Recent Progress in Medicinal Plants 29 - Drug Plants III

Recent Progress in Medicinal Plants

Volume 29

Drug Plants III

J.N. Govil Former Principal Scientist Division of Genetics Indian Agricultural Research Institute New Delhi, India

V.K. Singh Former Deputy Director (Botany) Central Council for Research in Unani Medicine (Dept. ofAYUSH, Ministry of Health & Family Welfare) 61-65, Institutional Area, Janakpuri, New Delhi, India

2010

®

Studium Press LLC, U.S.A.

Series Editors: J.N. Govil and v.K. Singh Consulting Editor: N.K. Goyal, National Medical library, Ansari Nagar, Ring Road, New Delhi, India.

©2010 Series Editors & Publishers

This book contains information obtained from authentic and highly regarded sources. Reprinted material from authentic sources which are acknowledged and indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the editors and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. All rights are reserved under International and Pan-American Copyright Conventions. Apart from any fair dealing for the purpose of private study, research, criticism or review, as permitted under the Copyright Act, 1956, no part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means-electronic, electrical, chemical, mechanical, optical, photocopying, recording or otherwise-without the prior permission of the copyright owner.

ISBN: 1-933699-19-1 SERIES ISBN: 0-9656038-5-7

Published by:

STUDIUM PRESS, LLC P.O. Box-722200, Houston, Texas-77072, USA Tel. 713-541-9400; Fax: 713-541-9401 E-mail: [email protected]

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COVER PHOTOGRAPHS 1. Azadirachta indica A. Juss. (Family: Meliaceae) (Left Top

Photograph) 2. Catharanthus roseus (L.) G.Don (Family: Apocynaceae) (Right Top Photograph) 3. Pongamia pinnata (L.) Pierre (Family: Fabaceae) (Left Bottom Photograph)

4. Artocarpus altilis Fosb (Family: Moraceae) (Right Bottom Photograph)

SERIES ISBN: 0-9656038-5-7

RECENT PROGRESS IN MEDICINAL PLANTS: Series Editors: J.N. Govil and V.K Singh

VOLUMES PUBLISHED Vol. 1 :

Ethnomedicine and Phannacognosy (2002) Eds. V.K. Singh, J.N. Govil & Gurdip Singh

Vol. 2:

Phytochemistry and Phannacology (2003) Eds. Surender Singh, J.N. Govil & V.K. Singh

Vol. 3:

Aesthetics (2004) Eds. Asha Khanna, V.K. Singh & J.N. Govil

Vol. 4:

Biotechnology and Genetic Engineering (2004) Eds. J.N. Govil, P. Ananda Kumar & V.K. Singh

Vol. 5:

Crop Improvement, Production Technology, Trade and Commerce (2002) Eds. J.N. Govil, Jitendra Pandey, B.G. Shivakumar & V.K. Singh

Vol. 6:

Diseases and Their Management (2002) Eds. P. Sinha, J.N. Govil & V.K. Singh

Vol. 7:

Ethnomedicine and Phannacognosy II (2003) Eds. V.K. Singh, J.N. Govil, Shamima Hashmi & Gurdip Singh

Vol. 8:

Phytochemistry and Pharmacology II (2003) Eds. D.K. Majumdar, J.N. Govil & V.K. Singh

Vol. 9:

Plant Bioactives in Traditional Medicine (2005) Eds. D.K. Majumdar, J.N. Govil, V.K. Singh & Rajeev Kr. Sharma

Vol. 10: Phytotherapeutics (2005) Eds. S.K. Sharma, J.N. Govil & V.K. Singh

Vol. 11: Drug Development from Molecules (2006) Eds. J.N. Govil, V.K. Singh & C. Arunachalam

Vol. 12: Globalisation of Herbal Health (2006) Eds. Anil K. Sharma, V.K. Singh, J.N. Govil & N.K. Goyal

Vol. 13: Search for Natural Drugs (2006) Eds. J.N. Govil, V.K. Singh & C. Arunachalam

Vol: 14: Biophannaceuticals (2006) Eds. J.N. Govil, V.K. Singh & Khalil Ahmad

Vol. 15: Natural Products (2007) Eds. V.K. Singh, Rakesh Bhardwaj & J.N. Govil

Vol. 16: Phytomedicines (2007) Eds. J.N. Govil, V.K. Singh & Rajeev Kr. Sharma

Vol. 17: Phytochemicstry and Pharmacology III (2007) Eds. V.K. Singh, J.N. Govil & C. Arunachalam

Vol. 18: Natural Products II (2007) Eds. J.N. Govil, V.K. Singh & Naila T. Siddiqui

VoL 19: Phytopharmacology & Therapeutic Values 1(2008) Eds. V.K. Singh, J.N. Govil & Rajeev Kr. Sharma

VoL 20: Phytopharmacology & Therapeutic Values II (2008) Eds. J.N. Govil, V.K. Singh & S.K. Mishra

Vol. 21: Phytopharmacology & Therapeutic Values III (2008) Eds. V.K. Singh & J.N. Govil

VoL 22: Phytopharmacology & Therapeutic Values IV (2008) Eds. J.N. Govil & V.K. Singh

VoL 28: Phytopharmacology & Therapeutic Values V (2009) Eds. V.K. Singh & J.N. Govil

VoL 24: Standardization of Herbal I Ayurvedic Formulations (2009) Eds. J.N. Govil & V.K. Singh

Vol. 25: Chemistry and Medicinal Value (2009) Eds. V.K. Singh & J.N. Govil

VoL 26: Cumulative Index to Abstracts Vols 1-25 (2010) Eds. J.N. Govil & V.K. Singh

Vol. 27: Drug Plants I (2010) Eds. Amani S. Awaad, J.N. Govil & V.K. Singh

Vol. 28: Drug Plants II (2010) Eds. Amani S. Awaad, V.K. Singh & J.N. Govil

Vol. 29: Drug Plants III (2010) Eds. J.N. Govil & V.K. Singh

VOLUME IN PRESS Vol. 80: Drug Plants IV (2010) Eds. V.K. Singh & J.N. Govil

About the Series Medicinal plants are value added for the content and chemical composition of their active components. Therefore, the demand on plant based therapeutics has increased many fold in both developing and developed countries due to the growing recognition that they are natural products, being non-narcotic, having no side-effects, easily available at affordable prices. In a wider context, there is a growing demand for plant-based medicines, health products, pharmaceuticals, food supplements, cosmetics etc. International market of medicinal plants is over US $ 60 billion per year, which is growing at the rate of7% and expected to be US $ 5 trillion by 2050. Herbal remedies would become increasingly important especially in developing countries. Progress in medicinal plants research has undergone a phenomenal growth during last two decades. The input of biochemistry to pharmacology has grown. Molecular pharmacology puts more emphasis on the mode of action of drugs. Worldwide trend towards the utilization of natural plant remedies has created an enormous need for information about the properties and uses ofthe medicinal plants. Based on this rationale, the present series Recent Progress in Medicinal Plants broughtout eight volumes, in the first phase, providing edited information from over 225 original and review papers by eminent scientists and researchers from India and abroad on a wide range of topics in the areas of Ethnomedicine, Pharmacognosy, Phytochemistry, Pharmacology, Aesthetics, Biotechnology, Genetic Engineering, Crop Improvement, Production Technology, Trade and Commerce, Diseases and their Management etc. In continuation to these foregone efforts, further eight volumes (9-16) viz., Plant Bioactives in Traditional Medicine; Phytotherapeutics; Drug Development from New Molecules; Globalisation of Herbal Health; Search for Natural Drugs; Biopharmaceuticals; Natural Products; Phytomedicines, providing recent research data in the areas of medicinal plants investigations, aimed at discovering new drugs of plants origin, were presented. Continuing with the ongoing efforts and over-whelming response, the Series editors have been hard pressed to bring out further nine volumes (Vols: 17-25) of the series on herbal drugs containing recent researches on bioreactive components based on their phytochemistry and phytopharmacology in order to discover potential drugs coupled with their therapeutic values. In this direction, nine volumes (17-25) on Phytochemistry and Pharmacology III, Natural Products II, Phytopharmacology and Therapeutic Values I, II, III, IV & V, Standardization of Herbal! Ayurvedic Formulations and Chemistry and Medicinal Value were published. Thus the publication of25 volumes of "Recent Progress in Medicinal Plants" (2002-2009) provides a comprehensive account of nearly 1800

important medicinal plants for producing drugs, cosmetics, perfumery etc. Hence, it was felt that there is an urgent need to document these 25 volumes in a more condensed form for scientist's desk reference in day to day research activity. Considering the importance of such a resource book, it was planned to bring out Vol. 26 containing the abstracts of papers published in 25 volumeset ofRecent Progress in Medicinal Plants. The Vol. 26- "Cumulative Index to Abstracts, Vols. 1-25"- provides information on some 1282 abstracts of original and review papers published in the aforesaid volumes. Considering the fact that many traditional remedies are back to therapeutic use, including plants as such, or extracts prepared in accordance with the pharmacopoeia of the country where they are used. These medicinal plants are increasingly used as (i) source of direct therapeutic agents; (ii) as a raw material base for the elaboration of more complex semi-synthetic chemical compounds; (iii) as models for new synthetic compounds; and, (iv) as taxonomic markers for the discovery of new compounds. In addition to these applications in developed countries, naturally, the medicinal plants will continue to be used increasingly in developing countries, where they are a traditional source of medicine for generations. This has created renewed interest of scientists in medicinal plants and research is at phenominal rate. We have received excellent studies for publication. It was, therefore, felt desirable to bring out further four Volumes 27-30 of the series, covering recent global updates in medicinal plants researches. It is hoped these volumes will open new vistas of knowledge and the information presented will lead to further research in the discovery of new drugs of natural origin and serve as good source of material for future work.

J.N. Govil and V.K Singh

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Recent Progress in Medicinal Plants Foreword

"Save plants to save lives" was the call given by the World Health Organisation a few years ago to stress the role of medicinal plants in achieving the goal of "health for all". Unfortunately, a high percentage of plant species used in the Indian Systems of Medicine like Ayurveda, Unnani and Siddha are still being collected from forests and from natual vegetation. With a rapid rise in the national and global understanding of the importance of herbal medicines in preventive and curative medicine, the pace of exploitation of medicinal plants from the wild state has increased. Consequently, several important medicinal plant species occurring in forest canopies are being threatened with extinction and are being listed in the Red Data books of IUCN and the Botanical Survey of India. Our first task is to bring about a paradigm shift from collection to cultivation. Species occurring in the wild should be domesticated and cultivated in accordance with market demand. Conservation, sustainable use and equitable sharing of benefits are all vital for developing a sustainable medicinal plant industry. At the same time, we should accelerate our efforts in the areas of validation and identification of the biomolecules responsible for specific medicinal properties. Medicinal plants are equally important in veterinary medicine and our vast livestock wealth can be made more productive only by attending to their health and nutrition. Dr. J.N. Govil and Dr. V.K. Singh deserve our gratitude for their painstaking efforts to compile 30 volumes containing a wealth of information on all aspects of medicinal plants with particular reference to the formulation of both traditional and novel drugs. Volumes 13 to 30 in the series Recent Progress in Medicinal Plants contain valuable ideas on the botanical, biochemical and pharmaceutical aspects of herbal drugs. Volume 16 deals with recent work on medicinal plants, including information on bioprospecting. This timely series of books reinforce the views expressed by

Charaka centuries ago that there are no useless plants in our planet. We must preserve our heritage in herbal medicine and also add to scientific knowledge relating to their properties and active principles. Dr. J.N. Govil, Principal Scientist, Indian Agricultural Research Institute, New Delhi and Dr. v.K. Singh, Assistant Director (Botany), Central Council for Research in Unani Medicine, New Delhi, have rendered valuable service in drawing attention to the vast scope in medicinal plants research and drug development. I hope these books will be widely read and used by all interested in promoting sustainable health security.

f).p.~ (M.S. Swaminathan) New Delhi Dated: 4 th October, 2005

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Foreword to Volume The drugs of herbal, herbo-mineral and animal origin have been used by the traditional healers to maintain health and treat diseases since antiquity. Such medicines are widely used in Africa and Asia including India and China. Due to the adverse side-effects and also the development of resistance against the synthetic drugs, the use of plant-derived drugs is becoming popular in the developed countries also. In recent years the search for natural products as potential therapeutic agents has been an important approach to discovery of new drugs. A multidisciplinary approach to drug discovery involving the generation of truly novel molecular diversity from the natural product sources, combined with total and combinatorial synthetic methodologies provides the best solution to increase the productivity in drug discovery and development. The input of biochemistry to pharmacology puts more emphasis on the mode of action of drugs, albeit it comes clear that the activities of most drugs are not confined to single mode of action. This has led to generating lot of new researches and there is great need for exchange ofthis scientific information for wider use by the scientific community engaged in the search of new drugs of natural origin. The present volume (Volume 29: Drug Plants III of the series Recent Progress in Medicinal Plants) is based on this rationale and presents excellent research and review articles on medicinal plants contributed by eminent scientists from far and wide. I am delighted to write foreword of this important and relevant volume and congratulate the editors Dr. J.N. Govil and Dr. V.K. Singh for their

painstaking efforts in producing this commendable work. I am sure the volume will prove to be an excellent exposition of current research efforts on new promising drugs of natural origin to combat many diseases and conditions, hitherto incurable in modern medicine. The volume should attract a wide audience engaged in the discovery of new drugs of natural origin.

(R.8. Gupta)

Preface Despite convincing progress in synthetic chemistry and biotechnology, plants are the most important source for preventive and curative medical preparations. The search for biologically active compounds to combat new and existing diseases is ongoing and potential plants having vast chemical diversity, have often been targeted as part ofthis search because they contain abundance of potentially active secondary metabolites which may emerge into new lead pharmaceutical compounds. In terms of modern research endeavour, drug developments from plants must necessarily imply a multidisciplinary approach. Recent investigations in specialized areas of biological activity have nevertheless confirmed that plants are a reservoir of chemical agents with therapeutic potential. Since new diseases as well as drug-resistant strains of known pathogens continue to emerge, the search for novel compounds from drug plants is ongoing process and there still would be many potential pharmaceutical components yet to be discovered. Currently there had been many advances in the strategies for discovery and evaluation of drugs, particularly plant-based. Research in herbals and discovering new valuable plants drugs bring together many disciplines including pharmacology, phytochemistry, pharmacognosy, clinical pharmacology, biochemistry, ethnopharmacology, microbiology and other medical sciences. Recent investigations have further widened the scope of medicinal plants encompassing it disciplines like pharmacokinetics, phytopharmacy, chemotaxonomy etc. This has led to generating considerable scientific data world-wide, and there is enormous need for exchange of this information for wider use by scientific community working in the area of discovery of new drugs of natural origin. In view of this global interest in herbal medicines, the present volume 29 : Drug Plants III of series Recent Progress in Medicinal Plants has been compiled. The volume presents 23 chapters based on the contributions received from the scientists from far and wide including Argentina, Brazil, Canada, China, Cuba, Denmark, France, India, Iran, Poland, Serbia, Taiwan, u.K., U.S.A., Zimbabwe. Thus, advanced scientific investigations on a number of medicinal herbs included in this volume have amply demonstrated their remarkable efficacy in combating many of the common and chronic diseases and conditions (owing to their innate medicinal properties and action) viz. Alcohol abuse and dependence (Hypericum perforatum, Pueraria lobata, Salvia miltiorrhiza, Tabernanthe iboga, Panax ginseng); Bone loss (Traditional Chinese Herbal Medicine: Hochuekkito, Ogikenchuto, Ninjin yoeito (containing 12 species of herbs); Schistosomiasis (47 species of medicinal plants are used); Diabetes mellitus (58 species of plants used); Chemopreventive and Radioprotective (Crataegus laevigata, C. microphylla, Citrus aurantum,Crocus sativus, Allium hirtifolium); Coronary Artery Disease (Xuefuzhuyu, Shengmaisan, Guanxin-Er-Hao, Suxiao jiuxin Wan, Gualou xiebai banxia); Gastrointestinal

disorders (Alchornea glandulosa, Byrosnima fagifolia, Mouriri pusa); Osteoarthritis (Zingiber officinale); Diabetes (Terminalia arjuna); Antihypertensive (Catharanthus rose us, Pongamia pinnata, Azadirachta indica, Tamarindus indicus, Artocarpus altilis, Morus alba); Pesticidal (Azadirachta indica, Melia azedarach, Chrysanthemum coronarium, Stellaria chamaejasme, Daphne tangutica, Xanthium sibiricum, Bidens pilosa, Xanthopappus subacaulis); Antiretroviral, Anti-HIV, Prostate carcinoma, Alzheimer's & Parkinson's diseases (Radix Scutellariae - Chinese medicine); Memory retention (Huperzia saururus); Antioxidants (Pterocarpus angolensis, Clausena anisata, Ziziphus mucronata, Turraea nilotica, Vangueria infausta, Annona stenophylla, Ximenia caffra, Dicoma anomalia); Antifertility, Antithrombotic, Anti-cancer (Daucus carota). The findings presented are likely to contribute valuable material for further research leading to development of new drugs. It is hoped the publication will attract a wide audience in the area of drug research, particularly those of Phytochemists, Pharmacologists, Biochemists, Microbiologists, Medical scientists and others in allied disciplines engaged in the search of natural drugs. Weare indeed grateful to all our valued contributors from India and abroad, for their overwhelming response to our request in contributing research papers included in this volume. We thank Dr. H.S. Gupta, Director, IARI, New Delhi for encouragement and writing a foreword to this publication. We also thank our publisher, Studium Press LLC, Texas - 277072, USA and the staff for a timely and expeditious job rendering the manuscript press-ready. 27.12.2009

Editors New Delhi

About the Editors Dr. J.N. Govil (b. 1945): Obtained his Masters and Doctorate degrees from Agra University, Agra, India. In his career span of 41 years research experience at the Indian Agricultural Research Institute, New Delhi, Dr. Govil has been involved in the breeding of cross-pollinated, often cross-pollinated, and selfpollinated crops. His research is mainly focussed on breeding for better quality, disease resistance, and for higher productivity in Pennisetum, Sorghum, maize, chickpea, and pigeonpea. Dr. Govil has been well exposed to the international scientific community through various training programmes. He took his training in "Plant exploration and collection techinques" through IBPGR in 1982. He was also awarded the prestigious FAO I IBPGR Fellowship in "Genetic resources, evaluation, and data preparation and management" at the University of Birmingham, UK. In 1983, he made visits to gene banks located in Europe . He also participated in various international seminars and conferences, including "Food and Legume Improvement for Asian Farming Systems" in Thailand in 1986. Dr. Govil is credited with more than eighty research papers in various journals of national and international repute in various aspects of genetics, crop breeding, and topics on general agriculture. He has written and edited a number of books on medicinal Plants (2 Vols each of two Titles) and other books with international authors. A new series "Recent Progress in Medicinal Plants" has been published by Studium Press, LLC, USA in 25 volumes under Dr. Govil's Chief Editorship. Dr. Govil has been Editor-in-Chief ofthe Journals, New Botanist (An International Journal of Plant Research) and Glimpses in Plant Research, since 1988. He has a lso guided more than a dozen post-graduate students. Dr. J.N. Govil was actively engaged through his leadership on pigeonpea breeding with special emphasis on "Breeding short duration pigeonpea varieties for improved management and low input conditions ." Through his intensive efforts over the last 20 years, nine varieties of early pigeonpea in arhar-wheat rotation have been released at national level. Currently, Dr. Govil has retired from ICAR and has joined as Publishing Director and Managing Editor with Studium Press LLC, USA. E-mail : [email protected] Dr. V.K. Singh (b. 1948): Formerly, Deputy Director (Botany) at Headquarter Office of the CCRUM, is responsible for execution of projects on ethnobotanical surveys, medicinal plants cultivation, development of Herbal gardens, and pharmacognostic studies of crude drugs. Dr. Singh received his Ph.D. in plant taxonomy (1971). He has been a plant explorer, ethnobotanist and conservationist and has taken a series of medicinal plants collection trips in different tribal areas, particularly in North

India, for over 27 years (1971-1997). Based on his studies, he has to his credit over 85 research papers published in various scientific journals in India and abroad, 26 books dealing with medicinal plants and folk medicines of India including edited volumes. Of recent, Dr. Singh has been conferred CCRUM Award (2005) and received first prize from Union Minister of Health & Family Welfare, Govt. of India, for his outstanding research contributions in the area of Survey and Cultivation of Medicinal Plants including development of Herb Gardens. Earliar, Dr. Singh was adjudjed for "Award for Medical Research (1972)" from erstwhile Central Council for Research in Indian Medicine and Homeopathy (CCRIMH) for his contribution to the botanical identity of controversial Ayurvedic drugs. Nominated as Referee for various scientific journals on medicinal plants, and also on the panel of experts committee on WHO studies on indigenous drugs in India. Recently, Dr. Singh was nominated by department of AYUSH, Govt. ofIndia to participate and present his paper in an international symposium on medicinal and nutraceutical plants held at Georgia, USA, during March 2007. Between 2001-2002, Dr. V.K Singh served in the National Medicinal Plants Board, Government of India, New Delhi and was actively associated in the policy formulations and guidelines for promotion & development of medicinal plants sector in the country. He contributed in a book on agrotechniques of medicinal plants entitled "Cultivation Practices of Some Commercially Important Medicinal Plants". During 2002-2007, Dr. Singh headed a Project "Awareness, Training and Cultivation of Medicinal Plants in Western u.P." Dr. V.K Singh happens to be the pioneer in giving the concept of medicinal plants cultivation and marketing among the farmers of Western UP. districts in India. E-mail: [email protected]

ISBN: 1-933699-19-1

Table of Contents About the Series Foreword to the Series Foreword to the Volume Preface Table of Contents of Volume 29

vu lX

xi xiii XVll

1.

Flaveria bide ntis and Flaveria haumanii-Effects and Bioactivity of Sulphated Flavonoids A.M. AGNESE, H.A. GUGLIELMONE AND J.L. CABRERA (ARGENTINA)

1-17

2.

Phytotherapeutic Approach to Alcohol Dependence

19-29

LUDOVICO ABENAVOLI, FRANCESCO CAPASSO AND GIOVANNI AnDOLORATO (ITALY) 3.

Effects of Chinese Herbal Medicines on Bone Loss in Castrated Female Rats SHUJI SASSA, NAHOKO NEMOTO, HITOMI OKABE, SATOE SUZUKI, HmEKI KUDO AND SHINOBU SAKAMOTO (JAPAN)

31-40

4.

Production ofET743, Bryostatin, and Taxol Using a Mineral Based Microbial Amplification System THOMAS J. MANNING, GISO ABADI, KARLY BISHOP, KrusTEN MCLEOD, GUNTER BULLOCK, GREG KEAN, DEVIN GRANT, STUART ANDERSON, KATRICE COOPER-WHITE, SHANDA SERMONS, OM PATEL, DENNIS PHILLIPS, THOMAS POTTER, JAMES NIENOW, PAUL KLAUSMEYER AND DAVID NEWMAN (USA, UK)

41-59

5.

Screening of Natural Products to Drug Discovery LIANET MONZOTE (CUBA)

61-68

6.

Ethnomedicines Used in Trinidad and Tobago for Eye, Dental Problems and Headaches CHERYL LANs (CANADA)

69-78

79-91

7.

Phytomedicinal Agents for Treatment of Schistosomiasis DIVYA HAmDAS AND WILLIAM N. SETZER (USA)

8.

Chemical Composition and Biological Activity of Salvia officinalis L. (Lamiaceae) N IKO RAoULOVIC, ALEKSANDRA DORDEVIC AND RAoOSAV PALIC (SERBIA)

93-111

9.

Evaluation of Medicinal Plants Used to Diabetes Treatment N.H. KAWASHITAANDA.M. BAVIERA(BRAZIL)

113-157

10. Cyclodextrins, Structures, Properties Useful for Treating Diseases and Revitalizing Body Systems

159-181

WIESLAWA MISIUKAND J.N. GOVIL (POLAND, INDIA) 11. Chemopreventive and Radioprotective Effects of Medicinal Plants from Iran SEYED JALAL HOSSEINIMEHR (IRAN)

183-205

12. Chinese Herbal Medicine for Coronary Artery Disease FENG QIN AND XI HUANG (CHINA)

207-215

13. Non-commercial Plants of Medicinal Purposes from the Brazilian Biomes for the Treatment of Gastrointestinal Diseases

217-236

CLAUDIA HELENA PELLIZZON, ARIANE LEITE ROZZA, PAULO CESAR DE PAULA VASCONCELOS, MARCIO ADRIANO ANDREO, WAGNER VILEGAS AND CLELIAAKIKo HIRUMA-UMA (BRAZIL) 14. The Treatment Period-Dependent Effects of Ginger Extract (Zingiber officinale) and Ibuprofen in Patients with Osteoarthritis

237-246

MASOUD HAGHIGHI, Au KHALVAT AND TAYEBEH TOLIYAT (IRAN) 15. Exploring the Anti-diabetic Effect ofTerminalia arjuna in In vivo Animal Model

247-267

MANoNMANI GANAPATHY, K BALAKRISHNAAND C.S. SHYAMALA DEVI (USA, INDIA) 16. Inhibition of Angiotensin Converting Enzyme (ACE) by Medicinal Plants Exhibiting Antihypertensive Activity JALAHALLI M. SIDDESHA, CLETUS J .M. D'SOUZA AND BANNIKUPPE S. VISHWANATH (INDIA)

269-308

17. Pesticidal Activities of Some Important Chinese Medicinal Plant IiANHONG Xu (CHINA)

309-328

18. The Pharmacokinetics and Pharmacodynamics of the Active Ingredients in Radix Scutellariae CHRISTOF KARRICK ARNOLD, CHAO-FONG CHIEN, JEN-CHIH CHANG, YU-TSE Wu AND TUNG-HU TSAr (TAIWAN)

329-343

19. Huperzia saururus: Anticholinesterase Activity and Action on Memory and Learning M.G. ORTEGA, M.G. VALLEJO, A.M. AGNESE AND J.L. CABRERA (ARGENTINA)

345-362

20. Total Phenolic Content and Antioxidant Activity of Some Zimbabwean Traditional Medicinal Plants TAFADZWA MUNODAWAFA, LAMECK S. CHAGONDA, IKLIM VIOL, MAUD MUCHUWETI AND SYLVESTER R. Moyo (ZIMBABWE, SOUTH AFRICA)

363-373

2l. Natural Products as Therapeutic Agents: Past, Present and Future LIANET MONzOTE (CUBA)

375-383

22. Daucus carota L.: A Common Plant with a Potentially Large Medicinal Application-field E. GUINOISEAU, A. LUCIANI, J. CASANOVA, F. TOMI, J .M. BOLLA AND L. BERTI (FRANCE)

385-411

23. Capsaicin: A Spice Derived Phytochemical that Modulates Calcium Homeostasis, Energy Inter-conversion and Cellular Metabolism YASSERAHMED MAHMMOUD (DENMARK)

413-429

INDEX

431-439

"This page is Intentionally Left Blank"

1 Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity of Sulphated Flavonoids A.M. AGNESE 1,2, H.A. GUGLIELMONE 3,4 AND J.L. CABRERA 1,2*

Abstract Flaveria bidentis and F. haumanii are two species from the Asteraceae family, widely distributed from Southern United States to the central area of Argentina. In this country, these species are used in traditional medicine as digestive stimulants, emmenagogues, antiseptic, antifebrile, vermifugue, for skin diseases and against snake poisoning. Even though the genus Flaveria is characterized by synthesizing sulphated flavonoids, these two species in particular are distinguished by the highest grade of sulphation presented by their derivatives of quercetin (tetra and trysulphated). Although, the different biological actions of the flavonoids with aglyconed structures or glycosides in general are well known, there is scarce information about these substances, especially those from the family of sulphate derivatives. In this chapter, we present our investigations related to their inhibitory activity against aldose reductase (enzyme involved in the formation of cataracts in diabetic patients) and their activity and mechanism of action as anticoagulant and antiplatelet agents. Key words: Flaveria bidentis, F. haumanii, Sulphated flavonoids, Aldose reductase inhibition, Anticoagulant, Antiplatelet activity 1. Farmacognosia, Departamento de Farmacia - Facultad de Ciencias Quimicas -

Universidad Nacional de Cordoba, Argentina. 2. IMBIV-CONICET. Ciudad Universitaria, Cordoba, Argentina. 3. Departamento de Bioquimica Clinica-CIBICI-CONICET-Facultad de Ciencias Quimicas - Universidad Nacional de Cordoba, Argentina. 4. Laboratorio de Hemoderivados - Universidad Nacional de Cordoba. Ciudad Universitaria, Cordoba, Argentina. * Corresponding author: E-mail:[email protected]

2

RPMP Vol. 29 - Drug Paints III

Introduction Flavonoids are the most important group of colouring matters in plants, and usually present as aglycons or glycosides. In 1937, a new group of flavonoids was reported, the sulphate derivatives, after the discovering of Persicarine (Isorhamnetin 3-sulphate) from Polygonum hydropiper (Harborne, 1975). Later on, especially in the seventies, additional structures were recognized, particularly within the flavones and flavonols, where distribution reaches more than 250 species of dicotyledonous and monocotyledonous families (Hannoufa et al., 1991); for this reason, these are not considered rare constituents of the flavonoid family, as it was in the past. Within this significant distribution of sulphated flavonoids (SF) in the plant kingdom, a remarkable fact is the ability of polysuI phation developed by two species of the genus Flaveria, compared to other species that contain these substances. These are Flaveria bidentis (L.) Kuntze and Flaveria haumanii Dim. et Orf. (= F. bidentis var. angustifolia O.K.). Flaveria Juss (Asteraceae) is an essentially American genus composed by 21 species, most ofthem native from the Northern hemisphere (Powell, 1978).

Flaveria bidentis and F. haumanii These are the only two representatives of their genus that are extensively distributed in the Argentinean territory. These are still not cultured or commercialized plants, recognized with their ordinary name "Fique", "Balda", "Matagusanos" and "Chasca", between others. Leaves of both species are used in traditional medicine as digestive stimulants, emmenagogues, antiseptic, antifebrile, vermifugue, for skin diseases and against snake poisoning (Ariza Espinar, 2006). They were also used as textile dye (Zhang et al., 2007).

Chemical constituents The aerial parts and roots of both species were studied in our lab and up to this moment, seven SF have been identified as main constituents of the polar extract of aerial parts (Fig 1), and two thiophenic derivatives from the apolar extract of the roots and aerial parts (Fig 2). These results are resumed in Tables 1 and 2 (Agnese et al., 1999).

Bioactivity Even though flavonoids are natural compounds recognized by their variety of activities, such as antioxidant, free-radical scavenging, antiviral, antimicrobial, anti-inflammatory, anticancer, and others, there are scarce assays that have specifically analyzed SF bioactivity. Herein, we describe the studies performed in order to evaluate their action as inhibitors of the aldose reductase enzyme, as well as their anticoagulant and antiplatelet properties.

3

Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity

K0 3SO OCOCR,

o

o

Quercetin tetrasulphate

Quercetin 3 acetyl trisulphate

OS03K OR

OS03K

<7

~I

o Quercetin 3, 7, 4' trisulphate

OS03K

Quercetin 3, 7, 3' trisulphate

OR OS03K

OR <71 ~

o Quercetin 3, 4' disulphate

Quercetin 3 sulphate

OCR 3 OR

~

""I

o

RO

o

Isorhamnetin 3, 7 disulphate

Isorhamnetin 3 sulphate

Fig 1. Sulphated flavonoids isolated from Flaveria bidentis and F. haumanii R

S

S

S

o-DrG Alpha terthienyl

/S"-........../S~

UU"

CR,

5 (3 buten 1 ynyl) 2, 2' bithienyl

Fig 2. Thiophene derivatives isolated from leaves and roots of Flaveria bidentis and

F. haumanii

Sulphated flavonoids as aldose reductase (AR) inhibitors AR is found in human and animal lens and is responsible for the transformation of the aldehyde groups into their corresponding primary alcohol. This reaction, performed by an enzymatic route, is involved in processes that lead to cataracts in experimental or clinical diabetes. This disease is characterized by the accumulation of sorbitol from glucose, which

RPMP Vol. 29 - Drug Paints III

4

Table 1. Sulphated flavonoids isolated from Flaveria bidentis and Flaveria haumanii

F. bidentis

Flavonoids (leaves) Quercetin 3,7,3',4'-tetrasulphate Quercetin 3-acetyl-7 ,3' ,4'-trisulphate Quercetin 3,7,3' -trisulphate Quercetin 3,7 ,4'-trisulphate Quercetin 3-sulphate Isorhamnetin 3,7-disulphate Isorhamnetin 3-sulphate

F.haumanii

+++ +++

+

+ + +++ ++

Table 2. Thiofenic derivatives isolated from leaves and roots of Flaveria bide ntis and F. haumanii Tiophenes a-terthienyl 5-(3-buten-l-ynyD-2,2' -bithienyl

F. bidentis Leaves Roots +

+

+

+

F.haumanii Leaves Roots

+

is accumulated in the lens in elevated amounts compared to normal values, but differently from glucose, this polyalcohol has a diminished ability of membrane permeability. This fact produces, in a first stage, myopia due to the increase ofthe lower diameter of the lens as consequence of an augment of its internal osmotic pressure, and in a second stage, the vast accumulation of sorbitol leads to events that generate opacity of the lens, this means cataracts formation (Dvornik et ai., 1973). One of the first structures that inhibits AR is 1,2 dioxo-1H-benz[delisoquinolin 2-3H acetic acid (AY-22,284) (Dvornik et ai., 1973). This is a synthetic structure that was used as reference in the first studies related to the inhibitory action offlavonoids (Varma et ai., 1975), evaluating later on numerous derivatives within the flavonoid family (flavones, flavonols, anthocyanins, chalconas, etc.) showing a marked non competitive inhibitory activity (Varma et ai., 1976). The finding in our lab of numerous SF isolated from F. bidentis and F. haumanii, derivatives from quercetin (Qc) and isorhamnetin (Pereyra & Juliani, 1972; Cabrera & Juliani, 1976; Cabrera & Juliani, 1977; Cabrera & Juliani, 1979; Cabreraet ai., 1985) has attracted the interest of the investigators, since these substances have not been studied as AR inhibitors and are highly soluble in biological systems. These studied were performed in the Department of Ophthalmology, University of Maryland, School of Medicine, Baltimore, Maryland (USA) under the direction of Dr . Shambu D. Varma. The studies to evaluate the potential efficacy of SF were performed in several stages:

Flaveria bide ntis and Flaveria haumanii - Effects and Bioactivity

5

Inhibitory activity of sulphated flavonoids over AR in homogenized rat lens (spectrophotometric method) To determine the inhibitory activity of flavonoids, we used AR from a homogenized of rat lens, using as substrate glyceraldehyde and analyzing spectrophotometric ally at 340 nm, the advance ofthe reaction compared to the corresponding sample without inhibitor (Dvornik et al., 1973), and determining the oxidation speed ofNADPH according to the following reaction:

AR Gliceraldehyde + NADPH

Glicerol + NADP

The results on chemical structures, concentration and inhibition values are shown in Table 3. In addition to the flavonoids obtained fromF. bidentis and F. haumanii, other semisynthetic derivatives were assayed (acetyl derivatives), taking Qc and AY-22,284 as references (Cabrera et al., 1980). Table 3. Percentage of inhibition of AR by SF at different concentrations Spectrophotometric technique Flavonoids

% Inhibition

10· M 7

10-8M

10·oM

AY-22,284 'a ) 0 0 0 Quercetin la) 15 0 0 Isorhamnetin-3-sulphate Ib) 62 20 0 Isorhamnetin 5,7 ,4'tri acetyl-3-sulphate Ie) 47 30 0 Isorhamnetin 3,7-disulphate Ib) 29 95 0 Quercetin 3,4'-disulphate Ib) 20 62 0 Quercetin 3,7, 3',4'-tetrasulphate Ib) 50 21 90 Quercetin 3-acetyl 7, 3', 4'-trisulphate Ib, 100 68 39 Quercetin 3,5-diacetyl 7, 3', 4'-trisulphate Ie) 76 50 90 Quercetin 5,7 ,3'-triacetyI3,4'-disulphate Ie) 20 90 75 a =reference compounds; b = natural flavonoids; e = semisynthetic flavonoids' • Semisynthetic flavonoids were obtained by traditional acetylation methods (pyridine-acetic anhydride).

Inhibitory activity of SF over AR in incubated rat lens The second stage of the study was performed incubating intact lens of rats weighting 100 g in a culture media with high contents of galactose (30 nM) (AR has the same behavior against glucose, but the use of galactose facilitates the process) maintaining it for 1:30 h at 37°C in an atmosphere of5% 02C in the air. The culture media with addition of inhibitor have their respective white assays of reference (without inhibitor). After the incubation, the lens were homogenized, deproteinized, centrifuged and the supernatant was lyophilized containing the polyalcohol (dulcitol) that was derived as silyl derivative and quantified by GC. In this manner, the inhibition produced by SF over AR was established, but in these assays, differently from the previous ones, the inhibitor had to go beyond the

RPMP Vol. 29 - Drug Palnts III

6

Table 4. Percentage of inhibition of SF over AR in cultures of rat lens % Inhibition

Flavonoids

Quercitrin (al Quercetin 3-acetyl 7, 3', 4'-trisulphate (bl Quercetin 3,5-diacetyl 7,3' ,4'-trisulphate (el Quercetin 3,7, 3',4'-tetrasulphate (bl Quercetin 5,7,3'-triacetyI3,4'-disulphate lei Quercetin 3,4'-disulphate (bl Quercetin -5-acetyI3. 7 ,3' ,4'-tetrasulphate (el a

lO-6M

10·7 M

20 65 46

0 23 8 0 0 0 0

40 24 11

9

= reference compound; b = natural flavonoids; e = semisynthetic flavonoids

barrier represented by the lens membrane to contact the enzyme. The results obtained taking the glycoside of Qc as reference are shown in Table 4 (Cabrera et aI., 1980).

Inhibition of AR from human lens. Comparative study of SF with other aglycons, glycosides and non flavonolic derivatives (spectrophotometric assay) This study was related to the activity of SF and other flavonoid derivatives, comparing them to other structures active against AR, which are in addition, recognized anti-inflammatory agents (sulindac, indomethacin and aspirin). The new information, more than the comparative fact of the inhibitory activity, is the use of AR from human lens to correlate it with material obtained from animals. It is necessary to remark that in these assays, we used samples purified by gel filtration (Sephadex G-75) instead of AR obtained directly from the homogenized of lens (Chaudhry et al., 1983). The more significant results are exposed in Table 5. Table 5. Inhibition of AR from human lens by different compounds Inhibitor Quercetin Taxifolin Quercetin-3-0-glucoside Quercitrin Quercetin-3-acetyl-7 ,3' ,4' trisulphate Myricetin Myricitrin Indomethacin Sulindac Aspirin

% Inhibition 10'5M I0-6M 10'1M I0-6M 10·9M

59 60 42 76 100 47 74 34 76 18

32 22 0 50 85 26 50 20 59 0

0 0 0 0 50 0 12 0 17 0

0 0 0 0 42 0 0 0 0 0

0 0 0 0 12 0 0 0 0 0

Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity

7

In vivo assays: Treatment of galactosemic rats with instillation of ophthalmological preparations based on sulphated flavonoid During the last phase ofthe study, intends to diminish the accumulation of dulcitol in galactosemic rats were performed. This disease was easily induced by a 5 days therapy, in which the rats received regular feeding during the first 3 days and a diet with 50% galactose during the last 2 days. Instillation through an ophthalmological solution was considered the most appropriate route of administration, since SF are esters that tend to hydrolyze when administered orally. The ophthalmological solution was prepared with one ofthe derivatives that had provided better results in previous in vitro assays, quercetin 3-acetyl-7,3',4' trisulphate (ATS) 10%, methylcellulose 0.3%, benzalkonium 1: 50.000; pH was adjusted at 7.4 with boric acid. We worked with groups of rats weighting 100 g each (n=6). Instillation was repeated hourly during 12 h in which the animals were kept in the dark and during the following 12 h the rats stayed at light without medication. The treatment was repeated during 5 days, in which each rat received the ophthalmological solution in one eye and placebo in the other. At the end ofthe fifth day, the animals were sacrificed and the lens extracted and processed in the same manner as detailed in (B), quantifying the remnant dulcitol, previously silylated by GC. These results are resumed in Table 6. Table 6. Percentage of inhibition of dulcitol by quercetin 3-acetyl-7,3',4' trisulphate (in vivo assay) Animal N° 1 2 3 4 5 6

Treated eye nM duicitolllens

Not treated eye nM dulcitolllens

% Dulcitol decrease

201 126 161 177 153 231

337 219 276 278 260 350

40.3 42.4 41.6 36.3 41.1 34.0

Average ofinhibition= 39.2%; other assays performed provided similar results (data not shown)

Results and Conclusions The first studies that analyzed the inhibition of AR by natural substances included flavonoid derivatives, which generated a special interest, not only by their condition of effective inhibitors achieving earlier and higher effects than the previously known synthetics, but also because of their non toxic condition. This fact generated expectations and further investigations towards new structures, especially those with different characteristics than substances previously studied, as SF. The studies previously described on AR inhibition by quercetin polysulphated derivatives were performed in this context. The in vitro results shown in Tables 3 and 5 demonstrate clear differences between previously non-sulphated known compounds,

8

RPMP Vol. 29 - Drug Palnts III

standing out especially ATS; for this reason, other acetylated derivatives were assayed, but did not provide better results. However, the differences as inhibitory agents were not very substantial compared to other aglycons and glycosides previously used, when the assays were performed in lens cultures (Table 4) where the tissular membrane constitutes a limiting barrier for the effectiveness of the inhibitor. This fact is more significant and clearly observed in in vivo assays with a preparation containing 10% SF (Table 6). A decrease of the effectiveness of this system over AR could be due, between other factors, to the high polarity of these compounds, which would act adversely when considering their ability to penetrate membranes. This fact was observed for the first time in assays with lens cultures and accentuated when administered in vivo, where the number of tissues necessary to enter the lens and act over AR was higher. The above mentioned assays were only preliminary studies, which main limitation was the limited available amount ofthe sulphate derivatives under study, and for this reason, the results shown are only illustrative.

Sulphated flavonoids: Anticoagulant and anti platelet effects Haemostasis or the physiological stop of bleeding involves a series of complex mechanisms including cellular (endothelial cells and platelets) and plasmatic components (coagulation proteins, fibrinolysis and its inhibitors). When a blood vessel is injured by traumatic or other causes, three different mechanisms act locally to stop bleeding: the contraction of the blood vessel wall, the formation of a platelet plug and finally, the stabilization of the thrombus by the formation of a fibrin network. The platelets that adhere to collagen expose compounds including ADP, serotonin and thromboxane~, which potentiate vasoconstriction. The seal of the damaged blood vessel begins after the adhesion of the platelets to the exposed subendothelial tissues. This process occurs through complex reactions that involve subendothelial macromolecules, in addition to circulating and integral membrane proteins over the platelet surface; these cells, once attached, expose the contents of their granules and accumulate to each other until producing a platelet plug. Haemostasis is reached if platelets are enough to stop bleeding. However, this effect is transitory since the platelet thrombus is not stable and the formation of a fibrin mesh is necessary to prevent further bleeding. This process is mediated by the coagulation system, which begins when a protein (tissue factor) that are not usually present in blood are exposed; these come from the injured sites of the vessels and as a consequence of the impaired anticoagulant function of the endothelium. This system is mediated by a cascade of enzymatic reactions that activate the coagulation factors present in plasma as proenzymes, which, when activated under the presence of cofactors, cellular surfaces and calcium ions, produce thrombin, the main enzyme of the coagulation system. Finally,

Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity

9

the transformation of fibrinogen into fibrin, mediated by thrombin and the following polymerization process, generate a compact mesh that involves in addition, the platelet thrombus. Haemostasis is regulated by a group of physiological inhibitors that act at different levels, being antithrombin III and heparin cofactor II, the most important factors within this group. Different situations can induce to failures in the regulatory mechanism of the coagulation system; this is translated into a tendency to develop thrombotic events of different magnitude, according to each defect. For this reason, several drugs with antithrombotic effects have been developed for years and classically can be divided in two groups. One ofthem includes drugs that act at platelet level, avoiding their activation (antiplatelet agents, as aspirin and clopidogrel) and the other group includes anticoagulants like heparin (a highly sulphated polyanion) and coumarinic derivatives that inhibit the activation of the coagulation system at different levels. In spite of diverse mechanisms of action, the final function of such drugs is to avoid the formation of thrombus inside the blood vessel.

Platelet anti-aggregant properties of SF isolated from F. bidentis Since the SF previously described are derivatives from Qc and in previous studies it was demonstrated that this flavonoid is capable of inhibiting platelet aggregation (Hubard et at., 2003), we developed studies with the purpose of determining if quercetin-3,7,3',4'-tetrasulphate (QTS) and ATS modulates the in vitro platelet activity and in such case, to establish the mechanism of action of this effect. For this reason, we used washed platelets from healthy humans without medication or history of hemorrhagic diseases (Guglielmone et at., 2000). The platelets were incubated with increasing concentrations of Qc, QTS and ATS (1 to 1000 ).lm) at different incubation times (0 to 90 min at 37°C), we took aliquots and stimulated them with different aggregant agents (ADP, adrenalin, collagen, ristocetin and arachidonic acid). The first assays were performed with the purpose of evaluating which was the concentration of SF that produced the highest anti platelet effect. Fig 3 shows that 250 ).lm was the optimum concentration to reach this effect. N ext, we evaluated the percentages of aggregation obtained with this flavonoid concentration compared to other aggregant agents, which were calculated from the records provided by a registering machine connected to a platelet aggregometer. These results are shown in Table 7. These data allowed us concluding that QTS has inhibitory effects over platelet aggregation in a similar manner than observed for Qc. On the other hand, the use of ATS only demonstrated a very mild effect over the percentage of platelet aggregation. In order to determine at what level this inhibitory effect was produced, we determined the percentage of platelet aggregation when platelets previously washed and incubated with different SF were confronted with U-46619 (a recognized platelet agonist that interacts over the receptor of thromboxane A2 ).

RPMP Vol. 29 - Drug Paints III

10 100 80

.S ....== bI)

os

60

~

40

bI)

os e"-

20 0

Control ~ 0

Qc

D

100 •

QTS

250

o

ATS

500

IiI!I

1000

Fig 3. The concentration-dependent inhibitory effects offlavonoids on platelet aggregation (expressed a s % of aggregation) induced by collagen 1 p.g/ml at different concentrations of Qc, QTS and ATS in p.m. The other agonists showed a similar pattern. Percentages of aggregation are presented as means ± SEM (n=3-4) Table 7. Effects ofQc, QTS and ATS on platelet aggregation induced by different agonists % Platelet aggregation

Flavonoids EP

ADP Control Qc QTS ATS

70.2 19.5 23.3 62.8

3.1 5.3** ± 4.5** ± 9.8 ± ±

74.5 14.5 19.7 58.5

Collagen

2.5 5.6** ± 6.7** ± 10.7

±

±

89.3 21.3 28.7 59.9

AA

5.4 86.6 ± 5.4 6 .5** 9.2 ± 3.4** ± 7.8** 20.3 ± 4.3** ± 8.4* 49.8 ± 8.9* ± ±

Ristocetin 88.7 79.6 75.4 80.4

3.3 4.4 ± 5.6 ± 10.3 ± ±

Values are expressed as means ± SEM of the % aggregation (n=25). *p<0.05, **p
The results shown in Fig 4 allowed us establishing that QTS developed antiplatelet effects through this link to the receptor ofthromboxane A2in a magnitude order similar to values observed for Qc, even though at higher concentrations (250 us. 500 pm). 100 80

.S ...==

~

~os e"-

60 40 20 0

Control ~O

Qc 0100

.250

QTS ~500

ATS .1000

Fig 4. Effects of flavonoids on platelet aggregation (expressed as % of aggregation) induced by U-46619 (1 m ) at different concentrations of Qc, QTS and ATS in m . Percentages of aggregation are presented as means SEM (n=3-4)

Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity

11

In order to corroborate these results and evaluate the formation of intraplatelet thromboxane A2 under the presence of SF, we performed measurements of thromboxane B2 (a stable metabolite of thromboxane A 2 ) in the supernatant of a suspension of platelets incubated with QTS, ATS and Qc and activated with arachidonic acid (AA) and/or collagen. These results are shown in Fig 5 and allowed us concluding that there was an important decrease in the production of thromboxane B2 by platelets incubated with QTS and Qc, and very low under presence of ATS. The results of this study clearly demonstrated that the flavonoid QTS isolated from F. bidentis inhibited in vitro platelet aggregation in a magnitude similar to Qc. According to the profile of aggregation obtained when U-46619 was used as platelet agonist, the mechanism of this inhibition was mediated through the receptor of thromboxane A2 or by interfering in the synthesis of prostaglandin endoperoxides. Additional studies destined to confirm this theory, determined the concentration of thromboxane B2 and these results demonstrated, once again, that QTS blocks the receptor ofthromboxane A2located in the platelet membrane. On the other hand, ATS, the other flavonoid isolated from the same plant but with lower degree of sulphation, did not significantly inhibit platelet aggregation, even at high concentrations (1.000 Ilm). Finally, none of the studied SF was able to inhibit platelet function induced by ristocetin. This fact demonstrated that other factors ofthe platelet-platelet interaction with plasmatic factors that participate in this process, like the glycoprotein complex of platelet membrane CIb-IXo lIb-IlIa) and even the von Willebrand factor, were not involved in this mechanism of inhibition (Guglielmone et al., 2005).

Anticoagulant properties ofSF isolated from F. bidentis From the numerous SF isolated from F. bidentis, (Fig 1), ATS and especially QTS have electro negativity; this is not usually found in other flavonoid derivatives; consequently, the structural requirements to associate them to the physiological glycosaminoglycans could be fulfilled. In view of these considerations, we initiated studies on the possible anticoagulant activity of SF. One of the first assays performed included determining of SF interfered with in vitro assays that evaluated the coagulation system, such as prothrombin time (PT), activated partial prothrombin time (APTT) and thrombin time (TT) and establishing of this effect was timeconcentration dependent. For this purpose, we incubated increasing concentrations (1 pM to 1 mM) of QTS and ATS with a pool of normal human plasma at 37 °e, at different periods of time (0, 60, 120 and 180 min). These results are shown in Fig 6, in which an important prolongation in the clotting time is observed, (expressed as relation oftime in seconds under presence or absence of SF) of APTT, a mild increase of PT and a null response ofTT in concentrations ofnM for QTS, and very low response in presence of ATS.

12

RPMP Vol. 29 - Drug Paints III A

120 ~

g:)

x

100

Eo<

80

f

60

=

40

~

20

.s ~

~

0

Qc

Control

~O

0

250

QTS

.500

ATS ~ 1000

B ~

g:)

x Eo<

... ~

CIS

"" ~

= ~

1:111

~

Qc

Control

~O

0

250

.500 QTS

ATS ~ 1000

Fig 5. Thromboxane B2 production in washed platelet incubated with Qc, QTS and ATS and induced by Collagen (a) and AA (b)

The following objective was to establish if the anticoagulant effects were really due to an inhibitory effect over the coagulation factors. For this purpose, factors II, V, VII, VIII, IX, X, XI and XII were assayed at different incubation times in normal human plasma incubated with 1 mM of QTS; the results did not show any modifications of the factors level (Figs 7 & 8). According to these results, in a second stage we analyzed if the activity of thrombin enzyme was modified by their natural inhibitors, antithrombin III and/or heparin cofactor II. For this purpose, we evaluated if these inhibitors could be activated by SF in a similar and comparable manner than by their natural activators (heparin for antithrombin III and dermatan sulphate for heparin cofactor II), in terms of potency. To measure the functional activity of both inhibitors we used chromogenic specific substrates for each of them and the results obtained are showed in Fig 9. As it can be observed, the ability of QTS to activate heparin cofactor II is manifested at different concentrations ofthe inhibitor. As a matter of fact, at levels 1.0; 0.5 and 0.25 UI of heparin cofactor II, the percentage of activation of this molecule by QTS is mildly inferior to values produced by its natural activator, the dermatan sulphate. On the other side, the other SF analyzed, ATS, showed a mild ability to activate this inhibitor but on the

13

Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity 2.S

2.S

2.4

2.4

n

TT

2

2

1.6

1.6

1.2 O.S

.-. 0'

1.2 O.S 60'

120'

ISO'

2.S

0'

120'

ISO'

2.4 PT

PT

2

1.2

60'

2.8

2.4

1.6

.

..-

2

..

1.6

....... . . . .- ...~ ±-..

1.2

-

O.S

~ ..,

~

~-~4---

0.8 0'

60'

120'

180'

0'

2.8

2.8

2.4

2.4

2

2

1.6

1.6

1.2

1.2

O.S

0.8 0'

60'

120'

ISO'

60'

120'

ISO'

~~ ............ ~

.-_.... 0'

-

.....-_ ... -..

