ Streptococcus group N. transforms caffeic acid to dihydrocaffeic acid. The ability of these gnotobiotic rats to reduce caffeic acid can be correlated with the capacity of their intestinal microflora to perform this reaction when cultivated on artificial media. Bacteria grown in culture, however, are capable of reactions that were not necessarily detectable in gnotobiotic rats infected with these bacteria. Lactobacillus sp, 1 transforms caffeic acid to vinylcatechol in culture, however, gnotobiotic rats infected with this organism do not excrete vinylcatechol when fed caffeic acid. Gnotobiotic rats infected with four strains of bacteria excrete ethylcatechol and m-HPPA in their urine when fed caffeic acid. These metabolites are not detectable in cultures of the four bacteria grown in the presence of caffeic acid nor, as described above, in the urine of germ-free rats given the appropriate metabolic precursors. A more likely explanation for these findings is that these transformations may be attributed to one or more of the four bacteria whose metabolic capacities may be altered by conditions in the animal host. These studies on germ-free and gnotobiotic rats further support the role of the intestinal microflora in converting caffeic acid either to m-HPPA or to ethylcatechol in conventional animals. Conclusion

The principal transformations of caffeic acid mediated by microorganisms are reduction of the unsaturated aliphatic side chain, dehydroxylation, and decarboxylation. Metabolism of Quinic Acid by Microorganism The ingestion of quinic acid by humans leads to the appearance of

928

hippuric acid in the urine [24-27]. The administration of neomycin in a dose sufficient to inhibit bacterial multipHcation in the intestine may prevent the conversion of quinic acid into hippuric acid in humans and rats [28, 29]. The fates of quinic acid in 22 species of animals have been investigated [14]. In three species of Old World monkeys, orally administered quinic acid may be extensively aromatized (20-60%) and excreted in the urine as hippuric acid. In three species of New World monkeys, in three species of lemurs, in the dog, cat, ferret, rabbit, rat, mouse, guinea pig, hamster, lemming, fruit bat, hedgehog and pigeon, oral quinic acid may be not extensively aromatized (0-5%). The observed species difference in the aromatization of quinic acid depends on variation in the gut flora rather than on variation in enzyme activity in the tissues of these animals. In rhesus monkeys, shikimic acid given orally is excreted as hippuric acid (25-56%)), but not in rats. It is known that certain bacteria convert quinic acid into aromatic compounds. Davis has established a route for the biosynthesis of aromatic amino acids from carbohydrates using biochemical mutants ofE. coli [30]. Shikimic acid forms a central metabolite. The three aromatic amino acids; phenylalanine, tyrosine and tryptophan are derived from a seven carbon straight chain compound 3-deoxy-7-phospho-Darabinoheptulosonic acid. This compound then cyclizes to give 3dehydroquinic acid. The remaining biosynthetic steps common to the three aromatic amino acids may be regarded as the successive introduction of further double bonds until aromatization is complete. 3Dehydroquinic acid is dehydrated to 3-dehydroshikimic acid, which is then reduced to shikimic acid. It is found that the grov^h requirements of auxotrophs of E. coli requiring all three aromatic amino acids may be satisfied by shikimic acid, thus making it the first precursor of aromatic acids to be identified. Carr et al [19] reported that quinic acid may be reduced to dihydroshikimic acid by Lactobacillus during cider fermentation. It thus appears that the steps in the aromatization from quinic acid to shikimic acid and beyond shikimic acid on the way to benzoic acid are carried out by the gut bacteria (Fig. (4)). Benzoic acid formation is not the sole pathway in aromatization of quinic and shikimic acids as their administration to rats increases the excretion on urinary catechol [31]. In addition, vanillic acid is also excreted. When rats feed quinic acid mixed in the purified diet, catechol may be readily detected free (unconjugated) as well as

929

HO^^COOH

Quinic acid

Shikimic acid

CONHCH2COOH

H2NCH2COOH Hippuric acid

Fig. (4)

Glycine

Benzoic acid

Metabolism of quinic acid

conjugated in the urine. Several studies on the site of catechol formation have shown that this metabolite is also produced from quinic and shikimic acids by intestinal microorganisms [32, 33]. The microorganisms responsible have not been identified but quinic acid is known to be converted to protocatechuic acid by strains of Pseudomonas [34]. The further decarboxylation of protocatechuic acid to catechol occurs when incubated with rat intestinal contents [32]. Conclusion

Quinic acid is metabolized to aromatic substances by gut flora when ingested by animals. However, the microorganisms responsible have not been identified. Species difference of aromatization of quinic acid may be related to gut flora. We are not aware of any reports about enzymatic transformations of quinic acid in body tissues. This subject evidently needs to be studied further.

930

BIOLOGICAL ACTIVITY Chlorogenic acids are ubiquitously distributed in plants. These compounds are considered to be the active principles in many medicinal plants. They may be constantly taken as food and beverage, and play a role in human health. Here we review the biological effects of chlorogenic acids in this section. The Antioxidant Properties of Chlorogenic Acids There are several endogenous and exogenous compounds (a-tocopherol, ascorbic acid, glutathione, uric acid, etc.) that offer antioxidant protection against free radicals. Notwithstanding the action of these substances, molecules suffer free radical oxidant damage. This damage may be responsible, at least in part, for aging and age-related diseases [35-37]. This damage can be caused by environmental insults such as ultraviolet radiation. It may also result from physical stress such as excessive exercise, psychological stress, or exposure to toxins. Currently, considerable attention is being paid to investigation of chlorogenic acids as an antioxidant. Here we will deal with the mechanisms of their action as an antioxidant. Chlorogenic acids as an Antioxidant

Despite the fact that oxygen is required for aerobic organisms to live, once metabolized, oxygen can be potentially harmful. The majority of the O2 (molecular oxygen, dioxygen) that is drawn into an organism's body is used to produce energy in the form of adenosine triphosphate (ATP); however, up to 5% of the O2 in an organism is converted to reactive oxygen species (ROS) or free radicals. Free radicals are molecules or parts of molecules which have one or more unpaired electrons. They often have an independent, yet very short-lived existence. It should be noted that through a dismutation reaction a radical can often self-oxidize; consider the reaction in Equation 1:

931

02 + 02 + 2 i r -> H2O2 + O2

(Eq. 1)

In this reaction (demonstrated in vitro), one of the two radicals is oxidizing while the other is reducing. In vivo, this reaction is catalyzed by one of several isoforms of an enzyme known as superoxide dismutase (SOD). As shown above, hydrogen peroxide may form as a result of the superoxide anion's dismutation reaction; however, it may also be produced from a bivalent reduction of O2. The addition of the second electron leads to the formation of hydrogen peroxide, which is a powerful oxidizing agent. Due to the unpaired electrons in their outer shells, free radicals are favored to pair with other molecules during bimolecular collisions. The term ROS is used to classify the products of O2 which do not contribute to the synthesis of ATP. They include oxygen free radicals such as superoxide anion (O2), hydroxyl radical (•OH), peroxyl radical (ROO*), and alkoxyl radical (R0»). Nonfree radicals that may be classified as ROS include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), single oxygen (^02), and ozone (O3). The superoxide anion is produced by the monovalent reduction of molecular oxygen. This occurs when O2 picks up one lone electron with its accompanying supply of energy. In vivo, superoxide anion may cause damage indirectly, as superoxide anion is a precursor of the highly toxic hydroxyl radical. This results from the Haber and Weiss reaction (Equation 2): O2 + H2O2 ^ 0 2 + OH" + -OH

(Eq. 2)

In another example of free radical formation known as the Fenton process, H2O2, in the presence of ferrous ions (Fe^^), decomposes into a harmless OH" ion and a powerful •OH radical. ^OH has the ability to attack stable organic structures (Equation 2, 3). This slow reaction process requires the presence of ferric ions which act as a catalyst. 02 + Fe'^ -^ 02 + Fe'^ Fe'^ + H2O2 -> Fe'^+ OH" + -OH

(Eq. 3) (Eq. 4)

The reactivity of hydroxyl radicals is high in biological media, and

932

their reactions with neighboring molecules results in the production of secondary radicals, either by the loss of a hydrogen atom or by transfer of the lone electron. Alkoxyl radicals and peroxyl radicals are produced from polyunsaturated fatty acid (PUFA) chains through the actions of oxygen free radicals, such as superoxide anions or hydroxyl radicals. Peroxyl radicals are known to be more selective and less reactive than hydroxyl radicals. They are responsible for chain reactions such as the basic process for lipid peroxidation in cell membranes. In vitro evidence has been reported that chlorogenic acids are voracious scavengers of ROS (Table 1) [39-49]. Chlorogenic acids have the capability of scavenging superoxide anions or hydroxyl radicals. The rate constants of the reactions of chlorogenic acid with superoxide anions and hydroxyl radicals are 1.67 X 10' M'^S"^ and 3.34 X 10' M'^S ^ respectively, while those of caffeic acid with superoxide anions and hydroxyl radicals are 0.96 X 10' M'^S'^ and 3.24 X 10' M'^S^ respectively [39]. Chlorogenic acids not only scavenge the very toxic hydroxyl radical directly, but utilize a second mechanism to neutralize hydroxyl radicals, as superoxide anion is a precursor of much of the •OH generated in vivo (Equation 3, 4). Table 1.

Free Radicals and Chlorogenic Acids

Free Radical

Chlorogenic acids

Reference

Superoxide anion

Chlorogenic acid, Caffeic acid,

[38-42]

Dicaffeoylquinic acids, Ferulic acid Hydroxyl radical

Chlorogenic acid, Caffeic acid,

[39, 43-45]

Peroxyl radical

Chlorogenic acid, Caffeic acid,

[38, 39, 42, 43, 46-48]

Dicaffeoylquinic acids, Ferulic acid Hypochlorous acid

Chlorogenic acid, Caffeic acid,

[49]

Protection of Lipids by Chlorogenic acids

Consider the peroxidation of PUFA. This has been shown to occur in three stages: initiation, propagation, and termination (see below).

933

Initiation starts with the removal of hydrogen atoms from the lipid molecule which results in the formation of carbon-centered free radicals that contain conjugated diene bonds. This can be caused by a variety of initiating radicals. Redox-active metals (Fe, Cu, etc.) have been found to assist in this process. In the propagation phase of lipid peroxidation, oxygen reacts with the carbon-centered free radicals, resulting in the formation of peroxyl radicals. These peroxyl radicals can then react with another PUFA, thus propagating the chain reaction and forming lipid peroxides (also called lipid hydroperoxides). It has been demonstrated in vitro that until it is stopped, this chain reaction has the potential to damage all accessible PUFA. This reaction can therefore be considered substrate limited. It should be noted, however, that radicalradical interactions do occur that result in the termination of the chain reaction that causes the formation of lipid peroxide. It has been shown that if two lipid radicals unite, nonradical products will be formed. The same chain termination occurs when a lipid radical interacts with a peroxyl radical. Initiation; formation of R» Propagation; R-+ O2 -^ ROO* R 0 0 - + R H (substrate) -^ ROOH + R* Decomposition;

ROOH + Me'^-> RO«+OH + Me'^ ROOH + Me'^ -> ROO«+ir + Me'^ ROOH + ROOH ^ ROO'+ROO'+H^O Termination; R*+ R» -^ nonradical products R»+ R00» -^ nonradical products R00»+ ROO* -^ nonradical products According to the theory of free radical oxidation, antioxidizing activities of chlorogenic acids are stipulated by their participation in reaction with free radicals. Chlorogenic and caffeic acids have high stoichiometric numbers and reactivity with peroxyl radicals as compared with trolox, the water-soluble analogue of tocopherol [48]. Considering

934

that trolox traps 2 peroxyl radicals per molecule, the stoichiometric number of peroxyl radicals trapped by each compound is as follows: chlorogenic acid, 3.1, caffeic acid, 2.7 [48]. The stoichiometric number of chlorogenic and caffeic acids show a good correlation with their scavenging activity on the l,l-diphenyl-2-picrylhydrazyl (DPPH) radical of our examination [38]. A free radical scavenger will come to possess an unpaired electron once it has contributed an electron to neutralize a free radical. Paradoxically, the free radical scavenger becomes a free radical. When chlorogenic acids neutralize a free radical they become phenoxyl radicals. However, products of chlorogenic and caffeic acids formed by reaction with free radicals are rapidly broken down further to products that are not able to generate any free radicals. This is the beneficial nature of antioxidants, because other antioxidants are not necessary for the reduction of one-electron oxidation products of these compounds [39]. It is currently believed that the oxidative modification of low density lipoprotein (LDL) is an important initial event in pathogenesis of atherosclerosis [50]. LDL peroxidation initiated by metmyoglobin/hydrogen peroxide is inhibited by chlorogenic and caffeic acids [51]. This is accomplished by a mechanism involving the oneelectron transfer reaction between chlorogenic acids and ferrylmyoglobin, with formation of metmyoglobin and corresponding phenoxyl radicals from chlorogenic acids [52]. Regarding the suggested role of ferrylmyoglobin as a contributing factor in the pathogenesis of ischemia reperfusion and atherosclerosis, and the suggested antioxidant activity of the chlorogenic acids in vivo, these findings provide new insights into the biochemical mechanisms underlying their antioxidant activity. In another demonstration of the ability of chlorogenic acid to reduce (in a dose-dependent manner) the level of lipid peroxidation products, carbon tetrachloride (CCI4, a widely used solvent and cleaning chemical) or CCI4 plus chlorogenic acid are administrated. When ingested, CCI4 severely damages liver tissue, and this damage is considered to be freeradical based. When chlorogenic acid is administered, however, beneficial results similar to those previously described above are obtained against CCI4 [53, 54] With respect to in vivo studies investigating the antioxidant potential of chlorogenic acid and caffeic acids, oral administration of these

935

compounds have been found to inhibit the elevation of serum glutamic oxaloacetic transaminase and glutamic pyruvic transaminase induced by feeding peroxidized oil [55]. Another study has used paraquat, a highly toxic xenobiotic. The activities of erythrocytes and liver glutathione peroxidase, and of both liver catalase and glutathione reductase, which are increased by feeding paraquat, decline to the levels in the control rats by supplementing chlorogenic acid to the paraquat diet [41]. It is well known that paraquat and CCI4 are metabolized by cytochrome P-450, resulting in the generation of the superoxide anion and other active oxygen species [5658]. Accordingly, it is suggested that part of the protection against CCI4 and paraquat hepatotoxicity afforded by chlorogenic acid in vivo may be due to its ability to act as a radical scavenge, analogous to various demonstrated effects in vitro. From these observations it is showed that the antioxidant activities of chlorogenic acids are preserved by inhibiting the formation of reactive oxygen species or by scavenging them. As a result, chlorogenic acids may play a beneficial role in the prevention of some oxidative diseases. Other Evidence ofAntioxidative Properties of Chlorogenic acids

Free radicals can also originate in an exogenous fashion, being formed by X-ray and y-ray ionizing radiation. In an effort to test the antioxidant properties of chlorogenic acids against potent •OH, ionizing radiation has been administered to bone marrow cells with or without chlorogenic acids. Ionizing radiation has been chosen, as it is known to cause the formation of oxygen-based radicals that can damage genetic material. Oral administration of chlorogenic acid (50, 100 and 200 mg/kg) to mice may significantly reduce the chromosomal damage induced by yradiation. The protective effect of chlorogenic acid has been observed in bone marrow cells sampled 24, 30 and 48 h after exposure to yradiation [59]. Intraperitoneal administration of caffeic and ferulic acids to mice has showed the survival effects. Skin injury induced by yradiation has been protected by intraperitoneal injection of ferulic acid [60]. Due to their powerful antioxidative potential, it has been postulated that chlorogenic acids help to preserve the overall integrity of DNA and in so doing reduce the likelihood of cancer.

936

Possible Mechanisms of the Chlorogenic acids Antioxidizing Action

Low-molecular-weight substances are well known to have the ability to strengthen the action of other antioxidant. The mechanism of this phenomenon is an antioxidant radical reduction up to the initial condition. In organisms and in their biological membranes, the natural antioxidants are always present. The antioxidizing effect of chlorogenic acids in vivo and in vitro can consist in strengthening already existing antioxidant actions. In LDL enriched with a-tocopherol, caffeic acid inhibits the initiation of oxidation longer than expected from the sum of discrete periods characteristic of caffeic acid and a-tocopherol. An effect similar to caffeic acid has been observed on a Triton-100 micellar system. These results suggest that caffeic acid may act synergistically with a-tocopherol, extending the antioxidant capacity of LDL by recycling a-tocopherol from the a-tocopherol radical [61]. In the same system, a synergistic antioxidant effect of caffeic and p-coumaric acids with ascorbic acid has been indicated [62]. The antioxidizing effect of chlorogenic acids synergistically increases in the presence of a-tocopherol or ascorbic acid [61, 52]. The other important property affecting lipid oxidation is the chelating effect of chlorogenic acids. It is important to keep in mind that the influence of biometals (Fe, Cu etc.) on lipid free radical oxidation is essential. It is well known that iron can react with hydrogen peroxide by the Fenton reaction (Equation 3). The hydroxyl radical formed in the Fenton reaction is capable of reacting with lipid and PUFA as the initiation stage. Iron can also participate in alkyl peroxide or lipid peroxide decomposition. Therefore, the nature of iron chelation in a biological system is an important aspect in disease prevention. Chlorogenic and caffeic acids have suppressed the formation of hydroxyl radical via the Fenton reaction, probably due to chelation of these acids with iron [44]. Indeed, recent studies report that chlorogenic acid shows chelating activity or reducing activity on iron required for the production of superoxide and hydroxyl radicals, resulting in the inhibition of lipid peroxidation induced Fenton reaction [45, 63].

937

The Antimutagenetic and Anticarcinogenic Activities

Chlorogenic and caffeic acids have been reported to react with reactive species of nitrogen in vitro [39, 49, 64]. In these reports, chlorogenic and caffeic acids have a good relationship between the ability to scavenge free radicals and to inhibit N-nitrosation. Since reactive intermediates of nitrogen have been reported to be important mediators of mutagenesis and carcinogenesis, these studies suggest that chlorogenic and caffeic acids may be effective not only in protecting against oxidative damage in vitro but also in preventing potentially mutagenic and carcinogenic reactions in vivo. It has been reported that chlorogenic acid is effective in reducing the mutagenic activity of the nitrosation products in Salmonella typhimurium TA 1535 [65]. Dietary administration of chlorogenic, caffeic and ferulic acids during the initiation phase of 4-nitroquinoline-l-oxideinduced rat tongue carcinogenesis clearly suppressed tumor development [66]. Chlorogenic, caffeic and ferulic acids inhibit the mutagenicity of the ultimate metabolite of benzopyrene in Salmonella typhimurium TA 100 [67]. Caffeic acid has been observed to diminish the incidence of forestomach tumors in mice treated with benzopyrene [68] and chlorogenic acid has been shown to block the formation of mutagenic compounds resulting from pyrolysis of protein [69]. Huang et al [70] have evaluated the effects of chlorogenic acids on tumor promotion in an animal study using CD-I mice. Chlorogenic, caffeic and ferulic acids inhibit the induction of ornithine decarboxylase by 12-0-tetradecanoylphorbol-13-acetate (TPA). TPA-mediated DNA synthesis has been weakly inhibited, but TPA-induced skin tumor promotion has been markedly inhibited by these compounds. Caffeic and ferulic acids inhibit superoxide anion production, when phorbol-12-mysristate-13-acetate (PMA) or mezerein interact in vitro with murine peritoneal macrophages. Thus, it seems that there is a close correlation between the ability of tumor promoters to induce the production of superoxide anion by peritoneal macrophages and their tumor-promoting activity [71]. The role of these phenols has been investigated in the promotional phase of carcinogenesis. Topical application of these compounds simultaneously with PMA or mezerein has resulted in significant protection against 7,12-dimethylbenz[a]anthracene-induced skin tumors in mice. Caffeic acid is a strong

938

inhibitor of tumor promotion with respect to tumor incidence at both stages of skin carcinogenesis. Further, caffeic and ferulic acids are stronger inhibitors of PMA- and mezerein-induced superoxide anion radical than ellagic acid in vivo and in vitro conditions [72]. Thus, it is possible that chlorogenic, caffeic and ferulic acids, may even inhibit the production of arachidonic acid metabolites in addition to superoxide anion generation by PMA, thereby reducing the tumor incidence. Immuno-regulatory Activity

Inflammation is always connected with the production of free radicals by inflammatory cells, so that, substances acting as radical scavengers have some anti-inflammatory capabilities. In this section, the activities of chlorogenic acids on cells of the immune system, namely mast cell, macrophage and neutrophil, are discussed. It is well known that a primary reaction of allergy and inflammation is the release of histamine from the tissues. Histamine release from isolated mast cells is a useful in vitro model for studying allergic and inflammatory diseases. Most of the drugs used in the treatment of allergy, asthma and inflammation are effective inhibitors of in vitro histamine release. It is well known that compound 48/80 or concanavalin A plus phosphatidylserine causes histamine release from mast cells. So, the inhibitory effects of chlorogenic acids on the histamine release from rat mast cells induced by compound 48/80, and on the histamine induced by concanavalin A plus phosphatidylserine have been determined by Kimura et al [73]. Chlorogenic and caffeic acids exhibit over 50% inhibition of the histamine secretion induced at compound 48/80 from mast cells at a concentration of 25 |uiM. 3,4-, 3,5and 4,5-di-O-caffeoylquinic acids (DCQAs) exhibit over 50% inhibition of the histamine secretion induced by compound 48/80 from mast cells at a concentration of 50 |jiM. Chlorogenic acid, caffeic acid and 3,5-di-Ocaffeoylquinic acid (3,5-DCQA) exhibit over 50% inhibition of the histamine secretion induced by concanavalin A plus phosphatidylserine from mast cells at a concentration of 10 (jiM. Antioxidants are useful for treating allergic disease [74, 75], since active oxygen species such as superoxide and hydroxyl radicals induce histamine release from mast cells [76, 77]. Therefore, allergic reactions may be prevented by scavenging active oxygen species. Ito et al [78] examined the

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relationship between anti-allergic capabilities and the radical scavenging activities of chlorogenic acids. Chlorogenic and caffeic acids inhibit the activation of hyaluronidase, which is known as one of the enzymes involved in allergic effects [79], migration of cancer cells [80], inflammation [81, 82], and increased permeability of the vascular system. The superoxide anion radical scavenging activity of chlorogenic acids correlates linearly to the hyaluronidase-inhibitoiy activity. ^-Hexosaminidase release from rat basophilic leukemia cells induced by antigen has been inhibited by chlorogenic and caffeic acids. These compounds do not inhibit phexosaminidase activity itself Oxygenation of arachidonic acid catalyzed by either cyclooxygenase or lipoxygenases initiates the biosynthesis of eicosanoids. Eicosanoids can be synthesized by all cells of the immune system, especially by monocytes and macrophages which are the first line of defense against infection. Macrophages contain both cyclooxygenase and lipoxygenase activities and are capable of generating large amounts of prostanoids, leukotrienes and different hydroxyl fatty acids. In a recent study, the effects of caffeic acid on eicosanoid production by mouse peritoneal macrophages in vitro and in vivo have been investigated [83]. Caffeic acid inhibits the production of leukotriene B4 by isolated mouse macrophage. Caffeic acid also suppresses the synthesis of leukotriene C4 in vivo, Chlorogenic acid has enhancing effects on the spreading and mobility of murine macrophages [84]. Chlorogenic acid, caffeic acid and DCQAs above 10 jxM enhance macrophage spreading in a dosedependent manner. There are no significant differences between monocaffeoylquinic acids and DCQAs. Macrophage spreading and mobility are preliminary steps, which precede macrophage infiltration into tissues affected by injury or infection. Macrophages are the first cells recruited to fight injury and infection and present a first line of defense in most tissues. The spreading and mobility of macrophage are thought to be two important markers of macrophage activation. It is therefore important to identify those compounds, that are active in the early stimulation of macrophages. Moreover, the effects of chlorogenic acids on arachidonate metabolism in human peripheral neutrophils have been investigated [85]. The results show that the formation of leukotriene B4 induced by calcium

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ionophore A 23187 in human peripheral neutrophil leukocytes has been inhibited by 3,4-, 3,5-, 4,5-DCQAs and caffeic acid. Chlorogenic acid had no effect. Koshihara et al [86] have found that caffeic acid is a selective inhibitor for 5-lipoxygenase and leukotrienes. Consequently, the anti-allergic capabilities of chlorogenic acids are correlated with the radical scavenging activity against the superoxide radical. Also, chlorogenic acids inhibit 5-lipoxygenase in arachidonate metabolism, so that the formation of the pro-inflammatory leukotrienes has been blocked. The recent accumulation of data clearly indicates that chlorogenic acids can exhibit potent antioxidant properties, significantly benefiting the defensive of organisms. Conclusion

Chlorogenic and caffeic acids may play a role in the body's defense against carcinogenesis and mutagenesis by their antioxidant properties. The antioxidant and antiradical influences of chlorogenic acids on lipid peroxidation are not yet clear. Research has indicated that chlorogenic acids are an antioxidant, however, their complex antioxidant properties require systematic investigation in diverse directions. To determine the influence of chlorogenic acids on various stage of lipid free radical oxidation in vivo and in vitro, it is necessary to use a highly sensitivity method, such as the chemiluminescence method. Antiviral activity It is assumed that chlorogenic acids in plants possess antiviral properties and the concentration increases after infection. Indeed, chlorogenic acid is an inhibitor of Botjytis spore germination [87]. Previously, chlorogenic acid was reported to have antiviral activity [88, 89]. In a recent investigation of phenolic compounds tested, using herpes simplex virus type 1 (HSV-1) infected Vero cells, caffeic acid has been reported to inhibit virus replication [91]. In a related study [92], chlorogenic acid significantly inhibited acyclovir-resistant HSV-1 replication without any cytotoxicity. However, flavonoids have exhibited cytotoxicity at the same concentration [92]. The human immunodeficiency virus (HIV) has been identified as the

941

cause of acquired immunodeficiency syndrome (AIDS), which is considered a lethal infectious disease. This disease is distributed around the world and is transmitted by sexual contact, by blood or blood products. Drug therapy for AIDS is currently limited to several classes of drugs. Drugs that can effectively treat or prevent AIDS are in demand. It has been reported that extracts of several medicinal plants and foods show inhibitory effects on HIV [93-95]. Also, HIV inhibitory substances have been investigated. Caffeic acid has been exhibited to inhibit HIV-induced cytopathogenicity in MT-4 cells and giant cell formation [96]. Schols et al have reported that this action seems to be due inhibition of interaction of the virus with the cellular target CD4 receptor. In another experiment, chlorogenic acid shows inhibitory activity against HIV-protease [94]. Dose-response study yields IC50 of 100 Jig/ml. Since HIV-protease plays an important role in the process of maturation and infections of the virus, it is considered to be a good target for the development of anti-HIV drugs. Today, most HIV drugs target two essential viral enzymes, reverse transcriptase and protease. Inhibiting these enzymes protects virus replication. In addition to these enzymes, another therapeutic target is HIV integrase. Integration of the cDNA copy of the HIV type-1 (HIV-1) genome is mediated by an HIV-1-encoded enzyme, integrase, and is required for productive infection of CD4+lymphocytes. This enzyme represents a novel target to which antiviral agents might be directed. Robinson et al [97] isolated 3,5-DCQA from an aqueous extract of Baccharis genistelloides and searched for effects against HIV-1 integrase or HIV-1 replication in tissue culture. Anti-HIV-1 activity has been measured as 50% protection against HIV-1-induced cytopathic effect 72 hr after addition of MT-2 cells. The anti-HIV-1 activity of 3,5-DCQA is 1 jxg/ml. Furthermore, it inhibits HIV-1 integrase in vitro and blocked HIV-1 replication in tissue culture. The toxic concentration of this compound is fully 100-fold greater than its antiviral concentration. Thus, 3,5-DCQA represents a potentially important new class of antiviral agents. In a related study [98], 1,5-, 3,4- and 4,5-DCQAs have been tested for inhibition of HIV-1 integrase in vitro and inhibition of HIV-1 replication in tissue culture. The activities of DCQAs against HIV-1 integrase are less than 1 |xM. Indeed, all of DCQAs have been found to inhibit HIV-

942

1 replication at concentrations ranging from 1 to 6 fxM in T cell lines, whereas their toxic concentrations in the same cell lines are all greater than 1.2 |jiM. Mahmood et al [93] reported that inhibition of virus infection by 3,4-DCQA was due to interaction of its compound with gpl20, preventing virus binding to the CD4 receptor. Furthermore, McDougall et al [99] measured the specificity of DCQAs against HIV-1 integrase. Clearly, DCQAs are potent and selective inhibitors of HIV-1 integrase. This result indicates that DCQAs are a potentially important class of HIV inhibitors. In an effort to develop more potent and selective inhibitors of HIV-1 integrase, analogues of DCQAs have been synthesized. The length of the side chains, the spatial arrangement of the phenolic hydroxyl groups, the size and structure of the central molecular core structure, and the requirement of one or more free carboxyl groups have been all studied. The effects of these changes have been assayed against HIV-1 integrase in the disintegration reaction as well as against HIV-1 replication and cell growth in tissue culture [100-104]. Conclusion

Chlorogenic acids are produced by plants as defense mechanisms in response to viral infection. Moreover, dietary intake of these substances may have an antiviral effect in humans. Bioability of Medicinal Plants As described above, chlorogenic acid and DCQAs are widely present in various plants. It is likely that many of the alleged effects of medicinal plants are linked to the functions of their constituents. In this context, several plants have been investigated for their biological activities and their active substances. This section briefly outlines the occurrence and role of chlorogenic acids in medicinal plants. Plants

The herb Cusciita reflexa has traditionally been used to treat blood infections. The active compounds have been isolated from the crude

943

extract and identified as flavanone and caffeoylquinic acid derivatives. Crude water extracts of Ctisciita reflexa exhibit HIV activity. The flavanone and caffeoylquinic acid derivatives isolated from Cusaita reflexa inhibit virus infection by different systems. The anti-HIV activity in crude extract of Ctiscuta reflexa may be the result of combined effects from these different modes of action [93, 105, 106]. For the purpose of finding anti-HIV agents from natural sources, various plant extracts have been screened for their inhibitory activity against HIV-protease, an enzyme essential for viral proliferation by Matsuse et al. [94]. The bark or roots of Swietenia mahagoni are used for treatment of gonorrhea, to halt diarrhea and as a febrifuge in America. The methanol extract of the bark of Swietenia mahagoni has shov^n inhibitory activity against HIV-protease. The butanol fraction of the methanol extract of the bark of Swietenia mahagoni affords chlorogenic acid methyl ester, which inhibits HIV-protease activity. Catechin and gallocatechin have also been isolated from the same plant extract, however, there were no inhibitory effects shown. Chlorogenic acid and its methyl ester inhibit HIV-protease in a concentration dependent manner, giving IC50 of 100 |ig/ml and 40 jxg/ml, respectively. Moreover, the same extract has inhibited the replication of HIV-1 in infected MT-4 cells [107] and moderately inhibited avian myeloblastosis virus-reverse transcriptase [108]. These investigations suggest that the anti-HIV effect of the methanol extract of Swietenia mahagoni seems to be due to inhibition of HIV-protease by chlorogenic acids. An infusion ofPersea americana leaves (Lauraceae) strongly inhibits HSV-1, Aujeszky's disease virus and senovirus type 3 in cell cultures. Chlorogenic acid (14.16 mg/ml) has been reported to be the main constituent in an infusion oi Persea americana leaves (10% w/v) [92]. Chlorogenic acid significantly inhibits HSV-1 replication. However, chlorogenic acid is less active than the infusion. The flavonoids isolated from the leaves of Persea americana show higher activity against acyclovir-resistant HSV-1. However, they are present at very low concentration in the infusion. This study suggests that the antiviral activity of the infusion may be due to a synergistic effect between chlorogenic acid and flavonoids. Extract from artichoke, Cynara scolymus L., has been used in folk medicine against liver complaints and such extracts or several constituents thereof have been claimed to exert a hepatoprotective effect

944

[109, 110]. Artichoke extracts show marked antioxidant and protective potential [HI]. This result suggests that chlorogenic acid and 1,5DCQA account for only part of the antioxidative principle of these extracts. Recently, Maruta et al [112] have found that methanol extracts of roots of burdock show a significant antioxidant activity in an in vitro lipid peroxidation assay, and have isolated five caffeoylquinic acid derivatives (CQAs) from the roots of burdock {Arctium lappa L.), an edible plant in Japan. Antioxidant activities of DCQAs and related compounds have been investigated by measuring the hydroperoxidation of methyl linolate via radical chain reaction. This study indicates that in this particular system caffeic acid and CQAs are more effective than atocopherol. These results approximately agree with our findings [38]. Additionally, CQAs as the principle antioxidative substance in burdock root have been characterized. Moreover, DCQAs (1,5-, 3,4- and 4,5-DCQAs), with antioxidative activity, have been isolated from the leaves of garland {Chrysanthemum coronaritmi L.) [113]. The garland {Chrysanthemum coronarium L.) has been regarded as a health food in East Asia because the edible portions, such as leaf and stem, contain abundant jj-carotene, iron potassium, calcium, and dietary fiber. In addition to these common nutrients, some compounds responsible for the chemoprevention of cancers and other diseases are thought to be contained in garland. The antioxidative activity of DCQAs has been assayed by the decay curves of p-carotene. The antioxidative ability of 1 (xg/ml these compounds are nearly equal to that of 0.1 |ig/ml 3-/^r/-butyl-4-hydroxyanisole (BHA). Both garland {Chrysanthemum coronarium L.) and burdock {Arctium lappa L.) belong to the same family o^Asteraceae. These facts suggest that various DCQA derivatives contributing to the plant protection systems are contained in the family Asteraceae. Chlorogenic acids have been isolated from other plants (flower and leaf) in the family Asteraceae [114]. Okuda et al. [11] have studied the medicinal property of Artemisia family and found that it relates to the composition of chlorogenic acid derivatives (chlorogenic acid and DCQAs) in the Artemisia species. Research on the distribution of DCQAs (3,4-, 3,5and 4,5-DCQAs) in the species oiArtemisia shows that the species used as haemostatics generally contain CQAs of composition similarly to those oi Artemisia montana P. and Artemisiaprinceps P.

