Advances in Phytomedicine Series, Volume Three Editors: Mahendra Rai and Marı´a Cecilia Carpinella
Naturally Occurring Bioactive Compounds
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Advances in Phytomedicine 3
Naturally Occurring Bioactive Compounds Mahendra Rai Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India
Marı´a Cecilia Carpinella Laboratoria Quı´mica Fina y Productos Naturales, Facultad de Ciencias Quı´micas, Universidad Cato´lica de Co´rdoba, Co´rdoba, Argentina
Amsterdam – Boston – Heidelberg – London – New York – Oxford – Paris San Diego – San Francisco – Singapore – Sydney – Tokyo iii
Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2006 Copyright r 2006 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
[email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN-13: 978-0-444-52241-2 ISBN-10: 0-444-52241-7 ISSN: 1572-557X (series)
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Contents Preface to the Series
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Preface
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1. Natural compounds as antioxidant and molting inhibitors can play a role as a model for search of new botanical pesticides Carlos L. Ce´spedes A., J. Guillermo Avila, J. Camilo Marin, Mariana Domı´nguez L., Patricio Torres and Eduardo Aranda
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2. Pesticides based on plant essential oils: from traditional practice to commercialization Murray B. Isman and Cristina M. Machial
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3. Natural substrates and inhibitors of multidrug resistant pumps (MDRs) redefine the plant antimicrobials George P. Tegos
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4. New concept to search for alternate insect control agents from plants Isao Kubo
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5. Role of Melia azedarach L. (Meliaceae) for the control of insects and acari: present status and future prospects Marı´a C. Carpinella, Marı´a T. Defago´, Graciela Valladares and Sara M. Palacios
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6. Bioactivity of fabaceous plants against food-borne and plant pathogens: potentials and limitations Deepak Acharya, Aniket Gade and Mahendra Rai
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7. Screening of plants against fungi affecting crops and stored foods Olı´via C. Matos and Caˆndido P. Ricardo
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8. Opportunities and potentials of botanical extracts and products for management of insect pests in cruciferous vegetables Tong-Xian Liu, Han-Hong Xu and Wan-Chun Luo
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9. The potential for using neem (Azadirachta indica A. Juss) extracts for pine weevil management in temperate forestry Jonathan R.M. Thacker, Wendy J. Bryan, Robin H.C. Strang and Stuart Heritage
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10. Plant allelochemicals in thrips control strategies Elisabeth H. Koschier
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11. Importance of plant secondary metabolites for protection against insects and microbial infections Michael Wink
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12. Naturally occurring house dust mites control agents: development and commercialization Young-Joon Ahn, Soon-Il Kim, Hyun-Kyung Kim and Jun-Hyung Tak
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13. The search for plant-derived compounds with antifeedant activity Monique S.J. Simmonds
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14. An overview of the antimicrobial properties of Mexican medicinal plants Diana Jasso de Rodrı´guez, Jose´ Luis Angulo-Sa´nchez and Francisco Daniel Herna´ndez-Castillo
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15. Promissory botanical repellents/deterrents for managing two key tropical insect pests, the whitefly Bemisia tabaci and the mahogany shootborer Hypsipyla grandella Luko Hilje and Gerardo A. Mora
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16. Naturally occurring anti-insect proteins: current status and future aspects Tzi Bun Ng 405 17. Antifungal natural products: assays and applications Doris Engelmeier and Franz Hadacek
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Contributors
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Subject Index
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Preface to the Series The systematic study of herbal medicinal products and the investigation of the biologically active principles of phytomedicines, including their clinical applications, standardization, quality control, mode of action and potential drug interactions have emerged as one of the most exciting developments in modern therapeutics and medicine. Studies in phytomedicine have moved from purely descriptive analytical studies to conceptual inquiries on the pharmacodynamic advantages and limitations of plant medicines for the treatment of moderate or moderately severe diseases and prevention. Healthcare practitioners and medical scientists have come to accept herbal medicinal products as drugs that are different from the pharmacologically active molecules that they may contain. Several comparative clinical studies have been published to show that these plant medicines could have full therapeutic equivalence with chemotherapeutic agents, while retaining the simultaneous advantage of being devoid of serious adverse effects. Developments in molecular biology and information technology have now made it possible for us to begin to understand the mechanism of action of many herbal drugs and the associated phytomedicines, which differ in many respects from that of synthetic drugs or single chemical entities. Herbal medicinal products are now generally available in both industrialized countries and traditional societies. With the current lack of standardization and regulation of herbal products, it is important to develop common criteria for judging safety and efficacy of phytotherapeutic agents. This ‘new’ science demands different approaches to the classical methods of drug analysis, dosage formulations, manufacturing and claims substantiation. The therapeutic response observed with most herbs and phytomedicines are often not fully explainable using the currently available methods. Their activity usually characterized as polyvalent and interpreted as an aggregate or additive outcome of several constituents in the plant medicines are subjects of intense pharmacological studies. In most cases, a rationale does not even exist for the observed pharmacodynamic effects of very low doses of phytomedicines after prolonged or long-term application. The public press is replete with lay information and claims about the use of herbal remedies, however, there is scarcity of scientifically accurate reviews and guides on the efficacy and safety of plant medicines. The time therefore seemed ripe to broaden the communication on the use and benefits of phytomedicines as safe and useful natural health care products aimed at the health professionals and scientists. It is also important to provide a broader dissemination of the extant scientific literature on phytomedicines, to enable the conventional medical community to fully appreciate the fact that plant extracts specifically, and natural products generally, offer valuable and needed benefits in the treatment and prevention of diseases, especially for conditions where there is no effective or generally acceptable drugs. Although there are many papers published yearly on the use and analysis of plants as sources of biologically active molecules and some published materials on the use of plants as medicinal substances, volumes specifically addressing the needs of vii
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scientists and clinicians in the use of herbal products and standardized extracts as medicinal agents are far-in-between. We considered it therefore appropriate to fill this gap by producing an entirely new multi-volume series on the sourcing, selection, standardization, safety and clinical application of herbal medicinal products. As the name of the series implies, emphasis will be on those herbal medicinal products that are well characterized, standardized and substantiated as phytomedicines. The series will also provide timely reviews on the industrial production, regulatory and policy issues related to the use of phytomedicines. Although the literature in this field is evolving so rapidly that books on the subject become obsolete soon after they leave the press, the volumes in this series will aim at capturing the fundamental framework of each topic while remaining thoroughly up-to-date and comprehensive in scope. The aim of this series is to present to the scientists and clinicians the state of current knowledge in various fields of phytomedicine research, development and use. The approach is to provide the historical background to each topic, discuss methodological issues and illustrate current trends with case studies and critical examples, and when ever possible, the authors will indicate those plant medicines that are available for immediate use in clinical settings. The series is not intended to serve or replace the many excellent journals in this field but it will rather attempt to distill information from primary references and introduce elements of medicinal plants research and development that are in transition from speculative knowledge to standard practice. Advances in Phytomedicine is also not meant to be a textbook of the various topics covered in the series. It will, however, provide a guide to specialized articles and books on the topics that are relevant to scientific research and development of plant medicines, as well as information on the regulatory issues, clinical trials and application of phytomedicines for healthcare. Advances in Phytomedicine will therefore serve as a platform for reviewing recent developments in the use of herbal medicinal products. The coverage will include reviews of studies and use of all plant medicines, phytotherapeutic agents, nutraceuticals, plant cosmetics and therapeutically important molecules derived from these plant medicines. The first volume in this series has been devoted to exploring the ethnomedical approaches to drug discovery. It is indeed a very important starting point in addressing the relationship between plants and human health. Subsequent volumes will deal with other aspects of the use of herbal medicinal products. Selection of subjects will be through consultations with experts in the various fields of interest. We shall be guided by the principles of the so-called 6S, that is herb selection, sourcing, structure, standardization, safety and substantiation. Acknowledged experts and authorities in the various aspects of phytomedicine will be invited to assemble and edit specific volumes in the series. The series is the outcome of extensive consultations among several biomedical scientists and clinicians who participated in the workshops and conferences organized by the Bioresources Development and Conservation Program (BDCP) on related subjects. I am immensely grateful to these colleagues for their support in developing the original concept. Many thanks go also to Ms. Kim Briggs and Ms. Joke Zwetsloot of Elsevier Science for their suggestions and help in producing this series. I am indebted to my colleagues at BDCP and the International Centre for Ethnomedicine and Drug Development for their contribution; and to my wife, Kate for her love and support. I acknowledge the International Cooperative Biodiversity
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Group (ICBG) of the Fogarty International Centre, United States National Institutes of Health for providing financial support to my research group at the Walter Reed Army Institute of Research and BDCP. Maurice M. Iwu M.Pharm., Ph.D. Se´ries Editor
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Preface There has been a long history of success in discovering drugs from natural sources, particularly in tropical countries like Japan, China, India and Nepal. Unfortunately, natural products have fallen out of favour in current high-throughput screening. However, the diversity of three-dimensional shapes of natural molecules still surpasses that from synthetic compounds, and this ensures that natural products will continue to be important for drug discovery. Owing to the excessive use of the chemicals, the environment has been polluted. Consequently, there is a pressing need of search for new and natural chemicals for the sustainability of the environment. The plant-based drugs provide the natural and harmless system for the cure of various diseases. Majority of the drugs are prepared from the plants or natural products. There are a number of new and emerging diseases and new spectrum of microbes which has warranted the scientists to go for new natural drugs. Since time immemorial, herbal drugs have been in use all over the world in general and tropical countries like Japan, China and India, also Mexico and South Africa, in particular. Natural dyes and colours are health-friendly and responsible for maintaining the sanctity of the environment. The medicinal use of plant parts and extracts has been traditionally practiced since ancient times. However, a scientific approach to understand the relation between active chemicals and cure of diseases is recent. The use of plants in medicine has evolved because, from the biochemical point of view, they produce different chemical compounds against bacteria and fungi, the number of chemical structures is large and only a few are well known. Secondary metabolites obtained from plants have diverse functions into the plant such as defensive and protective process. The phytochemical studies are biodirected with an aim to find botanical origin biopesticides. Diterpenes, limonoids, triterpenes, sesquiterpene lactones, coumarins and flavonoids from Agavaceae, Asteraceae, Cactaceae, Celastraceae, Meliaceae and Zygophyllaceae families and also some chemical derivatives from them are important. These compounds possess antioxidant, antifungal, insect growth regulator (IGR) or insecticidal activities. The acetylated and carbonyl a-b-unsaturated derivatives showed mainly a low activity, while those that contain hydroxyl, carbonyl and oxy groups are more active as well as antifungal, IGR or insecticides. The pests and diseases of plants as well as animals have developed resistance as a result of which the existing antibiotics do not give the expected results. Therefore, there is a great need to develop the natural product-based drugs. The book will not only describe the effects of natural extracts and/or their isolated compounds, but also an update of its use and commercialisation. In some cases, the results related to the effects are new or they are an update of known data. The book will also help to make the use of effective and environmentally safe botanical pesticides, a new weapon to control these negative pests and diseases, which means a considerable alternative to the dangerous and excessive use of synxi
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thetic pesticides. Although there are several books on natural drugs, the present book covers multiple aspects of natural products, which is the need of the hour. We are indebted to Professor R.C. Rajak, director of Basic Sciences, Bundelkhand University, Jhansi, Uttar Pradesh; and to Professor S.C. Agrawal, head, Department of Applied Microbiology and Biotechnology, Dr. H.S. Gaur University, Sagar, Madhya Pradesh, who graciously offered suggestions for this book. We thank all the contributors for submitting their valuable manuscripts. Thanks are also due to my colleagues – Drs. Anita Patil, N.J. Chikhale, P.A. Wadegaonkar, P.V. Thakare, Aniket Gade and S.D. Kove. MKR thanks Deepak Acharya and Ravindra Ade, who checked manuscripts and helped in type-setting. Our special thanks to Drs. Susanna Zacchino, Donatella Mares, Michael Wink, George Tegos, N.P. Manandhar and Shubhangi Ingole, who went through the chapters and offered helpful suggestions. We are grateful to Dr. Kamal Singh, Vice Chancellor, SGB Amravati University, Amravati, for inspiration. Finally, and most importantly, MKR thanks Shivangi, Shivani and Aditya for their unconditional love, support and patience during the editing process. Mahendra Rai, Maria Cecilia Carpinella
Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.
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CHAPTER 1
Natural compounds as antioxidant and molting inhibitors can play a role as a model for search of new botanical pesticides CARLOS L CE´SPEDES A, J GUILLERMO AVILA, J CAMILO MARIN, MARIANA DOMI´NGUEZ L, PATRICIO TORRES, EDUARDO ARANDA
Introduction Nowadays, pesticides of synthetic origin have been widely used, producing a strong impact on the environment with the emergence of resistant strains of microbes and insects to these types of compounds. New plant protection chemicals are needed for modern pest management due to insect resistance and ecological disorders associated with numerous currently used pesticides (Castillo et al., 1998). One area of investigation that is not so much an innovation as it is a return to an old approach with new technology is the characterization of secondary plant products (Berenbaum, 1989, 2002), and this can help in plant defense through new biotechnological approaches like metabolomic, genomic, and proteomic (Berenbaum, 1995, 2002; Kessler and Baldwin, 2002; Sumner et al., 2003). Most of them have potent effects on insect pests, low mammalian toxicity, lack of neurotoxic activity, low persistence in the environment, and biodegradability (Jacobson, 1989; Singh et al., 1997). Thus, organic molecules of botanical origin may offer a safe source of compounds for pest management, being environmentally friendly, and an excellent alternative to persistent synthetic insecticides (Berenbaum, 1989; Miyazawa et al., 1997; Castillo et al., 1998; Crowley et al., 1998). Plants in general produce a great variety of secondary metabolites that do not have apparent function in physiological or biochemical processes; these compounds (or allelochemicals) are important in mediating interactions between plants and their biotic environment (Berenbaum, 1989, 1991, 1995, 2002; Kessler and Baldwin, 2002). There is a widespread effort to find new pesticides, and currently it is focused on natural compounds such as flavonoids, coumarins, terpenoids, and phenolics from diverse botanical families from arid and semi-arid lands of Mexico and Americas. Additional experimental work has been carried out with natural products, which are potential models for defensive substances against insect and fungal predators (Kubo et al., 1994, 1995; Crombie, 1999; Kubo et al., 2000) and as enzyme inhibitors,
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tyrosinase or acetylcholinesterase (AChE) for instance (Kubo, 1997; Keane and Ryan, 1999; Ortego et al., 1999; Kubo et al., 2000; Ce´spedes et al., 2001a, 2001b; Kubo et al., 2003a, 2003b). The increasing interest in the possible application of secondary metabolites for pest management has directed the investigation towards search for new sources of biologically active natural products, with new mode, sites, and mechanisms of action, selectivity, and specific action (Jacobson, 1989; Gonza´lezColoma et al., 1997; Singh et al., 1997; Conner et al., 2000; Eisner et al., 2000; Eisner and Meinwald, 1995); these characteristics may enhance their value as commercial pesticides (Gonza´lez et al., 1992; Isman et al., 1995, 1996; Valladares et al., 1997; Gonza´lez and Este´vez-Braun, 1998; Gonza´lez et al., 2000). Recent studies have demonstrated that many plant species produce and accumulate a large variety of secondary metabolites that provide defense against insect predators (Berenbaum, 1989; Guella et al., 1996; Marvier, 1996; Berenbaum, 2002). Among several efforts to find new pesticides, current research is focused on limonoids from the Meliaceae family due to their potent effects on insect pests and their low toxicity to non-target organisms (Koul and Isman, 1992; Kumar and Parmar, 1996; Singh et al., 1997). Some examples are Azadirachta indica (Meliaceae) and Derris elliptica (Fabaceae), which produce very well known insecticides azadirachtin and rotenone, respectively (Gomes et al., 1981; Kraus et al., 1993, 1995). The main characteristics that account for the successful use of these secondary metabolites as natural insecticides are mentioned above, which make them lesser aggressive to the environment than the synthetic ones (Camps, 1988; Berenbaum, 1989; Castillo et al., 1998). Although the members of the family Meliaceae are widely distributed in the world, only Melia, Toona, Cedrela, and Swietenia have been studied (Arnason et al., 1987; Champagne et al., 1992; Kubo, 1992; Arnason et al., 1993; Kraus et al., 1993; Kubo, 1993; Govindachari et al., 1995; Chan and Taylor, 1966; Ce´spedes et al., 2000), and have afforded some limonoids such as azadirachtin, gedunin, toosendanin, cedrelanolide, mexicanolide, odoratol, anthothecol, nomilin, bussein, entandrophragmin, among others. Azadirachtin is the best known example of these limonoids (Champagne et al., 1989; Ramji et al., 1996). This compound and their analogues are potent insect antifeedant and ecdysis inhibitors (Govindachari et al., 1995; Kraus, 1995). However, the structural complexity of this compound precludes its synthesis on a commercial scale (Isman et al., 1996). These facts have led us to search for new simple secondary metabolites with insecticidal activity from other families including plants of Agavaceae, Asteraceae, and Meliaceae, such as Yucca spp., Parthenium spp., Roldana spp., Tagetes spp., and Cedrela spp., respectively, specially from tropical and subtropical lands of Mexico. As mentioned earlier, tyrosinase also known as polyphenol oxidase (PPO) (Meyer, 1987) is a copper containing enzyme widely distributed in microorganisms, animals, and plants. It catalyzes two distinct reactions of melanin synthesis (Robb, 1984), the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to the corresponding o-quinone (diphenolase activity). Tyrosinase is responsible for browning in plants and is considered to be deleterious to the color quality of plant-derived foods and beverages. In addition, sclerotization and molting regulation processes in insects show that tyrosinase is one of the key enzymes in the insect metamorphoses process (Andersen, 1990).
Natural compounds as antioxidant and molting inhibitors
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AChE, which is the enzyme contained in nerve tissues, plays an exceedingly important role in the transmission of a nerve impulse. The free acetylcholine in the inactive form bound to proteins accumulates in the ending of a nerve in vesicles. The consumed acetylcholine is constantly replenished by its synthesis (by the acetylation of choline). All these processes are occurring when an impulse is transmitted through a cholinergic synapse. Thus, the process of synaptic transmission is an involved biochemical cycle of acetylcholine exchange. AChE has a key role in this cycle because the inhibition of activity leads to the accumulation of free acetylcholine in the synaptic cleft, producing the disruption of nerve impulses, then convulsive activity of the muscles can be transformed into paralysis, and other features of selfpoisoning by surplus acetylcholine appear. It occurs when AChE is inhibited by some terpenoids (Ryan and Byrne, 1988; Miyazawa et al., 1997). On other hand, modifications to AChE can confer resistance to insecticides (Fournier et al., 1992). In addition to many chemicals (flavonoids, stilbenoids, phenylpropanoids, and phenolics, among others) that show tyrosinase inhibitory activity, these compounds also show a strong antioxidant (AOX) activity against diverse reagents as model of AOX measurements (i.e., DPPH, ABTS, TROLOX, TRAP, ORAC, etc.). Mainly the activity is due to the presence of diverse moieties of the chemical structure of the molecules, for instance, orcinol or catechol groups, or hydroxyl bonded to an aromatic system (Gallic acid, Gallates in general, resveratrol, quercetin, etc.); in these cases, it is possible to correlate the AOX activity with tyrosinase inhibition and insect growth regulatory (IGR) activity. All these data are important for allelopathic, antifungal, IGR, and insecticidal studies, and are being accepted as direct or indirect measures of different physiological processes affected by the assayed chemicals (Baldwin et al., 2001; Schultz, 2002; Kubo et al., 2003a, 2003b; Torres et al., 2003). Some investigations on sites and mechanism of action of insecticidal or IGR report that different phenolic compounds are enzymatic and metabolic inhibitors (Klocke and Kubo, 1982; Kubo and Klocke, 1986; Kubo et al., 1994, 1995; Hammond and Kubo, 1999; Kubo and Kinst-Hori, 1999a, 1999b; Kubo et al., 2000; Shimizu et al., 2000; Caldero´n et al., 2001; Panzuto et al., 2002; Kubo et al., 2003a, 2003b). In addition, many of these compounds are polyphenolic secondary compounds that are ubiquitous in angiosperms and that have antifeedant effects on phytophagous insects (Feeny, 1968, 1976; Rhoades and Cates, 1976; Swain, 1979; Champagne et al., 1989, 1992; Simmonds, 2003). It has been assumed that phenols bind to proteins, acting as nutritional protein precipitating agents, thus reducing their digestibility (Feeny, 1976; Rhoades, 1979). We have previously demonstrated that diverse secondary metabolites have different sites of action and different molecular targets when they interact with enzymes and metamorphosis processes (Ce´spedes et al., 1999, 2000; Caldero´n et al., 2001; Ce´spedes et al., 2001a, 2001b; Ce´spedes et al., 2002; Torres et al., 2003; Ce´spedes et al., 2004). Our field observations indicate that these botanical species from arid and semiarid lands possess a strong resistance to the insect attack. The aim of this work is to correlate this phenomenon between the phytochemical composition with the inhibitory behavior on growth and development of Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae). The role of these compounds as ‘‘chemical messengers’’ has proven important to our understanding of many ecological problems and has led to the development of ‘‘chemical ecology’’ (Seigler, 1997).
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Aspects such as insecticide and growth regulatory activities, rate of development, pupation time, adult emergence, and deformity were evaluated and compared with those of gallic acid 10, gedunin 19, toosendanin 21 (Chen et al., 1995), anisic acid 38, and Cedrela-MeOH extract known growth inhibitors of S. frugiperda and tyrosinase, respectively (Ce´spedes et al., 2000; Caldero´n et al., 2001; Ce´spedes et al., 2001a, 2001b; Kubo et al., 2003a, 2003b). S. frugiperda is a very common insect pest in North- and South-American crops. These bioassays were used as model systems of insect pests. Our data also indicate that it is possible to correlate some AOX activities (i.e., crocin, 2,2-diphenyl-1-pycrilhydrazyl (DPPH)) with growth and development of fungus and insects; these data are important for allelopathic and chemical ecology studies (Ce´spedes et al., 2002). In addition to gedunin 19, toosendanin 21 (Figure 3), parthenolide 1, santamarin 2, 20 ,40 ,60 -trihidroxy acetophenone 3, rutin 4, and (7)-naringenin 5 were used as natural pattern substances and internal standard, together with THQ 6, BHA 7, quercetin 8, caffeic acid 9, gallic acid 10, and tocopherol 11 (Figure 1) in tests concerned with the reduction of DPPH and other AOX measurements. In the present chapter, we present a review of extracts and bioactive compounds isolated by our group from selected endemic botanical species, and their AOX and biological activities. The chapter includes antifeedant activities shown by assayed compounds, i.e., insecticidal or IGR activities and their activities on some enzymes.
Insecticidal or IGR (insect growth regulator) activity Agavaceae Yucca periculosa F. Baker, known commonly as ‘‘palmitos’’ or ‘‘izote’’ is a tree, endemic to Mexico, that grows in the semi-arid regions of Tehuacan–Cuicatlan Valley, Puebla–Oaxaca States. In the wild, these plants survive under different environmental stress conditions (Casas et al., 2001), with a lifespan of up to 100 years or so. Our field observations indicate that this specie has a strong resistance to insect attack. From methanol extract were isolated 4,40 -dihydroxstilbene 12, resveratrol 13, and 3,30 ,5,50 -tetrahydroxy-4-methoxystilbene 14 (Figure 2). The most active compound was stilbene 14 which showed the highest larval mortality together with MeOH extract of Y. periculosa (Me-Yuc), producing 100% of larval mortality at concentrations greater than 25.0 and 50 ppm, respectively, being noteworthy insecticidal. Thus, compound 14 and the Me-Yuc extract showed relative growth index (RGI) values of 0.25 and 0.45 at 10 and 15 ppm, respectively (Table 1). In addition, this compound was more active than gedunin and the MeOHCedrela extract used as positive control. The LD50 values for 14 and Me-Yuc extract were 5.4 and 7.18 ppm, respectively. In addition, compounds 12, 13, 14, gedunin 19, and the Me-Yuc and Me-Ced extracts inhibited each larval stage and diminished considerably the percentage of larvae that reached pupation; 14 and Me-Yuc showed the highest larval growth inhibitory activity, significant delay in pupation, and reduction of the percentage of emergence (Torres et al., 2003).