-

60'

120'

ISO'

incubation time (min) - - . - - 1 pM

_

1 jJ.M

----.,---. 1 mM

Fig 6. Modification of the coagulation assays in the presence of flavonoids at different concentrations and incubation times expressed as ratio (vs. control plasma)

contrary, none ofthe SFs was capable of activating antithrombin III, which demonstrated high substrate specificity for these natural products. As conclusion, we can assure that QTS has anticoagulant properties since they extend in vitro some coagulation tests and that this phenomenon is not due to a direct effect over coagulation factors, but to their property of inhibiting the thrombin enzyme through activation of one of its natural inhibitors, heparin cofactor II. This effect is highly specific, since the same behaviour is not observed under the presence of antithrombin III and is

RPMP Vol. 29 - Drug Palnts III

14 1.4 1.2 1

~

0.8 0.6 0.4 0.2 0

II

•

0 min

VII V Coagulation factors

[J 60 min III 120 min

X

EJ

180 min

Fig 7. Level of coagulation factors II, V, VII, and X in normal pooled plasma incubated with 1 mM QTS at 0, 60, 120 and 180 min 1.6 1.4 1.2 1

~

0.8 0.6 0.4 0.2 0

VIII

•

0 min

XI IX Coagulation factors

[J 60 min

III 120 min LJ

XII

180 min

Fig 8. Level of coagulation factors VIII, IX, XI and XII in normal pooled plasma incubated with 1 mM QTS at 0, 60, 120 and 180 min

possibly related to the high grade of sulphation ofthis flavonoid (Guglielmone et al., 2002), as has been demonstrated with other sulphated macromolecules (Maimone et al., 1990).

Future perspectives The disorders ofhaemostasis and the alterations of the blood vessels walls lead to their obstruction and the formation of thrombus. These are generated by the activation ofthe platelets that expose adhesive glycoproteins (Ib-IX) that stick to the damaged vessel wall and aggregate to each other

Flaveria bidentis and Flaveria haumanii - Effects and Bioactivity

...... U == .....

0 I:

120 100

m m

80

.~

60

;. :;l

40

<

20

....os

mQIS •

CJ

If

15

ATS

0 1.00

0.50

0.25

Hell concentration (UlIml)

120

......

100

E= < ..... 0

80

...=

60

...

40

.~

os

.:! CJ

< If

20 0

)c )c

)c )c

:x»c

~ ~

»c »c

~ ~ ~>c

'?>c

»c =-= »c 1.00

K1

Heparin

mQIS .ATS

~

~~ 0.50

0.25

ATIII concentration (UlIml)

*p=NS for QTS vs. DS; *p< .001 for ATS vs. DS; (b) *p< .001 for QTS and ATS vs. Heparin Fig 9. Hell and ATIII activation by flavonoids QTS and ATS compared to DS (a) or heparin (b)

(glycoproteins lIb-IlIa) forming a platelet thrombus. On the other side, the activation of the mechanism of coagulation initiated by the expression of the tissular factor initiates a series of enzymatic reactions ofthe coagulation factors that originates thrombin and finally the formation of fibrin with consequent stabilization of the thrombus . The recanalization of the obstructed blood vessel obstructed by this thrombus is conducted by the fibrinolytic system (through its enzyme plasmin) that degrades it and allows blood recirculation. When this final process is not appropriately achieved, it is necessary to use pharmacological methods including drugs that block the activation of the platelets or the coagulation system. Currently, there are several drugs that fulfil these objectives, but none of them can inhibit both, the platelets and the coagulation system as well. Preliminary in vitro assays performed by our team allow us stating that QTS isolated from F. bide ntis is an effective inhibitor of the platelet aggregation and has anticoagulant effects as well. This dual property confers it potential abilities as anti thrombotic agent. Dl,.le to these promising results, our next objectives would be a) to evaluate the effects of these SF over the trigger of the

16

RPMP Vol. 29 - Drug Palnts III

coagulation system: the tissular factor, b) to study if these natural products affect in any manner the enzymes ofthe fibrinolytic system that participate in dissolution of the thrombus, and c) to demonstrate in animal models the antithrombotic properties of these flavonoids.

References Agnese, A.M., Nunez Montoya, S., Ariza Espinar, L. and Cabrera, J.L. 1999. Chemotaxonomic features in Argentinean species of Flaveria (Compositae). Biochem. Syst. Ecol. 27: 739-742. Ariza Espinar, L. 2006. Asteraceae. In : Barboza, G.E., Cantero, J.J., Nunez, C.O. and Ariza Espinar, L. eds., Flora Medicinal de la Provincia de Cordoba, Argentina. Museo Botanico Universidad Nacional de Cordoba, Argentina, pp. 373-375. Cabrera, J .L. and Juliani, H.R 1976. Quercetin-3 acetil-7,3',4', trisulphate from Flaveria bidentis. Lloydia (J. Nat. Prod) 39(4): 253-254. Cabrera, J .L. and Juliani, H.R 1977. Isorhamnetin-3,7-disulphate from Flaveria bidentis. Phytochemistry 16(3) : 400-400. Cabrera, J .L. and Juliani, H.R 1979. Two new quercetin sulphates from leaves of Flaveria bidentis. Phytochemistry 18(3): 510-511. Cabrera, J.L. , Juliani, H.R and Gros, E.G. 1985. Quercetin 3,7,3' trisulphate from Flaveria bidentis . Phytochemistry 24: 1394-1395. Cabrera, J .L., Juliani, H .R, Pohl, M.G. and Varma, S.D. 1980. Inhibition of rats aldose reductase by flavonoid esters. In : ARVO ed .. Invest. Ophthal. and Vis. Sci ., Supp. April 1980, p.150. Annual Spring Meeting, May 4-9, 1980, Orlando, Florida, USA. Chaudhry, P .S., Cabrera, J.L. , Juliani, H.R and Varma, S.D. 1983. Inhibition of human lens aldose reductase by flavonoids, sulindac and indomethacin. Biochem. Pharmacal. 32(13) : 1995-1998. Dvornik, E., Simard-Duquesne, N., Krami, M., Sestanj, K., Gabbay, K.H., Kinoshita, J .H., Varma, S.D. and Merola, L.D. 1973. Polyol accumulation in galactosemic and diabetic rats: control by an aldose reductase inhibitor. Science 182(117) : 11461148. Guglielmone, H., Agnese, A.M., Nunez, S.C. and Cabrera, J .L. 2005. Inhibitory effects of sulphated flavonoids isolated from Flaveria bide ntis on platelet aggregation. Thromb. Res. 115(6): 495-502. Guglielmone, H., Daniele, J ., Bianco, I. and Fidelio, G. 2000. Inhibition of platelet aggregation with gangliosides. Thromb. Res. 98: 51-59. Guglielmone, H ., Nunez, S.C. , Agnese, A.M. and Cabrera, J .L. 2002. Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis. Thromb. R es. 105(2): 183-187. Hannoufa, A. , Varin, L. and Ibrahim, RK. 1991. Spatial distribution of flavonoid conjugates in relation to glucosyltransferase and activities in Flaveria bidentis. Plant. Physiol. 97: 259-263. Harborne, J .B. 1975. Flavonoid sulphates: A new class of sulphur compounds in higher plants. Phytochemistry 14: 1147-1155. Hubbart, G.P., Steveus, J.M., Cicnil, M. , Sage, T. , Jordan, P .A. and Williams, C.M. 2003. Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway. J . Thromb. Haemost. 1: 1079-1088. Maimone, M. and Tollefsen, D.M. 1990. Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. J . Biol. Chem. 265: 1826318271.

Flaveria bide ntis and Flaveria haumanii - Effects and Bioactivity

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Pereyra, O.J. and Juliani, H.R. 1972. Isolation of quercetin 3,7,3',4'-tetrasulphate from Flaveria bidentis O.K. (Compositae). Experientia 28: 380-380. Powell, A.M. 1978. Systematics of Flaveria (Flaveriinae-Asteraceae), Ann. Missouri Bot. Gard. 65: 590-636. Suarez, S.S., Cabrera, J.L. and Juliani, H.R. 1979. Flavonoides en Flaveria bidentis (L.) O.K. y Flaveria bidentis var. angustifoha O.K. (Compuestas). An. Asoc. Quim. Argent. 67: 229-230. Varma, S.D. and Kinoshita, J.H. 1976. Inhibition oflens aldose reductase by flavonoids: Their possible role in the prevention of diabetic cataracts. Biochem. Pharmacol. 25: 2505-2513. Varma, S.D., Mikuni, I. and Kinoshita, J.H. 1975. Flavonoids as inhibitors oflens aldose reductase. Science 188(4194): 1215-1216. Zhang, X., Boytner, R., Cabrera, J.L. and Laursen, R. 2007. Identification of yellow dye types in some pre-columbian textiles. Anal. Chem. 79: 1575-1582.

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2 Phytotherapeutic Approach to Alcohol Dependence LunOVICOABENAVOLI 1*, FRANCESCO CAPASS0 2 AND GIOVANNI AnDOLORAT0 1

Abstract Alcohol abuse and dependence represent a worldwide problem from both medical and social points of view. In Italy, it is estimated that there are about one million of alcohol dependent subjects. The pharmacological treatment of patients with alcohol dependence, play a key role in order to achieve alcohol abstinence and prevent relapse. At present, the possible utility of the Complementary Medicines in the treatment of alcohol dependence, is controversial. In the last years, pre-clinical and clinical data from traditional medicines, suggest that novel pharmacological approaches for treatment of alcoholism and alcohol abuse, may stem from natural substances. The present review summarizes the findings of the effects of phytotherapy in alcohol addiction. Key words : Alcohol dependence, Addiction, Complementary medicine, Plants phytotherapy

Introduction Alcohol abuse and dependence hold an important role in the public health since both the medical consequences and economical costs 1 • The pharmacological treatment of patients with alcohol dependence playa key role to achieve alcohol abstinence and prevent relapse, especially if it is conceived together with the psychosocial interventions already used for many years 2 ,3. Within pharmacological approaches, some recent small preliminary data suggest the possible utility of the Complementary Medicines (CMs) in the treatment of alcohol dependence. CM is defined as "diagnosis, 1. Digestive Physiopathology Unit, Department of Experimental and Clinical Medicine, University Magna Graecia, Catanzaro, Italy. 2. Department of Experimental Pharmacology, University Federico II, Naples, Italy. * Corresponding author: E-mail: l.abenavoli®Unicz.it

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treatment and/or prevention which complements mainstream medicine by contributing to a common whole, by satisfying a demand not met by orthodoxy or by diversifying the conceptual frameworks of medicine"!. In spite of the utility of the CM is described in different diseases, the data concerning its possible use in alcohol dependent patients are controversial4 and do not permit to draft final conclusions. For several centuries, in particular in China, medicinal plants have been used for the treatment of alcohol dependence 5,6 (Table 1). Table 1. Herbal drugs and herbal preparations traditionally used to help alcoholism Common name

Latin name

Part(s) of plant used

St. John's wort

Hypericum perforatum

Leaves and flowering tops

Kudzu

Pueraria lobata (daidzin) Salvia miltiorrhiza Tabernanthe iboga Panax ginseng

Flowers and roots Roots

Oenothera biennis Silybum mananum Scutellaria laterifolia (catalpol)

Oil

Danshen Tabernanthe Ginseng Evening primrose Milk thistle Scullcap

Fruits Aerial parts

Key constituents Phloroglucinol derivatives (hyperforin, adhyperforin), anthraquinone derivatives (hypericin, pseudohypericin) Isoflavons derivatives (daidzein) Diterpene compounds (tanshinones, miltirone) Roots Ibogaine Roots Ginsenosides GLA (an omega 6 fatty acid) Silymarin, a complex of 5 flavonoids Flavonoids (scutellarin, scutellanein), iridoids

SKV* Agaricus** * An ayurvedic formula of 12 herbal ingredients. It is used to help alcoholism and other addictions ** An homeopathic product. It is recommended in cases of acute alcoholism and is a potent antidote against the ravages of a hangover

A recent study by our group7, highlighted that 16.50% ofItalian Alcohol and Drug Addiction Services, use CMs for alcohol dependence treatment, and in these services 10.08% of the patients, are treated by phytotherapy.

Hypericum perforatum L. (Fam. Clusiaceae) The antidepressant properties of the St. John's wort -Hypericum perforatum L. (HPE) - are well known since Hippocrates time. Recent pre-clinical and clinical studies (8) have demonstrated that HPE is effective in the treatment of mild to moderate the therapy of anxiety.

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HPE contains several biologically active compounds, including naphthodianthrones (hypericin and pseudohypericin), fluoroglucynol derivatives (hyperforin, adhyperforin), several flavonol glycosides, biflavones, phenylpropanes, proanthocyanidins, tannins, xanthones and some amino acids as the gamma-amminobutyric acid (GABA)9. Several experimental and clinical studies identified hyperforin (Fig 2A), as the major active principle for antidepressant action. Hyperforin is known to inhibit the uptake of aminergic transmitters such as serotonin and noradrenaline into synaptic nerve endings lO • It also increases the extracellular levels of other transmitters including acetylcholine, glutamate, and GABA (Fig 1). These effects may be secondary to an increase of the intra-cellular sodium concentration mediated by openings of non-selective cation channels in the synaptosomal membrane l l . Finally, hyperforin also interacts with a variety of receptors and ion channels including glutamatergic and GABA ergic receptors and calcium channels l 2 •

Presynaptic

••

• • •

.. - ... •• • a.

~----~U1fU--~--~

Postsynaptic

• Neurotransmitters

1 Increased response

Fig 1. Mechanism of the antidepressant action ofSt. John's wort. Hyperforin inhibits the neuronal re-uptake of a number of brain neurotransmitters (serotonin, noradrenaline, dopamine, glutamate and GABA) into presynaptic nerve terminal. By blocking the major route of neurotransmitter removal, hyperforin leads to increased concentrations of neurotransmitters in the synaptic cleft (From Capasso et al., 2006, Phytotherapy A quick reference to herbal medicine. Springer-Verlag, Berlino)

According to the high comorbidity, between depressive states and alcohol dependence, some studies have investigated HPE efficacy in the alcohol-seeking behaviour13. In particular, recent studies showed the ability of St. John's wort extracts to halve voluntary alcohol intake in different lines of selectively alcohol-preferring rats 5,6, and one pre-clinical study has

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suggested that hyperforin (5 mglkg) may be the active principle for this effect1 4 • This effect could be due to the block of the reuptake of serotonin . and dopamine with the consequent increase of these neurotransmitters in the synaptic cleft. Moreover, it has also been showed that hyperforin inhibits GABA uptake l5 and HPE blocks the GABA reuptake l6 . Opioid receptor antagonists, such as naloxone and naltrexone, have shown their efficacy to reduce alcohol intake in both rats and humans l7. A pre-clinical study evaluated the effect on alcohol intake by the combined administration of HPE and opioid receptor antagonists. When naloxone (1 mglkg) or naltrexone (0.5 mglkg) were given before different intra-gastric doses ofHPE, the attenuation of alcohol intake was more pronounced than HPE was given alone l8 receptor antagonists and HPE in reducing alcohol intake in animals. Since the crude extracts have been given only by the intra-gastric or intra-peritoneal route, the best site of action remains to be detected. This results, however, imply that HPE may be a therapeutic potential in the clinical treatment of alcohol abuse dependence.

Pueraria lobata Owhi (Fam. Fabaceae) The anti-drunkenness properties of the extracts of Pueraria lobata (PL), also known as kudzu, have been known since the traditional Chinese medicine. A experimental study demonstrated that the daily intra-peritoneal administration of a crude extract of PL (1.5 g kgl x day·l) roots halved alcohol intake in alcohol-preferring Syrian Golden hamsters, when a choice between alcohol solution and water was given l9. In this study, two putative active principles have been identified. Indeed, the administration of the two major isoflavones present in PL extracts (daidzin and daidzein) reduced ethanol intake in Syrian Golden hamsters with an efficacy similar to the one observed using the PL extract. The ability of PL to reduce alcohol consumption in animals has been also shown by testing a herbal mixture (intra-peritoneal injection of 0.5, 0.75, and 1.0 glkg; and orally administration of 1.5 glkg), comprising PL20. Interestingly, this mixture is commonly used in China to prepare the so-called "tea of sobriety". Daidzin (Fig 2B) is also a potent and selective inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH-2). Some authors showed a direct correlation between ALDH-2 inhibition and ethanol intake suppression and raise the possibility that daidzin may suppress ethanol intake of golden hamsters, by inhibiting ALDH-221. Puerarin (Fig 2C) represents the most concentrated isoflavonoid in kudzu although it is not as potent as daidzin. The beneficial effects of puerarin on alcohol intake in alcohol-preferring rats reported in literature also suggest the potential utility of puerarin as an anti-craving agent5.6. According to the animal data, a preliminary clinical study explored the effect of kudzu root extract on thirty-eight patients affected by alcohol dependence and randomly assigned to receive either kudzu root extract

Phytotherapeutic Approach to Alcohol Dependence

23

(1.2 g twice daily) or placebo22 . Sobriety level and a visual analogic scale to assess alcohol craving were assessed. Kudzu root appeared to be no better than placebo in reducing alcohol craving and/or promoting sobriety. Unfortunately the authors did not report the concentrations of the active isoflavones in their kudzu extract. More recently a study have tested the efficacy of a kudzu extract in a group of "heavy" alcohol drinkers, treated with either placebo or a kudzu extract (500 mg three times daily for 7 days)23. Mter the 7-day period, subjects had the opportunity to drink their preferred brand of beer in a naturalistic laboratory setting. Kudzu treatment resulted in significant reduction in the number of beers consumed, an increase in the number of sips and the time to consume each beer and a decrease in the volume of each sip. These changes occurred in the absence of a significant effect on the urge to drink alcohol. The authors concluded that kudzu may be a useful adjunct in reducing alcohol intake although the exact mechanism by which kudzu suppresses ethanol intake remains to be clarified.

Salvia miltiorrhiza Bge. (Fam. Laminaceae) The dried roots of Salvia miltiorrhiza (SM) are used in traditional Chinese medicine for the treatment of several pathologies (e.g., insomnia). Preclinical data suggest that extracts from the SM: tanshinone IIA, cryptotanshinone and miltirone (Figs 2D & 2E) are effective in reducing voluntary alcohol intake in animal models of excessive alcohol drinking24. Specifically, extracts of SM have been found to (a) delay the acquisition of alcohol-drinking behaviour in alcohol-naive rats given alcohol under the home-cage 2-bottle "alcohol versus water" choice regimen 25 ; (b) reduce voluntary alcohol intake under the 2-bottle choice regimen in rats that were alcohol experienced at the time of extract administration; and (c) suppress the temporary increase in voluntary alcohol intake occurring after a period of deprivation from alcohol26 • Recently the same study Group27 have found that miltirone is the possible active chemical component responsible for the reducing effect of SM extracts on alcohol intake in Sardinian alcohol-preferring rats. The authors have assessed the effect of 100 mg/kg (intra-gastric administration) of 4 extracts of SM, differing in miltirone content (0, 2, 3, and 7%, respectively), on alcohol intake in alcohol-experienced sP rats exposed to the 2-bottle "alcohol (10%, volume in volume) versus water" choice regimen. Subsequently, the effect of pure miltirone (2.5-10 mg/kg, intra-gastric, i.e., a dose range comparable to its content in the effective doses of the active extracts) on acquisition and maintenance of alcohol-drinking behavior was evaluated in alcohol-naive and alcohol-experienced sP rats exposed to the 2-bottle choice regimen. The effect ofmiltirone (10 mg/kg, intra-gastric) on blood alcohol levels was assessed after the intra-gastric and intra-peritoneal administration of alcohol. Finally, the effect of miltirone (30-100 mg/kg, intra-gastric) on the severity of alcohol withdrawal syndrome was evaluated in Wistar rats made physically dependent on alcohol by the repeated

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administration of intoxicating doses of alcohol. The authors reported that: reducing effect of 4 different extracts ofSM on alcohol intake was positively and significantly correlated with their miltirone content. Pure miltirone reduced alcohol intake in alcohol-experienced rats and delayed acquisition of alcohol-drinking behavior in alcohol-naive rats. Similar to SM extracts, miltirone markedly reduced blood alcohol levels when alcohol was administered intra-gastric but not intra-peritoneal, suggesting that miltirone hampered alcohol absorption from the gastrointestinal system. Finally, miltirone failed to affect the severity of alcohol withdrawal syndrome in alcohol-dependent rats. The ability ofmiltirone to reduce alcohol intake in rats, could be explained by the anxiolytic effect previously reported in literature28 • Future studies are needed to clarify this mechanism.

Tabernanthe iboga H. Bn. (Fam. Apocynaceae) Ibogaine, is a naturally occurring, psychoactive indole alkaloid derived from the roots of the rain forest shrub Tabernanthe iboga (TI). Indigenous peoples of Western Mrica use ibogaine in low doses to combat fatigue, hunger, and thirst and in higher doses as a sacrament in religious rituals. The stimulating effects ofTI are well-known for centuries. Ibogaine has been claimed to be effective in treating multiple forms of drug abuse, including morphine, cocaine, heroin and nicotine 5,6. However it has been proposed that ibogaine exerts, its anti-craving effects by stimulating dopaminergic and serotonergic systems 29 • Accordingly, TI seems to be able to markedly reduce voluntary alcohol intake in alcohol-preferring rats 6 • This effect was not related to a possible interaction between TI and alcohol, as showed by the virtually equal blood alcohol levels in both ibogaine- and placebo-treated rats. It is also of interest that the reducing effect on alcohol intake has been observed only when ibogaine was injected intra-peritoneally or intra-gastric ally but not when it was injected subcutaneously. Intra-peritoneal administration of 10, 30 and 60 mg/kg ibogaine, induced 8, 13 and 25% reduction in alcohol preference in rats 30 • This feature suggests that the active principle of ibogaine could be a metabolite produced by the liver. Because ibogaine, at high doses, can be toxic and cause side effects that may limit its therapeutic applications, an attempt has been made to design an ibogaine analog with no toxicity but with the same inhibitory action on reinforcing drugs. 18-Methoxycoronaridine (18-Me) (Fig 2F) appears to be such an analog. In animal models, 18-Me reduced intra-venous morphine, cocaine, methamphetamine and nicotine self-administration, oral alcohol and nicotine intake, and attenuated signs of opioid withdrawal, but had no effect on responding for a non-drug reinforcer and produced no apparent toxicity31. Another study 32 showed that a single injection (intra-peritoneal) of 5,20 or 40 mg/kg 18-Me significantly reduced alcohol intake and preference in a dose-dependent manner in preferring rats. It has been hypothesized that ibogaine and its analog exert their suppressant effect on alcohol intake by modulating several neuronal ways,

Phytotherapeutic Approach to Alcohol Dependence

25

in particular dopaminergic and serotonergic systems. The true mechanism of action of these compounds in attenuating alcohol intake is not fully understood. A firm conclusion awaits further pharmacological and behavioral studies.

Panax ginseng Hayer (Fam. Araliaceae) There are some accounts of the effects of ginseng Meyer and its derivatives on the alcohol intoxication. Early works recorded that ginseng saponines (Fig 2G), increased the rate of oxidation of ethanol in alcohol-fed rats 33 and red ginseng extract prevented memory failure and excitation in alcoholintoxicuted mice 34 . Afterwards using healthy human volunteers Lee and coworkers demonstrated that in 10 out of 14 cases ginseng extract accelerated alcohol clearence by 31-51%35. Ginseng saponines apparently stimulate the microsomal ethanol-oxidising system and the aldehyde dehydrogenase (ADH) enzyme action and therefore there is faster removal of acetaldehyde with rapid shunting of excess hydrogen into lipid biosynthesis 36 . It has been also shown that in rats plasma levels are lower (-20%) when alcohol is administered orally with red ginseng extract than when alcohol is given alone. However, further studies 34 supporting the idea that ginseng may promote faster disposal and elimination of alcohol from blood after drinking. Obviously further studies are needed concerning the value of ginseng in the treatment of alcoholism and associated problems, e.g. memory loss and nervous reactions.

Conclusions Alcohol abuse and alcoholism represent a world-wide problem, both from a medical and a social point of view. In the past the therapy for patients affected by alcoholism was based mainly on the psychological approach. In recent years the use of pharmacotherapy together with psychosocial interventions have enhanced the percentage of success in maintaining alcoholic patients in remission 1. Medical interventions in the field of alcoholism are primarily aimed at: relieving the consequences of alcohol withdrawal syndrome and arresting alcohol drinking, maintaining sobriety for as long as possible 2 ,3. Pharmacotherapy is conceived to provide a substantial contribution to these goals, facilitating the psychological support and social rehabilitation of alcoholic patients 37 . Recent experimental evidence and critical re-examination of empirical data from traditional medicines, suggest that novel pharmacological approaches for treatment of alcoholism and alcohol abuse may stem from natural substances. Several plant-derived compounds have been shown to significantly reduce alcohol intake mostly in animal studies. Although several neurotransmitter systems seem to be involved in their effects on alcohol-seeking behaviour, the exact mechanisms of action of these compounds remain to be clarified. Until extensive clinical studies are

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carried out, it will be difficult to extrapolate the findings on animal models of alcohol dependence to a human cohort. The role of these compounds in the treatment of alcoholism will ultimately depend on the outcome of carefully conducted clinical trials. Nevertheless, the extensive positive findings in animal models suggest that the outcome of clinical trials is likely to be positive as well especially when pharmacological treatment is combined with psychological support counselling. Phytotherapy can be a new old way to treat alcohol addiction. OH

OH 0 OH HO HO

OH A

o

B

c

E

F

~

CH3

1

f?

""I

H,C

CH 3

OH

D

HO

H,C

G Fig 2. Chemical formulas ofhypoforin (A), daidzin (B), puerarin (C), tanshinone IIA (D), miltirone (E), 18-methoxycoronaridine (F) and a general structure ofthe ginsenosides (G)

Phytotherapeutic Approach to Alcohol Dependence

27

References 1.

2.

3.

4. 5.

6. 7.

8.

9.

10.

11. 12.

13. 14.

15.

16.

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Perfumi, M., Santoni, M., Cippitelli, A., Ciccocioppo, R., Froldi, R. and Massi, M. 2003. Hypericum perforatum CO 2 extract and opioid receptor antagonists act synergistically to reduce ethanol intake in alcohol-preferring rats. Alcohol Clin. Exp. Res. 27: 1554-1562. 18. Overstreet, D.H., Kampov-Polevoy, AB., Rezvani, AH., Braun, C., Bartus, R.B. and Crews, F.T. 1999. Suppression of alcohol intake in P rats: Tolerance development and elevation of opiate receptor binding. Alcohol Clin. Exp. Res. 23: 1761-1771. 19. Keung, W.M. 2003. Anti-dipsotropic isoflavones: The potential therapeutic agents for alcohol dependence. Med. Res. Rev. 23: 669-696. 20. Overstreet, D.H., Lee, Y.W.,Rezvani,A.H., Criswell,H.E. and Janowsky, D.S. 1996. Suppression of alcohol intake after administration ofthe Chinese herbal medicine NPI-028, and its derivatives. Alcohol Clin. Exp. Res. 20: 221-227. 21. Keung, W.M. and Vallee, B.L. 1993. Daidzin and daidzein suppress free-choice alcohol intake by Syrian golden hamsters. Proc. Natl. Acad. Sci. USA. 90: 10008-10012. 22. Shebek, J. and Rindone, J.P. 2000. A pilot study exploring the effect of kudzu root on the drinking habits of patients with chronic alcoholism. J. Altern. Complementary Med. 6: 45-48. 23. Lukas, S.E., Penetar, D., Berko, J., Vicens, L., Palmer, C., Mallya, G., Macklin, E.A and Lee, D.Y. 2005. An extract of the Chinese herbal root kudzu reduces alcohol drinking by heavy drinkers in a naturalistic setting. Alcohol Clin. Exp. Res. 29: 756-762. 24. Carai, M.A, Agabio, R., Bombardelli, E., Bourov, I., Gessa, G.L., Lobina, C., Morazzoni, P., Pani, M., Reali, R., Vacca, G. and Colombo, G. 2000. Potential use of medicinal plants in the treatment of alcoholism. Fitoterapia 71(Supp11): S38-42. 25. Brunetti, G., Serra, S., Vacca G., Lobina, C., Morazzoni, P., Bombardelli, E., Colombo, G., Gessa, G.L. and Carai, M.AM. 2003. IDN 5082 a standardized extract of Salvia miltiorrhiza delays acquisition of alcohol drinking behavior in rats. J. Ethnopharmacol. 85: 93-97. 26. Serra, S., Vacca, G., Tumatis, S., Carrucciu, A., Morazzoni, P., Bombardelli, E., Colombo, G., Gessa, G.L. and Carai, M.AM. 2003. Anti-relapse properties ofIDN 5082, a standardized extract of Salvia miltiorrhiza, in alcohol preferring rats. J. Ethnopharmacol. 88: 249-252. 27. Colombo, G., Serra, S., Vacca, G., Om, A, Maccioni, P., Morazzonim P., Bombardelli, E., Riva, A, Gessa, G.L. and Carai, M.A 2006. Identification ofmiltirone as active ingredient of Salvia miltiorrhiza responsible for the reducing effect of root extracts on alcohol intake in rats. Alcohol Clin. Exp. Res. 30: 754-762. 28. Lee, C.M., Wong, H.N.C., Chui, K.Y., Choang, T.F., Hon, P.M. and Chang, H.M. 1991. Miltirone, a central benzodiazepine receptor partial agonist from a Chinese medicinal herb Salvia miltiorrhiza. Neurosci. Lett. 127: 237-241. 29. Glick, S.D., Rossman, K., Steindorf, S., Maisonneuve, I.M. and Carlson, J.N., 1991. Effects and after effects of ibogaine on morphine self-administration in rats. Eur. J. Pharmacol. 195: 341-345. 30. Rezvani, AH., Overstreet, D.H. and Lee, Y.W. 1995. Attenuation of alcohol intake by ibogaine in three strains of alcohol preferring rats. Pharmacol. Biochem. Behav. 52: 615-620. 31. Maisonneuve, I.M. and Glick, S.D. 2003. Anti-addictive actions of an iboga alkaloid congener: A novel mechanism for a novel treatment. Pharmacol. Biochem. Behav. 75: 607-618. 32. Rezvani, A.H., Overstreet, D.H., Yang, Y., Maisonneuve, I.M., Bandarage, U.K., Kuehne, M.E. and Glick, S.D. 1997. Attenuation of alcohol consumption by a novel nontoxic ibogaine analogue (8-methoxycoronaridine) in alcohol-preferring rats. Pharmacol. Biochem. Behav. 58: 615-619.

Phytotherapeutic Approach to Alcohol Dependence 33.

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Joo, C.N., Koo, J.H., Lee, H.B., Yoon, J.B. and Byun, Y.S., 1982. Biochemical studies on the absorption of ginseng saponin and its effect on metabolism in the animal body. Hanguk Saenghwa Hakhoe Chi. 15: 189-199. 34. Lee, Y.J., Pantuck, C.B. and Pantuck, E.J. 1993. Effect of ginseng on plasma levels of ethanol in the rat. PlantaMed. 59: 17-19. 35. Lee, F.C., Ko, J.H., Park, J.K. and Lee, J.S. 1987. Effects of Panaxginseng on blood alcohol clearence in man. Clin. Exp. Pharmacol. Physiol. 14: 543-546. 36. Kwak, H.S. and Joo, C.N. 1988. Effect of ginseng saponin fraction on ethanol metabolism in rat liver. Koryo Insam Hakhoechi. 12: 76-86. 37. Addolorato, G., Leggio, L., Ferrulli, A., Cardone, S., Vonghia, L., Mirijello, A., Abenavoli, L., D'Angelo, C., Caputo, F., Zambon, A., Haber, P.S. and Gasbarrini, G. 2007. Effectiveness and safety ofbaclofen for maintenance of alcohol abstinence in alcohol-dependent patients with liver cirrhosis: randomised, double-blind controlled study. Lancet 370: 1915-1922.

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3 Effects of Chinese Herbal Medicines on Bone Loss in Castrated Female Rats SHUJI SASSAi, NAHOKO NEMOTOi, HITOMI OKABE 2 , SATOE SUZUKI 3 , HIDEKI KUD0 3 AND SHINOBU SAKAMOT0 1

*

Abstract Traditional Chinese herbal prescriptions, Hochuekkito (HET), Ogikenchuto (OKT), and Ninjin'yoeito (NYT) have been used for the treatments of many clinical disorders in Japan, i.e. HET, which is involved in supplementary prescriptions, has been prescribed for the treatment of oligospermia and as a postoperative medication. OKT and NYT have been used for the treatment of weakness with a loss of appetite and delayed healing of wound, and as a postoperative medication. In the present study, we investigated the effects of HET, OKT, NYT and 17a-ethynylestradiol (EED) on circulating levels of estradiol (E,) and dehydroepiandrosterone sulfate (DHEA-S), and the tibial bone mineral density (BMD) in castrated female rats. Castration lowered the wet weights of adrenals and uterus, and decreased the serum levels of calcium and E 2 • Oral administration of EED markedly elevated the reduced serum levels of DHEA-S by castration to approximately 2-fold that in the castrated rats. Serum levels of DHEA-S were enhanced to 134.4% of the castrated rats by the additive treatment using HET. Castration reduced the BMD in the whole tibia and a proximal metaphysis of the tibia to 91.5 and 72.0% of that in normal control rats. On the other hand, HET, but not NYT and OKT, enhanced the BMD in the whole tibia and a proximal metaphysis of the tibia to 105.7 and 117.6% of that in the castrated rats, respectively. The bone histology in the castrated rats was characterized by a diminished area of the trabecular bone around the growth plate-metaphyseal junction 1. Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. 2. Department of Obstetrics and Gynecology, School of Medicine, Juntendo University, Tokyo 113-8421, Japan. 3. Department of Clinical Laboratory Medicine, Faculty of Health Science Technology, Bunkyo Gakuin University, Tokyo 113-8668, Japan. * Correspondence author: E-mail: [email protected]

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in the proximal tibia. However, the reduced area ofbone mass in the proximal tibia was prevented and / or replaced by the additive treatments using EED and HET, but not NYT and OKT. Key words: Traditional herbal medicine, Ovariectomy, Dehydroepiandrosterone sulfate, Bone mineral density, Osteopenia

Introduction Traditional herbal prescriptions are being reevaluated in the clinical fields because of their relatively few side effects and suitability for long-term administration compared to synthetic drugs. Traditional Chinese herbal medicines, Hochuekkito (HET), Ogikenchuto (OKT), and Ninjin'yoeito (NYT) have been used for the treatments of many clinical disorders in Japan, i.e. HET for dysfunction of the digestive system, weakness of muscles, weak physical condition, fatigue due to summer heat, during recovery, tuberculosis, loss of appetite, gastric ptosis, common cold, hemorrhoid, prolapsus ani, ptosis of the uterus, impotence, hemiplegia and hyperhidrosis, OKT for exhaustion, tiring easily, night sweat, abdominal pain, loss of appetite, dyspnea eruptive eczema, dermatitis and chronic festering wounds, and NYT for recovering after surgery, physically weak constitution, exhaustion due to surgery, loss of appetite, night sweat, anemia and cold hands and feet. HET, which is involved in supplementary prescriptions in Chinese herbal medicines, has been prescribed for the treatment of oligospermia (Amano et al., 1996) and as a postoperative medication. It was reported that HET suppressed the production of IgE (Kaneko et al., 1997) and growth of tumor in mice (Harada et al., 1995; Haranaka, 1989). OKT and NYT have been used for the treatment of weakness with a loss of appetite and delayed healing of wound, and as a postoperative medication. We have experienced a clinical case in which the progress of the patient from 66 to 76 years of age could be monitored. The diagnosis at the first medical examination of the patient was postmenopausal osteopenia and senile colpitis, i.e. the bone mass was 71.7% ofthe age-matched average value. The bone mass increased to 90.7% of the age-matched average value 2 years after the beginning of HET treatment, with body weight gain (+5 kg) for 5 years (Sakamoto et al., 1999). The chronic administration of a gonadotropin-releasing hormone agonist offers a means oftreating patients with symptomatic endometriosis, uterine adenomyosis and leiomyoma. The reason why such a potent agonist is used due to a reversible hypo-estrogenism via a desensitization of the pituitary gland to hormonal stimulation, possibly by the down-regulation of gonadotropin-releasing hormone receptors in women. However, the gonadotropin-releasing hormone agonist treatment induces adverse effects,

Effects of Chinese Herbal Medicines on Bone Loss

33

particularly increased bone remodeling and bone loss. As previously reported, we investigated the effects ofHET on femoral bone mineral density (BMD) in female rats chronically treated with the long-acting gonadotropinreleasing hormone agonist, buserelin acetate. HET enhanced the BMD to 106.2% ofthe chemically castrated rats (Sakamoto et al., 2000). The finding indicates that HET could be useful when combined with careful monitoring of the biochemical markers of osteoblastic activity or bone resorption and the BMD of the patients with bone mineral disorders. In the present study, we investigated the effects ofHET, OKT, NYT and 17a-ethynylestradiol on circulating levels of estradiol and dehydroepiandrosterone sulfate, and the tibial BMD in castrated female rats.

Materials and Methods Chemicals, animals and treatments Herbal extracts of3 Chinese herbal prescriptions, i.e. HET, OKT and NYT, are all gifts from Tsumura Co., Tokyo. They are composed of 10, 6 and 12 medicinal plants, respectively, as shown in Table 1. Three groups of mixtures consisting of each of the chopped components in the ratio in Table 1 were extracted with hot water, filtered, lyophilized, and stored at 4°C. In the present study, 48 female Sprague-Dawley rats (Sankyo Laboratory Service Co., Tokyo) were employed. Throughout the experiment, all rats were kept under controlled lighting and temperature, given tap water ad libitum, and weighed every 7 days. Forty rats underwent castration at age of 9 weeks, while the remaining 8 rats were intact. All the animals were fed a commercial diet (CE-2, CLEA Japan, Co., Tokyo) containing 1.18% calcium and 1.03% phosphorus, and given drinking water ad libitum. Beginning at 35 weeks of age, the castrated animals were divided into 5 groups of 8 rats each. The rats were orally given distilled water as a control vehicle (group 1), 17a-ethynylestradiol (EED; 0.1 mg/kg of b. wt.; Sigma chemical, St. Louis, USA) dissolved in distilled water (group 2), HET, OKT and NYT (0.5 g/kg of b. wt., an approximately 5-fold dose compared with that in clinical use; a gift from Tsumura & Co., Tokyo, Japan) suspended in distilled water and administered by gastric tubes once a day for 8 weeks (groups 3, 4 and 5). The normal control rats with sham operation were orally given distilled water, too (group 6). At autopsy at the age of 43 weeks, all the animals were bled by cardiac puncture under deep anesthesia with urethane (1.5 g/kg b. wt.; Merck, Darmstadt, Germany) and the bilateral tibias were removed. Each tibia was fixed and stored in 99.5% ethanol. All experimental procedures conformed to the regulations described in the Guide to the Care and Use of Laboratory Animals ofthe u.s. National Institutes of Health (NIH).

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Table 1. Components of Kampo medicines (Chinese herbal preparations): Hochuekkito (RET), Ogikenchuto (OKT) and Ninjin'yoeito (NYT)

HET: 5.0 g of a water extract offollowing raw materials a 1 Astragali radix (Ougi) 2 Atractylodis lanceae rhizoma (Soujyutsu) 3 Ginseng radix (Ninjin) 4 Angelicae radix (Touki) 5 Bupleuri radix (Saiko) 6 Zyzyphi fructus (Taisou) Aurantii nobilis pericarpium (Chinpi) 7 8 Glycyrrhizae radix (Kanzou) 9 Cimicifugae rhizoma (Shouma) 10 Zingiberis rhizoma (Shoukyou) OKT: 4.75 g of a water extract offollowing raw materials a 1 Paeoniae radix (Shakuyaku) 2 Astragali radix (Ougi) 3 Cinnamomi cortex (Keihi) 4 Zyzyphi fructus (Taisou) 5 Glycyrrhizae radix (Kanzou) 6 Zingiberis rhizoma (Shoukyou) NYT: 6.0 g of a water extract of following raw materials a 1 Rehmanniae radix (Jiou) 2 Angelicae radix (Touki) 3 Atractylodis rhizoma (Byakujutsu) 4 Hoelen (Bukuryou) 5 Ginseng radix (Ninjin) 6 Cinnamomi cortex (Keihi) 7 Polygalae radix (Onji) 8 Paeoniae radix (Shakuyaku) 9 Aurantii nobilis pericarpium (Chinpi) 10 Astragali radix (Ougi) 11 Glycyrrhizae radix (Kanzou) 12 Schizandrae fructus (Gomishi) aEach value (g) was represented as dry weight

4.0g 4.0g 4.0g 3.0g 2.0g 2.0g 2.0g 1.5g 1.0g 0.5g 3.0g 4.0g 4.0g 4.0g 2.0g 1.0g 4.0g 4.0g 4.0g 4.0g 3.0g 2.5g 2.0g 2.0g 2.0g 1.5g 1.0g 1.0g

Serum levels ofcalcium, estradiol and dehydroepiandrosterone sulfate Serum calcium (Ca) concentration was determined with commercial kit (Calcium C-test from Wako Pure Chemical Industries, Osaka, Japan). The serum levels of estradiol (E) were determined using radioimmunoassay kits (DPC estradiol kit from Japan DPC Coop., Tokyo). The interassay coefficient of variation was less than 4.8% in this analysis. The serum levels of dehydroepiandrosterone sulfate (DREA-S) were determined using radioimmunoassay kits (DREA-S kit from Mitsubishi Chemical Eng. Coop., Tokyo). The interassay coefficient of variation was less than 7.6% in this analysis.

35

Effects of Chinese Herbal Medicines on Bone Loss

Bone mineral density in tibia Each fixed tibia was dissected free from adhering soft tissues, and microradiographed (Softex, Softex Co., Tokyo; at 90 kV, 1 rnA for 60-90 sec) together with a standardized step-wedge made of synthetic hydroxyapatite (HA; Mitsubishi Kasei Co., Ltd., Tokyo). Since there is a linear relationship between the logarithms ofHA density (pg/mm2) of the step-wedge and gray levels (256 steps) of the microradiographic image of the step-wedge, the bone mineral density (BMD; pg HAlmm 2 ) in the whole tibia was determined by analyzing the gray level of the objective bone area in the microradiograph with an image analyzer (Winroof, Mitani Corp., Fukui, Japan), and expressed as pg Ca/mm2 after calculation.

Results Body growth and organ weights Castration (groups 1-5) enhanced the body weight to 112.7%, on average, compared to that in normal control rats (group 6) (p < 0.05) (data not shown). Oral administration ofEED reduced the body growth though not significantly (group 2) (data not shown). Adrenals and uterus were markedly lowered by castration (group 1) compared with the normal control (group 6) (p < 0.01 and 0.05, respectively), but the oral administration ofEED (group 2) increased those weights despite castration (p < 0.01 and 0.05, respectively) (Table 2). There were no differences in the wet weights of spleen among groups. Table 2. Wet weights of organs (mg/100 g ofb . wt.) Groups

(n)

Adrenals

1. OVX-Control 2. OVX-EED

(8) (8)

16.2 ± 0.8 26.4 ± 1.5**

3.0VX-NYT 4.0VX-OKT 5. OVX-RET 6. Normal-Control

(8) (8) (8) (8)

16.4 16.3 17.7 26 .5

± 0.9 ± 0.6 ± 0.8 ± 1.0**

Uterus 20.0 187.6 20.0 20.0 20.0 20.0

± ± ± ± ± ±

0.9 16.5** 0.6 0.9 1.0 14.6*

Spleen 157.1 163.0 157.1 157.1 157.1 157.1

± ± ± ± ± ±

6.3 9.1 7.1 8.1 11.6 8.7

OVX: castrated, EED: 17a-ethynylestradiol, NYT: Ninjin'yoeito, OKT: Ogikenchuto, RET: Rochuekkito Data are the mean ± SEM. ** and *Significantly different from that ofOVX-Control at p < 0.01 and 0.05

Serum levels ofcalcium (Ca), estradiol (E~ and dehydroepiandrosterone sulfate (DHEA-S), and alkaline phosphatase activity Castration decreased the serum levels of Ca and E 2 compared with that in the normal control rats (p < 0.01) (Table 3). However, oral administration ofEED markedly elevated the serum levels ofCa and E2 (p < 0.01) (group 2). Additive treatments by using NYT, OKT and HET little influenced the serum levels of Ca and E2 in the castrated rats. On the other hand, serum levels of

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36

DHEA-S were markedly reduced to 37.0% ofthat in the normal control rats by castration (p < 0.01) (Fig 1), but oral administration of EED markedly elevated the serum levels of DHEA-S to approximately 2-fold that in the castrated rats (p < 0.01). Serum levels ofDHEA-S were enhanced to 134.4% ofthe castrated rats by the additive treatment using HET (p < 0.05). Table 3. Serum levels of calcium (Ca) and estradiol (E 2 )

Groups

(n)

Ca(mg/dL)

Eg

1. OVX-Control

(B)

9.B4±0.11

ND

2. 0VX-EED

(B)

10.6± 0.3**

35.3 ±3.5**

3. 0VX-NYT

(B)

9.91±0.11

2.1B±0.lB

4. 0VX-OKT

(B)

9.94 ± 0.1O

2.0B±0.21

5.0VX-HET

(B)

9.93 ± 0.16

2.00±0.22

6. Normal-Control

(B)

10.4±0.1**

27 .B±7.7**

OVX: castrated, EED: 17a -ethynylestradiol, NYT: Ninjin'yoeito, OKT: Ogikenchuto, HET: Hochuekkito Data are the mean ± SEM. **Significantly different from that ofOVX-Control at p < 0.01 12

i!

•• ••

8

rn I

< ~

•

~

Ei

e

4

~

rn

o OVX

E,

NYT

- Control

OKT

HET

Normal - Control

Fig 1. Serum levels of dehydroepiandrosterone sulfate (DHEA-S) (mg/mL) ** and *Significantly different from that ofOVX-Control at p < 0.01 and 0.05

Bone mineral density (BMD) in tibia Castration significantly reduced the BMD in the whole tibia and a proximal metaphysis ofthe tibia to 91.5 and 72.0% (p < 0.01) ofthat in normal control rats, respectively (Fig 2). The 8-week oral administration ofEED markedly elevated the BMD in the whole tibia and a proximal metaphysis ofthe tibia to 107.0% (p < 0.01) and 123.8% (p < 0.05) of that in the castrated rats,

37

Effects of Chinese Herbal Medicines on Bone Loss Bone Mineral Density MgCa/cm'

Whole

150

100

50

0

0

Metaphysis

100

200

OVX -Control

**

E.

*

NYT

OKT

*

HET

Normal -Control

* **

Fig 2. Bone mineral density (BMD) in tibia (mg Ca/cm2) ** and *Significantly different from that ofOVX-Control at p < 0.01 and 0.05

respectively. Although NYT and OKT did not affect the tivial BMD values, HET significantly enhanced the BMD in the whole tibia and a proximal metaphysis ofthe tibia to 105.7% (p < 0.05) and 117.6% (p < 0.05) ofthat in the castrated rats, respectively.

Histology ofthe tibia The bone histology in the castrated rats was characterized by a diminished area ofthe trabecular bone around the growth plate-metaphyseal junction in the proximal tibia (Fig 3.2) compared with that in normal control rats (Fig 3.1). However, the reduced area of bone mass in the proximal tibia was prevented and/or replaced by the additive treatments using EED (Fig 3.3) and HET (Fig 3.4), but not NYT and OKT (data not shown).

Discussion In natural products, botanical or not botanical, there are many beneficial substances for human life. We previously demonstrated that a new clerodane diterpenoid isolated from propolis, a resinous material gathered by honey bees from the buds and bark of certain trees and plants, suppressed the incidence and growth of 7,12-dimethylbenz(a)anthracene-induced skin tumors in mice (Mitamura et al., 1996). In Japan, Chinese herbal medicines have been used for the treatment of postmenopausal osteoporosis and osteopenia. Bussabarger et al. and Sarasin reported bone loss in gastrectomized puppies (Bussabarger et al., 1938) and patients (Sarasin, 1941), respectively. It was reported that an administration of HET reduced the bone loss induced by gastrectomy in

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Fig 3. Histology of the tibia 1: normal control rats, 2: castrated rats, 3: supplemented with EED in castrated rats, 4: supplemented with HET in castrated rats

patients (Sugiyama, 1994) and rats (Suzuki et al., 1996). We previously reported that the treatments with conjugated estrogens, bisphosphonate and vitamin D3 analog cured the osteopenia induced with a gonadotropinreleasing hormone agonist in rats (Sakamoto et at., 1999). Futhermore, we demonstrated that an administration with HET prevented the femoral bone loss with a slight elevation of the serum estradiol levels in the chemically castrated rats (Sakamoto et at., 2000). These findings suggest that HET may increase bone mass via the increase of circulating estrogen levels, i.e. suppression of the bone resorption, and via the gain of appetite, i.e. increase ofthe intestinal calcium absorption. We have experienced a clinical case of an old female patient with a postmenopausal osteopenia and senile colpitis, and monitored for 11 years from 66 till 76 years of age. 2-year administration of HET elevated the bone mass from 71.1 to 90.7% of the age-matched average value and increased the body weight (+2 kg) (Sakamoto et at., 1999). HET is known to enhance appetite and body weight. Thus, the additive HET may be more effective in recovering the bone loss. Traditional Chinese herbal medicines such as HET, OKT and NYT have been used for the treatment of weakness with a loss of appetite and delayed healing of wound, and as a postoperative medication, i.e . these prescriptions are known to activate physical functions and cure the disorders. In the present study, we investigated the effects of HET, OKT, NYT and 17a-ethynylestradiol on circulating levels of estradiol and dehydroepiandrosterone sulfate, and the tibial BMD in castrated female rats. Castration enhanced the body weight to 112.7%, on average, lowered the wet weights of adrenals and uterus, and decreased the serum levels of Ca and E 2 • Oral administration ofEED markedly elevated the reduced serum levels ofDHEA-S by castration to approximately 2-fold that in the castrated

Effects of Chinese Herbal Medicines on Bone Loss

39

rats. Serum levels of DHEA-S were enhanced to 134.4% of the castrated rats by the additive treatment using HET. Castration reduced the BMD in the whole tibia and a proximal metaphysis of the tibia to 91.5 and 72.0% of that in normal control rats. On the other hand, HET, but not NYT and OKT, enhanced the BMD in the whole tibia and a proximal metaphysis of the tibia to 105.7 and 117.6% ofthat in the castrated rats, respectively. The bone histology in the castrated rats was characterized by a diminished area of the trabecular bone around the growth plate-metaphyseal junction in the proximal tibia. However, the reduced area of bone mass in the proximal tibia was prevented and/or replaced by the additive treatments using EED and HET, but not NYT and OKT. The present results, together with the previous findings (Sakamoto et al., 2000), suggest that HET enhances the reduced bone mass and causes a slight elevation of the serum levels of E2 and/or DHEA-S in castrated rats.

Acknowledgements We are grateful to Mr. Makoto Nomura, Mr. Kazuki Netsu, Miss. Ran Murao and Miss. Ai Katoh from Tsumura Co., Tokyo for their cooperation in this study.

Funding The present study was supported by the Foundations from Koihei Co. Ltd., Saitama, Japan and Japan Royal Jelly Co., Ltd., Tokyo, Japan. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References Amano, T., Hirata, A. and Namiki, M. 1996. Effects of Chinese herbal medicine on sperm motility and fluorescence spectra parameters. Arch. Androl. 37: 219-224. Bussabarger, RA., Freemann, S. and Ivy, A.C. 1938. Experimental production of severe homogenous osteoporosis by gastrectomy in puppies. Am. J. Physiol. 121: 137147. Harada, M., Seta, K, Ito, o. et al., 1995. Concomitant immunity against tumor development is enhanced by the oral administration of a kampo medicine, Hochu-ekki-to (TJ-41: Bu-Zhong-Yi-Qi-Tang). Immunopharmacol Immunotoxicol. 17: 687-703. Haranaka, K 1989. Traditional Chinese medicines as biological response modifiers. Mol. Biother. 1: 175-179. Kaneko, M., Kishihara, K, Kawakita, T. et al., 1997. Suppression of IgE production in mice treated with a traditional Chinese medicine, bu-zhong-yi-qi-tang (Japanese name: hochu-ekki-to). Immunopharma-col. 36: 79-85. Mitamura, T., Matsuno, T., Sakamoto, S. et al., 1996. Effects of a new clerodane diterpenoid isolated from propolis on chemically induced skin tumors in mice. Anticancer Res. 16: 2669-2672. Sakamoto, S., Sassa, S., Mitamura, T. and Zhou, Y.F. 1999. Does Hochu-ekki-to prevent bone loss? Kampo Igaku 23: 158-160 (In Japanese).

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Sakamoto, S., Sassa, S., Mitamura, T. et al., 1999. Prevention of osteopenia induced with a gonadotropin-releasing hormone agonist in rats. Calcif Tissue Int. 65: 152-155. Sakamoto, S., Sassa, S., Kudo, H., Suzuki, S., Mitamura, T. and Shinoda, H. 2000. Preventive effects of an herbal medicine on bone loss in rats treated with a GnRH agonist. Eur. J. Endocrinol. 143: 139-142. Sarasin, C. 1941. Osteomalacic und hypochrome anaemie nach magenresektion. Gastroenterologia 66: 182-197 (In Germany). Sugiyama, M. Osteoporosis and Kampo 1994. Sanhujinka-Kampo-Kenkyu-no-Ayumi 11: 1-16 (In Japanese). Suzuki, Y., Takaoka, T., Kashiwagi, H. and Aoki, T. 1996. Experimental study of TJ-41 on disorders of bone in gastrectomized rats. Pro. Med. 16: 1514-1516 (In Japanese).