945 Propolis

Propolis is a mixture of compounds obtained from beehives and has been a popular folk medicine. Dimov et al. [115] reported on the immunomodulatory function of propolis. They suggest that the water-soluble constituents of propolis contribute to macrophage activation and thus propolis exerted preventive effects against infection. Tatefuji et al [84] isolated six compounds from the water-soluble fraction of propolis and identified these substances as DCQAs which enhance macrophage spreading and mobility. Since chlorogenic components of propolis (chlorogenic acid, caffeic acid and DCQAs) appear to stimulate macrophage spreading and mobility, these properties might partly explain the immunomodulatory effects of propolis. Konig et al. [88] have reported that propolis shows preventive activity against the Herpes viruses. Also, they have described chlorogenic acids (chlorogenic acid, caffeic acid and DCQAs), which have been found in propolis, as antiviral active compounds. The water-soluble fraction of propolis shows a strong hepatoprotective activity against toxicity induced by CCI4 in rats [116]. Chlorogenic acid, caffeic acid, 3,4- and 3,5-DCQAs protect injury CCl4-induced in cultured rat hepatocytes. The active constituents have been found to be chlorogenic acid derivatives (chlorogenic acid, caffeic acid, 3,4-, and 3,5DCQAs). Probably the hepatoprotective activity of propolis may be due to the protective effect of chlorogenic acid derivatives. Elsewhere, the effects of propolis and its components on eicosanoid production during the inflammatory response have been investigated. This investigation demonstrates that both propolis and its component, chlorogenic acids, inhibit eicosanoid production which can strongly affect the immune and inflammatory response. Consequently, the effect on eicosanoid production by propolis may be due to chlorogenic acids of propolis component [83]. Conclusion

The observed pharmacological activities of medicinal plants relate to the chlorogenic acids constituents of the plant. However, medicinal plants are a complex mixture of chemically different compounds.

946

Consequently, study of pharmacological effects should focus on interaction with other components occurring in medicinal plants. Prooxidant and Mutagenic Activity Despite the abundance of biological data demonstrating antioxidant activities of chlorogenic acids, the controversy whether these compounds are potent antioxidants or pro-oxidants, remains. The pro-oxidant characteristics of chlorogenic and caffeic acids have been suggested in the several papers [44, 52, 117-121]. Chlorogenic and caffeic acids stimulate the formation of hydroxyl radicals in the Fenton reaction [117]. However, they have reported that chlorogenic and caffeic acids result in a decrease in hydroxyl radical formation in the different condition [44]. Yamanaka et al [121] have observed that chlorogenic and caffeic acids exert accelerate effects on the propagation phase of Cu^^-induced LDL oxidation at 0.5 jjiM. In contrast, chlorogenic and caffeic acids inhibit LDL oxidation in the initiation phase at the same concentration. Moreover, an elevated concentration of caffeic acid inhibits oxidation even in the propagation phase. Chlorogenic acid indicates the same tendency. In this experiment, these compounds display antioxidant and pro-oxidant activities depending on the oxidation state of LDL. This pro-oxidant activity is also observed for ascorbic acid [122, 123] and a-tocopherol [124]. Indeed, other studies have shown that chlorogenic acid is a potent cocarcinogenic agent [125-129] and an inducer of DNA damage [130, 131]. The cytotoxicity and mutagenicity of chlorogenic acids may be attributed to the generation of hydrogen peroxide, the reduction of transition metals (Cu^^ and Fe^^) and the catalyst of free radical formation [132], Stadler et al. [133] suggest that dual effects of chlorogenic acids are due to the high oxygen tension and large quantities of iron used for measurement. Conclusion

The above-described observations indicate that chlorogenic and caffeic acids switch from anti- to pro-oxidant activity, depending on their concentration, on the presence of free transition metal ions, or on their

947

redox status. CONCLUSIONS The extent of absorption of chlorogenic acids from dietary sources is not completely understood; for instance, data on the chlorogenic acids of coffee, a major dietary source, are virtually absent. Information on absorption is limited to caffeic acid and quinic acid. The absorption of caffeic acid estimated by measuring its urinary metabolites in humans was 24-30%. Caffeic acid gave rise to more than ten metabolites, then chlorogenic acid, which yielded an intermediate number, while coffee was the least effective [15]. Since caffeic acid occurs as a quinic acid ester in chlorogenic acid, this could tend to prevent a sudden increase in the concentration of caffeic acid per se in the animal body when chlorogenic acid is given. This would also be true of chlorogenic acid in coffee. The rate of absorption and total absorption of caffeic acid given in the form of chlorogenic acid could be considerably lower than that for caffeic acid itself The absorption of quinic acid in rats was estimated to be about 33% of ingested radioactive quinic acid based on the amount of radioactivity excreted into urine. The major sites of chlorogenic acids metabolism have been found to be the liver and the colonic flora. Only the liver has been investigated as a metabolic organ. Other tissues such as the intestinal wall and kidneys may play a role. The findings summarized in this review clearly show that gastrointestinal microorganisms can carry out a large number of metabolic reactions with chlorogenic, caffeic, and quinic acids in hydrolytic, reductive, oxidative, and decarboxylative pathways. Nonetheless, new routes of metabolism can be expected to be encountered in the future, especially among reactions of a pronounced degradation. Chlorogenic acids are shown to have some desirable biological activities. Most of their property relates to their function as antioxidants, evidenced by their activity to scavenge free radicals, to inhibit the formation of free radicals, and to block the oxidation reaction. However, other activities based on mechanisms other than scavenging capacity cannot be ignored. Also, further tests for the in vivo bioactivity of chlorogenic acids are to be needed. However, it is expected that chlorogenic acids may be beneficial to human health.

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ABBREVIATIONS /M-HBA /M-HPPA

m-HHA E. coli ATP ROS Eq. SOD PUFA DPPH LDL PMA TPA DCQAs DCQA HSV-1 HIV AIDS HIV-1 CQAs BHA

/w-Hydroxybenzoic acid m-Hydroxyphenylpropionic acid 7w-Hydroxyhippuric acid Escherichia coh Adenosine triphosphate Reactive oxygen species Equation Superoxide dismutase Polyunsaturated fatty acid 1,1 -Diphenyl-2-picrylhydrazyl Low density lipoprotein Phorbol-12-mysristate-l 3-acetate 12-0-Tetradecanoylphorbol-13 -acetate Di-0-caffeoylquinic acids Di-0-caffeoylquinic acid Herpes simplex virus type 1 Human immunodeficiency virus Acquired immunodeficiency syndrome Human immunodeficiency virus type-1 Caffeoylquinic acid derivatives 3-teA'/-Butyl-4-hydroxyanisole

ACKNOWLEDGEMENT We would like to express our thanks to Professor emeritus of Wakayama Medical College, Ryo Kido for critical reading of the manuscript. REFERENCES [1] [2] [3] [4]

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Matsuse, I.T.; Nakabayashi, T.; Lim, Y.A.; Hussein, G.M.E.; Miyashiro, H.; Kakiuchi, N.; Hattori, M.; Stardjo, S.; Shimotohno, K. Phytother, Res., 1997, 11, 433-436. Wigg, M.D.; Al-Jabri, A.A.; Costa, S.S.; Race, E.; Bodo, B.; Oxford, J.S. Antivir. Chem. Chemother., 1996, 7, 179-183. Schols, D.; Wutzler, R.; Klocking, R.; Helbig, B.; Declerq, E. J. AIDS, 1991, 4 677-685. Robinson, W.E.Jr.; Reinecke, M.G.; Abdel-Malek, S.; Jia, Q.; Chow, S.A. Proc. Natl. Acad. Sci. USA, 1996, 93, 6326-6331. Robinson, W.E.Jr.; Cordeiro, M.; Abdel-Malek, S.; Jia, Q.; Chow, S.A. Reinecke, M.G.; Mitchell, W.M. Mol. Pharm., 1996, 50, 846-855. McDougall, B.; King, P.J.; Wu, B.W.; Hostomsky, Z.; Reinecke, M.G; Robinson, W.E.Jr. Antimicrob. Agents Chemother., 1998, 42, 140-146. Nicklaus, M.C.; Neamati, N.; Hong, H.; Mazumder, A.; Sunder, S.; Chen, J. Milne, G.; Pommier, Y. J. Med. Chem., 1997, 40, 920-929. Artico, M.; Santo, R.D.; Costi, R.; Novellino, E.; Greco, G.; Massa, S. Tramontano, E.; Marongiu, M.E.; Montis, A.D.; CoUa, P.L. J. Med. Chem. 1998,41,3948-3960. Desideri, N.; Sestih, I.; Stein, M.L.; Tramontano, E.; Corrias, S.; Colla, P.L Antivir. Chem. Chemother., 1998, 9, 497-509. Vhetinck, A.J.; DeBruyne, T.; Apers, S.; Pieters, L.A. Planta Med, 1998, 64, 97-109. King, P.J.; Ma, G.; Miao, W.; Jia, Q.; McDougall, B.R.; Reinecke, M.G. Cornell, C ; Kuan, J.; Kim, T.R.; Robinson, W.E.Jr, J. Med Chem., 1999, 42 497-509. Mahmood, N.; Moore, P.S.; DeTommasi, N.; Desimone F.; Colman, S.; Hay A.J.; Pizza, C. Antivir. Chem. Chemother., 1993, 4,235-240. Mahmood, N.; Pizza, C ; Aquino, R.; DeTommasi, N.; Piacente, S.; Colman, S. Burke, A.; Hay, A.J. Antivir Res., 1993, 22, 189-199. Otake, T.; Mori, H.; Morimoto, M.; Ueba, N.; Sutardjo, S.; Kusumoto, I.T. Hattori, M.; Namba, T. Phytother. Res., 1995, 9,6-10. Kusumoto, I.T.; Shimada, L; Kakiuch, N.; Hattori, M.; Namba, T.; Supriyatna, S. Phytother Res., 1992, 6, 241-244. Adzet, T.; Camarasa, J.; Laguna, J.C. J. Nat. Prod, 1987, 50, 612-617. Wojcicki, J. Drug Alcohol Dependence, 1978, 3, 143-145. Gebhardt, R. Toxicol. Appl. Pharmacol, 1997, 144, 279-286. Maruta, Y.; Kawabata, J.; Niki, R. J. Agric. Food Chem., 1995, 43, 2592-2595. Chuda, Y.; Ono, H.; Kameyama, M.; Nagata, T.; Tsushida, T. J. Agric. Food Chem., 1996, 44, 2037-2039. Fontanel, D.; Galtier, C ; Viel, C ; Gueiffier, A. Z Naturforsck, 1998, 53c, 1090-1092. Dimov, v.; Ivanovska, N.; Bankova, V.; Popov, S. Vaccine, 1992, 10, 817-823. Basnet, P; Matsushige, K.; Hase, K.; Kadota, S.; Namba, T. Biol Pharm. Bull, 1996, 19, 1479-1484. Iwahashi, H.; Morishita, H.; Ishii, T.; Sugata, R.; Kido, R. J. Biochem., 1989, 105,429-434.

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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 25 © 2001 Elsevier Science B.V. All rights reserved.

955

EFFECTS OF ETHANOL EXTRACT OF CROCUS SATIVUS L. AND ITS COMPONENTS ON LEARNING BEHAVIOR AND LONG-TERM POTENTIATION H. Saitol, M. Sugiural, K. Abel, H . Tanaka^, S. Morimoto^, F. Taura^ and Y. Shoyama^* Department of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan ^Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University 3'1'1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Abstract: The increase of errors and the decrease in number of learned mice were significantly ameliorated by Crocus sativus ethanol extracts (CSE) in SD test. The LTP-blocking effect of ethanol was significantly improved by oral-, intravenous-, and intracerebroventricular-administration of CSE, respectively. When a single oral administration of crocin was given 10 min before the ethanol treatment the number of successful mice increased at a dose of 200 mg/kg. Crocin of 50 mg/kg ameliorated the blocking effect of ethanol on the LTP at 84% compared to the control. Qocetin gentiobiose glucose ester also antagonized the blocking effect of ethanol on the LTP dose-dependently indicating about a half of crocin. Crocetin di-glucose ester did not remove the inhibitory effect of ethanol on the LTP. It is concluded that two gentiobiose moieties are necessary for the appearance of pharmacological activity of crocin in the central nervous system.

1. INTRODUCTION Crocus sativus L. (Iridaceae) is cultivated for its red stigmatic lobes that constitute saffron. This plant blooms only once a year and the manual harvest of stigmas should be performed within a very short time. The manual cultivation methods practiced with saffron contribute greatly to its high price. Furthermore, weather conditions affect the quality of saffron. Author corresponding: Y. Shoyama Tel and Fax+92^642-6580 e-mail [email protected] ac.jp

956

Therefore, an indoor cultivation system was established in Japan nearly 80 years ago [1]. The stigmas can be collected from full blooming C. sativus in the room, Fig. (1) resulted that the concentration of crocin reached approximately 30 % in ethanol extracts of saffron [1]. This is the reason why the indoor cultivation method is advantageous for the achievement of a high quality and homogenous saffron and for saving time. Saffron finds use in medicine as well as a flavoring and a coloring agent. Saffron has three main chemical compounds as indicated in Fig. (2). TTie bright yellow coloring carotenoids; a bitter taste, picrocrocin; and a spicy aroma, safranal. The carotenoid pigments consist of crocetin di-(fi-Dglucose)-ester, crocetin-(B-D-gentiobiosyl)-(6-D-glucosyl)-ester and crocetin-di-(6-D-digentiobiosyl)-ester (crocin).

Fig. (1). Blooming of Crocus sativus L. in indoor-cultivation.

.CHO

HOOC

Picrocrocin

.coo

OOC RrOHgi

^^

R2-OH2I 0H2C OH

Ri«Glc R2»Glc

Crocin

Ri«Gic R2«H

Crocctin-(D-D-gcntiobiosc)-(D-D-glucosyl)-cstcr

Ri«H

Crocetin-di-(O-D-glucosyJ).ester

R2»H

Fig. (2). Structures of major compounds in C. sativus L.

Safranal

957

Concerning the pharmacological B ciooetin di-(p-D-g]uoow)-«tt«r activities of saffron components, D ciooetlo dl-(§-D-geiitlobi(Myl) •(p>D-gluoosyl)-e«tei the inhibitory effect for the increase • aoda of bilirubin in blood [2], and the deterioration activities or cholesterol and triglyceride levels in serum by crocin and crocetin have been reported [3]. Anti-tumor activity of saffron on mice transplanted with sarcoma-180, Ehrlich ascites carcinoma and Fig. (3). Seasonal variation of crocetin Dalton's lymphoma ascites glucose esters in C sativus L. tumours [4], inhibitory effects of saffron on chemical carcinogenesis in mice using two-stage assay system [5] and the effect of crocetin on skin papillomas and rous sarcoma [6] have been reported. Recently Escribano et al. [7] reported crocin inhibits the growth of HeLa cells and suggested apoptosis induction. More recentiy we reported the oral administration of ethanol extracts of saffron and crocin demonstrated an inhibitory effect on two-stage carcinogenesis of mouse skin papillomas [8]. From these results, crocetin and/or crocetin glucose esters in saffron are important constituents. Previously, we revealed that crocetin glucose esters increase from the period before blooming and reach maximum in the full blooming period, Fig. (3). They are sensitive for the presence of oxygen, light irradiation cause of the polyene structure, and for an indigenous 6-glucosidase [1] which hydrolyzes crocin to crocetin di-(B-D-glucose)-ester. These artificial pathway was indicated in Fig.(4). Moreover, it is evident that storage of saffron at -20 'C promotes the constant supply of saffron with a homogeneous pharmacological activity [1]. In order to control the quantity of saffron, we have ahready prepared a monoclonal antibody (MAb) against crocin, and set up a competitive ELISA using anti-crociu MAb [9].

Q-Glucosidtse

Unknown compounds Crocetin*(0*D*Kentiobiose)-(0-D>glucosyt)*ester W 0.Glucosid)ise Crocetin>di>(0-D*glucosyl)-ecter

Fig. (4). Artificial pathway of crocin

958

Development of natural products having the alleviation activity for the symptoms of leaming and memory impaimient has been expected. It is well-known that the hippocampus is very important in leaming and memory processes, and tiie long-temi potentiation (LTP) induced from its tissue is believed to be closely related to leaming and memory [10]. One of the authors, Saito investigated that the ginseng extract, ginsenoside Rbl and Rgl protected the increase of failure of ttie retrival of memory and prolonged the extinction of memory in hanging stressed mice, also antagonized the electro convulsive shock-induced inhibition of the retention of memory, and showed a tendency to facilitate the acquisition of short term memory in the step down test [11]. Recently Chang et al. [12] found that red ginseng water extract at 0.5 g/kg improved cycloheximide-induced amnesia in rats. Furthermore, they reported ginsenosede Rbl purified from ginseng at 0.1 mg/kg significantly improved cycloheximide-induced amnesia and at 1 mg/kg completely augmented. Recently several groups found the induction of LTP by natural products. Smriga et al. reported that Hoelen (Porta cocos Wolf) and ginseng {Panax ginseng C.A.Meyer) promoted the hippocampal LTP in vivo [13]. Abe et al. found the differential effects of ginsenoside Rbl and malonyl ginsenoside Rbl [14]. More recently Dunwiddie et al. [15] and Pockett et al. [16] found the LTP stimulation activity of forskolin isolated from Coleus forskohlii, and now drug in Germany, India and Japan. When compared these compounds, it is quite difficult to mle out some theory dependent upon the relation between structure and activity. As our ongoing study on leaming and memory of folk medicines we herein review the effects of ethanol extract of C. sativus (CSE) and its purified chemicals on the central nervous system in terms of leaming behaviors in mice and the LTP in the dentate gyrus of hippocampus in anesthetized rats and in the CAl region of rat hippocampal slices.

2 . MATERIALS AND METHODS 2.2. Extraction and separation of saffron components

Dried saffron (500 g) was extracted with 50% of EtOH. EtOH extracts of C. sativus (CSE) (311 g) were separated by Silica gel column chromatography using EtOAc-EtOH-H20 gradient solution (9:3:1 to 7:3:2) to separate crocetm glucose esters containing fraction (275 g) and noncrocetin glucose ester fraction (36 g). The crocetin glucose ester containing fraction was repeatedly purified by Silica gel column chromatography using EtOAc-EtOH-H20 gradient solution as same with the above, and finally purified by MCI gel column chromatography eluting with H2O-

959

MeOH gradient solution (1:0 to 0:1) to yield crocetin di-glucose ester (0.41 g), crocetin gentiobiose glucose ester (5.10 g) and crocin (5.69 g). CSE was dissolved in saline. Qocin and its analogues were dissolved in saline containing 30% dimethylsulfoxide. Their solutions were demonstrated for animals and hippocampal slices. 2.2. Learning performance test

Step through (ST) and step down (SD) tests were performed using 5week-old male ddY mice according to the method employed in our laboratory [9]. They were used in the experiments after 1 week acclimation to the environment. 2.3. Long-Term potentiation (LTP) experimental procedure

Anesthetized male Wistar rats 7-9 weeks old were used. Extracellular recording of population spike amplitude in the dentate gyrus in hippocampus were performed according to the method employed in our laboratory [10]. CSE was administered orally, ethanol was administered via three different routes, i.e. orally, intravenously or intracerebroventricularly. Crocin and its analogues were injected intracerebroventricularly. To summarize and compare several data sets of time-course curves of potentiation, the area under curve (AUC) from 5 to 60 min after tetanus was calculated. 2.4. LTP experimental procedure using rat hippocampal slice

Hippocampal slices (400-500 fim) were quickly prepared from male Wistar rats (8- to 9-weeks-old) and maintained in a chamber at 35 °C, where they were continuously perfused with artificial cerebrospinal fluid as described in our previous paper [11]. A bipolar tungsten electrode was placed in the stratum radiatum to stimulate Schaffer collateral and commissural afferents. The evoked potential was extracellularly recorded from the pyramidal cell layer of the CAl subfield with a glass capillary microelectrode. A single test stimulation (0.05 msec duration) was applied at intervals of 30 sec. Drugs were delivered by perfusion. To induce potentiation of the evoked potentials, tetanic stimulation was applied at the same intensity through the same stimulating electrode as used for the test stimulation. The magnitude of LTP was evaluated by the population spike amplitude 30 min after tetanic stimulation.

960

3. RESULTS J. 1, Effect of CSE on learning behavior [12]

There were no differences between control and CSE treated groups in either ST or SD test, suggesting that CSE had no effect on memory restoration in nomial mice. It is well-known that the oral administration of ethanol induced impairment of memory acquisition in ST and SD tests Fig. (5). The increase of errors (C) and the decrease (D) in number of leamed mice were significantly ameliorated by CSE in SD test dose-dependently, but not in ST test. The improving effects were dose-dependent.

Step-through test

Step-through test

CSE (mg/kg) + 30% EtOH 10 ml/kg

CSE (mg/kg) + 30% EtOH 10 ml/kg

Step-down test

Step-down test

300H

CSE (mg/kg) • 30% EtOH 10 ml/kg

CSE (mg/kg) •»• 30% EtOH 10 ml/kg

Fig. (5 ). Effect of CSE on memory acquisition in 30% ethanol-treated mice in ST and SD tests. D=Learning trial; attesting trial. A: The latency which indicated the time mice entered the dark compartment in ST test. B: Mice which did not enter the dark compartment within 300 s were termed successful mice. C: The number of errors was the number of times of stepping down to the floor in SD test. D: The number of mice which made no errors in SD test. *p < 0.05. **p < 0.01 vs. control group '*'p < 0.05 vs. ethanol group in Mann-Whitney's Utest. Mean + SEM. n=12.

961 3.2. Effect of CSE on LTP [13]

The induction of LTP after tetanus was significantly blocked by oral administration of ethanol in a dose-dependent manner Fig. (6). Thirty % of ethanol completely blocked the induction of LTP, Fig. (6-A).

H-H4i

30 Time (min) 3000

B c E

K

o <

Control

10%

20%

30%

EtOH tol lOml/kg p.o.

Fig. (6). Blockade of LTP by oral administration of ethanol. A: Time-couise of potentiation of population spike amplitude induced by application of tetanic stimulation (30 pulses at 60 Hz). Saline ( # , n=9) or ethanol (10%, A, n=5; 20% , D , n=5; 30%, 0 » n=8) was administered orally 20 min prior to the tetanus. B: The dosedependent, inhibitory effect of ethanol on the magnitude of LTP was summarized by calculating the area under the curve of time-course of potentiation from 5 to 60 min after tetanus. All data are represented as mean ± SEM of n observations. Asterisks indicate significant differences &om the saline group (control), ""^p < 0.01; Duncan's multiple range test.

962

Fig. (7) shows the effect of CSE on blockade of LTP by oral administration of ethanol 20 min prior to application of tetanus. The basal responses before tetanus were not significantly changed. The LTPblocking effect of ethanol was, however, significantly improved by oral administration of CSE (125-250 mg/kg). Fig. (7-A).

Time (mIn) 3000

B

c

e <3

##

##

O <

Control

125

250

CSE (mg/kg p.o.) -I- 30% EtOH lOfiH/kg p ^ .

Fig. (7 ). Effect of CSE on blockade of LTP by oral admlnistrtation of ethanol. A: Time-course of potentiation of population spike amplitude. Saline ( # , 0 ) or CSE (250 mg/kg, A) was orally administered 30 min before tetanus, and then saline ( # ) or 30% ethanol (0»A ) was orally administered 10 min later (20 min prior to tetanus). B: The dosedependency of the influence of CSE on the LTP-blocking effect of ethanol. The numbers of observations are as follows: saline alone (control), n=9; ethanol alone, n=8; ethanol and 125 mg/kg CSE, n=5; ethanol and 250 mg/kg CSE, n=ll. All data are represented mean ± SEM of n observations. •*p<0.01 vs. saline group (control). ##p<0.01 vs. ethanol alone (Duncan's multiple range test).

963

When 30 % of ethanol (20 ml/kg) was administrated by the intravenous injection 10 min prior to tetanus blocked the induction of LTP. By the oral CSE (125 and 250 mg/kg) administrations 20 min prior to ethanol injection, the LTP-blocking effect of intravenously injected ethanol was significantly attenuated dose-dependently Fig. (8-A). Fig. (8-B) indicated the dose-dependency of the influence of CSE on the LTP-blocking effect of ethanol. The effect of CSE was also observed when ethanol was injected intracerebroventricularly [13].

^ 150 a E a 1001 0

30 Time (min)

3000 F e E

60

B

-t

##

K

##

-i-

<

o

•f-

<

Control

125

250

CSE (mg/kg p.o.) -I- 30% EtOH 2iiil/kg l.v.

Fig. (8). Effectof CSE on blockade of LTP by intravenous injection of ethanol. A: Time-couise of potentiation of population spike amplitude. Saline ( # , 0 ) or CSE (250 mg/kg, A ) was orally administered 30 min before tetanus, and then saline ( # ) or 30% ethanol (Of A) was intravenous injected at a volume of 2 ml/kg 10 min prior to tetanus. B: Hie dose-dependency of the influence of CSE in terms of the magnitude of LTP. ITie numbers of observations are as follows: saline alone (control), ns5; ethanol alone, n=:8; ethanol and 125 mg/kg CSE, n=6; ethanol and 250 mg/kg CSE, n=5.

964 J.J. Effect ofcrocin

on leanung behavior [14]

A single oral administration of crocin had no effect on memory acquisition in normal mice in both ST and SD test (data not shown). In ST test, when crocin was given 10 min before the ethanol treatment the latency increased Fig. (9-A) and the number of successful mice increased at a dose of 200 mg/kg compared to ethanol-treated group in the testing trial Fig. (9-B). In SD test, crocin decreased the number of errors and increased the number of success dose-dependently Fig. (9-C and D).

Crodn (mo/kg) • 90%EUMnel10nU/kg

D ^

.100-j

Control Crocin (mg/kg) ••- 30H Ethanol lOir^/kg

0

M

100

200

Cradn (mo^kg) • 30M Cthanol 10 ml/kg

Fig. (9 ). Effect of crocin on 30% ethanol-induced impairment of memory acquisition in ST and SD tests. Open columns show the results in the learning trial. Hatched ones show the results in the testing trial. Crocin was given by a single oral administration 30 min before the learning trial. Then, 30% ethanol was given orally 20 min before the test. Mice in control and ethanol groups were orally given only saline or ethanol, respectively. All data are represented as the means ± SEM of 10 mice. A: The latency in ST test *p<0.05, •*p,0.01 vs. control group, '^<0.05 vs. ethanol group in Mann*Whitney's U test. B: The number of successful mice (%) in ST test ••p<0.01 vs. control group, *p<0.05 vs. ethanol group in chi-square test. C: The number of enors in SD test •p<0.05, **p<0.01 vs. control group, *p<0.05 vs. ethanol group in Duncan's multiple range test. D: Hie number of successful mice (%) in SD test. **p<0.01 vs. control group, '^p<0.05 vs. ethanol group in chi-square test.

965 3.4. Effect ofcrocin and its analogues on LTP [15]

We already indicated that intravenous injection of ethanol blocked the LTP induced by tetanic stimulation Fig. (6). However, when CES were injected intracerebroventricularly, the blocking effect of ethanol on the LTP decreased dose-dependently Fig. (8). Moreover, crocin prevents the ethanol-induced impairment of memory acquisition in ST and SD tests. From these results it is easily suggested that crocm antagonized the blocking effect of ethanol on the mduction of LTP. Qocin of 50 mg/kg ameliorated the blockmg effect of ethanol on the LTP at approxhnately 84% compared to the control as indicated in Fig. (10). Crocetin gentiobiose glucose ester also antagonized the blocking effect of ethanol on the LTP dose-dependently. But ttie intensity is not so strong, about a half of crocin when compared the mtensity of 50 mg/kg. On the other hand, crocetin di-glucose ester did not remove the inhibitory effect of ethanol on the LTP.

3,000

(6) 2.500

I 2.000 H ^ 1.600H

?

O 3 1.000

(5)

(13)

<

500 H

Control

A EtOH alone

10

50 Crocin

50 100 Crocedn gentiobiose glucose ester

(6)

50 100 (mg/kg) Crocetin di-glucose ester

Fig. (10 ). Effects of crocin and its analogues on the LTP-blocking effect of ethanol. The vehicle or drug was intracerebroventricularly injected 20 min before tetanus, and saline or 30% ethanol was intravenously Injected at a column of 2 ml/kg 15 min before tetanus. Hie AUG from 5 to 60 min after application of tetanus was calculated and defined as an index of magnitude of LTP in each group. The data are represented as the means ± SEM of the number of observations shown in parentheses. **p<0.01 vs. control group. +p<0.05, ++p<0.01 vs. ethanol group in Duncan's multiple range test.