Natural compounds as antioxidant and molting inhibitors
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OH
O HO
OH
CH 2
O O
CH 2
H O
O
(1)
OH
O
(2)
(3)
OH OH HO
O OH O
HO
OH
O
O
CH 3 O
O OH
OH
O
OH
OH
OH
OH
O
OH
(5)
(4)
OH OH
O HO
HO
OH
O OH
HO
OH
OH O
OCH 3
(6)
(7)
OH
HO
O
HO
O
(8)
O HO
OH
HO
OH HO
(9)
(10)
CH 3 HO H H3C
O CH 3
CH 3
H
CH3
CH 3 CH 3
CH 3
(11)
Fig. 1. Chemical structures of pattern and standard compounds used for antioxidant and bioassays.
In order to establish correlation between IGR and acute toxicity activities with the AOX properties of these phenolic compounds, crocin and DPPH radical scavenging tests of these stilbenes were carried out. In 14 the presence of a methoxyl group increases the strength of these compounds upon inhibition of DPPH. We suggest that insect growth inhibitory activity of Me-Yuc extract could be caused by a
Naturally occurring bioactive compounds
6 H HO
C
C
OH
H
12 HO H C
C
OH
H HO
13 HO
OH H C
CH3O
C H
HO
14
OH
Fig. 2. Stilbenes from Y. periculosa.
synergistic effect of the stilbene composition. The inhibition of DPPH activity by stilbenes is well known (Burns et al., 2000, 2002; Rimando et al., 2002). Therefore, the plant stilbenes may be considered as efficient IGR and radical scavengers (Stivala et al., 2001; Kim et al., 2002; Rimando et al., 2002). The AOX activity of these compounds was also evaluated spectrophotometrically on the bleaching of the H2Osoluble crocin (Bors et al., 1992). Compounds 12, 13, 14, and Me-Yuc extract were all active, with activities comparable to gallic acid (positive control). Resveratrol 13 and the methoxylated stilbene 14 had more potent insecticidal inhibitory activity. This shows that nature of the substituent at C-3 plays an important role for the insecticidal activity (Torres et al., 2003). Meliaceae Cedrela is a genus belonging to the Meliaceae family. This genus includes several species like C. odorata, C. oaxacensis, C. dugessi, and C. salvadorensis, among others. Particularly we were interested in these species, because it is known that several of these species contain limonoids of gedunin-type (i.e., gedunin, photogedunin, cedrelanolide, mexicanolide, etc.), which have been isolated from Mexican species of the Genus Cedrela, and limonoids are noted because of their potent insect antifeedant characteristics and its potential for the development of ‘‘green pesticides’’ in insecticidal and chemical ecology studies. C. salvadorensis Standley is a small tree that grows on the dry pacific slope ranging from Jalisco to Chiapas in Mexico, through Central America, and up to the north of Panama (Rzedowski, 1972; Huerta, 1981; Rzedowski, 1991, 1993). Previously, we reported the isolation of photogedunin epimers 15, 17 as a mixture from a CH2Cl2 extract of this plant and the synthesis of its acetate derivatives 16 and 18 (Figure 3) (Ce´spedes et al., 1998).
Natural compounds as antioxidant and molting inhibitors
7
C. dugessi is also a small tree that grows on the dry pacific slope ranging from Michoacan to Oaxaca states in Mexico, in a similar form to C. salvadorensis. C. odorata is a tree of 4–5 m height that grows on the cost of Gulf of Mexico, throughout Veracruz and Tabasco states. C. oaxacensis is a tree of 4–5 m height that grows on the slope of Sierra Madre Mixteca, throughout Michoacan, Oaxaca, Veracruz, and Tabasco states (Rzedowski, 1972; Huerta, 1981; Rzedowski, 1991, 1993). Limonoids, photogedunins 15, 17, gedunin 19, cedrelanolide 20, and photogedunin epimeric mixture (15+17) were isolated from young trees of C. salvadorensis and C. dugessi (Ce´spedes et al., 1998, 2000). Photogedunin epimeric acetate mixture (16+18) showed the highest insecticidal activity at 10.0 ppm with 17% survival; the photogedunin epimeric mixture (15+17) with 50% survival at 10.0 ppm in a similar form to toosendanin 21 was next; gedunin 19 showed this effect at 39.0 ppm. Cedrelanolide 20 induced only moderate larval mortalities (o40%) while photogedunin epimeric mixture (15+17), gedunin 19, photogedunin acetates (16+18), and toosendanin 21 generally produced higher mortalities (>45%). The surviving larvae produce deformed pupae, which did not survive (Ce´spedes et al., 2000). Table 1 Insect growth regulatory activity of the isolated compounds, mixtures, and extracts, isolated from mentioned plants against S. frugiperda larvae in a no-choice bioassaya 7 Days Treatment 12 13 14 Epimers (15+17) Epimers (16+18) Gedunin 19 Cedrelanolide 20 Toosendanin 21 Agarofuran 22 Agarofuran 23 24 25 26 27 28 29 Argentatin A 30 Argentatin B 31 32 33 34 35 36 37
21 Days
Pupation
GWI50b
GLI50c
LD50d
GI50b
EI50b
pI50e
26.54 8.75 6.97 5.0 7.5 2.87 25.0 1.75 3.84 7.55 9.7 13.5 30.3 N.D. 3.1 4.0 o1.0 >5.0 5.1 24.7 5.9 6.8 31.7 57.8
32.74 11.6 7.24 10.0 15.0 5.53 75.0 29.0 o6.5 o12.0 5.28 27.3 8.14 N.D. 3.1 8.36 17.8 36.1 21.3 20.6 5.6 5.8 14.7 N.D.
27.6 6.4 5.4 10.0 8.0 30.08 N.D. 8.5 7.5 8.2 3.9 N.D. 27.8 N.D. 10.7 3.46 21.3 37.0 19.12 20.76 33.31 5.77 62.02 81.81
9.24 5.94 3.45 o5.0 o4.0 1.90 >5.0 2.68 0.37 0.43 o1.0 6.5 6.5 9.5 o1.0 >2.0 o5.0 5.0 8.07 14.65 4.20 4.90 18.08 24.41
N.D. 12.29 4.83 N.D. N.D. 3.95 N.D. N.D. o1.5 o2.5 0.55 4.68 4.46 6.91 0.74 3.05 o15.0 o15.0 4.32 22.60 4.59 10.54 39.91 7.07
0.96 0.77 0.54 N.D. N.D. 0.40 N.D. N.D. N.D. N.D. 0.26 0.67 0.64 0.84 0.13 0.48 N.D. N.D. 0.90 1.16 0.62 0.69 1.26 1.39
PI50f 38.26 7.19 9.02 10.0 o5.0 14.01 >60.0 o5.0 o1.5 o2.5 3.46 N.D. N.D. N.D. 2.11 4.62 o15.0 o5.0 3.84 24.27 6.04 14.21 23.01 7.61 (Continued )
Naturally occurring bioactive compounds
8 Table 1 (continued ) 7 Days
21 Days
Pupation
Treatment
GWI50b
GLI50c
LD50d
GI50b
EI50b
pI50e
PI50f
M Ma Me-Yuc Hex-G. microcephala Me-G. microcephala Me-Ced Me-P. argentatum Me-Maytenus Hex-Maytenus
4.1 19.2 14.99 3.2 5.5 3.56 o10.0 14.0 7.3
33.4 33.2 13.22 12.82 14.45 10.87 6.4 13.5 7.5
17.76 27.51 7.18 7.5 7.95 8.22 6.9 9.4 6.0
2.44 5.17 5.13 10.0 12.0 14.17 7.5 4.8 0.37
8.92 3.60 5.79 10.37 13.85 1.43 3.5 o5.0 o5.0
0.39 0.71 0.78 1.01 1.14 0.99 N.D. N.D. N.D.
13.90 4.78 18.82 5.91 12.4 3.84 o0.5 o5.0 o5.0
Notes: M and Ma correspond to a mixture of the compounds 32, 35, and 38 and its acetylated form, respectively. a The parameters are in ppm values. b The GWI50 and GI50 correspond to the inhibitory concentrations for reduction of 50% of larval growth in weight at 7 and 21 days, respectively, in ‘‘no-choice’’ test (po0.05) and EI50 correspond to concentration producing 50% of emergence and were calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by PROBIT analysis and ANOVA (po0.05) corresponding to the growth inhibition at 7 and 21 days, respectively, under Microcal Origin 6.0. c GLI50 correspond to the growth inhibition in length at 7 days, and was calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by PROBIT analysis and ANOVA (po0.05) under Microcal Origin 5.1. d LD50 is the concentration producing 50% of lethal mortality at 7 days in ‘‘no-choice’’ test calculated by PROBIT analysis and ANOVA (po0.05). e pI50 correspond to log GI50. f PI50 correspond to concentration producing 50% of pupation, and was calculated as the dose corresponding to midpoint between complete inhibition (100% of control) and no effect by the computer program ANOVA (po0.05) under Microcal Origin 6.0.
We do not ignore the fact that these compounds could act as antifeedant because we did not do the election test, but as pupation did not take place (100% of inhibition above 52 ppm), we suggest that the mechanism by which these compounds act may be due to physiological effects in a similar form to other limonoids (Nihei et al., 2002). We are investigating to elucidate the target, mechanism, and mode of action of these compounds and probably corresponds to a combination of antifeedant action and/or postdigestive toxicity, as found for other limonoids (Champagne et al., 1992; Isman et al., 1995). Celastraceae Celastraceae family embraces shrubs and trees of moderate height occurring in southern Chile. Plants from this family belonging to Maytenus genus have antifeedant and anti-inflammatory effects. These species show high allelopathic and antifeedant effects on plant and insect species in their habitat, respectively (Gonza´lez et al., 2000; Ce´spedes et al., 2001a). Maytenus genus is characterized by the occurrence of different bioactive compounds, especially b-dihydroagarofurans (Itokawa et
Natural compounds as antioxidant and molting inhibitors
9
OR
OR
O
O
O
O
O O
O O
O
O
O O
O
O
O
OAc
O
OAc
15 R = H 16 R = Ac
O
OAc
17 R = H 18 R = Ac
19
O OAc
H O O
O
OH
OAc COEt OHC
O O
O AcO
O
OH
O CO2Me
20
HO H
21
Fig. 3. Limonoids from Cedrela spp., Meliaceae.
al., 1993; Backhouse et al., 1994; Shirota et al., 1994). Until now, we focused mainly on two South-American species of this genus, Maytenus disticha (Hook) Urban and M. boaria. The former, commonly known as ‘‘maitencito’’ or ‘‘romasillo,’’ is a small tree that grows in rainfall forests in the South Pacific slope ranging from Araucanian Region to ‘‘Tierra del Fuego’’ in the Patagonian Region in Chile. M. boaria, the sole tree among the Celastraceae family in Chile, usually grows in the arid climate in the Slope Mountains. Considering that in Celastraceae family the main biologically active compounds are b-agarofuran type sesquiterpenoids (Figure 4), we selected these plants due to their high resistance to insect attack and no insecticidal work has been carried out on this plant. Moreover, we investigated the inhibition of AChE, a key enzyme in the insect nervous system in which the colynergic system is essential. From the aerial parts of M. disticha we isolated six agarofurans (Alarco´n et al., 1991, 1995, 1998) and from the seeds of M. boaria, four b-agarofuran polyesters were obtained and chemically characterized (Alarco´n et al., 1991; Ce´spedes et al., 1999). Insecticidal bioassays showed that a hex/EtOAc (HE) extract produced the highest mortality (79.1%) at 12.0 ppm, similar to 22, 23, and MeOH extract (>65%) and at 15.0 ppm, 23 and HE extract showed the highest larval mortality (100%). When larvae were fed with a diet containing 25.0 ppm or higher doses of all extracts and compounds tested, all the larvae died. Both b-agarofurans 22 and 23 inhibited larval growth after 7 days, and showed a significant growth reduction after 23 days. Compound 23 was more active than the positive control (toosendanin) and
Naturally occurring bioactive compounds
10 CH2OAc
OAc
OBz
OAc AcO
OFu OAc
OAc
O
O OAc 22
OAc 23
Fig. 4. b-Agarofuran type sesquiterpenoids from Maytenus spps.
compound 22, decreasing larval growth, total length of the larvae, and percentage of larvae that reached pupation and emergence. It is noteworthy that b-agarofuran 22, although was not so effective as 23, showed an appreciable reduction on larval length growth compared with the control, for this reason the activity of these compounds is comparable to the commercial insecticide toosendanin 21 (Chen et al., 1995). In all cases the adult weight was clearly reduced. Moreover, these compounds and extracts showed dose-response dependent activity. In order to determine the site of action of compounds and extracts from both plants, we carried out an AChE activity assay. MeOH and HE extracts, and 22 and 23 showed inhibitory AChE activity in a dose-dependent manner confirming that these compounds are active inhibitors of AChE in Maytenus spp. The activity of extracts may be due to a synergistic effect more than an activity of a single compound, and the presence of a furanoxy ring substituent increases the strength of these compounds on inhibition of AChE (Ce´spedes et al., 2001a). Asteraceae Our interest is centered on the study of possible insecticides of desert shrubs belonging to the Asteraceae family, due to their strong resistance against insect attack observed in nature. We have worked on four species of this family – Gutierrezia microcephala, Parthenium argentatum, Roldana barba-johannis, and Tagetes lucida – and evaluated their insecticidal and IGR activities on FAW. Gutierrezia microcephala G. microcephala A. Gray is commonly known as broom wood and grows in arid regions of the Central and North of Me´xico and in the southwestern region of the United States (Roitman and James, 1985). The plant collected in Saltillo, Coahuila State, is rich in flavonoids (Wollenweber et al., 1997). Four flavones 24–27 were obtained from the acetonic and methanolic extracts that were isolated previously by Fang et al. (1985, 1986). n-Hexane extract afforded bacchabolivic acid 28 (Zdero et al., 1989, 1992), a new ent-clerodane reported on this plant, which was lately submitted to esterification to yield the methyl ester 29. Insecticidal effects of 24–29
Natural compounds as antioxidant and molting inhibitors HO
11 OH
OMe OMe
OMe HO
HO
O
O
OMe OMe
MeO OH
MeO
OMe OH
O 24
O 25
OMe HO OH
OMe
OMe
OMe MeO HO
O
O
OMe OH OMe
MeO
MeO
OMe OH
OH
O
O 26
27
O CO2R
28 R = H 29 R = Me
Fig. 5. Flavonoids and clerodane from G. microcephala.
(Figure 5), and hexane and methanol extracts against larvae were evaluated (Caldero´n et al., 2001). Although 24, 28, and MeOH and Hex extracts produced significant larval mortalities (>49%) at a low concentration (10 ppm). Compound 29 produces higher mortality (81%). It is relevant that 29, 24, hexane, MeOH extracts, and 28 were more active than control (gedunin) with MC50 (50% lethal concentration of larvae at 7 days) of 3.46, 3.9, 7.5, 7.95, and 10.7 ppm, respectively. Compounds 24, 26, 28, 29, and hexane and MeOH extracts inhibited specifically each larval stage. Compounds 28, 29, and hexane and MeOH extracts produced higher inhibition (upto 90% of weight). Clerodane 28 showed the highest inhibition (100% of length and weight) at 50.0 ppm and flavonoids had lower larval inhibition at the same concentration. Growth reduction at 21 days was produced mainly by 24 and 28. Although 28 produced a significant delay in pupation, most important effect was observed by 24, 28, and hex extract. Significant delays in pupation time were observed at 10.0, 15.0, and 50.0 ppm for 28, 29, and hexane extract. Furthermore, hexane extract
12
Naturally occurring bioactive compounds
significantly reduced pupae weights at 50.0 ppm. Related to percentage of emergence, all the compounds and extract tested showed further reductions. Nevertheless, 24, 29, and hexane extract drastically reduced the percentage of adult emergence to 0% at 25.0, 15.0, and 50.0 ppm. In many of the treatments, mean adult weight was significantly delayed in the average time to reach the adult stage relative to control larvae. Growth index (GI) and RGI clearly showed that the stronger effect was shown by 28, 29, and hexane extract. It is possible to infer that the substitution of polymethoxy flavones induces an increase on the activity of these flavones and a furan ring seems to be necessary for insecticidal activity of ent-clerodane type diterpenes. In order to determine the site of inhibition on the IGR activity and the acute toxicity of the compounds and extracts, the effect on AChE activity was studied. In a similar form to the acute toxicity, 28, 29, MeOH, and hexane extracts showed the greatest inhibitory effect at 50.0 ppm and flavonoids 24–27 displayed a lower inhibitory effects. At minor concentration (25.0 ppm), 28 and hexane extract showed stronger activity level than MeOH extract and 29. In the same way the last two were more active than flavonoids. In addition, both extracts and ent-clerodanes 28 and 29 inhibited AChE activity in a dose-dependent manner (Caldero´n et al., 2001). In addition, the presence or absence of a methyl ester group increases or decreases, respectively, the strength of these compounds on inhibition of AChE in clerodanes. It is obvious that the nature of the ester substituent at C-8 plays an important role for the insecticidal activity of ent-clerodanes. The most active compound 28 contained a small and relative hydrophilic acid group at C-8, whereas 29 with a bulky and more lipophilic ester group exhibited a lower activity level. This is according to previously reported QSAR analysis performed by Rodriguez et al. (1999), where they proposed that antifeedant activity of the respective natural products depends on the polarity of ring B and the size of the ester substituents. With respect to the flavonoids, the presence of an extra methoxyl substituent in the A ring seems to be the cause of growth inhibitory activity (length and weight) shown by 24 and 25. In a similar form to polymethylated flavonoids where the introduction of a methyl ether excluding the B-ring in the flavonoids structure increased the antifeedant activity (Morimoto et al., 2000). Parthenium argentatum P. argentatum, commonly known as ‘‘guayule,’’ is a shrub endemic to the Chihuahua desert and is used as an important source of natural rubber. Plant was collected in Matehuala, State of San Luis Potosi, Me´xico. After collection, aerial parts were milled and extracted exhaustively with methanol and partitioned with n-hexane and ethyl acetate. Preliminary assays showed that MeOH extract had a considerable insecticidal activity. Chromatography purification afforded mainly two cycloarten-type triterpenes named Argentatin A and B (Rodrı´ guez-Hahn et al., 1970; Matsubara and Romo de Vivar, 1985; Martı´ nez-Va´zquez et al., 1994). As both compounds were purified in sufficient amount these were used in bioassays. Insecticidal effects of the Argentatin A 30, B 31, toosendanin 21 as a control, and methanolic extract against larvae of first instar of S. frugiperda were performed. Argentatin A produced higher mortality (80.2%) at 35 ppm; however, MeOH extract
Natural compounds as antioxidant and molting inhibitors
13
shows the highest insecticidal activity (96.5%) at a lower concentration (10.0 ppm). It is important to note that when larvae were fed in a diet containing 15.0 ppm or higher doses of methanolic extract all larvae died. Argentatin A and B inhibited specifically each larval stage. At 23 days this growth reduction was clearly significant between 25.0 and 50.0 ppm. However, only toosendanin 21 showed the highest larval growth inhibition at the same concentrations. All tested compounds in comparison to control decreased the percentage of larvae that reached pupation. Although compounds 30 and 31 showed significant delay in survival after pupation, pupation time, and percentage of emergence, the most important effect was observed by MeOH extract and the control (Figure 6). Furthermore, 30 and MeOH extract significantly reduced pupae weights, but toosendanin 21 showed the greatest effect (Ce´spedes et al., 2001b). GI and RGI clearly showed that the stronger effect was shown by MeOH extract and toosendanin. These parameters together with LD50 values corroborate the highest effect that showed methanolic extract. Compounds 30 and 31 showed moderate acute toxicity, nonetheless toosendanin and MeOH extract showed a potent acute toxicity on larvae of last stage at 35.0 and 15.0 ppm, respectively. The LD50 values of 21 and MeOH extract were 1.5 and 3.1 ppm, respectively. In order to determine the site of inhibition on the IGR activity and the acute toxicity of compounds and extracts tested, AChE activity was studied. Argentatin B showed a moderate AChE inhibition activity, unlike Argentatin A, MeOH extract, and toosendanin that are strong inhibitors of AChE activity in a dose-dependent manner, confirming that these compounds are the active inhibitors of AChE. The presence of a ring with seven members, as in Argentatin B, decreases the strength of this compound on inhibition of AChE, opposite to the presence of a furan ring. Strong activity showed by MeOH extract may be due to a synergistic effect (Ce´spedes et al., 2001b). Roldana barba-johannis The methanol extract from the aerial parts of R. barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and the corresponding acetylated and methylated derivatives showed
OH O OH OH
O
O
O
31
30
Fig. 6. Triterpenes from P. argentatum.
Naturally occurring bioactive compounds
14
insecticidal and IGR activities against FAW. The most active compounds were sargachromenol 32, and its acetylated derivative 34; sargahydroquinoic acid 35 and its acetylated derivative 37; and a mixture of sargachromenol 32, sargahydroquinoic acid 35, and sargaquinoic acid 38 (6:3:1) and the acetylated form of this mixture; 33 and 36 showed a minor activity. All these compounds and mixtures had significant effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect (Figure 7). The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of C. salvadorensis and Y. periculosa (Ce´spedes et al., 2004). Tagetes lucida T. lucida known as ‘‘perico´n,’’ is a medicinal plant used from pre-Columbian times by Aztecs and other Mesoamericans. This specie is distributed from Northern Me´xico to Northern Nicaragua (Rzedowski, 1972, 1991). Their leaves and inflorescence are used for stomachache and for the treatment of diverse anti-inflammatory ailments. From a dichloromethane extract, we isolated several coumarins: scopoletin
O CO2R
R'O
32 R, R' = H 33 R = CH3 R' = H 34 R = H R' = Ac
CO2R
OR'
35 R, R' = H 36 R = CH3 R' = H 37 R = H R' = Ac OR'
O
CO2R
38
O M = 32 + 35 + 38 Ma = M methylated
Fig. 7. Plastoquinones and tocotrienols from R. barba-johannis.
Natural compounds as antioxidant and molting inhibitors CH3O
15 HO
HO
HO
O
O
HO
39
O
O
40
CH3O
O 42
O
O
O
O
O
41
AcO
CH3O
CH3O
CH3O
AcO
O
O
43
AcO 44
Fig. 8. Coumarins from T. Lucida.
(6-methoxy-7-hydroxycoumarin) 39, esculetin (6,7-dihydroxycoumarin) 40, 7-methoxy-6-hydroxycoumarin 41, scoparone (6,7-dimethoxy-coumarin) 42, and their derivatives 6,7-diacetoxy coumarin 43 and 6-methoxy-7-acetoxycoumarin 44 (Figure 8). The compounds 39, 40, 41, and dichloromethane extract showed high mortality effects between 5.0 and 15.0 ppm and an IGR activity between 1.0 and 5.0 ppm; 40 being the most active compound. The compounds 42, 43, and 44 showed IGR activity at concentrations higher than 70.0 ppm. A complete inhibition of the pupation and emergence above 70.0 ppm was observed. In the literature there are few information about insecticidal effects of coumarins. Nagaiah et al. (1992) reported about the IGR and antifeedant effects of some coumarins from the bark of Xeromphis uliginea on the growth of Spodoptera litura. These compounds are formed by the action of phenylalanine ammonia lyase (PAL) and are then converted to ortho-coumaric acid by the action of cinnamic acid o-hydrolase (Seigler, 1997). Coumarins are located within specialized structures such as secretory ducts called vittae (Berenbaum, 1991). Araucariaceae In the continuation with our search for natural products with possible insecticide activity, we have studied trees of the rain forest of southern Chile, due to their strong resistance against insect attack observed in nature. We have evaluated the insecticidal activity of Araucaria araucana and IGR activities on FAW. Araucaria araucana (Mol.) K. Koch This conifer is endemic to rain forest of southern Chile and Argentina. It has high commercial, ethnobotanical, taxonomic, and ecological value derived from its long biogeographical and remote occurrence. These characteristics offer a unique opportunity for the search of secondary metabolites, especially those that have a defensive role against pathological and phytofagous pests. Five lignans (secoisolariciresinol 45, lariciresinol 46, pinoresinol 47, eudesmin 49, and methyl-pinoresinol 48) were
16
Naturally occurring bioactive compounds
isolated from MeOH extracts of bark and wood of A. araucana and their structures were determined with spectroscopic methods. In addition to antifeedant, insecticidal, and IGR activities against S. frugiperda (FAW), the antibacterial activity of these compounds was determined against Gram (+) and Gram () bacteria and the antifungal activity against Fusarium moniliforme, Aspergillus niger, Trichophyton mentagrophytes, Mucor miehei, Paecilomyces variotii, Ceratocystis pirifera, Trametes versicolor, and Penicillium notatum. These lignans exhibited antifungal and antibacterial activities (Ce´spedes et al., 2006a) and antifeedant and IGR activities on FAW (Ce´spedes et al., 2006b), in the range between 1.0 and 50.0 ppm. Against FAW the strongest compound was pinoresinol 47 with an LD50 19.0 ppm, the MeOH extract from wood shows an LD50 of 400.5 ppm, and the MeOH extract from bark shows an LD50 of 92.1 ppm, these values show the antifeedant action of A. araucana against a Lepidoptera as FAW. In addition, these results show that the larval mortality of all compounds – below 50 ppm – is stronger than the bark MeOH extract and this suggests that this effect could be attributed to the lignin occurrence in this extract, since these lignans were isolated from this extract and all these measurements were twice lower than natural positive control – MeOH extract from bark of Y. periculosa – that showed a 98% of mortality at 50 ppm, as was previously observed (Torres et al., 2003; Ce´spedes et al., 2004). In addition to the above effects these lignans showed a strong AOX activity against DPPH radical reduction, with very low concentrations, between 1 and 10 ppm; almost all lignans showed an I50 inhibition at around 4.5 ppm, except methyl pinoresinol 48 and eudesmin 49, which did not show inhibition (Figure 9).