4 Production of ET743, Bryostatin, and Taxol Using a Mineral Based Microbial Amplification System THOMAS J. MANNING1*, GISO ABADI 2 , KARLY BISHOP!, KRISTEN McLEOD!, GUNTER BULLOCK!, GREG KEAN!, DEVIN GRANT!, STUART ANDERSON!, KATRICE COOPER-WmTE!, SHANDA SERMONS!, OM PATEL!, DENNIS PHILLIps 3 , THOMAS POTTER\ JAMES NIENOW5 , PAUL KLAUSMEYER6 AND DAVID NEWMAN6

Abstract Bacterial amplification chambers (BACs) are artificial media that allow marine bacteria to colonize a receptive surface. The composition of BAC's are derived from analytical measurements of an ecosystem. Marine bacteria are notoriously difficult or impossible to cultivate in a laboratory setting so a method of farming the microbes in their home environment was sought. Specifically, the BAC is left in a respective ecosystem for an extended period of time (days, weeks) and then harvested. We originally applied this to a set of marine natural products found in the Gulf of Mexico (bryostatin, ET743). From that work we adapted the methodology to the production of taxol in the Florida yew tree. We've identified six groups of chemicals that are used in constructing a BAC. (1) Trace inorganic species that may playa nutrient role (2) Organic based structures found in the sediment (3) Organic based nutrients (4) Naturally occurring polymers (5) Bulk inorganic species found in the local ecosystem (6) Components of the host organism. Preliminary results for the production of the pharmaceutical agents ET743 in Sarasota Bay (Fl) and Dickerson Bay (Fl), Taxol in Torreya State Park (Fl) and Bryostatin at Alligator Point Harbor (Fl) are discussed in this paper. 1. 2. 3. 4. 5. 6.

Department of Chemistry, Valdosta State University, Valdosta, Ga, 31698, USA. Sunderland University, Sunderland, UK. Mass Spec facility, Department of Chemistry, University of Georgia, Athens, Ga, USA. Watershed Lab, United States Department of Agriculture, Tifton, GA, USA. Biology Department, Valdosta State University, Valdosta, GA, USA. Natural Products Group, SAlC-Frederick, Inc., NCI-Frederick, Frederick, MD 2170, USA.

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Key words: Bryostatin, ET743, Taxol, Natural product, Mass spectrometry, Bacterial amplification chamber

Introduction The coastal waters of Florida are well known for their biodiversity but little is known or understood about the multitude of microbes that reside in the water column or sediment. Natural Products chemistry follows a similar pattern for the development of most compounds. The extract of an organism is tested for toxicity or medicinal activity against a known cell line. If the results are encouraging, additional large scale extracts are made of the host organism and an attempt is made to isolate the compound responsible for the activity. Once enough of the compound is isolated and purified, techniques such as mass spectrometry, infrared spectroscopy and nuclear magnetic resonance spectroscopy are used to identifY its structure. Assuming the organism can only provide a small quantity of the medicinal agent, organic chemists attempt to find an economical route to the total or semisynthesis of the compound. If an economical synthetic route is found and the compound is successful in Phases I, II and III clinical trials, it has a chance to be brought to market as a pharmaceutical agent. Because the synthesis of larger molecular species can often be difficult and expensive, the price and subsequent availability of many pharmaceutical agents is limited, particularly in the Global market. This preliminary study focuses on three pharmaceutical agents found in Florida that have enjoyed different levels of medicinal success; the bryostatins, ET743, and taxol. The bryostatins are a large macrocyclic lactone characterized by a bryophan ring (Fig 1)1. To date twenty variations of the marine natural product bryostatin have been extracted from the bryozoa Bugula neritina, with bryostatin-l being the first structure identified in 19822. The complex total synthesis ofbryostatin-2 and bryostatin-7 are not viewed as economical solutions for the large scale production of the marine natural product3. Early work in this lab focused on studying bryostatins distribution in the same north Florida-Gulf of Mexico ecosystem that the Bugula containing bryostatin was harvested 4•7 • It was demonstrated that a host of marine organisms as well as sediment samples contained different bryostatins. We performed a similar study on the distribution of ET743 (Fig 2) in a Florida keys ecosystem that was home to the sea squirt Ecteinascidia turbinata. Like the bryostatins distribution in the Bugula ecosystem, we identified ET743 in other marine organisms and sediment samples in the sea squirt's ecosystem7 • Both the bryozoa and the sea squirt are filter feeders which raised the possibilities that each organism was acquiring the bacteria and marine natural product from the water column. In addition to studying the distribution of the marine natural products in the host ecosystems, we also performed fairly large scale analytical measurements which included ICP-AES (Inductively Coupled Plasma- Atomic

Production of ET743, Bryostatin, and Taxol

43

Fig 1. Bryostatin-l (C47H6S017) is characterized by a bryophan ring. Different bryostatins have different Rl and R2 groups

Fig 2. The molecular species ET743 is extracted from a sea squirt that resides in warm waters, including the Gulf Coast of Florida

Emission Spectrometry) and ICP-MS (Inductively Coupled Plasma - Mass Spectrometry) studies to help understand the mineral composition in which the microbes thrived. Techniques such as Fourier Transform-Ion Cyclotron Resonance (FT-ICR), Liquid Chromatography-Mass Spec (LC-MS), Fourier Transform-Infrared Spectroscopy (FT-IR), Laser Diffraction, Multiangle Laser Light Scattering (MALLS) and Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) were used to gain a better insight into the composition and form (particle sizes) of sediment phase organics where the MNP's were identified 8 •9 • These measurements contributed to the compilation of chemicals and materials in our bacterial amplification chambers. They also led to a model that correlates our observations with the dispersion ofthe MNP in an ecosystem

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(see Figs 3A & 3B). As will be discussed in this paper, we have developed a method for farming marine microbes from those quantitative and qualitative measurements. More recently we investigated the ecosystem of the Florida yew tree found in a few stands on the banks of the Apalachicola River in Torreya State Park (Florida). Taxol was identified in the sediment surrounding the tree (Fig 4)1°. Considering taxols' short half-life in a number of physiological conditions, it is doubtful that there could be a significant long term build up in soil surrounding the tree l l • The Pacific yew tree, found in the northwestern portion of the United States, achieved fame for its production of the cancer drug taxol. It was also shown by researchers at Montana State University that a species of fungi was correlated with the production oftaxoP2-14. Past research that blended natural products production and aquaculture has focused on the growth ofthe host organism (i.e. Bugula, Ecteinascidia) to produce a natural product (bryostatin and ET743)15,16. In each case the systems utilized were high capital cost endeavors that produced low yields of the desired marine natural products. Analogous to the total synthesis of each molecule, aquaculture of the host organism has limited potential due to economic factors. In the case ofBugula, it has been postulated that a bacteria called Candidatus Endobugula sertula produces the bryophan ring17 ,lB. It has also been postulated that different genetic species of Bugula are responsible for the different variations ofbryostatin 19 . We have shown that the ester bonds in bryostatin are quite reactive and can be easily substituted by carboxylic acids routinely found in marine sediment20-22 . Also, from our work, we have found no conclusive evidence that suggests a single species of bacteria is responsible for the production ofbryostatin23. In electron microscope studies of the colonized BAC's from the marine environment we observed a range of shapes and sizes in the microbial colonies that have produced bryostatin (Fig 5). Experiments described in this study are focused on developing a versatile and economical natural products synthetic route that combines elements of biogeochemistry, marine and sediment science and microbiology as a model to colonize and grow the desired microbes.

Methods The bryostatin-l (C 47 H 6P17) used as a calibrant in this work was obtained from LCLabs (Boston, Mass). The ET743 used in this work was obtained from the National Cancer Institute (NCD repository. The first identification of bryostatin in marine sediment occurred in the summer of 2000 at the delta of the Suwannee River in the Gulf of Mexico. We have sampled ecosystems of interest at Alligator Point harbor (Fl), Florida State University Marine Science Center bay (St. Teresa beach), Dickinson Bay (Panacea, Fl), Shell Point (fl), Keaton Beach (Fl), Fort Desoto State park (Fl), Sarasota Bay (Fl), Pine Island (Fl), and at several locations in the Florida Keys. The locations shared two basic similarities; Bugula neritina and/or Ecticidean

Production of ET743, Bryostatin, and Taxol

45

Bacteria

Fig 3A. Past work in this lab has established a model for the distribution of microbial species and their associated marine natural products. (1) A bacterial species resides in the sediment, active or inactive. (2) It enters the water column through tidal and wave action in very low concentrations. (3,4) The bacteria find a favorable surface to colonize. We've identified bryostatin in a number of marine organisms in the Bugula ecosystem. (5) The surface has a specific chemical characteristic that allows the bacterial colony to thrive. Channel r"'"marker

Fig 3B. Through a series of analytical measurements, the chemical composition is determined and used to construct a bacterial amplification chamber. Given that marine bacteria are difficult or impossible to grow in a lab, BAC's were developed as a method to farm marine microbes in their home ecosystem.

o

HO

of;"r0° H .f O''"

~OH

~O'''',,-

00NH ~ ~

o

0

0);-

"-

Fig 4. The cancer drug taxol was originally extracted from the bark of the Pacific yew tree. It is studied here as a test for terrestrial natural products.

46

RPMP Vol. 29 - Drug Plants III

Fig 5. A scanning electron microscope image of bacteria from a BAC that produced bryostatins

turbinate had been identified in the area and they were in relatively shallow «10 feet), protected water that was high in organic content. Solvent extraction was accomplished using the optimized DN ratio method developed in this labx. A Shimadzu Reverse Phase C18 column method was used for purification with some samples. The samples were analyzed on a Bruker (Billerica, MA) Autoflex MALDI-TOF mass spectrometer using refleckon mode. 2,5Dihydroxybenzoic acid (DHB) or sinapic acid were dissolved in 50:50 acetonitrile:water with 0.1 % trifluoroacetic acid to form the MALDI matrix. Solvent extracts were analyzed by high performance liquid chromatography (HPLC) - tandem mass spectrometry (MS) using a Thermoquest LCQ® DECA system (Thermoquest - Finnigan, San Jose, CA) equipped with an electrospray ionization (ESI) interfaceoThe HPLC column, 150 by 4.6-mm Gemini (5Mm C18, 1l0A), was purchased from Phenomenex (Palo Alto, CA). HPLC flow rate during gradient elutions with 0.1% formic acid (A)-methanol (B) was 1 mL min-I. Initial conditions, 90% A and 10% B, were increased linearly to 10% A and 90% B in 15 min and held isocratic for 9 min. Prior to analysis of each sample set, MS response was optimized for caffeine's (M+H)+ by infusing a methanol solution (10 Mg MLol) at 5 ML min-l into the HPLC column effluent upstream ofthe ESI interface. During analysis the mass filter was scanned from m/z+=100 to 1000. Throughout this work marine organisms (Bugula, Ecteinascidia turbinata) and the Florida Yew tree are used as markers for locations to place BAC's and not as primary source of the natural products.

Results and Discussion We are proposing a new method to produce marine natural products and have used bryostatin, and ET743 as examples24026. Taxol is studied as a preliminary representation of a terrestrial natural product. Typical agars and microbial broths involve taking a water or sediment sample back to the

Production of ET743, Bryostatin, and Taxol

47

lab and then cultivating the microbes on/in these media's. Our approach differs from these well practiced techniques in two ways. First we acknowledge that the chemical composition ofthe marine environment cannot be replicated over a period of time in a lab setting. This takes into account factors such as (a) The steady state concentration of trace organic and inorganic nutrients (b) Symbiotic microbes that only thrive in a specific set of physical, chemical and biological conditions (c) The colonization time for marine bacteria is not understood but may vary from hours to weeks, depending on the conditions and (d) The routine fluctuations in parameters such as sun light, dissolved oxygen, pH, suspended organic and inorganic material, and temperature are difficult to simultaneously replicate in a lab setting. Second, rather than using standard agar or broth compositions 27 , we conducted analytical measurements ofthe ecosystem to better understand the chemical environment that the marine bacteria we sought would colonize and thrive under. For example, the surface of Bugula neritina is coated with a thin layer of CaC0 3 so our BAC's have a high composition of this compound. These parameters are complimented by local knowledge involving CaC0 3 acquired from years of observations by our group and the staff at Gulf Specimen marine Lab (Panacea, Fl). In the area where Bugula is most frequently found there exist large deposits of calcium carbonate, dolomite and gypsum as well as fresh water springs feed by the Floridian aquifer and that percolate from the Gulf floor carrying Ca+2 (aq)28. Marine bacteria are well known to be difficult or impossible to grow in a laboratory setting. We overcome this hurdle by raising them in their home ecosystem29 . The inorganic components of the BAC's were partially derived from ICP-AES and ICPMS measurements of marine sediment which contained trace levels ofbryostatin or ET743. Also used were environmental sampling kits that measured parameters such as nitrates, nitrites, ammonia, phosphates, sulfides, sulfates and pH. In addition scanning electron microscope (SEM) and transmission electron microscope (TEM) studies were used to understand the cationic form (i.e. particle size) in nature 30. Metals were often in the form of oxides or hydroxides nanoparticles so species such as Fe, Al or Zn where added as commercially available metal oxide nanoparticles. In developing the organic component, FT-ICR measurements were used to identify key sediment components suggested by the ICR measurements such as squalene (C 30 H 50 ), tetracosanoic acid (C24H4802)' docosanoic acid (C22H4402)' eicosanoic acid (C2oH4002)' stearic acid (C18H3402)' and palmitic acid (C 16 H 320Yl. Some of these compounds have become staples in our BAC material. Because FT-ICR analysis suggests thousands of different structures in marine humic substances 32, which is the decay product of plant and animal matter, we also include low levels of commercially available humic acid in some mixtures. The BAC has a biodegradable support material such as wood chips or cellulose sponges. The chemical species discussed below are absorbed into

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this material before being encased in a perforated PVC tube, perforated bucket or another container and left in the ecosystem for a period of time. Additional quantities of easily solubilized materials are placed in smaller containers within the BAC to allow a slow dissolution. For example, 50 g of the BAC material (CaC0 3 , Si0 2, sugar, protein, stearic acid, ethanoic acid, etc.) is placed in a plastic tube that is capped with cheese cloth and inserted within the BAC . In this study we tested different mixtures and the composition of specific BAC's evolved according to the location, time of year, geometry of the container, etc. The chemical groups that compose our BAC's include: (1)

Inorganic species that may playa nutrient role (i.e. Fe+3 , N0 3 -, NH/, S-2, PO/ , etc.) are added to the composite as salts. Some of these match analytical measurements of the area. For example, we identified elevated levels of iron in the ecosystems where Bugula resides. Also, species have been added to the BAC's in different forms . For example, Fe+3 was originally added as a FeCl 3*6H 20 but couldn't be evenly distributed throughout the matrix. We later switched to Fe 20 3 nanoparticles, which distributed more evenly and replicated the form we found iron in the host environment. Other key species may be added in more than one form because multiple species are found in the ecosystem. As an example, sulfur has been added as elemental sulfur, sulfides (i.e . Na 2S), sulfites (Na 2S0 3 ) and sulfates (i.e. CaS0 4 ) .

(2)

The second group is composed of organic based structures found in the sediment (stearic acid, acetate, octanoic acid, squalene, etc.). Our selections in this area have been drawn from our FT-ICR measurements and a number of published studies that examined the bulk chemical composition of marine sediment. While an ICR study can identify thousands of potential structures, we typically added between five and fifteen species depending on their commercial availability. We also used commercially available humic acid, the product of plant and animal decay, which is comprised of a large number of organic structures 33 -38 •

(3)

The third chemical group incorporated in BAC's are organic based nutrients such as vitamins, amino acids, alcohols and sugars . In a typical BAC, it is common to have twenty different amino acids, sixeight different vitamins, two or three sugars and one or two different alcohols. By mass, the sum of these constituents would be 1-2% of the total BAC material. One concern is the dissolution of these water soluble components in the marine environment. In the BAC's, we utilized containers that held water soluble salts (i.e. NH 4N0 3 , NaAc) and organic nutrients (sugars, vitamins) mixed in with an insoluble mineral paste (CaC03 , Si02) which allowed them to dissipate at a slower rate than the chemicals absorbed on the support material.

Production of ET743, Bryostatin, and Taxol

49

(4)

The fourth group added to BAC's are naturally occurring polymers and have included DNA, proteins, cellulose, chitin, and lignin. In some cases materials such as wood chips and sponges have served as the support material for the other chemicals as well as a source of cellulose and lignin. Over time, the cellulose, lignin, or chitin based materials are consumed. Specially, chemicals such as salts, sugars and amino acids are soaked in wood chips or sponges for a period oftime before the BAC is deployed into the specific ecosystem in a perforated container. Chitin from marine organisms, such as shrimp and crab shells, have been pulverized and incorporated into the mix. Proteins and peptones are purchased commercially and added in low quantities «0.1% by mass) to the BAC.

(5)

The fifth groups incorporated into the BAC matrix are bulk inorganic species found in the local ecosystem. These species are typically the highest contributor to the total mass percent of the BAC material. Compounds such as CaC0 3 , CaS0 4 , and Si0 2 have been added as powders, pressed into pellets or mixed with cement to form a favorable colonization surface.

(6)

The sixth potential component of the BAC is a sample of the host organism that is known to contain the natural product. For example, Bugula is chopped in to small pieces and incorporated in the BAC matrix when attempting to grow bacteria that produce bryostatin. This has been added for two reasons: it may contain a limiting nutrient and there may be colonies of bacteria already flourishing within the organism. Fig 6 shows a bacteria colony on the surface of Bugula chopped for addition to a BAC that was deployed at Alligator Point (Fl). Although some were successful, a number of successful BAC's did not contain the host organisms.

In addition we have used support materials in the construction of different geometric shapes and sizes of BAC's. For example, BAC material

Fig 6. A bacterial colony found on the surface of Bugula about to be used in a BAC

RPMP Vol. 29 - Drug Plants III

50

was mixed with quick dry cement and supported with a stainless steel mesh. The sheet was left in a specific location in the Gulf of Mexico, removed and its surface examined for bacteria by scanning electron microscopy and a surface film extracted and analyzed. A number of prototypes , such as burying BAC material in the marine sediment to pumping seawater through material on the surface, were tested for bacterial growth, natural product production and devices long term stability. From empirical data it was concluded that for a marine bacterial colony to successfully colonize a favorable surface or material, it had to stay in the host ecosystem for days or weeks. Work in this lab showed that naturally occurring carboxylic acids can undergo different esterfication reactions with bryostatin-1 to form new structures under different conditions (acidity, basicity, UV exposure, etc.)20,21. In our mass spectrometry analysis of solvent extracts ofBAC's that were colonized in a Bugula ecosystem we have infrequently identified bryostatin-1 (or bryostatin-1+Na+). Of eighty-seven BAC's deployed and analyzed in the Alligator Point and Dickinson Bay region of Florida since 2001, only three have shown mass spectral features that correspond to bryostatin-1 (C47H6S017Nal; 927 amu's). Bryostatin-1 was used as a control in our mass spectrometry studies and is almost exclusively observed as a Na+ adduct (Fig 7A). Typically the bryostatins are observed as Na+ adducts unless we utilized an aminocarboxylate such as EDTA or DTPA in the extraction. Fig 7B is the mass spectra of an LC-MS analysis and shows bryostatin-1 extracted from a BAC with an ethanol solution containing trace amounts DTPA. The most prominent bryostatin we have consistently identified is bryostatin-11 (C 39H 5P15' 766.37; +Na+ 789.37 amu's). This spectral feature has appeared in 38 solvent extracts (out of 87) studied since 2001. In some cases it has been identified as a single spectral feature (with isotopic peaks) or a collection of features in the 786 to 790 region that indicates the gain! loss ofH's on the structures (Figs 8A & 8B). We have measured these gain! loss patterns of H's in past studies involving bryostatin-1 under different chemical conditions. Fig 9 provides spectral features for both bryostatin's 11 and 13 and ET743, identified by the spectral features at 743 and 761. This extract was taken from a BAC situated in Dickinson (Panacea) Bay.

60000000 . . - - - - - - - - - - - - - ,

40000000

927.4

20000000

o+-~~~~~~~~~~

500550600650700750 SOO 850 900 9501000

Fig 7A. Bryostatin-l is used as a calibrant for LC-MS analysis (r30)

Production of ET743, Bryostatin, and Taxol

8.0 E + 07

51

j

...

=

/

'; 4.0 E + 07

"il ~

_IL 0.0 E + 00 800 825 850 875 900

..AM.. 925 950 975 1000

m/z

Fig 7B. LCMS analysis of a BAC (cellulose sponge support material) submerged at Alligator Point (Fl) and extracted with ethanol containing trace levels of the chelating agent DTPA, resulting in the parent ion (C 47 H 6 P 1 7' 904 m/z; and the +Na+ adduct at 927 m/z). When chelating agents such as DTPA and EDTA are used, the Na+ ion decreases.

3750

... .s

3000 2250

~ 1500 ~

750 0 750

J•.

.tIL 770

790

810

830

mJz

--

850

Fig SA. Mass spectral analysis reveals bryostatin-ll+Na+and the less intense parent ion (-Na+) at 766 m/z. Exposing bryostatins to conditions such as acidity and basicity can result in the gain and loss of protons and result in a complex spectra

1200 817.816 900

... = 600

.....

~

300 0 700

750

800

850

900

950

1000

mJz

Fig SB. Bryostatin-ll and bryostatin 13 at 817 m/z extracted from a BAC placed at Alligator Point. These are the two most common bryostatins identified

RPMP Vol. 29 - Drug Plants III

52

450

.....= ~

789 817

300 7 3

761

150

0 700

750

800

850

900

950

1000

mJz

Fig 9. A TOF-MS analysis of a BAC placed in Dickinson Bay reveals bryostatin-ll (C39H58015Na1), bryostatin-13 (C41H62015Na1) and ET743 in the same ecosystem, which does not contain Bugula neritina .

This bay, which has been closely monitored by the staff at Gulf Specimen marine lab for over 40 years, produces sea squirts but has never supported Bugula. There are chemical and physical differences between the two locations including salinity levels and the quantities of dissolved organic matter in the water. Fig lOA is a mass spectra ofthe ET743 standard, Fig lOB is the 761 and 743 spectral features of the extract of a BAC set out in Sarasota Bay during the summer of 2007, and Fig 10C is the result of a mass spectral analysis of a BAC set out in Dickinson Bay during the summer of 2007 also. Observations of this nature support the model outlined in Fig 3A that the microbe producing the marine natural product travels through the water column and can colonize a favorable surface. What is not known is the degree or range of microbes in the sediment or their ability to travel distances stimulated by tides and currents. Most studies involving the distribution of a specific marine natural product are limited to a single species. In the fall of2007 the Florida Department of Environmental Protection issued our group a permit (permit Number 07092611) to study the soil surrounding a stand of Florida Yew trees located in Torreya State Park on the Apalachicola River. The Florida Yew (Taxus (loridana), described by 2500 ~

2000

...

....= ai ~

1500 1000

J

Il

500 0 720

728

736

744

I, 752

760

768

776

mJz

A

Fig 10. (Contd.)

Production of ET743, Bryostatin, and Taxol

'="

.~ ::l

:§

...

....=

~

53

70 60 50 40 30 20 10 0

600

-

700

800

900

mJz

B

6.E + 07 5.E + 07

...

....= 'i ~

4.E + 07 3.E + 07 2.E + 07 I.E + 07 O.E + 00 500

600

700

800

900

1000

mJz

c

mJz

D

Fig 10. (Contd,)

RPMP Vol. 29 - Drug Plants III

54

2500 2000 ...l

.s

1500

ai

1000

Cl::

500 0 750

754

758

762

766

770

774

778

m/z

E Fig 10. (A) TOF-MS analysis ET743 obtained from the NCI repository shows both peaks associated with the molecule at 743 and 761 (C 39 H 43 NPllS), The 743 m/z spectral feature is the result of a loss of a water molecule from the parent ion. (B) LC-MS analysis BAC placed in Sarasota Bay illustrates a strong mass spectral feature for the 761 m/z peak. (D). TOF- Mass spectral features corresponding ET743 extracted from a BAC located in Sarasota Bay. (E) TOFMS analysis illustrates ET743 spectral feature from BAC located in Dickinson Bay (Fl).

botanist as one of the rarest trees in the world and listed as endangered, is only found along a small stretch of the Apalachicola River in the Florida panhandle. While taxol is one ofthe most used cancer drugs of all time and the economics of producing it synthetic are conducted in an economical fashion, we chose to examine this system as a preliminary proof - of concept for our amplification chambers in a terrestrial environment. Specifically we wanted to see if the microbial amplification concept could be extended to land based natural products, whether they be the product of bacteria or fungi. Taxol production has been correlated with a fungus symbiotic with the Yew tree. In this experiment, sediment material collected around a yew tree stand was mixed with BAC material in a moist environment. IIi a mass spectrometric study the original sediment extract showed no evidence for the presence oftaxol (see Figs llA, B & C). Whether a fungus or bacteria, we believed an organism that produced taxol resided in the sediment at a very low density (i.e. organism/cm3 ). After mixing the sediment with BAC material and allowing it to stand for 2 weeks, it was extracted and analyzed by mass spectrometer. The extract contained mass spectral features that correspond to taxol. It should also be pointed out that extracts of the Yew leaves and bark showed low levels of taxol in some samples. While this is preliminary work with terrestrial organisms and natural products, it does show that an agriculture approach to raising microbes holds potential as a simple method of production of natural products.

Conclusions BAC were placed in the same ecosystems where the known host organism is found on an annual basis over a number of years. While bryostatin-l, because

Production of ET743, Bryostatin, and Taxol

55

1600 1400 · 1200 '

~

:.:

1000 · 800 ' 600 . 400 ·

*liliiii1 .~tlnt..... n"

200 •

o

850

860

870

880

7'

tr l' 890

mlz

A

10000

7500

.... oS

~

5000

2500

0 850

~

I. 860

870

880

la, 890

mlz

B 1000 -

750

...1:1

....

~

500 '

c Fig 11. (A) MS analysis of soil extract revealed no detectable taxol (B) the taxol standard used (C) taxol from mineral paste combined with sediment sample in Florida Yew tree ecosystem shows evidence oftaxol production.

of its pharmaceutical status, is the desired bryostatin, we routinely identified other bryostatins in our BAC extracts (Fig 12). Because of the fairly rapid decay of the marine natural products, the absorbance and build up of the

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RPMP Vol. 29 - Drug Plants III

1250 1000 789.612

750

875.009 8 17.694

500

I~J

250

.1 , .....11

o I"'" ",' 700

740

780

L 820

.... 860

900

.l

940

980

Fig 12. Bryostatin 11 (789 m/z), bryostatin 13 (817 m/z) and bryostatin 6 or 9 (875 m/z) were extracted from a BAC located in the Gulf of Mexico at Alligator Point in the winter/spring of2007 .

chemical species from the local environment is not a viable explanation of the results observed. For bryostatin, whose studies started in 2000 and continue today in our lab, it was identified in BAC's year round in our BAC's but had the highest concentrations in the spring. As the Gulfwaters warmed the amount ofliving biomass expanded tremendously into the summer months, this presumably made the selective growth of the bacteria more difficult. Also, we could find no consistency with the time of year or BAC material used as to which produced a specific bryostatin or how much of it was produced. Other environmental factors, from cold fronts to hurricanes, can impact the BAC productivity but we avoided dispersing them during these events . Quantitatively, the highest yield ofbryostatin measured was 0.005% of mixed bryostatins from a series of small mineral based tablets allowed to colonize for a week in February of 2002. In all extractions we undoubtedly had lower than 100% extraction yields due to the complexity of the matrix. In our testing of different materials, geometries, and locations we've had a number ofBAC's that have produced no bryostatins (",,50%). We did develop an extraction technique to optimize the quantity and selectivity of the natural products removed from the BAC material. This study focused on locations, water depths , BAC geometries and delivery methods, time scales and chemical compositions.

Acknowledgements We'd like to thank Mr. Jack Rudloe, Dr. Ann Rudloe and the staff of Gulf Specimen marine lab (Panacea, Fl) for all ofthe discussions and insights to the marine environment. Professor Alan Marshall and Dr. Tu Lam of the National High Field Magnet Lab (Tallahassee, Fl) for measuring the hydrocarbon content of marine sediment via FT-ICR. We'd like to thank grants from NOAA (SBIR Phases I and II to MIC Systems, Inc, Valdosta, Ga) and NSF-NUE (TJM PI) who supported different parts of this work, which started in the summer of 2000. We'd like to acknowledge NSF-MRI grant to VSU that supported the SEM we use on a regular basis. We'd like

Production of ET743, Bryostatin, and Taxol

57

to thank the VSU chemistry department, the VSU Center for International Programs (CIP), and Sunderland University School of Chemistry for support of this project throughout its life. We'd like to thank the State of Florida for allowing us access to Torreya State park and to Dr. Richard Carter for insight to the location of the Florida Yew tree populations. We would like to thank the VSU Professional Development Fund (Barbara Gray and Helen Morgan) for support and we would like to thank the Florida State University Marine Lab (Prof. Felicia Coleman, Mr. Dennis Tinsley and Ms. Sharon Thomas) for access to their facilities and expertise.

References 1.

Abadi, G., Palen, W., Geddings, J., Irwin, T., Kasali, N., Colyer, J., Goodsen, F., Smith, J., Jones, K., Hester, J., Noble, L., Groundwater, P.W. and Manning, T.J. 2006. A history of the Bryostatins: A Prominent Marine Natural Product, In: "Recent Progress in Medicinal Plants Vol. 15 - Natural Product" 2006. 2. Pettit, R. George, L. Herald, L. Cherry Doubek, L. Dennis Herald, Delbert, Clardy, Jon. Arnold and Edward. 1982. Isolation and structure ofbryostatin-1. Journal of the American Chemical Society 104 (24): 6846-8. Kageyama, M., Tamura, T., Michael H. Nantz, John C. Roberts, Somfai, P., David 3. C. Whritenour and Masamune, Satoru. 1990. Synthesis ofbryostatin-7. Journal of the American Chemical Society. 112(20): 7407-8. 4. Manning Thomas, J., Land Michael, Rhodes Emily, Chamberlin Linda, Rudloe Jack, Phillips Dennis, Lam Tukiet, T., Purcell Jeremiah, Cooper Helen, J., Emmett Mark, R. and Marshall Alan, G. 2005. IdentifYing bryostatins and potential precursors from the bryozoan Bugula neritina. Natural Product Research 19(5): 467-91. 5. Manning, Thomas J., Umberger, Tice, Strickland, Stacy, Lovingood, Derek, Borchelt, Ruth, Land, Michael, Phillips, Dennis and Manning James C. 2003. Naturally occurring organic matter as a chemical trap to scan an ecosystem for natural products. International Journal of Environmental Analytical Chemistry 83(10): 861-866. 6. Tice Umberger and Manning, T. 2001. Mass Spectral Analysis of marine Sediment reveals Bryostatins, Valdosta State University, Council for Undergraduate Research, Spring. 7. Manning, T.J., Rhodes, E., Loftis, R., Phillips, D., Demaria, D., Newman, D. and Rudloe, J. 2004. Chemical Analysis of the Sea Squirt Ecteinascidia turbinate Ecosystem. Vol. 20, Number 5,10 May 2006, pp. 461-473 (13) Natural Products Research. 8. Manning, T., Michael Land, Emily Rhodes, Rick Loftis, Crystal Tabron, Giso Abadi, Leslie Golden, Helen, J., Cooper, T. TuKiet. Lam, G. Alan, Marshall, R. Phillips Dennis and Jack Rudloe. 2005. Analysis of Ulmic Acid by Mass Spectrometry. Georgia Journal of Science. 63: 97-114. (8b) Manning, T., T. Umberger, S. Strickland, D. Lovingood, R. Borchelt and D. Phillips. 2003. Correlating Civil War Folklore with a Natural Products Discovery. Georgia Journal of Science. 61(2): 117. 9. Manning, Thomas, J., Hardeman, Crystal, Olsen, Katie, Rhodes, Emily; Parkman, Render; Land, Michael, North, Suzanne, M., Riddle, Kim and Phillips, Dennis. 2004. N anoparticles in the environment: Let's start at the bottom of the Gulf of Mexico! Chemical Educator 9(5): 276-280. 10. Kean, Greg, Smith, Justin, Ogden, Magan, Abadi, Giso, Barbas, John, Manning and Thomas, J. 2007. The Florida Yew Tree and Taxol. Abstracts, 59 th Southeast Regional Meeting of the American Chemical Society, Greenville, SC, United States, October 24-27 (2007).

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11. Wiernik, P.H., Schwartz, E.L., Strauman, J.J., Dutcher, J.P., Lipton, R.B. and Paietta, E. 1987. Phase I clinical and pharmacokinetic study of taxol. Cancer Research 47(9): 2486-93. 12. Stierle, Andrea, Stierle, Donald, Stroble, Gary, Bignami, Gary and Grothaus, Paul. 1995. Bioactive metabolites of the endophytic fungi of Pacific yew, Taxus brevifolia. Paclitaxel, taxanes, and other bioactive compounds. ACS Symposium Series, 583 (Taxane Anticancer Agents), pp.81-97. 13. Strobel, Gary A 2002. Useful products from rainforest microorganisms. Part 1. Endophytes and taxol. Agro-Food-Industry Hi- Tech 13(2): 30-32. 14. Strobel, Gary A, Torczynski, Richard and Bollon, Arthur. 1997. Acremonium sp.A leucinostatin a producing endophyte of European yew (Taxus baccata). Plant Science (Shannon, Ireland), 128(1): 97-108. 15. Mendola Dominick. 2003. Aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: Process developments and economics. Biomolecular Engineering 20(4-6): 441-58. 16. Van Kesteren, Ch., de Vooght, M.M.M., Lopez-Lazaro, L., Mathot, R.AA, Schellens, J.H.M., Jimeno, J.M. and Beijnen, J.H. Yondelis. 2003. (trabectedin, ET-743): The development of an anticancer agent of marine origin. Anti-cancer Drugs 14(7): 487-502. 17. Sudek, Sebastian, Lopanik, Nicole B., Waggoner, Laura E., Hildebrand, Mark, Anderson, Christine, Liu, Haibin, Patel, Amrish, Sherman, David, H. and Haygood, Margo G. 2007. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. Journal ofNatural Products 70(1): 67-74. 18. Davidson, S.K., Allen, S.W., Lim, G.E., Anderson, C.M. and Haygood, M.G. 2001. Evidence for the biosynthesis ofbryostatins by the bacterial symbiont Candidatus Endobugula sertula of the bryozoan Bugula neritina. Applied and Environmental Microbiology 67(10): 4531-4537. 19. Davidson, Seana K. and Haygood, Margo G. 1999. Identification of sibling species ofthe bryozoan Bugula neritina that produce different anticancer bryostatins and harbor distinct strains ofthe bacterial symbiont Candidatus Endobugula sertula. Biological Bulletin (Woods Hole, Massachusetts) 196(3): 273-280. 20. Manning, Thomas J., Rhodes, Emily, Land, Michael, Parkman, Render, Sumner, Brandy, Lam, Tukiet T., Marshall, Alan G. and Phillips, Dennis. 2006. Impact of environmental conditions on the marine natural product bryostatin-1. Natural Product Research, Part A: Structure and Synthesis 20(6): 611-628. 21. Manning, T. et al., 2007. Naturally occurring esterification reactions with bryostatin, Natural Products Research (in-press). 22. Thomas, Jessica, Stoney, Tiffany, Sermons, Shanda, McLeod, Kristin, Roberts, Sheena, Manning, Thomas, Abadi, Giso, Potter, Thomas, Phillips, Dennis, Rudloe, Jack, Marshall, Alan G., Barton, Ike, Bryant, Jon and Newton, Joe. 2006. Computational and experimental studies of the hydrolysis of bryostatin. 58 th Southeast Regional Meeting of the American Chemical Society, Augusta, GA, United States, November 1-4 (2006). 23. Geddings, Jason, Irwin, Tucker, Manning, Thomas, Abadi, Giso, Phillips, Dennis, Nienow, Jim, Noble, Lyn and Groundwater, Paul. 2006. Tracking bacterial growth in a bryostatin microbial broth. Abstracts of Papers, 231't ACS National Meeting, Atlanta, GA, United States, March 26-30, 2006. 24. Are precursors to marine natural products ubiquitous in the ocean? Rhodes, E, Manning, T, Lam, Tu, Purcell, J, Marshall, A, Phillips, D, Newman, D. Chern. Dep., Valdosta State Univ., Valdosta, GA, USA 55 th Southeast Regional Meeting of the American Chemical Society, Atlanta, GA, United States, November 16-19, (2003).

Production of ET743, Bryostatin, and Taxol 25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

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Manning, T.J., Land, M., Rhodes, E., Rudloe, J., Phillips, D., Lam, TuKiet T., Purcell, J., Cooper, H., Emmett, M.R. and Marshall, A.G. 2004. Elemental analysis and nanoparticles in the synthesis ofbryostatin: Is there a connection? Abstracts of Papers, 227th ACS National Meeting, Anaheim, CA, United States, March 28-April 1,2004. Manning, T.J., Rhodes, E., Land, M., Loftis, R., Phillips, D., Newman, D., Marshall, A.G. and Lam, T. 2004. The Role of Marine Geochemistry in Designing a Marine Natural Products Aquaculture Experiment. Abstracts, 56 th Southeast Regional Meeting of the American Chemical Society, Research Triangle Park, NC, United States, November 10-13 (2004). Mikalsen Jarle, Skjaervik Olaf, Wiik-Nielsen Jannicke, Wasmuth Marit, A. and Colquhoun Duncan, J. 2008. Agar culture of Piscirickettsia salmonis, a serious pathogen offarmed salmonid and marine fish. FEMS Microbiology Letters 278(1): 43-47. Toth, David, J. and Katz Brian, G. 2006. Mixing of shallow and deep groundwater as indicated by the chemistry and age ofkarstic springs. Hydrogeology Journal 14(6): 1060-1080. Koenig, Gabriele M., Kehraus, Stefan, Seibert, Simon F., Abdel-Lateff and Ahmed, Mueller, Daniela. 2006. Natural products from marine organisms and their associated microbes. Chem. Bio. Chem. 7(2): 229-238. Manning, Thomas J., Hardeman, Crystal, Olsen, Katie, Rhodes, Emily, Parkman, Render, Land, Michael, North, Suzanne M., Riddle, Kim and Phillips, Dennis. 2004. Nanoparticles in the environment: Let's start at the bottom of the Gulf of Mexico! Chemical Educator 9(5): 276-280. Manning, Thomas, Land, Michael, Rhodes, Emily, Loftis, Rick, Tabron, Crystal, Abadi, Giso , Golden, Leslie, Cooper, Helen J. , Lam, TuKiet T. , Marshall, Alan G., Phillips, Dennis R. , Rudloe. 2005. Jack Analysis of ulmic acid by mass spectrometry. Ga. J. Sci, June 2005. Stenson, Alexandra C., Landing, William M., Marshall, Alan G. and Cooper, William T. 2002. Ionization and fragmentation of humic substances in electro spray ionization fourier transform-ion cyclotron resonance mass spectrometry. Analytical Chemistry 74(17): 4397-4409. Manning, Thomas J., Sherrill, Myra Leigh, Bennett, Tony, Land, Michael and Noble, Lyn. 2004. Effect of chemical matrix on humic acid aggregates. Florida Scientist 67(4): 266-280. Manning, Thomas, Strickland, Stacy, Feldman, Amy, Umberger, Tice, Lovingood, Derek, Coulibay, Mamadou, Elder, John and Noble, Lyn. 2003. Infrared studies of Suwannee River humic substances: Evidence of chlorination of humics in salt water. Florida Scientist 66(4): 253-266. Manning, T.J., Bennett, T. and Milton, D. 2000. Aggregation studies of humic acid using multiangle laser light scattering. Science of the Total Environment 257(2-3): 171-176. Fiskus, Warren C. and Manning, Thomas J. 1998. Effects of humic acid on the solubility product constants of some environmentally significant calcium compounds. Florida Scientist 61(1): 46-51. Gravley, Eddie D. and Manning, Thomas J. 1995. Determination of the thermodynamics of the calcium- humic acid complexation by an ion selective electrode. Florida Scientist 58(4): 320-26. Hayes, D., Carter, J. and Manning, T.J. 1995. Fluoride binding to humic acid. Journal of Radioanalytical and Nuclear Chemistry 201(2): 135-41.

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5 Screening of Natural Products to Drug Discovery

Abstract Natural products have inspired chemists and physicians for millennia. Their rich structural diversity and complexity has promoted the discovery of new entities against several diseases. Analysis of bioactive compounds from different sources; including plants, animals and microorganism are in advance. Several positive distinctions have been identified in natural products, which can explain their success in the pharmaceutical industry, but the way is arduous, hard and some limitations can be found. Here, an examination of concept, sources, advantages and limitations of natural products are discussed. Key words : Natural product, Natural sources, Advantages, Limitations

Introduction Natural products still playa major role as drugs, and as lead structures for the development of synthetic molecules. About 50% of the drugs introduced to the market during the last 20 years are derived directly or indirectly from small biological molecules. Therefore, the interfacing of biological and chemical assessment becomes the critical issue (Vuorelaa et al., 2004). Various reasons have been put forward to explain the success of natural products in drug discovery, but the way to obtain a new chemical entity is arduous and hard. This review will focus the concept and sources of natural products, as well as the advantages and limitations during drug discovery process. 1. Institute of Tropical Medicine "Pedro Kouri". Apartado Postal No. 601, Marianao 13. Ciudad

*

de la Habana, Cuba.

Corresponding autlwr : E-mail: [email protected]

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Concepts of natural products Natural products include extracts, fractions, pure compounds or minerals, which are biosynthesized in nature. They can be isolated from living terrestrial or marine organism. Different sources can be found; including microorganisms, plants and animals (Hartmann, 1996; Rollinger et al., 2006). Generally, they are classified as primary and secondary metabolites. The primary metabolites are universal, uniform and conservative compounds, which are indispensable for the live. The secondary metabolites are singular, diverse and adaptive compounds, which are not essential for growth and development, but indispensable for survival (Hartmann, 1996). The major biodiversity result of secondary metabolites, which are under continuous process related to defence, protection, attraction and signalling. These vital events enrich the structural diversity and provoke favourable pharmacokinetic properties (Bajorath, 2002).

Natural sources of drug discovery Despite the impact of natural product and their incredible success stories as potent remedies from the commencement of human therapeutic activity to modern research and drug development, scale up to research with potential activity. As natural source are considered among the minerals, bacteria, fungi, protozoan, insects, plants and animals, as well as the humans (Rollinger et al., 2005), they can be selected by their traditional uses in the population, such as the plants or as new sources explored since the last century. The plants have been the natural source more used by the humans for healing purpose. Herbs have been used as remedies for thousands of years and about 80% of the world's population report using the plant to treat or alleviate the symptoms of several diseases (Fransworth et al., 1985). However, it has been estimated that only 5 to 15% of the approximately 250000 described higher plant species have been tested for some type of biological activity, and the marine and/or inferior plant have been less explored (Verpoorte, 1998). Numerous studies have demonstrated the manifold utilization of structures from plants as sources to treat several diseases or plants and their purified products that showed pharmacological potentialities and biological properties. Paclitaxel (1) is a diterpine plant derived compound isolated from the bark of Taxus brevifolia, which was the first new agent to have confirmed single agent activity in breast cancer (Arbuck et al., 1994). In recent years, the attention has been concentrated on isolating novel species of microorganism, being maintained in cultures and purified novel compounds with relevant therapeutic activity, particularly of cyanobacteria. Some service companies are offering to provide extracts or living strains of microorganism marine species (http://www.cyanobiotech.com/and http://www.marine-organism.com!) (Lam, 2006).

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The marine environment is frequently recognised as the largest potential source of biodiversity, and it is being increasingly searched for novel chemicals with useful bioactivity. In 2005,812 new marine compounds were described in literature (Blunt et al., 2007) and they have been demonstrated anticancer, antimicrobial and anti-inflammatory effects (Lam, 2006). Marine environment are largely unexplored in the actuality (Lam, 2007). An example is the (+ )-discodermolide (2), an antitumor polyketide from Caribbean sponge Discodermia dissolute, which was first isolated and characterized in 1990 (De Souza, 2004).

52

o

NR ____

)=0

o 0

~.".,,/

1

OR

o OR

2

Chemical Structures of(1) Paclitaxel (2) (+)-discodermolide

Advantages and limitations of natural products for drug discovery It is clear that the search of natural products as a potential therapeutic agents is an important approach to the overall drug discovery process. Recently, there has been much attention paid to the high rate of pharmaceutical industry failure in drug development and low rate of production of new chemical entities approved as medicines. For that reason, it would seem instead that the decision to move away from natural extract screening was made due to an increasing dependence on high-throughput biochemical screening technologies that are appropriated for natural products (Rishton, 2008). Several general distinctions have been identified which can explain the success of natural products:

1.

They offer unmatched high chemical diversity with structural complexity and biological potency.

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2.

The effects of evolutionary pressure to create biological active molecules structurally similar to target different species. Typically have more stereogenic centres and more architectural complexity. Contains relatively more carbon, hydrogen and oxygen, and less nitrogen and other toxic elements. Have molecular masses in excess of 500 daltons abs high polarities (greater water solubility and better biodisponibility, per example to administer by oral route). A natural preorganizing form which don't need additional energy. In many cases, a long history of efficacy and safe has been traditionally known.

3. 4.

5.

6. 7.

Nevertheless, it is obvious that the difficulties of natural products approach and the reasoning are debatable. The principal obstacles or limitations to natural products drug discovery can be listed: 1.

2.

3. 4.

5.

6.

7. 8.

9.

The extract or compounds from natural sources may vary by part of organism used, time of collection and type of extract. They are present as complex mixtures in extracts, which require labor-extensive and time-consuming purification procedures. The presence of synergistic, antagonist and neutralizing combination of compounds are frequent. The long process between the collection of natural product and the development as a pharmaceutical form (10-20 years). The cost involved in the different process to develop a new product based on natural source. The impact on novelty of natural product. In several times the bioactive compounds or lead may be a known compound, as the number of described natural products increased the probability to rediscovery a compound. Many natural compounds can not be obtained by synthetic ways or it's complicate to obtain other derivatives. It is difficult to obtain amounts to scale up. In general, the natural products are often synthesized in small quantities, which difficult largescale to develop preclinical and clinical studies. Intellectual property complications.

However, the inherent limitations of natural product screening can be decreased with the new technologies, such as the availability of extensive compound libraries, spectroscopic techniques (particularly in NMR technologies). Currently, several researchers, institutions and organizations have been on an effort to standardize proceeds to extract and purify compounds from natural sources. In parallel, library of natural product have been beginning to develop in order to use modern techniques in the search of potential natural products, such as the high-throughput (Lam, 2007).

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Other important aspect is the deficient and/or inadequate date about clinical studies using natural products, which present poor methodology quality or incomplete reporting of trials. The report of adequate clinical trials to validate the efficacy and safe of natural products is a need (Gagnier et al., 2006).

Technologies for natural-products discovery The typical process of drug discovery from natural sources is showed in Fig 1. In this general approach, the natural product is extracted from the source, fractionated and purified as a single biological active compound (Koehn & Carter, 2005). In each step a rigorous pharmacological verification of the activity is a need. Precise detail about the selected source should be described. A characterization of the specie used, including the scientific and common name is the first step, together with the part of the source used, as well as time and zone of collection. A previous knowledge about pharmacological or toxicity studies of the source is important to select the correct material (Gagnier et al., 2006). The crude extracts are prepared by maceration or percolation offresh or dried powdered material in water or organic. Different methodologies have

,,

I

.,

I

I

Inactive fractions

,

Pharmacological evaluation and structure determination

,

I

.,

,

I

Known compounds

Pure bioactive compound ~PharmaCOlOgiCal and I toxicological evaluation I I

,:

Validation oflead

Toxic compounds

I

Synthesis of

.~vatives

I

Preclinical studies '---_ _ _ _---'~le-up Clinical studies Increasimg amount of"v<J"p~.""""u. ~ ''''f.''

Fig 1. General approach to drug discovery from natural sources

I

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66

been described to increase the yield ofthe extract, such as the use of heat (4060°C). In general, the aqueous extract presents the advantage that can extract hydrophilic compounds; while the organic extracts are richer in chemical structures and prevent of possible contaminations with microorganism. For hydrophilic compounds, polar solvents can be used, such as: methanol, alcohol or ethyl-acetate. For lipophilic compounds butanol, chlorophorm or dichloromethane are used. In some times, combination between organic solvent have been described or between both aqueous and organic phase such as the hydro alcoholic extract 70 or 80% (Cos et al., 2006). Several methods have been described to obtain fractions from crude extracts. A general and simple approach is shown in Fig 2, which separated from less-polar to polar constituents by sequential use of solvent, which will be evaporated. This approach is easy to be carried out and permit better discrimination between fractions. The rapid identification of known compounds can be performed by high-performance liquid chromatography (HPLC) coupled with mass Crude extract Treatment with hexane

FRACTION!

RESIDUE Treatment with ethylacetate

FRACTION 2 Treatment with butane

FRACTION 3 Treatment with methanol

FRACTION 4 Fig 2. Scheme to obtain fractions from crude extracts

Screening ofNatural Products to Drug Discovery

67

spectrometer and the availability of natural product databases. The second major hurdle in the process is the determination of new structures of compounds. This step is possible thanks to the revolution and advances in spectroscopic techniques, particularly in high-resolution nuclear magnetic resonance (NMR). Determination of molecular formula is crucial to develop a new drug. One of the most powerful techniques is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) (Koehn & Carter, 2005). The discovery of natural products aimed the availability of chemistry modification or synthesized totally. The knowledge of the target can be addressed to obtain structures with better properties; including pharmacological and pharmacokinetics. The confluences of these technologies offer exciting new possibilities to exploit the remarkable chemical diversity of nature in the quest for new drugs (Koehn & Carter, 2005).

Conclusions The successful of natural products are not disputed, taking into account the large number of compound currently used, the potential traditional and new sources and the different advantages previously mentioned. A several new technologies are now available to explore the natural products to treat different diseases, based on molecular biological approaches and combinatorial biosynthesis of drug-like compounds. If modern drug development can benefit the natural product as source of new products, unlimited of compounds can become to playa central role in the treatment of diseases.

References Arbuck, S.G., Dorr, A and Friedman, M.A 1994. Paclitaxel (Taxo}) in breast cancer. Hematol. Oncol. Clin. North. Am. 8: 121-140. Bajorath, J. 2002. Integration of virtual and high-throughput screening. Nat. Rev. Drug. Discov. 1: 882-894. Blunt, J.W., Copp, B.R., Hu, W.P., Munro, M.H., Northcote, P.T. and Prinsep, M.R. 2007. Marine natural products. Nat. Prod. Rep. 24: 31-86. Cos, P., Vlietinck, AJ., Berghe, D.v. and Maes, L. 2006. Anti-infective potencial of natural products: How to develop a strongr in vitro 'proof-of-concept'. J. Ethnopharmacol. 106: 290-302. De Souza, M.V. 2004. (+)-discodermolide: A marine natural product against cancer. Scientific World Journal 11: 415-436. Fransworth, N.R., Akerele, 0., Bingel, AS., Soejarta, D.D. and Eno, Z. 1985. Medicinal plants in therapy. Bull. WHO 63: 965-981. Gagnier, J.J., Boon, H., Rochon, P., Moher, D., Barnes, J. and Bombardier, C. 2006. Recommendations for reporting randomized controlled trials of herbal interventions: Explanation and elaboration. J. Clin. Epidemiol. 59: 1134-1149. Hartmann, T. 1996. Diversity and variability of plant secondary metabolism: A mechanistic view. Entomol. Gen. Appl. 80: 177. Koehn, F.E. and Carter, G.T. 2005. The evolving role of natural products in drug discovery. Nature 4: 206-220.

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Lam, K.S. 2006. Discovery of novel metabolites from marine actinomycetes. Curro Opin. Microbiol. 9: 245-25l. Lam, K.S. 2007. New aspects of natural products in drug discovery. TRENDS Microbiol. 15: 279-289. Rishton, G.M. 2008. Natural products as a robust source of new drugs and drug leads: Past successes and present day issues. Am. J. Cardiol. 101: 43D-49D. Rollinger, J.M., Langer, T. and Stuppner, H. 2006. Srategies for efficient lead structure discovery from natural products. Curro Med. Chern. 13: 1491-1507. Verpoorte, R. 1998. Exploration of nature's chemodiversity: the role of secondary metabolites as leads in drug development. Drug Discov. Today 3: 232. Vuorelaa, P., Leinonenb, M., Saikkuc, P., Tammelaa, P., Rauhad, J.P., Wennberge, T. and Vuorelaa, H. 2004. Natural products in the process of finding new drug candidates. Curro Med. Chern. 11: 1375-1389.