966 3.5. Effects of ethanol and crocin on the induction of LTP in the CAl hippocampal slices [16]

region of rat

In the control experiments, strong tetanic stimulation induced robust LTP. Ethanol (30%; 10-15 ml/kg) did not show any significant effect on the baseline synaptic responses, but suppressed the induction of LTP following strong tetanic stimulation in a concentration-dependent manner Fig. (11). 200r

Il50| a E m

I

-10

-!— Time (min)

15

30

w 6

Crodn (mg/kg) +30%EtOH(15ml/kg) Fig. (11). Effects of ethanol and crocin on LTP induced by strong tetanic stimulation in the CAl region of rat hippocampal slices. The inset in A is a representative evoked potential recorded from the CAl pyramidal cell layer. Calibration hards: vertical 2 mV, horizontal 10 msec, llie population spike amplitude was defined as an average of the amplitude from the first positive peak 1 to the succeeding negative peak 2 and the amplitude from the negative peak 2 to the second positive peak 3. A Time-course of potentiation induced by strong tetanic stimulation in the control slices (0> n=23) and in the slices treated with 30% of ethanol (15 ml/kg) ( # , n=:27) and in the slices treated with 30% of ethanol (15 ml/kg) and 20 mg/kg crocin (A, n=57). (1) and (2) indicated 30% ethanol of 15 ml/kg and 10 ml/kg, respectively. Ethanol or crocin was added in the perfusing ACSF from 15 or 20 min, respectively, before tetanic stimulation. The ordinate indicates the population spike amplitude expressed as a percentage of the baseline values immediately before tetanic stimulation. B; Summary of the effects of ethanol and crocin on the induction of LTP. TTie magnitude of LTP was evaluated with the population spike amplitude 30 min after tetanic stimulation. Tlie numbers of observations in each group are shown in parentheses. All data are represented as the mean ± SEM. **p<0.01 vs. control, #p<0.05 vs. 30% of ethanol (15 ml/kg) alone. Duncan's multiple range test.

967

The effect of crocin on the LTP-suppressing effect of ethanol was mvestigated Fig. (11). The potentiation induced by strong tetanic stimulation in the presence of 20 mg/kg crocin and 30% of ethanol (15 ml/kg) was significantly larger than that in the presence of 30% of ethanol (15 ml/kg) alone, indicating that crocin clearly attenuates the action of ethanol. 4. DISCUSSION A single oral administration of CSE ameliorated the ethanol-induced impaimients of memory. The increase of errors and the decrease in number of leamed mice were significantly improved. It had no effect, however, on acquisition or retrieval in normal mice, and did not improve the scopolamine-induced impaimient of memory registration in mice in either ST or SD test [12] suggesting that CSE has a specific ameliorating effect on ethanol-mduced memory deficiency or the toxic effects of alcohol. It is well-known that the hippocampus is very important in leaming and memory processes. The appearance of LTP by high-frequency afferent stimulation is suggested to be closely related to the cellular basis of leammg and memory [17]. The relation between LTP phenomenon and its inhibition by ethanol using the rat hippocampal slices has been reported by several groups [18-20]. The most importantfindingin the present study is that CSE antagonizedtiieLTP-blocking action of ethanol. TTie dose of CSE effective in antagonizing the LTP-blocking effect of oral administration of ethanol is consistent with that effective in improving the memory impairment induced by ethanol. The fact that CSE alone does not facilitate the generation of LTP but antagonizes the LTP-blocking effect of ethanol provides direct evidence that CSE has a specific antagonizing action against ethanol action, although the mechanism is still obscure. Since nearly 30% of CSE is crocin, it is easily suggested that crocm functions as an important factor on improving the memory impairment induced by ethanol and antagonizing the LTP-blocking effect of ethanol. An oral administration of crocin had no effect on memory acquisition in normal mice but improved the ethanol-induced impairment of leaming behaviors of mice in passive avoidance performance tasks. This phenomenon was resemble to that of CSE. The tendencies of effect between CSE and crocin are similar each other. From these results it can be easily speculated that crocin is the most important principle in CSE. Other crocetin glucoside ester weakly antagonized the blocking effect of ethanol on the LTP compared to crocin. The LTP-suppressing action of ethanol is reduced by the presence of crocin in rat hippocampal slices in in vitro experiment. Crocin alone dose

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not affect the baseline responses nor the potentiation induced by weak tetanic stimulation. It becomes evident that crocin attenuates the action of ethanol within the hippocampus supporting with in vivo experiments as abready documented. Although impairaient of ethanol for brain functions mcludmg learning and memory is well known [21,22], the mechanism is still obscure. Several groups reported the LTP-blocking effect of ethanol using rat hippocampal slices [18-20]. We confirmed that crocin prevents the LTP-suppressing effect of ethanol in the CAl region of rat hippocampal slices [13]. More recently we studied that crocin removes the inhibitory effect of ethanol for NMDA receptor-mediated responses in rat hippocampal neurons using rat hippocampal slices and cultured rat hippocampal neurons [23]. ITiis result suggests that the function of crocin may be able to be concentrated to NMDA receptor level in our on-going investigation of crocin. In this review we confirmed the structure and activity relation regarding crocin and other crocetin glucose esters. Since the elimination of glucose in a crocin molecule decrease its activity, it is easily suggested that two gentiobiose moieties are important for the induction of activities. Of course the effect of crocin was not mimicked by gentiobiose or glucose alone (data not shown). These results clearly suggest that gentiobiose attached to the crocetin moiety are important for crocin to exert the biological activity. This tendency was observed in the study of the inhibitory effect on two-stage carcmogenesis of mouse skin papillomas [8] On the other hand, we have reported that crocin easily changes into crocetin di-glucose ester which has almost no activity, by hydrolysis of two gentiobioses by an indigenous 6-glucosidase [1]. Therefore, the storage conditions of saffron are also important for the maintenance of crocin concentration resulting in higher pharmacological activity of saffron. References [1] [2] [3] [4] [5] [6] [7] [8] [9]

Morimoto, S.; Umezaki, Y.; Shoyama, Y.; Saito, H.; Nishi, K.; Irino, N. PlantaMed., 1994,60,438. Miwa, T. Jap. J. Pharmacol, 1954, 4, 69. Gainer, J.; Jones, J.R. Experimentia, 1975, 31, 548. Nair, S.C; Pannikar, B.; Panikkar, K.R. Cancer Lett., 1991, 57, 109. Salomi, M.J.; Nair, S.C; Panikkar, K.R. Nut. Cancer, 1991, 16, 67. Gainer, J.L.; Wallis, D.A.; Jones, J.R. Oncology, 1976, 33, 222. Escribano, J.; Alonso, G.L.; Coca-Prados,; M.; Femandes, J..A. Cacer Lett., 1996,100,23. Konoshima, T.; Takasaki, M.; Tokuda, H.; Morimoto, S.; Tanaka, H.; Xuan, L.J.; Saito, H.; Sugiura, M.; Molnar, J.; Shoyama, Y. Phytotherapy Res., 1998, 12, 400. Ishihara, A.; Saito, H.; Ohta, H.; Nishiyama, N.; Jpn. J.Pharmacol, 1991, 57, 329.

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[10] Ishiyama, J.; Saito, H.; Abe, K. Neurosci, Lett., 1991, 12, 403. [11] Abe, K.; Xie, F.; Saito, H. Brain Res., 1991, 547, 171. [12] Zhang, Y.; Shoyama, Y.; Sugiura, M.; Saito, H. Biol. Pharm. Bull, 1994, 17, 217. [13] Sugiura, M.; Saito, H.; Abe, K.; Shoyama, Y. Phytotherapy Res., 1995b, 9, 100. [14] Sugiura, M.; Shoyama, Y.; Saito, H.; Nishiyama, N. ProcJapan Academy, 1995a, 71, Ser.B, 319. [15] Sugiura, M.; Shoyama, Y.; Saito, H.; Abe, K. J. Pharmacol Exp. Therap., 1994, 271, 703. [16] Sugiura, M.; Shoyama, Y.; Saito, H.; Abe, K. Jpn. J. Pharmacol, 1995c, 67, 395. [17] Teyler, T.J.; Discema, V. Annu.Rev.Neuroscu, 1987, 10, 131. [18] Durand, D.; Carlen, P.L. Brain Res., 1984, 308, 325. [19] Sinclair, J.G.; Lo, G.F. Gen. Pharmacol, 1986, 17, 231. [20] Blitzer, R.D.; Orland, G.; Emmanuel, L.M. Brain Res., 1990, 537, 203. [21] Mair, G.R.; McEntee, J.W. Behav. Brain Res., 1983, 9, 1. [22] Butters, N. /. Clin. Exp. Neuropsycol., 1985, 7, 181. [23] Abe, K.; Sugiura, M.; Shoyama, Y.; Saito, H. Brain Res., 1998, 787, 132.

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971

SUBJECT INDEX AlandA2A-adenosine 895 ABC transporter gene 322 Aberrant crypt foci (ACF) 65 Abiesenonic acid 60 methyl ester 60 Abietane Abisapogenol 95 AbriHerba 99 antihepatotoxic effect of OGs from 99 Abrisapogenol A 96 Abrisapogenol B 96 Abrisapogenol C 96 Abrisapogenol D 96 Abrisapogenol F 97 Abrisapogenol I 97 Abrisapogenol L 96 Abrisaponin 97 Abrisaponin A 96 Abrisaponin C 96 Abrisaponin D | 96 Abrisaponin D2 96 Abrisaponin D3 96 Abrisaponin 1 97 Abrisaponin L 96 Abrisaponin SB 95 Abrisaponin SO] 95 Abnis 180 Abrus contaniemis 93 Abrusprecatoriiis 93 Abscisic acid 367 Absidiasp 160 Acanthaceae 249 Acanthela klethra 848,852 Acanthela pulcherrima 852 Acanthela sp 852,853 Acanthella 688,847 Acanthella cavernosa 689 Acanthella acuta 848,852,853 Acanthella of cavernosa 858

(-)-Acanthene B from Acanthella pukherrina 853 Acanthifolicin 832 Acanthodendrilla sp 704 Acanthosterol sulfate A-J 704 Acarniis bergqiustae 724 Acer sdcchanimn Marshall 666 Acelaminophenone 219,614 Acetate/mevalonate pathway 130 Acetophenones 666 7/J-Acetoxy-8,13-epoxy-1 a,6j3,9atrihydroxylabd-14-en-11 -one 256 6j3-Acetoxylabd-8( 17) en-15-oic acid 250 10-A cetoxy menthol 160 3-0-Acetylerythrodiol 59 3-(9-Acetyloleanane 59 Acetyl CoA carboxylase 525 Acetyl kaikasaponin 95 Acetylastrogaloside I 193 Acetylcholine 516,519,520,527,530,547 nictotinic receptors 519 vasodilator effect of 595 Acetylcholine-gated Na'^/K'^channels 527 Acetylcholinesterase 536 Acetyloleanolic acid 59 Acetylsoyasaponin 95 Acid catalysed epimerization of indole alkaloids 3 Acid catalysed epimerization 21,33 Acifluorfen 669 Aconitiim alkaloid sangorine 531 A cor OSp ora gob tens is 8 02 ACP-malonylated enzyme 506 Acquired immunodeficiency syndrome (AIDS) 734,941,948 Actaea 180 Acteoside 468,543 Actinase E 438 Actinomadura spiralis 776 Actinomycin D (Act D) 58,461,479 Actinoplanes sp 777 K-cells-IL2-activated killer cells 273 Acute phase proteins 461

972

Acyclovir 116 Acyl carrier protein (ACP) 501,509 Acylovir-resistent HSV-1 940 3-O-Acylquinic acids 921 5-O-Acylquinic acids 921 Adenocorticorophic hormone (ACTH) 265 Adenohypophysical hormones 265 Adenosine A1^ receptors 532 Adenosine A2A 532 Adenosine triphosphate 948 5-Adenosyl-L-methionine (SAM) 506 Adenosylmethinonine 828 Adenylate cyclase 260,262,265, 270,489 495,527,532,539 Adenylate cyclase activator 488 Adenylate cyclase system 493 Aderostenedione 419 Adocia sp 695,723,861 Adociaquinone A 862 Adociaquinone B 862 Adociaquinones 861 Adociasulfates 695 ADP-ribosylcyclase 535 Adrenergic antagonists 530 Adrenergic receptor 271,530 a-Adrenoceptors 30 Aerobacter 925 Aeromonas salmonishida 259 Aeroplysinin 718 Aeropyrum pernix 720 Aeruginosin 205 A 799 Aeschynomene indica 93 Aframoniiim danielli (Zingiberaceae) 257,260 Aframonium aulacocarpos 259,266 Agathis robusta 252 Agelas 779,709,712,722 Agelas clathordes 860 Agelas dendromorpha 792 Agelas dispae 106,110,122,711 Agelas marritiane 688,722,784,870 Agelas nakamurai 688,712,780,860 Agelas novacaledoniae 110 Agelas oroides 703

Agelas sponge species 688 Agelas wiedenmay^eri 712 Agelasidine B 861 Agelasidines A-D 860,861 (+)-Agelasidines B and C 860,861 Agelasimine A 688,792, 793 Agelasimine B 688 Agelasimines 687 Agelasphins 722 Ageliferin 779,780 Aggregation of heme into hemozoin 360 Aggregation of porphyrin and metalloporphyrin systems 358 Agmatine (l-amino-4-guanidinobutane) 530 A2-agonists 265 A] adenosine agonists 265 somatostain 265 opiates 265 Agroastragalaside I 197 Agroastragaloside II 197 Agroastragalaoside IV 197 Agrobacteriiim sp 395,659,711,878 Agrobacteriiim tiimefaciens 395,399 Agrochelin 711,878 Akuammigine 31 Alachlor 669 Alacocarpines A & B 266 Alanine 264,400 Alanine transaminase (ALT) 475 Alanine aminotransferase 91,121 Albioside 1 95 Albioside 11 95 Albizziajidibrissin saponins 208 Alcaligenes faecalis 718 Alcohol and D. Gal N models 463 Aldose reductase 263,264 Alexandroside I 214,198 Algoane 804 Alginate lyases 719 Alizarin 543,544 Allelopathy 658 AUoyohimbane 5,20

973

Allyl formate-induced liver injury 466 A Iphidium pliciferan 895 Alpinia galanga 257,260 Alternaria alternata 305,307,672 Alternaria solani 302,303,307,316 Alternatamide A 774 Alternatamide B 774 Alternatamide C 774 Alteromonas rawa sp 820 Alteromonas sp 717,719 Altertogorgia sp 695 Altohyrtin 761 AltohyrtinA 727 Alzheimer's Disease therapy 530,536,710 Amanita mushroom oxazole alkaloids 528 muscimol 528 dihydromuscimol 528 Amarillidaceae alkaloid 536 galanthamine (galantamine) 536 Amathaspiramide E 782 Amathia alternata 776 Amathia convohita 786 Amathia wilsoni 782 AmbigolA 792 Ambigol B 792 Ambliofuran 688 Amblyomma americanum 795 Ambrosia bQQi\Q 138 Ambrox 253 Amentoflavone 545 a-Amino-3-hydroxy-5-methyl-4-isoxazole prionic acid (AMPA) 529 1 - Aminocyclopropane-1 -carboxy late (ACC) 372 2-Aminoindan-2-phosphonic acid (AIP) 674 2-Amino-3-[5-deoxy-5-(dimethylassinoy)P-D-[ribofuranosyloxy]-2-Hydroxypropene-1-sulfonic acid 867 y-Amino butyric acid (GABA) 519,516, 520 y-Amino butyric acid 548 Aminoimidolium salt of steroid trisulfate from Topsentia sp 704

4-Aminoquinoline compounds 343,345 quinolinemethanol derivatives 343 Amodiaquine 344,348 Amopyroquine 344 Amorphane 848,849 Amoxycillin 617 5'-AIVIP-dependent protein kinase 524, 541,547 Ampelosin 470 Amphetamine 537,538 Amphicarpeae edgeworthii 93 Amphilectane-type diterpenoids 690 Amphimedon sp 709,710 Amphiphilic triterpenoids 541 Ampicillin 258 AMPK phosphorylates 525 Amylase 719 p' Amyrin 58 A nabaena cylindrica 117 Anabaenba sp 794 Anchinoe tenacior 862 Andalusol 249,250 Andrographis panicidata 251,270 ANF-activated membrane bound guanylate cyclase 523 Angelicafiircijuga 471,476 Angiotensin Il-induced hypertension 595 Anguibactin 877 Ankyrin 496 Annonaceae 249 Annonaine 531 Anomalin 471,476 Anopheline mosquito 328 Antagonists cannabinoid receptors 532 Antemisin 349 Antharufm 543 Anthocidarios crassispina 868 Anthocyanins 513 Anthocyridin IdB 616 Anthopleiira elegantissima 873 Anthraflavic acid 543,544,541 Anthraquinone-based PKA inhibitors 544 PKC inhibitors 544 Anthraquinones 509,513,541 alizarin 541

974

anthrarufin 541 chrysazine 541 chrysophanic acid 541 emodin 541 purpurin 541 quinalizarin 541 quinizarin 541 Anti TNF-a antibody 461 Anti-ARS2 antibody 435 Anti complementary activity of OG 17 Anti-crocin mAb 957 Antibody dependent cell mediated cytotoxicity (ADCC) 273 Anti-CAM IgG 491,492 Antifungal isocoumarin 484 Antifungal isoflavone 319 Antihepatotoxic effect of OGs 98,99 Antihistaminic dispacamides C andD 870 Anti-HRP monoclonal antibodies 334 Anti-IFN-Y 464 Anti-IL-1 antibody 464 Anti-IL-6 antibody 464 Antimalarial drugs 327,360 Antimalaial therapies 361 Antimetastic immunity 429 Anti-mu stimulation 272 Anti-pathogenic phytoalexin compounds 652 Anti-slgs and anti CD40 272 Antitumor immunopotentiation 454 Anti-TNF-a 464 Anti-TNFa-antibodies 461 Antitopoisomerase II 693 Antitumor-promotion activity 46 Antitumor-promotor 44,45,60 from edible plants fungi crude herbal drugs 44 antioxidant activites of 59 Antiviral activity of OGs 114 Apicularens 728 Apigenin 541,544,594 Apiin 595 Aplidhim gemis 837

Aplidiiim meridiamim 764 Aplysia 724 Aplysia brasiliana 702,717,785,801 Aplysiafistiilaris lil Aplysia ptmetata 769 Aplysina aerophoba 712 Aplysina archeri 720 Aplysina cauliformis 766 Aplysina sp 707,17 Aplysiotoxin 771 Apocynaceae 249 Apomorphine 538,541,543,544 Apomorphine-binding sigma receptors 538 Apoplastic fluids 400 Apoptic body formation 466 Apoptosis in in cerebellar granulosa cells 269 in primary granulosa cells 269 in susceptible Burkitt's lymphoma (BL) 272 Apoptotic bodies 469 Apoptotic bodies formation 468 Aporphine alkaloids apomorphine 544 boldine 544 Aporphine isoquinoline alkaloids xylopinine 530 isocorydine 530 Aporphine isoquinoline alkaloid 527,543 Arabidopsis genes mutated 414 Arabidopsis 368,391,395,413,422,426, 660 Arabidopsis dwarf 414 Arabidopsis dwarf4 mutant 420 Arabidopsis mutant dwfl/stel 416,417 Arabidopsis mutant cpd 421 Arabidopsis plant 395 Arabidopsis RLK 384 Arabidopsis thaliana 385,377,496 Arabidopsis mutant detz 419 Arabidopsis mutant sax I 418

975

Archaebacteria metabolism 939 Arachidonic acid 263,274,275,333,596, 597,616 Arachis hypogaea 93 AragusterolC 11 \ Araucaria bidwilli 249 Araiicaria excels 249 Arborinane 54 Arbus cantoniemis 91 Archaebacteria 686 Arctuin lappa L. 944 Arecoline 530 Arenastatain A 114,111 Arsenic-containing ribofuranosides (ACRs) 867 Arginomonas non-fermantam 155 Aristeguetia btilleaefolia 251 Arjuno acid triacetate 59 Aromadendrane 848,850 ARS2 435,451 NMR spectra of 435 antitumor activity 438,439 chemical characterization of 435 Artemisia family 944 Artemisia montana^ 944 Artemisiaprinceps p 944 Arthrobacter ilicis 719 Artimisinin 350 Arxulla adeninivorans 150 Ascidiacyclamide 879 Ascidian alkaloids 706 Ascidian metabolites 758 Ascidians 707,709 Ascomycetes 129 Ascorbic acid 236,349,930,936 Asemestioside A 195 Asemestioside B 195 Asemestioside C 195 Asiatic acid 541 Asimilobine 531 AskendosideA 198 AskendosideB 194 AskendosideC 198 AskendosideD 194 AskendosideC 198

Aslcaligenes entrophiis 162 Asparaginase 719 Asparagus officinalis 311 Asparagus sulfokine-a and /? 37 Aspartate transaminase 477 Aspaitic proteases 330,331,375 Aspegillus niger 136,140-143,145,148, 149,152-155,157,158,165,166,168 172,173,777 Aspergills nidulam 301,321 Aspergillus celliilosae 150 Aspergillusfumigatus 764 A spergillus ochraceus 13 9 Aspergillus oryzae 780 Aspergillus sp 150,151,156,729 Assay for enod 40 activity 381 Assay for systemin activity 373 Assay method for hepatoprotective effects 100 Aster spathidifolius 251 Asteraceae 235,249,250,252,944 Asteremyces cruciatus 828 Asternia pectinifera 698 Astrachyside A 194 Astragalloside 104 Astragallus gQnus 188,190 Astragaloside 179 Astragaloside I 193,224,227 Astragaloside II 193,220,221,223, 224,227 Astragaloside 111 194 Astragaloside IV 193,220,221,224, 226,227 Astragaloside V 195 Astragaloside VI 194,224,227 Astragaloside VII 194 Astragaloside VIII 95,115,116,118,203, 222,120 Astragalus 180,183,219,186,187,190 adsurgens 184 alexandrimis 184,189,192,193,214, 224 alopecurus 184,201 amarus 194,195 babatagi 184

976

basineri Trautv. 184,194 hethlehemiticus 188,189 boeticiis 192 breachypterus 184,192,193,197 cephalotes Bank & Sol. 184,197 chrysoptenis 184,194,203 coliiteocarpus 184,192 complanatus R.B. 93,184,203,204 dasyanthus Pall 184,199 disseetus 184,192 ephemerotonim 184 ernestii 184,195 exilis L. 184 falcatus 196 furcijuga 472 galegiformis L. 184,196 glycyphyllos L. 184,203 gummifer 188 hamosus L. 184 hepatoprotective mechanism of coumarins from 472 polyacetylenes from 472 illiemis L 184,195 kahiricus 189 kuhitangi 184, 192,194 kidabemis 184 melanophrurius Boiss 184,192,193, 194,197 membranaceiis BungQ 184,192,193, 194,195,205,219,220,221 microcephalus W\M 184,193,200 monghoUcits mongholicus Bunge 184,192,193, 194,202,219 oleifolhis 199 oleifolius DC 185,199 orbiculatiis LedQb 185,202 /?a/w/>*e«575 Oviz and Rassulova 185 192,200,197,202,203 penegrimis Woh] 185,195,216,225 pterocephaliis Bunge 185,192,193 pycnanthus 185,193,198 qidsqualis Bunge 185 saponins 186,219,224,227

schachirudensis Bunge 185,192,193, 194 siculns 220 sieberi DC 185,92,225 sieversiamis Pall 185,192,193,194, 195,219 simcits L. 185,203 spinosiis Vahl 185,188,192,193,221, 224 synthetic 200 tagacantha 192,197 taschkendicits BungQ 185,194,198 tomentosiis Lam. 185,199,225 tragacantha 185 tribidoides 203,204 trigomtsDC 185,188,193,194,200, 203,226 unirwdus 185,192,193 verrucosus 185,194,195,220 vUlosissimtis 185 Astragalus sp 179 Astragalus saponin I 226 Astragalus s^.\)OgQmx\s 186 Astragersianin VH 193 Astrailienin A 195 Astramembranin I 193,220 Astraverrucin 1 194 Astraverrucin II 194 Astraverrucin III 194 Astraverrucin IV 195 Astraverrucin V 195 Astraverrucin VI 195 Astraversian nl 193 Astraversian nil 193,224 Astraversian nIV 193 Astraversian nV 193 Astraversian nVI 193,224 Astraversian n X 192,224,227 Astraversian nIX 194 Astraversian nXI 194,226 Astraversian nXII 195 Astraversian nXIlI 195 Astraversian n XIV 193,194,224 Astraversian ns 179 Astrojanoside A 203

977

Atherosclerosis 934 Atisane 241 ATP hydrolysase 612 ATPase 780 ATP-binding cassette (ABC) membrane transport proteins 321 Atrazine 669 Atrial natriuretic factor (ANF) 519,540,547 Aulacocarpines A & B 259 Auiacorarpinolide 259,266 Aurainvilleol 787 AurantosideA 793,763 Aurantoside B 763 Aurantoside C 793 Aurantoside F 763 Aurantosides 757,763 Aureomycin 757 Aurilol 763 Auriside 757 Aurisides A and B 762 Auristain 716 Ausantoside E 780 Austrovenus stutchburgi 864 Aiixarthron unberium 19A Auxins 367,379,652,656,657 Avarol 697 Avarone 697 2-AVD treatment 462 AvenacinA-1 304-306,314 Avenacinase 304-306,308 Avirulence gene product 393 Avnacinase cDNA 308 avr (avirulence) gQiXQ 397 avr Bs2 gene 399 avr gene products 398,399 avr gene-encoded enzyme 397 avr genes 399,400 avr proteins 401,399 avr9 peptide 400,401,402 Avrainvillea longicaulis ISl AvrPto recognition 399 Axane skeleton 848,850 Axinella 847 Axinella cannabiena 848,852,853 Axinella carteri 714

Axinella sp 705 Axinella weltneri 700 Axinellamines 778 Axinellamine B 778,779 Axinellida 847 Axinysaa tenestratus 852 Axinyssa (Trachyopsis) aplysinoides 852, 853,858 Axinyssa 694,847,848,859 (+)-Axisothiocyanate-2 853 1-Axisothiocyanate 852 3-Axisothiocyanate 848 4-Axisotiiiothiocynate 852 6-Azaspiro (4,5) decane 871 Azepine-type bromopyrrole 799 Azukisapogenol B 97 Azukisaponin 95 Azukisaponin II 95,105,104,115,116, 226,118,203 Azukisaponin V 104,115,116,118,120, 203,226 Azukisaponin VI 97,104 B-90063 842 Baccatin III 245 Baccharis gaiidichaudiane 251,269 Baccharis genistelloides 941 Baccharis pedunelata 251 Baccharis scoparia 251 Baccharis petiolata 251 Bacillus calmette-Guerin (BCG) 462,497 Baciliis cereiis 259 Bacillus %^ 155 Bacillus subtilis 220,258-260,765,778, 770,776,777,779,782,789,792,863 Bacterial toxins 388 Baekhousia citriodora 132 Baeyer-Villiger monoxygenase 156 Baicalein 593,595 Baker's Yeast 143,145 Balanus amphirib 783,784,785 Baptisiasaponin 95 Barbamide 757,785,786,889,890 Barbatol 250

978

Barbiturates 528 Bartlett method 435 Basidiomycetes 150,129 Bastadin 719,720 Batzella sp 708,760,774,790,822,852 Batzelladine A 709 Batzelladines 708 BatzellineA 822 BatzellineB 822 Batzellines 707 Batzellines-isobatzellines 822 BCA method 433 BCG/LPS induced hepatotoxicity 471 Beesia 180 Bencimidazol 2-il methyl carbamete 669 Benthiozepines 534 diltiazem 534 Benzazepines 534 Benzoacetonitriles 719 Benzodiazapines 528 Benzoic acid 655,660,928,929 Benzoic acid-2 hydroxylase 660,669 Benzopentathiepin lissoclinotoxin B 833 Benzopyranopyrrole chromphore 776 Benzotrithiane 833 Benzotrithiepin 834,835 Benzotrithiepin S-oxides 834 Benzotrithiepin-5-(methylthio) varacin 834 Benzthiazole 896 Benzylisoquinoline alkaloids papaverine 541 isoquinoline 530 Benzyltetrahydroisoquinoline 824 Benzylthiocrellidone 832 Berbamine 472 Berbamine alkaloids 476 Berberine 528 Berberis vulgaris 476 Bethanecol 612 Betulin 60 Betulinic acid 59-61,66,541 BHT 792 Bidem aiirea 614 Bidesethylchloroquine 344

Biemma sp 703 Biflavones 125,126,127,131,539 amentoflavone 539 bilobetin 539 sequoiaflavone 540 ginkgentin 540 Biflustra pregragilis 825 Bioactive oleanane glucumoides 89 Bioactive peptide 367 Bioactive sponge peptides 757 Bioactivity of phenolic compounds 668 Biocides 668 Biological response modifiers (BRMs) 431,434 Biomineralization 335 of heme 334 ofhemozoin 360 process 327 Biomphalaria glabrata 7S5 Bionucleating tempUates (BNTI and II) 334,336,337 Biopholaria glabrata 792 Biosilica 717 Biosynthesis of brassinosteroids 413 enzyme involved in 413 Biota orientalis 275 Biotransformation of triterpenoids 138,125 Bis(6-formyl-4-hydroxy-2-(2'-n-pentyloxazol-4'-yl)-4-pyridon-3-yl] 841 Bisabolane 848 Bisbenzylisoquinoline alkaloid oxycanthine 530 Bisbenzylisoquinoline alkaloids 530 berbamin 530 decricine 530 isochondroendrine 530 macoline 530 rodiasine 530 Bisbenzyloisoquinoline alkaloids tubacurarine 530 Bisdesmosyl saponin 108,120 (-)-5,10-Bisisothiocyanakalihinol G 857 a,co-Bisisothiocyanates 857 Bismuth citrate 617

979

Bisobolane skeleton 849 ^/i:-quinoline antimalarial compounds 346 5/5-quinolines 345 Bis-t\\\?LZo\Q metabolite 790 Bistratamide D 903 Blastobacter sp 841 Blatella germanica 795 Blatellastanoside 757,795 Blepharispenum zangubariciim 251 Blossom oils 165 Blue cheese flavour 129 Blumenkrantz method 435 Boophiline 706 Boophilus microplus 706 Bordetella pertussis 521,538 Boric acid 674 Borjatriol 249,250 Bomeol 128,154 Botrylus leactis 706 Botrylus schlosseri 719 Botrytis cinerea 138,305,313,314,316 Botryodiplodia malorum 143 theobromae 320 Botryospheria obtusa 318 Botrytic cinerea 136,137,142,293,307, 662 Botzella sp 790 Bovine aortic endothelial cells 598 Bovine endothelin 841 Bovine aortic endothelial cells (BAEC) 593 Bovine famesyl-proteine transferase 790 Bovine serum albumin 334,337 BrachyosideA 197 BrachyosideB 192 BrachyosideC 197 Brassica (SRK) 368,386 Brassica napus 413 i5ra55/ca5-locus receptor kinase 384 Brassin 413 Brassino steroid precursors 415 Brassinolide 415-416 Brassinosteroid 316,413,420,425,367 Brassinosteroid biosynthesis 425,414,420

Brassinosteroid hormone 413 Brassinosteroid intermediates 414,417 Brassinosteroid pathway 421 Brassinosteroid precursor biosynthesis 416 Brassinosterol pathway 426 Brevetoxin 864,730 Brevetoxin B i 864 Brevetoxin B2 864 Brewer's wort 139 Briareitm excavatum 670 Brickellia lemmonic 230 BRMs (biological response modifiers) 452-455 Brominated aminoimidazolinylindole 763 Brominated indole alkaloids 712 Brominated phenylethylamine 765 Bromoagelifterin 779,780 6 Bromo-5-hydroxyindole 792 6-(p-Hydroxypheny l)-2H-3,4-dihydro-1,1,dioxo-l,4-thiazine 862 18-Bromo-octadeca-5,7,17-triyonoic acid 802 18-Bromo-octadeca-15,17-diene-15-ynoic acid 802 Bromobenzene 460 p-Bromobenzoyl derivative 816 Bromopyrrole alkaloids 762 5-Bromopyrrole-2-carbamide 780 Bromopyrrols 711,712,779,784 Bromotyrosin derivative 766 Bromotyrosine 642 derivatives 766 Bromotyrosine alkaloids 712 Bryostatin 684,726 derivatives 727 Bryostatin 1 726 Bryozoan 825 Bt2 cAMP 489,496 Bt2 cAMP-treated carrot cells 490 BufadienoHde 535 Bufadienolide Scillaren A 535 Bufotenin 531 Bugiila neritina 726 Bulgarian rose oil 132