Experimental part Bioassays with fall armyworm (FAW), Spodoptera frugiperda Larvae used for the experiments were obtained from the culture at the Centro de Investigacio´n en Biotecnologı´ a at the Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos, Me´xico, maintained under previously described conditions (Aranda et al., 1996; Caldero´n et al., 2001; Ce´spedes et al., 2000, 2001a, 2001b, 2004; Torres et al., 2003). Acute toxicity on FAW Acute toxicity was elucidated by topical application to last stage larvae of S. frugiperda. The larvae of S. frugiperda were iced to stop their movement and treated on their abdomens with each one of the test compounds. The solvent used was 10.5 ml of acetone in topical form with 50 ml microsyringe, and control was only treated with 10.5 ml of acetone. After 24 h, survival was recorded. Five larvae were used for each concentration, respectively (Torres et al., 2003; Ce´spedes et al., 2004). Relative growth index and growth index The RGI and GI were calculated according to Zhang et al. (1993).
Natural compounds as antioxidant and molting inhibitors
17 OCH3 OH
OH
HO
O H 3CO
OCH3
HO
OH
45
H3CO
O
46
R3 COOH
O O
R1
OH
HO
R4
O O OCH3 R2
O
50 47 R1=R3=CH3; R2=R4=H 48 R1=R3=R4=CH3; R2=H 49 R1=R2=R3=R4=CH3
Fig. 9. Lignans from A. araucana and anisol from other sources.
Reduction of 2,2-diphenyl-1-picrylhydrazyl [ ¼ 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl; DPPH] radical TLC autographic assay After developing and drying, TLC plates were sprayed with a 0.2% DPPH solution in MeOH. The plates were examined 30 min after spraying. Active compounds appear as yellow spots against a purple background. In a similar form, TLC plates were sprayed with 0.05% b-carotene solution in CHCl3. The plates were examined under UV254 light until the background had become discolored (bleached). Active compounds appeared as pale yellow spots against a white background. Spectrophotometric assay (Bors et al., 1992; Cuendet et al., 1997, 2000): A solution (50 ml) containing the compound to be tested was added to 5 ml of a 0.004% MeOH solution of DPPH. The measurement was done after 30 min, and the percent of activity was calculated (Torres et al., 2003). Inhibition of acetylcholinesterase An enzyme extract containing AChE was obtained according to the method of Grundy and Still (1985). Inhibition of AChE was determined according to the Ellman’s procedure (colorimetric method) (Ellman et al., 1961) using both the control (MeOH) and test solutions (compounds and extracts). All AChE inhibition was carried out according to procedures described previously in Ce´spedes et al. (2001a, 2001b).
18
Naturally occurring bioactive compounds
Discussion The sites and mode of action of these compounds is being investigated and probably correspond to a combination of diverse antifeedant actions as midgut esterase inhibition and postdigestive toxicity, as found for other limonoids (Champagne et al., 1992; Nakatani et al., 1994; Isman et al., 1996) and extracts (Feng et al., 1995) or as enzyme inhibitors such as estearases, proteases, tyrosinase, or AChE (Miyazawa et al., 1997; Ortego et al., 1999; Kubo et al., 2003a, 2003b). There are mainly three types of tyrosinase inhibitors based on their inhibition mechanisms. First, the inhibitors directly interact with the binuclear copper active site of the enzyme, known as either copper chelators or substrate analogues. The inhibitors belonging to this category are mostly aromatic compounds and usually act as competitive inhibitors; the inhibitors disrupt the tertiary structure of the enzyme rather than directly interact with the active site. These inhibitors are not only aromatic but are also non-aromatic compounds, and generally act as noncompetitive inhibitors. Second, some molecules have a hydrophobic part and this section interacts with the hydrophobic domain in the enzyme. These subunits associate in a geometrically specific manner, known as a quaternary structure. The hydrophobic moiety which is exposed on the other side of the molecule may disrupt tyrosinase’s quaternary structure. The low conformational stabilities of native proteins make them easily susceptible to denaturation by altering the balance of the weak non bonding forces that maintain the native conformation (Kubo et al., 2003c). Third, the inhibitors do not interact with the enzyme directly or indirectly but act as reducing agents for dopaquinone. In addition to these three main types, there are some additional types of inhibitors. For example, inactivators of the enzyme and some tannins belong to this type of inhibitors. Hence, tyrosinase inhibitors might ultimately provide clues to control insect pests by inhibiting tyrosinase, resulting in incomplete cuticle hardening and darkening (Kubo, 1997, 2000; Kubo et al., 2002). In the case of AChE, the action is directly related with the interaction between the inhibitors and acetylcholine, the inhibition of the enzyme occurs with detachment of the acid residue of the toxic compound, and the nature of acid residue does not affect the structure of the inhibited enzyme, but acts strongly on the process of its inhibition. Natural compounds carrying a charge in an acid residue are very active inhibitors of cholinesterase, such as alkylammonio, alcohols (Brestkin et al., 1992), alkaloids (physostigmine, phenserine, tolserine, among others) (Yu et al., 2002), some terpenoids (Ortego et al., 1999; Caldero´n et al., 2001; Ce´spedes et al., 2001a, 2001b), and monoterpenoids (Gracza, 1985; Grundy and Still, 1985; Miyazawa et al., 1997). Some investigations on sites and mechanism of insecticidal or IGR action report that different phenolic compounds are enzyme and metabolism inhibitors (Hammond and Kubo, 1999; Kubo and Kinst-Hori, 1999a; Kubo, 2000; Kubo et al., 2000; Shimizu et al., 2000; Caldero´n et al., 2001; Panzuto et al., 2002). It is important to note that similar IGR activity on S. litura (Common cutworm) was studied by Morimoto et al. (2000). These authors reported that flavonoids of Gnaphalium affine (Asteraceae) have insect antifeedant properties against this insect. It is possible to infer that the substitution pattern of flavonoids induce an increase in the activity
Natural compounds as antioxidant and molting inhibitors
19
of those phenolic compounds (Caldero´n et al., 2001). Until our report (Torres et al., 2003), there were no reports about insecticidal activity of stilbenes, but only those having tyrosinase inhibitory (Shimizu et al., 2000), and antifeedant activities of similar phenolic compounds (Kubo and Kinst-Hori, 1999a; Kubo, 2000; Kubo et al., 2000, 2003a, 2003b). The presence of hydroxyl, methoxyl, and furan moieties seems to be necessary for insecticidal activity as in limonoids containing several of these chemical groups (Nakatani et al., 1994; Ce´spedes et al., 2000, 2001a, 2001b; Caldero´n et al., 2001) and in other phenolic compounds as alkanols, tannic, gallic, and anisic acid, respectively (Hammond and Kubo, 1999; Panzuto et al., 2002; Kubo et al., 2003a, 2003b). It is worth mentioning that resveratrol 13, the methoxylated stilbene 14, photogedunin epimeric mixture (16+18), toosendanin 21, agarofurans 22 and 23, the flavonoid 24, the diterpenes 28 and 29, sargahydroquinoic acid 35, 6,7-dihydroxycoumarin 40, and pinoresinol 47 showed the most potent insecticidal activity. It is obvious that the nature of the substituents as well as hydroxyl and methoxyl groups in the aromatic ring plays an important role for the insecticidal activity. These results prove previous findings on the quantitative structure-activity relationship of stilbene derivatives for instance, namely that the inhibitory tyrosinase activity of the respective natural products depends on the polarity of ring A and on the size of the substituent (Gorham et al., 1995; Shimizu et al., 2000). These facts show that the antifungal activity showed by stilbenoids (Schultz et al., 1990, 1992; Pacher et al., 2002), acute toxicity, and growth inhibition may be due to the inhibition of tyrosinase, and this target was demonstrated also for other stilbenes and phenolics from natural sources, such as gallic 10 and anisic acid 50 (Shimizu et al., 2000; Gilly et al., 2001; Kim et al., 2002; Kubo et al., 2003a, 2003b). In addition to the enzymatic activities, IGR correlate very well with AOX measurements of the reduction of DPPH radical, in many of our cases mainly when the phenolic compounds show AOX activity they also show IGR activity. Our phenolic compounds could be considered as efficient IGR and radical scavengers (Ce´spedes et al., 2000, 2001a, 2002, 2003, 2004; Stivala et al., 2001; Kim et al., 2002; Rimando et al., 2002). Thus, it is possible to infer that when some compounds show AOX properties they can also show IGR or tyrosinase inhibitory activity. In our case the compounds that showed these activities were stilbenes, sargachromene and sargahydroquinoic acid, flavonoids such as quercetin, naringenin, and kaempferol, and coumarins. Thus, it can be said that natural compounds are AOX, antifungal, and molting inhibitors, which can play a role as a model for search of new botanical pesticides, and nutraceutical and bioactive compounds. The percentage of larvae that reached pupation decreased in all tested compounds in comparison to the control (Table 1). The most important effect was observed with stilbene 14, gedunin 19, toosendanin 21, b-dihydroagarofurans 22, and 23, clerodane 28, Argentatin A 30, sargachromenol 32, sargahydroquinoic acid 35, 6,7-dihydroxycoumarin 40, and pinoresinol 47, photogedunin epimeric mixture, photogedunin acetate mixture, which reduced successful pupation into a range between 0% and 25% and caused a significant delay in pupation time. The effects of these substances on reducing insect growth, decreasing the percentage of emergence, and increasing mortality of S. frugiperda are similar to those of other natural products (Arnason et al., 1987, 1993).
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Inspection of the structure of the most active compounds isolated from these plants suggests that the presence of oxygenated function was necessary for the activity displayed by the gedunin-type and phenolic molecules against S. frugiperda. Almost all tested compounds except cedrelanolide and methoxylated flavones showed comparable activity to the commercial insecticide toosendanin, which suggests potential for further development of these materials. However, none of these substances has been found with the outstanding activity of azadirachtin (Govindachari et al., 1995; Kraus, 1995).
Concluding remarks In summary, the insecticidal activity of extracts may be due to synergistic effects shown by the phenolic components of the mixtures in the test system used in this investigation. These facts are indicative of the potency of the methanol extracts. Thus, the effect of compounds on reducing insect growth, increasing development time, and increasing mortality of S. frugiperda is similar to that of gedunin and more potent than the MeOH extract from C. salvadorensis (Ce´spedes et al., 2000; Caldero´n et al., 2001). The sites and mode of action of these compounds and extracts are being investigated and probably correspond to a combination of antifeedant action as midgut phenol oxidase, proteinase, AChE, tyrosinase, or other PPOs and cuticle synthesis inhibition, as well as molting, sclerotization, toxicity, and nerval system inhibition, as has been found for other phenolics and terpenoids (Miyazawa et al., 1997; Kubo and Kinst-Hori, 1999a, 1999b; Kubo, 2000; Kubo et al., 2000; Simmonds, 2003) and extracts (Feng et al., 1995). In addition, the presence of an orcinol or catechol group seems to be important for these activities as shown for the most potent compounds in this chapter. Furthermore, a great percentage of larvae that reached pupation decreased with the application of phenolics in comparison to control, which might be due to the inhibition by tyrosinase as well or to the accumulation of proteinase inhibitors (Tamayo et al., 2000). The activity of these plants, their metabolites, and MeOH extracts is comparable to the insect growth regulators gedunin and toosendanin, which suggests potential for further development of these materials.
Future perspectives The plant kingdom offers a rich source of a wide range of structural biodiversity of natural secondary metabolites. One of the most recent trends in fungal and insect pest control is to reduce heavy reliance on synthetic pesticides and to move towards biodegradable substances. Synthetic pesticides of broad spectrum have been widely used as the main tools for controlling weeds, and fungal and insect pest, which are highly toxic to many living organisms as well as to the environment. Hence, new biorational and specific trends to pest control should be developed. In this chapter, we presented an update of our findings in this field, those approaches resulting from our studies on inhibition of growth (including larval growth, pupation, and emergence) and of the enzymes involved in key processes of insect life, specially modifying the apolysis during molting, sclerotization, pupation, and
Natural compounds as antioxidant and molting inhibitors
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emergence. Since these approaches refer to control of insect pests, many of them can be extrapolated and also considered suitable for medicinal chemistry studies, because the mechanism of action of these inhibitors is similar to that from human and other animals (Guerrero and Rosell, 2004, 2005). The study of AChE and other phenol oxidases inhibitors, as well as IGR substances, can help in the search of new natural compounds for the treatment of Alzheimer’s disease (Cummings, 2000) and Parkinson’s disease (Weinstock et al., 2003). Therefore, there is an increasing expectation about the research on enzymes inhibition by those compounds of botanical origin that could serve as lead compounds for the development of important substances with agrochemical and pharmacological properties. Thus, by studying the plant organisms that protect themselves against the pest attack, we can learn to control this attack in an ecological way and in addition can get pharmacologically active substances.
Acknowledgments We are very grateful to Prof. Isao Kubo (Department of ESPM, U. C., at Berkeley), Prof. Murray B. Isman (Faculty of Agricultural Sciences, U. British Columbia, Vancouver), and Prof. David S. Seigler (Plant Biology Dept. University of Illinois, at Urbana-Champaign), for the great and valuable help given to authors. The bioassays involving insecticidal to FAW larvae were skillfully conducted by Mrs. Laura Lina (Biological Control Lab., CEIB, UAEM, Cuernavaca, Morelos State) and in similar form the antifungal activity by Mrs. Ana Ma. Garcı´ a (Phytochemistry Lab, FESIztacala, UNAM) whose technical support is greatly appreciated. The works were partially supported by grants: IN243802 and IN211105 from DGAPA-UNAM.
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Schultz TP, Hubbard TF, Jin L-H, Fischer TH, Nicholas DD. (1990) Role of stilbenes in the natural durability of wood: fungicidal structure-activity relationships. Phytochemistry 29:1501–1507. Seigler DS. (1997) Plant secondary metabolism. Boston: Kluwer Academic Publishers, p. 790. Shimizu K, Kondo R, Sakai K. (2000) Inhibition of tyrosinase by flavonoids, stilbenes and related 4-substituted resorcinols: structure-activity investigations. Planta Medica 66:11–15. Shirota O, Morita H, Takeya K, Itokawa H, Iitaka Y. (1994) Cytotoxic aromatic triterpenes from Maytenus ilicifolia and Maytenus chuchuhuasca. J Nat Prod 57(12):1675–1681 (17 ref.). Simmonds MSJ. (2003) Flavonoid-insect interactions: recent advances in our knowledge. Phytochemistry 64:21–30. Singh M, Khokhar S, Malik S, Singh R. (1997) Evaluation of neem (Azadirachta indica A. Juss) extracts against American bollworm, Helicoverpa armigera (Hubner). J Agric Food Chem 45:3262–3268. Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, Forti L, Pagnoni UM, Albini A, Prosperi E, Vannini V. (2001) Specific structural determinants are responsible for the antioxidant activity and the cell effects of resveratrol. J Biol Chem 276(25):22586–22594. Sumner LlW, Mendes P, Dixon R. (2003) Plant metabolomics: large-scale phytochemistry in the functional genomics era. Phytochemistry 62:817–836. Swain T. (1979) Tannins and lignins. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interactions with secondary plant metabolites. New York: Academic Press, pp. 657–682. Tamayo MC, Rufat M, Bravo JM, San Segundo B. (2000) Accumulation of a maize proteinase inhibitor in response to wounding and insect feeding and characterization of its activity toward digestive proteinases of Spodoptera littoralis larvae. Planta 211:62–71. Torres P, Avila JG, Romo de Vivar A, Garcı´ a AM, Marı´ n JC, Aranda E, Ce´spedes CL. (2003) Antioxidant and insect growth regulatory activities of stilbenes and extracts from Yucca periculosa. Phytochemistry 64:463–473. Valladares G, Defago MT, Palacios S, Carpinella MC. (1997) Laboratory evaluation of Melia azedarach (Meliaceae) extracts against the Elm leaf beetle (Coleoptera: Chrysomelidae). J Econ Entomol 90:747–750. Weinstock M, Gorodetsky E, Poltyrev T, Gross A, Sagi Y, Youdim M. (2003) A novel cholinesterase and brain-selective monoamine oxidase inhibitor for the treatment of dementia camorbid with depression and Parkinson’s disease. Prog Neuro-Psychopharmacol Biol Psych 27:555–561. Wollenweber E, Do¨rr M, Fritz H, Papendieck S, Yatskievych G, Roitman JN. (1997) Exudate flavonoids in Asteraceae from Arizona, California and Mexico. Z Naturforsch 52c:301–307. Yu Q-Sh, Zhu X, Holloway HW, Whittaker NF, Brossi A, Greig NH. (2002) Anticholinesterase activity of compounds related to geneserine tautomers. N-Oxides and 1,2-oxazines. J Med Chem 45:3684–3691. Zdero Ch, Bohlmann F, Niemeyer H. (1992) Furolabdanes and linear diterpenes from Gutierrezia resinosa. Phytochemistry 31:1723–1726. Zdero Ch, Bohlmann F, Solomon JC, King RM, Robinson H. (1989) ent-Clerodanes and other constituents from Bolivian Baccharis species. Phytochemistry 28:531–542. Zhang M, Chaudhuri SK, Kubo I. (1993) Quantification of insect growth and its use in screening of naturally occurring insect control agents. J Chem Ecol 19:1109–1118.
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Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.
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CHAPTER 2
Pesticides based on plant essential oils: from traditional practice to commercialization MURRAY B ISMAN, CRISTINA M MACHIAL
Introduction With very few exceptions, botanical insecticides in commercial use have been used in traditional practices dating back at least 150 years, and often much longer (Jacobson and Crosby, 1971; Philoge`ne et al., 2005). Not unlike pyrethrum, rotenone and neem, plant essential oils or the plants from which they are obtained have been used for centuries to protect stored commodities or to repel pests from human habitations. Unlike other botanicals however, plant essential oils have a long history of human uses apart from pest control, notably as fragrances, flavourings, condiments or spices, as well as medicinal uses. It is ironic then, that development of plant essential oils as commercial insecticides has only occurred within the past decade. In noting the disparity between the vast number of plants shown to poison, deter or repel insects and the actual number of botanical insecticides in commercial use, I previously suggested that there are three barriers to commercialization of botanicals (Isman, 1997). These are: (1) availability of the plant material on a sustainable basis; (2) standardization of extracts based on quantification of active ingredients; and (3) governmental approval, normally requiring costly toxicological evaluation of the candidate product. Given the worldwide use of essential oils in the perfume, food and beverage industries, oils from many species are available in abundance yearround, and their major constituents are either well characterized or are themselves available in a relatively high degree of purity. Thus the major barrier to their use as pesticides may be government approval, but in the United States, several plants and their derivatives are exempt from registration owing to their widespread use in foods and generally acknowledged safety to humans. In the present chapter, the chemistry of plant essential oils, their biological activities in arthropods and fungi, and their potential as botanical pesticides in commercial practice is discussed.
Naturally occurring bioactive compounds
30
Sources and chemistry of essential oils Plant essential oils are obtained through steam distillation of the foliage or other plant parts of terrestrial plants in a limited number of taxa. Among the better-known families from which essential oils are frequently obtained are the mint (Lamiaceae), myrtle (Myrtaceae), citrus (Rutaceae) and carrot (Apiaceae) families. Most essential oils are highly complex mixtures of mono- (C10) and sesquiterpenes (C15), including biogenetically related phenols (phenylpropenes and cinnamates). These compounds are usually responsible for the characteristic odours and/or flavours of the plants from which they are obtained. The predominant group, the monoterpenes, are often cyclic, with a saturated (e.g. pulegone, Figure 1) or unsaturated hexacycle (e.g. limonene) or an aromatic system (e.g. thymol). Bicyclic (e.g. 1,8-cineole) and acyclic examples (e.g. citronellal) are not uncommon. CHO
O O
(+)-Limonene
(+)-Pulegone
1,8-Cineole
Citronellal
OH OCH3
OH
OH CHO Eugenol
Cinnamaldehyde
Thymol
Carvacrol
OCH3
O
Menthone
trans-Anethole
Fig. 1. Structures of some essential oil constituents with documented insecticidal and/or antifungal activities.
Pesticides based on plant essential oils
31
Functional groups include alcohols (e.g. thymol), aldehydes (e.g. citronellal) and ketones (e.g. menthone), but simple hydrocarbons also occur (e.g. limonene). Owing to their generally low boiling points (140–180 1C for monoterpenes, >200 1C for sesquiterpenes), all are relatively volatile, accounting for their use as fragrances and as insect repellents. While many of the monoterpenoids are chemically stable at ambient temperature, their volatility usually renders them nonpersistent in the environment. Among the best known essential oils with bioactivity against insects and other pests are clove oil (eugenol), thyme oil (thymol, carvacrol), mint oil (menthol, pulegone), lemongrass oil (citronellal, citral), cinnamon oil (cinnamaldehyde), rosemary oil (1,8-cineole) and oil of oregano (carvacrol). It is important to emphasize the intraspecific variability in chemical composition of plant essential oils. Sources of compositional variability can include the plant part extracted, phenological state of the plant, and time of year, as well as climatic and soil variations. Numerous studies have also confirmed that some species have distinct chemotypic races or populations, often separated geographically (Salgueiro et al., 1997; Pascual-Villalobos and Ballesta-Acosta, 2003; Angioni et al., 2004). In an investigation of the essential oils isolated from two rosemary ecotypes from Italy, the major constituent, 1,8-cineole, ranged from as low as 7.3% by weight to a high of 55.3% and a-pinene ranged from 11.5% to 30.3% (as isolated from apical, intermediate and lower foliage) (Flamini et al., 2002).
Biological activity Mode of action in arthropods The rapid onset of toxicity from essential oils or their constituents in insects and other arthropods points to a neurotoxic mode of action. In the American cockroach this is manifest as hyperactivity followed by hyperextension of the legs and abdomen culminating in rapid knockdown or immobilization, often in less than 30 s (Enan, 2001). Several essential oil monoterpenes have been shown to be weak inhibitors of acetylcholinesterase (AchE) in insect and mammalian preparations (Ryan and Byrne, 1988; Miyazawa et al., 1997), but toxicity in vivo did not correlate with AchE inhibition in vitro. More recently Enan (2001, 2005a) has provided evidence that many essential oil constituents poison insects by blocking octopamine receptors. Octopamine, synthesized from tyramine, is a neurotransmitter and neuromodulator in arthropods and may have neurohoromonal influences as well. Several essential oil constituents were demonstrated to be mild antagonists of octopamine binding activity in the cockroach nervous system, however, other such compounds were found to be agonists and yet others were without effect. Notable among the last group was thymol, one of the most toxic essential oil compounds to insects (Table 2). Enan (2005b) has also provided evidence that at least some essential oil constituents may act via the tyramine receptor cascade in the fruit fly Drosophila melanogaster. Physical effects, viz. dissolution and disruption of cell membranes, or blockage of the tracheal system in insects cannot be ruled out as alternative mechanisms of toxicity.