6 Ethnomedicines Used in Trinidad and Tobago for Eye, Dental Problems and Headaches

Abstract This paper focuses on the nineteen plants used for eye and dental problems and headaches. Thirty respondents, ten of whom were male were interviewed from September 1996 to September 2000. The respondents were obtained by snowball sampling, and were found in thirteen different sites, 12 in Trinidad and one in Tobago. A preliminary validation of ethnomedicinal practices was conducted as a preliminary step to establish which plants are safe or effective and which uses should be discontinued. Three plants are used for eye problems (Capraria biflora, Kalanchoe pinnata, Ocimum gratissimum), five for headaches (Acnistus arborescens, Lepianthes peltata, Musa sp., Ricinus communis, Senna occidentalis), five for problems in the mouth (Aristolochia rugosa, Chrysobalanus icaco, Cocos nucifera, Spondias mombin and Tagetes patula), one for ear problems (Tagetes patula), one as a brain tonic (Rosmarinus officinalis) and one as a narcotic (Datura stramonium). Four of the plants used may produce unwanted side effects. Key words: Eye problems, Headaches, Nerves, Dental problems, Sleep aids, Trinidad and Tobago

Introduction A study of ethnomedicinal plants used in Trinidad and Tobago was undertaken from 1995 to 2000. This study was part of a larger research program investigating ethnoveterinary medicine. A substantial body of research published since 2000 has provided sufficient data to conduct a preliminary evaluation of these plants in the discussion section of this paper. 1. PO Box 72045, Sasamat, Vancouver, BC V6R4P2, Canada.

*

Corresponding author: E-mail: [email protected]

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There is little knowledge about the medicinal plant traditions ofthe people living in the Caribbean before the arrival of Columbus except for the practices documented by Father Bartolome de las Casas. Caribbean folk medicine incorporates knowledge from Africa, Europe, India, and South America; a product of inter-group borrowing or medical syncretism (Lans, 2007; Morison, 1963).

Methods This study adhered to the research guidelines and ethical protocols of Wageningen University in the Netherlands. Thirty respondents, ten of whom were male were interviewed from September 1996 to September 2000. The respondents were obtained by snowball sampling, and were found in thirteen different sites, 12 in Trinidad and one in Tobago. Snowball sampling was used because there was no other means of identifying respondents. The chief objective of the sampling method was to identify knowledgeable respondents; no priority was given to extrapolating the data to the wider population to establish prevalence of use. No statistical analysis is applied to the data since this would have required the use of a random sample thus increasing the risk of not identifying knowledgeable respondents, and reducing the efficiency of the research. Twenty respondents were interviewed once, the other ten (who were healers) were interviewed three or four times. Healers were also asked to reconstruct the circumstances and contexts of the plant uses so that the means of administration of the plants could be identified. No interview schedule of questions was used but a more qualitative, conversational technique. Plants were collected when available to verify that the common names used by each respondent were the same in each ethnic group as those recorded in the literature. The majority of the plants were identified at the Herbarium of the University of the West Indies but voucher samples were not deposited. This ethnomedicinal study was part of a larger research project on ethnoveterinary medicine; other data collecting techniques were used in the larger study (Lans, 2007).

Validation of practices A preliminary validation of ethnomedicinal practices is considered a preliminary step to establish which plants are safe or effective and which uses should be discontinued. It also ensures that clinical trials are not wasted on plants that are used for cultural or religious reasons. The validation of the remedies was conducted with a non-experimental method (Heinrich et al., 1992). This method consists of: 1. 2.

Obtaining an accurate botanical identification. Determining whether the folk data can be understood in terms of bioscientific concepts and methods.

Ethnomedicines Used in Trinidad and Tobago

3.

71

Searching the chemicallphannaceuticallphannacologicalliterature for the plant's known chemical constituents and to determine the known physiological effects of either the crude plant, related species, or isolated chemical compounds that the plant is known to contain. This information is used to assess whether the plant use is based on empirically verifiable principles or whether symbolic aspects of healing are of greater relevance. If ethnobotanical data, phytochemical and pharmacological information supports the folk use of a plant species it can be grouped into the validation level with the highest degree of confidence.

Four levels of validity were established (Heinrich et al., 1992): 1. 2.

3.

4.

If no information supports the use it indicates that the plant may be inactive; or no research has been done on the plant. A plant (or closely related species of the same genus), which is used in geographically or temporally distinct areas in the treatment of similar illnesses, attains the lowest level of validity, if no further phytochemical or pharmacological information validates the popular use. Use in other areas increases the likelihood that the plant is active against the illness. If in addition to the ethnobotanical data, phytochemical or pharmacological information also validates the use in Trinidad, the plant may exert a physiological action on the patient and is more likely to be effective than those at the lowest level of validity. If ethnobotanical, phytochemical and pharmacological data support the folk use ofthe plant, it is grouped in the highest level of validity and is most likely an effective remedy.

Results Three plants were used for eye problems (Capraria biflora, Kalanchoe pinnata, Ocimum gratissimum), five for headaches (Acnistus arborescens, Lepianthes peltata, Musa sp., Ricinus communis, Senna occidentalis), three for nervous conditions (Annona muricata, Musa sp., Piper hispidum), three to aid sleep (Annona muricata, Citrus nobilis, Crescentia cujete), five for problems in the mouth (Aristolochia rugosa, Chrysobalanus icaco, Cocos nucifera, Spondias mombin and Tagetes patula), one for ear problems (Tagetes patula), one as a brain tonic (Rosmarinus officinalis) and one as a narcotic (Datura stramonium). The plants represent 17 plant families. The ethnomedicinal plants used in Trinidad and Tobago for eye and dental problems and headaches are summarised in Table 1.

Table 1. Ethnomedicinal plants used for eye problems, headaches and dental problems Scientific name

Family

Common name

Acnistus arborescens Annona muricata Aristolochia rugosa Capraria biflora Chrysobalanus icaco Citrus nobilis Cocos nucifera Crescentia cujete Datura stramonium Kalanchoe pinnata Lepianthes peltata Musa species

Solanaceae Annonaceae Aristolochiaceae Scrophulariaceae Chrysobalanaceae Rutaceae Arecaceae Bignoniaceae Solanaceae Crassulaceae Piperaceae Musaceae

Wild tobacco Soursop Mat root, anico Du the pays Ipecak Portugal Coconut Calabash Datur Wonder of the world Sun bush Banana

Ocimum gratissimum Piper hispidum Ricinus communis Rosmarinus officinalis Senna occidentalis Spondias mombin Tagetes patula

Lamiaceae Piperaceae Euphorbiaceae Lamiaceae Caesalpiniaceae Anacardiaceae Asteraceae

Fonbazin Candle bush Castor oil leaf Rosemary Wild coffee Hogplum Marigold

Plant part used Leaves Root Leaves Bud Root Leaves Leaves Leaves Young leaf, green fruit Seeds Leaves Leaves Leaves Leaves

Use Headache Nerves, Sleep aid Toothache Eye wash Tonsils Sleep aid for babies Bleeding gums Sleep aid Narcotic Eye problems Headache Tie on head for headache, Boil with skin for nerves, 'run down' Clears eyes Nerves Tie on head for headache Brain tonic Tie on for headaches Mouthwash, tonsils, sore throat Pain in ear, Toothache

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Discussion Non-experimental validation of plants used for eye problems, headaches and dental problems For each species or genus the ethnomedicinal uses in other countries are given; then follows a summary of chemical constituents, in addition to active compounds if known.

Acnistus arborescens leaves have been used traditionally to treat cancerous growths (Kupchan et al., 1965). Alcoholic extracts of dried leaves of Acnistus arborescens contained a novel steroidal tumour inhibitor (Kupchan et al., 1965). Annona muricata fruit and leaves are used in Caribbean traditional medicine for their tranquillizing and sedative properties (Hasrat et al., 1997). Bourne and Egbe (1979) found that an alcoholic extract from the ripe fruit of soursop (Annona muricata) decreased the motor activity and prolonged the barbiturate (thiopentone sodium) sleeping time of rats. The study supported local claims of sedative properties (Bourne & Egbe, 1979). Studies showed that the fruit of Annona muricata possesses antidepressive effects (in contrast to sedative properties), possibly induced by alkaloids, benzyltetrahydroisoquinoline, annonaine, nornuciferine, asimilobine or reticuline (Hasrat et al., 1997). In the French West Indies, PSP and atypical Parkinsonism predominated in patients who consumed herbal tea and fruits of the Annonaceae (custard apple or pawpaw family). Benzyltetrahydroisoquinolines (alkaloids), present in Annonaceae, are neurotoxic to the basal ganglia in animals (Caparros-Lefebvre & Elbaz, 1999). This analysis was based on small numbers of cases. Aristolochia species are used in western Panama as analgesics (Joly et al., 1987). Capraria biflora is used as a bath tonic in Belize and Cura\(ao (Morton, 1968; Arnason et al., 1980). The use ofChrysobalanus icaco as an astringent in Trinidad has been previously recorded (Wong, 1976).

Citrus aurantifolia was found to be active against Staphylococcus aureus (Facey et al., 1999). Cocos nucifera nut shell is used as a rubefacient in India (Kapoor, 1990).

Crescentia cujete is used in Panama as a tranquiliser (Duke, 2000). Datura stramonium is used as a narcotic in Pakistan and in the republic of Niger, alkaloids in the plant have an atropine-like effect (Djibo & Bouzou, 2000; Shinwari & Khan, 2000). Kalanchoe pinnata is used for headaches by the Caribs in Guatemala (Gironetal., 1991).

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In the Caribbean and South America, warm Lepianthes peltata leaves are tied to the head and forehead to relieve headaches (Hodge & Taylor, 1957; Lachman-White et al., 1992). Lepianthes peltata leaves are also applied to other areas for the relief of arthritic pains, hernia pains, liver pains and other inflammatory disorders (Lachman-White et al., 1992; Mongelli et al., 1999). Lepianthes peltata and Lepianthes umbella showed no mutagenicity (Felzenszwalb et al., 1987). A catechol derivative (4-nerolidylcatechoI) was isolated from the methanolic leaf extract (Mongelli et al., 1999).

Musa paradisiaca is used for epilepsy in India and for fevers in Barbados (Handler & Jacoby, 1993; Ahmad & Beg, 2001). Heated leaves of Musa species are used for eye infections in Brazil and Indonesia (Milliken & Albert, 1996). Ocimum micranthum was used as a wash for bloodshot eyes when the condition was caused by a blow (Asprey & Thornton, 1953-1955). Ocimum species seeds were put into the eye in Belize and Mexico (Arnason et al., 1980; Ankli et al., 1999). Ocimum species grown in Rwanda were found to be antimicrobially active against Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Trichophyton mentagrophytes var. interdigitale (Janssen et al., 1989). The essential oil (EO) and leaf extracts of Ocimum gratissimum inhibited Staphylococcus aureus, Shigella species, Aeromonas sobria, Salmonella species, Plesiomonas shigelloides, Escherichia coli, Klebsiella species and Proteus mirabilis. The endpoint was not reached for Pseudomonas aeruginosa (>=24 mg/mI). Eugenol was responsible for the observed antibacterial activity (Ilori et al., 1996; Nakamura et al., 1999). Combinations with antibiotics potentiated the antibacterial activity of Ocimum gratissimum (Jedlickova et al., 1992). In Costa Rica Piper marginatum leaves are boiled and the tea is drunk to treat headaches (Hazlett, 1986). The plant and leaf contain ascorbic acid, beta-carotene, minerals, cepharadione-B, riboflavin, safrole and thiamin (Duke, 2000). Aqueous and ethanol extracts of aerial parts of Piper auritum have produced spasmogenic uterine stimulant and vasodilator effects (Gupta et al., 1993).

Ricinus communis is put on the head for headaches in Belize (Amason et al., 1980). Stems contain flavonoids, phenolic acids, triterpenes and phytosterols (Cambie, 1997). Rosmarinus officinalis is used as a tonic in Venezuela (Morton, 1975). Spondias mombin contains long-chain phenolic acids, a long-chain phenol, two antivirally active ellagitannins and five 6-alkenylsalicylic acids (Corthout et al., 1990a, b; Corthout et al., 1994). Spondias mombin has antibacterial and molluscicidal properties (Ajao et al., 1985; Corthout et al., 1994). Spondias mombin leaves were extracted with aqueous, methanol and ethanol solvents and tested on hexobarbital-induced sleeping time and novelty-induced rearing (NIH) behaviours in mice and rats (Ayoka et al.,

Ethnomedicines Used in Trinidad and Tobago

75

2006). The leaf extracts of Spondias mombin possessed sedative and antidopaminergic effects.

Tagetes patula contains polyacetylenes, ellagic acid and thiophene derivatives. The leaves contain flavonoids (quercetagetin, patuletin, patulitrin, mannitol) (Cambie, 1997).

Conclusions More data are necessary to evaluate the safety of the plants used for eye problems, headaches, dental problems and other conditions related to the head. Annona muricata, Aristolochia rugosa and Datura stamonium have validity for the folk uses described but also potentially serious side effects. The following plants have been understudied and therefore few claims can be made about their validity: Lepianthes peltata, Musa species, Ocimum gratissimum, Piper hispidum, Ricinus communis, Senna occidentalis and Tagetes patula. More studies are needed to establish the validity of the following plants for their respective uses: Acnistus arborescens, Capraria biflora, Chrysobalanus icaco, Citrus nobilis, Cocos nucifera, Crescentia cujete, Kalanchoe pinnata, Rosmarinus officinalis and Spondias mombin. All of the ethnomedicinal plants are biologically active but more research is needed before their efficacy can be established or invalidated. Some of the plants such as Annona muricata, Aristolochia trilobata, Chrysobalanus icaco and Datura stramonium may produce minor to serious side effects.

References Acosta, S.L., Muro, L.V., Sacerio, AL., Pena, AR and Okwei, S.N. 2003. Analgesic properties of Capraria biflora leaves aqueous extract. Fitoterapia 74(7-8): 686-688. Ahmad, I. and Beg, AZ. 2001. Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. J. Ethnopharmacol. 74(2): 113-123. Ajao, AO., Shonukan, O. and Femi-Onadeko, B. 1985. Antibacterial effect of aqueous and alcohol extracts of Spondias mombin and Alchornea cordifolia - two local antimicrobial remedies. Int. J. Crude Drug Research 23(2): 67-72. Akinpelu, D.A 2000. Antimicrobial activity of Bryophyllum pinnatum leaves. Fitoterapia 71(2): 193-194. Ankli, A, Sticher, O. and Heinrich, M. 1999. Medical ethnobotany of the Yucatec Maya: healers' consensus as a quantitative criterion. Econ. Bot. 53(2): 144-160. Amason, T., Uck, F., Lambert, J. and Hebda, R 1980. Maya medicinal plants of San Jose Succotz, Belize. J Ethnopharmacol. 2(4): 345-364. Autore, G., Rastrelli, L., Lauro, M.R, Marzocco, S., Sorrentino, R, Sorrentino, U., Pinto, A. and Aquino, R 2001. Inhibition of nitric oxide synthase expression by a methanolic extract of Crescentia alata and its derived flavonols. Life Sci. 70(5): 523-534. Ayoka, AO., Akomolafe, RO., Iwalewa, E.O., Akanmu, M.A and Ukponmwan, O.E. 2006. Sedative, antiepileptic and antipsychotic effects of Spondias mombin L. (Anacardiaceae) in mice and rats. J. Ethnopharmacol. 103(2): 166-175.

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Bourne, R.K and Egbe, P.C. 1979. A preliminary study of the sedative effects of Annona muricata (Soursop). West Indian Med. J. 28: 106-110. Cambie, R.C. 1997. Anti-fertility plants of the Pacific. CSIRO Publishing, Australia, pp. 181. Camporese, A., Balick, M.J., Arvigo, R., Esposito, R.G., Morsellino, N., De Simone, F. and Tubaro, A. 2003. Screening of anti-bacterial activity of medicinal plants from Belize (Central America). J. Ethnopharmacol. 87(1): 103-107. Caparros-Lefebvre, D. and Elbaz, A. 1999. Possible relation of a typical parkinsonism in the French West Indies with consumption of tropical plants: A case-control study. Lancet 354(9175): 281-286. Corthout, J., Pieters, L., Claeys, M., Geerts, S., Berghe, D. and van den Vlietinck, A. 1994. Antibacterial and molluscicidal phenolic acids from Spondias mombin L. Planta Medica 60(5): 460-463. Corthout, J., Pieters, L., Janssens, J. and Vlietinck, A.J. 1990a. The long-chain phenolic acids of Spondias mombin. Planta Medica 56(6): 584. [Poster] Corthout, J., Pieters, L., Claeys, M. and Vlietinck, A.J. 1990b. Isolation and characterisation of geraniin and galloyl-geraniin from Spondias mombin. Planta Medica 56(6): 584585. [Poster] Djibo, A. and Bouzou, S.B. 2000. Acute intoxication with "sobi-Iobi" (Datura). Four cases in Nige:r. Bull. Soc. Pathol. Exot. 93(4): 294-297. Duke, J.A. 2000. Phytochemical and Ethnobotanical Databases. USDA-ARS-NGRL, Beltsville Agricultural Research Center, Beltsville, Maryland, USA. Facey, P.C., Pascoe, KO., Porter, R.B. and Jones, A.D. 1999. Investigation of plants used in Jamaican folk medicine for anti-bacterial activity. J. Pharm. & Pharmacol. 51: 1455-1460. Felzenszwalb, I., Valsa, J.O., Araujo, A.C. and Alcantara-Gomes, R. 1987. Absence of mutagenicity of Potomorphe umbellata and Potomorphe peltata in the salmnellal mammalian-microsome mutagenicity assay. Braz. J. Med. Biol. Res. 20(3-4): 403405. Fernandes, J., Castilho, R.O., da Costa, M.R., Wagner-Souza, K, Coelho Kaplan, M.A. and Gattass, C.R. 2003. Pentacyclic triterpenes from Chrysobalanaceae species: cytotoxicity on multidrug resistant and sensitive leukemia cell lines. Cancer Lett. 190(2): 165-169. Ferreira-Machado, S.C., Rodrigues, M.P., Nunes, A.P., Dantas, F.J., De Mattos, J.C., Silva, C.R., Moura, E.G., Bezerra, R.J. and Caldeira-de-Araujo, A. 2004. Genotoxic potentiality of aqueous extract prepared from Chrysobalanus icaco L. leaves. Toxicol. Lett. 151(3): 481-487. Gir6n, L.M., Freire, V., Alonzo, A. and Caceres, A. 1991. Ethnobotanical survey of the medicinal flora used by the Caribs of Guatemala. J. Ethnopharmacol. 34(2-3): 173-187. Gore, M.A. and Akolekar, D. 2003. Evaluation of banana leaf dressing for partial thickness bum wounds. Burns 29(5): 487-492. Gupta, M.P., Mireya, D., Correa, A., Solis, P. N., Jones, A., Galdames, C. and GuionneauSinclair, F. 1993. Medicinal plant inventory of Kuna Indians: Part 1. J. Ethnopharmacol. 40(2): 77-109. Handler, J.S. and Jacoby, J. 1993. Slave medicine and plant use in Barbados. J. Barbados Museum & Historical Society 41: 74-98. Hasrat, J.A., De Bruyne, T., De Backer, J.P., Vauquelin, G. and Vlietinck, A.J. 1997. Isoquinoline derivatives isolated from the fruit of Annona muricata as 5-HTergic 5HT1A receptor agonists in rats: unexploited antidepressive (lead) products. J. Pharm. and Pharmacol. 49(11): 1145-1149. Hazlett, D.L. 1986. Ethnobotanical observations from Cabecar and Guaymi settlements in Central America. Econ. Bot. 40(3): 339-352.

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Heinrich, M., Rimpler, H. and Antonio-Barrerra, N. 1992. Indigenous phytotherapy of gastrointestinal disorders in a lowland Mixe community (Oaxaca, Mexico): ethnopharmacological evaluation. J. Ethnopharmacol. 36(1): 63-80. Hodge, W.H. and Taylor, D. 1957. The ethnobotany of the island Caribs of Dominica. Webbia 12(2): 513--644. Hosseinzadeh, H. and Nourbakhsh, M. 2003. Effect of Rosmarinus officinalis L. aerial parts extract on morphine withdrawal syndrome in mice. Phytother. Res. 17(8): 938--941. nori, M., Sheteolu, AO., Omonigbehin, E.A and Adeneye, AA 1996. Antidiarrhoeal activities of Ocimum gratissimum (Lamiaceae). J. Diarrhoeal Dis. Res. 14(4): 283285. Janssen, AM., Scheffer, J.J., Ntezurubanza, L. and Baerheim Svendsen, A 1989. Antimicrobial activities of some Ocimum species grown in Rwanda. J. Ethnopharmacol. 26(1): 57--63. Jedlickova, Z., Mottl, O. and Sery, V. 1992. Antibacterial properties of the Vietnamese cajeput oil and ocimum oil in combination with antibacterial agents. J. Hyg. Epidemiol. Microbiol. Immunol. 36(3): 303-309. Joly, L., Guerra, S., Septimo, R., Solis, P., Correa, M., Gupta, M., Levy, S. and Sandberg, F. 1987. Ethnobotanical inventory of medicinal plants used by the Guaymi Indians in Western Panama. Part I. J. Ethnopharmacol. 20(2): 145-171. Kapoor, L.D. 1990. Handbook of Ayurvedic medicinal plants. CRC Press Inc., Boca Raton, Florida, pp. 416. Kirszberg, C., Esquenazi, D., Alviano, C.S. and Rumjanek, V.M. 2003. The effect of a catechin-rich extract of Cocos nucifera on lymphocytes proliferation. Phytother. Res. 17(9): 1054-1058. Kupchan, S.M., Doskotch, R.W., Bollinger, P., Mcphail, AT., Sim, G.A and Renauld, J.A 1965. The isolation and structural elucidation of a novel steroidal tumour inhibitor from Acnistus arborescens. J. Am. Chem. Soc. 87(24): 5805-5806. Lachman-White, D.A., Adams, C.D. and Trotz Ulric, O'D. 1992. A guide to the medicinal plants of coastal Guyana. Commonwealth Science Council, London, pp. 350. Lans, C. 2007. Creole Remedies of Trinidad and Tobago. Lulu.com, pp. 225. Milliken, W. and Albert, B. 1996. The use of medicinal plants by the Yanomami Indians of Brazil. Econ. Bot. 50(1): 10-25. Mongelli, E., Romano, A, Desmarchelier, C., Coussio, J. and Ciccia, G. 1999. Cytotoxic 4 Nerolidylcatechol from Pothomorphe peltata inhibits topoisomerase I activity. Planta Medica 65(4): 376--378. Morison, S.E. 1963. Journals and other documents on the life and voyages of Christopher Columbus. New York: The Heritage Press, pp. 417. Morton, J.F. 1968. A survey of medicinal plants ofCurac;ao. Econ. Bot. 22(1): 87-102. Morton, J.F. 1975. Current folk remedies of northern Venezuela. Quarterly J. Crude Drug Res. 13: 97-121. Murakami, A, Nakamura, Y., Ohto, Y., Yano, M., Koshiba, T., Koshimizu, K., Tokuda, H., Nishino, H. and Ohigashi, H. 2000. Suppressive effects of citrus fruits on free radical generation and nobiletin, an anti-inflammatory polymethoxyflavonoid. Biofactors 12(1·4): 187-192. Nakamura, C.v., Veda-Nakamura, T., Bando, E., Melo, AF., Cortez, D.A and Dias Filho, B.P. 1999. Antibacterial activity of Ocimum gratissimum L. essential oil. Mem. Inst. Oswaldo Cruz 94(5): 675--678. Ngassoum, M.B., Essia-Ngang, J.J., Tatsadjieu, L.N., Jirovetz, L., Buchbauer, G. and Adjoudji, O. 2003. Antimicrobial study of essential oils of Ocimum gratissimum leaves and Zanthoxylum xanthoxyloides fruits from Cameroon. Fitoterapia 74(3): 284-287.

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Rojas, G., Levaro, J., Tortoriello, J. and Navarro, V. 2001. Antimicrobial evaluation of certain plants used in Mexican traditional medicine for the treatment of respiratory diseases. J. Ethnopharmacol. 74(1): 97-101. Rutten, T., Kruger, C., Melzer, M., Stephan, U.W. and Hell, R 2003. Discovery of an extended bundle sheath in Ricinus communis L. and its role as a temporal storage compartment for the iron chelator nicotianamine. Planta 217(3): 400-406. Shinwari, M.1. and Khan, M.A. 2000. Folk use of medicinal herbs of Margalla Hills National Park, Islamabad. J. Ethnopharmacol. 69(1): 45-56. Sosa, S., Balick, M.J., Arvigo, R, Esposito, RG., Pizza, C., Altinier, G. and Tubaro, A. 2002. Screening of the topical anti-inflammatory activity of some Central American plants. J. Ethnopharmacol. 81(2): 211-215. Veras, M.L., Bezerra, M.Z., Lemos, T.L., Uchoa, D.E., Braz-Filho, R, Chai, H.B., Cordell, G.A. and Pessoa, O.D. 2004. Cytotoxic withaphysalins from the leaves of Acnistus arborescens. J. Nat. Prod. 67(4): 710-713. Wong, W. 1976. Some folk medicinal plants from Trinidad. Econ. Bot. 30: 103-142.

7 Phytomedicinal Agents for Treatment of Schistosomiasis

Abstract Parasitic infections such as schistosomiasis are a great cause of human morbidity and mortality. This disease affects millions ofpeople especially in Africa and Asia. So far only a single drug, praziquantel, has been effective against this disease. Recent research, however, has shown the efficacy of the drug to be decreasing. Natural sources could not only provide new antischistosomal agents with promise to combat this disease, but also afford lead structures for synthetic modification and optimization of biological activity. This review presents a summary of recent ethnopharmacological surveys for antischistosomal plants, an overview of potential biomolecular targets in Schistosoma species, as well as a summary of antischistosomal agents from higher plants. The most important plant family for potential antischistosomal agents revealed in this survey is the Fabaceae. Key words :Schistosomiasis, Chemotherapy, Ethnopharmacology, Phytochemical, Natural products

Introduction Schistosomiasis is a parasitic infection of increasing importance today. Also known as "bilharzia", the disease is prevalent in Asia, Africa, and South America, in areas that have contaminated fresh-water bodies with the presence of snails, which may carry the parasite. This disease is a major public concern as an estimated 200,000,000 people are infected, mostly in Africa (Chitsulo et al., 2000). It is considered second only to malaria as a 1. Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA. * Corresponding author: E-mail: [email protected]

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cause of morbidity by the World Health Organization (Caffrey, 2007). There is a pressing need for new anti-schistosomals. Praziquantel is a drug that has been heavily relied on for the past 25 years and it is found to be effective against all species of Schistosoma (Caffrey, 2007). This drug is safe and effective but a recent study showing the decreasing efficacy of praziquantel against the immature parasites when compared to the adult worms has raised major concerns as treatment failure could arise (Cioli, 1998; Doenhoff et al., 2002; Caffrey, 2007). Plant products and their derivatives have been rich sources for drugs for many years. This review presents a brief summary of recent ethnopharmacological surveys of antischistosomal plants and phytochemicals. We do not review molluscicidal agents for control of schistosomiasis; this has been thoroughly reviewed (Marston et al., 1993; Perrett & Whitfield, 1996; Singh et al., 1996).

Pl ~ 'Llj~ 0

o

Praziquantel

Schistosomiasis Schistosomiasis is caused by trematode parasites of one of five species: Schistosoma mansoni, S. haematobium, S. japonicum, S. intercalatum, or S. mekongi. It spreads via skin contact with fresh water containing infectious larvae (Fig 1). After 1-8 weeks of exposure, symptoms such as dermatitis, fatigue and fever arise. Long term infection, ifleft untreated, leads to anemia and eventually to liver fibrosis and hydronephrosis (Salvana & King, 2008). This disease is a particular threat as it readily spreads through travelers from Africa and can become epidemic with the availability of fresh water bodies. Schistosomiasis is now prevalent in more than 76 countries (Yoon, 2007). The main organism that spreads this disease is the freshwater snail Lymnaea luteola. These snails rapidly reproduce at optimum temperatures of 25-35 °C (Parashar et al., 1983). As illustrated in Fig 1, humans are the typical hosts for the parasite. The adult worms lay eggs that hatch into miracidia. These penetrate the snail tissue where they form sporocysts that then develop into cercariae that are released into the surrounding fresh water. They enter the human physiological system through skin contact and circulate through the blood stream till they finally enter the liver, intestines, or bladder, and mature into adults.

In vitro antischistosomal screening Antischistosomal screening against S. mansoni generally involves in vitro testing of materials against the adult (bloodstream) form of the parasite.

81

Phytomedicinal Agents for Treatment of Schistosomiasis

Schistosomiasis Cercariae lose tails during penetration and become

Cercariae released by snail into water and

~

"~""=:, Circulation

Miracidia penetrate snail tissue, develop into sporocysts, and then cercariae

EggS~~l/"r releasmg miracIdia

A

-,-.,.....-,,.,.---'1

B

--:-:-:-:-;i~oU -===---....,IoiiI

c

'or

S Japonlcum S. mansoni

B

A

Migrate to portal blood in liver and mature into adults

Paired adult worms migrate to: A, B: Mesenteric venules of bowel/rectum (laying eggs that circulate to the liver and shed in stools) C: Venous plexus of bladder

~----------------------__

S. haematobium

C

Fig 1. Schistosomiasis life cycle (CDC, 2008)

The adult worms can be obtained directly from the blood of infected animals and maintained in appropriate media (Abdulla et al., 2007; Xiao et al., 2007). Alternative in vitro screening has utilized schistosomules, obtained from cercariae by sheering stress with a syringe (M0lgaard et al., 2001; Sparg et al., 2000) or miracidia hatched from eggs (Madhina & Shiff, 1996). In vivo antischistosomal screening has generally involved oral (gavage) treatment of rodents previously infected with Schistosoma cercariae (Xiao et al., 2007; El-Ansary et al., 2007). An ex-vivo screening method using spleen cells from infected mice have also been utilized (Aboul-Ela, 2002).

Biomolecular targets in Schistosoma spp. In order to achieve selective chemotherapy against parasites, differences between key parasitic metabolic pathways and those ofthe host need to be exploited (Frearson et al., 2007). The biomolecular target of praziquantel remains uncertain, but the drug apparently alters Ca2+ homeostasis in the schistosomes (Cioli, 1998) in addition to binding to adult worm actin (Tallima & El Ridi, 2007). Recently identified Schistosoma biomolecular targets that are being explored for potential chemotherapy include superoxide dismutase (SOD) (Mkoji et al., 1988; Nare et al., 1990; Xiao et al., 2002), glutathione S-transferase (GST) (Scott & McManus, 2000; Xiao et

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al., 2002; Sayed et al., 2008), thioredoxin glutathione reductase (TGR) (Kuntz et al., 2007), and cathepsin Bl cysteine protease (Caffrey, 2007; Abdulla et al., 2007).

Ethnopharmacological aspects of antischistosomal phytotherapy An examination of plants that are used as traditional herbal medicines for the treatment of schistosomiasis may well lead to new medicinal agents to cure this parasitic disease. A compilation of plant species that have been used in traditional medicine as well as those showing good antischistosomal activity is presented in Table 1. This table reveals that 47 species of plants are distributed over 29 families. The most important families are Fabaceae with 11 species and Asclepiadaceae, Asteraceae , Capparidaceae, Combretaceae, Euphorbiaceae, Hyacinthaceae, Liliaceae, and Zingiberaceae, with two species each.

Antischistosomal phytochemicals Alkaloids The isoquinoline alkaloid emetine has been shown to be moderately effective in treating schistosomiasis (Cioli, 1998), but the compound is a cumulative cardiotoxic poison (DNP, 2008). Eomecon chionantha alkaloids, including sanguinarine, chelerythrine, protopinen and allocryptopine, have exhibited anthelmintic properties that are effective against S. japonicum cercariae (Peng et al., 2003). Triclisia sacleuxii, used as a traditional herbal medicine for treatment of schistosomiasis and ascariasis, has yielded the biologically active bisbenzylisoquinoline alkaloids pheanthine, N-methylapateline, 0methylcocsoline, 1,2-dehydroapateline, 1,2-dehydrotelobine, and gasabiimine (Murebwayire et al., 2006, 2008).

H3CO H3CO

H' C O :/ 'OCH 3

I

N

~ Emetine

OCH 3

Sanguinarine

Phytomedicinal Agents for Treatment of Schistosomiasis

83

CR,O OCR,

Celerythrine

Protopine OCR, OCR,

o o Allocryptopine

Phaenthine

OCR,

o o o OCR,

N-Methylapateline

Gasabiimine

o o o O-methylcocsoline

o o o 1,2-Dehydroapateline

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84

o

o o 1,2-Dehydrotelobine

Neolignans Virolin and surinamensin, isolated from the leaves ofVirola surinamensis, have shown efficacy in blocking the penetration of S. mansoni cercaria into mice (Alves et al., 1998).

H,cOdo~ I

H3 CO

OH

~

Virolin

Surinamensin

Terpenoids The monoterpenoid quinone, thymoquinone, is the principal constituent of Nigella sativa seeds, and has demonstrated protective effects on mouse cells infected with schistosomiasis (Aboul-Ela, 2002). Artemisinin, a sesquiterpene lactone isolated from the aerial parts of Artemisia annua, is not only an effective antimalarial agent, but has also been found to be effective against schistosomiasis (Weathers et al., 2006). Semisynthetic

o

fAy

o Thymoquinone

Artemisinin

OCH3

Artemether

yo HO

Goyazensolide

OH

trans-O-14,15-epoxygeranyl-geraniol

Table 1. Ethnopharmacological survey of Schistosomicidal plants Plant(Family)

Origin

Activity

Reference(s)

Abrus precatorius (FabaceaelPapilionoideae)

Zimbabwe South Mrica

Ndamba et al., 1994;Sparg et al., 2000;M~lgaard et al., 2001

Afromomum latifolium (Zingiberaceae) Afzelia quanzensis (FabaceaeiCaesalpinoideae) Alltum cepa (Liliaceae)

Mali

Mali

Aloe buettneri (Lilaceae)

Mali

Annona senegalensis (Annonaceae) Anogeissus leiocarpa (Combretaceae) Balanites aegyptiaca (Zygophillaceae) Berkheya speciosa (Asteraceae) Cadaba farinosa (Capparidaceae) Calotropis procera (Asclepiadaceae) Capparis tomentosa (Capparidaceae) Cassia italica (FabaceaelPapilionoideae)

Mali

Stem and root extract active against S. haemotobium and S. mansoni in vitro Fruit used to treat urinary and intestinal schistosomiasis Root extract active against S. haemotobium in vitro Bulb decoction used to treat urinary schistosomiasis Root decoction used to treat urinary schistosomiasis Root powder used to treat urinary schistosomiasis Leaf decoction used to treat urinary schistosomiasis Root powder used to treat urinary schistosomiasis Root extract active against S. haemotobium in vitro Leaf decoction used to treat urinary schistosomiasis Root decoction used to treat urinary schistosomiasis Leaf/root decoction used to treat urinary schistosomiasis Leaf decoction used to treat urinary schistosomiasis

South Africa

Mali Mali

South Mrica Mali Mali Mali Mali

Bah et al. , 2006 Sparg et al., 2000 Bah et al. , 2006 Bah et al. , 2006 Bah et al., 2006 Bah et al., 2006 Bah et al., 2006 Sparg et al. , 2000 Bah et al. , 2006 Bah et al. , 2006 Bah et al. , 2006 Bah et al., 2006

Table 1. (Contd.) Plant(Family)

00

0)

Origin

Activity

Reference(s)

Cassia nigricans (FabaceaelPapilionoideae) Cassia sieberiana (FabaceaelPapilionoideae) Cissus quadrangularis (Vitaceae)

Mali

Bah et al., 2006

Citrus aurantifolia (Rutaceae) Cochlospermum tinctorium (Cochlospermaceae) Combretum micranthum (Combretaceae) Commiphora molmol (Burseraceae)

Mali

Whole plant powder used to treat urinary schistosomiasis Leaf decoction used to treat urinary schistosomiasis Whole plant decoction used to treat urinary and intestinal schistosomiasis Leaf and fruit decoction used to treat urinary schistosomiasis Leaf decoction used to treat urinary schistosomiasis Leaf decoction used to treat urinary schistosomiasis Resin/oil extract active against S. mansoni in vivo; activity disputed, however Oil extract active against S. mansoni in vivo Root extract active against S. mansoni in vitro Stem bark extract active against S. mansoni in vitro Root powder used to treat urinary schistosomiasis Root extract active against S. haemotobium in vitro

Curcuma longa (Zingiberaceae) Dicoma anomala (Asteraceae) Elephantorrhiza goetzei (FabaceaelMimosoideae) Entada africana (FabaceaelMimosoideae) Euclea natalensis (Ebenaceae)

Mali Mali

Mali Mali

Egypt

Egypt Zimbabwe Zimbabwe Mali

South Mrica

Bah et al., 2006 Bah et al., 2006

Bah et al., 2006 Bah et al., 2006 Bah et al., 2006 Badria et al., 2001 Fenwick et al. , 2003

EI-Ansary et al., 2007; EI-Banhawey et al., 2007

~

Ml'llgaard et al. , 2001

"d

Ml'llgaard et al. , 2001

r-o

Bah et al., 2006 Sparg et al., 2000

~

~ ~

I

....t:l ~

~ '" ;:s .,.,.

~

Table 1. (Contd.) Plant(Family) Euphorbia hirta (Euphorbiaceae) Ficus thonningii (Moraceae) Ledebouria ovatifolia (Hyacinthaceae) Leptadenia hastate (Asclepiadaceae) Lonchocarpus laxi{lorus (FabaceaelPapilionoideae) Leucas martiniensis (Lamiaceae) Nigella sativa (Ranunculaceae)

"i::I

Origin

Activity

Reference(s)

Mali

Whole plant decoction used to treat urinary schistosomiasis Leaf decoction used to treat intestinal schistosomiasis Aqueous bulb extract active against S. hamotobium in vitro Aerial part decoction used to treat urinary schistosomiasis Bark decoction used to treat urinary schistosomiasis Whole plant decoction used to treat urinary schistosomiasis Seed extract active against S. mansoni both in vivo and ex vivo Whole plant decoction used to treat urinary schistosomiasis Leaf extract and root bark extract active against S. mansoni and S. haematobium in vitro Aerial part infusion used to treat urinary schistosomiasis Berry extract active against S. mansoni miracidia in vitro Bark extract active against S. mansom and S. haematobium in V[tro

Bah et al., 2006

Mali

South Africa Mali Mali Mali

Egypt

Nymphea micrantha (Nympheaceae) Ozoroa insignis (Anacardiaceae)

Mali

Peristrophe bicalyculata (Acanthaceae) Phytolacca dodecandra (Phytolaccaceae) Pterocarpus angolensis (FabaceaelPapilionoideae)

Mali

Zimbabwe

Zimbabwe Zimbabwe

~ ..... 0

;3 C\)

~ ~.

Bah et al., 2006

S·

Sparg et al., 2002

~

Bah et al., 2006

'C..,'">

Bah et al., 2006 Bah et al. , 2006 Aboul-Ela,2002

.....

I;l

;::I .....

~

C\)

I;l

.....

;3 C\)

;::I

..... .Q, ~

'"'

;:,1;;'

Bahetal.,2006

C

'"0

;3

Ndamba et al., 1994; M(lJlgaard et al., 2001

S·

'"

1;;'

Bah et al., 2006 Madhina & Shiff, 1996 Ndamba et al., 1994; M(lJlgaard et al., 2001

00

-l

Table 1. (Contd.) Plant(Family)

00 00

Origin

Activity

Reference(s)

Saba senegalensis (Apocynaceae) Scilla natalensis (Hyacinthaceae) Securidaca Ion gepeduculanta (Polygalaceae) Securinega virosa (Euphorbiaceae) Senna petersiana (FabaceaelPapilionoideae) Stylosanthes erecta (FabaceaelPapilionoideae)

Mali

Bah et al., 2006

Trichilia emetic (Meliaceae)

South Mrica

Vitellaria paradoxa (Sapotaceae) Ximenia amaricana (Olacaceae)

Mali

Zea mays (Poaceae)

Mali

Leaf extract used to treat urinary schistosomiasis Aqueous extract active against S. haemotobium in vitro Root maceration used to treat urinary schistosomiasis Root decoction used to treat urinary schistosomiasis Plant extract active against S. haemotobium in vitro Aerial part decoction used to treat urinary and intestinal schistosomiasis Plant extract active against S. haemotobium in vitro Root powder used to treat urinary schistosomiasis Root decoction used to treat urinary and intestinal schistosomiasis Spike extract used to treat intestinal schistosomiasis

South Mrica Mali Mali

South Mrica Mali

Mali

Sparg et al., 2002 Bah et al., 2006 Bah et al., 2006 Sparg et al., 2000 Bah et al. , 2006

Sparg et al., 2000 Bah et al. , 2006 Bah et al., 2006

Bah et al., 2006

Phytomedicinal Agents for Treatment of Schistosomiasis

89

derivatives of artemisinin (e.g., artemether) have shown promise (Utzinger et al., 2001). Artemether has been shown to inhibit both glutathione Stransferase as well as superoxide dismutase of S. japonicum CXiao et al., 2002). The sesquiterpene lactone goyazensolide, isolated from Eremanthus goyazensis has exhibited in vitro antischistosomal activity against S. mansoni (Barth et al., 1997). The diterpenoid trans-( -)-14, 15-epoxygeranylgeraniol, isolated from Pterodon emarginatus fruit essential oil, has shown prophylactic activity against S. mansoni (Mors et al., 1967).

Conclusions Higher plants continue to serve as valuable sources of pharmacological agents. Recent ethnobotanical surveys have revealed at least 47 plant species that may yield promising new antischistosomal agents. The most important family in the ethnopharmacological surveys was the Fabaceae, but many more families are important. Thus, there are abundant higher plant sources for new chemotherapeutic agents to treat schistosomiasis.

References Abdulla, M.H., Lim, KC., Sajid, M., McKerrow, J.H. and Caffrey, C.R. 2007. Schistosomwsis mansoni: Novel chemotherapy using a cysteine protease inhibitor. PLoS Medicine 4: e14. doi:1O.13711journal.pmed. 0040014. Aboul-Ela, E.!. 2002. Cytogenetic studies on Nigella sativa seeds extract and thymoquinone on mouse cells infected with schistosomiasis using karyotyping. Mutation Res. 516: 11-17. Badria, F., Abou-Mohamed, G., El-Mowafy, A, Masoud, A and Salama, O. 2001. Mirazid: A new schistosomicidal drug. Pharm. Bioi. 39: 127 -131. Bah, S., Diallo, D., Dembele, S. and Paulsen, B.S. 2006. Ethnopharmacological survey of plants used for the treatment of schistosomiasis in Niono District, Mali. J. Ethnopharmacol. 105: 387-399. Barth, L.R., Fernandes, AP.M., Ribeiro-Paes, J.T. and Rodrigues, V. 1997. Effects of goyazensolide during in vitro cultivation of Schistosoma mansoni. Mem. Inst. Oswaldo Cruz 92: 427-429. Caffrey, C.R. 2007. Chemotherapy of schistosomiasis: present and future. Curro Opin. Chem. Bio!. 11: 433-439. Centers for Disease Control and Prevention 2008. http://www.dpd.cdc.gov/dpdx/html! schistosomiasis.htm (accessed 8-31-08). Chitsulo, L., Engels, D., Montresor, A. and Savioli, L. 2000. The global status of schistosomiasis and its control. Acta Tropica 77: 41-51. Cioli, D. 1998. Chemotherapy of schistosomiasis: An update. Parasitol. Today 14: 418-422. Dictionary of Natural Products 2008. CRC Press, Boca Raton, FL USA Doenhoff, M.J., Kusel, J.R., Coles, G.C. and Cioli, D. 2002. Resistance of Schistosoma mansoni to praziquantel: is there a problem? Trans. Roy. Soc. Trop. Med. and Hyg. 96: 465-469. El-Ansary, A.K, Ahmed, S.A. and Aly, S.A. 2007. Antischistosomal and liver protective effects of Curcuma longa extract in Schistosoma mansoni infected mice. Indian J. Exp. Bioi. 45: 791-801. El-Banhawey, M.A., Ashry, M.A., El-Ansary, A.K and Aly, S.A. 2007. Effect of Curcuma longa or praziquantel on Schistosoma mansoni infected mice liver-histological and histochemical study. Indian J. Exp. Bioi. 45: 877-889.

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Fenwick, A, Savioli, L., Engels, D., Bergquist, N.R. and Todd, M.H. 2003. Drugs for the control of parasitic diseases: Current status and development in schistosomiasis. Trends Parasitol. 19: 509-515. Frearson, J.A, Wyatt, P.G., Gilbert, I.H. and Fairlamb, AH. 2007. Target assessment for antiparasitic drug discovery. Trends Parasitol. 23: 589-595. Kuntz, AN., Davioud-Charvet, E., Sayed, AA, Califf, L.L., Dessolin, J., Amer, E.S.J. and Williams, D.L. 2007. PLos Medicine 4: e206. doi:l0.13711journal.pmed. 0040206. Madhina, D. and Shiff, C. 1996. Prevention of snail miracidia interactions using Phytolacca dodecandra (L'Herit) (endod) as a miracidiacide: an alternative approach to the focal control of schistosomiasis. Trop. Med. Int. Health 1: 221-226. Marston, A, Maillard, M. and Hostettmann, K 1993. Search for antifungal, molluscicidal and larvicidal compounds from African medicinal plants. J. Ethnopharmacol. 38: 215223. Mkoji, G.M., Smith, J.M. and Prichard, R.K 1988. Antioxidant systems in Schistosoma mansoni: Correlation between susceptibility to oxidant killing and the levels of scavengers of hydrogen peroxide and oxygen free radicals. Int. J. Parasitol. 18: 661666. Ml'llgaard, P., Nielsen, S.B., Rasmussen, D.E., Drummond, R.B., Makaza, N. and Andreassen, J. 2001. Anthelmintic screening of Zimbabwean plants traditionally used against schistosomiasis. J. Ethnopharmacol. 74: 257-264. Murebwayire, S., Diallo, B., Luhmer, M., Vanlaelen-Fastre, R., Vanhaelen, M. and Duez, P. 2006. Alkaloids and amides from Triclisia sacleuxii. Fitoterapia 77: 615-617. Murebwayire, S., Frederich, M., Hannaert, V., Jonville, M.C. and Duez, P. 2008. Antiplasmodial and antitrypanosomal activity of Triclisia sacleuxii (Pierre) Diels. Phytomedicine 15: 728-733. Nare, B., Smith, J.M. and Prichard, R.K 1990. Schistosoma mansoni: Levels of antioxidants and resistance to oxidants increase during development. Exp. Parasitol. 70: 389-397. Ndamba, J., Nyazema, N., Makaza, N., Anderson, C. and Kaondera, KC. 1994. Traditional herbal remedies used for the treatment of urinary schistosomiasis in Zimbabwe. J. Ethnopharmacol. 42: 125-132. Parashar, B.D., Kumar, A and Rao, KM. 1983. Effect of temperature on embryonic development and reproduction of the freshwater snail Lymnaea luteola Troshel (Gastropoda), a vector of schistosomiasis. Hydrobiologia 102: 45-49. Peng, F., Huang, Q.Y. and Liu, N.M. 2003. Experimental study on the effects of alkaloids from Eomecon chiorantha in eliminating Schistosoma japonicum cercaria and protection against schistosomiasis. China Tropical Medicine 3: 734-735. Perrett, S. and Whitfield, P.J. 1996. Currently available molluscicides. Parasitol. Today 12: 156-159. Salvana, E.M.T. and King, C.H. 2008. Schistosomiasis in travelers and immigrants. Curro Infect. Dis. Rep. 10: 42-49. Sayed, AA, Simeonov, A, Thomas, C.J., Inglese, J., Austin, C.P. and Williams, D.L. 2008. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nature Medicine 14: 407-412. Scott, J.C. and McManus, D.P. 2000. Molecular cloning and enzymatic expression ofthe 28kDa glutathione S-transferase of Schistosoma japonicum: evidence for sequence variation but lack of consistent vaccine efficacy in the murine host. Parasitol. Int. 49: 289-300. Singh, A, Singh, D.K, Misra, T.N. and Agarwal, R.A 1996. Molluscicides of plant origin. Biol. Agricult. and Horticulture 13: 205-252. Sparg, S.G., van Staden, J. and Jager, AK 2000. Efficiency of traditionally used South African plants against schistosomiasis. J. Ethnopharmacol. 73: 209-214. Sparg, S.G., van Staden, J. and Jager, AK 2002. Pharmacological and phytochemical screening of two Hyacinthaceae species: Scilla natalensis and Ledebouria ovatifolia. J. Ethnopharmacol. 80: 95-101.

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Tallima, H. and El Ridi, R 2007. Praziquantel binds Schistosoma mansoni adult wonn actin. Int. J. Antimicrob. Agents 29: 570-575. Utzinger, J., Xiao, S., N'Goran, E.K, Bergquist, R and Tanner, M. 2001. The potential of artemether for the control of schistosomiasis. Int. J. Parasitol. 31: 1549-1562. Weathers, P.J., Elkholy, S. and Wobbe, KK 2006. Artemisinin: The biosynthetic pathway and its regulation in Artemisia annua, a terpenoid-rich species. In vitro Cell and Developmental Biology-Plant 42: 309-317. Xiao, S.H., You, J.Q., Gao, H.F., Mei, J.Y., Jiao, P.Y., Chollet, J., Tanner, M. and Utzinger, J. 2002. Schistosomajaponicum: effect of artemether on glutathione S-transferase and superoxide dismutase. Exp. Parasitol. 102: 38-45. Xiao, S.H., Keiser, J., Chollet, J., Utzinger, J., Dong, Y., Endriss, Y., Vennerstrom, J.L. and Tanner, M. 2007. In vitro and in vivo activities of synthetic trioxolanes against major human schistosome species. Antimicrob. Agents and Chemother. 51: 1440-1445. Yoon, S.S. 2007. Geographical information systems: A new tool in the fight against schistosomiasis. In: De Lepper, M.J.C., Scholten, H.J. and Stern, RM. eds., TheAdded Value of Geographical Information Systems in Public and Environmental Health 24: 201-213.

"This page is Intentionally Left Blank"

8 Chemical Composition and Biological Activity of Salvia officinalis L. (Lamiaceae)

Abstract Salvia L. is an important genus consisting of about 900 species in the family Lamiaceae. Some species of Salvia, especially S. officinalis L., have been cultivated worldwide for use in folk medicine and culinary purposes. Common sage (S. officinalis) is also known as: Garden, Kitchen and Dalmatian sage. The Latin name for sage, salvia, means "to heal". This species has been recommended for almost every illness or problem by different herbalists. Perhaps the most frequently cited effects of sage are its antihydrotic (antiperspiration), antibiotic, astringent, antispasmodic, estrogenic and tonic properties. Each of these effects has received some experimental support. Sage is commonly used to remedy leucorrhea, amenorrhea and dysmenorrhea. In a double blind, randomized and placebQ-controlled trial, sage was found to be effective in the management of mild to moderate Alzheimer's disease. This plant has also moderate but extensive bacteriostatic, antifungal and antiviral properties. Biologically active components of sage are partially within its essential oil, which contains mainly 1,8-cineole, borneol and thujone. Sage leaf, an approved herb by the German Commission E for internal and external use, contains carnosol, carnosic, oleanoic, ursolic, caffeic acids, flavones, flavone glycosides and polysaccharides as pharmacologically active constituents. The alcoholic extracts of S. officinalis L. leaves also possess antioxidant properties. These and a large number of the other researches support the traditional use of sage as a popular home remedy. Key words :Salvia officinalis L., Medicinal use, Chemical composition, Biological activity 1. Department of Chemistry, Faculty of Science and Mathematics, University of Nis,

*

Visegradska 33, 18000 Nis, Serbia. Corresponding author: E-mail: [email protected]

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94

Name of the Herb Salvia officinalis L.

Common names Common Sage, Garden sage, Meadow sage, Scarlet sage, True sage, Kitchen sage, Red sage, Dalmatian sage, Broad-leaved Sage, Narrow-leaved Sage and Salviae folium. English German French Spanish Italian Portuguese Greek Dutch Lithuanian Polish Serbian Russian Ukrainian Georgian Turkish

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

Sage, Old english = Sawge, Salbei, Konigssalbei, Sauge, Salvia, Salvia grande, Salvia, Alisfakia, Faskomilo, Salie, Salavijas, Szawia, Zalfija, Kadulja, Pelin, Kaloper, Slavulja, Schalfej, Schal'wija, Aptetschny, Shavliya, Salbi, Adacayi

Botanical name Salvia officinalis L. (syn. Salvia tomentosa Mill.)

Family Lamiaceae L. (Labiatae)

Description of different parts Herbs perennial. Stems erect, woody at base, minutely white tomentose, much branched. Petiole 0-3 cm; leaf blade oblong to elliptic or ovate, 1-8 x 0.6-3.5 cm, papery, finely corrugate, minutely white tomentose, base rounded or subtruncate, margin crenulate, apex acute to mucronate, rarely acute. Verticillasters 2-18-flowered, in terminal racemes 4--18 cm; upper bracts broadly ovate, apex acuminate. Pedicel ca. 3 mM. Calyx campanulate, 1-1.1 cm in flower, dilated to 1.5 cm in fruit, minutely tomentose on veins and margin, sparsely golden yellow glandular, ± tinged purple, 2-lipped to ca. 1/2 its length; upper lip shallowly 3-toothed, teeth subulate; lower teeth triangular, apex acuminate. Corolla purple or blue, 1.8-1.9 cm, minutely tomentose; tube imperfectly pilose annulate inside, straight, ca. 9 mM;

Chemical Composition and Biological Activity of Salvia officinalis

95

upper lip straight, obovoid, ca. 6 x 5.5 mM; lower lip ca. 1 x 1 cm. Filaments ca. 5 mM; connectives ca. 3 mM, arms equal. Nutlets dark brown, subglobose, ca. 2.5 mM in diam. Fl. Apr-Jun. (Flora of China). Salviae officinalis folium, European pharmacopoeia, 2007.