980

Bupleuroside IIIJV, Xlli 474 hepatoprotective activity 474 Bupleiirum falicatum 474 Buplenrum Radix 474 Bupleiirum scorzonerifolium 474,476 triterpene glycosides of 474 Butein 546 N-t-Butylamodiequine 344 C-14 reductase 304 C6 oxidation pathway 421 Ca^-ATPase 516 Ca^"^- and phospholipid dependent protein kinase (PKC) 522,547 Ca^"^- dependent protein kinase 547 Ca^'^-and Cu^"*'-CaM-dependent enzyme Ca^^-ATPAse inihibtor 536 Ca2-^-ATPase 492,493,524,536 Ca^'^-calmodulin (CAM-dependent protein kinase) 261,524 Ca^"*'-Cam-activated adenylyl cyclase 538 Ca^'^-CaM-dependent protein (CaM PK) 522,546 Ca^"^-CaM-depdendent protein kinase 522,547,544 Ca^"^-channel blockers 489 Ca^^^-channels 521 Ca2"^-dependent CAM 491,540 Ca^"^-dependent enzymes 517 Ca^'^-dependent process 487 Ca^"*"-dependent K^ channel 534 Ca^"^-induced LDL oxidation 946 Ca^^^-inhibitors 487 Ca2+-ionophoreA23189 490,487,509 Ca2~^-mediated signalling 526 Ca^^^-mediated signalling targets 542 Ca^"^-permeable ion channel 397 Ca2"^-pumping ATPase 492 Ca2+ -tansporting systems 492

Ca2+-translocating ATPase 492 activity of 492,496 Ca^+ZCalmodulin 261 Ca2+/CAM -dependent protein kinases 489 volatge-dependent 498 Cabreuvaoil 162 Cacospongia linteiformis 698 Cacospongiolide B 697 CAD 666 cADPR 527,535 Caenoshabditis elegans 785 Caesalpiniaceae 92 3-0-Caffeoyl-4-0-feruloylquinic acid 920 3-(9-Caffeoylquinic acid 920,921 4-0-Caffeoylquinic acid 920 5-0-Caffeoylquinic acid 919,920 Caffeic acid 919,920,922-927,932-934, 936-938,655,657,663,671 Caffeine 532,539 Caffeoylquinic acid 943,944 derivatives 465,662 in body tissues 924 Caissarone 713 Calarene 170 Calceolareoside A 543 Calceolsareoside B 543 Caledonin 813,814 Calendonian 697 Calendula officinalis 58 Calicheamicin y 796 Caiicheamicin yl 838 Calicheamicin Q 796 Calicheamicins 796,838 Calicoferols 705 Calicogorgia granulosa 670 Callipeltasp 775 Callipltoside A 775 Callipeltosides 757,775 Callyspongia 724 Callyspongia johnstorii 111 Callyspongia truncota 723 CallystatinA 724

981

Calmodulin (CAM) 373,487,509,517, 522,524,547,489,494,543 Ca^"^-binding protein 487 Calmodulin antagonist activity 758 Calmodulin domain-containing protein kinase (CDPK) 544 Calmodulin-dependent phosphodiesterase (CAM-PDE) 509 Calvularia virdis 704 Calyculin derivative 731 CalyculinJ 732 CaM antagonists 489,492 CaM inhibitors 491 CaM dependent kinases 543,544 CaM dependent MLCK CaM-dependent isoformofPDE (CAM-PDE) 490 CaM-dependent PKs I-IV 524 CaM-dependent protein kinases 493,527 Camellia sinensis 472,478 cAMP-dependent protein phosphorylation 494 cAMP-dependent protein kinase (PKA) 522,547 cAMP-gated Na"^ channels 517 cAMP-dependent protein kinase 488,489, 522,548 cAMP-dependent channels 495 cAMP-gated N^"^ channels 523,524,546 cAMP-inducedCa2+-influx 491 cAMP-induced influx of K"^ 497 cAMP-phosphodiesterase 539 cAMP-response element binding (CREB) 523 cAMP-response element binding protein 548 cAMP-sensitive ion channels 490,496,497 Campesterol 415,416,417 formation of 419 Camphor 125,128,154,156,547 D-(±)-Camphor 156 Campylotorpis hirtella 94 Canavalia gladiata 93 Cancer chemopreventive agents 43

Candida albicans 258,759,764,773,780, 790,822 Candida pellicula 308 Cannabis sativa 532 Cannobinoid receptors 533 Canton iensistriol 96 Caprifoliaceae 249,251,259 Carbachol 613 Carbendazein B 6772 Carbendozin 669 4-Carbohydroxypatchoulol 172 (/?)-Carvane 858 8-Carboxylinalool 140 j8-Carboline 33,782,902 /J-Carboline alkaloid eudistomidins 817 /J-Carboline derivative 10,14 /?-CarboIine marine alkaloids 819 j8-Carboline metabolites 818 j3-Cardene 944 jS-Carotene 236 /J-Carotene hydrolase 718 Carbomanoyl oxide 261 Carbon catabolite 313 Carbon tetrachloride 460 Carboxypeptidase 375 Carcinogen MNU 66 Carcinoma Ehrlich cells 836 Carcinus maenas 718 Cardenolide 535 Cardiac steroid 55,59 Caribbean coral 690 Caribbean sponge 698 Carnitine synthetase 525 Carolisterol A-C 872 Carotenoids 44,237 Cairageenan-induced edema 220 Carveol 145,147,149 cis 2iX\A trans 148,150 Carvone 145-149 Caryophyllene 125,168 (-)-Caryophyllene-4,5-oxide 168 Casbene synthesis 486 Casein kinase 545 Casein spleen 431 Casopongia 695

982

Caspase inhibitor 2-VAD 462 Caspase-3 activation 471 Caspase-3 protease 469 Cassane 241 Casteriospongia sp 713 CAT 674 Catabolite repression 313 Catabol ism of haemoglobin 332,348 Catechin 541,612 Catechin derivatives 612 (+)-Catechin 612 Catechin-based flavones 541 Catechol 613,662,929 Catechol oxidase 654 Catecholamines 516 dopamine 520 epinephrine 520 horepinephrine 520 Catechol-O-methyltransferase 923,924,927 Cathasterone 415,420 Cathansterone 23a-hydroxylation 421 Catharantheus roseus 384 Cativic acid 254 Cavemothiocyanate 858 CB2 receptors 532,533 CCl4-induced cytotoxicity 468 CCl4-induced liver injury 466,469 CCl4-induced liver injury model 471 CD 18 antigens 454 CD20 antigen 272 CD25 positive cells 454 CD4 positive cells 454 CD4-antigens 454 CD69 positive cells 454 CD71 positive cells 454 CDg positive cytotxic T lymphocytes 454 Cd-induced liver injury model 471 cDNA clone encoding tomatinase 308 cDNA coding 61 cDNA copy of the HIV type-1 (HIV-1) genome 941 CDPK 542,546 inhibitors of 544 Cefriaxon 258

Ceftazidine 258 Celenamide 757 CelenamideE 778 Cell division protein kinases (CDKs) 526 Cellobiose degradation 308 Celosia argentea 476 Celosian 476 Cenarchaeum symbiosum 118 Cenebellar granule cells 268 apoptosis 268 Centrifugal partition chromatography (CPC) 207 Cephalomannine 245 Cephalosporium aphidicola 153,155 Cephalotoside A 197 Ceratinamides A and B 620,784 Ceratocystis 129 Ceratocytis fimbriata 485 Ceratodictyor spongiosum 902 Ceratosoma brevicaudatum 815 C-erbB-2-kinase 797,799,870,871 Ceric ammonium nitrate 821 Cervical carcinoma 268 /w-Coumaric acid 923,925 cGMPPDEs 523 cGMP phosphodiesterase 521 cGMP synthase 540 cGMP-dependent protein kinase (PKG) 522,523,548,517 cGMP-gated ion channel 541 cGMP-gated Na"^ channels 517,521 a-Chaconinase 305 a-Chaconine 300,305,311 Chaetomium cochiliodes 168,170 Chalcone isomerase 668 Chalcone reductase 653 Chalcone synthase 655 malonyl-CoA 655 Chalcone-synthase 504 Chamaecyparis obtiisa 246 Chamigrane-type derivative 701 Chamigrene-type, halogenated 702 Chemiiuminescence 940 Chemokines 535

983

Chemo-perception system of plant cell 394 Chemotactic tripeptide 275 Chitin 396 Chitosan 396 Chlamys hastata 813 Chloramphenicol 757 Chlorella 429 Chlorella kessleri 430 Chlorella vulgaris 430,431,450,452,453 antitumor activity 452 Chlorella vulgaris strain CK 22 (CVs) 429-433,436,437,440,450,447 antitumor immuno-activity 432 anti-peptide ulcer effect 430 characteristics 429 glycoprotein from 429 host-mediated activity 431 immunopotentiating effects 429 Chlorinated cylindrol Bi 790 Chlorinated malyngamides K 788 2-Chloroethylphosphonic acid 509 Chlorogenic acid 663,655,657,667,919921,930,932-935,937-940,942-947 antioxidative properties of 919, 930,935 Chloroissoclimide 270 Chloroorcinols 757 Chloroperoxidase 767 Chloropyridine 792 Chloroquine 336,337,343,344,345,346, 347,348,349,352,360 Chloroquine Fe(III) PPIX 346 Chloroquine-pyrollidinyl 344 Cholera toxin 521,538 Cholesterol acyltransferase 797 Cholesterol biosynthesis 722 Cholesterol synthesis inhibition 525 Choline 475,476 Choloroquine 335 Choloroquine-resistant parasites 352 Cholynotus senperi 893 Chondocurine 472,476 Chondodendron autumnal 476 Chondria californica 842

Chondria tenuissima 249 Chondromyces sp 730 Chromaggin granules 536 Chromodris hamitoni 735 Chronic viral hepatitis 463 Chrysanthenum coronarium 944 Chrysazine 543,544 Chrysocephalum ambiguum 251 Chrysophanoic acid 543 CoumaroyI CoA Chymo trypsins 388 Cicer arietium 93 Ciguatoxins 732,733 Cimetidine 612 Cimicifuga 180 Cinachyrolide A 759 Cinchona officinalis 476 1,8-Cineole 140 Cinnamate-4-hydroxylase 653,660 Cinnamic acid 925,652,653 Cinnamomum camphora 140,156 Cinnamonin 395 Cinnamoyl CoA 655 Ciona savignyi 784 Cis and /ra«5-deethyleburnamonines 19 Cis decalin-tetrahydrofuranyl kalihinol 855 C/5,c/^-ceratospongamide 904,905 C/5-5-hydroxy-4(4'hydroxy-3'-methoxyphenyl)-4-2"-imidazoyl)-l,2,3-trithiane 837 Cw-deethylburnamonine 6 C/5-deethyldihydroebumamenine 6 Cistaceae 249,252,253 Cistanche deserticola 468,476 Cistus clusii 250 Cistus creticiis (cistaceae) 246,249,250, 252,255,256,257,266 Cistus creticiis subsp erioocephatus essential oils of 259 Cistus incanus sub sp. creticus 266,258 Cistus ladaniferus 252 Cistus laurifolius 250 Cistus palinhae 250 Cistus symphylifolius 250

984

essential oils 255 subspecies 255,257 Citral 132,133,136,137,143 Citranellae 132,140 Citranellol 132,133,136,137,140 Citrellamine 837 Citrol 136,137,140 Citronellal 128 Citronellic acid 133,136 Citronellyl acetate 136 Citrus limonum 594 Citrus sp 128,266,267 Citrus aurantium 476 Citrus peel oils 145 Cladosoporium sp 145 Cladosporium cucumerinum 116 Cladosporium coccodes 305 Cladosporiumfulvum 302,308,398,399, 400 Clarithromycin 617 Clary sage oil 252 Clathramides 151,111 Clathrina clathrus 870 Clavelina cylindrica 859,793 Clerodane 241 Cliona chilensis 11S Clonidine 530 Clostridium perfringens 925 Clostridium tyrobutyricum 147 Cnemidocarpa bicormita 165 CoA ligas 653,666 Corynantheine-type oxindole alkaloids 26 Cochliobolus lunatus 318 Codium arabium 704 Codium dworkense 725 Colchicum autumnale 473 hepatoprotective effect 472 serum ALT activity 473 Coleosol 252 Colestane 49 Coleusforskohlii 256,252,261,958 Coleiis sativus 956 Collectotrichum coccodes 307 Colon-38 tumor cells 761

Cowbretaceae 180 Combretum 180,183 C/.s-Communic acid 275 Comoramide A 883 Comoramide B 883 Complogenin 101,184,185,186,204 Comploside I 95 Comploside II 95,203,104 Comploside III 97 Comploside IV 97 Comploside V 97 Compositae 921 Compylotropis hirtella 93 Con A 227,271,272,436,463,464,455, 477 Con A agarose 436 Con A hepatotoxicity 464 Con A induced liver injury 464,465 Con A model 463 Condensation of/?-coumaroyl-CoA 655 Conifery 2 alcohol 666 Coniine 528 Coniiim macidatiim 528 Conyza steiidellii 252 Conyza trihecatactis 252 Conns 732 Copaiba oWs 168 Copaifera L. 168 Corallistersfluvodesmus 829 Corallistine 829,830 Corculigo 180 Coriloiis versicolor 452 Coronarins (A-D) 265,266 Coronary artery ligation-reperfusion 594 Coronopifolia 111 Corriander oil 140 Corticum sp 707 Cortisone 516 Corymbiu villosum 251 Corynantheidol 24 Coiynantheine-type indole alkaloids 31 Coryne cassiicola 149 Corynebacteinim sp 145 Coiynebacterium kutschei 452 Corynespora cassiicola 149,162,166

985

Corynoxine 27 Corynoxine B 27 Coscinoderma mathewsi 699 Costasiella ocellifera 1^1 4-Coumarate 653,664 p-Coumaric acid 655,657,920,936,925 w-Coumaric acid glucuronide 923,925 4-Coumaric acid 652,660 4-Coumaroyl-CoA 652 3-0-Coumaroylquinic acid 920 4-(9-Coumaroylquinic acid 920 5-O-Coumaroylquinic acid 920 Coumarine isoepoxypteryxin 476 Coumaryl glycosides vanicosideA 543 vanicoside B 543 Counter-current chromatography (CCC) 205 Coumaric acid 657 Creatine kinase 527 Crella sp 704 Crella spinulata 832 Crellastatin 704 Creulide 690 Cribicellina cribraria 819 Cribochalina sp 900 Cristaria plicata 868 CRO 46 Crocin 964 effect of 964 Crocetin 958 Crocetin di-glucose ester 955 Crocetin gentiobiose glucose ester 959, 966 Crocetin di-(jS-D-digentiobiosy)-ester (crocin) 956 Crocetin di-(j3-D-gentiobiosyl)-(j3-Dglucosyl)-ester 956,957 Crocetin di-(/J-D-glucose)-ester 956,957 Crocetin gentiobiose glucose 955 Crocetin glucose esters 957 Crocetin-di-glucose ester 959 Crocin 956,957,959,964,965,968 Crocetin glucose esters 968 Crostis sativa L. 955,956

Crotalaria albida 93 Croton 242 Croton oil 45 Croton oil-induced ear edema 556 Croton oil induced edema (CRO) 45 Croton oil induced edema inhibition 46 Croton tigUiim 242 Cryptotrienolic acid 259 Cryptochlorogenic acid 921 Cryptococcus fagisiiga 662 Cryptogein 395 Cryptomeria japonica 257,275 Cryptophycin I 789 Cryptophycins 789 Cry^ptotethya crypta 712 CSF (colony stimulating factor) 455,960, 963 Cubebane skeleton 51,848 Cucumis melo 390 Cucumisin 390 Cucurbitacin F 59 Cucurbitane 50,57,59 23,24-dihydrocururbitacin F Cunnighamia lanceolalta 251 Ciinninghamella 172 Cimmnghamella blakesleana 172,173 Cupressaceae 246,249 Cupressus sempervirens 253 CuracinA 732,733,876 Curacin B 876 CuracinC 876 Curacin D 876 Curacins 876 Curculigo orchioides 227 Curculigosaponin G 227 Curcuma longa 476 Curcuma zedoaria 172 Curcumin (turmeric yellow) 44,469,470, 541 Curcimua longa L. 469 hepatoprotective activity 469 serum transaminase activity 469 Curvularia liinata 254,261,260,263 Cuscuta reJJexa 944,945

986

CVS 443,445,447,450 antimetastatic effect 441 CVS induced antitumor immunity 454 antitumor effect 440 restortive effect on progenitor cells 448,449 tumor specificity 442 Cyanobacteria 728 Cyasterone 59 Cycas cricinalis 529 Cycleanine 472,476 Cyclic adenosine monophosphate 260, 509,598 Cyclic adenosine-5'-diphosphateribose 547 Cyclic adenosine-5'-diphosphateribose (CADPR) nicotininc acid adenine dinucleotide phosphate (NAADP) 516 Cyclic ADPR 517 Cyclic AMP phosphodiesterase 539 3*,5'-Cyclic AMP-dependent protein kinase (PKA) 512 Cyclic AMP-dependent protein phsophorylation 483 3'-5'-Cyclic AMP-sensitive cation channels 483 Cyclic GMP-phosphodiesterases 541 Cyclic nucleotide phosphodiesterase 512 Cyclic dependent kinase 4 798,799 Cyclo-(L-Pro-L-thiopro) 829 Cyclo-(L-Pro-Met) 829 Cycloacanthaside F 197 Cycloalpegenin 179,184,187,190,201 Cycloalpigenin A 184,201 Cycloalpigenin B 201 Cycloalpigenin C 201 Cycloalpioside A 201 Cycloalpioside B 201 Cycloalpioside C 201 9,19-Cycloanost-24-en-ol 190 Cycloanthogenin 187 Cycloaraloside A 194 Cycloaraloside B 195 Cycloaraloside C 195 Cycloaraloside D 195

Cycloaraloside E 194 Cycloaraloside F 195 Cycloarbicoside A 202 24/?-Cycloartan-3j3,6a,16j3-24,25-pentaol, 6-dehydroxycycIoastragenol 184 Cycloaitane 61,52 Cycloartane glycoside 221 Cycloartane saponins 183 Cycloartenol ferulate 61 Cycloasgenin C 185 Cycloastragenol 179,184,185,186,187, 190,191,192,215,217,225 Cycloastragenol-6-O-glucoside 224 Cyclocanthaside D 197 Cyclocanthogenin 184,185,197 Cyclocanthoside A 197 Cyclocanthoside G 197 Cyclocanthoside E and G 227 Cyclocarposide 193 Cyclocarposide B 193 Cyclocephalosdie I 200 Cyclocephaloside 11 193 Cyclocinamide 757 Cyclocinamides A 761,762 Cyclodercitin 893,894 Cyclodesipeptides 768,884 Cyclogalegenin 179,184,187,191,196, 218,225,227 Cyclogalegenoside A 196 Cyclogaleginoside B 196 Cyclogaleginosides 191 Cyclohexane-l,2,-diamine 345 Cycloheximide 58 Cycloheximide-induced amnesia 958 9j3,19-Cyclolanost-24-en-3/3-ol 181 Cycloorbicoside G 202 Cycloorbigenin B 185,187,202 Cyclooxgenase 597,616,790,939 Cyclooxygenase pathway 263,593 Cyclooxygenase inhibitor 593 Cyclooxygenase/Lipooxygenase 615 Cyclophosphamide 431 Cyclophosphamide treatment 447 Cyclopycnanthoside I 198 Cyclorenierins 694

987

Cyclosieversioside A 193 Cyclosieversioside B 194 Cyclosieversioside C 193 Cyclosieversioside D 194 Cyclosieversioside E 192 Cyclosieversioside F 193 Cyclosieversioside G 194 Cyclotheonamide B 715 Cyclotheonamides 715 Cycloxygenase pathway 274 Cyctodytes 894 Cyditol 920 Cylindricine A and B 793 Cylindricines I and J 859,860 Cylindricins 859,857 Cylindrines F,G and H 859,860 Cylindrol B] 790 Cylindrospermopsis raciborskii 732 Cymbastela 847 Cymbastela coralliophila 704 Cymbastela hooperi 689,814,856 Cymbastela si^ 714,768,792 /7-Cymene 145 Cynara scolymus 943 CynidinldB 613 a-Cyperone 170,171 Cysteine sulfoxide 854 Cysteine protease falcipain 331 Cysteine proteinase 375 D-Cysteinolic acid 872 Cystodytes genus 820 Cystodytess^ 820 Cystodytes violatinacus 820 Cystodytins 821 Cytochalasin 874,875 Cytochrome P-450 421,424,935 Cytochrome P-450-dependent monooxygenase 413 Cytochrome P-450 monooxygenase 318, 597 Cytochrome oxidase-inhibiting cyanide 513 Cytokine gene 470 Cytokines 463

Cytokinins 367,379 Cytolytic lymphocytes (CTL) 273 Cytolytic T cells 453 Cytolytic T lymphocytes 271 Cytomegalo virus infections 431 Cytoplasmic guanylate cyclase 382 Cytosolic PIA2 717 Cytosolic dehydrogenase activity 423 Cytosolic sphingosine kinase 264 Cytostatic T cells 453 Cytotoxic guanidine-alkaloids 706 Czapek-Dox medium 154 D-3-(3,5-Dihydrophenyl) alanine 824 Dactylospongia elegans 694 Dakaira subovoidea 831 Dalbergia hupeana 93 Dalbergia parviflora 162 Damarane 50 Damirones 707 Damnacanthal 546 Damacits carota 377,669 Dansyl-CaM fluorescence 544 Daphnane 246 Daphnoretin 545 Dasyanthogenin 199 Dalton's lymphoma ascites 957 3,4-and 3,5-DcQAs 945 1,5-DCQA 944 3,5-DCQA 942,941 l,5-3,4-and4,5-DCQAs 941 3,4,-3,5-4,5-DCQAs 940 De novo DNA synthesis 227 D^wovo generation of thymocytes 454 De «ovo-biosynthesis 374 Denovo synthesis 128,129,294,300, 329,401,659,661,662 of bioflavours 125 10-DeacetylbaccatinIll 245 10-Deacetyl cephalomannine 245 10-Deacetyltaxol 245 Debromoeudistomin K 816,817 Debromohymemialdisine 791 10-Dechlorodysideathiazole 890

988

10-Decholoro-N-methyldyrideathiazole 890 Decriamide 893 Decumbesterone A 59 Defence gene activation 396 Defense proteins 370 serine proteinase inhibitor I 370 Dehydro 7-o:-hydroxy-tomatidenole (D^-d) 318 3-Dehydro-24-e/>/-teasterone 422,423 3 -Dehydro-24-^/7/-teasterone-reductase 422 3-Dehydro-6-deoxoteasterone 415 Dehydro-6-hydroxymellein 498,499 Dehydroeburiconic acid 57 Dehydroepiandrosterone 419 5-Dehydroepisterol 415 Dehydrojuvabione 546 Dehydrometenolic acid 57 Dehydropachymic acid 57 Dehydroquinic acid 928 Dehydrosaponin 1 105 3-Dehydroshikimic acid 928 Dehydrosoyasaponin I 97,104,115,116, 118,120 3-Dehydro-teasterone 415,422,423 4'-Dehydroxy-4'-flourotebquine 344 4'-Dehydroxytebuquine 344 3-Dehyrdo-6-deoxoteasterone 415 13-Demethylisodysidenin 889,890 Demissidine 536 Demospongic acid 721 Dendrimeric multiple antigenic peptides 335 Dendrimedric peptide 336 Dendroamides 716 6-Deoxocastasterone 415 C6 oxidation 424 6-Deoxoteasterone 415 3-Deoxotyphasterol 415 3-[5-Deoxy-5(dimethylarsinoyl)-/J-Dribofuranosy loxy]-2-hydroxypropene-1 sulfonic acid 867 3-Deoxy-7-phospho-D-arabinoheptuiosonicacid 928

6-Deoxyandalusol 250 Deoxyglycyrrhetol 58 Deoxygylulose 130 Deoxyparguerol acetate 788 Dephosphoglycogenyl synthase 525 Dephosphory lation of retinoblastoma protein 270 Dercitamide 893,894 Dercitin 893,894 Dercitus 893 Dermasterias imbricate 824 A^-Desacetyllappaconitine 528 Deserpindine 8 4-Desmethyl sterols 56,57 3,4-Desmethylvariacin 834 Desmal 546 Desm odium styracifloiiim 93 2-Desoxy-D-glucose 613 Destruxin-A4 chlorohydrin 800 Det2-cDNA 419 Detoxification of heme 334,335,343 Detoxification of tomatine 313 3- Deuteroisoreserpine 8 Deuteroporphyrin IX (DPIX) 355 Dextran induced edema 46 24-Dexyoxytrogenin 97 Dezoxiben 593 D-Gal N/LPS model 468 D-GalN/LPS 459-461 liver intoxication with 462 D-Gal N/LPS-induced liver injury 460, 467,472-474 D-galN/TNF-a 460,461 liver intoxication with 462 ^1,6-D-galactopyranose 429 D-galactosamine (D-GalN) 219,473,465, 477,461,463,468,471 D-GalN/LPS model 464 D-GalN/LPS-intoxication 468 D-GalN-induced cytotoxicty 468 D-GalN-induced liver injury 463,469 j3-l,2-D-glucosidase 306 DI dopamine receptor 529 Diabrotica iindecimpimctata 792 Diacarms 688

989

Diacyglycerol (DAG) 271,483,487,488, 516,542, l,2,-Diacyl-3a-D-glucuronospyranosyl-snglycerol taurieamide 873 Diacylglycerol acyltransferase 797 Diacy Iglycerol-1 -oleoly 1-2-acetyl-mc glycerol 488 Diarylheptanoids 543 Diatriba 186 Dibromoageliferin 779,780 Dibromophakellstatin 770 Dibromosceptrin 779 Dibromo-tyrosine derivatives 719 Dibromotyrosine-derived metabolites 719 4,5-Dibrompyrrole-2-carbamide 784 DibutylcAMP 271,488,509 Dicaffeoyl tartaric acid 663,667,932 9,10-Dichloro-N-methyldysideathiazole 890 2,4-Dichlorophenol 767 2,6-Dichlorophenol 765 Dicholorolissoclimide 269,769 Dictyopteria genus 841 Dictyostelium discoideum 523 Didemnum sp 765 Didemnoline A 765 Didemnoline C 765 Didemmun voeltakowi 713 Didemnin 684,714 Didemnin B 713,757,816 Didemnoline 818,863 DidemnolinD 818,863 Didemnolines 757,816 Didemnum 878 Didemnum conchylatum 802 Didemnum rodriguesi 813,897 Didemolin A 818 Didenumsp 818,874 Didemum molle 883 Dideoxyforskolin 269 10,12-Dideuterated reserpine 10 Didemnimides B 802 Didemnaketal 874 8,13-Di-^/?/-sclareol252 Dietyosphaeriafarulosa 898

Diginea symplex 846 Digitalis lanata 535 Digitalis purpurea 535 /^-Digitatum 148,149 Digitonin 308 Digliicoside 200 Digioxigenin-3-O-tridigitoxide 535 23,24-Dihydrocucurbitacin F 59 (S)-(-)-9,10-Dihdyroxygeranylacetone 168 Dihydro alpha-agarofuran 171 2,3-Dihydroapigenin 541 Dihydrobetulinic acid 61 Dihydrocaffeic acid 923,926 (+)-Dihydrocarvone 145,146,147 Dihydrocinchonamine 28 Dihydrocitronellol 133 Dihydrocorynantheine 31,32 Dihydroeperutic acid 248 Diiiydroferulic acid 922-925 2,3-Dihydrofisetin 541 Dihydroflavanoids 469 (+)-ampelosin 469 dihydromyricetin 469 Dihydroflavonol reductase 653 Dihydrokoempferol 653 2,3-Dihydroluteolin 541 5,6-Dihydro-PGl2 595 Dihydropyridines 534 2,3,-Dihydroquercetin 541 6a,25-Dihydroxy-3,16-dioxo-9j3,19cyclolanestane 184 2,3-Dihydroxy-24-norfrieadela-l,3,5,7tetraen-29-oic acid 59 diacetoxy derivatives 59 l,3-Dihydroxy-3,7-dimethyloct-6-ene-2one 134 Dihydroxyconiferyl-alcohol 657 (S)'{-)-10,11 -Diiiydroxyfarnesol 166 5,4'-Dihydroxyflavone 541,544 5,7-Dihydroxyflavone 544 5,7-Dihydroxyflavone 541 10,11-Dihydroxynerolidol 163 Dihydroxyphenolic compounds 657 Diketopiperazine 901

990

Dilodia gossypina 131,149 Dilophus 690 Dilophus ligulatus 690 Dilophus okamurai 690 3,4-Dimethoxy-6-(2'N,N-dimethylaniinoethyl)-5-(methylthio)benzotrithiane 833 (2E,5E)-3,7-Dimethyl-2,5-octadiene-1,7diol 138 3-Dimethylsulfoniompropionate(DMSP) 846 7,12-Dimethyl-benz [a]anthracene 937 3,7-Dimethyl-(E)-2,6-octadien-l -al 132 3,7-Dimethyl-l,6-octadien-3-ol 140 3,7-Dimethyl-l,7-octanediol 133,136 2,6-Dimethy 1-1,8-octanediol 138,135 3,7-Dimethyl-l-octene-3,8-diol 138 (E)-2,6-Dimethy 1-2,7-octadiene-1,6,-diol 138,142 (Z)-2,6-Dimethy 1-2,7-octadiene-1,6,-diol 142,138 (+)-(R)-3,3-Dimethylsulfonio-2-methoxypyropanoate 846 (E)-2,6-Dimethyl-6-hydroxy-2,7octadienoic acid 140 (E)-3,7-Dimethyl-2-octene-l,8-diol 138 (E)-3,7-Dimethy 1-5-octene-1,7-diol 13 8 (Z)-2,6-Dimethyl-2-octene-1,8-diol 136 (Z)-3,7-Dimethyl-2-octene-3,8-diol 138 3,7-Dimethyl-6-octene-l-al 132 3,7-Dimethyl-7-octene-1,6-diol 138 8-Y,Y-Dimethylallylwighteaone 541 3'-Y,Y-DimethylalIylwighteone 541 7-12-DiiTiethylben[a] anthracene (DMBA) 44 4,4,-Dimethylcholestane-3a,5a-diol 56 2E-6E-2,6-Dimethylocta-2,6,-diene-1,8diol 138 2E-6Z-2,6-Dimethylocta-2,6,-diene-1,8diol 138 3,7-Dimethylet-6-ene-1,2,3,-triol 134 4,4,-Dimethylsterols 56 5-Dimethylsulfonio-2-methoxypropanoate 846

3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide method (MTT) method 266 Dimethyl-j3-propiothein 846 (E)-2,6-Dimethyl-2-octene-l,8-diol 138 7,12-Dimethylbenz [a] anthracene (DMBA) induced skin tumors 46 4,4-Dimmethylcholestane 56 Dimnoline B 818 Dinophysisfortii 729 1,3-Di-O-Caffeoylquinicacid 920 3,4-Di-O-Caffeoylquinic acid 920 3,5-Di-O-Caffeoylquinic acid 920 3,6-Di-O-Caffeoylquinic acid (3,5-DCQA) 938 4,5-Di-O-Caffeoylquinic acid (AQAs) 920,938,939 Di-0-Caffeoylquinic acids 921,947,948 Di-0-hemiphtholates of oleana-12- ene-3 j3,30-diol (deoxyglycyrrhetol) 58 Diosmetin 618 2,7-Dioxabicyio [4,3.o] honane 803 Dioxapyrrolmycin 757,794 l,l,-Dioxo-l,4-thiazinering 861 0-Dipehnols 654,670 Diphenol-oxygen oxidereductase 654 1,1 -DiphenyI-2-picrylhydrazyl (DPPH) 934,948 Diplamine 820,821,893 Diplodia gossypina 162,166,168,170 Diplosoma sp 821 Diplosoma visens 888 Dipolar learsiae 788 Discodermia 867 Discodermia calyx 731 Discodermia polydiscus 763 Discodermin E 867 Discodermin-halicylindramide 864 Discodermis A-H 665,865 Discorhabdin 824 Discorhabdin A 760,761,822 Discorhabdin alkaloids 708 Discorhabdin B 760,761,822,823 Discorhabdin C 760,776 Discorhabdin D 822,823