Naturally occurring bioactive compounds
32
Efficacy of essential oils to insects Table 1 lists a number of studies in which plant essential oils have been screened for bioactivity against insects. It should be readily apparent that the majority of studies to date have utilized stored product pests, particularly beetles such as Sitophilus, Tribolium and Acanthoscelides species, in which case fumigant toxicity has been investigated (Shaaya et al., 1991; Regnault-Roger et al., 1995; Obeng-Ofori and
Table 1 Screening studies with plant essential oils against insects Essential oil
Insect tested
Reference
53 species 31 species 28 species
Choi et al. (2003) Singh et al. (1989) Shaaya et al. (1991)
11 species
Trialeurodes vaporariorum Sitophilus oryzae Sitophilus oryzae, Rhyzopertha dominica, Oryzaephilius surinamensis, Tribolium castaneum Drosophila auraria
22 species
Acanthoscelides obtectus
4 species
Sitophilus oryzae, Tribolium confusum, Ephestia kuehniella Pediculus humanus Cydia pomonella Musca domestica Acanthoscelides obtectus
5 species 27 species 31 species 22 species 13 species
23 species Vetiver, Vetiveria zizanioides Patchouli, Pogostemon cablin Chamaecyparis obtuse Minthostachys andina, Hedomea mandonianum Tansy, Tanacetum vulgare Garlic, Allium sativum Nutmeg, Myristica fragrans Tetradenia riparia Holy basil, Ocimum sauve
Oryzaephilus surinamensis, Rhyzopertha dominica, Sitophilus oryzae, Tribolium castaneum Lipaphis pseudobrassicae Coptotermes formosanus Coptotermes formosanus Callosobruchus chinensis, Sitophilus oryzae Rhodnius neglectus, Triatoma infestans Choristoneura rosaceana Tribolium castaneum, Sitophilus zeamais Tribolium castaneum, Sitophilus zeamais Zabrotes subfasciatus Sitophilus zeamais, Rhyzopertha dominica, Sitotroga cerealella
Konstantopoulou et al. (1992) Regnault-Roger and Hamraoui (1994) Sarac and Tunc (1995) Mumcuoglu et al. (1996) Landolt et al. (1999) Singh and Singh (1991) Regnault-Roger et al. (1993) Shaaya and Kostjukovsky (1998) Sampson et al. (2005) Maistrello et al. (2001) Zhu et al. (2003) Park et al. (2003) Fournet et al. (1996) Larocque et al. (1999) Ho et al. (1996) Huang et al. (1997) Weaver et al. (1994) Bekele et al. (1996)
Pesticides based on plant essential oils
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Table 2 Toxicity of some essential oil constituents to insects Compound
Insect tested
Effect/dose
Reference
Eugenol
Spodoptera litura
LD50 ¼ 157.6 mg/larva
Sitophilus granaries
LD50~2.5 mg/beetle
Coptotermes formosanus Musca domestica Diabrotica virgifera Drosophila melanogaster Aedes aegypti
LD50~40 ppm (sand)
Hummelbrunner and Isman (2001) Obeng-Ofori and Reichmuth (1997) Cornelius et al. (1997)
Pulegone
Periplaneta americana Musca domestica Diabrotica virgifera Peridroma saucia Spodoptera litura Ostrinia nubilalis
Thymol
Citronellal
d-Limonene
Menthone
Drosophila melanogaster Musca domestica Drosophila melanogaster Drosophila melanogaster Spodoptera litura Culex pipiens molestus Spodoptera litura Callosobruchus maculatus Musca domestica Drosophila melanogaster Musca domestica Diabrotica virgifera Spodoptera litura Callosobruchus maculates Sitophilus oryzae Blattella germanica Musca domestica Sitophilus oryzae Drosophila melanogaster Peridroma saucia
LD50 ¼ 77 mg/fly LD50 ¼ 12 mg/beetle LD50 ¼ 4 mg/cm2 LD50~0.5 mg/43 cm2 paper LC50 ¼ 148 mg/cm2 paper LD50 ¼ 39 mg/fly LD50 ¼ 38 mg/beetle LD50 ¼ 753.9 mg/larva LD50 ¼ 51.6 mg/larva LC50 ¼ 26.3 ppm (in diet) LD50 ¼ 170 mg/4 mm dia paper LD50 ¼ 29 mg/fly LD50 ¼ 2600 mg/4 mm dia paper LD50 ¼ 5 mg/cm2 LD50 ¼ 25.4 mg/larva LC50 ¼ 36 mg/l LD50 ¼ 111.2 mg/larva
Lee et al. (1997) Isman (unpubl. Data) Bhatnagar et al. (1993) Ngoh et al. (1998) Lee et al. (1997) Harwood et al. (1990) Hummelbrunner and Isman (2001) Lee et al. (1999) Franzios et al. (1997) Lee et al. (1997) Franzios et al. (1997) Isman (unpubl. Data) Hummelbrunner and Isman (2001) Traboulsi et al. (2002)
LC50 ¼ 5.5 ml/l
Hummelbrunner and Isman (2001) Don-Pedro (1996)
LD50 ¼ 66 mg/fly LD50 ¼ 27 mg/cm2
Lee et al. (1997) Isman (unpubl. Data)
LD50 ¼ 50 mg/fly LD50 ¼ 88 mg/beetle LD50 ¼ 273.7 mg/larva
Lee et al. (1997)
LC50 ¼ 8.3 ml/l LC50 ¼ 61.5 ml/l LC50 ¼ 23 ml/l LD50 ¼ 98 mg/fly LC50 ¼ 12.7 ml/l LD50 ¼ 1290 mg/4 mm diameter paper LD50 ¼ 1735 mg/larva
Hummelbrunner and Isman (2001) Don-Pedro (1996) Lee et al. (2001) Coats et al. (1991) Lee et al. (1997) Lee et al. (2001) Franzios et al. (1997) Harwood et al. (1990)
(Continued)
Naturally occurring bioactive compounds
34 Table 2 (continued ) Compound
Insect tested
Effect/dose
Reference
Trans-anethole
Tribolium castaneum Musca domestica
LC50 ¼ 48.1 ml/l LD50 ¼ 75 mg/fly
Drosophila melanogaster Spodoptera litura
LD50 ¼ 7 mg/cm2
Ho et al. (1997) Marcus and Lichtenstein (1979) Isman (unpubl. Data)
Blattella germanica Cinnamaldehyde
Tribolium castaneum Coptotermes formosanus Trichoplusia ni
LD50 ¼ 65.5 mg/larva 80% mortality at 159 mg/cm2 LC50 ¼ 0.70 mg/cm2 96% mortality at 1 mg/g paper LD50 ¼ 176.3 mg/larva
Hummelbrunner and Isman (2001) Chang and Ahn, 2001 Huang and Ho (1998) Chang and Cheng (2002) Isman (unpublished)
Reichmuth, 1997; Shaaya and Kostjukovsky, 1998; Lee et al., 2001). Blood-feeding insects (Pediculus, Triatoma) have also been screened (Muncuoglu et al., 1996). Surprisingly, fewer crop pests have been examined in this regard. When all the data are reviewed collectively, certain plant species emerge as being consistently active against insects, although their rankings by potency vary greatly between pest species. Among the more active essential oils are those from thyme, oregano, basil, rosemary and mint, but much more empirical testing using less common plant species and a wider array of pest species will undoubtedly reveal particularly valuable biological activities (Shaaya and Kostjukovsky, 1998; Isman, 2005). Efficacy of pure constituents to insects Eight of the more potent essential oil constituents (monoterpenes and phenylpropenes) are listed in Table 2, along with their toxicity to various insect species if reported. Structures of these compounds are shown in Figure 1. Comparisons of toxicity among insect species for any particular compound are seldom relevant because of the differences in route of administration or exposure method between investigations. On the other hand, some studies have made direct comparisons of different compounds in one or more pest species (Lee et al., 1997; Hummelbrunner and Isman, 2001; Sampson et al., 2005). Accordingly, thymol and pulegone emerge as the most potent individual compounds, at least based on bioassays against the house fly Musca domestica, the tobacco cutworm Spodoptera litura and the turnip aphid Lipaphis pseudobrassicae. However, relative toxicity of individual compounds among pests can also be idiosyncratic. For example, in comparing the phenols eugenol and carvacrol and the monoterpenes a-terpineol and terpinen-4-ol for toxicity against four insect pest species and the twospotted spider mite, eugenol was the most toxic to two species (fruit fly and corn rootworm), terpinen-4-ol to two others (rice weevil and the spider mite), and carvacrol to the fifth (tobacco cutworm) (Isman, 2000). Comprehensive investigation into structure–activity relations of the toxicity of monoterpenes to insects has yielded few insights (Rice and Coats, 1994a), and efforts to enhance potency of
Pesticides based on plant essential oils
35
selected terpenes and phenols through derivitization produced very few analogues more potent than the parent natural products (Rice and Coats, 1994b; Tsao et al., 1995). In general though, phenols (e.g. thymol, eugenol) tend to be more active, both as insecticides and as fungicides, than the related monoterpenes. Essential oils as insect repellents The major use of insect repellents is for personal protection against biting arthropods, particularly mosquitoes. For almost 50 years the most heavily used and reliable insect repellent has been DEET (N,N-diethyl-m-touamide) (Peterson and Coats, 2001). One of the few viable alternatives to DEET for application to human skin has been preparations containing oil of citronella ( ¼ lemongrass oil). A number of such products containing citronella, often mixed with mint oil, are marketed in the United States, in spite of research indicating that they are far less efficacious than DEET (Fradin and Day, 2002). Other oils used in personal repellents include cinnamon oil, clove oil and soybean oil. Presumably these products, with modification, could be useful for protection of domestic animals (e.g. equestrian applications). Another use of repellents is for the removal of cockroaches and the prevention of reinfestation in human habitations and other buildings such as hospitals and restaurants. Certain essential oil constituents clearly repel cockroaches in a laboratory setting (Ngoh et al., 1998) and it is likely that at least part of the efficacy of commercial pest-control products based on essential oils is a consequence of ‘‘flushing’’ cockroaches from their harbourages and discouraging their return. Impregnating repellents into packaging to prevent insect infestation represents another potential use of essential oils that has yet to be realized. It is important to note that repellents could also prove useful in integrated pest management of agricultural crops, particularly in the context of a stimulodeterrent diversionary strategy (SDDS) or ‘‘pushpull’’ strategy (Miller and Cowles, 1990). Finally, it should be mentioned that certain essential oil constituents are effective attractants for some insects. For example, geraniol and eugenol are used as lures in traps for the Japanese beetle Popillia japonica and methyl eugenol has been used to trap Oriental fruit fly Dacus dorsalis (Vargas et al., 2000). Cinnamyl alcohol, 4-methoxy-cinnamaldehyde, cinnamaldehyde, geranylacetone and a-terpineol are attractive to adult corn rootworm beetles (Diabrotica spp.) (Hammack, 1996; Petroski and Hammack, 1998). Efficacy of oils and pure constituents to mites Some studies of the effects of essential oils or pure constituents against both parasitic and free-living mites are listed in Table 3. The tracheal mite Acarapis woodi and the varroa mite Varroa jacobsoni are economically important parasites of the honeybee Apis mellifera and there are few viable treatments for infested bee colonies. Menthol, thymol and some other compounds have been shown to have moderate efficacy as mite fumigants in beehives, although the difference in toxicity between the mites and their host bees (as little as 2–3 fold) requires that these substances be used precisely in practice. Essential oils rich in 1,8-cineole (tea tree oil, eucalyptus oil) have been shown to be effective against house dust mites. Several oils and their constituents are
Naturally occurring bioactive compounds
36
Table 3 Acaricidal activity of some essential oils and constituents Effective oil or compounds
Species tested
Reference
Menthol, thymol Blend of thymol, camphor, menthol and eucalyptus oil Citronellal, pulegone, terpin4-ol, piperitone Lavender oil, menthol, linalool Thymol, eugenol, menthol Wormwood oil, tansy oil Eucalyptus oil, Thuja oil, Melaleuca oil
Acarapis woodi Varroa jacobsoni
Ellis and Baxendale (1997) Calderone and Spivak (1995)
Ornithonyssus sylviarum
Carroll (1994)
Tyrophagus longior
Perrucci (1995)
Psoroptes cuniculi Tetranycus urticae Dermatophagoides pteronyssinus, D. farinae Tetranychus urticae
Perrucci et al. (1995) Chiasson et al. (2001) Yatagai (1997)
Tetranychus urticae, Phytoseilus persimilis Tetranychus urticae
Choi et al. (2004)
Carvomenthenol, terpinen-4ol, eugenol 53 plant essential oils Rosemary oil
Lee et al. (1997)
Miresmailli et al. (2006)
effective against the phytophagous spider mites, as commercial miticides based on rosemary or cinnamon oils attest (Isman, 2000; Miresmailli et al., 2006). Efficacy of oils and pure constituents to fungi Many plant essential oils and their major constituents have demonstrated antifungal activity against a range of plant pathogenic fungi including those responsible for both pre- and postharvest diseases. Some examples are listed in Table 4. Unlike insects, different fungal species yield more consistent results – thyme oil and its major phenolic constituents thymol and carvacrol are clearly active against most fungal species tested. The mechanism of action of these compounds against fungi is unknown but may be related to their general ability to dissolve or otherwise disrupt the integrity of cell walls and membranes. As a cautionary note, many of these oils and pure compounds show considerable phytotoxicity to plants at concentrations only slightly above those required for control of plant pathogenic fungi, presumably because plant cells are affected by a similar mechanism. Toxicity to mammals and other nontarget organisms Given the widespread use of plant essential oils in fragrances, as flavouring agents or condiments in food and in aromatherapy, we might expect these materials to be without appreciable toxicity in mammals. For most of the essential oil constituents toxic to insects and fungi this is the case (Table 5), although some may cause irritation of the skin, eyes and mucous membranes in concentrated form. But there are some notable exceptions. Pulegone is considerably toxic to rats (i.p. LD50 ¼ 150 mg/kg), although data on oral, acute toxicity has not been published. Pennyroyal oil, the source of pulegone, has been implicated in both human and animal poisonings
Pesticides based on plant essential oils
37
Table 4 Antifungal activity of some essential oils and constituents Effective oil or compounds
Species tested
Reference
Palmarosa, red thyme, cinnamon, clove bud oils Thymol, carvacrol
Botrytis cinerea
Wilson et al. (1997)
Botrytis cinerea, Monilinia fructicola Rhizoctonia solani, Fusarium moniliforme, Sclerotinia sclerotiorum Fusarium oxysporum 22 species of fungi Aspergillus niger Aspergillus spp. Aspergillus flavus
Tsao and Zhou (2000)
Thymol, carvacrol
Clove oil Cymbopogon spp. oils Cymbopogon nardus oil Thyme, oregano oils Thyme, oregano, cinnamon, basil, peppermint oils Thyme, oregano, marjoram, dictamus oils Thymol, eugenol, citronellol, cinnamaldehyde Thyme, mint, lavender oils
Muller-Riebau et al. (1995)
Penicillium digitatum
Bowers and Locke (2000) Singh et al. (1980) De Billerbeck et al. (2001) Paster et al. (1995) Montes-Belmont and Carvajal (1998) Daferera et al. (2000)
Seven species of fungi
Kurita et al. (1981)
Fusarium solani, Rhizoctonia solani, Pythium ultimum, Colletotrichum lindemuthianum
Zambonelli et al. (1996)
Table 5 Toxicity of some essential oil constituents to rats Compound
LD50 (mg/kg)a
1,8-Cineole (eucalyptol) Cinnamaldehyde Citral (from citronella oil) Eugenol d-Limonene Menthol Pulegone Terpinen-4-ol Thymol trans-Anethole
2480 1160 (guinea pig) 4960 2680 4600 (lowest published lethal dose) 3180 150 (mouse, intraperitoneal) 4300 980 (mouse ¼ 1800) 2090
a
Oral, acute unless otherwise specified. Source: Food and Agriculture Organization of the United Nations, 1999.
(Anderson et al., 1996). a-Thujone, the toxic constituent of the liqueur absinthe (made from wormwood oil), is also very toxic to rats (i.p. LD5045 mg/kg) (Hold et al., 2000). On the other hand, an essential-oil-based insecticide produced no mortality in rats when fed at 2 g/kg, and the LD50 exceeded 5 g/kg, the normal upper limit for testing required by regulatory agencies (E. Enan, unpubl. data). Eugenol is approximately
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1500 times less toxic than pyrethrum and 15,000 times less toxic than the organophosphate insecticide azinphosmethyl to juvenile rainbow trout Oncorhynchus mykiss, based on 96-h static water tests (Stroh et al., 1998). Effects on other nontarget organisms, namely, pollinators and natural enemies, have yet to be evaluated under field conditions. In the laboratory, a rosemaryoil-based insecticide/acaricide was significantly less toxic to predatory mites (Phytoseilus persimilis) than to their spider mite prey (Tetranychus urtica), although the whitefly parasitoid Encarsia formosa was more susceptible than its whitefly hosts (Miresmailli and Isman, unpublished data). However, most essential-oil constituents degrade quickly in the environment or are rapidly lost from plant foliage through volatilization, minimizing residual contact and favouring immigration or reintroduction of biocontrol agents and the foraging activities of honeybees and other pollinators.
Commercial products and uses In spite of considerable research effort in many laboratories throughout the world and an ever-increasing volume of scientific literature on the pesticidal properties of essential oils and their constituents, surprisingly few pest control products based on plant essential oils have appeared in the marketplace. This may be a consequence of regulatory barriers to commercialization (i.e. cost of toxicological and environmental evaluations) or the fact that efficacy of essential oils toward pests and diseases is not as apparent or obvious as that seen with currently available products. Nonetheless, some U.S. companies have introduced essential-oil-based pesticides in recent years. Mycotech Corporation produced an aphidicide/miticide/fungicide for greenhouse and horticultural crops and for bush and tree fruits based on cinnamon oil with cinnamaldehyde (30% in the EC formulation) as the active ingredient. However, this product is no longer being sold. EcoSMART Technologies has introduced insecticides containing eugenol and 2-phenethyl propionate aimed at controlling crawling and flying insects, under the brand name EcoPCOR for pest control professionals. An insecticide/miticide containing rosemary oil as the active ingredient has recently been introduced for use on horticultural crops under the name EcoTrolTM. Another product based on rosemary oil is a fungicide sold under the name SporanTM, while a formulation of clove oil (major constituent: eugenol), sold as MatranTM, is used for weed control. All of these products have been approved for use in organic food production. Several smaller companies in the U.S. and the U.K. have developed garlicoil-based pest control products and in the U.S. there are consumer insecticides for home and garden use containing mint oil as the active ingredient. Menthol has been approved for use in North America for control of tracheal mites in beehives, and a product produced in Italy (Apilife VARTM) containing thymol and lesser amounts of cineole, menthol and camphor is used to control Varroa mites in honeybees.
Conclusion and future prospects Pesticides based on plant essential oils or their constituents have demonstrated efficacy against a range of stored product pests, domestic pests, blood-feeding pests and certain soft-bodied agricultural pests, as well as against some plant pathogenic fungi
Pesticides based on plant essential oils
39
responsible for pre- and post harvest diseases. They may be applied as fumigants, granular formulations or direct sprays with a range of effects from lethal toxicity to repellence and/or oviposition deterrence in insects. These features indicate that pesticides based on plant essential oils could be used in a variety of ways to control a large number of pests. In general, the efficacy of these materials falls short when compared to synthetic pesticides although there are specific pest contexts where control equivalent to that with conventional products has been observed. Essential oils also require somewhat greater application rates (as high as 1% active ingredient) and may require frequent reapplication when used out-of-doors. Additional challenges to the commercial application of plant essential-oil-based pesticides include availability of sufficient quantities of plant material, standardization and refinement of pesticide products, protection of technology (patents) and regulatory approval (McChesney, 1994; Isman, 2005). Although many essential oils may be abundant and available year round due to their use in the perfume, food and beverage industries, large-scale commercial application of essential-oil-based pesticides could require greater production of certain oils. Pesticides derived from essential oils of relatively rare plants would not be easily produced due to a lack of sufficient quantities of plant material, limiting the essential oils of use to those which are readily available or from those plants which can be easily cultivated (e.g. in plantations) (McChesney, 1994). In addition, as the chemical profile of plant species can vary naturally depending on geographic, genetic, climatic, annual or seasonal factors, pesticide manufacturers must take additional steps to ensure that their products will perform consistently. All of this requires substantial cost and smaller companies may not be willing to invest the required funds unless there is a high probability of recovering the costs through some form of market exclusivity (e.g. patent protection). Finally, once all of these issues are addressed, regulatory approval is required. Although several plant essential oils are exempt from registration in the United States, many more oils are not, and few countries currently have such exemption lists. Accordingly, regulatory approval continues to be a barrier to commercialization and will likely continue to be a barrier until regulatory systems are adjusted to better accommodate these products. Nonetheless, pesticides derived from plant essential oils do have several important benefits. Due to the rapid volatilization of these products, there is a much lower level of risk to the environment than with current synthetic pesticides. Predator, parasitoid and pollinator insect populations will be less impacted as a result of the minimal residual activity, making essential-oil-based pesticides compatible with integrated pest management programs. Further, while resistance development continues to be an issue for many synthetic pesticides, it is likely that resistance will develop more slowly to essential-oil-based pesticides owing to the complex mixtures of constituents that characterize many of these oils. Ultimately, it is in developing countries where the source plants are endemic that these pesticides may ultimately have their greatest impact. However, while commercial development of plant essential-oil-based pesticides may face a variety of problems, it is evident that where human and animal health are at a premium and minimal residues are desirable, it is worth the effort to surmount these challenges. It is therefore expected that these pesticides will find their greatest commercial application in urban pest control, public health, veterinary health, mosquito abatement
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and in stored product protection. In agriculture, these pesticides will be most useful for protected crops (e.g. greenhouse crops), high-value row crops and within organic food production systems where few alternative pesticides are available. As conventional, hazardous insecticides are removed from the marketplace and the cost of developing new products continues to rise, it is likely that pesticides based on plant essential oils will gain further acceptance.
Acknowledgment We thank Dr. E. Enan (Vanderbilt University, Nashville, TN, USA) and Mr. S. Miresmailli (University of British Columbia) for allowing us to include their unpublished data. Supported by grants from NSERC and EcoSMART Technologies Inc. to MBI.
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CHAPTER 3
Natural substrates and inhibitors of multidrug resistant pumps (MDRs) redefine the plant antimicrobials GEORGE P TEGOS
Introduction Efflux mechanisms have become broadly recognized as major components of resistance to many classes of antibiotics. Some efflux pumps selectively extrude specific antibiotics, while others referred to as multidrug resistance pumps (MDRs) expel a variety of structurally diverse compounds with differing modes of action. Based on sequence similarity, multidrug transporter systems are classified into six super-families (Paulsen et al., 2002) (i) ATP binding cassette transporters (ABC), (ii) major facilitators (MF), (iii) resistance nodulation division (RND), (iv) small multidrug resistance (SMR), (v) multidrug and toxic compound extrusion (MATE), and (vi) multidrug endosomal transporter (MET) family. The first five families of multidrug systems are found mostly in microorganisms (the MET family appears restricted to higher eukaryotes) but representatives of all these families are also present in mammalian cells. Multidrug resistant human pathogenic microorganisms are directly associated with serious recalcitrant infections such as cystic fibrosis, nosocomial infections, and infections in immunocompromised patients undergoing anticancer chemotherapy or infected with HIV. A novel and promising approach to deal with multidrug resistance is improving the clinical performance of various antibiotics by the inhibition of efflux pumps. Many companies and research laboratories have undertaken programs to discover and develop efflux pump inhibitors and in many cases their attempts were successful. In addition, it was clearly demonstrated that an efflux pump inhibitor (reserpine) inhibits effectively not only the bacterial but the mammalian MDR ABC-transporter P-glycoprotein (P-gp) as well (Beck et al., 1988; Wang et al., 2001), thus implying that the development of broad range MDR inhibitors is not a chimera. Plants produce an enormous array of secondary metabolites and it is commonly accepted that a significant part of this chemical diversity serves to protect plants against microbial pathogens (Dixon, 2001). These plant substances are classified as either phytoanticipins, compounds present constitutively, or phytoalexins, whose
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levels increase strongly in response to microbial invasion (Morrissey and Osbourn, 1999). Plant compounds are routinely classified as ‘‘antimicrobials’’ on the basis of susceptibility tests that produce minimal inhibitory concentrations (MICs) in the range of 100–1000 mg/ml, orders of magnitude weaker than typical antibiotics produced by bacteria and fungi (MICs of 0.01–1 mg/ml) (Cowan, 1999). Simply because a compound is synthesized in response to pathogen invasion, is required for protecting the plant from a pathogen but shows little activity in an in vitro susceptibility test it is not necessarily an antimicrobial. Such a substance might have a regulatory function, which indirectly increases plant resistance. This analysis suggests that we currently lack a solid rationale for making a functional assignment for the vast majority of plant compounds that have been classified as antimicrobials. Helpful evidence regarding the possible function of plant secondary metabolites is that these compounds often show considerable activity against Gram-positive bacteria, but not against Gram-negative species or yeast (Lewis et al., 2001). Both yeast and Gram-negative bacteria have evolved significant permeability barriers. Gram-negative species have an outer membrane that is a fairly effective barrier for amphipathic compounds and a set of MDRs that extrude amphipathic toxins across the outer membrane. The single membrane of Gram-positive bacteria is considerably more accessible to permeation by amphipathic toxins in regions where MDRs provide limited protection. In yeast, the presence of ergosterol, which decreases permeability, combined with a set of broad-specificity MDRs also provides an effective barrier (Lomovskaya and Lewis, 1992; Nikaido, 1999; Lewis, 2001). Several Gram-positive bacteria invade plants, but the majority of plant pathogens are Gramnegative bacteria, or yeast and related fungi (Rogers et al., 2001). In this chapter we shall emphasize the rationale for characterization of natural substrates of MDR pumps, reporting the identification of the first MDR substrates and inhibitors from plants against bacteria and yeasts. We shall discuss the methodology for the isolation of these compounds as well as the specificity of their activity. Finally we shall analyze the current and future perspectives to natural drug discovery based on this classification of plant antimicrobials.