Definition Whole or cut dried leaves of Salvia officinalis L.

Content: Minimum 15 mJ/kg of essential oil for the whole drug and minimum 10 mJ/kg of essential oil for the cut drug (anhydrous drug).

Characters Sage leaf (Salvia officinalis) oil is rich in thujone. The powder of S. officinalis is light grey to brownish-green. It is examined under a microscope using chloral hydrate solution. The powder shows the following diagnostic characters: very numerous articulated and bent trichomes with narrow elongated cells and a very thick cell at the base as well as fragments of these trichomes; fragments of the upper epidermis with pitted, somewhat polygonal cells; fragments of the lower epidermis with sinuous cells and numerous diacytic stomata; rare single glandular trichomes with a uni- or bicellular head and a stalk consisting of 1 to 4 cells; abundant glandular trichomes with a unicellular stalk and a head composed of 8 radiating cells with a raised common cuticle.

Origin, distribution, commercially cultivated or wild Sage is found in its natural wild condition from Spain along the Mediterranean coast up to and including the east side of the Adriatic; it grows in profusion on the mountains and hills in Croatia and Dalmatia, and on the islands of Veglia and Cherso in Quarnero Gulf, being found mostly where there is a limestone formation with very little soil (Grieve, 1971). Its native range extends through the Mediterranean parts of Yugoslavia and Albania. However, S. officinalis exists on the territory of Serbia as well, and it is an edificator of one plant community. From PanCiC's "Flora of the Principality of Serbia", published in 1874, we learn that S. officinalis grew in Sicevacka Klisura - gorge in southeastern Serbia late in XIX century. Sicevacka Klisura is in fact one of the Mediterranean oases in Serbia - a refugium, i.e., a relict habitat of tertiary age. It is assumed that the population of species S. officinalis in Serbia is witness to its continual natural range, which was once, during the torrid tertiary, wider by far (Vasic, 1997). When wild it is much like the common garden sage, though more shrubby in appearance and has a more penetrating odour, being more spicy and astringent than the cultivated plant. The best kind, it is stated, grows on the islands of Veglia and Cherso, near Fiume, where the surrounding district is known as the sage region. The collection of sage forms an important cottage industry

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in Dalmatia. During its blooming season, moreover, the bees gather the nectar and genuine sage honey commands there the highest price, owing to its flavour. The plant is cultivated and collected from the wild in certain former republics of Yugoslavia, Albania, Turkey, Italy, Greece, Spain, Crete and the USA (Grieve, 1971). In cultivation, sage is a very variable species, and in gardens varieties may be found with narrower leaves, crisped, red, or variegated leaves and smaller or white flowers. The form of the calyx teeth also varies, and the tube of the corolla is sometimes much longer. The two usually absent upper stamens are sometimes present in very small-sterile hooks. There are many cultivars. The red sage and the broad-leaved variety ofthe white (or green) sage - both of which are used and have been proved to be the best for medical purposes - and the narrow-leaved white sage, which is best for culinary purposes as a seasoning, are classed merely as varieties of S. officinalis, not as separate species. There is a variety called Spanish or lavender-leaved sage and another called wormwood sage, which is very frequent (Grieve, 1971). A Spanish variety, called S. candelabrum, is a hardy perennial, the upper lip of its flower is greenish yellow, the lower a rich violet, thus presenting a fine contrast.

Salvia lyrala and S. urticifolia are well known in North America. Salvia hians, a native of Simla, is hardy, and also desirable on account of its showy violet-and-white flowers (Grieve, 1971).

Parts used for medicinal purpose Leaves, fresh or dried, gathered before flowering in May and whole herb, fresh or dried, gathered just after flowering, in August.

Medicinal properties Antihydrotic (antiperspiration), antibiotic, astringent, antispasmodic, hypoglycemic, estrogenic, tonic, spasmolytic and antiseptic properties, nervine, sedative, hemostat, laxative, stimulant, carminative, leucorrheal, amenorrheal, dysmenorrheal; effective in the management of mild to moderate Alzheimer's disease, flatulent dyspepsia, vermifuge action, pharyngitis, uvulitis, stomatitis, gingivitis, hyperhydrosis, galactorrhoea. Chemical constituents of medicinal value their structures, formula and properties Up to now, the following classes of compounds (secondary metabolites) have been isolated and/or detected as constituents of S. officinalis: essential oil constituents, non-volatile di- and triterpenes, phenolic compounds of the shikimate metabolism and polysaccharides. Table 1 gives the structures and related data of the representative biologically active S. officinalis metabolites.

Chemical Composition and Biological Activity of Salvia officinalis

97

Table 1. Structures ofbioactive secondary metabolites identified from Salvw officinalis Structure of bioactive compound Borneol

a-Thujone

-

H

)( W Viridiflorol

-

~

H ---

OH

Camphor

olf

Chemical profile

Bioactivity

CA Index name: Bicyclo[2.2.11heptan-2-01, 1,7,7-trimethyl-, (lR,2S,4R) -relMolecular formula: C1oH1RO Molecular wt: 154

Antimicrobial (Tabanca et al., 2001)

CA Index name: 2-0xabicyclo [2.2.21 octane, 1,3,3-trimethylMolecular formula: ClOH1SO Molecular wt: 154

Antimutagenic (Vukovic-Gacic et al., 2006) Antimicrobial (Pattnaiket al., 1997)

CA Index name: Bicyclo[3.1.01hexan-3 -one, 4-methyl-l( I-methylethylJ-, (lS,4R,5RlMolecular formula: C 1o H 16 0 Molecular wt: 152

Antimicrobial (Blagojevic et al., 2006)

CA Index name: IH -Cycloprop [e1 azulen -4-01, decahydro-l,I,4,7tetramethyl-, (laR,4S,4aS, 7R, 7as, 7bS)Molecular formula:

Antifungal (weak activity) (Scher et al., 2004)

Antimutagenic (Vukovic-GaCic et al., 2006)

C 15 H 26 0

Molecular wt: 222

CA Index name: Bicyclo[2.2.11heptan2-one, 1,7,7trimethylMolecular formula: C lO H 16 0 Molecular wt: 152

AntImicrobial (Blagojevic et al., 2006)

Antimutagenic (Vukovic-GaCic et al., 2006)

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98

Table 1. (Contd.) Structure of biactive compound

Chemical profile

Bioactivity

Linalool

CA Index name: 1,6-0ctadien-3-ol, 3,7dimethylMolecular formula: C lO H 1S O Molecular wt: 154

Antimicrobial (Blagojevic et al., 2006)

Manool

CA Index name: I-N aphthalenepropanol, a-ethenyldecahydroa,5,5,8a-tetramethyl2-methylene-, (aR,IS,4aS, 8aS)Molecular formula: C2o H 34 0 Molecular wt: 290

Antibacterial (Ulubelen et al., 1994)

Royleanone

CA Index name: 1,4-Phenanthrenedione, 4b,5,6,7,8,8a,9,10octahydro-3-hydroxy4b,8,8-trimethyl-2(1-methylethyll-, (4bS,8aS)Molecular formula: C2oH2S03 Molecular wt: 304

Anticancer (cytotoxic) (Slamenova et al., 2004)

Carnosol

CA Index name: 2H-9,4a(Epoxymethano) phenanthren-12-one, 1,3,4,9,10,10a-hexahydro5,6-dihydroxy-l, I-dimethyl-7( I-methylethyl)-, (4aR,9S,lOaS)Molecular formula: CZOH2604 Molecular wt: 330

Sedative and hypnotic (lmanshahidi & Hosseinzadeh,2006) Antioxidant (lmanshahidi & Hosseinzadeh, 2006)

Chemical Composition and Biological Activity of Salvia officinalis

99

Table 1. (Contd.) Structure of biactive compound

Chemical profile

Bioactivity

Carnosic acid

CA Index name: 4a(2H)Phenanthrenecarboxylic acid, 1,3,4,9,10,10ahexahydro-5,6dihydroxy-l,ldimethyl-7 (1-methylethyl)-, (4aR,lOaS)Molecular formula: C2oH2S04 Molecular wt: 332

Sedative and hypnotic Omanshahidi & Hosseinzadeh, 2006) Antioxidant (lmanshahidi & Hosseinzadeh, 2006)

Rosmanol

CA Index name: 2H-I0,4a(Epoxymethano) phenanthren-12-one, 1,3,4,9,10,10ahexahydro-5,6,9trihydroxy-l,ldimethyl-7 (l-methylethyl)-, (4aR,9S,10S,10aS)Molecular formula: C2oH2605 Molecular wt: 346

Antioxidant Omanshahidi & Hosseinzadeh, 2006)

a-Ursolic acid

CA Index name: Urs-12-en-28-oic acid, 3-hydroxy-, (3~)­ Molecular formula: C30 H 4s 0 3 Molecular wt: 444

Antiinflammatory (Imanshahidi & Hosseinzadeh, 2006) Inhibition of proteases (Jedinak et al., 2006)

CA Index name: 2-Propenoic acid, 3(3,4-dihydroxyphenyl)Molecular formula: C 9 H S04 Molecular wt: 180

Antioxidant (Imanshahidi & Hosseinzadeh, 2006)

HO

Caffeic acid ~

~ 10

HO

OH

eOOH

100

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Table 1. (Contd.) Structure of biactive compound

Chemical profile

Rosmarinic acid

HO

~ I "" """ a

ro~ 0

OH

Apigenin

r--

H

~

HO

OH

0

Hispidulin H HO MeO OH

0

Cirsimaritin OH MeG MeO OH

0

Bioactivity

CA Index name: Benzenepropanoic acid, a-[[(2E)-3(3,4-dihydroxyphenyl)1-oxo-2-propen-1-yll oxyl-3,4-dihydroxy-, (aR)Molecular formula: ClsH160S Molecular wt: 360

Antibacterial, antiviral and anti oxidative (Park et al., 2008)

CA Index name: 4H-1-Benzopyran4-one, 5,7 -dihydroxy-2(4-hydroxyphenyl)Molecular formula: C15HI005 Molecular wt: 270

Sedative and hypnotic (Imanshahidi & Hosseinzadeh, 2006)

CA Index name: 4H -1-Benzopyran-4one, 5,7 -dihydroxy-2(4-hydroxyphenyD6-methoxyMolecular formula: C16H1206 Molecular wt: 300 CA Index name: 4H-I-Benzopyran4-one, 5-hydroxy-2(4-hydroxyphenyD6,7- dimethoxyMolecular formula: C17H1406 Molecular wt: 314

Terpenoids Volatile mono-, sesqui- and diterpenoids (essential oil constituents) The chemical composition of S. officinalis essential oils varies widely (Bernotiene et al., 2007). The dominant constituents in many sage essential oils (Table 2) are a-thujone ($; 65.5%), 1,B-cineole ($; 59.0%), camphor ($; 45.7%), P-thujone ($; 40.1 %), a-humulene (33.7%) and linalool ($; 35.0%). Germacrene D (32.9%) as the major constituent was found only in one sage oil sample from Cuba. Viridiflorol ($; 24.0%) dominated in the wild

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plant essential oils. The latter compound (13.4%) and manool (14.7%) were the major constituents in one sample of the essential oil of S. officinalis growing in Cuba. The sage essential oils rich in viridiflorol and manool were found only in the last decade. Some sage oils were rich in a-pinene (~24.6%), limonene (~20.3%) and borneol (~ 15.0%). ISO 9909 for medicinal uses regulates the amounts of the following constituents in the sage essential oils: a-thujone (1S.0-43.0%), camphor (4.5-24.5%), 1,S-cineole (5.5-13.0%), ~-thujone (3.0-S.5%), a-humulene (~ 12.0%), a-pinene (1.06.5%), camphene (1.5-7.0%), limonene (0.5-3.0%), bornyl acetate (~2.5%) and linalool + linalyl acetate (~ 1.0%). The German Drug Codex requirements differ from the above ISO and are the following: thujones (~ 20.0%), camphor (14.0-37.0%), 1,S-cineole (6.0-16.0%), borneol (~ 5.0%) and bornyl acetate (~5.0%). This Codex regulates the amounts of only five compounds, while ISO 9909 - of eleven constituents. The lower limit of camphor in the German Drug Codex is by -3 times and the upper limit by -1.5 times higher than recommended by ISO 9909. Table 2. The major constituents (%) of essential oil of Salvia officinahs chemotypes according to Bernotiene et al. (2007) I t-65.5 0.4-45.7 t-59.0 ~-Thujone 1.0-40.1 a-Humulene 0.1-33.7 a-ThuJone Camphor 1,S-Cineole

II a-Thujone S.0-43.0 Camphor 4.5-24.5 1,S-Cineole 5.5-13.0 3.0-S.5 ~-ThuJone a-Humulene 0-12 0

III Vmdiflorol a-Thujone a-Humulene Manool 1,S-Cineole

lS.5-24.0 9.3-15.6 10.2-13.6 10.0-13 3 9.2-10.9

IV a-Thujone 14.S-1S.0 Manool 10.0-13.3 a-Humulene 7 .6-S. 7 Viridiflorol 7.7-S.2 1,S-Cmeole 6.6-S.2

t-trace « 0.05%)

The essential oils of S. officinalis of various chemotypes (a-thujone, 1,S-cineole, viridiflorol, camphor etc.) exhibit antioxidant, anti-inflammatory, antispasmodic, antimicrobial and stimulant properties. Besides, the essential oil of a-thujone chemotype has antivirial and antifungal properties. The essential oil exhibits insecticidal properties (Bernotiene et al., 2007).

Diterpenes: The diterpenes royleanone, horminone, and acetyl horminone, isolated from the roots of S. officinalis abrogated the survival of colon carcinoma cell Caco-2 and human hepatoma cell HepG2, cultured in vitro with induction of DNA breaks (Slamenova et al., 2004). Carnosic acid, a tricyclic diterpene, occurs in the fresh leaf (Brieskorn, 1991) and to some extent in the dried leaf (Tada et al., 1997) and certain types of extracts (Cuvelier et al., 1994). However, carnosic acid is fairly unstable and readily auto-oxidises to form lactones, especially the bittertasting lactone carnosol (Brieskorn, 1991). In turn, carnosol can degrade further to produce other phenolic diterpenes with lactone structures, such as rosmanol, epirosmanol, 7-methoxyrosmanol and galdosol, which have been identified in sage leaf(Tada et al., 1997; Kavvadias et al., 2003) and/or

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sage oleoresin (Cuvelier et al., 1994). Safficinolide and sageone (Tada et al., 1997), methyl carnosate, the lactone sagequinone methide A (Tada et al., 1997), and other related diterpenes (Tada et al., 1997) have also been isolated. Some of these compounds may be artefacts formed during extraction and isolation. Three rare apianane diterpenoids were isolated from the leaves of S. officinalis as well (Miura et al., 2001).

Triterpenes: Pentacyclic triterpene acids, mainly ursolic acid and oleanolic acid and the triterpene alcohols a- and ~-amyrin (Brieskorn & Kapadia, 1980), cis- and trans-martynoside (Hohmann et al., 2003) were found to be present in S. officinalis. List ofmetabolites ofthe shikimate pathway found in sage (Lu & Foo, 2002)

Phenolic acids: 4-Hydroxybenzoic acid, 3-methoxy-4-hydroxybenzoic acid (vanillic acid). Caffeic acid and its monomers: Caffeic and ferulic acid. Caffeic acid dimers: Rosmarinic acid. Caffeic acid trimers: Salvianolic acid I, methyl salvianolate I, salvianolic acid K, sagecoumarin. Caffeic acid tetramers: Salvianolic acid L, sagerinic acid. Phenolic glycosides: cis-p-Coumaric acid 4-(2-apiosyl)glucoside, trans-pcoumaric acid 4-(2-apiosyl)glucoside, 6-feruloyl-a-glucose, 6-feruloyl-~­ glucose, 1-(2,3,4-trihydroxy-3-methyl)butyl-6-feruloylglucoside, 6-caffeoyl-lfructosyl-a-glucoside, 1-caffeoyl-6-apiosylglucoside, 1-p-hydroxybenzoyl-6apiosylglucoside, 4-hydroxyacetophenone 4-glucoside (picein), 4hydroxyacetophenone 4-(6-apiosyl)glucoside, 4- hydroxyacetophenone 4-(2(5-syringoyl)apiosyl)glucoside, 1-hydroxypinoresinol I-glucoside, isolariciresinol 3a-glucoside, 2-(3-methoxy-4-glucosyloxyphenyl)-3hydroxymethyl-5-(3-hydroxypropyl)-7 -methoxy-2,3-dihydrobenzofuran.

Flavonoids Flavone aglycones: 5,7 ,4'-Trihydroxyflavone (apigenin), 5,7,4'trihydroxyflavone-7 -methyl ether (genkwanin), 5, 7,4'-trihydroxyflavone-7 ,4'dimethyl ether, 5,7,3' ,4'-tetrahydroxyflavone (luteolin), luteolin-7-methyl ether. 6-Hydroxyflavones: 6-Hydroxyapigenin (scutellarein), 6-hydroxyapigenin6-methyl ether (hispidulin), 6-hydroxyapigenin-6, 7 -dimethyl ether (cirsimaritin), 6-hydroxyapigenin-5,6,7,4'-tetramethyl ether, 6hydroxyluteolin-6-methyl ether (nepetin or eupafolin), 6-hydroxyluteolin6,7-dimethyl ether (cirsiliol).

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8-Hydroxyflavones: 8-Hydroxyapigenin (isoscutellarein). Flavone glycosides: Apigenin-7 -glucoside (cosmosiin), luteolin-7 -glucoside (cinaroside), luteolin-7 -glucuronide, luteolin-3'-glucuronide. 6-Hydoxyflavone glycosides: 6-Hydroxyapigenin-6-methyl ether-7glucoside (homoplantagenin), 6-hydroxyluteolin-7-glucoside, 6hydroxyluteolin -7 -glucuronide. Flavone-C-glycosides: Apigenin-6,8-di-C-glucoside (vicenin-2). Polysaccharides: Crude fractions rich in watersoluble arabinogalactans and also high-MW pectin and glucuronoxylan-related polysaccharides have been isolated from aerial parts of sage (Capek et al., 2003).

Mechanism of action The hexane and ethylacetate fractions of garden sage (8. officinalis) were assayed for their effects on tumor necrosis factor-a (TNF-a) and interleukin6 (IL-6) production in LPS-stimulated RAW 264.7 macrophages. The extracts inhibited the protein and mRNA expression of TNF -a and IL-6 in LPS stimulated RAW 264.7 cells at a concentration of 100 mglmL. These results suggest that the extract of sage may have antiinflammatory activity through the inhibition of pro-inflammatory cytokines. The n-hexane and the chloroform extracts ofthe plant dose-dependently inhibited croton oil-induced ear oedema in mice, the chloroform extracts being the most active. Further investigation of this extract revealed ursolic acid as the main active component, with the antiinflammatory effect ofursolic acid (ID so =0.14 mmollcm2 ) being 2-fold more potent than that of indomethacin, the reference non-steroidal antiinflammatory drug (NSAID), (Imanshahidi & Hosseinzadeh, 2006). Phenolics such as flavonoids, tannins and caffeic acid derivatives are reported to inactivate herpes simplex viruses by blocking ligands or receptors on the surface of viruses and host cells, respectively (Schnitzler et al., 2008). Carnosol and carnosic acid are two diterpenes isolated from the leaves ofthis plant which inhibited the binding oft-butylbicyclophosphoro [3SS]thionate (TBPS) to the chloride channel ofthe GABAlbenzodiazepine receptor complex in brain tissue (with IC so values of 57 ± 4 JIm and 33 ± 3 JIm, respectively), but had no effect on the binding of [3H]-muscimol, [3H]-diazepam or [3H]-flunitrazepam. Therefore the site of action of these compounds appears to be directly on the chloride channel, and therefore differs from miltirone (Rutherford et al., 1992). In another study, a benzodiazepine receptor binding assay-guided fractionation of the methanol extract from sage leaves revealed three flavones and two abietane diterpenes functioning as benzodiazepine receptor-active components. The flavones, apigenin, hispidulin and cirsimaritin, competitively inhibited 3H-flumazenil binding to the benzodiazepine receptor with IC so values of 30, 1.3 and 350 mM, respectively. The IC so value of abietane diterpenes, 7 -methoxyrosmanol and galdosol, were 7.2

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and 0.8 mM, respectively (Kavvadias et al., 2003). Kavvadias et al. (2004) describe the positive allosteric modulation of recombinant GABA A receptors by the flavonoid hispidulin (4', 5, 7-trihydroxy-6methoxyflavone). They demonstrate that hispidulin crosses the bloodbrain barrier and relate this to the anticonvulsant action of hispidulin. Preparations of sage have been used widely in herbal medicine to assist memory (Perry et al., 2000) and an extract of Spanish sage has been shown to enhance memory in healthy young volunteers (Tildesley et al., 2003). Sage also contains a-thujone, a known GABAA receptor antagonist and a toxic component of absinthe (Hold et al., 2000), which may influence the GABA-enhancing effects ofhispidulin and related compounds in sage extracts. The levels of a-thujone in individual sage plants are known to vary considerably (Perry et al., 1999). Kavvadias et al. (2004) show that hispidulin (at 50 nM or higher; maximal effect at 10 mM) acts as a positive allosteric modulator across a range ofGABAA receptor subtypes, including a6~2y2S subtypes that are insensitive to positive modulation by diazepam. The benzodiazepine antagonist flumazenil reduced, but did not block the action ofhispidulin on any of the GABAA receptor subtypes tested - data indicating that a part of the positive modulatory action of hispidulin is mediated through flumazenil-insensitive sites on GABAA receptors. As hispidulin did not influence the action of GAB A on al~2 GABAA receptors, hispidulin does not interact with low-affinity flumazenil-insensitive benzodiazepine sites (Walters et al., 2000). Thus, there is more to hispidulin than actions on classical benzodiazepine sites on GABA A receptors consistent with flumazenil-insensitive actions of other flavonoids, for example, amentoflavone (Hanrahan et al., 2003), apigenin and quercetin (Goutman et al., 2003).

Whether antibacteriaVantifungaVantiviral etc. Pharmacological studies in humans In a double-blind, placebo-controlled, crossover study, 30 healthy young volunteers (17 males, 13 females; mean age 24 years) were given, on three separate days at 7-day intervals in accordance with a randomized scheme, different single-dose treatments in identical opaque capsules: 300 mg or 600 mg of dried sage leaf, or placebo. On each test day, pre-dose and at 1 h and 4 h post-dose, each participant underwent mood assessment, requiring completion of Bond-Lader mood scales and the State Trait Anxiety Inventory (STAI) before and after a 20 min performance on the Defined Intensity Stress Simulator (DISS) computerized multitasking battery. The results indicated that single doses of sage leaf can improve cognitive performance and mood in healthy young participants, although the lower dose (300 mg) appeared to fall somewhat below the level required for beneficial effects. It is possible that inhibition of cholinesterases by sage leaf (demonstrated only in vitro) could be involved in the mechanism causing these effects (Kennedy et al., 2006).

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Clinical studies In a randomized, double-blind, placebo-controlled study, patients aged 6580 years 'of age with a diagnosis of mild to moderate dementia and probable Alzheimer's disease were treated for 16 weeks with 60 drops/day of either a sage leafliquid extract (1:1,45% ethanol; n = 15) or a placebo liquid (n = 15). Compared with the placebo group, patients in the sage leaf group experienced significant benefits in cognitive function by the end of treatment, as indicated by improved scores in the Clinical Dementia Rating (CDR; p < 0.003) and the Alzheimer's Disease Assessment Scale (ADAS-Cog; p = 0.03). Within the limitations of a fairly small number of patients and short period of follow-up, the results suggested efficacy of the sage leaf extract in the management of mild to moderate Alzheimer's disease (Akhondzadeh et al., 2003). Several open studies, carried out mainly in the 1930s on patients or healthy volunteers but including a larger 1989 study (unpublished) on 80 patients with idiopathic hyperhidrosis (the secretion of an abnormally large amount of sweat), supported the longstanding belief that sage leaf aqueous extracts have anti-hyperhidrotic activity (Bradley, 2006). Essential oil of sage exhibited remarkable bacteriostatic and bactericidal activities against Bacillus cereus, Bacillus megatherium, Bacillus subtilis, Aeromonas hydrophila, Aeromonas sob ria and Klebsiella oxytoca (Longaray Delamare et al., 2007). It also posseses in vitro antibacterial activity against some bacteria commonly used in the food industry, Lactobacillus curvatus, Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus or related to food spoilage Enterobacter gergoviae, Enterobacter amnigenus, although, the effect is dose-dependent (ViudaMartos et al., 2008). The antimicrobial activity of S. officinalis essential oil can be attributed to the presence of high concentrations of isomeric thujones, 1,8-cineole and camphor, three monoterpenes with well documented antibacterial and antifungic potential (Longaray Delamare et al., 2007). The antifungal activity of sage essential oil is generally higher in the vapour phase than in liquid state against filamentous fungi, dermatophytes and Scopulariopsis brevicaulis and Fusarium oxysporum, which are often resistant to available antifungal agents and it opens important perspectives in alternative antifungal therapies (Tullio et al., 2007). Compounds from S. officinalis essential oil have been shown to exhibit high antibacterial activity against Staphyloccocus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, cytotoxic activity against Vero cells and virucidal activity against herpes simplex virus 1 and vesicular stomatitis virus (Sivropoulou et al., 1997; Tada et al., 1994). Aqueous and ethanolic extracts of S. officinalis, revealed a high antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) using a plaque reduction assay, although the ethanolic extracts

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revealed a much higher activity than the aqueous ones (Schnitzler et al., 2008). In aqueous and ethanolic extracts of different Salvia species oligomers of caffeic acid derivatives were identified exhibiting the antiviral activity (Schnitzler et al., 2008). Rosmarinic acid exhibits various pharmacological activities including prevention of oxidation of low density lipoprotein, inhibition of murine cell proliferative activity and of cyclooxygenase, and anti-allergic action. The biological activity ofrosmarinic acid is described as antibacterial, antiviral, and antioxidative. Its activity especially against rheumatic and inflammatory conditions makes it a sought-after substance for use in phytotherapy. More recently, rosmarinic acid was reported to have anti-HN activities (Park et al., 2008). Interesting results were found for ~-ursolic acid isolated from S. officinalis, which significantly inhibited all tested proteases in vitro in the micromolar range. ~-Ursolic acid showed the strongest inhibition activity to urokinase (IC 50=12 pm) and cathepsin B (IC 50 =10 pm) as proteases included in tumor invasion and metastasis indicated possible anticancer effectivity. ~-Ursolic acid significantly decreased the number of B16 colonies in the lungs of mice at the dose 50 mg/kg (p < 0.05) (Jedinak et al., 2006). The diterpenes carnosol, rosmanol, epirosmanol, isorosmanol, galdosol, and carnosic acid exhibited remarkably strong antioxidant activity, which was comparable to that of a-tocopherol. The activity of miltirone, atuntzensin A, luteolin, 7-O-methylluteolin, and eupafolin was comparable to that of butylated hydroxytoluene. The activity of these compounds was mainly due to the presence of ortho-dihydroxy groups (Miura et al., 2002). Lipid peroxidation in both enzyme-dependent and enzyme-independent test systems were inhibited more effectively by a dry 50%-methanolic extract from aerial parts of sage leaf than by a-tocopheryl acid succinate (as a positive control) (Hohmann et al., 1999; Zupko et al., 2001). It has recently been shown that water-soluble polysaccharides isolated from aerial parts of sage possess immunomodulatory activity (Capek et al., 2003; Capek & Hribalova, 2004).

Salvia officinalis essential oil is applied in the treatment of a large range of diseases such as nervous system, heart and blood circulation, respiratory, digestive, metabolic and endocrine diseases, while the S. officinalis infusion is commonly used for the haemostatic, estrogenic, anti perspiration, antineuralgic, antiseptic, hypoglycemic and many other therapeutic effects (Farcasanu & Oprea, 2006). Ethanolic extract of S. officinalis potentiated memory retention and also it has an interaction with muscarinic and nicotinic cholinergic systems that is involved in the memory retention process (Eidi et al., 2006).

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Sage oil had only a relatively weak spasmolytic effect on isolated guinea pig tracheal and ileal smooth muscle in comparison with oils from other Labiatae such as melissa leaf or thyme (Reiter & Brandt, 1985).

Uses/ailments where this product is used Uses based on experience or tradition Internal: Digestive disorders such as dyspepsia, flatulence, poor digestion and bloating; to reduce excessive perspiration, e.g. in the menopause. Also taken as a gentle, stimulating tonic (Bradley, 2006). Topical (as a gargle or mouthwash): Inflammations of the mouth or throat mucosa, such as pharyngitis, tonsillitis, stomatitis, gingivitis and glossitis (Bradley, 2006).

Dosage/mode of usage This herb has approval status by the German Commission E. Recommended daily dosages in Germany are as follows:

Internal: 0.4-6 gofthe herb, 0.1-0.3 g of essential oil, 2.5-7.5 g oftincture, 1.5-3 g fluid extract. For gargles or rinses: 2.5 g of herb in 100 ml of water, 2-3 drops of essential oil in 100 ml of water, 5 g of alcoholic extract in 1 glass of water. External: Undiluted alcohol extract. Note: This herbal preparation information is a summary of data from books and articles by various authors. It is not intended to replace the advice or attention of health care professionals (Blumenthal, 1998).

Adverse reaction/side effects: None reported. Contraindications Sage leaf should not be taken during pregnancy or lactation (except in amounts present as a flavouring in foods). Epileptics are also advised to avoid it due to the convulsant potential of thujones (Bradley, 2006).

Precautions for usage The amount of sage leaf consumed as a culinary herb in food presents no hazard, but a degree of caution is necessary with larger amounts due to the presence ofthujones and camphor in the essential oil. Recommended dosages should not be exceeded or taken over prolonged periods, and sage leaf preparations should be avoided during pregnancy and lactation. The pure essential oil should never be used (Bradley, 2006).

Processing needed in the use of this medicinal plant/Commercial products available already, composition, recipes, etc. (Bradley, 2006) Essential oil, tea, fresh leaves, extracts, flavouring.

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Medicines UK: Accepted for general sale, internal or external use.

France: Accepted for specified indications. Germany: Commission E monograph published, with approved uses.

Food USA: Generally recognized as safe (21 CFR 182.10 and 182.20).

Council of Europe: Permitted as flavouring, category N2 with provisional limits on the content ofthujones (a and ~) in the finished product (0.5 mg! kg, with some exceptions).

Scope for commercial production All of the common cultivars of garden sage make beautiful accents in borders and rock gardens. Sage often is grown in containers for ornamental and culinary use. Sage is used extensively in the kitchen to add a unique flavor to salads, egg dishes, soups, stews, meats, and vegetables. It is used to flavor vinegars and tea. It is one of the most important culinary herbs in western cooking. Sage, parsley, rosemary, thyme, basil, chives, garlic, dill, sweet marjoram, savory, oregano, and French tarragon are indispensable in the basic culinary herb garden. The young leaves and flowers can be eaten raw, boiled, pickled or used in sandwiches. Sage is used as an ingredient in soaps, cosmetics and perfumes. Smeared on the skin, sage is a useful insect repellent. Dried leaves among clothes and linen will discourage moths.

References Akhondzadeh, S., Noroozian, M., Mohammadi, M., Ohadinia, S., Jamshidi, A.H. and Khani, M. 2003. Salvia officinalis extract in the treatment of patients with mild to moderate Alzheimer's disease: A double blind, randomized and placebo-controlled trial. J. Clin. Pharmacy Ther. 28: 53-59. Bernotiene, G., Nivinskiene, 0., Butkiene, R. and Mockute, D. 2007. Essential oil composition variability in sage (Salvia officinalis L.). Chemija 18(4): 38-43. Blagojevic, P., Radulovic, N., Palic, R. and Stojanovic, G. 2006. Chemical composition of the essential oils of serbian wild-growing Artemisia absinthium and Artemisia vulgaris. J.Agric. Food Chem. 54: 4780--4789. Blumenthal, M. (Ed.) 1998. The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines. American Botanical Council. Austin, TX. Bradley, P. 2006. British herbal compendium 2, A handbook of scientific information on widely used plant drugs, BHMA, u.K. pp. 339-344. Brieskorn, C.H. 1991. Sage-its constituents and therapeutic value. Z. Phytotherapie, 12: 61-69. Brieskorn, C.H. and Kapadia, Z. 1980. Constituents of Salvia officinalis. XXIV. Triterpene alcohols, triterpene acids and pristan in leaves of Salvia officinalis L.. Planta Med. 38: 86-90.

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Capek, P. and Hribalova, V. 2004. Water-soluble polysaccharides from Salvia officinalis L. possessing immunomodulatory activity. Phytochemistry 65: 1983-1992. Capek, P., Hnoalova, V., Svandova, E., Ebringerova, A, Sasinkova, V. and Masarova, J. 2003. Characterization ofimmunomodulatory polysaccharides from Salvia officinalis L.. Int. J. Bioi. Macromol. 33: 113-119. Cuvelier, M.E., Berset, C. and Richard, H. 1994. Antioxidant Constituents in Sage (Salvia officinalis). J. Agric. Food. Chem. 42: 665-669. Eidi, M., Eidi, A and Massih, B. 2006. Effects of Salvia officinalis L. (sage) leaves on memory retention and its interaction with the cholinergic system in rats. Nutrition 22: 321-326. European phannacopoeia 2007. 6th edition, Salvia officinalis, pp. 2853, EDQM. Farcasanu, I.C. and Oprea, E. 2006. Ethanol extracts of Salvia officinalis exhibit antifungal properties against Saccharomyces cerevisiae cells. Analele Universitatli din BucurestiChimie, Anul XV 1: 51-55. Flora of China: http://www.efloras.orglflorataxon.aspx?flora_id=2&taxon_id=200020236 Goutman, J.D., Waxemberg, M.D., Donate-Oliver, F., Pomata, P.E. and Calvo, D.J. 2003. Flavonoid modulation of ionic currents mediated by GABAA and GABAc receptors. Eur. J. Pharmacol. 461: 79-87. Grieve, M. 1971. A modern herbal: The Medicinal, Culinary, Cosmetic and Economic Properties, Cultivation and Folk-Lore of Herbs, Grasses, Fungi, Shrubs & Trees with Their Modern Scientific Uses, Chapter concerning Salvia officinalis, Dover Publications. Inc. New York. Hanrahan, J.R., Chebib, M., Davucheron, N.M., Hall, B.J. and Johnston, G.AR. 2003. Semisynthetic preparation of amentoflavone: a negative modulator at GABAA receptors. Bioorg. Med. Chem. Lett. 13: 2281-2284. Hohmann, J., Redei, D., Mathe, I. and Blunden, G. 2003. Phenylpropanoid glycosides and diterpenoids from Salvia officinalis, Biochem. Syst. Ecol. 31(4): 427-429. Hohmann, J., Zupko, I., Redei, D., Csanyi, M., Falkay, G., Mathe, I. and Janicsak, G. 1999. Protective effects of the aerial parts of Salvia officinalis, Melissa officinalis and Lavandula angustifolia and their constituents against enzyme-dependent and enzymeindependent lipid peroxidation. Planta Med. 65: 576-578. Hold, K.M., Sirisoma, N.S., Ikeda, T., Narahashi, T. and Casida, J.E. 2000. a-Thujone (the active component of absinthe): y-aminobutyric acid type A receptor modulation and metabolic detoxification. Proc. Nat!. Acad. Sci. U.s A. 97: 3826-3831. Imanshahidi, M. and Hosseinzadeh, H. 2006. The pharmacological effects of salvia species on the central nervous system. Phytother. Res. 20: 427-437. Jedinak, A, Muekova, M., Kostalova, D., Maliarb, T. and Masterova, I. 2006. Antiprotease and antimetastatic activity of ursolic acid isolated from Salvia officinalis. Z. Naturforsch. 61c: 777-782. Kavvadias, D., Monschein, V., Sand, P., Riederer, P. and Schreier, P. 2003. Constituents of sage (Salvia officinalis) with in vitro afimity to human brain benzodiazepine receptor. PlantaMed. 69: 113-117. Kavvadias, D., Sand, P., Youdim, K.A, Rice-Evans, C., Baur, R., Sigel, E., Rausch, W.-D., Riederer, P. and Schreier, P. 2004. The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anti-convulsive effects. Br. J. Pharmacol. 142: 811-820. Kennedy, D.O., Pace, S., Haskell, C., Okello, E.J., Milne, A and Scholey, AB. 2006. Effects of cholinesterase inhibiting sage (Salvia officinalis) on mood, anxiety and perfonnance on a psychological stressor battery. Neuropsychopharmacol. 31: 845-852. Longaray Delamare, AP., Moschen-Pistorello, LT., Artico, L., Atti-Serafini, L. and Echeverrigaray, S. 2007. Antibacterial activity ofthe essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food Chem. 100: 603-608. Lu, Y. and Foo, L.Y. 2002. Polyphenolics of Salvia -A review. Phytochemistry 59: 117-140.

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Miura, K, Kikuzaki, H. and Nakatani, N. 2001. Apianane terpenoids from Salvia officinalis. Phytochemistry 58(8): 1171-1175. Miura, K, Kikuzaki, H. and Nakatani, N. 2002. Antioxidant activity of chemical components from sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) measured by the oil stability index method. J. Agric. Food Chem. 50: 1845-1851. Park, S.u., Uddin, M.R Xu, H., Kim, Y.K and Lee, S.Y. 2008. Biotechnological applications for rosmarinic acid production in plant. Afr. J. Biotechnol. 7(25): 4959-4965. Pattnaik, S., Subramanyam, V.R, Bapaji, M. and Kole, C.R 1997. Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios 89(358): 39-46. Perry, N.B., Anderson, RE., Brennan, N.J., Douglas, M.H., Heaney, AJ., Mcgimpsey, J.A and Smallfield, B.M. 1999. Essential oils from Dalmatian sage (Salvia officinalis L.): variations among individuals, plant parts, seasons, and sites. J. Agic. Food Chem. 47: 2048-2054. Perry, N.S., Howes, M.-J., Houghton, P. and Perry, E. 2000. Why sage may be a wise memory remedy: effects of Salvia on the nervous system. Med. Aromat. Plants - Ind. Profiles 14: 207-223. Reiter, M. and Brandt, W. 1985. Relaxant effects on tracheal and ileal smooth muscles of the guinea pig. Arzneim.-Forsch.lDrug Res. 35: 408-414. Rutherford, D.M., Nielsen, M.P., Hansen, S.K, Witt, M.R, Bergendorf, O. and Sterner, O. 1992. Isolation and identification from Salvia officinalis of two diterpenes which inhibit t-butylbicyclophosphoro[35S1thionate binding to chloride channel of rat cerebrocortical membranes in vitro. Neurosc. Lett. 135: 224--226. Scher, J.M., Speakman, J.-B., Zapp, J. and Becker, H. 2004. Bioactivity guided isolation of antifungal compounds from the liverwort Bazzania trilobata (L.) S.F. Gray. Phytochemistry 65(18): 2583-2588. Schnitzler, P., Nolkemper, S., Stintzing, F.C. and Reichling, J. 2008. Comparative in vitro study on the anti-herpetic effect of phytochemically characterized aqueous and ethanolic extracts of Salvia officinalis grown at two different locations. Phytomedicine 15:62-70. Sivropoulou, A, Nikolaou, C., Papanikolaou, E., Kokkini, S., Lanaras, T. and Arsenakis, M. 1997. Antimicrobial, cytotoxic, and antiviral activities of Salvia fructicosa essential oil. J. Agric. Food Chem. 45(8): 3197-3201. Slamenova, D., Masterova, I., Labaj, J., Horvathova, E., Kubala, P., Jakubykova, J. and Wsolova, L. 2004. Cytotoxic and DNA-damaging effects of diterpenoid quinones from the roots of Salvia officinalis L. on colonic and hepatic human cells cultured in vitro. Basic Clin. Pharmacol. Toxicol. 94: 282-290. Tabanca, N., Kirimer, N., Demirci, F. and Baser, KH.C. 2001. Composition and antimicrobial activity ofthe essential oils of Micromeria cristata subsp. phyrgia and the enantiomeric distribution of borneol. J. Agric. Food Chem. 49: 4300-4303. Tada, M., Hara, T., Hara, C. and Chiba, K 1997. A quinone methide from Salvia officinalis. Phytochemistry 45: 1475-1477. Tada, M., Okuno, K, Chiba, K, Ohnishi, E. and Yoshii, T. 1994. Antiviral diterpenes from Salvia officinal is. Phytochemistry 35: 539-541. Tildesley, N.T., Kennedy, D.O., Perry, E.K, Ballard, C.G., Savelev, S.AW.K and Scholey, A.B. 2003. Salvia lavandulae-folia (Spanish Sage) enhances memory in healthy young volunteer. Pharmacol. Biochem. Behav. 75: 669-674. Tullio, V., Nostro, A, Mandras, N., Dugo, P., Banche, G., Cannatelli, M.A, Cuffini, A.M., Alonzo, V. and Carlone, N.A 2007. Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods. J. Appl. Microbiol. 102: 1544--1550. Ulubelen, A, Topcu, G., Eris, C., Soenmez, U., Kartal, M., Kurucu, S. and Bozok-Johansson, C. 1994. Terpenoids from Salvia sclarea, Phytochemistry 36(4): 971-974. Vasic, O. 1997. A survey of the Mediterranean species ofLamiaceae family in the Flora of Serbia, Lagascalia 19(1-2): 263-270.

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Viuda-Martos, M., Ruiz-Navajas, Y., Fernandez-Lopez, J. and Perez-Alvarez, J.A. 2008. Antibacterial activity of different essential oils obtained from spices widely used in Mediterranean diet. Int. J. Food Sci. Tech. 43: 526-531. Vukovic-GaCic, B., Nikcevic, S., Beric-Bjedov, T., Knezevic-Vukcevic, J. and Simic, D. 2006. Antimutagenic effect of essential oil of sage (Salvia officinalis L.) and its monoterpenes against UV-induced mutations in Escherichia coli and Saccharomyces cerevisiae. Food Chem. TOXicol. 44(10): 1730-1738. Walters, R.J., Hadley, S.H., Morris, KD.W. and Amin, J. 2000. Benzodiazepines act on GABAA receptors via two distinct and separable mechanisms. Nat. Neurosci. 3: 1274-1281. Zupko, I., Hohmann, J., Redei, D., Falkay, G., Janicsak, G. and Mathe, I. 200l. Antioxidant activity ofleaves of Salvza species in enzyme-dependent and enzyme independent systems oflipid peroxidation and their phenolic constituents. Planta Med. 67: 366-368.

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9 Evaluation of Medicinal Plants Used to Diabetes Treatment N.H.

*

KAWASHITA 1 AND

A.M.

BAVIERAI

Abstract Diabetes mellitus is an endocrine disorder characterized by chronic hyperglycemia and alterations ofcarbohydrate, lipid and protein metabolism caused by defects in insulin secretion and / or action. Diabetes is rapidly becoming a global epidemic; the World Health Organization estimates that this disorder will affect 221 million people worldwide by the year 2010. Current therapeutic strategies for diabetes treatment include insulin and oral hypoglycemic agents, associated with some lifestyle adjustments (for example, diet and exercise), improving the glycemia control. However, the available drugs for diabetes treatment have some limitations, such as adverse effects, high rate of secondary failure and limited access to populations of the underdeveloped countries. In this way, considerable attention has been given to research the hypoglycemic effect ofmedicinal plants. There is growing trend towards using natural remedies in traditional and complementary medicine to diabetes treatment, representing an alternative therapy to the patients since herbal drugs are associated with positive effectiveness, less side effects and relatively low cost. Moreover, medicinal plants represent important sources for the development of new drugs in the treatment of diabetes. Nowadays, a large diversity of experimental design has been developed to evaluate hypoglycemic and antidiabetic effects and to understand the mechanism of action of herbal preparations, offering central information to advances in the development of original antihyperglycemic therapies. The objective of this work is to systematize the animal experimental models of diabetes mellitus and the specific methodologies that have been described in the scientific literature to investigate medicinal plants with potential antidiabetic properties. In addition, the work summarizes, based 1. Department of Chemistry, Federal University of Mato Grosso, Brazil.

* Correspondence author: E-mail: [email protected]

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on information obtained from international literature, some plants species which hypoglycemic effect has been scientifically demonstrated. Key words: Diabetes mellitus, Glycemia control, Anti-diabetic properties, Medicinal plants, Anti-hyperglycemic therapies

Introduction Diabetes mellitus is a congenital or acquired group of endocrine disorders characterized by defects in insulin production by the pancreatic beta cells and/or in insulin action on peripheral tissues, leading to abnormalities in carbohydrate, fat and protein metabolism. Increase in glucose blood levels (hyperglycemia) is the main well known consequence of this disorder, and its maintenance is considered a key factor in the development of several chronic diabetes complications, such as retinopathies (blindness, visual impairment), nephropathy (renal failure, for example), sensory neuropathy, foot ulceration and lower limb amputation, erectile dysfunction and cardiovascular complications, which contribute to the morbidity and mortality observed in diabetes. Diabetes can be classified into two major categories, types 1 and 2 diabetes. mellitus, although a patient may not present typical characteristics of one unique type of diabetes. Type 1 (insulin dependent) diabetes etiology can be explained by an abnormal autoimmune-mediated response or by idiopathic causes, both promoting pancreatic beta cells destruction and subsequent insulin deficiency. The main symptoms related to type 1 diabetes are acute hyperglycemia, polyuria, polydipsia, dehydration, weight loss and responses of diabetic ketoacidosis episodes, such as mental confusion, nauseas, abdominal pain. The disease occurs in 5% of the cases and manifested in childhood, adolescence or young adulthood. The great majority (80-90%) of diabetic patients present type 2 (non insulin dependent) diabetes mellitus that normally occurs in adults, but this incidence is growing in all age groups. The type 2 diabetes result from the combination between impaired insulin secretion and insulin resistance; it has an initial phase characterized by defects in the insulin secretion and compensatory hyperinsulinemia can be observed in the later phase. The insulin resistance occurs in liver, skeletal muscle and adipose tissues, impairing glucose uptake and increasing hepatic gluconeogenesis. Obesity and overweight are substantial risk factors for the development of type 2 diabetes. Excessive adipose tissue mass is associated with the release of increased amounts of inflammatory cytokines linked to insulin resistance, for example tumor necrosis factor-alpha and interleukin-6. Nowadays, the prevalence of diabetes across the world is estimated to be more than 171 million and projected to rise to 366 million in 2030 (Wild et al., 2004), indicating a situation of "epidemic diabetes". This raise can be attributed to several factors, such as population growth, urbanization, changing lifestyle (sedentary daily life and increased consumption of energy

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rich diets) and an increased prevalence of obesity. So, many global strategies must to be implemented to modify worldwide way of life for an adequate prevention and management of diabetes mellitus. In addition to this, pharmacological researches have become increasingly focused in their attempt to develop new drugs for diabetes treatment, preventing complications associated with this disease in its chronic state and enhancing patient quality oflife. The available therapeutic approach applied to diabetes mellitus attempts to maintain glycemia values close to normal and is based on diet, oral hypoglycemic drugs and insulin administration, used independently or in combination (Warren, 2004; Cohen & Horton, 2007). Despite the availability of multiple classes of hypoglycemic agents and insulin, these drugs promote some adverse effects, for example hypoglycemia due to higher doses, liver dysfunction, lactic acidosis and others. In addition, the excessive cost of the diabetes treatment arises as another disadvantage. These negative consequences in the conventional diabetes therapy stimulate the use of alternative medicines by diabetic patients, for example the treatment with herbal preparations and/or derivatives. In traditional practices, plant-based medicinal products are known since ancient times and have been used to control diabetes around the world. Crescent attention has been focused on the evaluation of the efficacy and safety of plant preparations for diabetes. Using different experimental models of diabetes and several methodologies, the ethnopharmacological research has been contributing to the selection of plants with confirmed antidiabetic effect as well as elucidating the mechanism of action that explains their hypoglycemic activity. These studies will generate essential information useful to the advances in the development of novel antidiabetic medicines. The objective of this review is to systematize the animal experimental models of diabetes mellitus and the in vivo/in vitro specific methodologies that have been used by the ethnopharmacological research to investigate preparations and/or compounds derived from plants with potential antidiabetic properties that are frequently used in traditional and alternative medicine.

General assays and methods applied to select plants with hypoglycemic effect Alternative medicinal plant based preparations have been used and also well accepted in the treatment of diabetes. Ethnopharmacological studies have reported several plants species with antihyperglycemic effect in experimental models of diabetes mellitus (Ivorra et ai., 1989; Yeh et ai., 2003). In these studies, an initial criterion must be selected for a correct plant trial, based on the elucidation of plant effects on general physiological parameters usually altered in diabetic animals, which will permit the continuity of further exploration. Nowadays, growing attention has been given to the elucidation of the mechanism of action that explains the hypoglycemic effect of these plants, using specific in vivo, ex vivo and in vitro methodologies applied on each tissue targeted by herbal preparations.

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These procedures are useful to bring about the isolation of underlying compounds and the development of drugs derived from plants. The following sections describe the most common physical and biological parameters measured in hypoglycemic plant trials.

Biochemical parameters The glucose plasma measurement is certainly the crucial biochemical analysis in the investigation of plant antidiabetic effects. In fact, glycemia values associated with other results are always presented in ethnopharmacological studies, evaluating the beneficial effects of herbal preparations administered to diabetic animals. The reduction of plasma glucose can be determined under different experimental conditions: for example the investigation of glycemia after chronic and/or subchronic treatments, acute experiments offasting and postprandial plasma glucose alteration, or glycemia evaluation in response to glucose tolerance tests (GTTs). These different determinations offer results that highlight several possibilities to propose the mechanism of action related to antidiabetic plants. However, the reduction in glycemia values after treatment with herbal preparation is not enough for the comprehension ofthe whole plant effect. Certainly, the next step is to use several other assays to clarify the plant beneficial action against diabetes symptoms. In addition, researches must be attempted to elucidate the antihyperglycemic effect of herbal preparations under different conditions of diabetes severity, which can vary according to some parameters, for example, animal age, dose of diabetogenic drugs and others. Herbal preparations that decrease glycemia may have several mechanisms of action, including stimulation of insulin synthesis and its release from pancreatic beta cells, correction of insulin resistance, improvement of peripheral glucose uptake, inhibition of endogenous glucose production, activation of liver and muscle glycogenesis, inhibition of liver glycogenolysis and/or inhibition of renal glucose reabsorption. The evaluation of some effects above cited will be later described in this review. Besides glycemia, determination of the urinary glucose can be an effective parameter investigated in the selection of herbal preparations with hypoglycemic effect. It is well known that blood glucose is continuously filtered in the kidney glomeruli and then reabsorbed by sodium glucose cotransporter type 2 (SGLT2) located in epithelial cells of the renal proximal tubules. Several studies have shown that glycemia reduction in diabetic animals treated with different herbal extracts directly reflects in the glycosuria values, also diminished after plant treatments (Pepato et al., 2002; Singh et al., 2007; Rajagopal & Sasikala, 2008). On the other hand, reduction in glycemia values after treatment with antidiabetic plants can be consequence of an increase in the glucose urinary excretion, since some plant preparations can promote reduction in the renal reabsorption of

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glucose. Studies have proposed the use of SGLT2 inhibitors as strong candidates for the treatment of diabetes (lsaji, 2007; Abdul-Ghani & DeFronzo, 2008; Jabbour & Goldstein, 2008). Thus, some herbal preparations have shown an antihyperglycemic effect through an inhibition in renal glucose reabsorption, since the urinary glucose excretion is markedly increased together with a decrease of blood glucose levels in diabetic and non-diabetic treated animals (Eddouks & Maghrani, 2004; Maghrani et al., 2005; Eddouks & Maghrani, 2008). Glycogen represents the primary storage form of glucose in the postprandial state, in skeletal muscle, adipose tissue and mainly in the liver, promoting maintenance of glucose homeostasis. On the other hand, in an initial fasting period, hepatic glycogen represents a major source of glucose for energy processes, avoiding hypoglycemia. It is known that gluconeogenesis process is increased and muscle and hepatic glycogen content is reduced in human diabetes (Shulman et al., 1990; Cline et al., 1994; Velho et al., 1996), leading to the development of hyperglycemia. Thus, an inhibition of endogenous glucose production, stimulation of hepatic glycogenesis and/or inhibition of glycogenolysis may be targeted by plant preparations to interfere in glycogen content and to promote an antihyperglycemic response. Consequently, quantification of muscle and mainly hepatic glycogen content is an adequate parameter analyzed in the trials of antidiabetic plants. Glycogen content can be quantified by simple methods. A first method estimates glycogen content in muscle or liver tissue samples through a colorimetric assay using a mixture of iodine-potassium iodidecalcium chloride, which binds to the glycogen molecule (Vysochina et al., 1968; Bobrova, 1986). However, the most usual method is based on initial tissue alkaline digestion and subsequent glycogen hydrolysis. The glucose liberated can be quantified by colorimetric assays for example, glucose oxidase method (Bergmeyer & Bernt, 1974) and anthrone reagent (Carroll et al., 1956) or by reaction with hexokinase and followed by spectrophotometric determination of the rate of nicotinamide adenine dinucleotide phosphate reduction. Several ethnopharmacological studies have been found enhance in the glycogen content in skeletal muscle and liver from diabetic animals treated with hypoglycemic plant extracts. So, the antihyperglycemic effect of some herbal preparations has been attributed, at least in part, to improvement in hepatic glycogen metabolism (see some examples in Table 1).