991

Discorhabdin P 760,761 Discorhabdin Q 760,761,822,823 Discorhabdins 823 Discorhaines 707 Dismosin 469 Dispuupehedione 715 Distratamide A 881,882 Distratamide B 881,882 Distratamide C 881,882 Distratamide D 881,882 Distyophaeria sericea 722 Distyostelium cAMP receptor protein 526 Distyota ciliolata 722 1,3-Disubsituted tetrahydro /J-carboline 16,17 Diterpene isothiocyanates 853 Diterpenoid alkaloid aconifine 534 aconitine from Aconitum 534 ajacine 534 avadharidine 528 bikhaconitine 534 cassaidine 535 cassaine 535 condephine 528 delcoline 528 delcorine 528 delphinine 534 falaconitine 534 indaconitine 534 jesaconitine 534 lappaconitne 534 mesaconitine 534 N-deacetyllappaconitine 534 pseudoconitine 534 raynodine 535 Dithiofurodysinin disulfate 815 Dithiomethyl lissochinotoxin C 833 Dithiothreitol 836 Dittrichia viscosa 613 DMBA 45,48,61,62,65 743-DNA 826 DNA electophoresis 267 DNA polymerase 895 DNA polymerase B 871

DNA polymerase inhibitors 868 DNA synthesis 526 DNA-damaging antitumor 796 Docetaxel 245 Dodeahydro benz[/]indolo [2,3,-a] quinolizidine 20 Dodonaea viscosa 276 DolabeUa auriculana 716,760,763,878, 887 Dolabellane 690,790,791,888 Dolastatin 10 716,802,886,887 Dolastatin F 887 Dolastatin I 887 Dolabella 724 Dopamine 516,530,536,535,538,662 Dopamine Dl receptors 531 Dopamine D2 receptors 532 Dopamine signalling 531 Dopamine receptors 531 Doris verrucosa 829 Down-regulators of TNF-a 465 PGE2 463 dexamethasone 463 DPY-4189 669 Dragmacidin 765 Dragmacidine D and E 798,799 Droplet counter current chromatography (DCCC) 205,206 Drosophila 389 Drupellafragiim 792 Deserpindine (11-demethoxyreserpine) 8 Dt-B 118 Dt-C 118 Dt-E 118 Ducan's multiple range test 93,960, 964-966 Dumas ia truncata 106 Dyrideathiazole 890 Dysamides 801 Dysamides K 801 Dysidea arenaria 714 Dysideaavara 693,814,815 Dysidea herbacea 691,692,697,776,801, 815

992

Dysidea sp 264,692,693,815,869,896,698 Dysidea sxiongQs 815 Dysideathiazole series 889 Dysidenin 889,890 Dysidia chlorea 801 Dysinin 814 EBA-EA activation inhibition assay 58 Ebumamonine 19 EBV 46 EBV assay 45 EBV-EA activation induced by TPA 59 Ecdysone 515 Ecdysone synthesis 242 Echmoclathria sp 814 Echinoclathrine B 814 Echinoclathrine C 814 Echinocystic acid (cochalic acid) 60 Echinodictyum sp 900 Echinosulfone A 900 Echinosulfonic acid A 900 Ecteinascidia turbinata 707,825 Ecteinascidin 736-type 827 Ecteinascidins 707,825 Ectinoscidin 812 Ectoprotein CD38 535 Edman degredation 437 Effective concentration (EC50) 265 EGF receptor kinase 797,870 EGTA 490 EGTA-Ca^-^ buffer system 490 EGTA-containing buffer 491,492 EGTA-treated plasma membrane 492 Ehrlich ascites carcinoma 957,939 Eicosanoid 274,520,693,939 Eicosanoid production 615 Eicosanoid signalling 374 eIF4E-dependent mRNA translation 526 Eilatin 532 Elatin 709 Eleutherobia aiirea 690 Elicitin receptor 396 Elicitins 395,396 Elicitor activity 397

Elicitor-active structure 394 Elicitors 393,394 Ellagicacid 545,938 inhibitor of PKA and PKC 550 Em eric alla iwiquis 780 Emodin 543,544,547,546 Enantiopinifolic acid 252 Enantio-j8-e7?/-lalbdanolic acid 252 Endogenou defense signal system 397 Endogenous hepatoprotective factor 471 Endogenous ligand anandamide arachidonylethanolamide 532 Endogenous ligands 384 Endogenous peptide signals 401,383,402 for plant defense 402 Endoplasmic reticulum 548 Endoproteinases 375 Endoproteolytic proceesing 387 Endothelin-1 841 EndothelinA 841 Endotoxemia 463 Endo-/J-l,4-galactanase 438 Endo-)3-l,6-galactanase 438 Endo-j3-galactosidase 436 Endorphin ligand 533 Enediyne chromophore 796 £-Nerolidol 164 Enkephalin ligand 533 Enkephalins 533 ENOD40 368,379 ENOD40gene 379 ENOD40 peptide 381 £«/-13-^)t?/-12a-acetoxymanoyl oxide 273 £/7/-13-epi-ketomanoyl oxides 260 £A7r-13-e/7/-manoyl oxide 267,253,261, 266 £A7M5,16-epoxy-9aH-labdane-13(16)-14diene 3j8,3a-diol 276 Ent' 16-hydroxy-13-epi-manoyl oxide 250 £A7r-19-hydroxy-13-^/)/-manoyl oxide 253 £m-3a-hydroxy-13-e/?/-manoyl oxide 250 £A7/-3/3,6j3-di hydroxy-13-ep/-manoyl oxide 261 £'w/-3/J-hydroxy 13-e/?/manoyl oxide 253,254,267,263

993

£^/-8alpha-hydroxy-lamba-13, 14dien 263 £m-8,9-di-6?/7/-sclareol 252 £A7/-8-e/?/-sclareol 252 £w/-8a-hydroxy-l 3,14-dien-18-oic acid methyl ester 261 Ent'Sa-hydroxy-labda-13 (16), 14diene 273,274 Entagenic acid 60 Enterobacter sp 170,171 Enterobacter cloaceae 258,259 Enterococcus faecalis 116J1S Enterococciis faecium 116 Ent'hydxoxy and e«/-acetoxy-3j3-monyl oxide 256 £«r-labd-8,13(£)-dien-15-ol 249 £«/-labdane oxides 249 £/7/-labdanes 251 £m-manoyl oxide-16-hydroxy-l 8-oic acid methyl ester 261 £/7/-manoyl oxides 264 £w/-j3-e/?/-concinndiol 249 Epialloyohimbane 5,20 Epibatidine 757,791,792 24-£/?/-brassinolide 425,426 24-£/?/-catasterone by Bayer-Villager oxidation 425 (-)-Epicatechin 541 (-)-Epicatechin gallate 612 13- Epicorynantheidol 24 Epidermal growth factor (EGF) 766 Epidennoid carcinoma 266 (-)-Epigallacatechin gallate (EGCG) 470, 546 Epidioxy-substituted non-diterpenes 688 Epidithodioxopiperazines 901 (-)-10-£/7/-isokalihinol4 856 (-)-10-£/?/-kalihinolI 856 (-)-10-£/?/-kaliinolH 854 13-£/>/-manool 252 Epimephrine 896 Epimerization of/3-carboline-type indole 5 Epimorphinan alkaloid from Sinomenium acutiim 412

Epinephrine 516,536,538,896 Epipedobates tricolor 791 (+)-Epiplasin-B 852 Epipolases kushimotoensis 898 Epipolasin-A 853 Epipolasis kushimotoensis 853 Epipolythiodioxopiperazines 839 Epiquinine complexes 347 13-£p/-sclareol 252 xylopyranoside 252 Episterol 415 Epitaondiol 690 24-£p/-teasterone 422,433 Epithelial NO synthase 525 24-£/?/-typhasterol 422,423 Epoxide hydrolase 150 (20(/?),24)5-Epoxy-9A 19-cyclolanostan3Al6i3-25,tetrol 217 (20(S),24)/?-Epoxycycloartane 227 8,13-Epoxy-labd-14-en-oic acid 249 6,7-Epoxy-Lat A 874 6,7-Epoxylinalool 141 EPR Spectroscopy 340 Epstein-Barr virus (EBV) 45 Ergine 531 Ergoline alkaloids 706 Ergostane 49,61 Ergosterol biosynthetic enzyme 304 Ergosterol peroxide 61 Ergotamine 531 Ergothioneine 813 Erica andevalensis 612 Ericaceae 246 Ericaceae family 242 ER ryanodine receptor Ca^"^ channels 517 Erwinia amylovra 394 Erwinia carotovora 394 Erwinia chrysanthemi 394 Erwinia coli 220,258,259,394,450, 821,928 Erythrina isoquinoline alkaloids 528 dehydro-j5-erythroidine 528 erysinine 528 eiysotrine 528

994

erythratidine 528 a-erythroidine 528 j8-erythroidine 528 Erythrocyte agglutination 464 Erythrodiol 57,60,64 Escherichia chrysanthemi 394 Escherichia coli 251,319 J65,115,925, 948 Escherichia faecilis 259 Esteinascidin 825 Estrogen-receptor antagonists 797 Et 743 N^2 oxide 825,827 Et722 826 EtN^^.oxide 826 Eteromorpha prolifera 691 [^H]-Ethanolamine 342 S,S'-l,2-Ethendiyl 0,0'-diisobutyl 898 Ethyl catechol 925-927 l-Ethyl-4-methylsulfone-p-carboline 819 Ethyl phenylpropiolate (EPP) induced edema 46 Ethylene biosynthesis 657 Etlatol 787 Et's 595,581,592,826 Eucalptus citriodora 132 Eucaryote signals-regulated protein kinase (PK's) 521,522 Eudesmane 848,849 Eudistoidin E and F 818 Eiidistoma divaceum 902 Eudistoma sp 820,834 Eudistoma glaiwiis 758,817 Eudistoma olivaceum 816 Eudistomidins 816 Eudistomidin A 758 Eudistomidins B 759 Eudistomidins C-D 758 Eudistomln C,E 782 Eudistomins C,E,F and L 816,817 Eudistomidin D 759 Eudistomidin F 863 Eudistomin K 815 Eudistomin K sulfoxide 863 Eudistomin sulphide 816,817

Eudistomins 815,817,782 Eudistomins D,J,N and O 782 Eugenol 126 Eiinicea toiirneforti 690 Eimicella cavolinii 690 Euphane 50 Euphol 61 Euphorbia 242 Euphorbia maddeni 594 Euphorbiaceae 242,243,245 Euryspongia arenaria 703 Euryspongia deliculata 694 Euthyroideones A-C 862,863 Euthyroideones and other sulfones 862 Euthyroides episcopalis 862 Evasoteria troschelii 813 Exifone 349,350,351 2,3,4,3',4',5'-hexahydroxybenzophenone 349 Exogenous peptide signals 393 Exogenous peptide el icitors 401 receptors 401 Exophilin A 723 Exophiola pisciphila 723 Exopolyphosphatase 718 Exoproteases 331 Exo-a-galactosidase 438 Exo-a-mannosidase 436 Exo-/J-galactosidase 438 Extracellular LRR domains 401 Extracellular signal regulated kinases 526 Fabaceae 92,168 Fabaceous crude drugs 98 j3-Fabatriose 94 Fabatrioside 89,109 Faboideae 92 Factor-a-dependent inflammatory liver injury 459 a-Fagarine 539 Faglis sylvatica 662 Falcarindiol 471,476 Falcipain 330,352 Famotidine 612

995

Faradiol 57,58,62,63 Faradiol-3-O-myristate 58 3-O-palmitate derivate 58 Farensyl pyrophosphate 237 Farmosan gorgonian coral 690 a-and j3-Famesene 546 Farnesol 125,162,165-168 Famesyl isothiocyanates 854 Fasciospongia cavernosa 688,697,703 Fasciospongia remosa 874 Fasicularin 860 Fatty acid oxidation 525 Fatty acid synthase type-I 500,501 Fe(IlI) protoporphyrin 332 a-Fenchol 154 Fenton process 931 Fenton reaction 634,644 Ferrylinyoglobin 934 Ferulic acid 56,57,653,662,665,671,920 Ferulic acid ester 655,665 Ferulic and p-coumaric acid 920 FeruloylCoA 653 3-(9-Feruloyl-4-0-caffeoylquinic acid 920 Feruloyllycine 923 3-O-Feruloylquinic acid 920 F-gitonin 308 Filter-sterilized pectinase 485 Fischerella ambigua 792 Fischerella major 802 Fischerella muscicola 802 Fisetin 541,543,544 Flagellin 394 Flagellin protein 394 Flavin-adeninedinulceotide-dependent oxidoreductase 417 Flavobacterium sp 868 Flavocristamides A and B 868,869,903 Fig 22 epitope 394,395 5-Flourouracil (5FU)-induced lethality protective effect of CVS 447,448 5-Fluorouracil-inducedmyelosuppression 429 5-Fluorouracil prophlylactic effect against mylosuppression 447,448

Flourinated erythromycin 780 Flow-cytometric analysis 442 Fluometuron 669 Fluorescent Ca^'^'-indicator (fluo-3) 489 Fluoresein 5'-isothiocynate (FITC) 446 Follicular mantle B Cells 272 (-)-10j3-Formamido-5 /J isothiocyanatokalihinol-A Formaldehyde induced arthritis 46 Formation of j3-hemetin 357 Formosanine 25,26 Formyl methionyl-leucyl phenylalanine (fMLP) 275 Forskolin 256,261,262,264,265,268,270272,275,276,489,490,496,539 Forsyth iaside 543 4-0-Freuloylquinic acid 920 5-0-Freuloylquinic acid 920 4,9-Fridodriane 867 Friedelane 54,59 Fructose-2,6-bipohsphate 523 Fructose-6-phosphate-2-kinase 523 5FU (5-Flourouracil) 450,453 5FU treatment 449 5FU-induced adverse effect 449 5FU-induced suppression of the bone marrow hematopoiesis 454 Fuciis vesiculosiis 711,125 Fungal glycoprotein 396 Fungal tomatinase 316,321 Fungal-tomato pathogens 295,312 Fungicide thiazole 321 Furanocoumarines 666 Furanoid 7/J-acetoxylabdanes 251 Furanoid linalool oxide 141 CIS and trans 143 Furanoterpene sulfates 688 Furanoterpenes 688 Ci2-Furanoterpene fiirospongolide 697 Furin 389 Furodysinin 815 Furodysinin lactone thioacetate 815 Furospongolide 698

996

Fusarhim oxysponim 293,295,296299, 305,311,312,313,317,319,320,322 f. sp gladioli 307 f. sp melonis 307,411 f. sp niveum 307 f. sp tuberosi 307 sp lycopersici 295,299,307, 312-314,316,318 Fusarium oxysporum tomatinase 312 Fiisariiim sambucinum 317 Fusarium solani 303,305,307,314,316, 317 Fusicoccin 374 GABA 30,536,537 GABA A receptors 529,895 GABA agonist 528 GABA gated Cl-channels 528 GABA receptors 531 GABA transporter 537 Gaeumannomyces graminis 304 jS-Galactosidase 210 Galangin 541 Galaxura marginate 704 Galeopsis angustifolia 251 Gallocatechin 943 Gamblerdiscus toxicus 730 Gametocyte 329 Gastric H^,K+-ATPase 612 Gastrin receptor 611 Gastrodia elata 529 Gastrointestinal disorders effect of flavonoids 607 Gastroprotective agents 614 Gastroprotective effect of flavonoids 615 Gaudichaudioside F 251 Gaudichaudol 269 Genanial 133 Genatiana macrophylla 476 Genista nimelia 612 Genistein 544,546,593,613 Gentiana sp 471 Gentianaceae 471 Gentiobiose 968

Gentiopicroside 471 serum ALT activity Gentiopicroside 471 serum transaminase activity 471 Geodia 768 Geodia cydonium 718 Geodiamolide A 769 Geodiamolide C 769 Geodiamolide D 769 Geodiamolide E 769 Geodiamolide F 769 Geodiamolides 757,768 Geodiamolides A-T 768 Geotrichum penicillatum 129 Gentiopioroside 476 Geranicacid 132,133,134 Geraniol 125,132,134,136-138,140,142, 143,163,165 Geranium oil 132 GeranyI acetate 136,164 Geranyl geraniol 239 Geranyl linalool 254 Geranyl linalyl pyrophosphate coaplyl PP 239,240 Gerany lacetol 168,169 (5H+)-Geranylacetol 163,164 GeranyIgeranyl pyrophosphate 237,240, 244 Germacrone 172,173 Gemaiol 132 Gernayl geranyl pyrophosphate 248 Geseneriaceae 251 Gibberella cyanea 162,166 Gibberelafujikuroi 253,255 Gibberella gaminis 313 \?ixavenae 306,308,310,304,305 Gibberella pluicaris 293,305,311,317, 319 Gibberella pulicaris 307 Gibberellane 241 Gibberellic acid 245 Gibberellin 367,657 Gibbonsia elegans ISl Ginseng 182 Ginsenoside Rbl 958

997

Ginsetioside Ryl 958 Gissampelos insular is 478 Galactose rich acidic glycoprotein 453,454 GLC method 435 Gleicheniajaponica 251,276 y-Gliadin 320 Gliovictin 828 Gloemerella cingulata 164,166-16 8 Glucans 396 Glucocorticoids 463,516 Gluconeogenic enzyme 523 6-0-j3-D-Glucopyranoside 224 jS-Glucoronidases 313 /J-Glucoronidase inhibitor 263 Glucosaccharo-l,4-lactone 263 j3-Glucosidase 210,211,308,957,968 Glucosinolates 513,514 j5-Glucosyl hydrolases 308 Glucuronic acid 89,94,102-104,119,465 j3-D-Glucronides 263 Glutamate (N-methyl-D-aspartate receptor (NMDA) 519 (non-NMDA receptors) 519 Glutamate transporters 536 Glutamic pyruvic transaminase 935 Glutamine 320 Glutathione 219,614,831,930 Glutathione-S-transferase 718 Glutin-5-ene 59 Glutinane 53,59 Giyceraldehyde-3-phosphate (GAP) 134 Glyceroglycolipids 431 Glycine as co-agonist 529 Glycine max L, 93,105,669 Glycine receptors 530 Glycine max cv. Kuromame 93 Glycine receptors 530 Glycine soya 93,101 Glycine-gated Cl-channels 530 Glycine-rich protein 394 Glycoaldehyde 163,164,296,314,318 Glycoalkaloid 305 from potato 305 Glycoalkaloids 296,311,317-319 Glycogen synthase kinase 3 525,546,548

Glycosyl hydrorolases 306,308,321 glycosylated 543 Glycycrrhizin 114 Glycyrrhetic acid 57,59,60,65 18a-Glycyrrhetinicacid 64,551 G18-j3-lycyrrhetinicacid 551 Glycyrrhiza glabra 465,476 Glycyrrhizin 90,99,103,465,467 hepatoprotective effect 466 \so\diXQA ixom glycyrrhiza glabra 466 Glyphosat 669 j3-Glycosyl hydrolase 304 Gm ENOD46 380 Gnathotricus retusus 143 Gnathotricus sp 143 Gnathrotriciis sulcatus 143 Gnidimacrin 543 Gnoderma applantum 131 Gogonane skeleton 850 Golgi-resident enzyme 389 Gomeraldehyde (ent-8,13,epoxy-labd-15-al) 249 Gomericacid 249,268 Gomerol 249 13-epigomerol 249 Gomojosidae 259 Gomojosides A-J 251 Gonyaulax polyedra 845 Gorgonane 848 Gossypol 541,543,544 G-protein-linked receptors 530,531,539 o2-adrenergic 530 dopamines (D2) 530 GABA(B) 530 opioid (m and d) 530 serotonin 530 somatostatin 530 Gracilaria 769 Gracilaria coronopifolia 729 Gralactose-rich acidic glycoprotein 451, 452 Gramine 531 Granulocyte macrophage colonystimulating factor (GM-CSF) 449 Grateloupiafilicina 862

998

Grayanotoxin 242 Greany lactone 166 Green fluorescent protein (GFP) 380 Green tea polyphenols 470 Grenadadiene 151 Jl\ Grindalia haverrdii 252 Grindella sp. 251 Griseofulvin 757 GTP-binding protein 385 GTP-bound stimulatory G protein 262 Guaiacol 126 Guaiacyl subunits 653 Guaiane skeleton 850 Guaiazulene 690 pigments of 690 Guaione 848 Guanosine triphosphate 598 Guanylyl nucleotide exchange factor 548 Gutierrezia grandis 251 Gutierrezia spathulata 251 Gutierrzia sphacrocephala 251 Gymnodinium 864 H"^, K"^-ATPase inhibitors 612 H"^,K"^-ATPase 613 Haber and Weiss reactions 931 Haemonchus contortus 794 Haemophilus influenzae 781 Halenaquinol 694 Halenaquinones 861 Halichlorine 799 Halicondria cylindrata 867 Halichondriajaponica 718 Halichondria okadii 799 Halichondria sp 716,847,848,852,854 Halichondria panicea 852 Halichondridae sp 263,699 Halichondrin 728 Halichondrin B 727 Haliclonacyclamines 710 Halicona oxigua 732 Halicona kormella 722 Halicona sp 694,695,710,725,709,728 Halicylindramide A 867

Halicylindramide B 867 Halicylindramides D 865,867 Halicylindramides E 866,867 Halimium genus 250 Haliotis discus hannai 788 (-)-Halipanicine 852 Halistanol disulfate B 704 Halistanol sulfates 703 Halistanol trisulphate 704 Halisulphate 263,799 derivative 699 Halocynthia roretzi 704,784 Halogen-free malyngamide J 788 Halomon 702 Halopappum parvifolus 251 Halopappus parvifolus {AstQvacQdLQ) 255 Halopappus sp 251 Haloperoxidase 758 Hamigera tarangaensis 803 Hamigeran B 803 Hamioea cymbalum ISl Hanacantha sp 705 Hansemda mrakii 129 Hapalosin 714 Hapatocyte necrosis 462 Hapatotoxins 461 Haplosamate A 898 Haplosamate B 898 Happlopappus arbutoides 251 Happlopappus pulchellus 251 Hapten-carrier protein conjugated 434 Haimaline 533 Harmine 533 Heat-inactivated proteases 439 Hederagenin 60 Hedychium coronarium (Zingiberaceae) 265 HeLa 301 cells 268 HeLa assay 45 HeLa cells 45,46 inhibition 45 HeLa S3 Cells 790 Helianthiis annuus 391,670 Helianthus occidentalis (Asteraceae) 251 Heliantriol C 57,62

999

Heliantrol B2 57 Helichrysum rupestre 259 Helicobacter pylori 611,617,618,778 Helicobactor pylori infection 617 Helicostylium piriforme 317 Helioporuns A-E 690 Heliothis verescens 768 Helix pomatia 210 Helminthosporium 828 jS-Hematin 343 Hematoporphyrin IX 355 Hematoporphyrin 354 Heme aggregation 329,343,349,351,352, 353,355,356 Heme aggregation activity 341 Heme aggregation assays 348,352,354 Heme aggregation efficacy 355 Heme aggregation inhibitor 352,329,343, 359 Heme aggregation inhibitors 327 Heme detoxification 329,331 Heme polymerase 30,333 Heme polymerization 334,348 Heme polymerization assay 334,354 Heme polymerization inhibitor 327,343, 347,348 Heme polymerization inhibitory activity 345 Heme proteolysis 331 Heme trophozoite 332 Heme-acetate complex 343 Heme-metalloporphyrin complex 358 Heme-thiolate monooxygenase 414 dehydrogenases 414 j8-Hemetin aggregation 357 jS-Hemetin assay 355 j3-Hemetin 31,38,332,333,340,356,358, 360, 11,15-Hemiacetal 266 Hemibiotrophic tomato pathogen 302,308 Hemin 337 Hemoglobin 330 Hemoglobin catabolism 327-331,341, 342,343,345,347-349,352,353,357,360

Hemoglobin proteolytic enzyme 360 Hemoglobinase 330 Hemolysis 180,464 Hemozoin aggregation 334,337 Hemozoin aggregation inhibition 342 Hemozoin aggregation inhibitors 342 Hemozoins composition 338 Hemozoin inhibition 341,342,356 Hemozoin inhibitor drugs 360 Hemozoin nucleation 334 Hemozoin polymer 347 Hepatic apoptosis 468 Hepatic DNA fragmentation 466,467,468, 469,472 Hepatic information 479 Hepatic intoxication with D-GalNandLPS 466 Hepatic mRNA induction 468 Hepatitis B virus 114 Hepatocellular uridine phosphate UDPandUTP 461 Hepatocyte apoptosis 459,460,462,464, 468,469 inhibtion of 477 Hepatocyte necrosis 466,468,476 Hepatoprotective activity of ornithine glucuronides 90,103 Hepatoprotective activity of palustroside III 106 Hepatoprotective agents 462,465 Hepatoprotective drugs 100,103 Hepatoprotective effects 106 of oleanolic acid-type glucuronides saponins 106 of plants 459 Hepatoprotective natural products 465 Hepatoprotective phytoconstituents 477 Hepatoprotective proteins 461 Hepatoprotective saponin 465 Hepatotoxicity 471 2,3,4,5,2',3',4'-Heptahydroxybenzophenone 351 5,9-Heptacosadienoic 721 Heptapeptide lissodinamide 878 Heptapeptides 715,883

1000

HerbamideA 900 Herbasolide 692 Herbicides 668 Herpex simplex 815,891 Herpex simplex vims 121,779,782 Herpex simples virus type 1 948 Hesperetin 469,618,541 Hesperidinase 210 Hetero-metalloporphyrin inhibition complex 359 Heterotrimeric proteins 520 hormones receptors linked to 520 Heteroyohimbine alkaloids 30 5,9-Hexacosadienoic 721 12-O-Hexadecanoyl-16-hydroxyphorbol13-acetate(HHPA) 45 induced edema inhibition (HPA) 46 Hexadentateethylenediammine-N,N'-bis [2-hydroxy-R-benzylimino] ligand (ENBPl) 351 Hexadeutrarated reserpine 9 Hexapeptide biotratamide 878 Hexapeptides 887 j3-Hexosaminidase 939 3-Hexyl-4,5-dithiacycloheptanone 842 /w-HHA 925 HHPA induced edema (HPA) 45 HHPA-induced inflammation 56 9,11 -Hidechloro-13-demothey lisodysidenin 889 Hippospongia A-F 688 Hippospongia sp 698,721,871 Hippuricacid 928,929 Hirsutanonol 543 Hirsutenone 543 Hirsutine 31,32 chromotropic and oxidole alkaloids 32 Histamine 516,520,532,536,538,612, 613,701,719 Histamine induced edema 46 Histamine production 611 Histamine and peptide leukotriene C4 (LCT4) 275 Histamine receptor 611,34

Histamine secretion 936 Histidine 334,335,526,813 Histidine decarboxylase 612 Histidine kinases 526 Histidine-rich protein (HRP) 334,342,360 Histidine-rich proteins II and 111 334 Histone HI 526 HIV inhibitors 942 HIVintegrase 941,942 HlV-protease 941,942 H202-induced apoptosis 269 Ho oils 140 Holoenzyme 522 Homanindole A 863 /J-Homochelidone 539 Homodolastain 3 902 Homphymia conferta 793 Hop-n-ene 59,65 Hordenine 531 Honnone-activated guanylate cyclase (GC) 519 Hormone-activated receptor tyrosine kinase 518 Hovenia dulcis Thunb. 469 Hoxaxinella sp 762 HPA 46 Y-HPKC 61 w-HPPA 925,927 H2 receptors 612 HRP II inhibition 336 HRP II mediated hemozoin synthesis assays 352 5HT3 class receptors 529 Huangquiyegenin A 195 Huangquiyegenin B 202 Huangquiyegenin I 195 Huangquiyegenin II 202 Huangquiyenin D 192 Huangquiyenin 184 Human colon adenocarcinoma cell cultures 903 Human immunodeficiency virus type-1 948 Humulene 168,170 Hyalurnidase-inhibitory activity 939

1001

Hyaluronidase 939 10-Hydroxypatchoolol 171,172 16a-Hydroxytrametenolic acid-3-Oaceate 61 Hydalhodes 391 Hydrazinolysis 438 Hydrocortisone 56,57 Hydrolysates from carrot cells 486 Hydrolyzable tannins 541 2-Hydroxydiscorhabdin D 823 6-Hydroxy-1,2,3,4-tetrahydropyrrole (1,2-a) pyrimidinum skeleton 829 6-Hydroxymellein biosynthesis 499 6-Hydroxymellein formation 503 6-Hydroxymellein synthase-catalyzing reactions 503 6-Hydroxymellein synthase 499-504 mechanisms of substrate channeling 505 7-Hydroxy-6-methylhept-5-en-2-one 138 7-Hydroxy-6-methylhept-5-ene-2-one-1 -ol 136 Hydroquinone 658 22a-Hydroxycampesterol 418 Hydroxy cinnamoyl-CoA esters 652 8-Hydroxy citronellol 136 4-Hydroxy isopiperitenone 151 Hydroxy methylglutarylcoenzyme A synthetase 525 3/J-Hydroxy sterols 306 3^Hydroxy[6a-acetoxy,23-ethoxy, 16^,23 (R) epoxy-24,25,26,27-tetranor]-9i8,19cyclolanostane 185 3pja 22-Hydroxy-16,23-epoxy-24,25,26, 27-tetranor-9/J, 19-cyclolanostane 184 3-Hydroxy-2,2,6-trimethyltetrahydropyran 144 C/5(3S,5R)-2-Hydroxy-2,2,6-trimethyltetrahydropyran 143 1 - Hydroxy-3,7-dimethyl-6-octen-2-one 133 l-Hydroxy-3-methoxyxanthone 467 3-Hydroxy-3-methylglutaryl CoA 130 5-Hydroxytryptamine 529, 531

(E)-IO- Hydroxy-6,10-dimethyl-5undecen-2-one 168 7-Hydroxy-6-methylheptan-2-one 138 (1R*,3S*,4R*,7S*,8S,12R*,13R*)-12Hydroxy-7-isothiocyanatoamphilectall(20),14-diene 853 4-Hydroxy-7[ 1 -hydroxy-7-[ 1 -hydroxy-2methy lamino)ethyl]-1,3-benzothiazole2(3H)-one 896 2-Hydroxy-8-p-mehthen-7-oic acid 149 Hydroxyxanthone-based drugs 349 Hydroxybenzoate 660 Hydroxybenzoic acid 655,660,661,922, 923,947 3jS-Hydroxybiformene 249 22a-Hydroxycampesterol 415,420 conversion of 418 6a-Hydroxycatasterone 424 Hydroxycinnamates 656 Hydroxycinnamic acid 652,657,666,919, 920 2-Hydroxydiscorhabdin D 823 20-Hydroxyecdysone 413 co-Hydroxy-£-nerolidol 162 (225'-24/?)-22-Hydroxyergos-4-en-3-one 418 12-Hydroxyfamesol 165,166 5-Hydroxyflavone 543 8-Hydroxygeraniol 140 11 -Hydroxygeranylacetone 165,168 3a-Hydroxy-gomeric acid 249 Hydroxyhexadecanoic acid (tricolorin A) 543 Hydroxyisopiperitenone 151 C/5(3S,5R)-2-(2'-Hydroxyisopropyl)-5methyltetrahydrofuran 143 3j3-Hydroxyl, A^'^ steroids 419 Hydroxylation of tomatidine 318 co-Hy droxylation 136,13 8,162,163,166, 168 8-HydroxyIinalool 136,140,142 3a-Hydroxymanool 275 6-Hydroxymellein synthase 483,506 6-Hydroxymellein 498,499,506,507