The quest for natural substrates – rationale Even though MDR pumps extrude structurally unrelated compounds, a general theme emerges if one considers the preferred artificial substrates of most MDRs. These substances are mostly amphipathic cations. It has been suggested that amphipathic cations represent the existing natural substrates of MDR pumps (Lewis, 1999). The simplest MDRs of the SMR family have amphipathic cations as their exclusive substrates, at least in Gram-positive bacteria (Chung and Saier, 2001). Most of the MDR members of the MF super-family extrude exclusively amphipathic cations. The QacA pump only extrudes cations (Mitchell et al., 1998); the NorA pump of the human pathogen Staphylococcus aureus extrudes cations and some quinolones (Kaatz et al., 2000); the BMR pump of Bacillus subtilis extrudes primarily cations and neutral chloramphenicol (Van Bambeke et al., 2000; VazquezLaslop et al., 2000). RND super-family MDRs have a broad substrate spectrum, including amphipathic cations (Elkins and Nikaido, 2002; Mao et al., 2002; Murata
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47
et al., 2002) that are preferential substrates in the case of ABC transporters (Lage, 2003). Simple MDRs like SMRs only export amphipathic cations (Krupka, 1999); MF and MATE MDRs export mainly cations; RND and ABC have broad spectra of specificity that include amphipathic cations as preferred substrates. It appears that a similar need to protect the cell from amphipathic cations evolved in different groups of MDRs (and in different organisms) in spite of a lack of overall homology or similarity in the action mechanism. Quite surprisingly, one does not find amphipathic cations in a general list of natural antimicrobials. (The known cationic antibiotics of the aminoglycoside group such as streptomycin and kanamycin are hydrophilic substances that get smuggled into the cell via specific translocases and are not general substrates of MDRs).
MDR inhibitors – proof of principle Research efforts in a series of academic and industrial laboratories led to the identification of numerous inhibitors of the NorA pump of S. aureus (Aeschlimann et al., 1999; Markham et al., 1999; Gibbons et al., 2003; Kaatz et al., 2003) and MexABOprM, MexCD-OprJ, MexEF-OprN MDRs of Pseudomonas aeruginosa (Renau et al., 1999; Lomovskaya et al., 2001). The inhibitors INF271 (NorA-Influx Co., Chicago, Illinois) and MC207110 (MexAB-OprM, MexCD-OprJ, MexEF-OprN P. aeruginosa Essential Therapautics-Mountain View, CA) have been extremely valuable tools for drug discovery. INF271 identified by screening a synthetic chemical library using ethidium bromide (EtBr) as a substrate in a sub-inhibitory concentration. On the contrary, in the case of MC207110 an array of synthetic compounds and natural product extracts was assayed using strains of P. aeruginosa over-expressing each of the three pumps in the presence of levofloxacin. To qualify as MDR inhibitors hit compounds had to meet several criteria: (i) must enhance the activities of levofloxacin and other antibiotics that are effluxes in strains containing functioning pumps, (ii) must not significantly potentiate the activities of antibiotics in a strain that lacks efflux pumps, (iii) must not potentiate the activities of antibiotics that are not effluxes, (iv) must increase the level of accumulation and decrease the level of extrusion of efflux pump substrates, and (v) must not affect the proton gradient across the inner membrane (Table 1).
MDR mutants and inhibitors valuable tools for natural drug discovery It seems plausible that MDRs evolved in response to natural antimicrobial amphipathic cations and this fact makes these substances difficult to discover in standard screens that employ cells carrying MDR pumps. The development of MDR mutants for the NorA of S. aureus gives an answer to this apparent paradox (Hsieh et al., 1998) and serves as a sensitive tool for drug discovery. The plant isoquinoline alkaloids, berberine, and palmatine resemble artificial MDR substrates such as EtBr and are widely spread among the plant world especially in species of Ranunculales (Schmeller et al., 1997). A susceptibility test to identify MICs for these compounds against the wild type and the NorA mutant of S. aureus was performed. Palmatine
Naturally occurring bioactive compounds
48 Table 1 Natural MDR inhibitors H N
O HN
H2 N
OMe O
O
H N
N H
O
HO
O
NH
OH OMe
O
OH
MeO
N H
INF271
NH2
OH O
MC207110
OMe
5-MHC Berberis sp., Mahonia sp. OH
O
MeO MeO OH
Pheophorbide a
R4O
OR5
2'
3' rha 1' 4'
R3O
OR OMe
O
Chrysoplenol D /Chrysoplenetin Artemisia annua
HO 3 2
O O
OR2 glu 4 1
O
5'
CH3
5
O 6
OR1
6'
1: R1 = R2 = R4 = R5 = H; R3 = CO(CH2)14 CH3 2: R1= -CO(CH2)14CH3; R2 = R4 = R5 = H; R3 = -COPr 3: R1 = R2 = R3 = -COiPr; R4 = -COPr 4: R1 = R2 = R3 = -COiPr; R4 = OAc; R5 = COPr
Neohesperidosides Geranium caespitosum
Dalea versicolor
Dalea versicolor
has very low (>200 mg/ml MIC) and berberine poor (250 mg/ml) activity against wild type S. aureus. The antimicrobial activity of the alkaloids increased sharply in a norA mutant, with an MIC of 50 mg/ml for palmatine and 7.5 mg/ml for berberine. Berberine resembles EtBr and binds to DNA (Jennings and Ridler, 1983). The DNA binding apparently contributes to the antimicrobial activity of berberine. Similar to EtBr, DNA-bound berberine has increased fluorescence and is used as a model plant antimicrobial for uptake studies. Efflux of berberine was more rapid than EtBr. Similarly, the differential rate of berberine accumulation in both strains indicated that these alkaloids represent the first identified natural plant substrates of the MFS MDRs of S. aureus (Stermitz et al., 2000a). The outer membrane of Gram-negative bacteria is a barrier to a broad range of amphipathic compounds, and to all large molecules, which allows for evolving of broadly specific MDRs that pump their various substrates across this barrier.
Natural substrates and inhibitors of MDRs
49
Similarly, Gram-positive species that lack an outer membrane have MDRs with a reported substrate spectrum largely limited to amphipathic cations. Apart from the usefulness of protecting the cell from this class of compounds that actively accumulate in the cell, the relatively slow permeation of amphipathic cations across the cytoplasmic membrane made the evolution of ‘‘cation-pumping’’ MDRs possible, as compared to neutral compounds of similar polarity.
A new series of promising plant antimicrobials Similar studies aimed at finding the basis of Gram-negative bacterial resistance to plant antimicrobials. In Rhizobium etli, there is an operon activated by a number of plant phytoalexins. The operon appeared to code for an RmrAB MDR pump (Gonzalez-Pasayo and Martinez-Romero, 2000). Mutant rmrAB had a diminished ability for root colonization, and increased susceptibility for phytoalexins naringenin and coumaric acid. However, the difference in susceptibility between wild type and mutant is very small, around 30%. In Agrobacterium tumefaciens, coumestrol, an antifungal phytoalexin of soybeans induces expression of an LfeAB MDR (Palumbo et al., 1998). Mutation in the pump increases accumulation of coumestrol in the pathogen, and the mutant was out competed by the wild type in colonizing the plant. Neither the wild type nor the mutant, however, is sensitive to coumestrol. In a slightly different approach a panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant and human pathogens and applying a combination of MDR mutants and MDR inhibitors (Tegos et al., 2002). One of the main observations was the strong potentiation of antimicrobial action against strains with disabled MDRs. The activities of the majority of plant antimicrobials were considerably greater against the Gram-positive bacteria S. aureus and B. megaterium and disabling of the MDRs in Gram-negative species led to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb (Agarwal et al., 2000), was potentiated 100–2000-fold (depending on the bacterial species) by disabling the MDRs. A similar effect was observed with plumbagin (Didry et al., 1994), resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of Gram-negative bacteria (Figure 1). Recently the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16 was identified and characterized (Barabote et al., 2003). The gene encodes a 51-kDa protein with 70% identity to its Escherichia coli homolog. The E. chrysanthemi gene functionally complements the E. coli tolC gene in MDR efflux pumps. A tolC mutant of E. chrysanthemi is extremely sensitive to an array of plant-derived chemicals including berberine, rhein, plumbagin, pyrithione, genistein (40 ,5,7-trihydroxyisoflavone), p-coumaric acid and t-cinnamic acid (phenolic acids), (2-mercaptopyridine-1-oxide) and esculetin (6,7-dihydroxycoumarin). This mutant is unable to grow in planta and its ability to cause plant tissue maceration is severely compromised and is defective in the efflux of berberine. The E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue conferring resistance to plant antimicrobials.
Naturally occurring bioactive compounds
50 200
A
150
RFU
RFU
150
100
50
0
B
200
100
50
0
5
10
15
20
25
0
30
0
5
Time (min)
C
20
25
30
D
200
150
150
RFU
RFU
15
Time (min)
200
100
50
0
10
100
50
0
5
10
15
20
25
30
Time (min)
0
0
5
10
15
20
25
30
Time (min)
200
E
RFU
150
100
50
0
0
5
10
15
20
25
30
Time (min)
Fig. 1. Accumulation of berberine by Gram-negative bacteria. Uptake of berberine without (o) or with MC207110 (i), INF271 (D), MC207110/INF271 (}) by cells of Salmonella typhimurium (A), Pseudomonas syringae (B), Xanthomonas campestris (C), Erwinia carotovora (D), and Sinorhizobium meliloti (E) was measured by fluorescence increase following binding to DNA and expressed as RFU (relative fluorescence units). Berberine was present at a concentration of 30 mg/ml; MC207110 and INF271 were added at the same final concentrations as for the MIC determinations.
Natural MDR inhibitors from plants – an increasing body of evidence, 50 methoxyhydnocarpin and synergy in Berberis plants The rationale behind detecting MDR inhibitory activity is to test the combined action of a plant extract (the nonalkaloid fraction) with a weak plant antimicrobial coming from the same plant added at a sub-inhibitory concentration (Table 2). Extracts that inhibit cell growth in the presence of the antimicrobial and have no
Natural substrates and inhibitors of MDRs
51
Table 2 Identified natural substrates or MDRs O
OCH H3 OCH H3
O
O OH
N +
N +
CH3O OCH3
OMe Berberine
OH O
O
Palmatine O
HO
OH
Plumbagin
O N
O
COOH
SH
O
O
OH
Rhein
Coumestrol
Zink-Pytothione OH
OHC
OH
OH
HO
HO CHO OH
HO
HO
O
O
Me
OH
Me
Resveratrol
Gossypol
Esculetin
O O
O
O
O O
Asarinin
activity when added alone are likely to contain an MDR inhibitor. A bioassay-driven purification is the most common technique used to detect MDR inhibitors. Several Berberis medicinal plants (Berberis repens, B. aquifolia, and B. fremontii) producing berberine were found also to synthesize an inhibitor of the NorA MDR pump S. aureus. The inhibitor 50 -methoxyhydnocarpin (50 -MHC), a flavonolignan, was previously identified in Hydnocarpus wightiana (Flacourtaceae) (Ranganathan and Seshadri, 1974) and reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy (Norton, 1994). 50 -MHC-D is an amphipathic weak acid and is
52
Naturally occurring bioactive compounds
distinctly different from the cationic substrates of NorA. 50 -MHC-D had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of EtBr and berberine from S. aureus cells was completely inhibited by 50 -MHC-D. The level of accumulation of berberine in the cells was increased strongly in the presence of 50 -MHC-D, indicating that this plant compound effectively disables the bacterial resistance mechanism against berberine (Stermitz et al., 2000a, 2000b). This first identified natural MDR inhibitor is abundant in other Berberis sp. (Musumeci et al., 2003).
Inhibitors against MFS MDRs Identification of 50 -MHC-D intensified the search for natural MDR inhibitors of plant origin and made the idea of synergy in plant antimicrobials more plausible. Bioassay-driven purification and structural determination from various plant sources yielded a number of new MDR inhibitors acting against Gram-positive bacteria with activity similar to 5-MHC pheophorbide a from Berberis sp., and Mahonia (Stermitz et al., 2000a, 2000b, 2001), crysoplenol and crysoplenetin from Artemisia annua (Stermitz et al., 2002), dicaffeoylquinic acids from A. absinthium (Fiamegos et al., 2003), polyacylated neohesperidosides from Geranium caespitosum (Stermitz et al., 2003), diterpenoids from Pinus edulis, (F.R. Stermitz personal communication), and chalcones and a stilbene from Dalea versicolor (Belofsky et al., 2003). Additionally, isoflavones such as genistein, orobol, and biochanin exhibit MDR inhibitory activity against the NorA pump of Gram-positive bacteria (Morel et al., 2003) and aminoquinolines in the AcrAB-TolC pump of E. coli (Mallea et al., 2003).
Inhibitors against ABC transporters P-gp the mammalian ABC transporter is mainly responsible for the unsuccessful chemotherapy of cancers through occurrence of MDR (Leonard et al., 2003). P-gp shares high structural homology with yeast ABC transporters (Saier and Paulsen, 2001). The development of screening methodologies has significantly enhanced the number and the efficiency of available inhibitors (Hiraga et al., 2001; Varma et al., 2003). The inhibitory activity of numerous plant compounds against P-gp such as alkaloids, polymethoxyflavones, diterpenoids, camptothecins (Ulukan and Swaan, 2002), polyphenols (Jodoin et al., 2002), ascorbic acid, vitamins, and others has been extensively studied (Chen and Waxman, 1995; Ikegawa et al., 2000; Foster et al., 2001; Motohashi et al., 2001; Perloff et al., 2001; Deferme et al., 2002; Appendino et al., 2003; Corea et al., 2003; El-Masry and Abou-Donia, 2003). Likewise, many plant sources and products seem quite promising for the identification of new P-gp specific inhibitors: sweet pepper ‘‘Anastasia green’’ (Motohashi et al., 2001), St. John’s Wort (Hypericum perforatum) (Perloff et al., 2001), caper spurge (Euphorbia lathyris) (Appendino et al., 2003), and apricots (Deferme et al., 2003). A novel and interesting inhibitor from G. caespitosum (Figure 2) shows interesting properties. It potentiates berberine in S. aureus, erythromycin in P. syringae pv. maculicola and fluconazole against Saccharomyces cerevisae and Candida albicans.
Natural substrates and inhibitors of MDRs
53
Fig. 2. Geranium caespitosum a paradigm of synergy between plant antimicrobials.
In addition, a structurally similar compound from the same source has no antimicrobial action alone, but combined with the inhibitor is active against P. syringae (a designated pathogen of Geranium sp.) and S. cerevisae (Tegos et al., 2006 (unpublished results)).
Conclusions and future perspectives The experimental data available demonstrate and reassure that plants produce MDR inhibitors against Gram-positive bacteria and yeasts. An effective plant MDR inhibitor of Gram-negative bacteria is still a missing part of the puzzle. Currently, there is only preliminary evidence for this, but the identification of such a compound is more than plausible. A comprehensive library of extracts from the National Cancer Institute (NCI-USA), which consists of over 50,000 extracts from approximately 35,000 plants from all over the world is currently being screened (Tegos et al., 2006 (unpublished results)). The collection represents most genera of vascular plants found in the tropics. The extracts are tested against a panel of representative microorganisms (S. aureus, E. coli, S. cerevisiae) for direct antimicrobial activity. Furthermore, they are tested for MDR inhibitory activity with a sub-inhibitory amount of antibiotic. A pilot screen produced extremely promising hit rates for direct and MDR antimicrobial activity (Figure 3). It is significant to find MDR inhibitory activity against Gram-negative bacteria of which no known natural inhibitors have
Naturally occurring bioactive compounds
54 0.19 0.57 0.76
0.57 0.38 0.19
1.32 2.65 6.25 7.95 10.41
68.75
Inactive
S. cerevisae[MDR]
S. aureus[D], E. coli[D]
S. aureus [D]
S. aureus[D], S. cerevisae[D][D]
S. aureus[D], E.coli[MDR]
S.aureus[MDR]
S. aureus[D], S. cerevisae[MDR]]
S. aureus[MDR], E. coli[MDR]]
S. serevisae [D]
S. aureus[MDR], S. cerevisae[MDR]]
S. aureus[D], E. coli[D], S.cerevisae [MDR]
Fig. 3. Hit rates of extracts from the Natural Products Repository Program (National Cancer Institute-USA, http://dtp.nci.nih.gov/branches/npb/repository.html). A pilot screen of 524 extracts (352 organic/176 aqueous) produced very promising hit rates for the identification of new MDR inhibitors and plant antimicrobials (see text). The majority of hits came from organic extracts. [D] – Direct, [MDR] – MDR-inhibitory activity.
been identified. Extracts with MDR inhibitory activity and without direct detectable activity suggest that antimicrobials from these plants exist. Interestingly, a number of instances exist where a specific part of the same plant produced direct activity against S. aureus; while an extract from another part exhibits MDR inhibitory activity. Closer examination may demonstrate both activities operating at different levels in the entire plant. Similar examples of synergistic activities coming from the same plant are observed in the case of E. coli and S. cerevisiae. The potential activity of plant antimicrobials when they gain access to the microbial cell is only part of the intriguing puzzle. Even if a plant uses a synergistic combination of an antimicrobial and an MDR inhibitor, this does not in itself explain why plants do not come up with self-sufficient antibiotics like aminoglycosides or tetracycline. Plants are stationary, slowly evolving organisms that confront mobile, rapidly evolving pathogens. Resistance to antibiotics such as streptomycin or tetracycline can be gained by a single mutation leading to target modification, or to over-expression of an MDR pump (Lewis, 2001). It is possible that plants employ an antimicrobial strategy aimed at limiting the probability of resistance development. This is apparently the case when plants produce MDR inhibitors, directly targeting the microbial resistance mechanism. Importantly, disabling MDRs also results in a dramatic decrease in acquisition of resistance based on target modification (Zheleznova et al., 1999; Lomovskaya and Watkins, 2001; Markham and Neyfakh, 2001; Lomovskaya et al., 2003). There is another intriguing possibility that plants may employ to limit resistance development. Resistance will not be easily achieved against a substance that hits
Natural substrates and inhibitors of MDRs
55
more than one target. Apparent low activity of an antimicrobial could then be a result of a trade-off – penetration capability is sacrificed in favor of additional functionality, an ability to hit two different targets, while access to the pathogen cell is ensured by a separate molecule, an MDR inhibitor. Do plant antimicrobials hit more than one target? Answering this question might be highly significant for combating the rapid rise of multidrug resistance. Antimicrobials able to act against more than one target would be extremely valuable as therapeutics. Interestingly, a large-scale ‘‘experiment’’ on resistance development to this type of compounds has been performed unwittingly. Synthetic fluoroquinolones act against two different targets – DNA gyrase and topoisomerase (Hooper, 2001, 2002). Resistance by target modification occurs in a series of steps and is a low-probability event. It was shown that blocking MDR pumps of pathogens by an inhibitor decreases resistant mutation development to fluoroquinolones below the limit of detection (o1011). Blocking MDRs increases the effective concentration of fluoroquinolone in the cell and makes the first step in the target modification process ineffective. In retrospect, this means that were fluoroquinolones applied originally in combination with an MDR inhibitor, we might have had a case of largely resistance-proof therapy. It is too late for fluoroquinolones, the resistance to this class of compounds has already developed. But we might still have a chance, or many chances, with potentially dual/multiple target acting plant antimicrobials combined with an MDR inhibitor. There is not sufficient information concerning the plant antimicrobial action mechanisms. Some useful indirect evidence, however, does point to more than one target for at least some compounds. Rhein is effective at mg/ml levels against yeast and Gram-positive bacteria (Cyong et al., 1987; Hatano et al., 1999) and was found specifically to inhibit NADH dehydrogenase in mitochondria (Kean et al., 1970). It is assumed that microbial NADH dehydrogenase is the target of action. At the same time, rhein was reported to be very effective against Gram-positive oral pathogens under anaerobic conditions (Didry et al., 1994) and is very effective against E. coli tolC (defective in major MDRs) under both aerobic and anaerobic conditions. Since NADH dehydrogenase in not functional under anaerobiosis, there should be a second target. Rhein is currently used as a systemic drug against arthritis-administered in the form of a prodrug diacerein (Spencer and Wilde, 1997).
Acknowledgments The author thanks Mahendra Rai for his invitation to participate in this text as well as Kim Lewis (Northeastern University) for continuous support and critical supervision and Frank R. Stermitz (Colorado State University) as a colleague and a mentor in the organic chemistry of natural products. Most of the author’s work was carried out in Kim Lewis Molecular Microbiology Laboratory, Department of Biology at Northeastern University and supported by the NIH grant RO1GM59903 (In search of Natural Substrates and Inhibitors of MDR pumps). Gil Belofsky (University of Tulsa) and John Fiamegos (University of Ioannina) offered valuable feedback information. Susan Harvey and Jennifer Freedman provided significant help revising this manuscript.
56
Naturally occurring bioactive compounds
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Mao W, Warren M, Black DS, Satou T, Murata T, Nishino T, Gotoh N, Lomovskaya O. (2002) On the mechanism of substrate specificity by resistance nodulation division (RND)type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition. Mol Microbiol 46:889–901. Markham PN, Neyfakh AA. (2001) Efflux-mediated drug resistance in Gram-positive bacteria. Curr Opin Microbiol 4:509–514. Markham PN, Westhaus E, Klyachko K, Johnson ME, Neyfakh AA. (1999) Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus. Antimicrob Agents Chemother 43:2404–2408. Mitchell BA, Brown M, Skurray RA. (1998) QacA multidrug efflux pump from Staphylococcus aureus: comparative analysis of resistance to diamidines, biguanidines, and guanylhydrazones. Antimicrob Agents Chemother 42:475–477. Morel C, Stermitz F, Tegos G, Lewis K. (2003) Isoflavones as potentiators of antibacterial activity. J Agric Food Chem 51:5677–5679. Morrissey JP, Osbourn A. (1999) Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiol Mol Biol Rev 63:708–724. Motohashi N, Kurihara T, Wakabayashi H, Yaji M, Mucsi I, Molnar J, Maruyama S, Sakagami H, Nakashima H, Tani S, Shirataki Y, Kawase M. (2001) Biological activity of a fruit vegetable, ‘‘Anastasia green,’’ a species of sweet pepper. In Vivo 15:437–442. Murata T, Kuwagaki M, Shin T, Gotoh N, Nishino T. (2002) The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component. Biochem Biophys Res Commun 299:247–251. Musumeci R, Speciale A, Costanzo R, Annino A, Ragusa S, Rapisarda A, Pappalardo MS, Lauk L. (2003) Berberis aetnensis C, Presl. extracts: antimicrobial properties and interaction with ciprofloxacin. Int J Antimicrob Agents 22:48–53. Nikaido H. (1999) Microdermatology: cell surface in the interaction of microbes with the external world. J Bacteriol 181:4–8. Norton S. (1994) Useful plants of dermatology. I. Hydnocarpus and chaulmoogra. J Am Acad Dermatol 31:683–686. Palumbo JD, Kado CI, Phillips DA. (1998) An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots. J Bacteriol 180:3107–3113. Paulsen IT, Chen J, Nelson KE, Saier Jr. MH. (2002) Comparative genomics of microbial drug efflux systems. In: ITPaK Lewis editor. Microbial Multidrug Efflux. Norfolk: Horizon Press, pp. 5–21. Perloff MD, von Moltke L, Stormer E, Shader RI, Greenblatt DJ. (2001) Saint John’s wort: an in vitro analysis of P-glycoprotein induction due to extended exposure. Br J Pharmacol 134:1601–1608. Ranganathan KR, Seshadri T. (1974) Minor phenolic components of seed hulls of HydnocarpusWightiana – Constitution of methoxyhydnocarpin. Indian J Chem 12:993–996. Renau TE, Leger R, Flamme EM, Sangalang J, She MW, Yen R, Gannon CL, Griffith D, Chamberland S, Lomovskaya O, Hecker SJ, Lee VJ, Ohta T, Nakayama K. (1999) Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin. J Med Chem 42:4928–4931. Rogers B, Decottignies A, Kolaczkowski M, Carvajal E, Balzi E, Goffeau A. (2001) The pleitropic drug ABC transporters from Saccharomyces cerevisiae. J Mol Microbiol Biotechnol 3:207–214. Saier Jr. MH, Paulsen IT. (2001) Phylogeny of multidrug transporters. Semin Cell Dev Biol 12:205–213. Schmeller T, Latz-Bruning B, Wink M. (1997) Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores. Phytochemistry 44:257–266. Spencer CM, Wilde MI. (1997) Diacerein. Drugs 53:98–106; discussion 107–108. Stermitz FR, Beeson T, Mueller PJ, Hsiang J, Lewis K. (2001) Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu strictu) species. Biochem Syst Ecol 29:793–798.