Physical parameters The preliminary investigation of herbal preparations with hypoglycemic effect is based on assays that evaluate the physical amelioration of diabetic animals after plant treatment. Increase in body weight and decreases in food and liquid intake and urinary volume are the simplest determinations related to diabetes improvement achieved with the administration of antihyperglycemic plant preparations. The use of these physical determinations can help in trials to select plants with hypoglycemic action on experimental diabetes and supply initial information about their

Table 1. Some beneficial effects observed in diabetes experimental models after hypoglycemic plants administration. Plant specie

Plant part(s) used to the preparation

Experimental model

References

Reduction of body weight Aegle marmelos Panax quinquefolius Parkia biglobosa Smallantus sonchifolius Vatairea macrocarpa

fruits berry juice seeds leaves stem-bark

Cissus sicyoides Heliotropium zeylanicum Vatairea macrocarpa

Decrease of food and liquid intake leaves STZ-diabetic rats STZ-diabetic rats whole plant stem-bark STZ-diabetic rats

Pepato et al., 2003 Murugesh et al., 2006 Oliveira et al., 2008

Cissus slcyoides Parkinsonia aculeata Siraitia grosvenori Vatairea macrocarpa

Reduction of urinary volume STZ-diabetic rats leaves alloxan-diabetic rats aerial part fruit preparation Goto-Kakizaki rats stem-bark STZ-diabetic rats

Pepato et al., 2003 Leite et al., 2007 Suzuki et al., 2007 Oliveira et al., 2008

STZ-diabetic rats ob/ob mice alloxan-diabetic rats STZ-diabetic rats STZ-diabetic rats

Kamalakkannan & Prince, 2005 Xie et al., 2007 Odetola et al., 2006 Aybar et al., 2001 Oliveira et al., 2008

Increase of liver and/or muscle glycogen content seeds Eugenia jambolana Tamarindus indica seeds Aegle marmelos, Murraya leaves koenigii, Ocimum sanctum Inhibition of intestinal glucose absorption Andrographis paniculata leaves and aerial parts Bougainvillea spectabilis, leaves Murraya koenigii, Ocimum tenuiflorum, Syzygium cumini

alloxan-diabetic rabbits STZ-diabetic rats STZ-diabetic rats

Sharma et al., 2003 Maiti et al., 2005 N arendhirakannan et al. , 2006

- inhibition of alpha-glucosidase activity Subramanian et al., 2008 in vitro yeast enzyme activity enzyme obtained from Swiss Bhat et al., 2008 mouse small intestine

Table 1. (Contd.) Plant specie

Plant part(s) used to the preparation

Experimental model

References

Marrubium radiatum, Salvia acetabulosa Pinus densiflora (pine)

whole plant

in vitro enzyme activity

Loizzo et al., 2008

pine bark and needles

Kim et al., 2005

Matricaria chamomilla L

flowers

Syzygium cumini (Eugenia jambolana)

seeds

enzyme obtained from porcine small intestine enzyme obtained from brush border membranes - rat small intestine in vivo - enzyme obtained from Goto-Kakizaki rats; in vitro - mammalian, yeast and bacterial enzyme activities

Ipomoea aquatica Myrcia uniflora Plantago ovata

Kato et al., 2008

Shinde et al., 2008

Inhibition of intestinal glucose absorption - in situ perfused small intestine preparation leafy stem STZ-diabetic rats Sokeng et al., 2007 leaves STZ-diabetic rats Pepato et al., 1993 husks STZ-diabetic rats Hannan et al., 2006

Cichorium intybus Momordwa charantia Piperbetle Smallanthus sonchifolius

Inhibition of hepatic whole plant seeds leaves leaves

Syzygium aromaticum

whole plant

glucose production STZ-diabetic rats STZ-diabetic rats STZ-diabetic rats in vitro assay - isolated rat hepatocytes in vitro assay hepatocytes and H4IIE hepatoma cells

Pushparaj et al., 2007 Sekar et al., 2005 Santhakumari et al., 2006 Valentova et al., 2004 Prasad et al., 2005

Table 1. (Contd.) Plant specie

Plant partes) used to the preparation

Trigonella foenum-graecum L

seeds

Experimental model alloxan-diabetic rats

References Mohammad et al_, 2006

Aegle marmelos, Murraya koenigii, Ocimum sanctum Catharanthus rose us Momordica charantia

Increase of hepatic glycogen synthase activity leaves STZ-diabetic rats

N arendhirakannan et aL , 2006

leaves, twigs and flowers seeds

Singh et al., 2001 Sekar et al., 2005

Brassica juncea, Murraya koenigii Momordica charantia

Inhibition of glycogen phosphorylase activity normal rats leaves seeds STZ-diabetic rats

Allium sativum, Azadirachta indica, Momordica charantia, Ocimum sanctum Annona squamosa L Artemisia absinthium, Camellia sinensis, Mentha piperita, Thymus vulgaris and other plant species Eucommia ulmoides Oliver Grifola frondosa Matricaria chamomilla L Phlomis anisodonta Quillaja saponaria, Yucca schidigera

STZ-diabetic rats STZ-diabetic rats

Khan et al., 1995 Sekar et al., 2005

Antidiabetic plus antioxidant properties leaves, fruits or fresh garlic STZ-diabetic rats bulbs

Chandra et al., 2008

leaves

Gupta et aL, 2008

leaves, flowers, roots or whole plant

leaves compound isolated from fruits aerial parts aerial parts plant power preparation

STZ-diabetic rats (type 2 diabetes) in vitro antioxidant assays (DPPH radical scavenging activity; scavenging of hydrogen peroxide) C57BUKsJ - db / db mice KK-Ay mice STZ-diabetic rats STZ-diabetic rats STZ-diabetic rats

Biiyiikbalci & EI, 2008

Park et al., 2006 Hong et al., 2007 Cemek et al., 2008 Sarkhail et aL, 2007 Fidan & Diindar, 2008

Table 1. (Contd.) Plant specie

Plant part(s) used to the preparation

Rosa rugosa Rosmarinus officinalis Scutellaria baicalensis Theobroma cacao

roots leaves roots cocoa powder

Experimental model STZ-diabetic rats alloxan-diabetic rabbits STZ-diabetic rats Obese-diabetic (Ob-db) rats

References Cho et al., 2004 Bakirel et al., 2008 Waisundara et al., 2008 J alil et al. , 2008

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antidiabetic efficiency. Trial antidiabetic studies have shown that, as a consequence of the improvement in carbohydrate metabolism in diabetic animals treated with several plant preparations, increase in body weight can be observed, mainly after chronic treatments. Another basic parameter investigated in the antidiabetic effect of herbal preparations is the decrease in food and/or liquid intake in treated diabetic animals. Some examples of pharmacological studies that measured these physical parameters in the treatment of diabetic animals with hypoglycemic plant species are described in Table 1. Reduction in the urinary volume have been achieved with antidiabetic plant treatment, showing an improvement of the diabetes state and protecting against the development of chronic renal complications related to this disease. The classical pathogenesis of diabetic nephropathy has two interacting components: the metabolic and the hemodynamic pathways, primarily based on long-term chronic hyperglycemia, microalbuminuria and an increased glomerular filtration (Leon & Raij, 2005; Schena & Gesualdo, 2005; Kanwar et al., 2008). Moreover, among the different reasons that lead to the development of diabetic nephropathy, the hyperfiltration seems to be an important factor in the genesis and progression of kidney disease (Mogensen, 1986; Dahlquist et al., 2001). Consequently, the urinary volume reduction in diabetic animals treated with herbal preparations seems to be a protective effect of this plant against diabetes renal complications. Table 1 presents some herbal preparations that promote decrease in urinary volume in experimental diabetic animals. First ethnopharmacological studies used normal, non diabetic animals to confirm the hypoglycemic effect of antidiabetic medicinal plants. However, the scientific literature showed progressive advances in the development of effective experimental diabetes models obtained from several genetic, pharmacological and nutritional manipulations. Now, these diabetic models have been currently used in scientific investigations to select antidiabetic plants and to characterize the hypoglycemic action of herbal remedies and/ or isolated plant compounds. The next topic systematizes various diabetic animal models frequently used by ethnopharmacological research.

Experimental diabetic models applied in studies to evaluate the effect of medicinal plants used in diabetes treatment Since medicinal plants have several mechanisms of action to promote antidiabetic and/or hypoglycemic effects, one single experimental diabetes model is not enough to evidence this diversity of plant actions. Moreover, the conclusions obtained from the achieved results will depend on the model used in the experiments. The knowledge of the experimental model features that will determine the diabetic state in a pharmacological investigation is very important to avoid misinterpretation and/or incorrect conclusions.

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The first experimental diabetes model was obtained by Mering after phlorizin administration in 1886. Later, Von Mering and Minkowski, in 1889, produced another permanent and severe diabetes model by pancreatectomy, initially in dogs and subsequently in other animal species. This discovery stimulated the investigation of the pancreatic function in diabetes generation and allowed the confirmation of insulin participation in pancreatectomy-induced diabetes. In 1932, Houssay and Biassotti showed the diabetogenic effect of adenohypophysis extracts administered in rats and dogs. In 1937, Young produced a new type of permanent metahypophyseal diabetes mellitus by repeated injections of anterior pituitary extracts. Following the Long and Lukens' demonstration in 1936, that adrenalectomy ameliorates diabetes in pancreatectomized animals, Ingle developed, in 1941, a type of temporary diabetes in rats through the administration of glucocorticoid hormones. Several other models of diabetes related to the administration of mammalian gland extracts (parathyroid, thyroid, adrenal medulla, etc) were suggested, although only a few have contributed to diabetes investigation. Further on, other different experimental diabetic models were developed, for example, genetically diabetic animals, animals obtained from nutritional manipulation and chemically induced diabetes models. Nowadays, many of these experimental models have been useful in the pharmacological investigation of plants with antidiabetic properties. In the experiments to test and select antidiabetic herbal preparations or to test the effect of plant drugs derivatives, the use of genetically modified diabetic models (knockout transgenic animals, animals with one single or double mutation, for example) has been limited. However, genetically modified animals have an important application in the advanced studies that evaluate the mechanism of action of antidiabetic plants. The excessively high cost for the development and maintenance of transgenic animals is another reason that justifies the absence in literature of studies applying genetically modified animals in trial experiments of antidiabetic plants.

Genetic diabetic models Animals presenting characteristics that resemble diabetes may be obtained from one or several genetic mutations transmitted through generations or by the selection of non-diabetic outbred animals by repeatedly breeding over several generations (Srinivasan & Ramarao, 2007). The homogeneous genetic background, unlike heterogeneity seen in human diabetes, reduces the variability in the results, so only a small sample size is required when genetically obtained diabetic animals are used. However, some problems can be observed in the use of these animals in diabetes studies, such as the high mortality of some models and the expensive cost oftheir maintenance. The most prominent genetically diabetic animal models resembling type 1 diabetes (TID) are the BB rats and NOD mice. Among genetically type 2 diabetes (T2D) animals that show diabetes and obesity,fa / fa and ZDF rats

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can be cited; ob/ob, db/db, KK and KK-Ay mice are the models frequently used in the investigation of compounds or drugs with antidiabetic effects. Nevertheless, some non-obese diabetic models are also used in the investigation of T2D, which allows the dissociation of confounding obesity factors, such as leptin deficiency, leptin resistance and other associated hypothalamic factors; the GK (Goto-Kakizaki) rats is an example of this model.

Type 1 Diabetes Genetic Model a) BB rat (Bio Breeding): In BB rats, autoimmune destruction of pancreatic beta cells and insulitis are present. Virtually they do not have beta cells, so these animals are not easy to handle since they do not produce insulin and manifest symptoms like hyperglycemia, glycosuria, weight loss and ketoacidosis (Whalen et al., 2001). BB rats spontaneously develop insulitis followed by impaired glucose tolerance (lGT) and/or an insulin-dependent diabetic syndrome like that in human (Poussier et al., 1982). Lines of BB rats designated as diabetes prone (DP-BB) and diabetes resistant or nondiabetes prone (DR-BB) have been used in autoimmune and/or diabetes studies. DP-BB rats spontaneously develop the disease within 55-120 days of age (Beaudette-Zlatanova et al., 2006). Intestinal dysfunction and deregulation ofthe gut immune system may playa role in the development of TID in BB rats (Malaisse et al., 2004). In addition to histological evidence of gut damage, altered intestinal disaccharidase activity, changes in intestinal peroxidase activity, glucagon-like peptide 1 anomalies, and perturbation of both intestinal permeability and mucin content in BB rats were also observed (Scott et al., 1997; Scott et al., 2000; Malaisse et al., 2004). b) NOD mouse (Non obese diabetic mouse): Diabetes in NOD mice developed abruptly after 100-200 days of age, with rapid weight loss, polyuria, polydipsia and severe glycosuria (Verspohl, 2002). TID incidence is significantly higher in NOD females and has a more widespread autoimmune disorder. Although TID occurs in humans with about equal frequency in males and females and, in addition, most human TID cases do not present widespread autoimmune disorders, the NOD mouse remains the most representative model of human TID, with similarities also in the target autoantigens, including glutamic acid decarboxylase IA-2, and insulin (Giarratana et al., 2007). In NOD mice, multiple islet auto antigens are recognized by T lymphocytes and autoantibodies before the development of immunemediated diabetes; there is evidence that autoimmunity to insulin may be central to disease pathogenesis since blocking immune responses to insulin prevents diabetes and insulin peptides can be utilized to induce diabetes (Zhang et al., 2008a). In addition to autoimmune destruction, the NOD mouse shows insulitis with pancreas leucocytic T cells (CD4 and CD6) infiltration (Verspohl, 2002). Hence, investigations using this model have provided not only essential information on TID pathogenesis, but also valuable insights

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into mechanisms of immunoregulation. The fact that TID incidence in the NOD mouse is sensitive to environmental conditions (Scott, 1990; Leiter, 2001), it is likely to render even more similar traits to human TID. Although it seems to be an interesting model for evaluation of antidiabetic plant effects resulting from pancreatic benefits, the NOD mouse has not been frequently used in these studies.

Type 2 Diabetes Genetic Model a) fa I fa rats (Zucker fatty obese rat) and ZDF rat (Zucker Diabetic Fatty rat): The Zucker fatty rat carries a spontaneous homozygous mutation (fa I fa) in leptin receptor gene that results in leptin signaling defects, impaired leptin appetite suppression and other actions of the hormone, promoting obesity and insulin resistance (Garnett et al., 2005). The ZDF rats were derived from the inbreeding of hyperglycemic Zucker fatty rats. At around 7 weeks of age they clearly displayed peripheral insulin resistance with glycemia slightly above normal that results in compensatory hyperinsulinemia. Subsequent decreased insulin levels and overt hyperglycemia can be observed when diabetes develops at around 10 weeks of age (Etgen & Oldham, 2000). In this period oflife, the ZDF are frequently used in experiments to investigate diabetes alterations. Female littermates are obese and insulin resistant but do not develop diabetes, thus they are frequently used in comparative testing assays (Srinivasan & Ramarao, 2007). Comparison between ZDF rats and fa I fa rats allows the study of progression of diabetes, separate from the effects of insulin and obesity (Garnett et al., 2005). In contrast to fa Ifa rats, the ability to oversecrete insulin to compensate peripheral insulin resistance is limited in ZDF rats. Studies suggest that the primary defect lies not in an ability of beta cells to proliferate but rather in an enhanced rate of secretory beta cell in response to glucose. Previous studies have demonstrated that high plasma levels of free fatty acid (FF A) and high triglyceride content in beta cells are responsible to the deleterious phenomenon on beta cell function (lipotoxicity) in ZDF rats (Shafrir & Ziv, 1998) and/or an enhanced rate of apoptosis (Pick et al., 1998), a phenomenon similar to human T2D. The evolution of diabetes in these animals replicates human diabetes, from insulin-resistant to a hyperglycemic insulin-deficient state. Decreased glucose transport activity associated with decreased GLUT-4levels is observed in adipose tissue and skeletal muscles ofZDF rats. The ZDF animals show hyperphagia, polyuria, polydipsia (Ktorza et al., 1997) and high levels of plasma triglyceride and cholesterol. This ZDF rat has been mostly used for the associated investigation with insulin resistance and beta cell dysfunction in T2D, as well as for testing insulin sensitizers, insulinotropic agents and others (Ramarao & Kaul, 1999; Nielsen, 2005; Zhang et al., 1996). In analogy to diabetic human patients, these animals also show accelerated gastric emptying (Green et al., 1997); so, substances that promote reduction in gastric emptying may improve glucose control in these animals. The ZDF rats are frequently used in studies related to diabetes general investigation;

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however, only few examples can be cited about the use of this diabetes experimental model in ethnopharmacological studies verifying the antidiabetic effect of plant preparations or derivatives (Huang et al., 2005; J anle et al., 2005; Banz et al., 2007). b) ob /ob mouse (Lepob): The ob /ob mouse (obese) is an autosomal recessive mutation in leptin gene in the C57BLl6J mouse strain. This model (homozygous mutant) shows early hyperphagia and energy expenditure reduction that result in excessive body weight gain and obesity about 4 weeks of age (Bell & Hye, 1983). Moreover hyperglycemia, impaired glucose tolerance and hyperinsulinemia by hypertrophy and hyperplasia of pancreatic islets (Velasquez et al., 1990; Srinivasan & Ramarao, 2007) are characteristic of these animals. Hyperinsulinemia develops after body weight increase and in adult ob /ob mouse is responsible for maintaining the plasma glucose levels almost normal. The insulin resistance can be result of the increased circulating cortisol as consequence ofleptin mutation. Antiobesity agents (herbal extract preparations or isolated plant compounds) improve peripheral insulin sensitivity and consequently show antidiabetic activity when assayed in this model (Attele et al., 2002; Xie et al., 2002; Xie et al., 2003; Xie et al., 2007). c) db / db (diabetic) mouse (Lepr ob): The db/ db (diabetic) mouse is derived from an autosomal recessive mutation to db gene, which encodes for the leptin receptor in mice of C57BLIKsJ strain. In db / db mice, the lack of leptin receptors results in hypothalamic disturbances and neuropeptide Y (NPY) abnormalities. This NPY abnormality induced in ob gene hyperexpression results in hyperleptinemia. They are hyperphagic, obese, hyperglycemic, hyperinsulinemic (due to insulin oversecretors) and insulin resistant within first month of age. Later on they develop hypoinsulinemia, hyperglycemia with a peak between 3-4 months of age together with progressive body weight loss. They not survive longer about 10 months and have been commonly used to investigate T2D, diabetic dyslipidemia and in screening assays of agents such as insulin mimetic and insulin sensitizers (Salituro et al., 2001; Wagman & Nuss, 2001; Lee et al., 2006; Jung et al., 2008; Tamrakar et al., 2008b). The db / db mouse is not, however, generally responsive to insulin secretagogues (Reed & Scribner, 1999). d) GK rats (Goto-Kakizaki rats): This model was obtained by selective inbreeding of Wi star rats with abnormal glucose tolerance. It is one of the best characterized animal models of spontaneous T2D without obesity (Miralles & Portha, 2001). Adult Goto-Kakisaki (GK) Wi star rats exhibit a spontaneous non-insulin-dependent diabetes characterized by impaired glucose tolerance (appears at 2 weeks of age) and insulin secretion, decreased beta cell mass, hepatic glucose overproduction, and moderate insulin resistance in muscles and adipose tissues (Picarel-Blanchot et al., 1996). Studies with antidiabetic compounds normally use 5-7 weeks old adult male

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rats, an age at which the beta cell mass and defective function in these cells decrease insulin store in 60% (Srinivasan & Ramarao, 2007). The lack of beta cell reactivity to glucose as seen during the adult period, when the GK rats are overtly diabetic, represents an acquired defect (perhaps due glucotoxicity). Several data suggest that the permanently reduced beta cell mass in the GK rat reflects a limitation of beta cell neogenesis during early fetal life (Portha et al., 2001). Antihyperglycemic, insulinotropic, glucagonostatic and insulin-like activities, associated with enhanced peripheral utilization, were responses observed after plant extract administration in GK rats (Jeppesen et al., 2002; Kar et al., 2006). e) KK mouse (Kuo Kondo mouse) and KK / Ay mouse (yellow mouse variant):

KK mouse is produced by selective inbreeding of animals with increased body size in Japan, also named as Japanese KK mouse (Velasquez et al., 1990; Srinivasan & Ramarao, 2007). These mice have polygenic defects that produce hyperphagia, hyperinsulinemia with increase in number and size of pancreatic islets, with histological degranulation of beta cells and hypertrophy of islets. This animal may become obese and hyperglycemic with age, dietary treatment or expression of the Ay gene (yellow obese gene), which attains maximum at 4-5 months (Reed & Scribner, 1999). Insulin resistance precedes the onset of obesity and there is selective failure of insulin to suppress gluconeogenic pathway in KK mouse. KKlAy mouse are preferentially used in the diabetes investigation since it carries both the diabetic gene present in KK mouse and the yellow obese gene (Ay). The animal shows severe obesity, hyperglycemia, hyperinsulinemia and glucose intolerance after 8 weeks of age (Srinivasan & Ramarao, 2007). The diversity of symptoms expressed in KK mice and their variants have allowed the use of this model to test many types of therapeutic compounds, so it is a genetic model frequently applied in pharmacological researches with antidiabetic plants, where several actions can be observed: glucanostatic effects (Yao et al., 2008a; Yao et al., 2008b), insulin like activity (Enoki et al., 2007; Zhang et al., 2007), increase in insulin sensitivity and amelioration in insulin resistance of peripheral target tissues (Miura et al., 1997; Miura et al., 2001a; Miura et al., 2001b; Manohar et al., 2002; Yao et al., 2008a; Yao et al., 2008b; Yajima et al., 2004), inhibition of alphaglucosidase activity, leading to a decrease in the intestinal glucose absorption (Kurihara et al., 2003; Takeuchi et al., 2001), stimulation of insulin biosynthesis (Waki et al., 1982) and reduction of gluconeogenesis (Ribnicky et al., 2006). TSOD mouse (Tsumura Suzuki Obese Diabetes mouse): TSOD mouse is a new model of T2D and obesity that results from the selective breeding of obese ddY mice that present specific characteristics, such as increased body weight and appearance of urinary glucose. In male TSOD mice, the body mass index (EM!) clearly showed moderate obesity and urinary glucose together with increase in food and water intake, increased body weight and f)

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some fat accumulation. Increases in blood glucose, insulin and lipids levels are also found in this experimental model. In histological studies, hypertrophic pancreatic islets from TSOD male animals were found without any signs of insulitis (Suzuki et al., 1999). TSOD mice developed glucose intolerance, hyperlipidemia, hypertension and hyperinsulinemia. The reduced insulin sensitivity in diabetic TSOD mice is presumably due, at least in part, to the impaired GLUT4 translocation by insulin in both skeletal muscle and adipocytes (Shimada et al., 2008). Miura et al. (2001c) demonstrated that TSOD mice treated with Bofutsushosan presented reduction in body weight gain and in visceral/subcutaneous fat accumulation, in addition to the decrease in plasma glucose and insulin levels. Abnormal glucose tolerance, elevation of blood pressure and peripheral neuropathy were significantly suppressed, accompanying the improvement of metabolic alterations.

Pharmacological models Alloxan (ALX) and streptozotocin (STZ) induced diabetic animals are most widely diabetes experimental models used for screening of compounds including natural products with different antidiabetic activities. Most studies in the literature that used these pharmacological models primarily referred to diabetic rats, followed by mice and rabbits. Animals of other species have been little used in research with antidiabetic plants.

Streptozotocin Streptozotocin (STZ), or 2-deoxy-2[([methyl-nitrosoaminol-carbonyD-aminolD-glucopyranose, is a toxic glucose analogue isolated from Streptomyces achromogenes (Fig 1). It has a broad spectrum of antimicrobial activity and antineoplastic properties and is often used to induce diabetes mellitus in experimental animals through its toxic effects on pancreatic beta cells. STZ induces diabetes in rats, dogs, hamsters, monkeys, mice and guinea pigs, but rabbits have reduced sensitivity to STZ (Rerup, 1970). In 1963, Rakieten et al. published the first observation made in dogs and rats about STZ diabetogenic properties. The deoxy-glucose group ofthe STZ molecule allows it to pass over the cell membrane through the GLUT2 glucose transporter. The importance ofGLUT2 in this process is also shown by STZ damages in other organs that express this transporter, particularly

Jl

If Nr

O~o o

Alloxan

OR

HO .....

CR3

O~""~

C~

00

R, OR Streptozotocin

Fig 1. Chemical structures of alloxan and streptozotocin

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kidney and liver. This antibiotic was found to be mutagenic in bacterial assays and eukaryotic cells; so STZ is also carcinogenic; a single administration induces tumors in rat kidney, liver, and pancreas (Bolzan & Bianchi, 2005). The high reactivity ofnitrosoureas in the STZ side chain is responsible for starting its cytotoxic action inside the cell, alkylating DNA bases. The transfer of the methyl group from STZ to DNA results in fragmentation of the DNA and poly (ADP-ribose) polymerase overstimulation, which in turn diminishes nicotinamide adenine dinucleotide (NAD+) levels and subsequently the ATP production, leading to beta cell necrosis (Lenzen, 2008b) (Fig 2). Reactive oxygen species generated during hypoxanthine metabolism may accelerate beta cell destruction, but do not playa crucial role (Lenzen, 2008b; Srinivasan & Ramarao, 2007). Alloxan ~_?'"-.:::--_ Dialuric acid 0,-

0,

+

20,- + 2H- -----. H,O, + 0,

Fe" ----. Fe"

- - - -••

OH-

Fig 2. Schematic representation ofthe ROS generation by alloxan in pancreatic beta cells to produce diabetes

STZ diabetes models have been particularly important for the characterization of antidiabetic compounds as insulin sensitizers (Pushparaj et al., 2007), insulin secretagogues (Dimo et al., 2007) and inhibitors of glucose absorption (Hamdan & Afifi, 2004), and to develop drugs to prevent diabetic complications, as aldose reductase inhibitors (Ueda et al., 2004), and inhibitors of renal advanced glycosilation (Yokozawa et al., 2008). The blood glucose level fluctuates after a diabetogenic dose of STZ with initial hyperglycemia, followed by severe hypoglycemia and finally permanent hyperglycemia. The hypoglycemia that follows the initial hyperglycemia may be associated with convulsions and death ifthe animals remain in a fasting state. Therefore, to prevent animal death, food may be offered about 2 h after STZ administration (Koren & Fantus, 2007). Among the procedures to prevent hypoglycemia, solution of glucose has been offered to the animals up to 24 h after STZ, avoiding convulsion and death (Chandramohan et al., 2008; Subash-Babu et al., 2008). There is a wide variety ofSTZ-diabetogenic doses and the susceptibility depends on animal age, species, strain and other factors. In dogs, diabetes may be produced by repeated daily injections of a lower dose of STZ, about 15 mg/kg per day during 3 days and the LD50 was estimated between 25-50 mg/kg (Rakieten et al., 1963). In adult rats, the i.v. injection of 50 mg/kg of STZ promotes

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mild diabetes and the dose of 70 mg/kg promotes severe diabetes (Sharma et al., 2008). The lower STZ dose with diabetogenic finality in adult rats found in the literature was 40mg/kg administered intraperitoneally (Heo et al., 2007; Geethan & Prince, 2008). The frequent STZ dose used to produce experimental diabetes in most studies is between 45-65 mg/kg, with i.p. or i.v. injection (Gondwe et al., 2008; Lee & Ku, 2008; Sangameswaran & Jayakar, 2008; Sharma et al., 2008; Shin et al., 2008), although doses of70 mg/kg are also found (Cemek et al., 2008). The resulting TID or T2D animals will depend on the dose and the conditions of the protocol for STZ application. The TID may be obtained when a single high dose or multiple low doses of STZ is applied in adult animals (Verspohl, 2002; Srinivasan & Ramarao, 2007). One other model that resembles TID is obtained through administration of STZ plus Complete Freund's Adjuvant (Verspohl, 2002; Snigur et aI., 2008). The injection oflyophilized isolated islets suspended in Freund's adjuvant in mice has also been found to produce lymphocytic infiltration and beta cell degeneration (insulitis) which later on resembles TID (Rossini et al., 1977). To guarantee an effective diabetogenic effect, the STZ has usually been administered after a fasting period of 12-24 h (Andallu & Varadacharyulu, 2007; Sharma et al., 2008). It was estimated that the STZ half life in mice is about 5 min and in rats 15 min, so i.v. administration is recommended, although the i.p. injection is the most common method used for STZ administration. The experimental use of diabetic animals following STZ administration occurs after 2 to 7 days, with a previous selection of these animals according to their blood glucose levels. In general, fed animals with plasma glucose values above 250 mg/dL are selected. However, literature offers studies where animals with higher glycemia values were selected, as well as some other studies which selected diabetic animals with fasting glycemia values higher than 90 mg/dL. It is clear that the limits of this selection depend on the objectives of the research. In several works, "standard drugs" as glibenclamide, metformin or insulin were used to compare the effects of the tested material. The criterion for this choice depends on the diabetic model used in the assessment. According to Rerup (1970), the STZ dose between 175 to 200 mg/ kg is required to induce in mice the same diabetogenic effect observed in rats when injected with 50 mg/kg, i.v., a dose used as a reference for several studies (Oliveira et al., 2005; Vijayakumar et al., 2005); however, other doses, as example 150 mg/kg of STZ, have been applied by other authors, as related by Ma et al. (2007). Lower multiple doses (40 mg/kg) during five consecutive days (Beppu et al. 2006) or 50 mg/kg during 2 consecutive days (Zheng et al., 2007) are also used to induce diabetes in mice. Rossini et al. (1977) observed that multiple STZ injections in mice produced mild hyperglycemia during the initial 5-6 days of the experiment, with a complete diabetic syndrome observed by the 8 th_11th

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day with intense initial insulitis and residual inflammation after a 3-4 weeks period, similar to those observed after a single diabetogenic dose of STZ or ALX. To induce the T2D model, several protocols based on STZ administration have been described in literature, among them: •

•

Single dose ofSTZ (80-100 mg/kg, i.v., i.p. or s.c.), in 1, 2 or 5 days old neonatal rats, is considered the best model to observe beta cell regeneration and defects in insulin action (Bonner-Weir et al., 1981; Fernandez-Alvarez et al., 2004); Nicotinamide injection (230 mg/kg) 15 min before STZ (65 mg/kg) administration in adult rats, developing moderate and stable nonfasting hyperglycemia without any significant change in plasma insulin levels, thus suitable for acute and chronic pharmacological investigations of insulinotropic agents (Masiello et al., 1998). Shirwaikar et al. (2006) used 120 mg/kg of nicotinamide and 65 mg/kg of STZ for this same protocol;

•

STZ injection into genetically modified animals, such as ZDF and SHR rats. These animals are genetically insulin-resistant and after STZ administration they will present beta cell destruction as an additional impairment (Reaven & Ho, 1991);

•

STZ treatment in rats and mice submitted to high fat or high fructose diets (Tobey et al., 1982; Hwang et al., 1987; Reaven & Ho, 1991). In these cases, hyperinsulinemia and insulin resistance promoted by hypercaloric diets is followed by beta cell damage as a result of STZ administration. These animals present a higher response to the action of insulin sensitizing and insulinotropic agents and exhibit stability in parameters related to diabetes, such as hyperglycemia, polyuria, polydipsia and polyphagia, and are useful in antidiabetic drugs screening studies CReed et al., 2000; Zhang et al., 2003).

Alloxan Alloxan (ALX), also known as mesoxalylurea, mesoxycarbamide, 2,4,5,6-tetraoxypyrimidine (Fig 1), is a uric acid derivative highly unstable in water at neutral pH, but reasonably stable at pH 3. The mechanisms associated with its diabetogenic action can be synthesized in two points: 1) ALX is reduced to dialuric acid that is then oxidized back to ALX resulting in the production of free radicals (Fig 3); 2) ALX reaction with protein thiol groups. During each cycle with thiol groups, an ALX-glutathione adduct is formed with gradual reduction of the reduced glutathione available in the cell and consequent decrease of the organism's natural defense against oxidation. The hypoglycemic action of ALX was first observed in 1937 by Jacob in

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STREPTOZOTOCIN (NitrosourE>as sidE> chain)

/

DNA alkylation

Hypoxanthine Mt>tabolism

I ROS g(>neration

I

Beta cell Destruction

~

NAD'

~ ATP

/

Fig 3. Schematic representation of the streptozotocin effects in pancreatic beta cells to produce diabetes

rabbits. Later on, Dunn et al. (1944) also observed some consequences of the ALX administration in rabbits, such as death caused by hypoglycemia and necrosis of renal tubules and pancreatic beta cells. Since that time, several studies related to ALX experimental diabetes have been published with information about this diabetogenic dose in some animals and its mechanism of beta cell damage. Many rodent and non-rodent animals can be made diabetic by ALX, although guinea pigs and musk shrews are resistant to the ALX action (Rerup, 1970; Ohno et al., 1998). Diabetes has been produced after ALX administration through many routes, although it is not effective when orally administered. Since ALX has a short half-life and acidic characteristics in solution, the i.v. route is preferred. According to Rerup (1970), the necessary ALX dose for the production of diabetes varies in different species between 40 and 200 mgt kg. Many factors may influence the relationship dose/effect, the same as observed with STZ; moreover, very young animals have high resistance to the diabetogenic effect of ALX. The most common ALX doses administered in adult rats to obtain the diabetogenic effect varied from 100 mg/kg up to 200 mg/kg i.p. (Fernandes et al., 2007; Patel et al., 2007), the 150 mg/kg dose being the most frequently used (Raut & Gaikwad, 2006; Leite et al. 2007; Cunha et al., 2008). Although the use of ALX diabetic mice is not frequent, the administration of ALX in doses of 65-160 mg/kg i.p. and 60 mg/kg i. v. to promote the diabetic state in mice is described in a few studies (Shan et al., 2006; Zhao et al., 2007). In some studies using rabbits, the animals become diabetic with 80-150 mg/kg of ALX administered into the

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marginal ear vein (Nammi et al., 2003; Sharma et al., 2003; Al-Azzawie & Alhamdani, 2006). The blood glucose level fluctuates after the administration of a diabetogenic dose of ALX, as observed with STZ treatment, with hyperglycemia, followed by hypoglycemia and permanent hyperglycemia. ALX acts selectively, destroying the pancreatic beta cells and leading to insulin deficiency, hyperglycemia and ketosis which result in high mortality, specifically in rats. Korec (1967) observed that after an intravenous ALX administration to fasting rats in a dose of 40 mg/kg, the mortality is about 20-50%; when the same dose is administered to fed rats, a mild diabetes or an absence of effect can be observed and the mortality falls to 20%. In some experimental protocols, however, diabetes by ALX is disadvantageous because the percentage of diabetes incidence is not proportionately related to increased doses, so severe diabetes without mortality is less predictable than if STZ is used. T2D with ALX was developed by Kodama et al. (1993), injecting 200mg/kg i.p. to 2, 4 or 6 days old male neonatal rats, becoming an adequate and useful model for studies about long term T2D complications.

Diet-induced diabetes models High fructose ingestion Fructose consumption induces insulin resistance, impaired glucose tolerance, hyperinsulinemia, hypertriacylglycerolemia, and hypertension in animals (Elliott et al., 2002; Hwang et al., 1987). Studies have shown that high fructose diets cause impairment of insulin action, particularly in the liver and muscles of rodents (Tobeyet al., 1982; Yadavet al., 2004). Nakagawa et al. (2006) proposed that fructose induces insulin resistance, at least in part, through induction of hyperuricemia, which in turn inhibits nitric oxide bioavailability. Nitric oxide is required for insulin to stimulate glucose uptake by skeletal muscle (Recchia, 2002). Tobey et al. (1982) suggest that the insulin resistance resulting from chronic fructose feeding is characterized by diminished ability of insulin to suppress hepatic glucose output, and not by a decreased insulin-stimulated glucose uptake in muscle. So, fructosefed rats provide a model of dietary-induced insulin resistance that has been used to investigate interactions between metabolic disorders. This model has been also used to assess the therapeutic efficacy of insulin sensitizing agents mainly in rats, but also in other animals (Leung et al., 2004; Yadav et al., 2004). The animals present in this model usually received fructose in the diet (20-70%) or fructose solution in drinking water during 3 to 10 weeks (Robbez Masson et al., 2008; Bell et al., 1998; Zamami et al., 2007; Zamami et al., 2008; Tobey et al., 1982).

High-fat diet (HFD) The main strategy to obtain diabeti~ animals through HFD administration is to associate genetically diabetic animals or chemically ind~ed diabetic

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animals and a high-fat diet. Zhanget al. (2008b) developed the best model of T2D, with the administration of multiple doses of 30 mg/kg STZ i.p., two injections a week in HFD-fed rats for 2 weeks, producing frank hyperglycemia. Insulin tolerance test (ITT) demonstrated insulin resistance in these animals. The HFD diet is composed of 22% fat and 44.3 KJ/kg total calories. Studies confirm that C57BU6J mice, through a genetic predisposition, developed symptoms assembling noninsulin-dependent diabetes mellitus on a regular schedule when fed solely on a diet with high fat, simple carbohydrates and low fiber contents (Huo et al., 2003). Countless other possibilities of T2D models that result from the combination of the HFD with other experimental diabetes models can be found in scientific literature.

Assays to elucidate the mechanism of action of hypoglycemic plants Since ethnopharmacological studies have demonstrated the hypoglycemic effect of several plant species in diabetic patients and/or animal experimental models through the application of methods that promote an accurate and trustworthy plant trial, further exploration is required to elucidate the pharmacological mechanism of action of these herbal remedies in order to explain their beneficial effect in diabetes symptoms. The purpose of the complete plant mechanism comprehension is to guide the isolation of new phytochemical compounds and the development of new synthetic drugs. Thus, there are a large number of methodologies applied by pharmacological researches to clarify the action of plants in specific targets to exert their antidiabetic effect. Some methods will be described as follows.

Investigation ofthe glucose intestinal absorption Antidiabetic plant studies have used several methodologies to elucidate the mechanism of action that is involved, isolated or combined, in the promotion of the plant hypoglycemic effect, such as stimulation of pancreatic cells insulin release, reduction of liver glucose production, enhancement of glucose uptake by peripheral tissues and/or inhibition of intestinal glucose absorption. There are two groups of enzymes directly involved in the intestinal carbohydrate digestion, the pancreatic alpha-amylase and the intestinal alpha-glucosidases. Dietary polysaccharides are initially degraded in the gut by the alpha-amylase activity, releasing oligosaccharides and disaccharides that are then converted into monosaccharides by intestinal alpha-glucosidases. Glucose is then absorbed and results in postprandial hyperglycemia. It has been demonstrated that the pharmacological inhibition of intestinal enzymes involved in the carbohydrate digestion could reduce glucose absorption and control postprandial hyperglycemia; consequently this strategy may be useful in the therapy for type 2 diabetes mellitus

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(Bischoff, 1995; Scheen, 2003). In this way, several studies have demonstrated the reduction in the intestinal absorption of glucose promoted by herbal preparations, showing beneficial effects on glycemia values in diabetic animals. The reduction of intestinal glucose absorption promoted by hypoglycemic plants can be assessed through methods based on in vivo and in vitro approaches. The in vivo methodology to determine intestinal absorption of glucose is based on the in situ small intestine perfusion technique in diabetic animals. The in vitro methods determine the activity of alpha-glucosidase and alpha-amylase enzymes. The intestinal alphaglucosidase enzymes can be assayed in brush border membrane preparations from the small intestine or in supernatant obtained from homogenization of animal gut. In both, the alpha-glucosidase activity can be measured using adequate carbohydrates as substrates. The glucose released can be quantified colorimetric ally. The alpha-amylase activity determination obtained from animal pancreas can be assayed using starch as substrate and measuring the remaining polysaccharide by colorimetric method. Bhat et al. (2008) demonstrated that leaf extracts from recognized hypoglycemic plants (Syzygium cumini, Ocimum tenuiflorum, Murraya koenigii, Bougainvillea spectabilis) inhibit the in vitro activity of alphaglucosidase isolated from murine small intestine and consequently are able to reduce intestinal glucose uptake. The hypoglycemic effect of Syzygium cumini seed extracts were also confirmed by Shinde et al. (2008) who used two different techniques: an in vitro method that demonstrated inhibition of alpha-glucosidase activity isolated from rat intestine by plant extracts, and an in vivo study using Goto-Kakizaki diabetic rats orally treated with plant acetone extract, in which maltose hydrolysis by alpha-glucosidase was significantly reduced when compared to untreated rats, explaining the antidiabetic action of Syzygium cumini. Many other researches used the in situ perfused small intestine preparation to demonstrate reduction in the glucose absorption in diabetic rats treated with different herbal preparations. In these studies, a significant improvement in glucose tolerance was observed in STZ-diabetic rats treated with different plant extracts, such as Plantago ovata husks (Hannan et al., 2006a) and Ipomoea aquatica leafY stems (Sokeng et al., 2007). The antihyperglycemic effect of these plants was, at least to some extent, consequence of the inhibition in the intestinal glucose digestion. Even though not yet explored, another possible mechanism of action presented by hypoglycemic plants that act at intestinal level is to promote inhibition of glucose absorption at two distinct targets: 1) classical carbohydrate absorption mediated by the Na+/glucose cotransporter, and 2) facilitative transport through glucose transporter type 2 (GLUT2) present in the apical membrane (see Review in Kellett et al., 2008). Recent work has shown that insulin reduces GLUT2 quantity in both apical and basolateral enterocyte membranes, promoting rapid traffic of this glucose transporter away from the plasma membrane and preventing GLUT2 insertion into the

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apical membrane, independently of the glucose amount in the luminal intestine (Tobin et al., 2008). Furthermore, apical membrane GLUT2 is dramatically increased in experimental diabetes characterized by hyperglycemia and insulinopenia (Burant et al., 1994). In this way, changes in GLUT2 quantity in apical enterocyte membrane from diabetic rats treated with hypoglycemic plant extracts cannot be ruled out.

Investigation ofgluconeogenesis and glycogen metabolism It has been proposed that the development of new drugs that inhibit the hepatic glucose production will be important to highlight new targets for diabetic treatment (McCormack et al., 2001; Link, 2003; Wu et al., 2005). Reduction of gluconeogenesis can be achieved through modulation of activity and/or gene expression ofthe main enzymes present in the regulatory steps of this pathway: pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase and glucose-6-phosphatase. Several studies have been demonstrated medicinal plant preparations that reduce hyperglycemia through inhibition of hepatic glucose production. Trigonella foenum-graecum L., Momordica charantia and Piper betle are some examples of well known hypoglycemic plants that act, at least in part, through inhibition of PEPCK, fructose-1,6-bisphosphatase and glucose-6phosphatase activities, respectively (Mohammad et al., 2006; Sekar et al., 2005; Santhakumari et al., 2006).

Since hypoglycemic plant studies have provided evidences of hepatic glycogen content improvement in trial experiments to identify antidiabetic plants, it seems interesting to elucidate the mechanism of action that promoted this effect. Glycogen synthesis and degradation are multi-step processes controlled by the activities of two key enzymes: glycogen synthase and glycogen phosphorylase, respectively. It is well known that the increased blood glucose levels observed in diabetes patients is consequence, at least in part, of the decrease in hepatic glycogen synthesis and/or raise in glycogenolysis. In attempting to clarify the mechanism by which some medicinal plants promote stimulation of glycogen synthesis, studies have investigated the increase in the activity of glycogen synthase in treated diabetic animals. N arendhirakannan et al. (2006) demonstrated that diabetic rats treated with Murraya koenigii, Aegle marmelos and Ocimum sanctum extracts presented an increased activity of the hepatic glycogen synthase, justifying the beneficial effect of these plants on glycogen metabolism, increasing hepatic glycogen content in these same animals (see Table D. An increase in the hepatic glycogen synthase activity was also reported by Sekar et al. (2005) in diabetic rats treated with aqueous extract of Momordica charantia seeds, explaining the mechanism by which this plant has a hypoglycemic action. Moreover, herbal medicines that improve the glycogen metabolism in diabetes can act through inhibition of glycogenolysis, and reduction of glycogen phosphorylase activity in plant-treated diabetic animals was also

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reported. Apart from increasing the activity of hepatic glycogen synthase, Momordica charantia seeds extract also reduced the activity of glycogen phosphorylase in STZ-diabetic rats (Sekar et al., 2005). A decrease in the glycogen phosphorylase activity was also observed in diabetic rats treated with Brassicajuncea and Murraya koenigii extracts (Khan et al., 1995).

Investigation of insulin secretion and glucose uptake in cell cultures Many laboratories around the world, where ethnopharmacological research is carried out, have been used specific and refined methods applied in cell cultures to confirm the in vivo antihyperglycemic effect of herbal medicines. Pharmaceutical industry researches present a clear trend to the application of these methodologies in the discovery and development of new phytochemical derivatives, providing both initial characterization of plant specific action and elucidation of cellular and/or molecular targets in advanced stages of drug development. Apart from providing clarification on plant mechanism of action on specific tissues in the promotion of antidiabetic effects, there are other benefits in using cell culture assays, such as the analysis of plant effects without the use of whole animals, investigation with no living animal tissue under conditions of ethical and financial limitations and in situations of much smaller amount of plant material necessary for the studies. Two main actions frequently studied in the clarification of plant antidiabetic effects can be cited when cell cultures are used: its effect on 1) insulin-secreting cells and 2) cellular glucose uptake. 1) The first explanation about the antidiabetic plant mechanism of action can be attributed to an insulinotropic pancreatic effect. Stimulation of insulin release can be investigated using either the in vivo perfused pancreas method or in vitro isolated pancreatic islets. However, recent works have applied some insulin-secreting cell lines, such as BRIN-BDll and RINm5F, to evaluate the action mechanisms of herbal preparations on insulin secretion.

The decrease in plasma glucose levels after treatment of diabetic rats and humans with Ocimum sanctum leaf preparations (Chattopadhyay 1993; Rai et al., 1997) can be attributed to stimulation of insulin secretion since ethanol extract and aqueous, butanol and ethylacetilate fractions of this plant promoted increase in the insulin secretion from perfused rat pancreas, isolated rat islets and rat BRIN-BDll pancreatic cell line (Hannan et al., 2006b). The BRIN-BDll cell line was also used by Gray and Flatt (1999) to confirm the antidiabetic effect of the Viscum album leaf and stem extract through insulin secretion stimulation, since consumption of diet containing Viscum album preparation had shown amelioration in some parameters of severely STZ-diabetic mice, as polydipsia, hyperphagia and body weight loss (Swanston-Flatt et al., 1989). Rat insulinoma cells (RINm5F) belong to a pancreatic cell line and are also used in the investigation of plant antidiabetic effects. However, RINm5F cells are often used in studies related to the protective action of plant extracts against pancreas oxidative

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damage observed in diabetes, since type 1 diabetes mellitus is characterized by an autoimmune disease resulting from the destruction of insulinproducing beta cells promoted by infiltrated immune cells in pancreatic islets (Nerup et al., 1988; Yoon & Jun, 2001). The islets insulitis is mainly caused by macrophages and dendritic cells, the first cell types to infiltrate the pancreatic islets, releasing interleukin-12 that activates and recruits Tlymphocytes involved in the destruction of beta cells. These cells release high levels of proinflammatory cytokines (interleukin-1, interferon-gamma, tumor necrosis factor-alpha), which stimulate the production of excess nitric oxide through nitric oxide synthase activation in pancreatic islets, which leads to apoptosis in rat and human beta cells (Cetkovic-Cvrlje & Eizirik, 1994; Rabinovitch, 1998). Recent studies have shown that the Scoparia dulcis plant extract, apart from evoking a stimulation of insulin secretion from isolated islets (Latha et al., 2004a), it also protected RINm5F cells against STZ-mediated cytotoxicity and nitric oxide production and completely abrogated apoptosis induced by STZ, suggesting the protective effect ofthis plant in the oxidative stress in diabetes (Latha et al., 2004b). Similar results were achieved with Artemisia capillaris, which presented an antihyperglycemic effect (Pan et al., 1998) and the mechanism of action can be attributed, at least in part, to its effect on the pancreas: RINm5F cells treated with Artemisia capillaris extract presented reduction in interleukin1 and interferon-gamma-mediated cytotoxicity and in cytokines-induced NO production, restoring insulin release from isolated islets (Kim et al., 2007). 2) The second important approach recently explored in antidiabetic plant studies is related to the insulin-like properties of herbal preparations. Several studies have evidenced an enhancement of glucose uptake in cell culture based assays. The maintenance of blood glucose homeostasis is consequence of the precise regulation of glucose uptake and its utilization and storage by specific tissues. Like insulin, some plant extracts can promote increase of glucose uptake on peripheral tissues, mainly adipose and skeletal muscle tissues, which express the glucose transporter type 4 (GLUT4). The mechanism of increased glucose uptake is associated with GLUT4 transport away from intracellular compartments with the subsequent fusion of the GLUT4-containing vesicles with the plasma membrane, driven by stimuli that promote exocytosis of GLUT4 and restrain transporter endocytosis, increasing the number of transporters into the plasma membrane (Thong et al., 2005). The increased glucose transporter intrinsic activity also seems to be important for glucose uptake enhancement (Furtado et al., 2002). Several cell lines representing adipose and skeletal muscle peripheral tissues have been used in the in vitro investigation of glucose uptake stimulation by antidiabetic plant preparations. Thus, plant extracts and/or derivatives that stimulate glucose uptake in peripheral tissues might be useful sources of new hypoglycemic agents for the development of pharmaceutical drugs or as complementary therapy of diabetes mellitus. Glucose transport assay is normally determined through the measurement of the 2-deoxy-D-[3Hl

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glucose uptake. The different culture cells are previously incubated in an adequate solution containing plant extract in the absence or presence of insulin. After that, cells are incubated with 2-deoxy-D-[3H] glucose to determine glucose uptake. The assay is interrupted and the disruption of the cells permits the determination of radioactivity.

Guazuma ulmifolia is a plant used in traditional medicine for the treatment of diabetes mellitus. Alarcon-Aguilara et al. (1998) showed that hyperglycemic rabbits treated with Guazuma ulmifolia aqueous extract presented decrease in the hyperglycemic peak and in the area under the glucose tolerance curve. The antidiabetic property of this plant is due to stimulation of glucose uptake in adipocytes, assayed in the murine 3T3F442A adipocyte cell line (Alonso-Castro et al., 2008). Other antidiabetic plants have their effects attributed to the glucose uptake increase in adipocytes; for example, Cichorium intybus methanolic extract (Muthusamy et al., 2008) and Cinnamomum zeylanicum aqueous extract (Roffey et al., 2006) stimulated glucose uptake in 3T3-L1 adipocytes. Roffey et al. (2007) demonstrated that 3T3-L1 adipocytes treated with a combination of Momordica charantia extract (fruit and seeds water extract) plus insulin presented further glucose uptake increase in comparison to insulin response; the plant extract had no effect in the absence of insulin. Using the same 3T3-L1 adipocytes, another study observed that methanol fractions of Cortidis Rhizoma extract promoted an insulin sensitizing action, stimulating glucose uptake (Ko et al., 2005). These results have turned the use of these plants, and others with similar effects, promising as complementary therapies for type 2 diabetes mellitus. Many ethnopharmacological studies have been investigated the herbal preparation changes in glucose uptake on skeletal muscle derivative cell lines. Since skeletal muscle accounts for the major portion of glucose disposal after infusion or ingestion of glucose, it is an important tissue for the glucose homeostasis maintenance (Koistinen & Zierath, 2002; Zierath & Kawano, 2003). The cell lines frequently used in these studies are C2C12 myoblasts and L6 myotubes. Martineau et al. (2006) showed that root, stem, and leaf extracts from Vaccinium angustifolium, an antidiabetic plant highly recommended by Canadian traditional medicine (Haddad et al., 2003), significantly enhanced glucose transport in C2C12 cells in the presence or absence of insulin. Pharmacological studies that used L6 cell line to demonstrate increase in the glucose uptake promoted by plant preparations can be cited, for example incubation with the n-hexane fraction of ethanolic extract from Ceriops tagal (Tamrakar et al., 2008a) and with Aegles marmelos and Syzygium cumini extracts (Anandharajan et al., 2006). Another plant species that seems to promote enhancement of glucose uptake in skeletal muscle is Momordica charantia; several studies have shown that its fruit has an effective hypoglycemic effect and therefore is widely used in the treatment of diabetes mellitus (Welihinda et al., 1986; Day et al., 1990; Ahmed et al., 1998). Cummings et al. (2004) showed that the

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incubation of L6 muscle cells with either the lyophilized extract or the chloroform extract of Mormordica charantia fruits stimulated glucose uptake into this cultured muscle cell, in a similar manner to the effect observed with insulin, a result that supports the beneficial use of this plant as a hypoglycemic agent. Together with the determination of glucose uptake by cell cultures, many researchers have studied the effect of herbal preparations on both GLUT4 translocation to the cell surface and the GLUT4 protein expression, analyzed through Western blotting method. As example, studies from Pinent et al. (2004) and Purintrapiban et al. (2006) used Western blotting analysis to verify alterations in GLUT4 protein content in adipocytes and skeletal muscle culture cells, respectively, after incubation with antidiabetic plant extracts.