1002

6-Hydroxymellein-O-methyltransferase 483 Hydroxymenthol 155 5-Hydroxy-N,N-dimehtyl tryptamine 531 7-Hydroxypeganine 532 Hydroxyphenolic acid 657 /w-Hydroxyphenylpropionic acid (m-HH A) 922,923,947 7a-Hydroxysitosterol 56 diacetate 56 3j3-Hydroxy-sterols 96,299,303 7-a-Hydroxy-tomatidine 318 16a-Hydroxytrametenolic acid 3-O-acetate lanostanes 63 [^H]-5-Hydroxytryptamine 31 Hydroxyxanthone 327,349,354,361 6-Hydrxy-10-formamido-15-isothiocyanokalihinene 856 Hymenaceae sp 252 Hymenaea ablongifolia 252 Hymenaea parvifolia 252 Hymenamide F 830,863 Hymeniacidon sp 713,797,798,799,830 Hymenialdisine 711,787,795 Hymeriacidon sp 870 Hypercytokinemia 463 Hyperforin 537,538 Hypericum perforatum 551 Hyperkinetic ventricular arrhythmias 594 Hypersensitive response (HR) 402,394 Hyphomonas jannaschiana 873 Hypocholorous acid 931 Hypolactin-6-glucoside 613 Hypolactin-8-glucoside 612,616 Hypooxidaceae 180 [G-H] Hypoxanthine 348 Hypoxylon oceanicum 1\1 Hypselodoris nana 815 Hyrtiomanzamine 710,819 Hyptis spicigora 276 Hyrtios altum 727 Hyrtios erecta 696,698,710,819 Hyrtios sp 716 Hystiomanzamin 819

125I-AVR9 400 lAA oxidase 657 lanthella 719 lanthella basta 719 Ibhaynid 802 Ibotenate 531 Ichthyotoxic malyngomides 757 Ichtyotoxic activities 768 Ichtyotoxic agent 730 Icosanoid 945 IFN-Y 465 IgE/anti Ig E complexes IL (Interleukin) 455 Imberic acid derivatives 183 Imidazole 335 Imidazole [4,5-e-]-l,2-thiazine 897 9-N-[2-Sulfoethyl) Imine 870 Immune-mediated cytotoxicity 467 Immuno-active acidic glycoprotein from C. vulgaris 455 Immunoaffinity chromatography 382 Immunoblotting 499 Immuno-deficiency virus 734 Immunohistochemical staining 442 Immunoreactive polypeptide 383 Immunosuppressive drugs 464 FK508 464 dexamethasone 464 amphetamine 537 calebassine 528 carcurine 528 coronaridine 533 eseramine 536 eseridine 536 harmaline 537 harmine 537 ibogaine 533,537 ibogamine 533,537 physostigmine 536 physovenine 536 reserpine 538 sapargine 528 strychnine 530 tabernanthine 533

1003

toxiferine 528 yohimbine 530 Indolizidine alkaloids 190 Indole hallucinogens 531 psilocin 531 Indoloquinolizidine 4,28 Indolo [2,3-a] quinolizidine 7,10,11 conformations 22 Indomethacin 56,58,593,595,613,616 Infetroban 593 Ingenane 243,246 Ingenol diterpenoids 17-hydroxy ingenol-20-hexadecanoate 543 ingenol-20-hexadecanoate 543 Inguinal lymph nodes 444 Inhibit LDL oxidation 946 Inhibition of 4-(methylnitrosamino)-1 -(3-pyridyl)1-butanone (NNK) induced lung tumors 46 5-lipoxygenase 47 12-lipoxygenase 47 15-lipoxygenase 47 adenylyl cyclase 532 adjurant induced polyarthritis 46 arachidonic acid 66 arachidonic acid induced edema 46 j5-hematin aggregation 342,354 bradykinin induced edema 46 capsaicin induced edema 46 carrageenan induced edema 46 cotton pellet granuloma 46 cycloxygenase 47 Epstein-Barr virus early antigen (EVB-EA) activitaion induced by TPA 46 eukaryate signal transduction componenets 512 falcipain 331 heme polymerizations 348 heme sequestration 348 hemozoin 354 histamine release 47 human leucocyte elastase 47

hylauronidase 47 inducible cyclooxygenase 47 inducible nitric oxide synthase 47 DMBA/TPA induced skin tumor 48 lyso PAF acetyltransferase activity 615 Inhibitors of 544 /J-hemetin 357 cahnodulin 706 human synovial PLA2 697 lAA oxidation 657 kinesin motors 695 PKA 541 PMA 938 protein phosphatases 707 sterol biosynthesis 303 Inhibitory effects on HHPA 55 TPA 55 croton oil-induced inflammator edema 55 Inones 125 Inositol-1,3,4,5-tetraphosphate acid 516 Inositol triphosphate (IP3) 483,487,488 radioactive 487 Inositol triphosphate (IP3) 509,524 Inositol-1,4,5-triphosphate (IP3) 522, 547,548 Insositol-1,4,5-triphosphate (IP3) receptor 534 Insulin B chain 390 Insulin signalling activated protein kinase 522 Insulin-responsive protein kinase (PKB) 517 Interaction of THS with a-TNF 468 Interferons 519 Interferon Y (IFN-y) 431,477,464 Interleukin-2 536 Interleukin 2-dependent (IL-2) 271 Interleukin-6 cytokine family 717 Interleukin-8 711 Interleukines 271,540 Intraerthrocytic antimalarial activity 343

1004

Intraerythrocytic cycle 329 Intraerythrocytic phase 327,328,329 Intrasinusoidal hematosis 464 ^^^ I-iodotyrosine 373 125 I-Tyr2, Ala 16-systemin 373 lodoperoxidase 758 Ion gradient generating ATPase 512 IonophoneA23189 275,274,940 lonophores 374 lP3-lonositol-1,4,5-triphosphate 516 lonotropic glutamate receptors 528 lonotropic receptors 528 lonotropic serotonin receptors 529 losobutylmethylxanthine 275 IP3 receptors Ca^"^ channel 534 IP3-gated Ca^"^ channels 517 lP3-induced release of Ca^"*" 488 Ircinia dendroides 885 Ircinia sp 688,703,705,709 Ircinia sponges 695 Ircinin 688 Ischaemia reperfiision 610,614 (15*,35*,4/?*,75*,85'M25*,135*)-7lsocyano-15-isothiocyanatoamphilect11(20), 14-diene 856 {\SMARJS,%SMSMSA2>S,\5Ra^R)20-Isocyanao-7-isothiocyanato-isocycloamphilectane 856 Isoamyl acetate 129 Isoastragaloside I 193 Fsobatzelline A 822,790 Isobatzelline B 822,790 Isobatzelline D 822 Isobazellines 707 Isobomeol 154 6-Isobutyloxycarbonyl derivative 829 Isochromophilones 797 Isococculidine 528 Isocycloamphilectanes 853 Isodaucane 848,850 Isodictya setifera 828 Isodysidenin 889 Isoepoxyteryxin 471

Isoflavone synthase 655 Isolaurenterol 787 Isoliquirtogenin 537,539 Isolissoclinotoxin A 833,834 Isomenthol 155 Isomitraphylline 25,26 Isonakafuran-type sesquiterpenes 692 Isonovaldal 152 Isopentyl diphosphate (IPP) 130 Isopiperitenol 150 (+)-lsopiperitenone 150 Isoprenoid unit 513 jS-Isopropenyl pimelic acid 145 j3-Isopropyl pimelic acid 154 Isoprostane ¥2a 596 Isoproterenol 594,896 Isopteryxin 41 \,416 Isopulegol 136 Isoquinoline 4,824,825 Isoquinoline alkaloids (-)-salsolinol 531 atherosperminine 540 dihydropyranocoumarins 540 dopamine 531 narceine 533 phenylpropanoid glycosides 540 Isoreserpic acid lactone conformation 24 Isoreserpine 3,13,29 conformation 23 Isorhynchophylline 26 (-)-(15,6/?,7/?, 1 OR)' 10-Isothiocynato-4amorphene 852 10-Isothiocyanato-5-amorphene-4-oI 852 Isothiocyanates 514 (-)-l 5-Isothiocyanato-l-e/?/-kalihinene 856 (+)-10-Isothiocyanato-4,6-amorphadiene 852 7-Isothiocyanato-7,8-dihydro-a-bisabolene 848 (+)-4-1 sothiocyanato-9-amporphene 852 (+)-1 -Isothiocyanatoaromadendrene 853 lO-lsothiocyanatobiflora-4,15 diene 854, 856

1005

(-)-13-Isothiocyanatocubebane 853 (+)-Isothiocyanatocubebane 853 (-)-10-Isothiocyanatoguaia-6-ene 852 (-)-lO-lsothiocyanatokalihinol C 852 (-)-lO-Isothiocyanatokalihiniol G 856 (+)-5-Isothiocyanatopapukeanane 853 (-)-9-Isothiocyanatotrachyopsane 853 (+)-(1 R,6SJS, I OR)' 10-Isothiocynato-4amoq^hene 852 (+)-10/^-Isothiocyanoallo aromadendrane 853 (+)-6a-lsothiocyano-5 aH,7aH, 1 Oaendesm-4-(14) ene 852 (-)-1 Oa-Isothiocyanoallo aromadendrane 853 (-)-4a-Isothioyanatogorgon-l 1-ene 852 (-)-11 -Isothiocyano-7-/JH-endesm-5ene 852 2-Isothiocynoto-6-axene 850 p-Italicum 145 IvyPNP 383 JAK/STAT- linked receptors 520 JAK/STAT pathway 519 Janus kinase 3 (JAK 3) 271 Janus kinase 522,547,548 Japonenyne A and B 802 Jasmonates 367,373 Jasmonic acid 374 Jaspamide 768,757 derivative 768 Jaspis 700,714 Jaspis coriaceae 725 Jaspis splendans 768 Jaspisamides 727 Jaspsamide A 727 Jessie acid 182 Jessie acid arabinoside 182 Jessie acid xyloside 182 Jinbricatime 824 Jospissp 704,719,727,731 Julibrosidel 208 Julibroside II 208 Julibroside III 208

Jimgennanniaceae 252 Jimiperns procera 257,259 Juvabione 546 Juvenile hormone 546 K"^ and Ca"^ channels 531 K"^ channels 530,534 K"^-channeI mediated ion transport 497 Kaempferol 541,544,546,594,615 Kaikasaponin I 111,112,115,116,118 Kaikasaponin III 95,99,104,115,116,118 Kainic acid 529 Kainic acid-binding glutamate receptors 529 Kakkasaponin I 95,104 Kakkasaponin III 111,112 Kalihinane diterpenoids 689 (-)-Kalihinol G 856 (-)-Kalihinol L 856 (+)Kalihinol 856 KalihinolA 757 Kalihinol metabolites 854 Kalihinols 854 Kalinanes 782 Kalinihol I 856 Kalinihol J 856 (-)-Kalinihol 856 Kalipyran B 783 Karacoline 528 p-Ketoamide 785 K A T P channels 534

Karounidiol 61 Kaurane 241,246 Kaurenic acid 242 Kedarcidin 796 Kedarcidin chromophore 797 KeenamideA 887,888 Keramadine IIIJIS Keramamides 885 Keramamides F 884 Keramamides G 884 Keramamides H 884 Keramamides J 884

1006

Keramamides K 884 j3-Ketoacid 165 3-Ketoadociaquinone A 861 6-Keto-PGF2a 595 Ketoreductase 501 19-Keto-soyasapogenol B 184 N-Keto-stypodiol diacetate 591 Kexin 390 Kexin-like subtilases 389,390 3-Ketodociaquinone A 862 Kievetone 546 Kiheisterone C 795 Klebsielapneumoniae 258,259 Kloechera corticis 143 Konbuacidin 798 Konbuacidin A 711,798 Kororamels 902 kP6 preprotoxin 389 KSD assay 862 Kudzusapogenol A 95 Kudzusapogenol B 97 Kuduzusapogenol C 96 Kuanoniamines A-D 893,894,995 Kudzusapogenol A glycoside 103,105 Kudzusapogenol Bi 97 Kudzusaponin Ai 95 Kudzusaponin A2 95,104 Kudzusaponin A3 95^104 Kudzusaponin A4 95,104 Kudzusaponin A5 95,104 Kudzusaponin C] 96,103 Kudzusaponin PA3 118 Kudzusaponin SAj 95 Kudzusaponin SA2 104 Kudzusaponin SA3 95 Kudzusaponin SA4 104 Kudzusaponin SBi 95 Kuepaloxane 787 Kupffer cells 464,475 Kyurenic acid 529 L(+)-Muscarine 530 Laurencia rigida 783

Labd-13(E)-en-8a,15-diol 267 8( 17), 12(E)-Labdadiene-15,16-diol 265,266 Labdadiene-15,16-diol 11,15-hemiacetal (ll-OH-8(17),12(E)-) 266 Labdadienoic acid 248 5RMM. 107?-Labdan-13(6)-ene-8d, 15diol 258 Labdan-8( 17), 13-dien-3j3-15,18-triol 251 Labdane 13-0-glycosides 251 Labdane 241,244,259,276,275,277,256, 266,261 activities 257 Labdane glycosides 251 Labdane S 246,249 Labdane xylosides 252 Labdane type diterpenes (Labiateae) 263, 265 isolated from Hytis spicigora 276 nitrogenous 269 Labdanum oil 253 Labiatae 235,246,252,256 Labiataetannin 921 Labiate 921 Labopghora variegata 729 Labophorins A and B 729 Lachmim papyraceiim 781,785 Lachnumol 785 Lachnumon 781,782 Lachnumore 757 Lactase phlorizin hydrolase 609 Lactobocillus sp 257,924,927,928 16-Oxo-8( 17), 12(E)-ladadiene-15-oic acid 266 Lamellarins 706 Laminae eae 171 Laminaria pedicularioides 868 Lam in aria sp 724 Langmuir-Blodgett films 334 Lanosprazol 612 Lanostane 49,57 16a-Lanostane carboxylic acids hydroxydehydrotrametenolic acid 57 dehydrotumulosic acid 57 dehydropachymic acid 57

1007

16a-hydroxtrametenolic 57 pachymic acid 57 Lanostanes 0-16a-hydroxytrametenolic acid-3acetate 61 pachymic acid 61 poricoic acid B 61 abiesonic acid methyl ester 64 LantadeneA 64 LantadeneB 59,64,66 LantadeneC 59 Z-antibiotic peptidase 388 Lappaconititine 528 jL-arginine-analogues 471 Lappaconitine 528 Laser desorption mass spectrometry 330,331 (+)-LatA 874 (+).LatB 874 L a t e 875 LatD 875 LatM 874,875 LatS 874,875 Laternula elliptica 723 Lathyrus satives 529 Lathyrus palustris war.pilosus 93 Latrunculi B 732 Latrunculia 822,758 Latrunulia monocytogenes 450 (cytomegalovirus) 450 Latrunculia apical is 708 Latrunculia brevis 822 Latrunculia magnifica 874 Latrunculia purpurea 760,822 Latrunculin A 733 Latrunculins 874 L a t s A a n d B 874,875 Laucopolargonidin 653 Lauraceae 943 Laurencia brongriartii 863 Laurencia clavidormis 110 Laurence genus 249 Laurenciajaponensis 803 Laurenica TQd Q\gaQ 701 Laurencia saitoi 788

Laurencia sp 701 Laurenica majusciila 702 Laurenica nidifica 702 Laurenica nipponica 702 LDL peroxidation 934 L-Dopa 824 LeSBTl 392 Leguminosae 92,180,186,188,246,248,465 Leguminous isolflavone reductase (IFRs) 319 Leishmania donovani 254,260 Lemnalia africana 695 Lendenfeldia chondrodes 703 Lens esculenta 93 Lentinan 450 Lentinus edodes Sing 450 Leonunis hereteropyllus 272 Lepidium salivum 781 Leptoclinides dub ius 119 LeptosinA 839,840 Leptosin B 839 Leptosin C 839,840 Leptosin D 839 Leptosin E 839 Leptosin F 839 Leptosin G 839 Leptosin Gi 839 Leptosin G2 839 Leptosin H 839 Leptosin 1 839 Leptosin J 839 Leptosin K 839,840 Leptosin Kl 839 Leptosin K2 839 Leptosins 838,839 Leptosphaeria sp 839,901 Leucetta microraphis 105 Leucettamine A 705 Lucosceptoside 543 Leucine 331 Leucine aminopeotidase 375 Leucine rich repeat (LRR) domain 398, 402

1008

Leukemia P-388 760 leukopenia induced by 431 Leukotriene B4 (LTB4) 275,617,815, 939 Leukotriene B4 production 615 Leukotriene B4 receptor 705 Leukotrienes 274,275,940 Leukotrinene 939 Leyssera guaphaloides 250 Libhim maximowiczii 788 Ligand-gated ion channel neurotransmitter receptor 527 Liginine 126 Ligmncola laenis 899 Lignothioglycolic acid 668 Lilium longiflonim 391 Limocitrin rhamnoside 594 Limonene 44,125,133,140,145-147,149, 150,151 (+)-Limonene-1,2, (-)/m«5-diol 147,150 Limonene-1,2-epoxide 150 Limonene-1,2-epoxide hydrolase 150 Linalool 125,136,140,142,162 Linalool epoxide 143 (R)-(.)-Linalool 143 Linalool-8-carboxylic acid 141 O-Linalool 141,147 Linalyl acetate 136,140,141 Lingbya majusciila 876,902 /3,1 -Linked galactose 295,316 Linone-1,2-diol 146 Linonene epoxide 146,147 Lintenolide F & G 700 Lipoperoxidation assay 940 Lipoperoxidation process 614 Lipooxygenase pathway 263 Lipooxygenase derived fatty acid hydroperoxide 595 Lipooxygenases 597,939,940 Lipopollysaccharide (LPS) 477,460 Lissoclin disulfoxide 834,863 Lissoclinamide 1 880 Lissoclinamide 2 880 Lissoclinamide 3 880

Lissoclinamide 4 880 Lissoclinamide 5 880 Lissoclinamide 6 880 Lissoclinamide 7 880 Lissoclinotoxin A 834 Lissochnotoxin B 835 Lissoclinotoxin C 835 tris(methylthio) derivatives 835 Lissoclinotoxin D 833,835 Lissoclins 821 Lissoclimim 707,821,833,834,862,878 Lissochlinum bistratum 881,882 Z/5'5oc//>7i/Aw cyclopeptides 881

L issoclinum fragile 182 Lissoclimim genes 833 Lissoclinum japonicum 833 Lissoclimim lisschlinotoxin A 833 Lissoclimim patella 878,891 Lissoclimim perforatum 833 Lissoclimim vareaii 871,833 Lissoclimim voeltakowi 769 Lissoclimim voeltzkowi Michaels on (Urochor data) 269 Lissodinum 833,834 Listeria infection 431 Listeria monocytogenes 431 Listeria-specific cell mediated immunity 431 Lithistida (theonelld) 847 L-itronellol 125 Litsea ciibeba oil 132 Liver-derived NO 471 L-norepinephrine 530 Longamide B UIJIS Longamides 757 Lotus corriculatus 93 Low density lipoprotein 948 Lowry method 435 L-Phenylalanine 652,655 LPS hepatotoxicity 461,462 LPS stimulation 475 LPS induced liver and lung injuries 475 LPS toxicity 461 effects on liver injury 461 LPS-beated macrophages 473

1009

LPS-elicited hepatocyte apoptosis 463 LPS-elicited liver injury 463 LPS-induced elevation of [Ca^+j 475 LPS-induced nitric oxide 471 LPS-induced production of TNFa 470 LPS-related liver injuiy models 463 LPS-stimulated TNF-a production 470 LTP-blocking effect ofethanol 966 L-Tryptophane 652 L-Tyrosine 652 Luffariella variabilis 696 Lupane triterpenoids 60,61 Lupanes betulin 61 Lupenyl cation 176 Lupeol acetate 62 Lupinoside PAi 95,104,105,118 Lupinoside PA2 95 Lupinoside PA3 118 Lupinoside PA4 95,104,105,118 Lupinoside PA5 95 Lupinus polyphylus 94 Lupinus polyphylus hydride 93 Luteolin 541,543,544,593,595,615 Luttariella sp 694 Lycopersicon esculentum 294,298,657 Lycopersicon penivianum 372-375 Lycopersicon pimpinellifoliiim 294,298 Lycopersicon sp 293 Lycotetraose 316,317 j3-Lycotetraose 295,296,297,300,306,315 Lymphocyte transformation 224,225,226 Lymphokines 271 Lyngbya bouillonii 891 Lyngbya genus 891 Lyngbya lagerheimii 868 Lyngbya majuscula 722,732,716,761, 785,787,889 Lyngbyapetin A 891 Lypooxygenase inhibition 617 Lypooxygenase pathway 274 Lysergic acid diethylamide (LSD) 531

Lysozyme 334 Maaline skeleton 851 mAb (monoclonal antibody) 465 Madura pomifera 391 Macrolactamization 712 Macrophyllosaponin A 199 Macrophyllosaponin B 198 Macrophyllosaponin C 199 Macrophyllosaponin D 199 Magnoflorine 528 Maititoxins 730 Majusculainides 716 Makaluramines 822,823 Makaluvamin F 824 Makaluvamine A-E 700 Makaluvamine F 700 Makaluvamine N 700 Makaluvamines 707,708,757 Malabarican triterpenoids 700 Malabaricane triterpenes 700 Malampsori lini 397 Malaria pigment 332 Malondialdehyde (MDA) 219 Malonic acid esters 251 Malonyl-CoA 499,501,503,506 Malonylcoenzyme A 525 Malyngamide K 787 Malyngamide L 787 Malyngamide N 787 Mann-Whitney's U Test 962 Manauealide A 771 Manauealide B 771 Manauealides 729,730 a-Mangostin 544 y-Mangostin 544 Manool 253,254,259 biosynthesis ManoyI oxide 245,248-250,253-255 fragmentation 255 derivatives 256 ManoyI oxide isomer 259 13-e/?/-Manoyl oxide 250 ManoyI oxide series 258,259 ManoyI oxides 255 ManoyI oxides epigomeric acid 267

1010

DL-Menthyl chloroacetate 155 l-/?-Manthene 145 [4,6-Meo-ENBPI]Fe(lII) complex 352 Manzamine 709 Mepacrine 344 MAPK 545 2-Mercaptobenzothiazole 816 MAPK Kinase 526 6-Mercaptopurin 266 phosphorylation 526 Meridian B 764 MAPK kinase kinase (Raf) 522,547 Meridian C 764 Marchantia polymorpha 423 Maremycins A and B 828 Meridian D 764 Marine pyridoacridine alkaloids 758 Meridian E 764 Marinomonas mediterranea 719 Merozoite stage 328,329 Marinone 780 Mescaline 531 MauritamideA 870 a-Mating factor pheromone 387 Mayotamide 884 Metabolism of caffeic acid by Mayotamides A 883 microorganism 924 Meciadanol 618 Metabotropic glutamate receptors 531 Medicago polymorpha 93 Metal free porphyrins 354,355,356 Medicago trunuclata 379 Metal homeotasis 327 a-Megaspermin 395 Metalloporphyrins 347 /J-Megaspermin 395 Metalloporphyrins complexes 354 Megnaporthe grisea 322 Metalloprotease 330,331 co-Methyl hydroxylation 158 Metalloprotoporhyrins 357,358 Melemelenose A and B 869 Metallothioneins 717 Melilotus officinalis 93,132 Metal-N402 Schiffbas complexes 327 Melittin 274 Metamorphosis 784 Mellilogenin 97 Meth A 433 Mellilotus-saponin O] 95 Meth 1 tumor cells 432 Mellilotus-saponin O2 97 Meth A tumor 432 Melodinus monogynus (Apocynaceae) 276 Meth A tumor cell antigen 444 Membranipora perfragilis 825 cytotstatic activity 433,434 j8-Menene 131 Meth A tumor growth 453 /7-Menth-l-ene-9-ol 138 /?-Methane-3,8-diol 136 ;?-Menth-8-ene-l,2,-diol 148 2-Methlbutyl acetate 129 Mentha piperita 155 4'-0-Methl-kaempferol 544 Mentha pulegium 156 Methotrexate 266 /7-Mentha,l,8-diene-4-ol 148 1-Methoxymethyl-'6-oxo-6H-anthra /7-Mentha-2,8-diene-l-ol 148 [l,9-bc]thiophene 831 cisdinA trans 148 6-Methoxymellein 486,498,499 l-/?-Menthen-6,9-diol 145 biosynthetic sequence of 498 1-Menthene 149 6-Methyl-5-hepten-one 163 8-/?-Menthene-l,2-c/5'-diol 145 6-Methoxy mellein synthesis 486 8-/?-Menthene-l-ol-2-one 145,147 Methoxy thiofurodysinin acetate lactone Menthol 125,133,155 815 (±)-Menthol esters 155 6-Methoxymellein biosynthesis 505 Menthyl acetate 156

1011

6-MethoxymeIlein production 485,487, 488 biosynthetic enzymes involved in 498 6-Methoxymellein 483,484-486,506,507 biosynthetic activity 483 stimulation in carrot cells 484 6-Methoxymellein-O-methyltransferase 506,507,508 (5)-Methoxyphenylacetic acid 813 Methoxy-substituted dihydrocinchonamine 28 2 Methyl-6-methylisoquinoline-3,5,8(2H)trione 825 3-Methylbutyl acetate 129 10-(/?)-O-MethyleudistomidinC 817 AR,9R, 10/?-[ 1 -(5-Iso-quinolinesulfoxy 0-2Methyl piperazine 488 Methyl 18-carboxy-labda-8,13-(£)-diene15-oate 260 24-Methylcholesterol 417 4'-(9-Mettiyl-kaempferol 541 4-Methyl sterols 56 3-0-Methyl-2,3-dihydroluteolin 541 (+)-2-Methylthio-1,2,-/mAW-cyclopropane dicarboxylic acid 845 3-Methyl-2-butenoic acid 133 3,4(4-Methyl-3-pentenyl)-3butenolide 134 4- (Methylnitrosamino)-1 -(3-pyridyl)-1 butanone (NNK) 65 5-Methylthiovaracin 834 O-Methylseryl thiazole 885 2-Methyl-2-vinyltetrahydrofuran-5-one 141,140 (iEr)-4-Methyl-3-hexenoic acid 140 7-Methyl-3-oxo-6-octenoic acid 134 6-Methyl-5-heptane-2-one 134 6-Methyl-5-heptanoic acid 133 6-Methyl-5-hepten-2-ol 143 6-Methyl-5-hepten-2-one 136,13 8,140, 143,144,145,165,166 (S)-(+)-6-Methyl-5-hepten-2-ol 143 (Z)-2-Methyl-5-isopropylhexa-2,5dien-1-al 152

Methyl-5-methylthio-3-oxopentanoate 845 l-Methyl-5-thiol-L-histidine disulfate 814 (24/?)-24-Methyl-5a-cholestan-3-one 419 2-Methy 1-6-methy len-7-octen-2-ol 131 7-(9-Methylapigenin 541 A^-Methylated systemin derivative 376 (9-Methylbufotenine 531 Methylcholanthrene-induced fibroscoma 432 (24/?)-24-Methylcholest-4-en-3-one 419 24-Methylcholesterol 416,417 1-Methylcyclohexene 150 24-Methyl-demosterol 417 N-Methyldysideathiazole 890 Methylene blue 349 24-Methylenecholestrol 417 Methyl lycaconitine 528 N-Methyl-nitrosourea (MNU) 65 (±)-Methyl-0-acetyl-isoreserpate 18 Methylsulfinylindoles 861 1 -Ethyl-4-methylsulfone-j3-carboline 819 Methyllycaconitine 528 Methylthioadenosine (MTA) 829 Methylthiotidine 813 0-Methyltransferases 505 Methylxanthine theophyline 532 2-Methyl-Y-butyrolactone 138,136 (+)-Methy-0-acetyl-isoreserpine 24 Metmyoglobin/hydrogen peroxide 934 Metolachlor 669 Metronidazol 617 Mevalonic acid 130 [^H] Myo-inositol 487 Mezerein 937 Mezerin-induced superoxide anion radical 938 Microalgal metabolies 758 Micrococcus sp 48,79,94,719 Micrococcus liiteus 258,722,777,778 Microcolins 718 Microcyctis aeruginosa 799 Microcystin 733 Microcystis microcystins 543

1012

Microgenin 299-A 799 Microginin 299-B 799 Micromonospora echiospora 796 Micromonospora sp 841 Microtubule-depolymerization 473 Mimosa hostilis 248 Mimosamycin 825 Mimososideae 92 Minalemines D-F 897,898 Mineralcorticoid aldosterone 516 Misoprostol 616 Mitaria coccinea 251 Mitariosides A-D 251 Mitomycin C 268 Mitogen-activated protein kinase (MAPK) 522,547 Mitogen-induced mouse splenocyte proliferation 271 Mitraphylline 25,26 MLCK 546 MNU inuduced mammary tumors 46 Molamola 872 Mollamide 883 MoUicacid 182 MoUic acid arabinoside 182 Mollie acid derivatives 183 Mollic acid glucoside 182 MoUic acid xyloside 182 Monamine oxidases (MAO-A and MAO-B) 536,537 Moneses iinijlora 111 Mongholicoside I 202 Mongholicoside V 202 Mono and di-oleolglycerol 333 Monoalide 696 Monoalkyl-substituted polysulfides 843 Monoamineoxidase activity 594 Monocaffeoylquinic acid 939 Monoclonal antibody (MAO) 464,955 Monocyte chemoattractant protein 1 598 Monodesethylamadiaquine 344 Monodesethylchoroquine 344 Monodesmosyl saponin 108 Mononuclear cells (MNCS) 462,477 Mono-p-bromobenzamide 847

Monophosphate dehydrogenase 836 Moracenins 594 Moretenane 54 Morin 541,544 Morphinan isoquinoline alkaloids morphine 533 3-methylmorphine ecodeine 533 /3-codeine neopine 533 Moms alba 594 Mosher's method 467 Mossbauer spectroscopy 357,340 mrTNF-a-induced hepatic apoptosis 468 MTT method 266 Mucor circinelloides 166 Mucormiehei 259 Miicor plumbens 253 Mucor sp 723 Mucuna sempervirens 93 Multiflor-8-ene 59 Multiflor-9-ene 59 Multiflorane 53,59 Muricella sp 704 Murine acquired immunodeficienty syndrome (MAIDS) 433 Muscarinic receptors 530,611 Miissaenda 180,183 Mutagen-induced genetic variation 395 c-wj'c and c-7WW pathways 271 Mycalanmide derivative 733 Mycale 698,727,831,892 Mycalolides 831 Mycaperoxide 260 Mycaperoxide A 598 Mycaperoxide B 598 Mycoarbizins 781 Mycobacterium rhodochorus 156 Mycobacterium tuberculosis 779 Mycobacterimi intracelluar 259 MycorrhijinA 782,785 Mycosporine taurine 873 Mycothiazole 896 Myocardial infaction 598 Myocardial ischaemia 594 Myosin light chain kinase (MLCK) 522,523,524,547