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Stermitz FR, Cashman K, Halligan KM, Morel C, Tegos GP, Lewis K. (2003) Polyacylated neohesperidosides from Geranium caespitosum: bacterial multidrug resistance pump inhibitors. Bioorg Med Chem Lett 13:1915–1918. Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA, Lewis K. (2000a) Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 50 -methoxyhydnocarpin, a multidrug pump inhibitor. Proc Natl Acad Sci USA 97:1433–1437. Stermitz FR, Scriven LN, Tegos G, Lewis K. (2002) Two flavonols from Artemisa annua which potentiate the activity of berberine and norfloxacin against a resistant strain of Staphylococcus aureus. Planta Med 68:1140–1141. Stermitz FR, Tawara-Matsuda J, Lorenz P, Mueller P, Zenewicz L, Lewis K. (2000b) 50 -Methoxyhydnocarpin-D and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J Nat Prod 63:1146–1149. Tegos G, Higginbotham A, Ball A, Stermitz FR, Lewis K. (2006) Neohesperidosides from Geranium caespitosum; versatile multidrug pump inhibitors Chemistry & Biology, submitted. Tegos G, Stermitz FR, Lomovskaya O, Lewis K. (2002) Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob Agents Chemother 46:3133–3141. Ulukan H, Swaan PS. (2002) Camptothecins: a review of their chemotherapeutic potential. Drugs 62:2039–2057. Van Bambeke F, Balzi E, Tulkens PM. (2000) Antibiotic efflux pumps. Biochem Pharmacol 60:457–470. Varma MV, Ashokraj Y, Dey CS, Panchagnula R. (2003) P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement. Pharmacol Res 48:347–359. Vazquez-Laslop N, Zheleznova EE, Markham PN, Brennan RG, Neyfakh AA. (2000) Recognition of multiple drugs by a single protein: a trivial solution of an old paradox. Biochem Soc Trans 28:517–520. Wang EJ, Casciano C, Clement RP, Johnson WW. (2001) Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors. Biochem Biophys Res Commun 289:580–585. Zheleznova EE, Markham PN, Neyfakh AA, Brennan RG. (1999) Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter. Cell 96:353–362.
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CHAPTER 4
New concept to search for alternate insect control agents from plants ISAO KUBO
Introduction There is no doubt that many plant secondary metabolites affect insect behavior, development and reproduction. Identifying these substances is an important first step in understanding the effect of plants on insect life at the molecular level. Besides its benefits to basic science, accumulation of this kind of knowledge may provide us with a more rational and scientific approach to insect pest control. Continuing search for alternate insect control agents from plants resulted in characterization of many active phytochemicals. Despite their excellent and interesting activities, the use of these botanical insect control agents is still limited. The reason for this limitation is caused mainly by the lack of availability of materials and/or complex molecules for synthetic optimization. In addition, plant defenses to insect attack usually involve multifunctional multichemicals (Kubo and Hanke, 1985). Preliminary artificial diet-feeding assay has indicated that the methanol extract of a medicinal plant Fuchsia tetradactyla (Onagraceae) contains two hydrolyzable tannins in varying quantities. Both tannins inhibited Pectinophora gossypiella larvae when ingested in small amounts. P. gossypiella larvae became much smaller in size from the control due to the ingestion of the tannins, and this activity is likely due to the prooxidant action. Tannins are estimated to be the fourth most abundant biochemical produced by vascular plant tissue after cellulose, hemicellulose and lignin. Tannins are known to play a role in a number of ecological processes in addition to herbivore defense. Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggests that application of tannins as insect control agents needs to be investigated from practical points of view rather than as leading compounds for synthetic optimization. In addition, both mosquito larvicidal triterpene saponins isolated from the root bark of Pachyelasma tessmannii (Leguminosae) (Nihei et al., 2005) and insect antifeedant limonoids characterized from Croton jatrophoides (Euphorbiaceae) (Kubo et al., 1990; Nihei et al., 2002; 2004) are other examples. The saponins isolated from
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P. tessmannii were found to exhibit potent mosquito larvicidal activity. However, these triterpene saponins have not been further studied as practical mosquito control agents because of the lack of availability. The limonoids isolated from C. jatrophoides showed potent antifeedant activity against two lepidopteran larvae: pink bollworm, P. gossypiella; and fall armyworm, Spodoptera frugiperda. However, these limonoids are complex and energetically costly molecules to synthesize, and are available in minute amounts. Hence, the application of natural products as alternate insect control agents needs to be investigated from a different aspect. The data have been reported as the result of sporadic research, so it is timely to review and synthesize in one paper all that is currently known.
Tannins Gallae Rhois is a unique natural medicine that is used in the manufacture of tannin, tannic acid and gallic acid. It is a kind of gall produced by the gall-forming aphid Melaphis chinensis on the leaves of Rhus javanica. Eggs of M. chinensis are artificially deposited on the fresh leaves of R. javanica to make commercial Gallae Rhois. After the aphid is colonized, the plant tissue undergoes rapid cell division and enlargement. The aphid, during the wingless stage of its complex life cycle, forms galls along the midrib of the leaves from which the aphid parthenogenetically produces winged offspring. The galls are highly irregular and lobed shape, grayish-brown in color, and have a thin but hard wall and large hollow interior (Figure 1). The wall is the source of gallic acid, tannin and tannic acid, and used as the commercial Gallae Rhois. This gall formation may also be a plant defense mechanism to minimize its damage by aphid invasion. The cavity contains numerous dead winged aphid bodies in white
Fig. 1. Gallae Rhois.
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powder that was removed prior to commercial extraction of tannins. It should be noted that the dead aphid bodies are a result of the heating process of Gallae Rhois before their escape. There has been little study to verify the biological activities and chemical analysis of this white powder. In a preliminary thin-layer chromatography (TLC) analysis, this white powder was found to contain mainly two types of chemicals: both nonpolar and polar compounds without anything in between. Non-polar compounds were found to consist mainly of fatty acids and di- and tri-glycerides by gas chromatography–mass spectroscopy (GC–MS) analysis. Interestingly, the ethanol extract of the white powder was found to exhibit growth inhibitory activity against the pink bollworm P. gossypiella in an artificial diet-feeding assay (Chan et al., 1978; Kubo, 1993). The ethanol extract of the commercial Gallae Rhois showed much more potent activity. Plants can defend themselves against insect attack by means of their secondary metabolites, which interfere with many biological activities. Since there is no way to anticipate a priori what effects specifically involve in the growth inhibitory activity against P. gossypiella larvae, the artificial diet-feeding assay (Kubo, 1991) was performed to detect as much of these activities as possible. Importantly, careful observation of the insects throughout the assay is essential. Any difference from the control should not be overlooked (Kubo, 1993). In the current experiment, P. gossypiella larvae became much smaller in size from the control caused by the ingestion of the ethanol extract, but any additional noticeable difference was not observed. Fractionation of the ethanol extract of the white powder of Gallae Rhois was guided by the artificial diet-feeding assay against the pink bollworm. Successive partitioning of the crude extract with n-hexane, CH2Cl2, EtOAc and H2O revealed the biological activity to be retained in the EtOAc layer. Further fractionation of the EtOAc fraction, using droplet counter-current chromatography (DCCC), gave three major compounds. Further purification with Sephadex LH-20 column chromatography gave three purified phenolic compounds (1–3) (see Figure 2 for structures). The 1H-NMR (nuclear magnetic resonance) spectrum of 1 is rather unusual in that it shows only a single peak, corresponding to the aromatic protons near 6.9 ppm, and the other two peaks present are due to the solvent (DMSO and H2O). The simplicity of the spectra reveals the highly symmetrical nature of the compound. The 13 C-NMR data confirm that compound (1) corresponds to gallic acid. Confirmation was also obtained by comparing 1 with an authentic sample. The 1H-NMR spectra of the two remaining compounds, 2 and 3, show the presence of a methyl and ethyl
1, R=H 2, R=CH 3 3, R=CH 2CH3
Fig. 2. Structures of gallic acid and its methyl and ethyl esters.
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group, respectively. In conjunction with their 13C-NMR and EI-MS data, they are characterized respectively as methyl gallate (2) and ethyl gallate (3). Since ethanol was used in the initial extraction, the latter compound is probably an artifact. This was confirmed by the fact that ethyl gallate was not detected when methanol was used for extraction. Similarly, methyl gallate may also be an artifact since methanol was used as a solvent during purification procedure, although this was not confirmed. As a result, the polar part of the ethanol-extractable fraction of the white powder probably consists almost exclusively of gallic acid (Kubo et al., 2003). Interestingly, gallic acid characterized in the white powder could not be detected in the fresh leaves of the original plant R. javanica by high-performance liquid chromatography (HPLC), prior to attack by the gall-forming aphid M. chinensis. In contrast, hydrolyzable tannin is abundant in the fresh leaves. More importantly, this tannin rapidly gets accumulated in the wall after an attack by M. chinensis and produces Gallae Rhois. This process can be termed as a proteinase inhibitor inducing factor (PIIF) (Ryan, 1979), since the aqueous extract of this Gallae Rhois much more strongly inhibited two digestive proteinases tested, chymotrypsin and leucineaminopeptidase, than that of the fresh leaves of R. javanica. It may be logical to assume that gallic acid was derived from hydrolyzable tannin during the gall-forming process in R. javanica by the insect, or by accompanying microorganisms within the parasites’ body, or the plant itself. It appears, therefore, that the releasing mechanism of gallic acid may be one of the key processes to understanding plant defense and/or gall formation. It should be pointed out that the ethanol extract of the white powder did not significantly inhibit the aforementioned two digestive enzymes. The chemicals involved in gall formation are undoubtedly numerous. However, as an initial step to understanding this process, we focused the current study on the biological activity of gallic acid, especially its effects on insects. To facilitate it, tannic acid isolated from Gallae Rhois was also studied for comparison. It should be noted that the structure of tannic acid has not yet been established, although it was reported as the mixture of at least seven related galloylglucoses by HPLC analysis. The basic structure is 1,2,3,4,6-penta-O-galloyl-b-D-glucose to which the depside galloyl group is randomly distributed among C-2, C-3 and C-4 positions (Nishioka, 1983). In the artificial diet-feeding assay against P. gossypiella larvae, both gallic acid and tannic acid showed growth inhibitory activity and the ED50 of gallic acid was found to be 2000 ppm while that of tannic acid was 50 ppm. In other words, tannic acid showed 40-fold more potent growth inhibitory activity in this feeding assay, indicating that P. gossypiella larvae are sensitive to ingested tannic acid but somewhat tolerate gallic acid. It appears that the gallic acid characterized is responsible for the original insect growth inhibitory activity of the ethanol extract of the white powder observed in the preliminary screening. Similarly, tannic acid is responsible for that of the Gallae Rhois ethanol extract. It should be borne in mind that plant secondary metabolites can act in general by a variety of different mechanisms in insects. The sensitivity of P. gossypiella to ingested tannic acid may be a consequence of its extensive chemical modification in the midgut and oxidation is the first thinkable chemical modification. If so, quinones should be the first oxidized structure. Needless to say, quinones are usually highly toxic to insects as well as to many other organisms. For example, quinones may readily react with biologically important
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nucleophilic groups such as sulfhydryl, amino or hydroxyl. Sulfhydryl groups in proteins and lower molecular weight compounds such as glutathione are known to play an important role. The quinones may cause depletion of cytoplasmic and mitochondrial glutathione which functions in eliminating reactive oxygen species (ROS). This depletion can be caused by a direct interaction between the enone moiety and the sulfhydryl group of glutathione by a Michael-type addition. Neither benzoic acid nor 2,4-dihydroxybenzoic acid (b-rosorcylic acid) inhibited insect growth. The result obtained is consistent with the previous work (Elliger et al., 1980). This observation may support that the oxidized products are responsible for the activity. In addition, oxidation also produces ROS in the gut lumen, and this ROS damages biological systems (Pardini, 1995). In fact, alkyl gallates are known to induce ROS production thereby triggering apoptosis pathway in the human leukemia HL60RG cells (Serrano et al., 1998; Sakaguchi, 1999). It seems that the detrimental effects of tannic acid on the non-adapted pink bollworm are the result of ineffective defenses in a consequence of chemical modification in the midgut and/or against ROS generated in the gut lumen (Barbehenn and Martin, 1994; Barbehenn et al., 1996). The possibility that their adverse effects are a consequence of their potential to act as a prooxidant may need to be considered. In fact, gallic acid is known to produce superoxide anion (Serrano et al., 1998). Furthermore, tannic acid may somewhat irreversibly inactivate enzymes (proteins) in the midgut by cross-links, a process known as tanning, prior to being oxidized. This process also needs to be taken into consideration since tannic acid may bind with proteins in the gut and, as a result, inhibit digestive enzymes as well as protein digestion (Feeny, 1976). This can be supported by the observation that tannic acid showed significant inhibitory activity against fungal protease. At the concentration of 3 mg/mL, tannic acid inhibited the enzyme activity 68%, while gallic acid did not show any inhibitory activity up to 6 mg/mL. In addition, tannic acid inhibited the two digestive enzymes, chymotrypsin and leucine-aminopeptidase. Gallic acid also inhibited the same enzymes but much weaker compared to tannic acid. In connection with this, tannic acid is also known to form complexes with heavy metal ions (Scalbert, 1991). For example, gallic acid is known to inhibit iron absorption to the same extent as tannic acid, per mol galloyl groups (Brune et al., 1989) (Table 1). The possibility of involving tanning effect is unlikely but cannot be entirely ruled out since tannic acid is relatively stable compared to other hydrolyzable tannins such as agrimoniin and davidiin (Kubo et al., 1995). Despite numerous investigations, the precise mechanisms of tannin–herbivore interactions are still largely unknown.
Table 1 Scavenging activity of gallic acid and tannic acid Compounds tested Gallic acid Methyl gallate Ethyl gallate Tannic acid
DPPH consumption 6.5170.30 6.3470.12 6.1870.18 78.874.8
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On the other hand, tannic acid isolated from Gallae Rhois was found to inhibit tyrosinase activity with an IC50 of 19 mM that is about 230-fold more potent than that of gallic acid (Kubo et al., 2003). Tyrosinase (EC 1.14.18.1), also known as phenol oxidase (PO), is a copper-containing enzyme widely distributed in microorganisms, animals and plants. In our continuing search for alternative insect control agents from plants (Kubo, 1993), tyrosinase inhibitors have recently been targeted (Kubo, 1997) because tyrosinase is one of the key enzymes in the insect-molting process (Andersen, 1979, 1990). Hence, tyrosinase inhibitors might ultimately provide clues to control insect pests by inhibiting tyrosinase, resulting in incomplete cuticle hardening and darkening (Kramer and Hopkins, 1987). For example, this enzyme is previously reported to highly correlate with aphid resistance of the Solanum plants (Ryan et al., 1982). Finding alternate insect control agents by inhibiting tyrosinase is one of our goals in our continuing search for tyrosinase inhibitors from plants. However, the in vitro results using fungal tyrosinase described is still far from our goal. For example, mushroom tyrosinase used for the initial screening differs somewhat from insects (Andersen, 1990). More importantly, it has not been intentionally overlooked but, as a result, the dynamic function of tyrosinase in insect cuticle has not been thoughtfully taken into account. Thus, tyrosinase does not always exist as the active form in insects (Gupta et al., 2005) and also its inhibitors cannot always reach to tyrosinase in cuticle in sufficient concentration. Tannic acid is a rather bulky polar compound and hence, it unlikely penetrates insect cuticle. Moreover, the reaction time and the amount of available oxygen need to be considered from a practical point of view since insect tyrosinase is an aerobic oxidase. As far as the artificial diet-feeding assay using P. gossypiella larvae is concerned, some tyrosinase inhibitors characterized inhibit insect growth but some do not. The fact that plant secondary metabolites affect by a variety of different mechanisms in insects needs to be kept in mind. The relevance of the results of in vitro experiments in simplified systems to the in vivo situation should be carefully considered. The further study using more appropriate bioassay methods (Londershausen et al., 1996) is currently under investigation (Li and Kubo, 2004). The gall-forming process in which the aphid M. chinensis makes the white powder still cannot be clearly explained on a molecular level, but it may not be illogical to assume that gallic acid is an end product resulting from the defense of the gallforming aphid against the induced defense mechanisms of R. javanica. This may be supported by the previous report that gallic acid is known to be the metabolite of tannic acid when the latter acid was administered to insects (Bernays and Chamberlain, 1980) and sheep (Zhu et al., 1995) orally. Although the effects of tannic acid against M. chinensis could not be directly tested, it should be reasonable to conclude that this aphid can detoxify, at least in part, ingested toxic tannic acid to relatively non-toxic gallic acid. This ability to tolerate ingested tannic acid may rely on its rapid and extensive hydrolysis, similar to being reported to the tolerance of polyphagous grasshoppers (Bernays and Chamberlain, 1980). This can be supported by the finding of gallic acid in large quantities in the white powder, indicating that hydrolysis is the predominant of possible chemical fates of ingested tannic acid. It seems that tannic acid plays an important role in the defense mechanisms of R. javanica as an antimetabolite, resulting from PIIF. Nevertheless, understanding the
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gall-forming mechanism or the defense mechanisms of R. javanica needs to be investigated not only from one aspect but also from a whole and dynamic perspective. In our earlier studies, gallic acid and its methyl ester were often characterized in large quantities from several plants such as a Guatemalan medicinal plant F. tetradactyla (Onagraceae) locally known as ‘‘patayuc’’ and Bersama abyssinica (Melianthaceae) (Kubo and Matsumoto, 1985). However, their weak inhibitory activity against insect’s growth was usually overlooked in the presence of more potent active compounds (Kubo and Matsumoto, 1984). On the other hand, tannins have seen limited success as speciality chemicals. Previous attempts to develop tanninbased chemicals have suffered because of our failure to appreciate fully economic requirements, purity and stability factors and application needs of industrial process. In addition, tannins may not be good leading compounds to develop more effective insect control agents through synthetic optimization because of their complicate structures. Thus, chemical structures of tannins were started to clarify not long ago but the mechanisms of tannin–herbivore interactions has not yet been much studied, especially on a molecular basis. However, tannins are still available as pest control agents for individual farmers, and need to be investigated from more practical points of view. F. tetradactyla contains two hydrolzsable tannins as well as gallic acid in quantities. Both the tannins also inhibited P. gossypiella larvae when being ingested. Tannins are known to play a role in a number of ecological processes in addition to herbivore defense (Kraus et al., 2003). Because tannins are complex and energetically costly molecules to synthesize, their widespread occurrence and abundance suggests that application of tannins as pest control agents needs to be investigated from practical points of view rather than as leading compounds. Extraction and isolation The white powder (350 g) used for chemical analysis was obtained from the galls and extracted with 95% aqueous EtOH for a week (3 ) at an ambient temperature. The solvent was concentrated in vacuo at 40 1C to give a slightly yellowish residue that weighed 41.8 g. The crude extract was successively partitioned between n-hexane CH2Cl2, EtOAc and H2O in this order and subsequent bioassay revealed the biological activity to be retained in the EtOAc fraction. The solvent was evaporated from the bioactive EtOAc fraction in vacuo to give a residue (11.3 g). Then, 2.02 g of this residue was dissolved in 5 mL of the mobile phase and were injected onto DCCC. The ascending method was used with one fraction equivalent to 300 drops. The solvent system consisted of CHCl3–MeOH–H2O–AcOH–MeCoEt (23:42:26:3:6, v/v/v/v/v). Fractions were collected and checked with SiO2 TLC plates using the stationary phase as the developing solvent. Three active compounds were obtained and the further purification by LH-20 column chromatography yielded, gallic acid (1) (83 mg), methyl gallate (2) (17 mg) and ethyl gallate (3) (134 mg). They were identical in all respects including spectroscopic data to authentic samples. Insect feeding assay The artificial diet-feeding assay was carried out as previously described (Chan et al., 1978) with some modifications (Kubo, 1991). The homogeneous incorporation of
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plant extracts or purified chemicals into the diet with the addition of as little heat as possible is an important concern in the artificial-feeding assay. Since there is no way to anticipate a priori what effects will appear, careful observation of the insects throughout the assay is important. Any difference from the control should not be overlooked. For example, P. gossypiella larvae failed their molting cycle caused by the ingestion of the crude extracts of some plants such as Ajuga (Kubo et al., 1981), Podocarpus (Kubo et al., 1984), and Vitex (Kubo et al., 1985; Zhang et al., 1992) species; the insect underwent normal apolysis, but failed to complete ecdysis (Kubo et al., 1983). If materials permit, plant extracts should be fractionated, at least between organic solvent-soluble and -insoluble fractions. In general, water-soluble substances, especially primary metabolites such as sugars and amino acids, are usually feeding stimulants, so they often mask interesting biological activity.