Investigation of insulin intracellular signaling Insulin represents the main hormone involved in glucose homeostasis and its release occurs after increases in blood glucose levels. The insulin effects occur through intracellular signal transduction after its binding to the insulin receptor (IR). The tyrosine kinase activity of the IR stimulates many intracellular intermediates, including insulin receptor substrates (IRSs), phosphatidylinositol 3-kinase (PI3K) and the downstream effector AKT or PKB. The AKT, a serineltreonine kinase activated by phosphorylation, accounts for several intracellular metabolic actions of insulin, including the GLUT4 translocation to the plasma membrane surface, increasing cellular glucose uptake. At the molecular level, defects on insulin postreceptor signaling promote resistance to hormone action in glucose uptake in skeletal muscle and adipose tissues, explaining the development oftype 2 diabetes mellitus (Kahn, 1998; Saltiel, 2001). Consequently, the discovery of new compounds that correct the disruption of insulin signal transduction in peripheral tissues is interesting for their application in diabetes mellitus therapy. Hence, a broad range of ethnopharmacological studies have been focused in the investigation about the improvements in insulin intracellular cascade promoted by antidiabetic plant medicines, allowing the isolation of phytochemicals for the development of new hypoglycemic drugs. Changes on insulin signal transduction can be investigated through Western blotting analysis of protein content and phosphorylation levels of the different intracellular intermediates. The increase of glucose uptake by 3T3-L1 adipocytes promoted by methanol fractions of Cortidis rhizoma, as previously cited, was associated with the stimulation of insulin signaling pathways: these plant preparations promoted further increase in the phosphorylation levels ofIRS-1 and AKT, both in the presence insulin (Ko et al., 2005). The well known hypoglycemic effect of Trigonella foenum-graecum L. (Raju et al., 2001; Basch et al., 2003; Devi et al., 2003) was also explained by the stimulation of insulin

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intermediates. Seed extract of Trigonella foenum-graecum L. activated insulin signal transduction, increasing the phosphorylation of IR, IRS-I and p85 subunit ofPI3K in 3T3-LI adipocytes and human hepatoma cells HepG2 (Vijayakumar et al., 2005). Besides activating insulin signal transduction, antidiabetic plant extracts can act inhibiting negative regulators of insulin signaling. Mter the demonstration of the Cichorium intybus hypoglycemic effect in STZinduced diabetic rats (Pushparaj et al., 2007), Muthusamy et al. (2008) showed that the methanolic extract of Cichorium intybus increased the glucose uptake in 3T3-LI adipocytes and this effect was attributed to inhibition of the protein tyrosine phosphatase IB (PTPIB) activity. Recent study by Wang et al. (2008) showed that ethanolic extract of Artemisia dracunculus L. stimulated glucose uptake in primary human skeletal muscle culture after increases in the protein content and/or phosphorylation levels of insulin intermediates, such as IRSs and AKT. In addition, the skeletal muscle culture incubated with this antidiabetic plant extract also presented reduction in the PTPIB content. It is well known that PTPIB has been implicated in the negative regulation of insulin signaling. Recently, many studies have indicated that increased PTPIB activity is associated with the development of insulin resistance and obesity (Kasibhatla et al., 2007; Koren & Fantus, 2007). Progress towards the elucidation of herbal preparations that inhibit PTPIB inhibition represents a novel possibility of intervention in the diabetes mellitus therapy.

Antioxidant properties of medicinal plants used in diabetes treatment In addition to the classic metabolic complications of diabetes mellitus, the overproduction of reactive oxygen species (ROS) is involved in the etiology and pathogenesis of diabetes (Evans et al., 2002; N ewsholme et al., 2007), leading to the development of diabetic complications. Prolonged exposure to hyperglycemia causes oxidative stress, which represents an imbalance between ROS formation and endogenous antioxidant defense activity and characterizes the glucose toxicity profile; evidences indicate that diabetic patients have both increased levels of markers from ROS induced damage and decreased antioxidant defenses. Several mechanisms explain the development of the oxidative stress in diabetes mellitus: a) the increased intracellular glucose metabolism promotes overproduction of NADH that, in excess, leads to an increased production of superoxide by the electron transport chain; b) the irreversible generation of advanced glycation endproducts, which occurs after the nonenzymatic covalent bonds of reducing sugars to proteins, induces ROS production; c) activation of the polyol pathway contributes to ROS generation through reduction ofNADPH (which reduces glutathione regeneration and NOS synthase activity) and increase of NADH availability (Jay et al., 2006; Robertson & Harmon, 2006). Other circulating factors that are elevated in diabetic patients, such as free fatty

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acids and leptin, also contribute to increased ROS generation. Hydrogen peroxides (HP2) and hydroxyl radicals (OHO) have been also implicated in the pathogenesis of diabetes (Oberley, 1988; Lenzen, 2008a). Efforts to promote oxidative stress reversion in diabetes mellitus represent a possibility in the improvement or treatment of this pathology. So, in addition to their hypoglycemic effect, the antioxidant potential of plant preparations seems to be a novel and pertinent discovery to decrease diabetes complications linked to oxidative stress. Several methodologies can be used to determine the plant antioxidant effect in experimental diabetes. The in vivo animal oxidative stress level can be determined by the measurement of plasma analytes, for example 8-isoprostane, a reliable stress biomarker that is increased in diabetes experimental models that can be quantified by specific enzyme immunoassay. Malondialdehyde, an end product oflipid peroxidation, can be quantified by colorimetric method from tissue samples. H 20 2 and reduced glutathione can be also colorimetrically measured. The activities of the antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, are frequently determined in plasma and tissue samples from diabetic animals treated with herbal preparations. Several studies have evaluated the antioxidant properties of plant preparations in experimental diabetic models. STZ-induced diabetic rats treated with Phlomis anisodonta aerial parts extract presented beneficial responses in the control of diabetes, such as reduction of blood glucose, increasing of insulin levels and also reduction of oxidative stress, through activation of hepatic antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), which leads to reduction of hepatic lipid peroxidation (Sarkhail et al., 2007). Waisundara et al. (2008) demonstrated similar results in diabetic rats treated with root extract of Scutellaria baicalensis, commonly prescribed in complementary medicine as a plant with efficient antioxidant properties (Wang et al., 2007). Reduction of hyperglycemia and oxidative stress was also observed in STZ-diabetic rats treated with Matricaria chamomilla L. aerial part extract (Cemek et al., 2008) and in Obese-diabetic (ob-db) rats treated with cocoa (Theobroma cacao) extract (Jalil et al., 2008).

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on hepatic cells lipid peroxidation and antioxidant enzymes in experimental diabetes, Pharmacological Research 56(3): 261-266. Scheen, A.J. 2003. Is there a role for alpha-glucosidase inhibitors in the prevention of type 2 diabetes mellitus? Drugs 63(10): 933-951. Schena, F.P. and Gesualdo L. 2005. Pathogenetic mechanisms of diabetic nephropathy, Journal of the American Socwty of Nephrology 16(SuppI1): S30-S33. Scott, F.W. 1990. Cow milk and insulin-dependent diabetes mellitus: Is there a relationship? The American Journal of Clinical Nutrition 51(3): 489-491. Scott, F.W., Cloutier H.E., Kleemann R., Wiierz-Pagenstert D., Rowsell P., Modler H.W. and Kolb H. 1997. Potential mechanisms by which certain foods promote or inhibit the development of spontaneous diabetes in BB rats: Dose, timing, early effect on islet area, and switch in infiltrate from Thl to Th2 cells, Diabetes 46(4): 589-598. Scott, F.W., Olivares E., Sener A. and Malaisse W.J. 2000. Dietary effects on insulin and nutrient metabolism in mesenteric lymph node cells, splenocytes, and pancreatic islets ofBB rats, Metabolism: Clinical and Experimental 49(9): 1111-1117. Sekar, D.S., Sivagnanam K and Subramanian S. 2005. Antidiabetic activity of Momordica charantia seeds on streptozotocin induced diabetic rats, Die Pharmazie 60(5): 383-387. Shafrir, E. and Ziv E. 1998. Cellular mechanism of nutritionally induced insulin resistance: the desert rodent Psammomys obesus and other animals in which insulin resistance leads to detrimental outcome, Journal of Basic and Clinical Physiology and Pharmacology 9(2-4): 347-385. Shan, J.J., Yang M. and Ren J.W. 2006. Anti-diabetic and hypolipidemic effects of aqueous-extract from the flower of Inula japonica in alloxan-induced diabetic mice, Biological & Pharmaceutical Bulletin 29(3): 455-459. Sharma, B., Balomajumder C. and Roy P. 2008. Hypoglycemic and hypolipidemic effects of flavonoid rich extract from Eugenw Jambolana seeds on streptozotocin induced diabetic rats, Food and Chemical Toxicology 46(7): 2376-2383. Sharma, S.B., Nasir A., Prabhu KM., Murthy P.S. and Dev G. 2003. Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxaninduced diabetic rabbits, Journal of Ethnopharmacology 85(2-3): 201-206. Shimada, T., Kudo T., Akase T. and Aburada M. 2008. Preventive effects of Bofutsushosan on obesity and various metabolic disorders, Biological & Pharmaceutical Bulletin 31(7): 1362-1367. Shin, M.S., Kim S.K, Kim Y.S., Kim S.E., Ko I.G., Kim Y.S., Kim C.J., Kim Y.M., Kim B.K and Kim T.S. 2008. Aqueous extract of Anemarrhena rhizome increases cell proliferation and neuropeptide Y expression in the hippocampal dentate gyrus on streptozotocin-induced diabetic rats, Fitoterapia 79(5): 323-327. Shinde, J., Taldone T., Barletta M., Kunaparaju N., Hu B., Kumar S., Placido J. and Zito S.W. 2008. Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats, Carbohydrate Research 343(7): 1278-1281. Shirwaikar, A., Rajendran K and Barik R. 2006. Effect of aqueous bark extract of Garuga pmnata Roxb. in streptozotocin-nicotinamide induced type-II diabetes mellitus, Journal of Ethnopharmacology 107(2): 285-290. Shulman, G.I., Rothman D.L., Jue T., Stein P., DeFronzo R.A. and Shulman R.G. 1990. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy, The New England Journal of Medccine 322(4): 223-228. Singh, S.K, Kesari A.N., Gupta R.K, Jaiswal D. and Watal G. 2007. Assessment of antidiabetic potential of Cynodon dactylon extract in streptozotocin diabetic rats,

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10 Cyclodextrins, Structures, Properties Useful for Treating Diseases and Revitalizing Body Systems *

WIESLAWA MISIUK 1 AND

J.N.

GOVIL

Abstract Cyclodextrins (CD) are natural products formed by bacterial digestion of starch. They are cyclic oligosaccharides consist of six, seven or eight a- (1-4) linked a-D- glucopyranose units and called a-, f3- and y- CD, respectively. The important functional ability of CD is forming inclusion complexes with various guest molecules, altering their physicochemical behaviour and reducing undesirables effects. Different aspects of utility of inclusion complexes includes solubilization, encapsulation, transport small molecules (toxins and drugs) are presented. Studies on cyclodextrin - based supramolecular architectures and complexes inspire progress on supramolecular biomaterials for drug and gene delivery and future functional molecular devices and nanoscience. The annual consumption ofcyclodextrins in wordwide is growing at high rate.CD are useful in medicine, pharmacy and foods for treating diseases and revitalizing body systems. Key words : Cyclodextrins, Inclusion, Aggregates, Biomaterials, Treating diseases

Introduction Cyclodextrins, their properties and structure constitute an intriguing and fascinating subject that, due to their complexity, lacked and extended coverage in monograph for long time. The field has grown extensively and immensely, thus discussing or even mentioning many significant works was not possible. 1. University of Bialystok, Department of Biology and Chemistry, Institute of Chemistry,

Bialystok, Poland. 2. Division of Genetics, Indian Agricultural Research Institute, New Delhi 1100012, India. * Corresponding author : E-mail: [email protected]

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Cyclodextrins is a group of cyclic oligosaccharides produced from starch by means of enzymatic conversion. Their properties is a very challenging field. The utilization of the research results in the form of products and technologies, expressed by the amount of products, impact ofthe results on the earlier technologies and value of the developed technologies inform about the real progress. Cyclodextrins are important and widely used in many fields oflife sciences. The negligible cytotoxic effects are an important attribute in cyclodextrins applications such as drug carrier and drug delivery, gene delivery, photosensitizers, flavous, encapsulation and environmental protection. The actual and potential uses of cyclodextrins in biomaterials, medicine, pharmacy, foods and agriculture are presented in research articles, the relevant reviews and recent CD monographs. The paper get information about important aspects of cyclodextrin research which play a major role in treating different diseases and revitalizing body systems.

Cyclodextrins, structure and physical, chemical and biological properties Cyclodextrins (CD) are a group of structurally related natural products formed during the bacterial digestion of starch. Starch is one of the major carbohydrates stored by plants. CD are obtained by starch bioconversion using cyclodextrin glucosyltransferase. This enzyme is produced by a variety of bacteria, mainly by several species of Bacillus, but also by Klebsiella pneumoniae, Klebsiella oxytoca, Micrococcus luteus, Thermoanaerobacter thermosulfurigenes, Thermococcus and other microorganisms. Different types of starch can be used as a substrate, but potato starch is the most commonly used for CD production. Among the used starches, cassava starch is a good raw material because it has a high content of amylopectin and a low liquefaction temperature. The starch derivatives are non-toxic ingredients, are not absorbed in the upper gastrointestinal tract and are completely metabolized by the colon microflora. Cyclodextrins (CD), which are produced from amylase fraction of starch by glucosyltransferases, consist of three well known family members which comprise six, seven or eight glucopyranose units, generally called, U-, [3-, yCD, respectively (Fig 1). Cyclodextrins are toroidal shaped cyclic oligo saccharides with a hydrophilic outer surface and in internal hydrophobic hollow interior, which can entrap a vast number of lipophilic compounds into their hydrophobic cavity, depending on their size and molecular structure. The conical structure can be attribiuted to the position of the glucose units in the cyclodextrin molecule and in particular to the primary hydroxyl groups, which are at times inclined towards the cavity due to the freely rotating carbon atom 6. The secondary hydroxyl groups located on the upper, broad edge of the cyclodextrin molecule are not mobile, and so the cyclodextrin

Cyclodextrins, Structures, Properties Useful for Treating Diseases

Fig 1. Molecular structure ofthe natural

(X- ,

~-,

161

y- cyclodextrins

molecule tapers only in lower section. The many outwardly projecting hydroxyl groups would suggest good water solubility. However, when the solubility of cyclodextrins is compared with each other, significant differences emerge which do not correlate with the ring size. ~-cyclodxtrin is approximately ten times less soluble than ex -, y - CD. The low solubility of ~-cyclodextrin can be explained by cyclic structure. This enables that the secondary hydroxyl groups of carbon atom 2 and 3 to come so close to one another and intramolecular hydrogen bonding occurs (Fig 2).

Fig 2. Intermolecular hydrogen bonds in the

~-

cyclodextrin molecule

Various derivatives of CD have been synthesized to improve their solubility and stability in aqueous solutions and their availability and cytotoxicity in biological systems. Ideally, CD derivatives for biological experiments have good solubility and little cyctotoxicity as well as hemolytic activity. Cyclodextrins has been considered as having one of the major roles in increasing the stability action, encapsulation and adsorption of contaminants by the formation of inclusion complexes. The remarkable

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ability of cyclodextrins to include hydrophobic compounds has been exploited in several fields, spanning from pharmaceutical to cosmetics, from food manufacturing to commodity. A novel functional surface treatment of cotton based on the permanent fixation of p-cyclodextrin on fabric is receiving increased attention. Cyclodextrins were also used in the cleavage of DNA. It was reported that some Cyclodextrins-C 6o conjugates were synthesized and used as photodriven DNA- cleavage reagents. Physicochemical and biological properties of novel cyclodextrin as the reaction products of maltose and p-cyclodextrin produced by the action of Klebsiella pneumoniae pollulanase were determined and examined in cultured Caco-2 cells. pcyclodextrins also provide a unique tool to modulate cellular cholesterol in living cells. It needs to be recognized that P-CD have a pleiotropic effects on the level and distribution of different membrane components. Basing on the results it can be suggested that substituting cholesterol with other sterols using P-CD as carriers, will provide additional insights into role of cholesterol in cellular function. a- Cyclodextrin was introduced as a new probe to study mechanism of adhesive interactions, mechanism of cellular interactions. Its use could help identify the active sites that control adhesive interactions in a variety of systems. Application of the inclusion of nonylphenol at a-CD cavity of a-CDalginate matrix could provide the positive effect of the biodegradation and could reduce the toxicity of high nonylphenol concentration. CD-alginate beads formation exhibit applicability for immobilization of microbial cells for bio-remediation application ofnonylphenyl contaminated water.

Inclusion c omplexes An important property of cyclodextrins is their ability to include in their hydrophobic cavity a large variety of guests molecules without formation of any covalent bond. The potential utility of inclusion complexes includes encapsulation, solubilization and transport of small hydrophobic molecules such as toxins and drugs . Cyclodextrins modified with hydrophilic substituents have enhanced solubilities in water compared to their native form . While chemical modification are often aimed at increasing the solubility of cyclodextrin or of the inclusion complex, the presence of substituents may also contribute to the complexation of the host.

The complex behaviour and the interdependence of several molecular parameters influencing the complexation were discussed. These parameters included contributions ofthe charge and hydrophobic character ofthe guest, type of the substituent at the cyclodextrin and possible inclusion complexation of the substituent inside the cavity. The chemical, physical and biological properties of guests molecules would be altered due to the formation of host-guest complex and thus, the character of guests molecules are modified. The influence factors of inclusion interactions such as host

Cyclodextrins, Structures, Properties Useful for Treating Diseases

163

. A

B

c Fig 3. The optimized structures of cina lukast (A) and inclusion complexes of cinalukast -(X- CD (B) and cina lukast - DMCD (C)

molecule, guest molecule and pH are discussed. The optimized structures ofthe inclusion complexes with a-CD and heptakis - (2,6,-di-O-methyl) - pCD ( DMCD), obtained by energy optimization, are displayed in Fig 3. Cyclodextrin structure, physicochemical and biological properties are useful in different disciplines of human life . Cyclodextrins with their properties had been widely used in various fields such a s medicine, pharmacy, chemistry, agriculture, etc. Investigation of the mechanism of inclusion compounds plays an important part in supramolecular chemistry. Scanning electron micros cope (SEM) method s and photogra phs of cyclodextrins and their inclusion complexes by SEM can be assumed as a proof of solid inclusion complexes formation . SEM photographs of pcyclodextrins and their complexes with L- tyrosine are given in Fig 4.

Cyclodextrin conjugates Another kind of cyclodextrins interactions with various molecules are conjugates 19. 22 . A new conjugates of p-cyclodextrin with homocarnosine were synthesized to better study the role of the bioconjugation on the activity of the peptide. The data on the scavenger ability of the homocarnosine conjugates against the OH'" provided also to investigate the oxidant activity of the carnosine and the homocarnosine conjugates in the copper (II) dependent LDL oxidation assay, where other free radical species are involved. The evaluation of the antioxidant activity of conjugates of cyclodextrins with biological dipeptides histidine containg carnosine and

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Fig 4. Scanning electron microscope photographs (Pt coated): ~ -CD x 500 (a), ~-C D x 3000 (b), L- tyrosine (TYN) (c), TYN- ~ CD complex (d), (Nagalakshimi et al., 2008)

homocarnosine is important objective of research. The investigation of bioconjugates of carnosine and homocarnosine can explain stabilizing the biological peptides towards the protease action. These conjugates are good candidates to carry out further studies devoted to characterize the pharmacological profiles in more complex cellular and animal models. A novel glicoconjugates based on chondroitin oligomer and y CD scaffolds were synthesis and characterization. Inhibition of pathogen -host recognition/interaction using carbohydrate- based pharmaceuticals is intensive development and presents a promising approach for the prevention of microbial infections. The chondroitin oligomer- based conjugates present their oligosaccharide ligands on a linear scaffold, which may mimic e.g. natural mucins and polylactosaminoglycans. Among the carries employed for constructing multivalent conjugates, the most common are cyclodextrins, dendrimers and calixarenes. The starburst polyamidoamine dendrimer (generation 3, G3) conjugate with aCD having an average degree of substitution of2,4 (a-CDE ) provided aspects as a gene delivery carrier. 2,4 (a-CDE) has some advantages for gene delivery such as efficient gene transfer activity into mammalian cells and low cytotoxicity. The a-CDE recommended as a new candidate of a novel carrier for siRNA. The potential use of a-CDE for an siRNA carrier in the transfection system was evaluated.

Thermodynamics host-guest complexation and modeling of cyclodextrins and their complexes In order to gain more information about the mechanism guest-host inclusion complexes involving modified cyclodextrins thermodynamic parameters are

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165

analyzed and determined. Molecular recognition based on cyclodextrins and their derivatives is of current interest and the process of supramolecular multirecognition interaction can be deduced by driving non -covalent force, such as hydrophobic interaction, van der Waals or hydrogen bonding force. Various thermodynamic parameters such as formation constant (K), enthalpy (~H), entropy (~S) are calculated for the purposing indicate what forces play an important role in the inclusion process and may also contribute to the complex formation. The details on the determination of thermodynamic parameters of host-guest complexation are important for understanding molecular recognition 23 -26 • The formations and structures of inclusion compounds are studied by molecular modeling using semiempirical PM6, RMI method and Hyperchem MM + molecular mechanics dynamic method. In Fig 5 complex formed between sulfadizine and hydroxypropyl ~-cyclodextrin (HP- ~CD) is presented from PM6 and RMI methods 27 • The theoretical calculations based on the sequential methodology using semi empirical PM3 and DFT (Density Functional Theory) approaches have been successfully tested for cyclodextrins geometries and were carried out to find the global minimum structures ofthe two probable their inclusion complexes. The sequential methodology: BLYP/6-31G(d ,p)//PM3 was successfully used in theoretical work involving single point calculations at the DFT level, permit to obtain more accurate electronic puIs nuclear repulsion energy (~E e'_nu) and define the most stable structure.

PM6

RM1

Vacumm

(a)

(b)

(e)

(t)

Water

(c)

(d)

(g)

(h)

Fig 5. SulfadizinelHP- ~ CD complex in two orientations: NH2 - in Ca, c, e, g) and NH2 out (b, d, f, h ) from PM6 and RMI methods calculated in vacuum and water medium

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More details concerning the modeling of cyclodextrins and their complexes are discussed in papers 27 -30 •

Methods for study of cyclodextrins and their complexes The methods preferred for study of cyclodextrins and their complexes include one and two- dimentional NMR, FTIR, Raman spectroscopy and microscopy for visualization of supramolecular structures. lD and 2D NMR techniques, FT-IR, Raman spectroscopy and current microscopies are used to demonstrate the formation of cyclodextrin complexes with different organic, inorganic and biomolecules. The methods are important tool to determine the structures of the formed complexes. 2 D NMR spectroscopy were used to probe interaction and conformational changes upon cyclodextrin interactions. Total correlation spectroscopy (TOCSY), heteronuclear singlequantum correlation (HSQC) and heteronuclear multiple band correlation (HMBC ) were used to solve resonance problems and to assign l3C NMR signals of the appropriate molecules during dynamic interaction with cyclodextrins in details 31-35 • ROESY NMR spectrum of inclusion complex and its geometry is presented in Figs 6 and 7. Modern microscopies such as STM, AFM, SEM, TEM and florescent are widely used to characterized cyclodextrin - based aggregates various types of native and modified cyclodextrins, inclusion complexes and their aggregates, cyclodextrin rotaxanes and polyrotaxanes, cyclodextrin nanotubes and their secondary assembly and high-order aggregates of H