1013

Myrcene 125,130,131,541,544,593 Myrtenic acid 154 Myrtenol 153 Myxoderma platyacanthus 872 N(10)-methyleudiotomin E 902 N,N'-Thiocarbonyldiimidazole 250 N,N-Dimethyl-5-(methylthio) varacin 833,834 N,N-Dimethyltryptamine 533 N-(p-hydroxyphenthyl) actividine 538 N-diethybiitrosamine induced hepatic tumor 46 N-formyl methionyl-lucyl-phenylalanine (FMLP) induced superoxide 47 N-methyl-N-nitrosurea (MNU) induced colon tumors 46 N ^ jN^-Bis (7-chloro-quinoline-4-yl) phenylenen-l,2-diamine 346 N4O2 SchifF-base coordination complexes 361 N4O2 Schiff-based metallodrugs 351 Na"^ and K"^-ATPase 517,535,542,836, 861 Na+,K+ ATPase inhibitor 535 Na"^/Ca2"^ antiporters 517 Na"^/Cr-linked monoamine neurotransmitter transporter 537 N-acetyl-beta-D-glucosaminidase 274 NAD (P)-02-oxidoreductase activity 811 NADDP receptor Ca^"^ channels 539 NADDP receptors 534 N ADH-dependent keto-reducing activity 500 NADPH synthase 502 NADPH-depdendent reduction 499 NADPH-dependent 6-hydroxy mellein production 502 NakmurolA 688 Nallanin 541 Namenamicin 837,838 Napyradiomycin Bi 798 Napyrodiomycins A and B 797,798

Naringen 615 Naringenin 614,613,618,653 Naringenin chalcone 653 Naringin 469,541,544,613,615,618 Na-sulfanomido ^carbolin 6 Natriuretic peptides (NPs) 367,381 Natural bolaphile 813 Natural cyclophanes 757 Natural killer cells G^K cells) 273 N-desacetylappaconitinee 528 Neamphine 897,898 Necrotizing agent 615,613 Neohop-12-ene 59 Neohop-13-ene 59 Neohop-13(18)-ene 65 Neoisomenthol 155 Neomenthol 155 Neomycin 925,928 Neoplastic B cells 270 Neosiphonia superstes 731 Neovasularization of solid tumors 593 Nephtea chabroli 708 Nephteis brasiliensis 875 Nephtesis facicularis 859 Neral 125,132,133,136,137,138 Nerol 140,143,165 Nericacid 133 Nerolidol 125,162,163,164,166,168 Nerylacetone 162,165,166,168,169 Netimicin 258 Neiirasapora crassa 304 ergosterol mutants of 304 Neurolathyrrism 529 Neuropeptide receptor 688 Neuropeptides 387,387 Neurotransmitter antagonists 515 Neurotransm itter receptors 512 Neurotransmitter signalling 517 Neurotransmitter-gated ion channels 519 Neurotransmitters 516 Neu's reagent 662 Neurotransmittor coverting protein 512 Neurotransmittor transporting protein 512 Neutraceuticals 597 NF-^B activation 470

1014

NF-A:B Nuclear factor kappa B 470,711 Nicandra physaloides 300 Nicotinic acetylcholine receptors 528 Nicotiana tabaccum L. 665,666 Niphates erecta 1X1 Niphatessp 732 Niphatevirin 717 Nippostronglyolus brasiliensis 815,896 Nippostronglyolus dubias 815 Nitraria tangutoriin 20 Nitric oxide (NO) 470,477,598 Nitrobenzene oxidation assays 666 Nitrogen oxides 665 Nitropropionic acid-glucose derivatives 190 3-Nitropropyl-gluosides 190 selniferous derivatives 190 4-Nitroquinoline-l-oxide 937 Nitrosamine 65 NMDA receptor-mediated responses 966 A^-Menthene 152 N-Methyl chloropyrrole 761 A^-Methylamino-L-alanine (L-BMAA) 529 N-methyl-D-aspartate binding glutamate receptors 529 N-nitro-L-arginine methyl esters 595,598 N-nitrosodiethylamine 66 NNK-induced lung tumorigenesis 65 NO production 471 NO synthase inhibitor 595 NO synthesis 595 Nocardia sp 153 Nocardioides sp 719 Nodule morphogenesis 379 Nodulin (ENOD) 379 Non-adrenergic imidazoline receptors 530 Non-insulin-dependent diabetes mellitus 534 Non-NMDA binding glutamate receptors 529 Nonsteroidal antiinflammatory drugs (NSAIDS) 610 Nootkatone 128,170,171 Noradrenaline 530,595 Norathyriol 543

Norcardia alba 162 Nordercitin 893,894 Norepinephrine 516,536,537,538 Norharmane 531 Nor-pachoulenol 171,172 Norsesquiteipene 163,166 NOS inhibitors 471 Nostoc linckia 794 Nostoc sp 789 Nostocyclophane D 794 Nostocyclophanes 757 Novalal 152,153 /J-7V-oxalylamino L-alanine (L-BOAA) 529 NSAlDs 613 Nuclear factor k B 598 Nuclear sterol regulatory element binding protein nSSREGP 388 Nucleating scaffold proteins 335 Nucleoidin glucoside 781 Nucleotide-gated ion channels 527 Oceanapamine 706 Oceanapia sagittaria 894 Oceanapia sp 706,894 Ocimene 125,130,131 Ocimenone 143,144 5,9-Octacosadienoic 721 Octalactin 728 Octalactin A and B 728 Octandecanoid pathway 374 Octapetide patellamide 878 Oestrogen 516 Ogebikase 654 Okadaicacid 9,10,731,832 from dinoflagellates 543 Okadaic acid-induced TNF-a-gene expression 470 18a-Olean-12-ene-3j3,23,28-triol 64 Olean- l2-ene-3A23,28-triol 60,64 Olean-12-ene-3A23,28-triol tri-Ohemiphathalate sodium 65,66 Olean-12-ene-3j8-23,28-triol 60 18a-Olean-12-ene-3i3-28-diol 60 Oleanane glucuronoide 89-92,94,121

1015

Oleanane saponins 183,203 Oleanane triterpenoids 60 Oleananes arjunolic acid 64 Oleanane-type saponins 207 Oleanene glucuronosides 117 Oleanene-type triteq^ene saponins 103 Oleanolic acid 59,61,541,107 Oleanolic acid 28-O-glucuronide 106, 107,108 Oleanolic acid 3-0-glucuronide 106,107, 108 Oleanyl cation 181 Olearia paniciilata 255 Oleic acid 333 Oleoylacetylglycerol 536 D-Oleoylphosphatidylcholine 333 Di-Oleoylphosphatidylethanolamine 333 Oleuropic acid 141 Olfaction 521 Oligoadenylate synthease 718 Oligogalacturonides 485,486,487 Oligonucleotides 390 Oligopeptide elicitors 396 Omeprazol 612 O-Methyltranferase 498,499 Oncogenes 545 Oomycete 299,303 phythhim 299 phytophthora 299 Open readingframes(ORFs) 379 Opiate receptors 533 Orange oils 169 Oregon in 543 Organochlorines 767 Oriamide 884,885 Orientin 594 Orina sp 712 Orinthine 937,66 Oripanine 533 agonist and antagonist effects 533 Ornithine 66 Ornithine decarboxylase (ODC) 45,66 TPA induced inhibition 45 Oroidine 783,785

Oroidin-family 870 Oryza sativa 311 Oscillatoria agardtii 799 Osteoporotic drug 711 Ostodes 242 Ouabagenin 3-0-L-rhamnoside 535 Ovoperoxidase 813 Ovothiol A-C 813 4-Ovothiol ARl 813 5-Ovothiol RBI 813 6-Ovothiol CRl 813 Oxaloacetic transminase 935 Oxazine alkaloid 542 Oxazoline 881 j3-Oxidation 166 Oxidative burst 396 Oxidative DNA damage 66 Oxinastatin-4 721 Oxindole alkaloids 3,4,25,26,27,33 Oxinellamides 757 6-Oxo-cycloartan-3,16 200 6-Oxocampestanol 415,420 6-Oxocativic acid 250 11-Oxo-manoyl oxide 256,252 Oxycorynia fascicularis 251.727 Oxystigma buecholtzii 165 Oxytrogenin 96 Oxytrpis 180 PI3 (phosphatidyl linositol)-kinaseindependent pathway 264 P-388 lymphocytic leukemia cell 764 P-388 model 823 P69 subtilases 391 Pachastrella sp 704 Pachymatismin 717 Pachymeniopsis lanceolata 722 Pachymic acid 63,64 Pachypellins 709 Pachyrhizziis erosus 93 Paclitaxel 245 Paecilomyces sp 725 PAF antagonist 275,615 Pachypellina sp 710

1016

Palauamine 110,111 Palindromic structure 371 Palmitic acid 333 Palmitoleic acid 333 r-(9-Palmitoyl-3'-0-(6-sulfox-D-quinovo pyranosylglycerol 868 Palmrosa oil 132 Palustroside I 97 Palustroside II 104 Palustroside III 104 Palythoa 735 Pan C auxotroph mutant 319 Panacene 786 Panax ginseng C.A. Meyer 956 Pandaros acanthifolium 832 Pantoisofaranoid A 802 Pantoisofaranoid B 802 Pantoisofaranoid C 802 Pantoneura plocamioides 801 Pantopyranoids A and B,C 801 Pantothenate synthetase 319 Papavarine 561 Papuamides A-D 714 Paramecium 494 Para-nitrophenyl-jS^D-glycopyranoside 306 Parasitmia 329 Parguerol 757 Parguerol triacetate 788 Parkinson's disease 531 Passiflora 180,183 Passiflora quadranglaris 205,206 Passifloraceae 180 Patchaulol 125,170 Patchouli alcohol 170 PatellamideA 879 PatellamideB 879 PatellamideC 879 PatellamideB 879 PatellamideG(l) 879 Patellazoles A-C 891 Pathogenesis-related gene expression 394 Pathogen-induced necrosis 660 PBS (phosphate buffered saline) 455 p, coumarate 655

PDE isoenzyme 490 Pectenotoxin 2 729 Pectin esterase 486 Pectinase 483,485 Pellina sp 709,723 Penaeus japonins 718 Penicillium 828,901 Penicillium digitatum 138-140,145,148, 149 Penicillium italicum 150 Penicillium notatum IIOJSO Penicillium roquefortii 129 Penicillium sp 797,155 Pennyroyal oil 156 2,3,4,5,6-Pentahydroxyxanthone 351,349 Pentabromopseudoline 776 Pentacyclic triterpenoids fridelane 55 glutinane 55 hopane 55 lupane 55 moretenane 55 multiflorane 55 taraxastane 55 taraxerane 55 Pentagastrim 613 2,3,4,5,6-Pentahydroxy xanthone 350 3/3, 6a, 16/J,24a, 25-Pentahydroxy-9/J, l9-cyclolanostane 184 Pentapeptide dolastatin 885 1,2,3,5,6-Pentathiepanes 843 (Poly) peptide hormoens 387 (Poly) peptide ligands 385 Peptide enod 40 379 Peptide elicitors 393,395,397 Peptide prohormones 387 Peptide signals 387,388 Peptidonimetic inhibitors 39 Peptostreptococcus sp 925 Perfragilins A and B 825 Perhydroquinolins 859 Pericentral hepatocyte necrosis 466 Periconia byssoides 790 Pericosine A 790,791 Perillaldehyde 146,147

1017

Perillicacid 145,146,147,150 Perillyl alcohol 146,147,148,149,150 Perna canaliculus 864 Peroxidase (POD) 657,663 Persa americana 943 Petrosapongia nigra 697 Petrosapongiolides 697 Petroselimum crispeum L. 666 Petrosia sp 722,723 Petrosia contignata 113 Petrosia steriata 694 Petrosia weinbergi 703 Petusis toxins 489 Peyssonelia sp 694 Peyssonols A 694 PHA 271 Phakellia 847 Phakellia costada 830 Phakellia mauritiana 770 Phakallistatin 5 830 Phakellistain 715,716 pharmacological effects 429 Phase transfer catalyst 357 Phasecoside IV 97 Phaseogenin 97 Phaseolus coccineus 93 Phaseolus vulgaris 489 Phaseoside I 95,104 Phasesidelll 97 Phenobarbitol induced Hepatic tumors 46 Phendoxidase 719 Phenobarbitol 66 Phenolase 654 Phenolic compounds 651,663 degradation of 651 Phenolic ellagic acid 543,546 Phenolic compound metabolism 651 regulations of 651 Phenolic oxidation 671 Phenolic oxidation (PPO and POD) 668 Phenol-sulfuric acid method 435 Phenylisothiocynate 435 Phenylpropanoid 541,543 curcumin 543

2-Phenylacetyl-^carbolines (R,SandT) 782 Phenylalanine 652,653,660,928 Phenylalanine ammonia lyase 489,652 Phenylalanine hydrolases 718 Phenylalkylamines 534 verapamil 534 (/?)-Phenylethanoid glycosides (1,7-Bis(3, 4-dihydroxyphenyl) heptan-3-ol) 543 Phenylethanoids 465 Phenylethylamine 538 Phenyllanine hydroxycinnamate 652 Phenylpropanoid 651 Phenylpropanoid alkaloids 540 D-cathinone 531 D-cathine 531 I-ephedrine 531 pseudoephedrine 531 Phenylpropanoid compounds 651,659 Phenylpropanoid bisoynthesis 663 Phenylpropanoid metabolism 652,658, 663,666,676 Phenylpropanoid pathway 651,652 Phenylpropanoid pathway enzymes 666 Pheophytin 691 Phloeodictine B 829 Phloeodictine C12 829 Phloeodictines 829 Phloeodycton sp 829 Phlomis medicinalis 251 Ph lorn is mendocina 133 Phlomis younghiisbandii 257 Phlomosides 251 Pholagacanthiis thyrsiflorus 251 Phoma sp 691 Phomopsin-A 886 Phorbas sp 759 Phorbol-12,13-dibutyrate(PDB) 542 Phorboal ester 726 Phorbol 242 Phorbol ester promotors 66 Phorbol esters 243,246,524,536,542,543 Phorbol l2-myristate j3-acetate 598 Phorbol 12-mysristate-13-acetate 488, 937,948

1018

Phorbol-12-myristate-13-acetate (PMA) 242 Phorbolbased pKC activators 524 Phorboxazoles 756 Phorboxazoles A & B 759,760 Phormidiiim temte 868 Phosphate buffered saline 434 Phosphatidyl inositol-3-kinase (P13K) 522,525 L-Of-Phosphatidylcholine 333 L-a-Phosphatidylethanolamine 333 Phosphatidylinositol-3,4,5-triphosphate (PI347P3) 516,517 Phosphatidyl inositol-3,4-kinase (PIP2) 522,547 Phosphatidyl inositol phosphate-activated protein kinase 522,547 Phosphatidyl linositol 475 Phosphatidylinositol -4,5-biphosphate (IP3) 521 Phosphatidylinositol 487 Phosphatidylinositol cycle 483,494 Phosphatidylinositol phosphate-activated proteins 517 Phosphatidylinositol-3,4,5-triphosphate (PIP3) 522,547 Phosphatidylinositol-3,4-biphosphate (PI34P2) 516 Phosphatidylserine 938 L-a-Phosphatidy-L-serine 333 Phosphodiesterase (PDE) 507,597 Phosphodiesterase (PDE) inhibitor 488 Phosphodiesterase inhibitor 275 Phosphoenol pyruvate carboxykinase 523 6-Phosphogluconic acid 670 6-Phosphogluconic dehydrogenase 670 Phosphoglycogen synthase 525 Phospholipase A2 263,270,374,734,688, 842,867,903 Phospholipase A2 inhibitor 47,593 Phospholipase B 333 Phospholipase C (PLC) 522,521,530, 548,696 Phospholipase C-y (PLCy) 545

Phosphoprotein (P-Pr) 522,548 Phosphoprotein phosphatases (PPs) 517,521 Phosphorylase kinase 264 Phosphorylated phsophotidylinositol 487,488 Phosphoserine 517 Phosphothreonine 517 Phosphotyrosine binding SH2 domains 525 Photobacterium damsela 719 Phthalide isoquinoline alkaloids (+)bicculine 528 bicuculline 532 N-methyl bicuculline 528 narcotoline 533 norbicuculline 537 a-narcotine 533 tetrahydropapaveroline 533 Phycornyces blakesleeanus 318 Phycopsis terpnis 858 PhyllactonH 695 Phyilacton I 695 PhyUidia ocellata 858 Phyllidia pmtulosa (nudibranch) 852 Phylloquinone 236 Phyllospongia sp 695 Phytoaiexins 484 Phytoalexin production 494 biosynthesis in carrot-cells 483 Phytoalexin r is hit in 319 Phytoalexin synthesis 507 Phytoaiexins 484 Phytoanticipin 294,321,662 Phytocanticipin-degrading enzymes 312 Phytochrome proteins 529 Phytoecdysone 55,59,546 Phytohantiones 367 Phytohemaglutinin (PHA) 267 Phytol 245 Phytolexin in carrot cells 483 biosynthesis 483 Phyto-pathogenic oomycete 395 Phytophthora 303, 395,396 Phytophthora elicitins 395

1019

Phytophthora megasperma 303,396 Phytoprotectants 320 Phytosulfokines 368,380 Phytosulfokines signal 379 Phytosulfokines receptor 378 Phytoxin pisatin 322 a-Pinene 125,145,149,151 ^Pinene 145,154 a-Pinene epoxide 153 Piceaabies L 666 Piceatannol 541,543,546 Picrocrocin 954 Pigment-producing parasites 25 Pilocarpine 530 Pilosine 530 Pimarane 241 Pinaceae 245,246,249 Pinane monoterpenoids 153 Pinifolic acid 258 Pinna muricata 871 Pinocarveol 153 Piniis nigra 249 Pinus caribaca 151 Pinus palustris 151 Pimis strobus 245 Pinus sylvestris 249 Pinusolide 275 Piocarvone 153 Piperidine alkaloids 528 (-)-lobeline 528 ^alanine-3-aminopropionic acid 537 anabasine 521 guvacine 537 isoguvacine 528 nicotine 528 nipecotic acid (3-piperidinecarboxylic acid) 537 nomicotine 528 Piperitenone 150 Piperidine alkaloids pseudoconhydrine 528 y-coniine 528 (+)-N-methylconiine 528 Pisumsatium 93,417

Pithomyces sp 172 (2/?,55)-Pitoyl 144 Pityrophtors pitygraphiis 144 PK or PP targetting proteins 521 PKA inhibitors 527,538,542 acridine based 542 isoquinoline based 542 phenanthrene based 542 PKA-catalyzed phosphorylation 529 PKA-gated Na"^ channels 521 PKC isozymes 61,517,524,726 PKC 271,526,536,543,721,726 diacylglycerol activated 66 PKG 523,527 PLA2 669,695,696,704,902 Plakelliasp 715 Plakoside A and B 721 Plakosides 722 Plamepsin I and II 330 Plankortide 724 Plankortis lits 724 Plankortis simplex 721 Plant proteases 389 Plant pyrolysins 392 Plant peptide signals 402 Plantainoside D 543 Plasminogen activator (t-PA) 221 Plasma membrance located receptors 518 Plasma membrance potential 374,396,397 Plasma membrane H"^-ATPase 374 Plasma membrane-located receptors 522 Plasma transaminase 472 Plasma-membrane-located signals receptors 518 Plasmepsin 352 Plasmepsin I 331 Plasmepsin II 331 Plasminogen 725 Plasminogen activator 221 Plasmodia 348 Plasmodia biochemical pathway 337 Plasmodial organelles 334 Plasmodial protQ'm^SQS 330 Plasmodium 327

1020

Plasmodium berghei 333,338,341 Plasmodium CD?K 544 Plasmodium falciparum 328,330-332, 334,336,338,340-342,345,348,350352,359,527,814 Plasmodium malariae 328 Plasmodium vivax 328 Platelet activating factor (PAF) 275,615 Platelet antiaggregant 595 Platelet-derived growth factor (PDGF) 593,598 Plaustroside I 104 Pleckstrin homology 525 Plectranthus {Ld!o\?iX2iQ) 238 Pleiotropic effect of TNF-a 460 Pleurobranchus forskalii 887 Pleuronectes platessa 118 Pleurotus glabellatus 131 Pleurotus sajor-caju 131 Pleurozia acinosa 252 Plocamadiene A 701 Plocamium 701 Plocamium cartilagineum 701 Plocamium castatum 701,781 /?-Methoxyphenacyl bromide 836 PM-located voltage-gated Ca^"^ channels 534 Podocarpaceae 246 Podocarpane 241 Podocarpus ferrugineus 246 Podocarpus macrophyllus 246 Poecillastra sp 731 Pogostemom cablin 111 Pokweed mitogen (PWM) 271 Polauamine 784 Polio vaccine 817 Polio wirus 782 corosolic acid 543 poliumoside 543 Polyalthia macropoda 260 Polyamine metabolism 66 Polyamines 66 Polyasparagine 334 Polybrominated methylthioindoles 863

Polycarpa 836 Polycarpa aureta 706,836 Polycarpamine A 836 Polycarpamine B 836,835 Polycarpamine C 836 Polycarpamine D 836 Polycarpine 836 Polychlorinated dibenzodioxins 757 Polychlorinated phenyl ether 792 Polycitorsp 834 Polycitorella mariae 837 Polyclonal antibodies against ARS2 435 Polydiscamide A 865,866 Polyene antibiotics 299 Polyfibrospongia australis 694 Plygalacturonase 374 Polygalacturonides 486 Polyhistidine 337 Polyhydroxylated phytohormone brassinolide 414 3 j3,5a,6 a, 15 a-Polyhydroxylated steroids 872 Polyketide synthase 502 Polymastiamides 704 Polymerase activity 826 Polymerase chain reaction (PCR) 390 Polymoi*phic S locus 386 Polymorphonuclear leukocytes 432 Polymostia boletiformis 704 Polyol ingenol 242 Polypeptide ligands 368 Polyphenoloxide (PPO) 657 Polyphenols acteoside 476 (+)-ampelopsin 476 curcumin 476 (-)-epigallocatechin gallate 476 gomisin A 476 hovenin 1 476 lithospermate B 476 tetrahydroswertianolin 476 naringin 476 Polypodine B 59 Polysine 334 Polystyrene latex particles 270 Polysyncaraton lithostrotum 837

1021

Polytheonamide B 715 Polyunsaturated fatty acid (PUFA) 932,948 Polyuronides esterification of 486 Ponax notoginseng 476 Poniciretin 618 Pontoneureins A and B 801 Porcirin 618 Porta cocos Wolf 956 Poricoci acid 64 Poricoci acid A 57 Poricoci acid B 57 Porphyrin 338,340 Porphyrin complexes 354,355 Porphyrins 327,359,361 Portieria harnemannii 702,767 CD4-Positive helper T cells 450 Post translational modification 367 Potato-saponin a-chaconine 319 Potomogetonnodosus 260 PP inhibitors 521 PPl inhibitors 543 PP2A inhibitors 543 PPO activity 656 PPO cytochemistry 654 PPO phenolic compounds 656 Purealidins J, K and Q 766,767 Pregnenolone 419 Prehispanolone effect on B cell proliferation 273 Prelissoclinamide 1 880 Prelissoclinamide 2 880 Premna oligotricha (Verbenaceae ) 257 Prenylated xanthones a-mangostin 541 y-mangostin 541 PrepANP 381 Prepatellamide B formate 879 Prepro proteins 400 Prepsammaplin A 844,845 Prepsammaplin B 844,845 Prepsammaplin C 844 Prepsammaplin D 844

Preulithiacyclamide 879 Priano melanos 822 Prianos sp 760,822 Prianosin A 823 Prianosins C and D 823 Prianosinsdischabdins 802 Prianosins-discorhabdins 802 Primaquine 349,350 Primaiy biliary cirrhosis 463 Proanthocyanidins 666 Probit-graphic interpolation method 47 Procyanidin 544 Procyanidin glycoside 594 Procyanidin dimer 541 Prodelphenidin 544 Progesterone 139,421 Proinflammatory cytokines 461 Prolactin (PRL) 265 Proleiis mirabilis 259 Proline 320,371,372 Prolysine 388 prophylactic effects 429 Propionibacterliim acnes/LPS'inducQd hepatocyte apoptosis 463,471 liver injury 463 Propionibacterium acnes 462,465 Propionibacteniim acensll.?^ model 462 Propionibacterium acneS'XndncQd mononuclear cell infiltration 463 Propolis 945 Proprotein coveitase (PC) 402,388 Proprotein processing protease 392 Prosapogenin 1 209 Prosapogenin 2 209 Prosapogenin 3 209 Prosapogenin 4 209 Prosapogenin 5 209 Prosapogenin 6 209 Prosapogenin 7 209 Prostacyclin 598 Prostaglandin E2-dependent cytoprotective effect offlavonoids 607 Prostaglandin D2 (PGD2) 275

1022

Prostaglandin E2 (PGE2) 58,271,273, 598,616 Prostaglandin endoperoxide receptor antagonist 593 Prostaglandin H2 (PGH2) 595 Prostaglandin production 617 Prostaglandin synthesis 616 Prostaglandins 274,367,463,516,616 related eicosanoids 516 Prosystamin cDNA 370 Prosystemin 373,374,375 Prosystemin polypeptide 375 Protaneous lignads 368 Protease 65,483,941 Protease hydrolyzing casein 718 Proteases 483 Protein (SpA) mitogens 271 Protein kinase 261,518 Protein phsophotyrosine phosphatase (PTPase) 264, 522,548 Protein kinase C (PKC) 60,61,243,598, 734 Protein kinase B (PKB) 264 Protein kinase C inhibition 46,488 Protein kinase domain 384 Protein kinase inhibition (PKC) 45,513, 540,545 Protein phosphotyrosine phosphatases 518 Protein tyrosine kinase 519 Proteinaceous receptor 371 Proteinase K 388 Proteolysis assocaited proteins aspartic proteinase 370 carboxypeptidase 370 cysteine proteinase 370 leucine aminopeptidase 370 ubiquitin-like protein 370 Proteolytic inactivation of systemin 375 Proton pump inhibitors (PPIs) 612 Protooncogenes 47,269 Y-myc and bcl-2 267 Protophyrin IX (PPIX) 335,354,355 Protopine alkaloid allocryptopine 539

Prototropic cyclization 141 Prymnerium parvum 794 Prymnesin-2 794,795 Psamaplin B 858 Psammaplysilla purea 712 Psammaplysilla purpurea 719 Psammaplysilla sp 833 Psammaplysin A 784 Psammocinia 761 Pseudaxinyssa sp 857 Pseudaxinyssa pitys 854 Pseudoceratidine 785 Pseudoceratina verrucosa 712 Pseudoceratina purpurea 720,774,784 Pseudoceretina 719 Pseudodistoma aurum 782 P, pseudomallai 154 Pseudomonas 145,146,148,155,929 Pseudomonas aeruginosae 133,257,258, 259, 394,828 Pseudomonas citronellolis 132,133,165 Pseudomonas clavata 836 Pseudomonas convexa 133 Pseudomonas eruciviae 168 Pseudomonas fluorescens 152,394 Pseudomonas incognita 140,133,141,147 Pseudomonas medicinalis (Labiateae) 257 Pseudomonas pseudomallei 140 Pseudomonas putida 131,15 6,661 Pseudomonas putida-arvilla 153 Pseudomonas sp 152,153 Pseudomonas syringe 399,394,398 Pseudo-nitzschia multiseries 729 Pseudoplatanus 376 Pseudopterasin 689,690 Pseudopterogorgia elisabethae 690 Pseudopterosin E 689 Pseudoyohimbine 29,30 Psilocybe mexicana 531 PSK derivatives 378 PSK in mitosis 377 PSK precursor 377 PSK purification 378 PSK-receptor 378 PSK-a activity 378

1023

PSK-a analog 378 Psoitis tabricci 701 Ptilotafilicina 718 Pto-avrPto system 399 PtO'OvrPto complex 399 Pueraria lobata 93 Pueraria thomsonii 93 Puerariae Flos 91,94,99,100 Puerarlae lobata 91,103 PUFA 933 Puleagone 125,156 Pupukeanane skeleton 851 Purealidins 757 Purealidins N,P and Q 766,767 Purine receptors 532 Purpurin 543,544 Purpurogallin 546 Putative receptor proteins 398 Putrescine 66 Puupophenone 698,699 Puwainaphycin C 793, 794 Pyranocoumarins decursin 543 decursin angelate 543 Pyranoid linalool oxide cis and trans 143 Pyridinebetaine B 873 Pyrido [4,3,2,-m,n] thiazolo [322-b] acridinum skeleton 893 Pyrido [4,3,2,-ni,n,]acridine skeleton 894 Pyridoacridine 707,709 Pyridone-oxazole B 90063 841 Pyrogallol 613 Pyroloiminoquinones 824 Pyrolomycins 757 Pyrolysin 390 Pyronaridine 344 Pyrrol [2,1-j] quinolines 859 Pyrrolintrin 757 Pyrrolinyl-j8-carbolines G,H,J,P & Q 782 Pyrrolomycin antibiotics 794 Pyrroloquinolines 705,707 Pyrrolyl-)8-carbolines A and M 782 Pythium 303 Pytressin-induced coronary spasm 594

Quadranguloside 206 Quassine 59,548 Quercetagenin 541 Quercetin 44,56,541,543,544,546,593, 595,597,608,610,613-616 glycosylation 544 Queretaroic acid 60 Quidine 344 Quinacrine 593 Quinalizarin 543,544 Quinic acid 920,922,924,928,929 by microorganism 927 metabolism of 929 Quinic acid esters 919,947 Quinidine 347 Quinine 344,478,347,546 Quinizarin 544 Quinoline antimalarial drugs 329,348,349 Quinoline drugs 348 Quinoline family of drugs 343,360 Quinoline polymerization 348 Quinoline-based antimalarials 344 Quinoline-ring antimalarial drugs 327 Quinolines 327,345,347,348 Quinolin-heme complex 348 Quinolizidine alkaloid (7-hydroxy peganine) vasicinol 536 Quinolizidine alkaloid spartein (lupinidine) 534 R(resistance) gene 397 Race-specific elicitor 397,398 Race-specific peptide elecitors 398,399, 400,401 Radioligands 373 Radopholus similis 662 Raji cells 58,59,60 Ranitidine 612,617 rANP-triggered signal transduction 382 Rapamycin 892 Raphisia pallida 705 Raspaciona aculeata 700 Raspacionins 700,701 Raubasine (ajmalicine) 30,31

1024

Raunculaceae 180 Rauwolfia alkaloids 8 Reactive oxygen species 948 Reactive oxygen metabolites (ROMs) 614 Receptor-like protein kinases 383 Receptor protein phosphotyrosine phosphatase (RPTP) 522,548 Receptor serine/threonine protein kinase (RSTK) 518,522 Receptor tyrosine kinase (RTK) 512,522, 548 Receptor-like kinases 384 ai-Receptors 30 a2-Receptors 29,30 Redox-active metals 933 reduction of adverse effects of 5FU 454 Reinera sp 723 related compounds 538 Renealmia alpinia 266 Reserplc acid lactone 18 conformation 24 Reserpine 3,11-13,16,22,29,33,610 conformation 23 total synthesis 17,23 Resiniferatoxin 242 Response decay mechanism 491 Resveratol 546 Retinoicacid 59,60,515 Retinoids 44,515 Reverse transcription-polymerase chain reaction (RT-PCR) 322,477 Reverse transcriptase 941 Rhamnacease 469 3-O-Rhamnosyl-quercetin 541 Rhizobia 395 Rhizobiaceae 379 Rhizobium meliloti 395 Rhizobium symbiosis 659 Rhizopus nigricans 253,261,318 Rhodococcus erythropolis 150 Rhodoccus rubropertinctus 162 Rhodococcus robropertinctiis 165,166 Rhododendron sp. 242 Rhoifolin 594,595