Saponins Despite significant advances in the techniques used to control mosquitoes during recent decades, they still pose a serious public health problem. In addition to the persistent irritation they cause to humans and animals simply by virtue of their bloodsucking behavior and the itching it causes, mosquitoes are also the principal vector of a variety of serious diseases, including malaria, yellow fever, dengue and encephalitis. Worldwide, approximately 2.7 million human deaths occur each year solely as a result of malaria transmitted by mosquitoes (Butler, 1997). By bioassay-guided fractionation using Aedes aegypti larvae, four novel saponins, pachyelaside A, B, C and D, were isolated as potent mosquito larvicidal principles from the root bark of a west African medicinal plant P. tessmannii (Leguminosae) (Nihei et al., 2005) by using recycling HPLC (Kubo and Nakatsu, 1990). Despite their excellent mosquito larvicidal activity, these triterpene saponins are difficult to obtain in quantities and energetically costly complex molecules to synthesize (Figure 3). To cross these hurdles, a series of aliphatic primary alkanols were tested as a model for their mosquito larvicidal activity, since saponins are usually considered as surface-active agents (surfactants). It should be noted that alkanols are among the most versatile of all organic compounds; free and esterified alkanols occur widely in nature. The use of a series of aliphatic primary alkanols applied to water as soluble solutions for the control of mosquitoes in their larval stage. The species used for this study is Culex tarsalis, the principal vector of the Western equine and St. Louis encephalitis viruses common throughout the western US, but preliminary assays against Culiseta incidens suggest that the alkanols tested show similar activity against members of other mosquito genera too (Hammond and Kubo, 1999). In an effort to understand the molecular basis of alkanol toxicity, a variety of naturally occurring unsaturated long-chain alcohols were also tested for comparison. Of these, farnesol was previously reported as acting in some insects as juvenile hormone mimics (Schmialek, 1961). The toxicity of the homologous series of primary alkanols against C. tarsalis larvae was recorded as mortality after 24 h and the results show that larvicidal activity at dodecanol, with an LD50 of 30 mM against the 1st instar larvae and 28 mM against mixed 3rd and 4th instar larvae. Methanol and ethanol were very
New concept to search for alternate insect control agents
Pt 72A C 54 H 88 O 23 Mol. Wt.: 1105.26
H COOH Glc I O
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OH
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H
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Glc I HO O O O HO OH O HO O Glc III HO HO O O OH HO Glc II HO O HO O HO HO OH Glc IV Pt 751 C 67 H 100 O 28 Mol. Wt.: 1353.49 Xyl
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H
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H
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Fig. 3. Structure of potent mosquito larvicidal saponins characterized from P. tessmannii.
weak agents and mortality did not reach 50% at the highest dose tested, 500 mM. Activity increases through the series as chain length is lengthened from propanol to dodecanol, tapers off slightly from dodecanol to pentadecanol, and then cuts off at hexadecanol. Alkanols with greater than 16 carbons were ineffective as mosquito larvicides because they did not display biochemical toxicity and did not consistently
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prevent larvae from surfacing; consequently, they did not produce 50% mortality up to the maximum practical dosage, even when assays were extended to 4 days. The values cited in Table 2 for tridecanol through eicosanol exceed literature values for their maximum solubility in water (Bell, 1973) but experiments at high doses were included because these compounds are far more soluble in cell membranes – the putative active site – than in water. By creating a supersaturated suspension in the aqueous medium, even temporarily, we were able to deliver a high dose to the mosquitoes themselves. Addition of the surfactant Tween-80 to improve solubility increased the toxicity of tridecanol and tetradecanol solubility, and of pentadecanol by nearly a full order of magnitude (Table 2). Activity of alkanols with less than 13 or more than 15 carbons was unaffected by the use of surfactant or sonication. Tween itself had no noticeable toxicity to larvae up to 400 mg/mL, four times the concentration used here. Sublethal doses provoked symptoms of anaesthesia in mosquitoes (e.g. unresponsiveness to outside stimuli such as tapping on the container and cessation of characteristic diving and feeding behaviors).
Table 2 LD50 values of alkanols against C. tarsalis larvae Alkanols tested
LD50 (mM) 1st instar
Methanol Ethanol Propanol Butanol Pentanol Hexanol Heptanol Octanol Nonanol Decanol Undecanol Dodecanol Tridecanol Tetradecanol Pentadecanol Hexadecanol Heptadecanol through Eicosanol Tridecanol+Tweena Tetradecanol+Tween Pentadecanol+Tween Hexadecanol through Eicosanol +Tween 1,10-Decanediol a
>500 >500 171 (727.8) 74.6 (73.66) 20.0 (74.10) 7.55 (71.18) 2.64 (70.322) 1.12 (70.081) 0.260 (70.061) 0.057 (70.015) 0.038 (70.005) 0.030 (70.004) 0.034 (70.003) 0.049 (70.010) 0.302 (70.058) Inactive up to 0.6 Inactive up to 0.6 0.029 (70.003) 0.034 (70.005) 0.034 (70.007) Inactive up to 0.6 Inactive up to 4.6
In combination with Tween-80 added at 100 mg/mL.
3rd and 4th instar
1.02 (70.139) 0.278 (70.026) 0.081 (70.007) 0.046 (70.004) 0.028 (70.002) 0.042 (70.006) 0.039 (70.005) 0.088 (70.028) 0.222 (70.047)
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Whereas there was no appreciable difference between the patterns of physiological toxicity of alkanols to larvae of different instars, the lethal effects of reduced surface tension were most evident against 3rd and 4th instar larvae, which were less able than 1st instars to tolerate prolonged period without air – note the potency of pentadecanol and hexadecanol against late instars as compared to 1st instar. In short, the effectiveness of hexadecanol against late instars only is apparently the result of its physical properties for temporarily lowering surface tension, and not of inherent biochemical toxicity. Statistical analysis of the data showed that the difference in potency of alkanols differing in length by one carbon was highly significant (Po0.01) for C3–C10 and for C14–C16. The difference between pentadecanol alone and pentadecanol+Tween was also highly significant (Po0.01). However, the differences among the most potent homologues (C10–C14) and the effects of combining each of these with Tween were not significant for this data set (n ¼ 5). In order to better understand the significance of the cutoff in activity after pentadecanol, several other alcohols of relevant structure and chain length were tested as well, all in combination with Tween to ensure maximum solubility. It was interesting to note that, whereas hexadecanol was not toxic to 1st instar mosquitoes at the highest concentrations tested, cis-11-hexadecanol was nearly as potent as the strongest of the saturated alkanols. Similarly, the introduction of two (in the case of linoleyl alcohol) or three (linoenyl alcohol) double bonds to octadecanol converted the compound from completely inactive into being among the most potent. The isoprenoid sesquiterpenoid farnesol, which has an overall length of 12 carbons, also showed toxicity comparable to that of dodecanol, leading us to infer the hydrophobic and hydrophilic moieties. The head—tail-type structure is apparently essential for larvicidal activity because decanol was toxic to mosquitoes in the range of 60 mM, while decane-1,10-diol was not toxic even at 4.6 mM. Although the differences in relative toxicity were consistent among the four unsaturated alkenols tested, they were not statistically significant (Po0.01 and n ¼ 5). The objective of the surface tension experiments was to test the hypothesis that the lethal concentration of a given alkanol can be positively correlated to the concentration required to lower surface tension enough to prevent larvae from surfacing for air. Dilute solutions of Ivory soap produce low, yet stable surface tension values, and were used to confirm the results of earlier researchers that some mosquito larvae have difficulty surfacing for air when surface tension (g) – normally about 72 dynes/cm – is decreased to 27–36 dyne/cm. For C. tarsalis we found variability among individuals, but most 1st- and 2nd instar larvae were unable to attach to the surface g when decreased to the range of 28–32 dyne/cm; 4th instar larvae were able to tolerate lower surface tensions, but none were able to surface when g decreased to about 26 dyne/cm. In contrast, the concentration of C3–C10 alkanol that caused 50% mortality in 1st instar larvae produced surface tensions of 62–47 dyne/cm, which allowed even the smallest larvae to surface without difficulty. The concentration of each C3–C10 alkanol necessary to lower g to 30 dyne/cm was on an average eight times than its respective LD50 concentration. Furthermore, for chain lengths up to 10 carbons, many of the dead larvae were found with their respiratory siphons still firmly suspended from the water’s surface, unequivocally eliminating surface phenomena as the cause of death.
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Alkanols with 11–16 carbons, on the other hand, were found to be superior surface tension-reducing agents and even at low doses, larvae attempting to surface for air simply bounce off the air–water interface and sink back downward. Nevertheless, the swiftness of death hinted that even these larvae were succumbing to a biochemical phenomenon long before they could be expected to display the effects of suffocation. For these long-chain alkanols, the role of a biochemical mode of action was demonstrated by using a method that prevented contact with the surface for the course of the assay. Mosquito larvae, especially early instars, can survive for extended periods without access to air, apparently by cutaneous respiration of dissolved oxygen in the water. A glass ceiling such as a Petri dish can be used to trap larvae without air. By creating a test system free of air and its associated interfaces, the container ceiling is effectively made identical to its walls and bottom and, most importantly, the larvae are specifically isolated from any surface-related phenomenon that might wet their siphon opening, reduce its hydrofuge properties or allow leaks into their respiratory siphons during attempts to surface. When we trapped 20 1st instar C. tarsalis larvae under glass without air as a control, 100% were alive and active 16 h later. Fourth instar populations survived only about 3–4 h, apparently due to their higher oxygen requirements and lower surface area-to-volume ratio. Survival time of 1st instar larvae under glass was approximately doubled to 32–48 h by lowering the temperature of water to 8–10 1C. In contrast, 1st instar larvae trapped without air in jars of water treated with decanol or undecanol were immobilized on the bottom and made no further efforts to surface after 5 min; the same result was observed with dodecanol after about 12 min, with tridecanol after an hour, with tetradecanol after 3 h, and with pentadecanol after approximately 9 h. Extrapolation of this time-dependency curve predicts activity for hexadecanol after about 18 h, but no mortality due to hexadecanol nor heptadecanol treatments occurred before control larvae themselves began to die of anoxia, after about 24–30 h. Nevertheless, we could not rule out the possibility that hexadecanol may act by a mechanism similar to – but weaker than – shorter chain homologues, for example as a partial anesthetic. In an effort to extend the survival of larvae trapped without air and thereby allow sufficient time for the possibility that hexadecanol might still show activity at longer exposures, the same air-free experiments were also carried out under refrigeration, at 6 1C, 120 1C and 14 1C. Control larvae were thus able to survive up to 48 h, but we were surprised to find that 260 mM pentadecanol+Tween, which had caused 100% mortality at 20 1C, caused just 20% mortality at 14 1C, and toxicity disappeared entirely at 10 1C or less. Comparatively, the toxicity and time dependency of tetradecanol and the shorter-chain alkanols were unaffected by these lower temperatures, suggesting that the loss of activity is at least partially a function of declining solubility. Subsequent experiments, in which larvae treated with lethal doses of C10–C15 alkanols were transferred to clean water well after the onset of anesthesia had become evident, confirmed that the effects were completely reversible. Within minutes or hours, again depending on chain length, larvae returned to behavior indistinguishable from that of untreated larvae.
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The significance of our findings are twofold: First, they challenge the previously accepted belief that alkanols act as mosquitocides only via suffocation provoked by surface phenomena; second, for studies measuring biological activity of the alkanol series, this is the first-documented instance of cutoff occurring in an animal at a chain length beyond C12. The speed with which alkanols pC15 act as mosquito larvicides is in sharp contrast to the conclusions of previous researchers using insoluble monolayers of dodecanol (McMullen and Hill, 1971), hexadecanol (McMullen and Hill, 1971), and lecithins (McMullen et al., 1977), which were specifically described as producing larval death only after the dissolved oxygen content of the water was depleted, usually overnight. The increasingly time-dependent nature of toxicity as chain length is increased offers further evidence of the biochemical mode of action for alkanols, independent of any suffocation mechanism resulting from reduced surface tension at the air–water interface. Action of farnesol was also rapid, as far as mosquito larvicidal activity is concerned, the rapid lethality may eliminate the possibility of previous conclusion that farnesol is a juvenile hormone mimics (Schmialek, 1961) since hormonal disturbances take time. The toxicity of farnesol against mosquito larvae is apparently via the same mechanism as for unbranched, saturated alcohols. When, for comparison purposes, mosquitoes were treated with Golden Oil, a commercial product sold as a surface-active mosquitocide, 4th instar larvae died after a period of several hours and 1st instar larvae were weakened, but still alive, 24 h later – both results consistent with an explanation of death via suffocation. Alkanols, in contrast, were generally observed to act more quickly on the 1st instar than on the 4th instar larvae. Also, whereas pupae have been shown previously to be more susceptible to monolayers than larvae (McMullen and Hill, 1971; Levy et al., 1982), our preliminary findings indicate that solutions of long-chain alkanols are more toxic to Culex larvae than to the pupae, possibly owing to the pupae’s thicker cuticle and concomitant capacity to resist penetration by foreign substances. Tests with animals in vivo are somewhat limited, but long-chain alkanols are known to produce anesthesia in fathead minnows (Veith et al., 1983), tadpoles (Alifimoff et al., 1989; Miller et al., 1989) and brine shrimp (Chiou et al., 1990), and to cause growth impairment in the ciliate protozoan Tetrahymena pyriformis (Schultz et al., 1990). In our own earlier studies, they showed activity against a variety of Gram-positive bacteria and fungi, but not against Gram-negative bacteria (Kubo et al., 1995). A persistent quandary facing researchers has been the ‘‘cutoff’’ phenomenon in the homologous series of alkanols, whereby potency increases with chain length until reaching a maximum, and the alkanol containing a single additional carbon shows no potency at all, even when duration of exposure lasts several days (Miller et al., 1989). Although attention has been focused on instances where cutoff occurs immediately after dodecanol, the exact length of carbon chain where cutoff occurs clearly varies among genera (Figure 4). Our review of the literature found no other animal (only microorganisms and a protozoan) for which cutoff has been reported at a chain length beyond dodecanol, which may be the result of differences in the membrane composition of test organisms. By comparison, tadpoles were fully anesthetized by nonanol after 30 min, by
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Fig. 4. Comparison of biological activity of alkanols against various organisms. Cutoff in activity against fish and tadpoles occurs after C12, but in mosquitoes not until after C15. Although the endpoints measured are not identical in all test systems, sublethal doses provoked symptoms of anaesthesia in mosquitoes and growth inhibition in microorganisms. &, Tadpole (Rana pipiens), loss of righting reflex, EC50 (Alifimoff et al., 1989); J, Minnow (Pimephales promelas), 96 h LC50 (Veith et al., 1983); r, Protozoan (T. pyriformis), inhibitory growth concentration, EC50 (Schultz et al., 1990); D, S. aureus, minimum bactericidal concentration (MBC) (Kubo et al., 1995); , Mosquito (C. tarsalis), 24 h LD50, Tween used to maximize solubility of alkanols ZC13; B, P. acnes, MBC (Kubo et al., 1995); +, Clostridium botulinum (Huhtanen, 1980), minimum inhibitory concentration.
decanol and undecanol after 60 min, by dodecanol after 120 min, but not by tridecanol, even when exposed for 96 h (Alifimoff et al., 1989). Mosquitoes are also unusual in that the loss of activity is gradual, tapering off from the strongest compounds before disappearing, whereas for most organisms, maximum activity occurs at a given chain length of n, and absolutely no activity is present at the alkanol of chain length n+1. For example, the minimum bactericidal concentration against Propionibacterium acnes was 1.56 mg/mL for hexadecanol, but heptadecanol was completely inactive, even when tested at 800 mg/mL (Kubo et al., 1995). The precise anesthetic mechanism is still not well understood, and attempts to explain it have covered a wide array of biological functions, mostly related to structure and function of lipid membranes and/or proteins (Franks and Lieb, 1994). The increase in activity with chain lengthening has been widely correlated to each compound’s octanol– or lipid–water partition coefficient, and thereby its relative tendency to accumulate in lipid regions of the cell membrane at concentrations
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sufficient to interfere with basic nutrient or iron transport processes (Treistman and Wilson, 1987; Elliott and Elliott, 1994; Weingart and Bukauskas, 1998). Nevertheless, it has been difficult for lipid-based theories to account for the cutoff effect and with this deficiency in mind, other researchers used alkanol inhibition of the lipid-free luciferase enzyme to build a case for anesthetics acting by building directly to a protein pocket of circumscribed dimension (Franks and Lieb, 1994; Abraham et al., 1991). More recently, the effects of alkanols on ligand-gated ion channels in wildtype and mutated neurotransmitter receptors have also led some to conclude that alkanols act directly on membrane proteins, and do not depend on lipid–protein interactions (Mascia et al., 1996; Mihic et al., 1997; Wick et al., 1998). Still, doubt remains as to whether these systems adequately model the site of general anesthesia in animals (Alifimoff et al., 1989; Moss et al., 1991), or for that matter whether anesthetic activity of alkanols is limited to a single mechanism. The fact that sonication, temperature and combination with a surfactant could be used to manipulate mosquitocidal activity nearly 10-fold around the cutoff, supports the notion that the solubility of alkanols in biological membranes and their capacity to perturb membrane lipids at the lipid–protein interface have some bearing on explanation of the cutoff phenomenon. Indeed, there are several recent studies to support this hypothesis. Anesthetic chain lengths were shown to cause perturbation of membrane lipids whereas non-anesthetic alkanols dissolved in membrane lipids without perturbing them (Miller et al., 1989). Fourier transform infrared spectroscopy (FTIR) studies of lipid membrane vesicles in D2O showed that hydrogen bonding of the alkanol hydroxyl to the phosphate moiety in reversed micelles increases up to decanol, and then declines sharply at tetradecanol (Chiou et al., 1990). If alkanols bond to the phosphate moiety in biological membranes the way they do in membrane vesicle, then certainly they would affect the conformation and function of proteins normally held in place by hydrogen bonds at the lipid–protein interface. The surfactant concept can be extended to answer many other questions related to membrane-bound proteins. For example, there has been a long-standing debate on whether alkanols produce their effects in the central nervous system by acting on lipids or on proteins. Alkanols are known to act as general anesthetics, with increasing potency correlated to increasing chain length until a point of cutoff is reached, usually at dodecanol, after which activity disappears entirely. Since anesthesia involves many membrane-bound proteins such as synaptosomal ATPases and acetylcholine receptor, alkanols may alter these membrane-bound proteins in similar fashion, by disrupting and disorganizing the hydrogen bonds at the lipid bilayer–protein interface nonspecifically. Knowledge that introduction of a double bond shifts the cutoff to a longer-chain length in tadpoles (Pringle et al., 1981) – and now in mosquitoes – is further evidence in support of explanations which consider the structural role of lipids rather than isolated action on a protein-binding site, because if cutoff were due to a compound’s hydrophobic moiety exceeding the limited size of a protein pocket, then since the addition of a cis double bond makes the compound even bulkier, it should theoretically provoke cutoff at shorter, rather than longer, alkyl chain lengths. We speculate that hydrogen bonding plays an important role in determining the molecular basis of alkanol activity in mosquitoes and other organisms.
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Alkanols are stable, colorless, inexpensive, biodegradable (Swisher, 1970), and essentially nontoxic to humans (Opdyke, 1973). The fact that they are already approved for use in food products at concentrations comparable to the doses used here may facilitate their approval as insecticides. If further experience bears out the conclusions of earlier workers who reported a cutoff in activity after dodecanol for fish and amphibians, then the narrower spectrum activity of tridecanol, tetradecanol, and pentadecanol against mosquitoes makes these promising candidates for environmentally sensitive pest management programs. The cutoff in anesthetic potency occurs at different chain lengths in different organisms, lending new perspective to the molecular basis of anesthesia. In brief, the mosquito larvicidal activity of alkanols against C. tarsalis larvae depends on the hydrophobic alkyl chain length from the hydrophilic hydroxyl group. The hydrophilic hydroxyl group can be replaced by any hydrophilic groups as long as the ‘‘head and tail’’ structure is balanced. On the basis of this surfactant concept, various phytochemicals previously isolated were tested for their mosquito larvicidal activity against C. tarsalis larvae. As expected, some phytochemicals showed even more potent and selective larvicidal activity compared to aliphatic alkanols. For example, phytochemicals possessing the phenolic moiety as the hydrophilic portions can be expected to exhibit mosquito larvicidal activity. Anacardic acids and cardols isolated from the cashew nut shell liquid (CNSL) are an excellent example for the surfactant concept (Kubo et al., 1986). Although CNSL is available in far greater tonnage, it is neglected in commercial terms, and there is considerable potential for its exploitation. Extraction and isolation of saponins The air-dried root bark (500 g) of P. tessmannii (Leguminosae) was cut into small pieces and extracted with MeOH (500 mL 3) at ambient temperature for 2 weeks. The MeOH extract (70 g) was evaporated to dryness and suspended in 500 mL of H2O, and then it was extracted successively with n-hexane, ethyl ether, CHCl3, EtOAc and n-BuOH to yield four fractions, n-hexane and ethyl ether (3 g), CHCl3 (14 g), EtOAc (10 g), and n-BuOH (30 g). A portion (4 g) of the n-BuOH fraction was subjected by a recycling HPLC (Kubo and Nakatsu, 1990). Column: 50 cm 2.5 cm Asahipack GS-320, 5 mm; mobile phase: MeOH; flow rate: 3 mL/min; detection: refractive index; amount of sample loaded: 500 mg (Nihei et al., 2005). Bioassays Bioassays were conducted at room temperature (2072 1C) using distilled water (20 mL) in 1 oz clear plastic containers made by Plastics Inc. (St. Paul, MN). Alcohol was added by solubilizing them first in acetone, then diluting in water to the appropriate concentration and briefly shaking to ensure mixing. Ten larvae were then pipetted into each 20 mL volume and observed for a minimum of 24 h, when mortality was recorded. Controls were treated with the maximum amount of acetone applied in each alcohol assay, usually 0.1 mL. Acetone itself was found to have no effect on the larvae up to a concentration of 1%.
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All compounds were assayed against 1st instar larvae and octan-1-ol through hexadecane-1-ol were also tested against mixed 3rd and 4th instar larvae. Assays were repeated at least five times for each compound. Compounds were tested at a minimum of five concentrations following range finding tests. Owing to their excessive clumping and general insolubility in water, alkanols with 15 or more carbons were tested only up to 0.6 mM. Although this dose exceeds the measured solubility of long-chain alkanols in water (Bell, 1973), the application of excess material was used as a way to supersaturate the water and thereby deliver a higher dose to the mosquito itself, in whose cell membranes these compounds are much more soluble. For alkanols greater than 10 carbons, bioassays were repeated with the addition of the commercial surfactant Tween-80 (100 mg/mL) to see if improved solubility might increase activity. For assays employing surfactant, shaking was impractical, so solutions were added while sonicating the test water, which helped to maximize solubility and mixing. Larvae were considered dead or moribund if they stopped moving for a prolonged period, were unable to resurface after sinking to the bottom of the container, and were still unable to respond after gentle probing with a small spatula. Mortality among controls was zero for more than 95% of the assays, and in no instance did it exceed 10%. In assays where larvae were trapped without air and prevented from any contact with the surface, each test solution was prepared as described above, then used to fill a 250 mL glass jar above its brim so as to form a meniscus; a Petri dish was then slid horizontally across the mouth of the jar so that no air bubbles remained. The edge was sealed with petroleum jelly to prevent drying and leakage. Time required to manifest toxicity is expressed as the time at which the last bodily movement could be detected from the last survivor among 10 larvae, and values cited are the mean of at least three experiments (Hammond and Kubo, 1999).
Acknowledgments The work was partly reported at the 15th International Plant Protection Congress in Beijing, China. I am indebted to Dr. D. G. Hammond, Dr. J. A. Klocke and Dr. S. Asano for performing mosquito larvicidal and artificial diet-feeding assays, Mr. S. Kawamichi for supplying Gallae Rhois; Dr. M. Takasaki for chemical analysis of Gallae Rhois at an earlier stage of the work, Dr. N. Masuoka for obtaining scavenging antioxidant data and Dr. C. Ce´spedes for assisting the final manuscript preparation.
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Ryan JD. (1979) Proteinase inhibitors. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interaction with secondary plant metabolites. New York: Academic Press, pp. 599–618. Ryan JD, Gregory P, Tingey WM. (1982) Phenolic oxidase activity in glandular trichomes of Solanum berthaultii. Phytochemistry 21:1885–1887. Sakaguchi N, Inoue M, Isuzugawa K, Ogihara Y, Hosaka K. (1999) Cell death-inducing activity by gallic acid derivatives. Biol Pharm Bull 22:471–475. Scalbert A. (1991) Antimicrobial properties of tannins. Phytochemistry 30:3875–3883. Schmialek P. (1961) Double identification in Tenebrio faces and in yeast of substances with juvenile hormone activity. Z Naturforschg 16b:461–463. Schultz TW, Arnold LM, Wilke TS, Moulton MP. (1990) Relationships of quantitative structure-activity for normal aliphatic alcohols. Ecotox Environ Safe 19:247–253. Serrano A, Palacios C, Roy G, Cespo´n C, Villar ML, Nocito M, Gonza´lez-Porque´ P. (1998) Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation. Arch Biochem Biophys 350:49–54. Swisher RD. (1970) In: Swisher RD, editor. Surfactant biodegradation. New York: Marcel Dekker. Treistman SN, Wilson A. (1987) Alkanol effects on early potassium currents in Aplysia neurons depend on chain length. Proc Natl Acad Sci USA 84:9299–9303. Veith GD, Call DJ, Brooke LT. (1983) Structure-toxicity relationships for the fathead minnow, Pimephales promelas: narcotic industrial chemicals. Can J Fish Aquat Sci 40:743–748. Weingart R, Bukauskas FF. (1998) Long-chain n-alkanols and arachidonic acid interfere with the Vm-sensitive gating mechanism of gap junction channels. Eur. J. Physiol. 435:310–319. Wick MJ, Mihic SJ, Ueno S, Mascia MP, Trudell JR, Brozowski SJ, Ye Q, Harrison NL, Harris RA. (1998) Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc Natl Acad Sci USA 95:6504–6509. Zhang M, Stout MJ, Kubo I. (1992) Isolation of ecdysteroids from Vitex stricheri using RLCC and recycling HPLC. Phytochemistry 31:247–250. Zhu J, Filippich LJ, Ng J. (1995) Rumen involvement in sheep tannic acid metabolism. Vet Hum Toxicol 37:436–440.
Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.
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CHAPTER 5
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari: present status and future prospects MARI´A C CARPINELLA, MARI´A T DEFAGO´, GRACIELA VALLADARES, SARA M PALACIOS
Introduction One of the biggest challenges that humanity confronts is providing enough food to the fast growing population of the planet, which is calculated to reach approximately 8 billions of people in the year 2020 (Knight et al., 1997). In order to materialize this, it is necessary to increase food production, which mainly depends on plants. These are often affected by many pests and diseases among which insects play a preponderant role. Commercially available synthetic pesticides are currently the most effective means of pest control. However, the continuous and indiscriminate use of these substances have not only caused adverse effects on mammals health, but have also affected other benefic members of the ecosystem (Theiling and Croft, 1988; C - elik et al., 2005; Chauhan and Gupta, 2005) and the environment in which they are immersed. They are also responsible for the development of resistance in pathogens (Thompson et al., 1993; Bass et al., 2004). Under these circumstances, new ways of pest control must be studied and established. There are many options, but the most promising is the one that involves the secondary metabolites produced by plants, many of which are highly toxic to a wide spectrum of insects and microorganisms. There is a great variety of families of plants that possess potent anti-insect compounds. From the Meliaceae family, strong insecticide molecules have been isolated, the limonoid azadirachtin obtained from Azadirachta indica or Melia azadirachta (neem) being the most potent and studied (Govindachari, 1992; Schmutterer, 1995). Another tree belonging to the Meliaceae family, far less studied than the previously mentioned, is Melia azedarach L., commonly named Paraı´ so, Chinaberry or Persian Lilac. Our research group has been studying the activity of extracts obtained from different structures of M. azedarach on a wide variety of pest insects, either in laboratory or in field experiments. A limonoid (compound 1) has been isolated from this tree as the most potent substance for controlling insects of agronomic importance. In
82
Naturally occurring bioactive compounds
the present chapter, information about the active principles isolated by various investigators from M. azedarach is provided together with their respective bioactivities. Moreover, a description of the effects on feeding, growth, larval and pupal development, fecundity, fertility, toxic effects, as well as alterations in the insect morphology of different extracts and some pure compounds from the tree is reported either in laboratory or in field trials.
Phytochemistry Many compounds have been isolated from different vegetal structures of M. azedarach collected in different places of the world. Most of them have been assayed as antiinsect and cytotoxic compounds showing excellent results. In relation to the first activity, most assays have been carried out on insects belonging to Spodoptera genus and in less measure on Epilachna. Other activities such as antifungal, antibacterial and anti-inflammatory have been less studied (Table 1). Phytochemical analysis of Argentinian M. azedarach ripe fruit extract revealed that alkaloids are absent (Carpinella et al., 1999a); however, Khan et al. (2001), reported the presence of this family of compounds in extracts from leaves, root and stem bark of M. azedarach from Papua New Guinea. Khan et al. (2001) also found tannins and flavonoids in the three mentioned tree structures. These last compounds were also found in the fruit extract from Argentinian M. azedarach (Carpinella et al., 1999a). Riba et al. (1996) followed a bioguided assay purification from a methanolic extract from M. azedarach seeds collected in Spain. After carrying out an HPLC purification, three main fractions were obtained. The first fraction (Fr. 1) obtained at the first 10 min with methanol/water 50:50 did not show any effect on Sesamia nonagrioides (Lepidoptera: Noctuidae) survival while the one obtained between 10 and 20 min of running (Fr. 2) exhibited the highest percentage of mortality till it reached pupation. The fraction obtained with methanol 100% (Fr. 3) showed a 46% of accumulated mortality till pupation on S. nonagrioides. From this fraction four majority products were obtained but only one showed effectiveness (Riba et al., 1996). From Fr. 2, a subfraction which exhibited the major activity was obtained. This subfraction contained two products in a relation 3:1. These products could not be separated; although successive purifications were performed similar chromatograms were obtained (Figure 1; Riba et al., 1996). The authors concluded that it was a decomposition of the majority compound. Carpinella et al. (2002) afforded the same mixture of compounds (compound 1) after carrying out a bioassay-guided isolation from the ethanolic ripe seed kernel extract from M. azedarach from Argentina. The fact that was interpreted as decomposition by Riba et al. (1996) was elucidated by Carpinella et al. (2002) as a mixture of two interchangeable isomeric limonoids, which occur/exist as an equilibrium between isomers A and B. The last one was named ‘meliartenin’ (Carpinella et al., 2002). Compound A was favoured in CH3CN (A/B ¼ 80–70:20–30) while the molar fraction of B increased in CHCl3 (A/B ¼ 53:47) (Figure 2a and b). Note the similarity between chromatograms of Figures 1 and 2a.
Table 1 Pure compounds isolated from extracts obtained from different structures of M. azedarach and their respective bioactivities Origin
Activity
Reference
1-cinnamoyl-3-hydroxy11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 1.5 mg/ml
Takeya et al. (1996b)
1-(2-methylpropanoyl)-3acetyl-11methoxymeliacarpinin
MeOH ext. of root of M. azedarach L. var japonica
No lethal activity on Artemia salina at 100 mg/ml
Fukuyama et al. (2000)
1,12-di-O-acetyltrichilin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Nakatani et al. (1994b); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.46 mg/ml
Takeya et al. (1996a)
1,3-dicinnamoyl-11hydroxymeliacarpin
Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.
Insecticide on S. littoralis, LC50 ¼ 2.36 ppm; EC50 ¼ 0.57 ppm
Bohnenstengel et al. (1999)
11-methoxy-20acetylmeliatinin
MeOH ext. of fruit of M. azedarach from Greece
12 a-acetoxyfraxinellone
Et2O ext. of stem bark of Japanese M. azedarach
Kraus et al. (1985) Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2
Nakatani et al. (1998)
Ichthyotoxic on Oryzias latipes at 50 ppm 12-acetoxyamoorastatin ¼ toosendanin ¼ 29deacetylsendanin
Cytotoxic against human tumour cells, ED50 ¼ 0.0007–0.04 mg/ml
Ahn et al. (1994)
Bark of Chinese M. azedarach
Antifeedant against S. litura
Chiu (1989)
Et2O ext. of root bark of Chinese and Japanese M. azedarach
Antifeedant against S. eridania (Boisduval), MIC ¼ 6 mg/cm2
Nakatani et al. (1998); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.026 mg/ml
Itokawa et al. (1995)
83
MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Compound
84
Table 1 (continued ) Origin
Activity
Reference
1-O-deacetylochinolide A
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 1449 mg/ml
Zhou et al. (2004)
1-O-deacetylochinolide B
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 58 mg/ml
Zhou et al. (2004)
1-O-deacetyl-1-Obenzoylochinolide B
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 22,638 mg/ml
Zhou et al. (2004)
1-O-deacetyl-1-Otigloylochinolide A
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 20,374 mg/ml
Zhou et al. (2004)
1-O-deacetyl-1-Otigloylochinolide B
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ 22,443 mg/ml
Zhou et al. (2004)
12-deacetyltrichilin I
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.011 mg/ml
Takeya et al. (1996a)
12-hydroxyamoorastatin ¼ 12-deacetyltoosendanin
MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica
Cytotoxic against human tumour cells, ED50 ¼ 0.01–0.25 mg/ml
Ahn et al. (1994)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.090 mg/ml
Itokawa et al. (1995)
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant S. eridania (Boisduval) 3 mg/cm2
Nakatani et al. (1998); Huang et al. (1995b)
EtOH ext. (CH2Cl2 fr.) of seed kernel of M. azedarach from Argentina
Antifeedant and insecticide against E. paenulata at 4 mg/cm2 and antifeedant against S. eridania at 1 mg/cm2
Carpinella et al. (2002)
Isomer A of compound 1
Naturally occurring bioactive compounds
Compound
MeOH ext. (EtOAc fr.) of stem bark of M. azedarach var. japonica
Cytotoxic against human tumour cells, ED50 ¼ 0.38–13.7 mg/ml
Et2O ext. of root bark of Chinese M. azedarach
Ahn et al. (1994) Nakatani et al. (1998)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
12-O-acetylazedarachin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
12-O-acetyltrichilin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Nakatani et al. (1994a); Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
1-acetyl-2-deacetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.66 mg/ml
Takeya et al. (1996a)
1-acetyl-3-deacetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.40 mg/ml
Takeya et al. (1996a)
1-acetyl-3-tigloyl-11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 3.3 mg/ml
Itokawa et al. (1995)
1-acetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.47 mg/ml
Takeya et al. (1996a)
1-cinnamoilmelianolona
MeOH ext. (CH2Cl2 fr.) of fruit of M. azedarach from USA
Insecticide against Heliothis virescens, EC50 ¼ 0.12 ppm; LC50 ¼ 1.50 ppm and S. frugiperda, EC50 ¼ 0.04 ppm; LC50 ¼ 1.30 ppm
Lee et al. (1987); Kraus et al. (1989); Lee et al. (1991) 85
12-O-acetylazedarachin A
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
12-hydroxyamoorastatone
86
Table 1 (continued ) Compound
Origin
Activity
Reference
1-cinnamoyl-3-11 dihydroxymeliacarpin
MeOH ext. (CH2Cl2 fr.) of fruit of M. azedarach from USA
Insecticide against H. virescens, EC50 ¼ 0.18 ppm; LC50 ¼ 3.50 ppm and S. frugiperda, EC50 ¼ 0.04 ppm; LC50 ¼ 1.60 ppm
Lee et al. (1991)
1-cinnamoyl-3-acetyl-11hydroxymeliacarpin
Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.
Insecticide against S. littoralis, EC50 ¼ 0.27 ppm; LC50 ¼ 0.48 ppm
Bohnenstengel et al. (1999)
1-cinnamoyl-3-acetyl-11methoxymeliacarpinin
Root bark of Japanese M. azedarach L.
Antifeedant against S. eridania (Boisduval), MIC ¼ 1 mg/cm2
Nakatani et al. (1994a); Huang et al. (1995b)
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 10.5 mg/ml
Takeya et al. (1996b)
MeOH ext. of fruit of M. azedarach from Greece
Growth regulator on E. varivestis
Growth regulator on E. varivestis No antifeedant against E. varivestis
Kraus et al. (1985, 1987)
Effect on moulting on Tessaratoma papillosa at 50 mg/ml
Chiu (1989)
Stem bark of Okinawan M. azedarach L. EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L. 23bmethoxymeliacarpin Kraus et al. (1985, 1987) MeOH ext. of fruit of M. azedarach from Greece
1-cinnamoyl-3methacrylyl-11hydroxy-meliacarpin
Me2CO and MeOH ext. (CH2Cl2 fr.) of leaves of Indian M. azedarach L.
Insecticide against S. littoralis, EC50 ¼ 0.57 ppm; LC50 ¼ 1.19 ppm
Bohnenstengel et al. (1999)
1-deoxy-3-methacrylyl-11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 47.0 mg/ml
Takeya et al. (1996b)
Naturally occurring bioactive compounds
1-cinnamoyl-3-feruloyl11-hydroxy-22,23dihydroNo antifeedant against E. varivestis 1-cinnamoyl-3-feruloyl11-hydroxymeliacarpin
Antifeedant against S. exigua (Boisduval) at 3 mg/cm2 and S. eridania (Boisduval), MIC ¼ 1 mg/ cm2
Nakatani et al. (1993); Huang et al. (1995b)
1-desacetylnimbolinin B
PE and Et2O ext. of fruit of M. azedarach var. japonica
Antifeedant against E. varivestis
Kraus and Bokel (1981)
1-O-detigloyl-1-Obenzoylohchinolal
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
1-O-detigloyl-1-Ocinnamoylohchinolal
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
1-methacrylyl-3-acetyl-11methoxymeliacarpinin
MeOH ext. root of M. azedarach L. var japonica
Lethal on A. salina, LC50 ¼ 19 mg/ml
Fukuyama et al. (2000)
1-tigloil-11-methoxy-20acetylmeliacarpin
Fruit
1-tigloyl-3,20-diacetyl-11methoxylmeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 100 mg/ml
Takeya et al. (1996b)
1-tigloyl-3-acetyl-11methoxymeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 3.2 mg/ml
Itokawa et al. (1995)
1,5-dihydroxy-8-methoxy2-methylanthraquinone3-O-a-Lrhamnopyranoside
Stem bark
Srivastava and Mishra (1985)
1,8-dihydroxy-2methylanthraquinone-3O-b-Dgalactopyranoside
Stem bark
Srivastava and Mishra (1985)
Kraus et al. (1987)
87
Et2O ext. of root bark of Chinese M. azedarach
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
1-deoxy-3-tigloyl-11methoxymeliacarpinin
Table 1 (continued ) Origin
Activity
Reference
29-isobutylsendanin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.034 mg/ml
Itokawa et al. (1995)
2b,3b-dihydroxy-5apregn-17(20)-(2)-en-16one
MeOH ext. of root of M. azedarach L. var japonica
Lethal on A. salina, LC50 ¼ 2.1 mg/ml
Fukuyama et al. (2000)
30-hydroxy-fraxinellone
AcOEt ext. of roots of Italian M. azedarach
3-deacetyltrichilin H
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.045 mg/ml
Takeya et al. (1996a)
3-tigloyl-1,20-diacetyl-11methoxylmeliacarpinin
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 48.0 mg/ml
Takeya et al. (1996b)
4,8-dimethoxy-1-vinyl-bcarboline
MeOH ext. (EtOAc fr.) of cortex of M. azedarach L. var japonica
Anti-inflammatory activity (suppression inducible nitric oxide synthase and cyclooxygenase-2 induction) 1.5–10 mM
Lee et al. (2000)
EtOH ext. (CH2Cl2 fr.) of seed kernels of Argentinian M. azedarach L.
Antifungal against Fusarium verticillioides, MIC ¼ 0.4 mg/ml
Anti-inflammatory activity (suppression inducible nitric oxide synthase and cyclooxygenase-2 induction) 2–10 mM
Lee et al. (2000)
Carpinella et al. (2003b) 4-methoxy-1-vinyl-bcarboline 6-acetoxy-3b-hydroxy-7oxo-14b, 15bepoxymeliac-1,5-diene3-O-b-Dglucuronopyranoside
methoxycinnamaldehyde
Cortex of M. azedarach L. var japo´nica
Roots of M. azedarach L. from India
D’Ambrosio and Guerreiro (2002)
Srivastava and Gupta (1985)
Naturally occurring bioactive compounds
4-hydroxy-3-
88
Compound
6-acetoxy-11-a hydroxy 7oxo 14 b, 15 b epoxymeliacin 1, 5 diene 3-O-a-L rhamnopyranoside
EtOH ext. (C6H6 fr.) of seeds of M. azedarach L. from India
6-hydroxy-7methoxycoumarin
EtOH ext. of leaves
Khalil et al. (1979)
6b-hydroxy-4-campesten3-one
PE extract of bark
Nair and Chang (1973)
6b-hydroxy-4stigmasten3-one
PE extract of bark
Nair and Chang (1973)
7a-acetoxy-14b, 15bepoxy-gedunan-1-ene-3O-b-D-glucopyranoside
Stem bark
Saxena and Srivastava (1986)
7-deacetyl-7-oxogedunin
Light PE ext. of stem wood
Okogun et al. (1975)
9a-hydroxyfraxinellone
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
9b-hydroxyfraxinellone
AcOEt ext. roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Aesculetin
EtOH ext. of leaves
Khalil et al. (1979)
Amoorastatone
Et2O ext. of root bark of Chinese M. azedarach
Nakatani et al. (1998)
Aphanastatin
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 4 mg/cm2
Nakatani et al. (1995b); Huang et al. (1995b)
Azedarachin A
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 4 mg/cm2
Huang et al. (1994); Nakatani et al. (1995b); Huang et al. (1995b)
Srivastava and Gupta (1985) Antibacterial activity
Srivastava (1986)
89
Roots of M. azedarach L. from India
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
6-acetoxy-7a-hydroxy-3oxo-14b, 15bepoxymeliac-1,5-diene
Table 1 (continued ) Origin
Activity
Reference
Azedarachin C
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval), MIC ¼ 8 mg/cm2
Nakatani et al. (1995b); Huang et al. (1995a, 1995b)
Azedarachol
Et2O ext. of root bark of M. azedarach var. japonica
Antifeedant against Ajrotis sejetum Denis at 10 mg/cm2
Nakatani et al. (1985)
Azedaralide
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2 Ictiotoxic O. latipes at 50 ppm
Nakatani et al. (1998)
Azedaric acid
Light PE ext. stem wood
Okogun et al. (1975)
Campesterol
Fruit
Hanifa Moursi and AlKhabitib (1984)
EtOH ext. of fruit of M. azedarach
Khalil et al. (1979)
Cinamodiol
MeOH ext. (EtOAc fr.) of seeds of Brazilian M. azedarach
Cinnamic acid
EtOH ext. of leaves
Khalil et al. (1979)
Cycloeucalenone
Light PE ext. stem wood
Okogun et al. (1975)
D-Glucuronic
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Keshri et al. (2003)
acid
Kelecom et al. (1996)
Deacetylsalannin
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant against S. eridania (Boisduval) at 20 mg/cm2
Huang et al. (1996a, 1996b)
Fraxinellone
Et2O ext. of stem bark of Japanese M. azedarach
Antifeedant against S. littoralis (Boisduval) at 10 mg/cm2
Nakatani et al. (1998)
Light PE ext. stem wood
Ictiotoxic on O. latipes at 10 ppm
Okogun et al. (1975)
PE ext. of trunk wood
Ekong et al. (1969)
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Naturally occurring bioactive compounds
Little antimoulting activity on R. prolixus at 10 mg/ml
90
Compound
Et2O ext. of stem bark of Japanese M. azedarach
No antifeedant against S. littoralis (Boisduval)
Gedunin
Light PE ext. of stem wood
Okogun et al. (1975)
PE ext. of trunk wood
Ekong et al. (1969)
Nakatani et al. (1998)
Ictiotoxic O. latipes at 50 ppm.
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Prevents the pregnancy and the number of implants at 50 mg/kg of rat/day
Keshri et al. (2003)
PE ext. of bark M. azedarach from Taiwan
Chiang and Chang (1973); Chang and Chiang (1969)
Kulinone
PE ext. of bark M. azedarach from Taiwan
Chiang and Chang (1973)
Kulolactone
PE ext. of bark M. azedarach from Taiwan
Chiang and Chang (1973); Chang and Chiang (1969)
Melain
Buffer ext. of fruit M. azedarach var. japonica
Protease
Kaneda et al. (1994)
Melain G
Buffer ext. green fruit M. azedarach var. japonica
Protease
Uchikoba et al. (1999)
Melazolide A
AcOEt ext. of roots of Italian M. azedarach
D’Ambrosio and Guerreiro (2002)
Meldenin
EtOH ext. (C6H6 fr.) seeds M. azedarach L. from India
Srivastava (1986)
Meliacarpinin A
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b)
Meliacarpinin B
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b) 91
Kulactone
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Fraxinellonone
92
Table 1 (continued ) Origin
Activity
Reference
Meliacarpinin C
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b)
Meliacarpinin D
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 1 mg/cm2
Nakatani et al. (1995b)
MeOH ext. root of M. azedarach L. var japonica
No lethal activity on A. salina at 100 mg/ ml
Fukuyama et al. (2000)
Meliacarpinin E
Et2O ext. of root bark of Chinese M. azedarach
Antifeedant against S. eridania (Boisduval) at 1 mg/cm2
Huang et al. (1996b)
Meliandiol
Fruit
Han et al. (1991)
Melianin A
Light PE ext. of stem wood
Okogun et al. (1975)
Melianin B
Light PE ext. of stem wood
Okogun et al. (1975)
Melianin B
MeOH ext. root of M. azedarach L. var japonica
Fukuyama et al. (2000)
Melianol
CHCl3 ext. of fruits
Lavie et al. (1967a); Han et al. (1991)
Melianolide
Root bark of Chinese M. azedarach L.
Huang et al. (1996a)
Melianone
CHCl3 ext. of fruits
Lavie et al. (1967a); Han et al. (1991)
Melianoninol
Fruit
Antifeedant against Pieris rapae L.
Han et al. (1991)
Meliantriol
CHCl3 ext. of fruits
Repellent against S. gregaria at 8 mg/ cm2
Lavie et al. (1967b); Ascher (1986)
Naturally occurring bioactive compounds
Compound
EtOH ext. (CH2Cl2 fr.) of seed kernel of M. azedarach from Argentina
Meliatoxin A1 ¼ trichilin D
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Meliatoxin A2
Meliatoxin B1
Antifeedant and insecticide against E. paenulata at 4 mg/cm2 and antifeedant against S. eridania at 1 mg/cm2
Carpinella et al. (2002, 2003a) Oelrichs et al. (1983); MacLeod et al. (1990)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2 (400 ppm)
Nakatani et al. (1994b, 1995b); Huang et al. (1994, 1995b)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 0.055 mg/ml
Takeya et al. (1996a)
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Antifeedant against S. litura at 400 ppm (480 mg/cm2)
Oelrichs et al. (1983); MacLeod et al. (1990)
Et2O ext. of root bark of Chinese M. azedarach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 8 mg/cm2
Huang et al. (1994); Nakatani et al. (1994b, 1995b); Huang et al. (1995b)
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Growth inhibitory effect against S. litura at 400 ppm
Oelrichs et al. (1983); MacLeod et al. (1990)
EtOH ext. (CH2Cl2 fr.) of root bark of Chinese M. azedarach L.
Cytotoxic activity against P388 lymphocytic leukaemia cells, IC50 ¼ 5.4 mg/ml
Takeya et al. (1996a)
EtOH ext. (Et2O fr.) of fruit of M. azedarach var. australasica
Oelrichs et al. (1983); MacLeod et al. (1990)
Methyl kulonate
PE ext. of bark of M. azedarach from Taiwan
Chiang and Chang (1973)
Methylene cycloartanone
Fruit and roots of M. azedarach L.
Schulte et al. (1979)
Myristic acid
EtOH ext. (CHCl3 fr.) of root of M. azedarach from India
Prevents the pregnancy and the number of implants at 50 mg/kg of rat/day
Keshri et al. (2003)
Nimbolidin A
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
93
Meliatoxin B2
Role of Melia azedarach L. (Meliaceae) for the control of insects and acari
Meliartenin (isomer B of compound 1)
94
Table 1 (continued ) Compound
Origin
Activity
Reference
Nimbolidin B
PE and Et2O ext. of fruit of M. azedarach var. japonica from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
Et2O ext. of root bark of Chinese M. azedrach L.
Antifeedant against E. varivestis at 0.005%
Bokel (1980)
Antifeedant against S. eridania (Boisduval) at 10 mg/cm2
Huang et al. (1996a, 1996b)
Nimbolin A
Okogun et al. (1975)
PE ext. of trunk wood
Ekong et al. (1969)
Nimbolin B
PE ext. of trunk wood
Ekong et al. (1969)
Nimbolinin B
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
Et2O ext. of root bark of Chinese M. azedrach L.
Antifeedant against S. exigua Hu¨bner (Boisduval) and S. eridania (Boisduval) at 20 mg/cm2
Nakatani et al. (1995a, 1995b) Huang et al. (1996a, 1996b)
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
PE and Et2O ext. of fruit of M. azedarach var. japonica and from Yugoslavia and Greece
Antifeedant against E. varivestis
Kraus et al. (1980); Kraus and Bokel (1981)
MeOH ext. of fruits of Brazilian M. azedarach L.
Cytotoxic activity against HeLa S3 human epithelial cancer cells, IC50 ¼ mg/ml
Zhou et al. (2004)
Ochinolid A
Ochinolid B
Naturally occurring bioactive compounds
Light PE ext. stem wood
Fruit of M. azedarach L. var. japonica Makino
Fukuyama et al. (1983)
Ohchinolal
Fruit of M. azedarach L. var. japonica Makino
Fukuyama et al. (1983)
Pinoresinol
MeOH ext. of fruits of Brazilian M. azedarach L.
No cytotoxic activity against HeLa S3 human epithelial cancer cells
Zhou et al. (2004)
MeOH ext. (EtOAc fr.) seeds of Brazilian M. azedarach
Inhibited moulting R. prolixus, ED50 ¼ 84 mg/ml
Cab