Rhopaloeides sp 698 Rhopaoloic acid A B and C 698 Rhynchophylline 26 Ribavirin 31 Ribenol 250 Ribosomal 56 protein phosphorylation 525 Rimsulfuron 669 Ritterella sigillinoides 782,816 RNA synthetase 841 Robinia pseiido-acecia 93 Robinioside A 97 Robinioside B 96 Robinioside C 96 Robinioside D 96 Robinioside E 96 Robinioside F 96 Robinioside G 96 Robinioside H 96 Robinioside I 95 Robinioside J 95 Ropholic acid A 698,699 Rosa bourbonia oil 132 Rose oxide trans dindcis 138 Rosmarinic acid 921 Roylea calycina 252 RTK tyrosine kinase 545 Rubiaceae 180 Rubrolides A,B and C 789,790 Rubrosides A-H 763 Rubusfoliolosiis 251 Rudea aurea 93 Rufigallol 349,350,351 1,2,3,5,7-hexahydroxy anthraquinone 349 RuleofRuzika 130 Russula siibnigricans 765 Russuphelin A 765 RussupheUn B 765 Russuphelin C 765 Russuphelin D 765 Russuphelins 757 Rutin 469,608,610,613,615 Ryanodine receptor (RyR) 534,535

1025

Ryanodine receptor Ca^"*" channels 535 S-(4-Hydroxybenzyl) glutathione 820 13-e/7/-Sclareol 246 S. locus cysteine-rich protein (SCR) 387,402 S locus genes 386 S locus glycoprotein 386 S /ocw5 receptor kinase 386 S locus receptor-like kinase (SRK) 384,402,387 Saccharomyces carlsbergensis 759 Saccharomyces cerevisiae 143,258,780, 782 Saccharomyces fibuligera 308 Saccorliza polyschides 758 S-Adenosyl-L-homocysteine (SAH) 509 S-Adenosyl-L-methionine (SAM) 509 Safracins 826 Saframycin 826 Safranal 956 Sagitol 894,895 Saikosaponins 90,474, Salicyclic acid 391,653,655,659-662, 664,666,669 Salicylate hydrosylase 661 Salicylihalamides A and B 728 Silymarin 614 Sanguinarine 541 Salmo salar 723 Salmonella typhimurium 837 Salvia officinalis 12 8 Salvia sclarea 246,253 Sapium 242 Sapogenol 89,94,116 Saponin avenacin A-1 320 Saponin detoxification by fungi 304 Saponin detoxyfying enzyme 313,295, 304 Saponinase 312 Sarcoaldesterols A & B 704 Sarcoma 182 tumors 432 Sarcophytum sp 704 Sarcosine (N-methylglycine) 537

Sargassum lacerifolium 867 Saxitoxin 684,730 Sayasaponin 1 120 Scalaradiol 695,696 Scalardysin 697,900 Sceptrin 779 Schaffer collateral 959 Schizophrenia 531 Schizophyllum commune Fries 452 Scilla maritima 535 Scillarenin 3-O-glusyl rhamnoside 535 Sclareol 246, 252,253,255,259,267 biosynthesis 254 Sclareol glycol 265 Sclareol isomers 266 Sclareolide 253 7a-hydroxy derivative 253 Scleritoderma 703 Scoparia dulics 263 Scoparic acids A,B and C 263 Scots pine (Pinus sylvestris) 666 SCR gene experession 387 SDS buffers 338 SDS denaturation 333 SDS PAGE gels 370 SDS-PAGE analysis 315 SEB staphylococcal enterotoxin B 465 Secoadociaquinones A and B 861 Secoiridoid glycosides 471 Secolactam 33 Second messenger 516 2,3-Secoreserpine 12,33 3,4-Secoreserpine 12,33 (-)-Securinine 528 Seed oil (Croton oil) 242 Self-incompatibility (SI) 402 Self-incompatibility in pollen 386,393 Senieramycin 826 Sepedonium ampullosporum 318 Septoria lycopersici 293,295,301,304, 302,307,308, 314-316 erg-3 gene 304 Serine proteases (subtilases) 388

1026

cysteine proteinase inhibtor 370 poly phenol oxidase 370 serine proteinase inhibitor IT 370 Serine/theorine or tyrosine-specific protein kinases 528 Serine/threonine protein kinases 399,545 Serine/threonine phosphorylation 520 Serine-threonine phosphatase 799 Serine-threonine-specific receptor protein kinases (S/T-RPKs) 520 Serine-type acetylcholinesterase 719 Serotonin (5-hydroxytryptamine) 536 Serotonin 516,519,520,536,537,538,610 Serotonin agonists 531 anticomplementary activity 46 cyclic AMP-dependent protein kinase 46 delayed-type allergy suppressant activity 46 serotonin induced edema 46 Serotonin receptors 531 Serum ALT activity 462 Serum ALT and AST levels 466 Serum TNF-a 468,475 Serum transaminase activity 469 Serum transaminase levels 466,477 Serum transminases 467 Serum tumor necrosis factors (TNF-a) 459 Sesquiterpene isothiocyanates 848 Setaria italia 781 Seubert's medium 153 Sheffe'stest 101 Shermilamine B 820 Shermilamines D & E 820 Shigella boytic 260 Shigella lycopersici 306,313 Shigella Shiga 260 Shikimate pathway 328 Shikimate/arogenate pathway 652 Shikimicacid 928,929 Shizandra chinensis 476 Sho-Saiko-To pretreatment 474 ShugChher 275 Sideritis arborescens 249,250,

Sideritis foetens 250,254 Sideritis gomerae 249 Sideritis javalambrensis 250,263,274 Sideritis lycopersici tomatinase 308 Sideritis mugronensis 250 Sideritis nutans 249 Sideritis shiga 260 Sideritis varoi 250 Sidnutol 249 Sieberosidel 191,196,225 Sieberoside 11 196,225 Signal pathway-associated proteins 370 acyl-CoA binding protein 370 calmodulin 370 lipooxygenase 370 prosystemin 370 nucleotide diphosphate kinase 370 Signal regulated protein kinases 540 Signal transduction mechanisms 518 Signal transduction pathways 514,515 Signal transductors and activators of transcription (STATS) 519 Silicatein 717 Siliquariaspongiajaponica 723,759 Silymarin 612,614 Sinapic acid 434,653,655,920 Sinapis alba 781 Sinapyl-alcohol 653 Singmadocia symbioltica 902 Sino-artrial node 32 Sinomenine 472,476 hepatoprotective effect 472 Sinomenium aciitum 476 Sinularia disseda 704 Similariaflexibilis 690 Sinularia nanolobata 690 Site-1 proteases 390 Skimiate pathway 652,653 Smith degradation 452,438,439 Snake 8 kDa polypeptide toxin abungarotoxin 528 Snechocystis sp 718 Sodium channel blocker 684

1027

Sodium dodecyclosulfate-polyacrylamide 499 Sodium metaperiodate 134 Solanaceae 249,369,370,386,400 Solanaceous sp 304,368 a-Solanine 300,305,311 Solarium sp 293 Solon 618 Somato tropin (STH) 265 Sophorajlavescens 93 Sophora subprostrata 93 Sophoradin 612 Sophoradiol 95,111,112,115,116,118 Sophoradiol OG 94,99 Sophoradiol glucoronides 111,114,119 Sophoradiol glycoside 115 Sophoradiol monoglucuronide (SoMG) 111,112,113,114,115,121 Sophoraflavoside 96 Sophorodiol glucoroides 119 Sophoroflavoside III 96 Sophoroflavoside IV 96 Sopongiacidin B 798,799 Sorangium cellulosum 779 Sorbitol dehydrogenase 472 Sorgassum tortile 839 Souliea 180 Sowdanones 705 Soyabean saponins 222,223 hypocholesterdemic effects 222 Soyapogenin III 95 Soyapogenin IV 95 Soyapogenin V 95 Soyasapogenol A glycoside 103 Soyasapogenol B 94,95,102-104,111, 112,115,118-120,184-186,203 Soyasapogenol B glucuronides 114,119 Soyasapogenol B monoglucuronide (SBMG) 102,121 Soyasapogenol B OG 99 Soyasapogenol E glucuronides 114 Soyasapegenol E 97,115,120 Soyasaponin 90 Soyasaponin A3 95,104

Soyasaponin B 115,116 Soyasaponin 1 95,99,102,103,112,114, 115-118,120,203 Soyasaponin I and II 222,223 Soyasaponin II 103,115,116,119 Soyasaponin III 103,110,112,114,116-118 Soyasaponin IV 103,203 Soyasaponin V 104 Soyasaponins I-IV 101 Soyasaponin Me ester II 203 Soyasaponin Me ester III 203 Sphigolipid receptors 534 Sphigosine-1-phosphate 535 Sphigosyl-phosporylcholine 535 Sphingolipid receptor Ca^"^ channels 535 Sphingosine derivative 721 Sphizomeline 333 Spingosine kinase 269 Spiphonodicyton coralliphagem 694 Spirastrella abata 722 Spirastrella sp 719 Spirastrella spinispirulifera 759 Spiro (4,5) decane skeleton 851 Spiro isoxazole derviatives 757 Spirodysin 692 Spiroiridal triterpenoid 28-deacetylbelamcandal 543 Spirostane 55 Spirulina platensis 725 Spodoptera exigea 792 Spongia mycofijiensis 896 Spongia sp 759 Spongia official is 688 Spongiacidin A 798,799 Spongiacidins A-D 711 Spongiidae 688 Spongistatine 1 and 9 759,760 Spongistatins 4 and 5 759 Spongistatins 757 Spongosorites sp 707,799 Spontaneous metastasis 440 Stachytarpheta cayennenis 611,613 Staphylcooccus aureus 253,257,259,260 271, 765,770,776-778,789,791,821

1028

Staphylococcus boUyosiim 305 Staphylococcus epidermidis 116 Staphylococcus haemolyticus 116 Staphylococcus hominis 258 Stearic acid 333 Stellaria media 781 Stelleta 5/70/7ge species 700 Stelletamine 893,895 Stellettanide A and B 705 Stellettazoles B and C 706 Stemphylium botryosum 307 StemphyUum epidermidis 258,259 Stemphylium solani 302,303,307 Stemphylium sonnei 260,305 Stephania tetrandra 476 Steroid hormones 515 Steroidal glycoalkaloid 293,295,297,302 303 Stevia selriana 251 Stigmastane 49 Stigmasterol 61 Stilbene phytoalexin 662 Stilbene synthase 502,655 Stimulus-response mechanisms 515 Stratospheric ozone concantractions 663 Streptococcus durans 259 Streptococcus faecHis 259,260 Streptococcus fecal is var liquifaciens 923 Streptococcusfecium 925 Streptococcus group 925 Streptococcus pneumoniae 780 Streptococcus pyogenes 452,776 Streptococcus sp 257 Streptokinase/streptodornase 271 Streptomyces aldus 781 Streptomyces erythraeus 780 Streptomyces lavendulae 825 Streptomyces sp 717,781,828 Stroglycentrotus purpuratus 813 Strongylocentrotus nudus 788 Strongylophora 690 Strphanthus grains 537 Stylissa carteri 712,791 Styloguanidines 784 Stylotella agminata 110

Stylotella aiirantium 113,783,784 StylotellaneB 783 Stypopodium 691 Stypopodium zondils 691 St)>popodiimi-flabelliforme 690 Styracaster caroli 872 Sitberea creba 719 Suberin 655,658,666 Suberosenone 695 Subprosidell 95 Subprosidelll 97 SubprosidelV 96 SubprosideV 95,104 SubprosideVl 95 Subproside VII 95 Subtilase (SBT) 388,390,391,392,402 Subtilisin 388 Subtilisin/kexin-isozymel (SKI 1) 390 Subtilisin-like serine protease 388 Succinyl betulinic acid 61 Sulcatol 143,144,145,163 5'-0-Sulfanoyl-2-chlorodensine 781 Sulfated pentapeptide 376,377 Sulfer-containing furanosesquiterpenes 814 Sulfide pyridoacridines 819 Sulfide isoquinolines 824 Sulfide pyrroloquinolines 822 Sulfide pyridoacridines 819 Sulfobus soifataricus 720 Sulfonic acid 900 Sulfonoceramides 868 Sulfonoglycolipids 868 Sulfonolipids 868 Sulfoquinovosyl diacylglycerols (SQDG) 868 Sulforhadamine B (SRB) method 267 6-Sulfo-a-D-quinovopyranosyl-(l-7 3)l',2-diacylclycerol 868 Sulfur-containing diketopiperazine cyclo(L-Pro-thiopro) 828 Sulphonylurea drugs 534 Superoxide dismutase 931 Sweroside 471,476 serum ALT activity

1029

Swertia sp 471 Swertiajaponica 476 Swietenia mahagoni 943 Swinholide 730,731 Symbiodinium sp 722 Symploca hydnoides 887 Symplostatin 1 886,887 Symplostatin 2 886,887 Synaptic dopamine transporters 539 Synaptic glycine transporters 539 Synaptic serotonin transporters 539 Synoicum prunum 723 Syringyl subunits 653 Systematic wound reponse proteins 370 in potato plants 370 Systemic acquired resistance (SAR) 40,394 Systemic wound response protein 402 Systemin 85,371,374,375,377,380 derivative 389 Systemin proteolytic cleavage 376 Systemin (II) 370 Systemin maturation 376 Systemin oligopeptide 371 Systemin polypeptide 371 Systemin signal 374 Systemin synthesis 374 Systemin translocation 375 System in-triggered alkalinization 375 Systemin-triggered changes 374 Tagetone 143,144 Tangutorine 20,21,33 Tanichthys alobonubes 794 Taonia atomaria 249 Taraxastanes 59,64 faradiol 61 heliantriole 61 -Taraxastenes 57 \|/-Taraxasterol 57,58,59,64 Taraxerane 59 Taraxastane 57 Tathy lyncuriiim 718 Tatraclinis articidata 246

Taurine 536,537,872,873 Tauroacidin A 798,713,870 Tauroacidin B 798,713,870 Tauropine dehydrogenases 718 Tauropinnaic acid 871 Taurospongin A 721,871 Tawicyclamide A 883 Tawicyclamide B 883 Taxaceae 244 Taxodiaceae 249 Taxol 244,245 Taxotene 245 Taxiis baccata 245 Taxtts brevifolia I^A^IAS T-cell activation 463 T-cell subsets antitumor-effect 442 T-cell mediated cytotoxicity 466 T-cell mediated liver injury 463 Tea tannins 44 Teasterone 415,421,422 Teasterone oxidative reductive epimerization 422 Tebuquine 344 Tedania ignis 814,828,896 Testosterone 419,516 Teleocidin B 59,65 Terpene isothiocyanates 847 a-Tei-pincol 135,140,142,141,154 Terrestrial haloperoxidase 758 Tert-butyl hydrogeroxide 171 3-Tert-butyl-4-hydroxyanisole (BHA) 944,948 7,8,3',4'-Tetrahydroxyflavone 546 Tetaracyclic polyol esters 242 Tetrachloradibenzofuran 767 Tetrachlorodibenzodioxin 767 12-(9-Tetradecanoyl phorbol-13-acetate 44,222,272,542,948 Tetradecanoylphorbolacetate TPA response elements (TREs) 524 12-(9-Tetradeceanoy Iphorbol-13-acetate(TPA-1) induced edema inhibition 46 Tetrahedroswertianolin (THS) 477 Tetrahydroalstonine 30,31

1030

Tetrahydrobellidifolin 467 A^-Tetrahydrocannabinol 533,532 from marijuana 532 Tetrahydroharmine, (+) and (-) 16 1,3,4,5-Tetrahydropyrdo [4,3,2,de] quinoline 822 Tetrahydroisoquinoline 826,828 (-)- Tetrahydroharmine 18 Tetrahydroswertianolin (THS) 467 hepatoprotective activity 467 Tetrahydroxanthone 465 3A6a,16A25-Tetrahydroxy-20(7?),24(S)epoxy-9,19-cycloalanostane 179 3j3,6a, 16/J,24a-Tetrahydroxy-20,25epoxy 9)3,19-cyclolanostane 184 la, 3 A 16A27-Tetrahydroxy-9i8,19cyclolanost-24£-ene 185 cycloanthgenin 184 la, 7j3, 24a,25-Tetrahydroxy-9j3,19cyclolanostane 184 12-(9-Tetradecanoyl phorbol-13-acetate 271 7,8,3',4'-Tetrahydroxyflavone 541 Tetrahydro-A-carboHnes 782,815 3A 6a, 16/J,24a-Tetrahydroxy-20,25epoxy-9/J,19-cycloIanostane 184 Tetralysine 336 Tetramer russuphelol 765 Tetramic acid glucosides 763 Tetrandrine 472,476 Tetrane isothiocyanates 854 Tetranoic acid 695 Tetrapeptide derivative 376,377 Tetrasaccharide 295 1,2,4,5-Tetrathianes 842,843 Thalassiosira weissflogii 731 Thalassoma bifasciatum 7S7 Thalepogane skeleton 688 Thalictrum 180 Thapsia sp 536 Thapsigargin 536,543 thebaine 533 Theobromine 539

Theonella sp 714,715,771,772,775,878, 884,900 Theonella s^ 892 Theonella swinchoei 115,724,728,731, 848 Theonellamide 771,772 Theonellin isothiocynanate 848 Theonezolides A-C 984 Theonhella 11 \ Theophyline 488,539 Thermitage 388 Thermococcus tadyiricus 842 Theronine deaminase 370 Thiazole alkaloid 711 Thiazoline 881 Thiocoraline 842 {lR*,2R*,5R*,6S*,7S*)-2-Thiocyanatopupukeanane 869 (15*,45'*,65*,7/?*)-4-Thiocyanato-9cadinene 858 Thiocyanotoneopupukeanane 858 2-Thiocyanatoneopupukeanane 859 (-)-4-Thiocyanatonepupukeanane 858 Thiocyantoneopupukeanane 858 Thiodepsipeptide thiocoraline 841 Thiofurodysinin 814 Thiofurodysinin acetate 814,815 Thiomarlnol A 840 Thiomarinol C 840 Thiomarinol D 840 Thiomarinol F 840 Thiomarinol G 840 Thiomarinols 838,840 Thiomycololides A and B 831 5-Thiothistidine derivative 813 2-Thiothistidines 813 Thorecta choanoides 694 Thorectopsamwa xana 844 Threonine dephosphorylation 526 Threshold excitory level 491 Throboxane A2 600 Thromboxane B2 (TXB2) 274,275,600 Thromboxane synthase inhibitor 593 Thromolysin 438,439

1031

THS effect on hepatocyte apoptosis 467 THS inhibited hepatocyte apoptosis 468 Thymelaceae 242,243,246 ^H-Thymidine method 266 Thyroid harmones 515 triidothyronine 515 tetraiodothyronine 515 thyroxine 515 Tigliane diterpenoids 542 Tigliane diterpenoids esters 543 4-deoxyphorbol 543 12-deoxyphorbol 543 4,20-dideoxy-5-hydroxyphorbol 543 Tigliane skeleton 242 Time-off-flight (TOP) mass spectrometer 434 Tirucallane 50 Tistularin 720 TMB (3,4,5,-trimethoxybenzoy) 4 cytotoxic action of 477 TNF-a 461,462-465,469,472,474 treated macrophages 473 dependent liver injury 470 oncolytic effects 460 inhibition by THS 468 TNF-a cytotoxicity 475,477 TNF-a dependent liver injury 472,473, 476,477 TNF-a induced lethality 474 TNF-a production 468,470,471-473,475 suppression of 472 inhibition of 475 TNF-a receptor (TNF-R) 477 TNF-a-depdendent inflammatory liver injury 460,461,465,466 TNF-a-depndendent models 476 TNF-a-gene expression 470 TNF-a-induced hepatocyte apoptosis 471 TNF-a-induced hepatotoxicity 469,471 TNF-a-mediated cytotoxicity 466 TNF-a-production 469,470,477 TNF-a-sensitive L929 cells 467 Tobacco protoplasts 380 a-Tocopherol 236,792,930,936,944,946

Tolypathrix nodosa 691 Tomatidine 293 detoxification 293 Tomatidine 295,296,297,300,301,306, 316-318 /J-glucosyl hydrolases 308 enzymatic detoxification 304 from Botrytis cinerea 314 mechanism of action 306 of Fosaniim oxysporum 314 of Sideritis lycopersici 308,314 Tomatinase degrading enzymes 321 Tomatinase gene 313 Tomatinase-encoding genes 293 Tomatinase-induced genes 322 Tomatinase-producing transformants 308 Tomatinases 306 Tomatinase-encoding genes 295 /}2-Tomatinase 306 Tomatine (lycopericin) 532 deglycosilation of Tomatine 295,296,294,297,298,299, 300,301,302,306,311,312,315-322 insecticidal effects 294 glycoalkaloid 304 deglycosylation 295 toxic action 299 detoxification 296 deglycosylation of 317 Tomatine subproducts 317 Tomatine -detoxyfying enzymes 293 jSl-Tomatine 295,314 j82-Tomatine 317 Tomatine-detoxifying enzymes 295,306 a-Tomatine 293,296,297,306 steroidal glycoalkaloid 294 ^3]-Tomatine 295,317 j32-Tomatine 295,301,306 5-Tomatine 296 Y-Tomatine 296,297 Tomato (pro) systemin 371 Tomatosaponin metabolism 293 by phytopathogen fungi

1032

Tomentoside 1 199 Tomentoside 11 199 Topoisomerase (TOPO) Il-mediated decateration of kinetoplast DNA 820 Topoisomerase I 721 Topoisomerase 11 708,709,862 Topoisomerase Il-sensitive (CHO) cell lineXrs-6 824 Toposiomerase I and 11 708 Topsenia sp 703,704 Topsentinols A-J 703 Torpane alkaloid atropine 530 cocaine 537 ecgonine 537 hyoscamine 530 hyoscine (scopolamine) 530 Tondopsis glabrata 258 Totarane 241 Touroacidins A and B 797 Toxic free heme 327 Toxic xenobiotic 935 TPA (12-O-tetradecanoylphorbol-13acetate) 43 TPA induced edema (TPA) 45 TPA induced ornithine decarboxylase inhibition (ODC) 45,46 TPA induced skin tumors 46 TPA stimulated -^^Pi incorporation in HeLa cells 60 TPA-induced assay 58,61 TPA-induced ear edema 57,58 TPA-induced edema inhibition (CRO) 45 TPA-induced inflammatory edema 56 TPA-induced inflammation 47,48,63 TPA-induced ODC accumulation 60 TPA-mediated DNA 937 TPA-stimulated ^^Pi incorporation in HeLa cell inhibition (HeLa) 46 TPA-stimulated Pi 45 TPA-type tumor promotors 61 Trachycladine 775 Trachycladus laevispirulifer 115 Trachylobhim verrucosum 252

Trachyopsane 848,851 Trachyopsis halichondraides 704 Tradescantia nndtiflora 382 7Va/75decalin-tetrahydropyranylkalihinols 855 rra/75,/m^5-ceratospongamid 902,903 Tram-1,2-dihydroxy limonene 150 Transacylase 501 Transcriptase 722 Transcriptional factor 470 Transcriptional inhibitors 461 Transcylase 502 Transducin 521 7>'aA75-epoxysqualene 181 G1 o-Transgenic plant 661 ZVara-pinocarveol 154 D-(+)- Tm^^-sobrerol 152 7>aA75"-squalene oxide 182 Tras Glogi area 114 Traxacum officinale 391 Trelox 933 2-[3a,7a, 11 a-Trihydroxy-24-oxo-5/Jcholan-24-yl] amino ethane-sulfonate 872 3,3',4'-Trihydroxyflavone 544 Triacetic acid lactone 498 Triacetyl-l,2,-dipalmitoyl-3-0-(6'-sulfo-22',3',4'-D-quinovopyranosyl) glycerol 868 Tribrominated bisindole alkaloid 765 l,8-Tribromo-3,4,7-trichloro-3,7dimethyl-l,5-octadine 783 Tricetin 541,544 methylation 544 Trichlorinated orcinol 786 Trichloroleucine metabolite 801 Trichoderma sp 155 Trichoderma longibrachiatum 725 Trichoderma reesei 308 Trichophyton mentagrophytes 780 Trichosporon sp 150 Tricyclic xanthone 349 Tridecagalacturonides 486 Tridecapeptide polydiscamide A 867 Tridentata marginata 896

1033

Tridentatol A,B and C 895,896 Trididemnum solidiim 718 Trididemmm sp 819,820 Trifloiiim sp 667 Triflouroacetic acid (TFA) 6,434 Trifluoperazine 487 Trifluralin 669 Triglyceride lipase 523 Trigonoside 1 192 ,224 Trigonoside II 194,224 Trigonoside HI 194,224 3A 6a, 16/J -Trihydroxy-9,19-cyclolanost-24-ene 185 3/3, 6a, 16j3-Trihydroxy-9Al9-cyclolanost-24-oxo,25-ene 185 Trihydroxylean-12-ene saponins 179,191 3 j3,16/J,22 a-Trihydroxytaraxastene 62 3,7,11 -Trimethy 1-1,6,10-dodecatrien3-ol 161 (2)-3,7-11 -Trimethy 1-1,6-dodecadiene3,10,1 l,triol 164 3,7,11 -Trimethy 1-2,6,10-dodecatrien-1 -o 1 165 (2£,6£)-3,7,11 -Trimethyl-2,6-dodecadien1,10,11-triol 166 Trioloeolglycerol 333 3,4,5-Tri-O-Caffeoylquinic acid 920 2,3,4,Tri-0-methylgalactose 438 3',4',5'-Tri-(9-methyltricetin 541 D-Tryptophan 885 Tristamine 819,820 Triterpene alcohol 43,44 Triterpenes 478 bupleuroside III, IV and XIII 476 ginsenoside Rs and Rgl 476 glycyrrhizin 476 scorzoneroside A,B,C, 476 Triterpenoids and sterols antitumor and antiinflammatory activities 43 Trithiane 837 1,2,4-Trithiolanes 840,843 Trojanoside A 193 Trojanoside B 194 Trojanoside C 198

Trojanoside D 198 Trojanoside E 198 Trojanoside F 198 Tropanyl compounds 29 Trophozoites 332,333 acetonitrile extract of 333 Trophozoite stage 329 Trophozoite extract 334,342,354 Tropono-1,2-dihydro-3,6-phenathroline 820 Trunkamide A 883 Trypsin 483 Tryptophan 823,885,928 Tsitsikammamine A and B 709 Tubercidin 712,713 Tumor metastasis 440,454 Tumor necrosis factor a (TNF-a) 459, 460,461,477,598 Tumor-promoting phorbol ester 190 Tunicate Lissoclinium patella 879,880 Turpentine 151 TXB2 production 593 Type-lII secretion system 394,398 Typhasterol 415,422,423 oxidative reductive epimerization of 422 Tyrosine ammonia lyase (TAL) 669 Tyrosine dephosphorylation 526 Tyrosine kinase inhibitors 720,691 Tyrosine kinase-mediated signalling 545 Tyrosine kinases 519,518,703 JAKl andJAK2 519 Tyrosine kinases 597 Tyrosine phosphatases (PTPases) 519 Tyrosine phosphorylated STAT dimer (P-STAT) 522,548 Tyrosine phosphorylation 519 U2 66 myeloma cell 273 UDP-galactosamine (UDP-GalN) 461 U-endo-^l,4galactanase 436 U-exo-)8-galactosidase 436 Ulicyclamide 880,882 Ulithiacyclamide 879

1034

Ulithiacyclamide B 879 Ulithiacyclamide E 879 Ulithiacyclamide G 879 Viva sp 725 Umbelliferae 471 Umbraculum mediaterraneum 831 Uncarine C,D,E and F 25 a,J^Unsaturated butenolide 276 A^-Unsaturated sterols schottenol 56 spinasterol 56 Urethanase 719 Uric acid 930 Uronic acids 435,486 Uronide elicitors 488 Uronide complex esterification of 486 Ursane 53,57,59 Ursolic acid 59,60,61,66,543 Vstilago maydis 389 Valencene 125,128,170,171 2-Vanilloyl imidazole 837 Vanadium haloperoxidase 758 Vancomycin 791 Vanilla planifolia 126 Vanillic acid 90,671,924,925 /?-Vanilliccinnamic acid 923 Vanillin 126 Vanilloylglycine 90,923 VaracinA 834 VaracinB 834,835 VaracinC 835 Varacin-lissoclinotoxin 833 Varamines 893 Variabilin 717,718 Varamines 821 Vasorelaxant chalcone 540 Veramines 821 Verapamil Ca^^^-channel blocker 487 Verapamil 673 Ca^"^-channel blocker 487 Veratridine 534

Verbascoside 468 Verbenaceae 249 Verbenol 152 C/.s-D-Verbenol 152 Verbenone 152,153 Verongamine 721 Verongida gigantea 721 Veronica persica 781 Verticillium albo-artrum 299,307,302, 311 Verticillium dahliae 305,307,311 Vescictdar stomatitis 779 Vesicular glutamate transporters 538 Vesicular monoamine transporters 538 Vexibinol 612 Viagra (sildenafil) 541 Vibrio angucillarum 877 Vibrio cholerae 521 Vibrio valnificiis 718 Viburnum suspensum 251,257,259 Viciafaba 93 Vicia sativa 93 Vigna angidaris 93 Vigna unguicidata 93 Vinblastine 269 4.Vinylcatechol 925,926 Vinylfurans 694 Vinylogous urethane 10,11,33,34 Viral glycoproteins 388 Virenamide A-E 888 Virenamide 888 Vitamin C,E,K,A,D 236 Vitexin 594,595 Vinylcatechol 927 Voltage-gated Ca^''" channels 517 Voltage-gated ion channels 520 Voltage-gated K"^ channel 531 Voltage-gated Na"*" channels 533 Volutamides 757 Volutamides A-E 786 Waitzia acuminata 251 Walsh-Krebs inhibitors protein 523 Warangolone 541 Wayakin 707

1035

Welwiltindolinone alkaloids 802 Welwitindolinoric A isonitrile 780 Winn assay 432 Wistaria brachybotrys 222 Wistariasapogenol A 97 Wistariasapon n A2 97 Wistariasapon n A3 97 Wistariasapon n B 2 95 Wistariasapon n B 3 95 Wistariasapon n C 222 Wistariasapon n D 97 Wistariasapon n D 116,118 Wistariasapon! n Y C i 96,104 Wistariasapon n YC2 96 Wistariasapon ns 222 Wisterai brachybotrys 93

X-ray absorbance spectroscopy (XAS) 359 X-ray absorption fine spectroscopy (EXAFS) 338 Xylanases 312 9-[5'deoxy-5'(methylthio)-j3-D-Xylofurano syl)adenine 829 6-0-j^D-Xylopiranoside 224 j3-Xylosidase 210

Xanthium spinosum 781 Xanthocephalum linearifolium 251 Xanthomonas oryzae 395,398 Xanthomonas compestris 399 pv vesicatoria 399 Xanthone 350,351 Xanthone a-mangostin 531,536 inhibitor of Ca^"*" ATPase 536 Xanthone hypothesis 351 Xanthone-a-mangostin 536 inhibitor of Ca2"^-ATPase 536 Xenia 690 Xenicane 690 Xenopus 294 Xestaspongins 732 Xestomycin 826 Xestoquinones 861 Xestoquinolide B 862 Xestospongia 694 Xestospongia genus 861 Xestospongia cf carbonaria 861 Xestospongia sp 703, 709, 710,769,859, 898 Xestospongia ashmorica 710 Xanathomonas adaptation 399

Zea mays 671 Zingiberaceae 249,266 Zinnia elegans 317 Zn(n) protoporpyhyrin (IX) 335 Zoanthellatoxin B 722 Zoanthid 711 Zoanthius sp 711 Zymogen activation 391 Zymosan-activated macrophages 274 Zyzzafuliginosa 708,760,770 Zyzza massalis 760,898 Zyzzya cf.marsailis 790,824

Yarrowinina lipolytica 150 Yeast kex 2 protease (Kexin) 387,388, 389 Yohimbine 20,29 Yohimibine-type alkaloids 3 Yunganogenin C 96

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