25,29

J \. imatinib

H

~~~

26,28 imatinib

F2 !ppm ; H 30 13.74 imatinib< 3.76

H 5 I3-CD H

6

,-' 3.78 '

~ 3.80 ./ 3.82

·- - 13-CD c --._

3.84

.

~.

' \ 3.86 j 3.88 ;

H 3 I3-CD

~) 3.90

-=-=""\ 3.92 .

~ 3.94

13.96 -

i 3.98 --,......-,--,.........,...,..~..,.....--""T"'"~,...,......-"""T'".........,J 7.857.807.757.707.657.607.557.507.45

Fl,ppm Fig 6. ROESY spectrum of aqueous sample imatinib and ~ -CD at 2: 1 molar ratio

Cyclodextrins, Structures, Properties Useful for Treating Diseases

167

H '*--------~---r-----------,OH

"tf

H£CH 2 ~6-28 H::'"

I

H 25-29

H-C3 /

H-C, • I H- 6

Fig 7. Proposed geometric arrangement ofimatinib in cavity of B- CD based on ROESY experiment

cyclodextrins. The investigations permit to obtain the direct morphology and structure of samples . Fluorescence microscopy is also applied for observing the momonolayers before and after ~-CD injection at initial surface pressure during CD interaction with different lipids. The methods applied for study of cyclodextrins and their complexes are shown in the papers 31 -43 •

Cyclodextrin - Based polypseudorotaxanes and polyrotaxanes The construction of supra molecular systems involves selective molecular combination between host and guest. Among all potential hosts, the cyclodextrins seem to be the most important ones. Cyclodextrin aggregates investigated range widely from the aggregate of native CD to high-order and complex ones. The aggregates can divide into the following types: aggregates of native and modified CD, inclusion complexes and their aggregates of CD, cyclodextrin rotaxanes nad polyrotaxanes, cyclodextrin nanotubes and their secondary assembly, and other high-order aggregates such as nanosphere and network aggregates. Recent progress in highresolution STM imaging has allowed the visualization ofthe arrangement, orientations and even inner structures of molecules in air, in ultrahigh vacuum (UHV) and in solution. Using the technology, self- organization of highly ordered molecular adlayers and lor two-dimentional (2D) supramolecular aggregations were investigated 43 .45-5 1. Description about cyclodextrin aggregates and then focus on their characterization with available modern microscopies such as STM, AFM, SEM, TEM and fluorescent are presented. STM is a convenient and widely applied tool for the detection of the microstructures and supramolecular aggregates. AFM is a relatively novel technique with which three- dimentional (3D) images can be obtained on the surface of insulating and conducting materials from nanometer to micrometer scale. AFM imaging of organic specimens is easier to perform as it does not require the specimen to be electron- or ion-

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conductive. AFM allows imaging under hydrated conditions without pretreatment of the samples, but the tip geometry and probe force usually lead to over - estimated lateral sizes of organic sample features and non- contact mode imaging has a maximum resolution of around 2 nm. TEM and SEM imaging has no source-sample contacts and allows much higher resolution, however, it is usually carried out in high vacuum and requires pre-treatment of samples. The difference between TEM and SEM is the surface topology can be obtained from SEM while more inner structure is shown by TEM. The supramolecular structures of cyclodextrin - based polypseudorotaxanes and polyrotaxanes are generally with some novel intriguing properties, and as a result of they have attracted more and more attention. Polypsedudorotaxanes and polyrotaxanes are constructed simply by incorporating pseudorotaxane/rotaxane moieties into polymers. According to how the cyclic and linear units are connected, different kinds of polypsedudorotaxanes and polyrotaxanes can be made. For polypsedudorotaxanes and polyrotaxanes, at least one covalent polymer should be used as a component. Polypsedudorotaxanes and polyrotaxanes posseses mechanically linked subunits, for which the connecting forces are non- covalent interactions. Because there is no covalent bond between their linear and cyclic components, polypsedudorotaxanes and polyrotaxanes can be viewed as composites at a molecular level. Due to their architectural differences from conventional polymers, polypsedudorotaxanes and polyrotaxanes have unique properties. Extensive studies have been made on polypseudorotaxanes and polyrotaxanes formation and application of various polymers with cyclodextrins (CD) in aqueous and non-aqueous media. Cyclodextrins extensively studied as host molecules in polypseudorotaxanes and polyrotaxanes, are a series of cyclic oligosaccharides of 1,4 - linked D( +)- glucose units. Supramolecular complexes formed by molecular selfassembly are promising candidates for future functional molecular devices and nanoscience. Supramolecular assemblies have attracted a great attention due to their topologies and their application in various fields such as nanodevices, molecular switches, drug delivery systems and sensors. Polyrotaxanes (PR) have attracted interests, due to their inherent scientific importance and potential applications as smart materials for hydrogels, carriers for drug delivery, fibber spinning, etc. PR comprise a linear polymer and numbers of cyclodextrins as rings stopped by bulky endcapping groups. A variety of bulky groups and end-capping reactions have been exploited to prepare PR from their precursors ofpolypseudorotaxanes (PPR). A kind of novel main-chainpolyrotaxanes has been prepared by lengthily tunable poly (2-hydroxyethyl methacrylate) (PHEMA) block as bulky end stoppers. Polypseudorotaxanes were made from the self-assemblies of a distal 2- bromoisobutryl end- capped PEG with a varying amount of (XCD in aqueous media and used as a macroinitiators in situ to initiate ATRP (Atom Transfer Radical Polymerization) of HEMA (hydroxyethyl methacrylate) catalyzed by Cu(I)Br/PMDETA (N ,N ,N' ,N' ,N' -

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169

pentamethyldiethylenetriamine) at room temperature. Holding the active Br end groups, they show the potential to be used as macroinitiator to initiate new ATRP polymerization. As the unique properties of general PR with that of block copolymers, these polyrotaxanes are promising to be used as smart materials for preparation of supra molecular sliding gels, biosensors, carriers for drug controlled and scaffolds for tissues engineering. The polypseudorotaxanes can also be prepared by supramolecular selfassembly of ~-cyclodextrins threaded onto the triblock copolymers in a ionic liquid (n-butyl-3-methylimidazolium hexafluorophosphate) with different manners. Study on PPR construction in non-aqueous media added new understanding on the assembly theories between CD and macromolecules. Pseudorotaxane-like supramolecular complex of coenzyme Q10 with ycyclodextrin formed by solubility method was examined 46 • The studies may be further required to elucidate the formation of supramolecular complexes with various isoprenoids and their structure and pharmaceutical properties. The study on the design and evaluation of the pegylated insulin/CD polypseudorotaxanes indicated that they can work as a sustained drug release system and the polypseudorotaxane formation with cyclodextrins may be useful as a sustained drug delivery technique for other pegylated proteins and peptides and to pegylated low-molecular weight drugs.

High-order aggregates of cyclodextrins Besides the above mentioned aggregates of cyclodextrin, other high-order aggregates of cyclodextrin, such as nanometer structural wire-shaped aggregates, nanospheres, polymeric micelles, network aggregates, starpolymers, self-assembled multilayer and cyclic daisy chains were reported 43 • A size - controlled 3D magnetic nonosphere, using ~-CD as surfactants, oleic acid and oleyl amine as cosurfactants for the assembly of magnetite nanoparticles is reported and SEM and TEM images of the samples stabilized with different amounts of ~-CD is given in Fig B. Fig B a is a SEM image of assembled magnetite prepared at higher temperatures, which has a regular spherical structure with the average size of 2 pm. The enlarged SEM images (Figs Bb,c) show that the spheres consist of many nanoparticles. In above preparation process, if ~-CD but not oleic acid and oleylamine existed, seispherical morphology were obtained (Fig B d). If oleic acid and oleylamine without !)--CD existed, isolated particles with an average size of 11 nm were synthesized (Fig Be ). The obtained results suggest that the assembly and size of magnetite particles are dependent on the amount of ~-CD . Large network aggregates were formed from water-soluble gold nanoparticles capped with thiolated y-CD hosts in the presence of C60 fullerene molecules 39 • This aggregation phenomenon was driven by the formation of inclusion complexes between two CD attached to different nanoparticles and one molecule of C60.

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Fig 8. SEM images of sample synthesized from ~-CD, oleic acid and oleylamine (a-c), TEM image of sample synthesized from only ~-CD (d), TEM image of sample synthesized from oleic acid and oleylamine (e) (He et al., 2008)

Fig 9. TEM images ofy - CD - capped gold nanoparticles (A) and C60 - induced aggregate (B)

From TEM images in Fig 9 can be seen that the addition of C60 makes the y-CD-capped gold nanoparticles (3.2 nm) to transform into large network aggregates (300 nm). Another netlike supramolecular aggregates were synthesized through the linkage of gold nanoparticles with cyclodextrinbased polypseudorotaxanes. TEM images were employed to characterized the sole gold nanoparticles and further aggregates with polypseudorotaxanes. Cyclodextrins can form different aggregates under different conditions . Microscopy, as one of the most important tools to characterize above assembly, has been used widely. Using AFM, single cyclodextrin can be easily manipulated. With the development of microscopy, cyclodextrin aggregates will be investigated more and more widely.

Cyclodextrin - Based supramolecular architectures for development of novel biomaterials for drug delivery and gene delivery The supramolecular architectures formed between cyclodextrines and

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polymers have inspired progress in studies on supramolecular biomaterials for drug and gene delivery. The design and synthesis of CD-based supramolecular hydrogels and biodegradable polyrotaxanes are interesting developments as biomaterials for potential controlled drug delivery. Cyclodextrins - containing cationic polymers and cationic polyrotaxaned were explored in developing a new class of cationic supramolecules for gene delivery. The development of CD-based supramolecular biomaterials for drug and gene delivery is an area which faces a lot of challenges in future. The supramolecular approach has opened up new possibilities for designing novel drug and gene delivery systems. For obtaining supramolecular self assemblies, cyclodextrins are potential candidates because of their ability to form complexes through supramolecular interactions with a great variety of substances. The structures of the supramolecules can be controlled with many different copolymers and CD derivatives. Controlling the assemblies is important for constructing new materials which are more functional and have higherordered structures. Useful of CD - based supramolecular architectures for developments of novel biomaterials are demonstrated 50-64 : • • • • • • •

In colonic delivery of drugs, CDIPEG based hydrogels for drug delivery, Polymer associated liposomes as a novel delivery system for CD-bound drugs, In macromolecules drug delivery system based on chitosan! CD nanoparticles, For a novel particulate system named beads using selfassembling system a-CD loil to bead formation , In synthesis of magnetite nanoparticles ~-CD complexes which made an interesting candidate for hyperthermia treatment, An osteotropic alendronate ~-CD conjugate as a bone targeting delivery system developed for improved treatment of skeletal diseases; this CD-conjugate was studied as a delivery system for prostaglandin E for treatment of bone defects.

A new concept in drug delivery system is based on using amphiphilic cyclodextrin molecules in preparing nanoparticules or nanocapsules. The hydrophilic outer surface of these molecules results in a week interaction with biological membranes. It was found that amphiphilic cyclodextrins were of considerable interest for pharmaceutical applications in view of their capacity for to self-assemble in water at physiological pH, to form micelles, nanospheres, nanocapsules and liposomes. The introduction of sulfate groups onto the hydroxyl groups of cyclodextrins gave rise to a new class of modified cyclodextrin. The sulfate groups confer to cyclodextrins an interesting biological activity, similar and sometimes superior to those of heparin on such derivatives. It is also mentioned that the biological activity of the sulfate compounds depend on number of sulfate groups introduced. It

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appeared very interesting to associate to cyclodextrins on the one side a biological activity by grafting sulfate groups on their primary hydroxyl face. And on other side, to render these compounds amphiphilic, hydrophobic chains were grafted on their secondary hydroxyl face. Preparation of nanospheres from amphiphilic l3-cyclodextrins formed by different acylation degree (DA) at the secondary hydroxyl face (DA = 14 and 21) followed by varying the sulfatation degrees (DS) at the primary hydroxyl face (DS = 0, 4 and 7)61. The nanoparticles prepared from amphiphilic f3-cyclodextrins were characterized by mean size, zeta potential and their morphology. Sulphated amphiphilic f3-cyclodextrins having HLB values higher than 8 were able to self-organize in water to form nanoparticles. The amphiphilic f3-cyclodextrins that HLB values lower 6.6. are insoluble in water but soluble in organic solvents rendering possible the preparation of nanoparticles by nanoprecipitation technique. The association of sulphated amphiphilic 13CD to the per acylated amphiphilic f3-CD was interesting, it led to improve the stability of nanospheres size and probably confer them a biological activity. Physicochemical properties of non-sulfated and sulfated amphiphilic cyclodextrins and their influence on the properties of produced nanospheres are given in Tables 1, 2. SEM photographs of amphiphilic f3-cyclodextrins nanosperes are shown in Fig 10.

Fig 10. SEM photographs of amphiphilic nanospheres of ~ - CD14C6

~-cyclodextrins:

(a) nanospheres of

~

-CD21C6, (b)

Cyclodextrins properties useful for treating diseases and revitalizing body systems Cyclodextrins, their structures and properties are useful in different disciplines, e.g. medicine, pharmacy, foods, agriculture. A mixture of Salacia reticulata (Kotala himbutu) aqueous extract and cyclodextrin reduce body weigh gain, visceral fat accumulation, and total cholesterol and insulin increases in male fatty rats, a model of type 2 diabetes mellitus. Salacia reticulata is the most popular herb in Sri Lanka, where many people use an aqueous extract of its stems or roots as an herbal therapy for diabetes mellitus. Recent pharmacological studies have demonstrated that the Salacia roots modulate multiple targets, including peroxisome proliferators-activated

Table 1. Physicochemical properties of amphiphilic cyclodextrins and their influence on properties of nanospheres Characteristics of amphiphilic cyclodextrins Characteristics of nanospheres Amphiphilic Acylation ~ - CD degree (DA) ~CD21C6 ~CD21C6S4 ~CD21C6S7

21

~CD14C6 ~CD14CnS4 ~CD14C6S7

14

Sulfatation degreee (DS) 0 4 7 0 4 7

Molecular weight (g/mol) 3193 3601 3907 2507 2915 3221

HLB' values 5.6 7.2 8.2 7.1 9 10

CMC

Diameter (nm)

P.I. •

(IJ-M)

Zeta potential (mY)

0.04± 0.038 0.06±0.03

- 20 ± 1.2 - 50.6 ± 0.7

10

37.2 ± 3.4 b 132 ± 1.5 41 -92c 159 ± 5.5 29 -86' 33 -84'

0.09 ± 0.074

-15

120 110

±1.7

. Hydrophile Lipophile Balance Polydispersity index b Unstable preparation and formation of aggregates after keeping in an aqueous suspension for 1 h 'Biomodal distribution of the size a

Table 2. Influence of mixing sulfated and non- sulfated amphiphilic ~ - cyclodextrin on nanoparticle properties B - CD 21 C 6 % (w/w) 85 70 50 -: 80 15

~-CD21

C 6S 7

% (w/w)

15 30 50 70 85

Mean size (nm) 84.4 71.0 69.4 76.6 80.4

Polydispersity Index 0.210 0.188 0.338 0.481 0.391

Zeta potential (mY) -31.5 - 33.2 - 35.8 - 33.6 - 30.15

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receptor a (PPAR - a) -mediated lipogenic gene transcription, angiotensin II/angiotensin II type 1 receptor, a-glucosidase, aldose reductase and pancreatic lipase. These action may contribute to the Salacia root introduced improvement of type 2 diabetes mellitus and obesity-associated hyperglycemia, dyslipidemia, and related cardiovascular complications seen in humans and rodents. Use of cyclodextrin with Salacia will reduce the development oftype 2 diabetes mellitus in obesity by egzaming the temporal change in blood glucose, triacylglycerol, total cholesterol, insulin, and adiponectin. Due to their properties cyclodextrins can contribute to protect human health. They playa significant role in solubilization of organic contaminants, enrichment and removal of organic pollutants and heavy metals from soil, water and atmosphere. CD are used in water treatment in increase of stabilizing action, encapsulation and absorption of contaminants. Using CD highly toxic substances can be removed from industrial effiuent by inclusion formation. Cyclodextrin complexation resulted in the increase of water solubility ofbenzimidiazole type fungicides, e.g. thiabendazole, fuberidazole and carbendazin, making them more available to soil. They also decrease the toxicity resulting in an increase in microbial and plant growth. CD accelerated the degradation of all types of hydrocarbons influencing the growth kinetics, producing higher biomass yield and better utilization of hydrocarbon as a carbon and energy souce. Thus ~-cyclodextrins can be a useful tool for bioremediation process. Application of cyclodextrin for testing of soil remediation is very useful. The soil test for determining bioavailability of pollutants using CD and its derivatives was discussed and given a good results. Use of cyclodxtrins is important to delay germination of seed. During grain treated with ~-cyclodextrins some of the amylases that degrade the starch supplies ofthe seeds are inhibited. It was found that improved plant growth yields a 20-45% larger harvest of plants. Recent developments involve the expression of cyclodextrin glucanotransferases into plants. The pharmaceutically useful cyclodextrins can serve as multifunctional drug carriers and drug delivery system through the inclusion complex formation or the form of CD/drug conjugates. The combine use of CD and pharmaceutical expicients can be of improving efficacy and reducing side effects of drug molecules. The potential use of CD in design and evaluation of CD-based drug formulation, is focusing on their ability to enhance the drug absorption across biological barriers, the ability to control the rate and time profiles of drug release, and the ability to deliver a drug to targeted site. Cyclodextrins and their derivatives can be applied in topical formulations to create new formulations with well known actives, advantages and limitations. The cyclodextrins can also be used as solubilizers and supramolecular photosensitizers in photodynamic therapy, PDT, in the treatment of tumors etc. Hydrophilic

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cyclodextrins have been used to form supramolecular aggregates with drugs although their external hydrophilicity can represent a drawback for cell internalization and result in a lack of affinity of the included molecule for biological membranes. This constitutes one of the reasons in developing cyclodextrin derivatives with a modulated external hydrophobicity. A lot of chemical modification have been carried out on CD thus obtaining nonionic or cationic derivatives able to form supramolecular nanoaggregates suitable for pharmaceutical applications. The potential of these molecules in engineering nanocarries able to deliver and especially target a drug is largely unexplored. Supramolecular aggregates have a size compatible with injection and could be used to optimize drug distribution in the body. Strategies involving nanocarries are well suited for the delivery of highly toxic anticancer drugs to solid tumors making use of their potential to extravasate at level of tumor defective capillary bed and deliver the drug at the site of action. Modified CD could give nanoparticles or nanocapsules able to entrap anticancer drugs such as tamoxifen citrate and paclitaxel with good efficiency. CDbased nanoassembles applied to enhanced oral delivery of organoantimonial drug. CD in nanoparticle - based drug delivery system have considerable potential in chemotherapy tuberculosis (TB). The important technological advantages of nanoparticles used as drug carries are high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances, and feasibility of variable routes of administration, including oral application and inhalation. N anoparticles can also be designed to allow controlled drug release from the matrix. These properties enable improvement of drug bioavailability and reduction of the dosing frequency, and may resolve the problem of nonadherence to prescribe therapy. Which is one of the major obstacles in the control of TB epidemics. Cyclodextrins also play important role in feeding of people and have health- promoting significance. Many people have an unhealthy diet. They eat too much bad fat and too little dietary fiber. The food industry uses cyclodextrins in a number of different ways. In the human diet the consumption of flavonoids is well known to prevent cardiovascular, antiinflammantory, antioxidants and inhibitors of platelet aggregation. Despite the health benefits produced by flavonoids compounds the therapeutic outcome is still dependent on improvement of the pharmacokinetic profile ofthese compounds after oral administration. The mechanism of gastrointestinal absorption flavonoids are complex. Flavonoids are poorly absorbed in their natural form in the intestine. It is belived that the flavonoids are extensively degraded by intestinal microorganisms or enzymes and different metabolites can be produced. These metabolites, if absorbed, are subjected to the hepatic enzymatic system and a new metabolites can be formed varying in bioactivity. Many flavonoids have poor water solubility, what limits their pharmaceutical use. Application of

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cyclodextrins to formation of inclusion complex increases the guest's in vivo stability against hydrolysis, oxidation, decomposition and dehydration, consequently increasing bioavailability. Complexation with 13-cyclodextrins confers oral activity on the flavonoid dioclein. Dioclein is a flavonoid present in the roots of Dioclea grandiflora Mart. Ex Benth. It have many beneficial effects on the cardiovascular system such as vasorelaxant, hypotensive, antioxidant and antiarrythmogenic activities. The mechanism underlying the increase in bioavailability is probably a consequence of a proactive effect of 13-CD against in vivo biodegradation by enzymes and possibly increased water solubility. Cyclodextrins are utilized in foods mainly as carries for molecular encapsulation of the flavours and other sensitive ingredients with cyclodextrins what improve the stability of the vitamins, flavours, colourants, unsaturated fats, etc. both in physical and chemical sense leading to extended product shelf -life. Accelerated and long -term storage stability test results showed that the stability of cyclodextrin-entrapped food ingredients surpassed that of the traditionally formulated ones. Advantages of the use cyclodextrins in foods are also manifested in improved sensory, nutritional and performance properties. At present the significant role of cyclodextrins is observed in foods and food processing. Natural cyclodextrins are used as nutraceuticals. This fact designed that cyclodextrins can promote good health and prevent diseases caused by dietary deficiencies. Application of cyclodextrins and their complexes in foods offers the following advantages: •

Protection of active ingredients against oxidation, heat-promoted decomposition, light-induced reactions, loss by volatility, sublimation,

•

Elimination or reduction of undesired tastes, odours, microbiological contaminations, fibres and other undesired components,

•

Inclusion typically stable, standardizable compositions, simple dosing and handling of dry powders, reduced packing and storage costs, more economical technological processes, manpower saving. Various aspects ofcyclodextrins useful in foods were discussed 65 ,66.

Conclusions Cyclodextrins, their structures and properties are useful in many fields of human life. Important functional property of cyclodextrins is ability to form inclusion complexes with different biomolecules. The utility of inclusion complexes includes encapsulation, solubilization and transport of small hydrophobic molecules. The supramolecular architectures based on cyclodextrins have inspired progress in research on biomaterials for drug and gene delivery. This is an area which faces a lot of challenges in future. The supramolecular approach has opened up a new possibilities for designing novel drug and gene delivery system. Amphiphilic cyclodextrin molecules are useful in preparing nanoparticles and nanocapsules which as drug delivery

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system have considerable potential in chemotherapy. Cyclodextrins also play important role in feeding of people. They can promote good health and prevent diseases caused by dietary deficiencies. Due to their properties CD are widely used in treating diseases and revitalizing body system.

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Jullian, C. et al., 2007. Studies of inclusion complexes of natural and modified cyclodextrin with (+) catechin by NMR and molecular modeling, Bioorg. Med. Chem. 15: 3217-3224. Beni, S., Szakacs, Z., Csernak, 0., Barcza, L. and Nosal, B. 2007. Cyclodextrin imatinib complexation - Binding mode and charge dependent stabilities, Eur. J. Pharm. Sci. 30: 167-174. Bergonozi, M.C., Bilia, AR, Di Bari, L., Mazzi, G. and Vincieri, F.F. 2007. Studies on the interactions between some flavones and cyclodextrins, Bworg. Med. Chem. Lett. 17: 5744-5748. De Sousa, F.B., Oliveira, M.F., Lula, I.S., Sansiviero, M.T.C., Cortes, M.E. and Sinisterra, RD. 2008. Study of inclusion compound in solution involving tetracycline and ~-cyclodextrin by FTIR -ATR, Vibrato Spectr. 46: 57-62. Stancanelli, R, Ficarra, R, Cannava, C., Guardo, M., Calabro, M.L. and Ficarra, P. 2008. UV-vis and FTIR-ATR characterization of 9-fluorenon-2-carboxyester/(2hydroxypropyl)-~-cyclodextrin inclusion complex, J. Pharm. Biomed. Anal. 47: 704-709. Barillaro, V., Dive, G., Ziemons, E., Bertholet, P. et aZ., 2008. Theoretical and experimental vibrational study of miconazole and its dimmers with organic acids: Application to the IR characterization of its inclusion complexes with cyclodextrins, Int. J. Pharm. 350: 155-165. Rajabi, 0., Tayyari, F., Salari, Rand Tayyari, S.F. 2008. Study of interaction of spironolactone with hydroxypropyl-~-cyclodextrin in aqueous solution and in solid state, J. Mol. Struct. 878: 78-83. Grauby-Heywang, Ch. and Turlet, J-M. 2008. Study of the interaction of ~­ cyclodextrin with phospholipids monolayers by surface pressure measurements and fluorescence microscopy, J. Coll. Interface Sci. 322: 73-78. He, Y., Fu, P., Shen, X. and Gao, H. 2008. Cyclodextrin-based aggregates and characterization by microscopy, Micron 39: 495-516. Tong, X., Zhang, X., Ye, L., Zhang, A. and Feng, Z. 2008. Novel main-chain polyrotaxanes synthesized via ATRP ofHEMA initiated with polypseudorotaxanes comprising BriB-PEG-iBBr and a -CDs, Polymer 49: 4489-4493. Jing, B., Chen, X., Hao, J., Qiu, H., Chai, Y. and Zhang, G. 2007. Supramolecular self- assembly of polypseudorotaxanes in ionic liquid, Colloids and Surfaces A: Physicochem. Eng. Aspects 292: 51-55. Nishimura, K., Higashi, T., Yoshimatsu, A., Hirayama, F., Uekama, K. and Arima, H. 2008. Pseudorotaxane-like supramolecular complex of coenzyme QlO with ~-cyclodextrin formed by solubility method, Chem. Pharm. Bull. 56: 701-706. Higashi, T., Hirayama, F., Misumi, S., Arim, H. and Uekama, K. 2008. Design and evaluation of polypseudorotaxanes of pegylated insulin with cyclodextrins as sustained release system, Biomaterials 29: 3866-3871. Higashi, T., Hirayama, F., Arima, H. and Uekama, K. 2007. Polypseudorotaxanes of pegylated insulin with cyclodextrins: Application to sustained release system, Bworg. Med. Chem. Lett. 17: 1871-1874. Belitsky, J.M., Nelson, A, Hernandez, J.D., Baum, L.G. and Stoddart, J.F. 2007. Multivalent interactions between lectins and supramolecular complexes: galectin1 and self-assembled pseudopolyrotaxanes, Chem. & Bioi. 14: 1140-1151. Li, J. and Loh, X.J. 2008. Cyclodextrin - based supramolecular architectures: Syntheses, structures and applications for drug and gene delivery, Adv. Drug Deliv. Rev. 60: 1000-1017. Denadai, AM.L. et al., 2007. Supramolecular self- assembly of [3-cyclodextrin: an affective carrier of the antimicrobial agent chlorhexidine, Carbohyr. Res. 342: 2286-2296.

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RPMP Vol. 29 - Drug Plants III Lu, D. , Yang, L., Zhou, T. and Lei, Z. 2008. Synthesis, characterization and properties of biodegradable polylactic acid-~-cyclodextrin cross- linked copolymer microgels, Eur. Polymer J. 44: 2140-2145. Adeli, M., Zarnegar, Z. and Kabiri, R. 2008. Amphiphilic star copolymers containing cyclodextrin core and their application as nanocarrier, Eur. Polymer J. 44: 19211930. Maestrelli, F., Zerrouk, N., Cirri, M., Mennini, N. and Mura, P. 2008. Microspheres for colonic delivery ofketoprofen-hydroxypropyl- ~- cyclodextrin complex, Eur. J. Pharm. Sci. 34: 1-11. Salmaso, S., Semenzato, A, Bersani, S. et al., 2007. CyclodextrinlPEG based hydrogels for multi-drug delivery, Int. J. Pharm. 345: 42-50. Bertolla, C., Rolin, S., Evrard, B. et al., 2008. Synthesis and pharmacological evaluation of a new targeted drug carrier system: ~-cyclodextrin coupled to oxytocin, Bioorg. Med Chem. Lett. 18: 1855-1858. Prabaharan, M. and Gong, S. 2008. Novel thiolated carboxymethyl chitosan-g-~­ cyclodextrin as mucoadhesive hydrophobic drug delivery carriers, Carbohyd. Polym. 73: 117-125. Lim, H.J., Cho, E. Ch., Shim, J., Kim, D-H, An, E.J. and Kim, J. 2008. Polymerassociated liposomes as a novel delivery system for cyclodextrin-bound drugs, J. Coll. Inter. Sci. 320: 460-468. Frezard, F., Martins, P.8., Bahia, AP.C.O., Le Moyec, L. et al., 2008. Enhanced oral delivery of antimony from meglumine antimoniate/~-cyclodextrin nanoassemblies, Int. J. Pharm. 347: 102-108. Krauland, AH. and Alonso, M.J. 2007. Chitosan/cyclodextrin nanoparticles as macromolecular drug delivery system, Int. J. Pharm. 340: 134-142. Abdelwahed, W., Degobert, G., Dubes, A, Parrot-Lopez, H. and Fessi, H. 2008. Sulfated and non- sulfated amphiphilic-~-cyclodextrins: Impact of their structural properties on the physicochemical properties of nanoparticles, Int. J. Pharm. 351: 289-295. Bochot, A, Trichard, L., Le Bas, G., Alphandry, H., Grossiord, J.L., Duchene, D. and Fattal, E. 2007. a-Cyclodextrinloil beads: An innovative self-assembling system, Int. J.Pharm. 339: 121-129. Cabos Cruz, L.A., Perez, C.A.M., Romero, H.A.M. and Casillas, P.E.G. 2008. Synthesis of magnetite nanoparticles- ~- cyclodextrin complex, J. Alloys Compounds 466: 330-334. Liu, X-M, Wiswall, A.T., Rutledge, J.E. et al., 2008. Osteotropic ~- cyclodextrin for local bone regeneration, Biomaterials 29: 1686-1692. AbelIan, C.L., Fortea, I., Nicolas, J.M.L. and Delicado, E.N. 2007. Cyclodextrins as resveratrol carrier system, Food Chem. 104: 39-44. Almenar, E., Auras, R., Rubino, M. and Harte, B. 2007. A new technique to prevent the main post harvest diseases in berries during storage: Inclusion complexes ~­ cyclodextrin - hexanal, Int. J. Food Microbiol. 118: 164-172. Martin Del Valle, E.M. 2004. Cyclodextrins and their uses: a review, Process Biochem. 39: 1033-1046. Loftsson, and Duchene, D. 2007. Cyclodextrins and their pharmaceutical applications, Int. J. Pharm. 329: 1-11. Uekama, K, Hirayama, F. and Arima, H. 2006. Recent Aspects of cyclodextrinbased drug delivery system, J. Incl. Phenom. Macrocycl. Chem. 56: 3-8. Dentuto, P.L., Catucci, L., Cosma, P., Fini, P. et al., 2007. Cyclodextrinl chlorophyll a complexes as supramolecular photosensitizers, Bioelectrochem. 70: 39-43. Brewster, M.E., and Loftsson, T. 2007. Cyclodextrins as pharmaceutical solubilizers, Adv. Drug Deliv. Rev. 59: 645-666.

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Cal, K. and Centkowska, K. 2008. Use of cyclodextrins in topical formulations: Practical aspects, Eur. J. Pharm. Biopharm. 68: 467-478. Lin, H-S, Leong, W.W.Y., Yang, J.A., Lee, P., Chan, S.Y. and Ho, P.C. 2007. Biopharmaceutics of 13-cis-retinoic acid (isotretinoin) formulated with modified ~-cyclodextrins, Int. J. Pharm. 341: 238-245. Tewes, F., Brillault, J., Couet, W. and Olivier, J-C. 2008. Formulation of rifampicincyclodextrin complexes for lung nebulization, J. Control. Release 129: 93-99. De Araujo, R.D., Tsuneda, S.S., Cereda, S.M.C., Carvalho, F. Del, G.F. et al., 2008. Development and pharmacological evaluation of ropivacaine -2- hydroxypropyl ~-cyclodextrin inclusion complex, Eur.J. Pharm. Sci. 33: 60-71. Li, S., Wu, F., Wang, E. and Li, L. 2008. Photoreactive inclusion complex of aryliodonium salt encapsulated by methylated-~-cyclodextrin, J. Photochem. Photobiol. A: Chem. 197: 74-80. Stancanell, R., Mazzaglia, A., Tommasini, S. et al., 2007. The enhancement of isoflavones water solubility by complexation with modified cyclodextrins: A spectroscopic investigation with implications in the pharmaceutical analysis, J. Pharm. Biomed. Anal. 44: 980-984. Yang, J., Wiley, C.J., Godwin, D.A. and Felton, L.A. 2008. Influence ofhydroxypropyl~-cyclodextrin on transdermal penetration and photostability of avobenzene, Eur. J. Pharm. Biopharm. 69: 605-612. Trichard, L., Le Bas, G., Duchene, D., Grossiord, J-L and Bochot, A. 2008. Formulation and characterization of beads prepared from natural cyclodextrins and vegetable, mineral or synthetic oils, Int. J. Pharm. 354: 88-94. Bouquet, W., Ceelen, W., Fritzinger, B., Pattyn, P. et al., 2007. Paclitaxel/~­ cyclodextrin complexes for hyperthermic peritoneal perfusion - Formulation and stability, Eur. J. Pharm. Biopharm. 66: 391-397. Makholf, A., Miyazaki, Y., Tozuka, Y. and Takeuchi, H. 2008. Cyclodextrins as stabilizers for the preparation of drug nanocrystals by the emulsion solvent diffusion method, Int. J.Pharm. 357: 280-285.

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11 Chemopreventive and Radioprotective Effects of Medicinal Plants from Iran *

SEYED JALAL HOSSEINIMEHR 1

Abstract Human are exposure to many toxic chemical substances and ionizing irradiation. Epidemiology studies showed that the frequency of cancers increased in population. It is established that the increasing of oxidative stress leads to be main reason for these diseases. Exposure of cells with elevated oxidative stress may contribute to various pathological conditions including tumorgenesis and cancer either by mechanism involving damage to DNA or modulating cellular signal transduction pathways. Chemopreventive agents are specific substances (natural or synthetic agent) or their mixtures to suppress or reduce process of carcinogenesis. Consumption of these agents is very benefitted to neutralize reactive oxygen species or their effect and then they protect cells and tissue against deleterious effect of oxidative stress and reduce the incidence of human cancer. Natural products mainly herbal medicines protect human against oxidative stress and can reduce incidence of cancer in human population. In this review, I have focused on potential chemopreventive and radioprotective roles of herbal medicine and chemically pure naturally compounds that are being investigative from Iran, including hawthorn, Citrus aurantium, saffron, Allium, Umbelliprenin. Key words : Radioprotective, Chemopreventive, Oxidative stress, Medicinal plant

Introduction Epidemiology studies showed that the frequency of cancer increased in population. It is established that the increasing of oxidative stress leads to be 1. Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. * Corresponding author: Email: [email protected]@mazums.ac.ir

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main reason for these diseases. Oxidative stress is inbalance of high level of reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidative defense system. ROS such as free radical and hydrogen peroxide produce excessively during stress conditions. Chemical hazardous substances and ionizing irradiation are served to generate ROS in the exposed cells. These toxic substances interact with critical macromolecules such as DNA, protein and membrane to promote irreversible oxidative damage and interfere with vital cellular function. Increased oxidative stress associated with oncogen induced transformation, increased metabolic activity, mitochondrial malfunction, promotion of mutation and genetic instability, alteration in cellular repair and cell death 1. Cells are equipped with an antioxidative defense system consisting of variety of enzymatic and nonenzymatic antioxidants to inert oxidative stress resulted substance, thereby protecting cellular macromolecules from side effects induced by ROS. Exposure of cells with elevated ROS may contribute to various pathological conditions including tumorgenesis and cancer either by a direct mechanism involving damage to DNA or indirectly by modulating cellular signal transduction pathways 2,3. With regard to increase of environmental carcinogens in the public life it is important to prevent cancer by chemoprotective agents. Chemopreventive agents are specific substances (natural or synthetic) or their mixtures to suppress or reduce process of carcinogenesis4 • Consumption of these agents is very benefit to neutralize ROS or their effect and then they protect cells and tissue against deleterious effect ofROS and reduce the incidence of human cancer. One ofthe main strategy is to use natural products particularly herbal medicine as chemoprotective and radioprotective agent for protection body system against oxidative stress produced by chemical and ionizing irradiation in cells. Many studies have been reported the benefit effects of excess consumption of vegetables and fruits on human health. These natural products protect human against oxidative stress and can reduce incidence of cancer in human population. In this review, I have focused on potential chemopreventive and radioprotective roles of herbal medicine and chemically pure naturally compounds that are being investigative from Iran. These herbs are many commonly used in the world with different indication.

Free radicals Oxygen is necessary for basic cell function in human. However oxygen can produce toxic substances during normal metabolism within the cell, such as peroxide, superoxide, hydroxyl radicals and "excited stage oxygen". Free radicals are oxygen molecules or atoms that have at least one unpaired electron in their outer orbit. They essentially have an electrical charge and tendency to transfer electron to near molecules or substance. Free radicals attach the other molecules with theirs free electrons. If these free radicals are not neutralized rapidly by biological defense system or antioxidant compound in the cells, they may cause damage to the nucleus (DNA), membrane, lipids, proteins and other cellular organelles (Fig 1). Free radicals are the most dangerous substance, other reactive oxygen species (ROS) are hydrogen peroxides. ROS and RNS (reactive nitrogen species) are many unstable and

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highly reactive molecules that they can attack other molecules and macro molecules and induce cell damage. ROS can be produced from both endogenous and exogenous substances. The main sources of reactive species in cells are mitochondria, cytochrome 450 and peroxisome. Under normal physiological condition, ROS at normal level are essential for life because of their role in many vital process such as signal transduction, cell cycle and bactericidal activity ofphagocytes5' 7 • The mitochondrial respiratory chain can lead to the formation of super oxide radical, the first molecule in the pathway responsible for production of reactive oxygen species (ROS) (Fig 1). For production of RNS, nitric oxide (NO) is synthesized in different cell types. Super oxide accelerates the destruction of NO by forming the potent oxidant peroxy nitrite (ONOO) and its conjugate acid peroxy nitrous acid. It is a potent oxidanF.

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The balance between production and removal ofROS and RNS lead to beneficial and/or harmful effects in these reactive substances on cells. The cellular defense systems are enzymatic and non enzymatic antioxidants that protect the cells against oxidative damage. Antioxidant enzymes systems are glutathione-s-tranferase. Antioxidant non enzymes compounds are vitamin C, vitamin E, Coenzyme Q10, glutathione, trace elements and cystein6 ,7. Public is also countered daily with many chemical hazardous compounds such as cigarette smoke, pollutants, toxins, heavy metals, ionizing radiation and drugs. Today, people are exposed to more chemical and pollutants in air, food, and water than ever before. Driving in the major city, people seeing many pollutants in air. Some of these chemicals are metabolized and excreted. Some toxins are stored, especially in our fat. All of these toxins significantly increase the levels of production of free radicals. Inflammatory cells secrete a large number of cytokines and chemokines. ROS and RNS are produced under the stimulus of pro-inflammatory cytokines in phagocyte and non phagocytic cells through the activation of protein-kinase signaling6. One of the main sources of excessive ROS is exposure to ionizing radiation. Ionizing radiation has different type of electromagnetic rays (e.g. gamma and x-rays) and particle (alpha, beta and neutron). These rays deposit

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radiation energy in the cells. Because water is the main constituent of cellular matter, it is primarily the ionization of water resulting to production of secondary species high reactivity and short life time substances, such as the OH radical or hydrogen atoms. Eq.l shows the radiolysis products that result from the ionizing of water:

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Eq.l The chemistry of production of free radicals and hydrogen peroxide H 20

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Oxidative stress particularly free radicals and hydrogen peroxides can directly attack DNA, resulting to the generation of oxidized bases (e.g., 8-oxo-dG). DNA strand breaks, DNA intra-strand adducts, and DNA-protein cross links. Oxidative modifications of DNA bases may result in mutations during DNA replication due to base pair mismatching replication errors and genomic instability, all of which are associated with carcinogenesis 7-9 • The hydroxyl radical is known to react with all components of the DNA molecule: damaging both the purin and pyrimidine bases and also the deoxyribose backbone. It is well established, in various cancer tissues free radicalmediated DNA damage has occurred. Signal transduction is triggered by extra cellular signals such as hormones, growth factor, cytokines and neurotransmitters 10. Signals sent to the transcription machinery responsible for expression of certain genes are normally transmitted to the cell nucleus by a class of proteins. These signal transduction processes can induce various biological activities, such as muscle contraction, gene expression, cell growth and nerve transmission 10. The role of ROS in the process of genes and signal transduction pathways depends on the type and concentration ofROS. The activation oftranscription factors including MAP-kinase/AP-l and NF-KB pathways has a direct effect on cell proliferation and apoptosis l1 . Abnormalities in growth factor receptor functioning are closely associated with the development of many cancer. ROS affected several growth factor receptors (EGF, PDGF, VEGF). Increased expression ofthese growth factor receptor contributes in lung and urinary cancer. Other way, expression of NF-KP has been shown to promote cell proliferations. Several studies established that several tumors related to express activated NF _Kpl1. The cell membranes contains unsaturated fatly acids, many ofthese being polyunsaturated and thus susceptible to oxidation when reacting with ROS. The formation ofthe free radical chain reaction in the polyunsaturated lipid layer of the all membranes is characteristic of ROS-induced lipid peroxidation. These chain reactions result further more in protein oxidation, loss of weakening of cell membrane structure and function, and generation

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of aldehyde products such as acrolien, crotonaldehyde, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE)12. In high concentrations, lipid-derived products are considered the more damaging species because they easily react with proteins, DNA and phospholipids, generating a variety of intraand intermolecular toxic covalent adduct that lead to the propagation and amplification of oxidative stress. This has been demonstrated in human carcinogenesis 6 • Lipid peroxidation is one of the most investigated consequences of ROS action on membrane structure and function. Lipid peroxidation is triggers of essential mediators of apoptosis 13 .

Carcinogenesis Carcinogenesis is a complex multi step process, in which numerous molecular mechanisms play to induce it. Carcinogenesis and tumor development studied in animal model showed it has three steps, initiation, promotion and progression. When normal cells exposed to carcinogen, cellular DNA damage, if the fixation of genotoxic damage happen, the cell has abnormal function. Promotion is a form of active proliferation of multicellular premalignant tumor cell population. Progression is a clone tumor cells with increased proliferative capacity and metastasis 3,14. It has been established for many years that excessive ROS concentration is increased in cancer cells. ROS are generally considered to be carcinogen, given their potential for induction of DNA damage. ROS induces DNA damage with several mechanisms including; directly attack DNA, production of lipid peroxidation, directly activate intracellular signaling pathways, mitochondrial dysfunction and mitochondrial DNA mutation and rearrangement. These mechanisms lead cells to tumorgenesis 5,14.

Mechanism of chemopreventive effects Many mechanisms contribute to prevent against carcinogenesis induced by oxidative stress which are subdivided into two major categories including blocking agents and suppressing agents. Blocking agents are especial compounds that can inhibit initiation by inhibiting the formation of carcinogens for precursor molecules or prevention of reactive possess antioxidant or free radical scavenging potential effect, these effects dependent to efficacy of antioxidant activity that particularly relate to chemical structures of compounds 3,15. Non-enzymatic antioxidants are represented by ascorbic acid (vitamin C), glutathione, vitamin E, herbal medicine, flavonoids and other natural products. These compounds scavenge free radicals and ROS, which can inhibit effects ofROS on critical macromolecules and reduce DNA damage and consequence on carcinogenesis (Fig 2). For inhibiting ofROS on DNA, the chemopreventive agent particularly should be presented in biological and cellular environment before ROS attacking. Numerous chemopreventive agents have suppression or elimination effects on tumor cells, which affect on growth inhibition by induction of cell cycle arrest or apoptosis. Apoptosis is programized death that has been characterized as a fundamental cellular

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pl~~

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Fig 2. The damaging induced by reactive oxygen species and preventive effects of natural compounds

activity to maintain the physiological balance of the organism. It is also involved in immune defense machinery and plays an important role as a protective mechanism against carcinogenesis by eliminating damaged cells or abnormal excess cells proliferated owing to various chemical agents' induction 16 • It is established that anticancer agents are involved in the induction of apoptosis, which is regarded as the preferred way to manage cancer. Chemopreventive agents can be suppress cancer cell proliferation, inhibit growth factor signaling pathway, induce apoptosis, inhibit growth factor signaling pathway, induce apoptosis, inhibit NF-KB, AP-l and JAKSTAT activation pathways, inhibit angiogenesis, suppress the expression of anti-apoptotic proteins, drug with these mechanism particularly use of therapeutic effects in cancer 17 • Curcumin, the yellow pigment isolated from the rhizomes of Curcuma Zonga Linn, inhibits chemically induced carcinogenesis in organs in various experimental models. This compound inhibited inflammation, tumor cell proliferation and generation ofROS and oxidative DNA stress2 • Curcumin potentially inhibited NF -KB pathway in many human cancer18.

Radioprotective effects Radioprotective agents are chemical or natural products to protect biological system when administered before or immediately after exposure to ionizing irradiation. These agents are useful in eliminating or reducing the severity of deleterious and cellular effects of ionizing radiation in cells. A radioprotective agent has several characteristics, including significant protection, preferable route of administration, low toxicity, and compatibility with other drugs that are administered to patients 19 • Free radical scavenging effects ofthese compounds remove and detoxifY products of water radiolysis and reactive oxygen species before these toxic substances interact with critical macromolecules. Radioprotective agents may be inducing hypoxia and consumption of oxygen in cells, which decrease the levels of reactive oxygen species and hydrogen peroxide. Thiol-containing compounds have

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these effects in radioprotection 20 . Although these agents had good radioprotection, the limited usage of these compounds is related to their's side effects. The search for finding of radioprotective effects is interested because less toxicity correlated to herbal medicines. The proposed radioprotective efficacy of plant extracts is as a result of their containing a large number of active constituents, such as antioxidants, immunostimulants, and compounds with antimicrobial activity. Most efficacy studies on plants have been on total extracts for their ability to protect against radiation-induced chromosomal aberrations, micronuclei formation. We showed that citrus extract at a dose 250 mg/kg mitigated genotoxicity induced by gamma irradiation, when administrated Ih prior to y-irradiation. Citrus extract protected mice bone marrow 2.2-fold, compared to the non drug-treated irradiated controJ21. Hawthorn fruit has been widely used for the treatment of cardiovascular disease. Administration of hawthorn extract at a dose of 100 mg/kg, Ih prior to gamma irradiation (2 Gy), reduced the frequency of MnPCES and its efficacy was comparable with amifostine at a dose of 100 mg/kg22.

Chemopreventive effects There have been extensive studies carried out to search for potential chemopreventive agent against cancer and some of them interest to use for human. Antioxidant vitamins have been investigated for their possible chemoprevention efficacy among normal or high risk (e.g., smokers, asbestos workers, etc) population. The most popular antioxidants inclued betacarotene, vitamin E and vitamin C. Carotenoids have been found to interfere with carcinogenesis and safe, non-toxic nutrients for prevention. There are mechanisms by which beta-carotene may work as a chemoprotector, and immune modulator. Vitamin E is a lipid-soluable antioxidant that inhibits oxidation which prevented colon cancer23. Experimental and epidemiologic studies supporting the beneficed effect of fruits and vegetables. These are studies to show the dietary supplementation of natural products with antioxidant activity to reduce frequency of cancer in public. Several herbal medicine and their constituents have been evaluated in animal model to have chemopreventive effect. These compounds particularly have antioxidant activity to scavenge offree radicals. These agents inhibited the effects of ROS on macromolecules. Vegetarian diet can lead to a slight decrease in oxidative DNA damage in lymphocytes 24 . A meta-analysis 25 included three cohorts and one population based study for green tea, were analyzed for a link between black tea and breast cancer. Results showed an approximately 20% statistically significant reduction in risk of breast cancer associated with high intake of green tea. No such protection was observed for black tea. The active constituents green tea is called E GCG (epigallocatechin gallate), which has been found to inhibit carcinogenesis. Green tea is of the most popular beverages in Asia, where it has been used as a medicine for over 4000 years.

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Garlic (Allium sativum) is herb which is useful for a lot more than flavor. Oral administration of garlic inhibited the growth of tumors and reduced mortality in mice with bladder cancer26 . Ally sulfur compounds found in garlic inhibit neoplasma and can suppress tumor cells 27 . In the Shandog province in China, stomach cancer mortality was 13 times lower in those that ingested 20 g of garlic daily than those that ingested only one gram daily28. Plant polyohenols, a large group of natural antioxidant ubiquitous in a diet high in vegetables and fruits, have chemopreventive effects. Polyphenols are an important part ofthe human diet, with flavonoids being the most constituent in medicinal plants 29 . Today flavonoids are interested due to their potential role in the prevention of chronic diseases and beneficial health effects including antioxidative, anti-inflammatory, gastroprotective, cardioprotective and anticancer effects 29 . Antioxidant activity of herbal medicine is relationship to content flavonoid herbs. Our research on herbs in plants showed the herbs with higher phenolic acid and flavonoid content to have more antioxidant activity. In this study, method for evaluation of antioxidant activity was diplenyl picrgh hydrazy (DPPH) method 30, 31. Phenolic compounds acting as antioxidants may act as terminators of free radical chains and as chelators of redox active metal ions that are capable of catalyzing lipid peroxidation. Phenolic antioxidants interfere with the oxidation of lipids and other molecules by the rapid donation their of hydrogen atom to radicals. Hydroxyl group is the main chemical group in the structure offlavonoids for biological activity8. One important structured feature offlavonoids involves the presence of2,3 unsaturation in conjugation with a 4-0XO group in the C-ring (Fig 3). The other functional groups involving both hydroxyl groups of ring-B and the 5-hydroxy group of ring-A are all important contributors in the ability offlavonoids to chelate redox-active metals. Recently several studies showed flavonoids act as modulator of cell signaling, which have chemopreventive effects 32 . Fistetin, or 3,7 ,3,4-tera hydroxyl flavone, belongs to flavonal subgroup of flavonoids can be found in many fruit, and vegetables including onion, apple, kiwi fruit and cucumber. Cell culture studies show that fisetin exerts OH OH

Fig 3. Chemical structure of quercetin as main flavonoid

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anti proliferative effect on human prostate cancer cells. Fisetin can alter the mitochondrial membrane function of prostate cancer cells there by including apoptosis, an important molecular target for chemoprevention of cancer. Fisetin and other flavonoids that contain a catechol group have been shown to be potent inhibitors ofthe type 5a-reductase activities, which may be useful for the prevention or treatment of androgen-dependent disorders including prostate cancer33. The catechins or other flavonols present in green tea are the epicatechin, epigallocatechin, epicatechin-3-gallate, and epigallocatechin-3gallate (EGCG). The EGCG is most attention with regards to preventive effects against cancer has potential anticarcinogenic activity. EGCG constitutes up to 50% of the total cetechin content and has a higher antioxidant activity than vitamins C and E. EGCG treatment has been shown to result the induction of apoptosis in cancer cells 33 . EGCG effectively inhibits 5a-reductase, this hormone is important for regulation of androgen action in several organs 33 ,34. There were several studies to show of green tea (high content ofEGCG) to reduce cancer and risk diseases in population 33 ,34.

Hawthorn Hawthorn is a common, thorny shrub that grows up to five feet tall on hillsides and in sunny wooded areas of Asia, North America, Europe, and North Africa. The Hawthorn plant produces small berries, called haws, which sprout each May after the flowers of the hawthorn plant bloom. Hawthorn berries are usually red when ripe, but may be much darker. Hawthorn leaves, while usually shiny, may grow in a variety of shapes and sizes.

Botanical classification Family: Rosaceae Genus and specie: Crataegus laevigata , C. microphylla, C. monogyna, C. oxyacantha Other names: May, quickset, haw, sorkhe valik, may blossom and mayflower Parts Used: Flowers, leaves, fruits Active Compounds: The leaves, flowers, and berries of hawthorn contain a variety of bioflavonoid-like complexes that appear to be primarily responsible for the cardiac actions of the plant. Bioflavonoids found in Hawthorn include oligomeric procyanidins, vitexin, quercetin, and hyperoside. Chlorogenic acid and cafffeic acid are the phenolic acid compound in this herb. The action of these compounds on the cardiovascular system has led to the development ofleaf and flower extracts

History Dioscorides, a Greek Herbalist, used Hawthorn in the first century A.D. It went out fashion as a medicine until the 19th century, when an Irish

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physician included them in a secret remedy for heart disease. Years later, the medicine was found to be made from hawthorn berries, which are still prescribed in folk medicine for a variety of heart-related problems - among them high blood pressure and over-rapid heartbeat.

Indication This herb has many pharmacological properties for treatment of several diseases including; angina pectoris, atherosclerosis, congestive heart failure, hypertension (high blood pressure), antispasmodic, cardiac, sedative and vasodilator. This herb is very good when treating either high or low blood pressure by strengthening the action of the heart, and helps many blood pressure problems. It is good for nervous tension and sleeplessness heart disease. Hawthorn may help the heart in several ways. It may open (dilate) the coronary arteries, improving the heart's blood supply. It may increase the heart's pumping force. It may eliminate some types of heart-rhythm disturbances (arrhythmias). It may help limit the amount of cholesterol deposited on artery walls. In Germany, three dozen hawthorn based heart medicines are available. It has become one of the most widely used heart remedies. It is prescribed by physicians to normalize heart rhythm, reduce the likelihood and severity of angina attacks, and prevent cardiac complications in elderly patients with influenza and pneumonia.

Dosage and administration Hawthorn products standardized to contain either 4 to 20 mg flavonoids/30 to 160 mg oligomeric procyanidins, or 1.8 vitexin rhamnoside/10 procyanidins, are recommended. When supplementing with hawthorn make sure to follow the manufacturer's recommendations. Hawthorn extracts standardized for total bioflavonoid content (usually 2.2%) or oligomeric procyanidins (usually 18.75%) are often used. Many people take 80-300 mg of the herbal extract in capsules or tablets two to three times per day or a tincture of 4-5 ml three times daily. If traditional berry preparations are used, the recommendation is at least 4-5 g/day. Hawthorn may take one to two months for maximum effect and should be considered a long term therapy. Physicians prescribe 1 teaspoon of hawthorn tincture upon waking and before bed for periods of up to several weeks. To mask its bitter taste, mix with sugar, honey, or lemon, or mix it into an herbal beverage blend. It can be used at 2 teaspoons of crushed leaves or fruits per cup of boiling water. Steep 20 min. Drink up to 2 cups per day. Hawthorn for heart failure or angina may require at least six weeks of use, three times per day before an effect is noticed.

Safety It is safe for long term use. There are no known interactions with prescription cardiac medications or other drugs. There are no known contraindications to its use during pregnancy or lactation. Large amounts

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of hawthorn may cause sedation and/or a significant drop in blood pressure, possibly resulting in faintness.

Chemopreventive and radioprotective effects ofhawthorn from Iran The genus Crataegus (Hawthorn) is considered as one of the oldest pharmaceutical plants in the world, and it has been widely prescribed or used in medicine 35 • Many pharmacological studies have proven that hawthorn extract have beneficial effects on the cardiovascular system. Hawthorn contains phenolic and flavonoids compounds including chlorogenic acid, epicatechin, rutin, hyperoside and vitexin 36-38 • In particular, antioxidant and radical scavenging activities are suggested as possible modes of action in these compounds 37 • Leskovac et al. showed that treatment of human peripheral blood lymphocytes in vitro with Crataegus monogyna Jacq., fruit extract reduced micronuclei induced by gamma irradiation, but they have tested this extract only in vitro and efficacy ofthis extract was not evaluated in vivo by oral administration 39 • With the many biological effects of hawthorn that these effects related to flavonoids and phenolic compounds in this herb. We studied several experiments for evaluation of this herb for chemoprotective and radioprotective effects in animal and human models. In these experiments we assessed the protective hawthorn extract on genotoxicity induced by cyclophosphamide or gamma irradiation in mice bone marrow cells, as we assessed this extract on genotoxicity induced by gamma irradiation in volunteers' human blood lymphocytes for radioprotective effects. This report is reviewing the recent results that obtained about hawthorn in my laboratory. The ripe fruits of Crataegus microphylla were collected from N eka in the north ofIran. This plant has voucher number is E-27-32 in Faculty of Pharmacy, Mazandaran University of Medical Sciences (Fig 4). Powdered extract was prepared by aqueous alcohol solvent of dried peel fruit. Administration of extract reduced the frequency of micronuclei in polychromatic erythrocyte (MnPCEs) in bone marrow cells induced by cyclophosphamide (CP)40. The frequency of micronuclei was increased in all groups of mice treated with CP compared with the control group. In mice treated with the extract and CP, the number of MnPCEs had decreased compared with those treated with only CPo Hawthorn extract at all doses

Fig 4. Image of Hawthorn tree with red fruit

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significantly reduced (p < 0.0001) the frequency of MnPCEs induced by CP treatment. The frequency of MnPCEs was lower in the hawthorn extract + CP group by factors 1.92, 2.48, 2.51, 2.30 and 2.84 for the five doses of extracts 25, 50, 100,200 and 400 mglkg b.w., respectively, than that of the CP treated group. Data showed that hawthorn should have a suppressive action on cyclophosphamide-induced clastogenic effects. Also, the extract was reduced in the spontaneous levels ofMnPCE in mouse bone marrow 40. Determination of PCE/PCE+NCE ratio in CP treated mice showed a pronounced cytotoxic effect of CP on bone marrow proliferation. Treatment of mice with hawthorn extract arrested the CP-induced decline in the PCEI PCE + NCE ratio. Increase in the PCEIPCE+NCE ratio in the extract + CP groups (at doses 100,200 and 400 mg/kg b.w.) was higher than that of CP alone group (p < 0.001). There was a dose-dependent effect of hawthorn extract at doses 50 and 100 in increasing the PCEIPCE+NCE ratio. Our studies showed that hawthorn has excellent antioxidant activity with diphenyl picry hydrazyl (DPPH) method. It was obtained in inhibition of 89 and 91 % at 0.2 mg/ml for BHT and hawthorn extract, respectively40. Consumption of fruits , vegetables and herbs or their phytochemical constituents is recommended as part of a cancer prevention diet. The most important target for ROS in the carcinogenesis process is probably DNA. It is believed that antioxidant properties of these phytochemical materials protects cell from ROS-mediated DNA damage that can result in mutation and subsequent carcinogenesis. The main mechanism for protective effects of hawthorn against DNA damage induced by cyclophosphamide to be phenolic and polyphenolic compounds with antioxidant activity. Ionizing irradiation is the one of the main oxidative stress that generates free radicals and ROS that attack DNA and induce genotoxicty and carcinogenesis. We were evaluated the efficacy of hawthorn extract on genotoxicity induced by gamma irradiation in mice bone marrow cells 22 . A single intraperitoneal (i.p. ) administration of hawthorn extract at doses of 25,50, 100 and 200 mglkg 1 h prior to gamma irradiation (2 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCEs). All four doses of hawthorn extract significantly reduced the frequencies of MnPCEs and increased the PCEIPCE+NCE ratio (polychromatic erythrocytel polychromatic erythrocyte + normochromatic erythrocyte) in mice bone marrow compared with the non drug-treated irradiated control (p < 0.020.00001). The maximum reduction in MnPCEs was observed in mice treated with extract at a dose of200 mglkg22. The radioprotective effect of hawthorn (Cratageus microphylla) fruit extract was investigated against genotoxicity induced by gamma irradiation in cultured blood lymphocytes from human volunteers. Hawthorn ingestion by five human volunteers at dose 500 mg. Peripheral blood samples were collected from human volunteers at 0 (10 min before), and at 1, 2 and 3 h after a single oral ingestion of hawthorn powder extract. At each time point, the whole blood was exposed in vitro to 150 cGy of cobalt-60 gamma irradiation, and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in

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cytokinesis blocked binucleated cell. The mean values of percentage of micronucleated binucleated cells in control samples were 1.6 ± 0.58, 1. 72 ± 0.41, 1.6 ± 0.62, 1.52 ± 0.27, at 0 (before extract ingestion), and at 1, 2 and 3 h after the ingestion of hawthorn extract respectively41. The total micro nucleated binucleated cells values were 44, 25 and 27% fold, less in the 1, 2 and 3 h after the oral ingestion of hawthorn extract. All three times after the administration of oral ingestion, showed its efficiency in significantly reducing the micronucleated binucleated cells. It was usually lower at 1 h, as compared with those at 2 and 3 h, after the oral dose of extract. In cytokinesis-block micronucleus assay, cells that have completed one nuclear division are blocked from performing cytokinesis using cytochalasin-B and are consequently readily identified by their binucleated appearance . Micronuclei are scored in binucleated cells only, which enables reliable comparison of chromosome damage between cell populations that may differ in their cell division. These results showed ingestion of hawthorn protected DNA damage in human lymphocytes against oxidative stress induced by gamma irradiation. The protective effects of hawthorn are related to phenolic acids and flavonoids. Hawthorn has high amounts of phenol and flavonoids compounds. The HPLC analysis showed the fruit Crataegus microphylla constitutes phenolic compounds that include chlorogenic acid (phenolic acid), hyperoside (flavonoid ) and epicatechin (proanthocyanins )41. Epicatechin exerts antioxidant activity and protects hepatocytes against oxidative stress induced by tert-butyl hydroperoxide. Epicatechin increases superoxide dismutase activity, and inhibits the lipid peroxidation and cell membrane damage 42 • Hyperoside has many kinds of biological function such as scavenging ROS, preventing the free radical induced oxidation and increased superoxide dis mutase activity43. Hyperoside has protective effects on myocardial cells 44 . Chlorogenic acid scavenged directly OH free radical in a dose-dependent manner, and it eliminated ROS induced by hydrogen peroxide. The chlorogenic acid has protective properties against oxidative stress induced in neural cell line 45 . Since hawthorn extract has been used extensively as an herbal drug for cardiovascular disease, in addition to being safe; with regard to protective effects of this herb against oxidative stress and genotoxicity induced by cyclophosphamide and ionizing irradiation, this herb may be a useful candidate agent for protection in oxidative stress induced by chemical hazardous compounds as well as protective effects in occupational radiological and radiotherapy personals.

Citrus aurantium Citrus aurantium is the name of a very popular plant in the world. The popular name is bitter or sour orange. It is about five meters tall, with

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scented white flowers. Citrus aurantium is too sour to be popular for eating, but the juice of ripe fruit is used for food sauce in Iran. The flowers of citrus aurantium are prepared as excellent jam in Iran. The flowers are used in tea. Essential oil from the dried peel called of unripe fruit flavors Curacao and Cointreau. The active compound of the fruits of this plant is called synephrine. Synephrine is a part of many cold and allergy medications. Most weight loss and energy supplements, which contain the active compound of Citrus aurantium 46 •

Botanical classification Family: Rutaceae Genus and specie: Citrus aurantium Other names: Bitter Orange, Ch'Eng, Chin Ch'Iu, Hua Chu Hung, Kuang Chu, Orange Bitters, Naranja Agria, Neroli, Petitgrain Parts Used: Fruit juice and Peel Active Compounds: (+ )-auraptenal,4-terpineol,5-hydroxyauranetin, acetaldehyde, acetic-acid, alpha-humulene, alpha-ionone, Alphaphellandrene, Alpha-pinene, alpha -terpineol, Alpha-terpinyl-acetate,alphaylangene,ascorbic-acid, Aurantiamene, aurapten, Benzoic-Acid, Betacopaene, Beta-elemene, Beta-ocimene, Beta-pinene, Butanol, Cadinene, Camphene, Caprinaldehyde, Carvone, Caryophyllene, Cinnamic-acid, Cisocimene, Citral, Citronellal, Citronellic-acid ,Citronellol, Cryptoxanthin, Dcitronellic-acid, D-limonene, D-linalool,d-nerolidol, Decanal, Decylaldehyde, Decylpelargonate, delta-3-carene, Delta-cadinene, Dipentene, Dl-linalool, Dl-terpineol, Dodecanal, dodecen-2-al-(l), Duodeclyaldehyde, EO, Ethanol, Farnesol, formic-acid, Furfurol, Gamma-elemene, Gamma-terpinene, Geranic-acid, geraniol, Geranyl-acetate, geranyl-oxide, Hesperidin, hexanol, Indole, Isolimonic-acid, Isoscutellarein, Isosinensetin, Isotetramethylether, L-linalool, L-linalylacetate, L-stachydrine, lauric-aldehyde, Limonene, Limonin, Linalool,linalyl-acetate, Malic-acid, Mannose, Methanol, Myrcene, Naringenin, Naringin, neral, nerol, Nerolidol, neryl-acetate ,Nobiletin, Nomilin, Nonanol, Nonylaldehyde, Nootkatone,octanol, Octyl-acetate, pcymene, p-cymol, Palmitic- Acid, Pectin, Pelargonic-acid, Pentanol, Phellandrene, Phenol, Phenylacetic- Acid ,Pyrrol ,Pyrrole,rhoifolin, Sabinene, Sinensetin, Stachydrine, Tangeretin, Tannic-acid, Terpenylacetate, Terpinen-4-01 ,Terpinolene, Tetra-O-methyl- Scutellarein, Thymol, trans-hexen -2-al-l, trans-ocimene, U mbelliferone, U ndecanal, Valencene, Violaxanthin

History The most common use of C. aurantium is medicinal rather than culinary. The dried, entire unripe fruit is used in Asian herbal medicine primarily to treat digestive problems. It is called Zhi shi in Chinese, Kijitsu in Japanese, and Chisil in Korean. Dried peel ofthe unripe fruit is also used in Western herbal medicine to stimulate appetite and gastric secretion.

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Indication Antifungal, antioxidant, sedative effects, digestion, anti-nausea, antistomachic, hypolipidemic effects tonic and losing weight effects. The most active components in C. aurantium fruit are synephrine and octopamine. The main claim about C. aurantium usage is weight-loss aid. Few clinical trials have examined the effects of C. aurantium alone or in combination with other ingredients on body weight. Several reviews were displayed that one study satisfied all inclusion criteria for evaluation of C. aurantium consumption on weight losing in 23 healthy subjects. A systematic review after assessment ofthese studies concluded that there is no evidence that Citrus aurantium is effective for weight 10ss46-48.

Dosage and administration There is no definitive knowledge as to the dose of C. aurantium that would be optimal for indication such as weight loss. Many health professionals recommend 1 to 2 g of dried bitter orange peel simmered for 10 to 15 minutes in a cup of water. 120 mg/day ofC. aurantium extract was recommended 48. Three cups are usually recommended as a daily dosage. As a tincture, 2 to 3 ml is usually recommended, also to be taken three times a day. Supplementing with pure bitter orange oil is usually avoided.

Safety Bitter orange is safe in the small amounts found in foods. However, bitter orange is not safe when used in high doses. Bitter orange, which contains synephrine and N -methyltyramine, can cause hypertension and cardiovascular toxicity. Ingestion of bitter orange by 15 healthy adults volunteers, a 900 mg dietary supplement extract standardized to 6% synephrine, or matching placebo, with a one week. Hypertension was higher for upto 5 h after a single dose of bitter orange 50 . A variant angina was observed in a 57 year old man with history of ingestion of bitter orange in a dietary supplement51 • They may interact with some other medicines and can cause adverse effects. Chemopreventive and radioprotective effects ofCitrus aurantium from Iran Citrus belongs to family Rutaceae and several commercial citrus varieties such as sweet orange, grape fruit, lime and lemon have been very popular. The fruits were considered to possess natural compounds with several health benefits. Citrus is containing of high amounts of vitamins, minerals and antioxidant compounds such as flavonoids . Flavonoids are a family of phenolic compounds which have many biological properties, including hepatoprotective, antithrombotic, antibacterial, antiviral and anticancer effects. These physiological benefits of flavonoids are generally thought to be due to their antioxidant and free radical scavenging properties. The main

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flavonoid found in the most cultivated citrus species is hesperidin. This compound can account for up to 5% of the dry weight of the leaf and fruit tissue. Citrus flavonoids were reported to decrease capillary fragility and to improve blood flow, and were actually labeled ''vitamin p". Other therapeutics usages are anticancer and antiulcer 52 ,53. With the excellent efficacy and regular eating ofthese fruits, we studied several experiments for evaluation of this herb for chemoprotective and radioprotective effects in animal. In these experiments we assessed the protective effects of Citrus aurantium extract on genotoxicity induced by cyclophosphamide or gamma irradiation in mice bone marrow cells. In our studies, the ripe fruits of Citrus aurantium var.amara were collected from Arnol in the north of Iran (Fig 5). The peels of the citrus were dried at room temperature and powdered in a grinder. Powdered extract was prepared by aqueous alcohol solvent of dried peel fruit. In this way, 25.5 g of extract powder was obtained (25.5% w/w).

Fig 5. Image of citrus fruit (Citrus aurantium var. amara)

Administration of extract reduced the frequency of micronuclei in polychromatic erythrocyte in bone marrow cells induced by cyclophosphamide (CP)54. The frequency of micronuclei was increased in groups of mice treated with CP compared with the control group. In mice treated with the extract and CP, the number ofMnPCEs had decreased compared with those treated with only CP. Citrus extract significantly reduced the frequency ofMnPCEs induced by CP treatment. The frequency of MnPCEs was lower in the citrus extract + CP group by factors 1.16, 1.5 and 2.8 for the three doses of extracts 100,200 and 400 mg/kg b.w., respectively, than that ofthe CP treated group. Data showed that citrus should have a suppressive action on cyclophosphamide-induced clastogenic effects. Also, the extract reduced the spontaneous levels of MnPCE in mouse bone marrow 55 • Determination of PCEIPCE+NCE ratio in CP treated mice showed a pronounced cytotoxic effect of CP on bone marrow proliferation. Treatment of mice with citrus extract arrested the CP-induced decline in the PCEIPCE+NCE ratio. Increase in the PCEIPCE+NCE ratio in the extract + CP groups (at doses 100, 200 and 400 mg/kg b.w.) was higher than that ofCP alone group (p < 0.001). In this study we showed that administration of citrus extract at doses of 200 and 400 mg/kg b.w. increased the hepatic GSH content up to 6.23 pmole/g

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ofliver. A single i.p. dose of citrus extract administered 1 h prior CP treated caused to increase GSH content which it reduced by CP treatment (p < 0.05 compared to CP administered group). The glutathione is a thiol antioxidant in the cells to help cell against oxidative stress. Our studies showed that citrus have excellent antioxidant activity with diphenyl picry hydrazyl (DPPH) method. It was obtained in inhibition of 80 and 89.2% at same concentration for BHT and citrus extract, respectively54. Oxidative damage is one of the many mechanisms leading to cancer and other chronic diseases. Many compounds found in fruits and vegetables include flavonoids. Flavonoids reduce toxic damage to the critical macromolecules such as DNA. Citrus species are extremely rich in flavonones, a class of compounds, which belong to the flavonoids family55. Our results have shown that citrus peel extract contained high amounts of flavonones. We evaluated the efficacy of C. aurantium extract on genotoxicity induced by gamma irradiation in mice bone marrow cells 21 • A single intraperitoneal (i.p.) administration of citrus extract at doses of 250, 500 and 1000 mg/kg 1h prior to gamma irradiation (1.5 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCEs). All three doses of hawthorn extract significantly reduced the frequencies of MnPCEs and increased the PCEIPCE+NCE ratio (polychromatic erythrocyte/ polychromatic erythrocyte + normochromatic erythrocyte) in mice bone marrow compared with the non drug-treated irradiated control. The maximum reduction in MnPCEs was observed in mice treated with extract at a dose of 250 mg/kg 2.2-fold against side effects of y-irradiation with respect to non-drug-treated irradiated controP. Isolimonic acid and its native form were identified in sour orange seed and these compounds were tested as cancer preventive effects against colon cancer cells. These compounds had potential chemopreventive properties. The significant arrest of cell growth was observed within the treatment of colon cancer cells by these agents56. Citrus peel is a rich source of polymethoxyflavones as major constituents, associated with antiinflammatory, antioxidant and antitumor activities 57 . Antiproliferative effects were observed with C. aurantium juice at concentration of 10% in leukemia cell lines. Citrus juice showed growth inhibition on cancer cellline 58 . Citrus family is contained high amounts offlavonoids. Antioxidative effects of flavonoids are approved but other protective mechanism of these compounds is investigated. Some of flavonoides interact directly with nucleophilic metabolites of polycyclic aromatic hydrocarbon, while others inhibit metabolic activation of prom utagens 59. In our research we have shown that citrus extract increased non protein thiollevel in the cells 54 . The intra cellular thiollevel is accepted to be important in determining the extent of cellular damage induced by stress shock. The intra-cellular GSH concentration may determine sensitivity of the cells to damage produced by the anticancer drug60 . The elevation of GSH level by citrus extract can participate to protect genotoxicity induced by CP in bone marrow cells.

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Flavonoids act either by trapping the initiating radicals or propagating lipid peroxyl radicals and recycling a-tocopherol. The strong antioxidative activity of citrus extract with elevation of GSH level in the cells contributed to reduce genotoxicity induced by toxic compounds. Since citrus has been used extensively as fruit, in addition to being safe; with regard to protective effects of this fruit against oxidative stress and genotoxicity induced by cyclophosphamide and ionizing irradiation, this fruit may be a useful agent for protection in oxidative stress induced by chemical hazardous compounds as well as protective occupational radiological and radiotherapy personals. It is noticed that consumption of high amounts of Citrus aurantium may cause side effects mainly on cardiovascular system that be cautioned usage in high risk patients.

Short view of other herbal medicine and natural products from Iran Saffron is the dry stigmas of the plant Crocus sativus L., belongs to the Iridaceae family and principally grows in Iran. It is widely used as spice for culinary purposes and food colorant. It is used in folk medicine with several pharmacological properties as antispasmodic, eupeptic, gingival sedative, anticatarrant, nerve sedative. It has antitumor, radical scavenging effects. Safranal is a main constituent of the essential volatile oil responsible for the characteristic saffron odor and aroma. It attenuated cerebral ischemia induced by oxidative damage in rat hippocampus and renal ischemiareperfusion induced oxidative damage in rat61 -63 . Tavakol-Mshari showed that ethanolic saffron extract caused apoptosis and cell death in cancerous cell line as HeLa and HepG2 cells. Flow cytometry histogram displayed that saffron induced toxicity and apoptogenic effects on cancer cells but not on L929 as non-malignant control cells64 . Administration of saffron extract reduced genotoxicity and DNA damage induced by methyl methanesulfonate in mice bone marrow cells with comet assay65. These results showed that while saffron has several properties in food production, it has protective effects against genotoxicity and oxidative stress. Hesperidin is a flavonone glycoside, belonging to the flavonoid family. This natural product is found in citrus species. Hesperidin was reported to have many biological effects including anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant effects and decreasing capillary fragility66. We showed that hesperidin reduced genotoxicty induced by gamma irradiation in mouse bone marrow cells when administrated prior gamma irradiation67 . A single intraperitoneal (i.p.) administration of hesperidin at doses of 10, 20, 40,80 and 160 mg/kg 45-min prior to gamma irradiation (2 Gy) reduced the frequencies of micronuleated polychromatic erythrocytes (MnPCEs). Hesperidin has powerful protective effects on the radiation-induced DNA damage and on the decline in cell proliferation in mouse bone marrow 67 • In other study from our laboratory, we showed that administration of hesperidin

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for five consecutive days reduced significantly genotoxicity induced by cylophosphamide as genotoxic and DNA damaging agent. The frequency of MnPCEs was lower in the hesperidin + CP group by factors 2.36 for hesperidin at dose 200 mg/kg. Histological examination of bone marrow showed that hesperidin affected on proliferation and hyper cellularity of immature myeloid elements in bone marrow that reduced by cyclophsopahmide68 • We showed that ingestion of hesperidin (single dose, 250 mg) by human volunteers reduced significantly genotoxicity and micronuclei induced by gamma irradiation on lymphocytes69 • Our results showed that hespridin as natural compound from citrus family has protective effects against genotoxicity induced by ionizing irradiation and hazardous chemical compounds. It is safe for usage in human for chemopreventive effects.

Umbelliprenin is a sesquiterpene coumarine that synthesized by various Ferula species such as Ferula szowitsiana. It has also been found in various plant species consumed as food or used for food preparation such as celery. It has many pharmacological properties such as antibacterial, anticoagulant, antileishmania and anti proliferative activity. The protective studies of umbelliprenin was performed against genotoxicity induced by hydrogen peroxide in human lymphocytes. Ferula szowitsiana was collected from the mountains of Golestan forest (the north of Iran) and umbelliprenin was purified. Human lymphocytes incubated at different concentration of with umbelliprenin and/or H 2 0 2 • The DNA damage induced by hydrogen peroxide was reduced significantly by Umbelliprenin70 • Allium hirtifoZium (Persian Sahllot) belongs to Allium genus (Alliaceae family). The several pharmacological properties were reported such as antibacterial, antifungal, antiviral, antiprotozal and antihelminitic effects. Alliums can be useful in diabetes, hemorrhoids, colds and flu. Allium hirtifolium with the common Persian name of'Moosir', a native edible plant in Iran has been widely used as medicine and condiment predates. It belongs to the same biological genus as Allium sativum (garlic) and other onions. Allicin (diallyl thiosulfonate) is the main chemical constituent in this family. Many organosulfur compounds, the major active extract might exert its chemopreventive effect by inducing apoptosis 71 • Ghodrati showed that anti proliferative activity of Allium hirtifolium extract on cancer cell lines. The bulbs of A. hirtifolium was collected from Isfahan province, Iran. After drying and powdering, this extract was obtained with chloroform solvent. The cultured tumors cells (Hela, MCF-7) were treated with Allicin and A. hirtifolium extract at different concentration. Apoptosis and DNA damage were determined. This herb extract was inhibited cell growth on cancer cell lines; it was stronger than Allicin 71.

Conclusions Human are exposure to several toxic chemical agents and ionizing radiation in the life time. The epidemiology studies showed that prevalence of cancer

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increased in public, therefore it is important to reduce the effects of oxidative stress in organs with daily consumption of herbal medicine. There are several studies to establish the herbal medicine with antioxidant activity reducing side effects particularly DNA damage induced by oxidative stress. It is recommended that the ingestion of herbal medicine with anti oxidative stress properties are beneficial in the life of public for reducing of different cancer in human.

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12 Chinese Herbal Medicine for Coronary Artery Disease

Abstract Despite coronary revascularization and standard antianginal therapy, many patients continue to experience symptoms of stable angina and progression of their disease. Chinese herbal medicine is increasingly being recognized and accepted by more and more people of the world in the treatment of coronary artery disease. Unlike standard antianginal agents, herbal medicine formula possess many pathways to the management of coronary artery disease. These herbal treatments are effective for coronary artery disease with minimal side effects. This article discusses the use of Chinese herbal medicines in the management of chronic stable angina through the use of combination herbal formula therapeutics. Peer-reviewed articles and abstracts were identified from MEDLINE and the China National Knowledge Infrastructure database using the search terms herbs, angina, and pharmacology. The result shows that a lots of Chinese herbal medicines, including simple herbal and combination formulae, are perhaps the ideal therapeutics of choice in the management of coronary artery disease. Key words : Traditional chinese medicinal, Herb, Coronary artery disease, Angina

Introduction Coronary artery disease (CAD) is expected to be the main cause of death globally owing to a rapidly increasing prevalence in developing countries and eastern Europe and the rising incidence of obesity and diabetes in the 1. Institute ofIntegrated Traditional Medicine and Western Medicine, Xiangya Hospital, Central South University, Changsha 410008, China. * Corresponding author: E-mail: [email protected]

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Western world (Venardos et af., 2007). Coronary artery disease causes 38 percent of all deaths in North America and is the most common cause of death in European men under 65 years of age and the second most common cause in women (Nabel, 2003). Despite treatment advances, it remains the leading cause of death (Thom et af., 2006). Approximately half of patients with ischemic heart disease demonstrate chronic stable angina as their initial symptom (Abrams, 2005; Thom et af., 2006). Angina is a symptom that results from an imbalance between myocardial oxygen supply and demand. Current treatments for chronic stable angina focus on increasing myocardial oxygen supply by vasodilation of the coronary artery (e.g., nitrates and calcium channel blockers) or reduction of coronary artery obstruction (e.g., antiplatelet agents), as well as reducing myocardial oxygen demand (e.g., ~-blockers and calcium channel blockers) (Cheng, 2006). Modification of cardiac risk factors to delay the process of atherosclerosis is also important (e.g., antihyperlipidemic, antihyperglycemic, and smoking-cessation therapy) (Cheng, 2006). Furthermore, percutaneous coronary interventions such as balloon angioplasty and stent placement help to revascularize the coronary arteries (Cheng, 2006). These therapies not only reduce angina symptoms, but also reduce the risk of myocardial infarction or death (Cheng, 2006). However, despite revascularization and standard antianginal therapy, 26% to 54% of patients continue to experience symptoms of stable angina and progression oftheir disease (Holubkov et af., 2002; Hueb et af., 2004). Many patients cannot tolerate combination antianginal therapy because of low blood pressure (e.g., combination ~-blocker and calcium channel blocker therapy) or an increased risk of bleeding (e.g., combination aspirin and clopidogrel therapy), and others may not be good candidates for percutaneous coronary interventions. Therefore, an antianginal agent that works through other pathways that can be used in combination with standard therapy or as monotherapy for patients unable to tolerate other agents would be useful. In some asian countries, traditional Chinese medicine (TCM) herbs are playing an indispensable role in the prevention and treatment of diseases due to their particular effectiveness in the orient public life for more than 2000 years (Wang et af., 2008). They are increasingly being recognized and accepted by more and more people of the world in the treatment of CAD (Miller,1998; Simon et af., 2003). These herbal treatments are effective for CAD with minimal side effects. This paper reviews traditional Chinese medical herbal therapies used for the treatment of CAD.

Methods Peer-reviewed articles and abstracts were identified from MEDLINE and the China National Knowledge Infrastructure database using the search terms herbs, angina, and pharmacology. Citations from available articles were reviewed for additional references. Abstracts presented at recent professional meetings were also reviewed.

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Traditional Chinese medicine treatment of CAD Traditional Chinese medicine differs from Western medicine not only in theory and diagnosis but also in interpretation of both normal physiological function and pathological changes in the human body. From Chinese medical theory, TCM emphasizes the harmony between the human body and the illness that is caused by the ''Yin'' and ''Yang'' imbalance resulting from invasion of exogenous factors (Jiang, 2005). TCM recognizes coronary artery disease as being symptomatic of a condition known as heart blood stasis, characterized by sharp pains in the chest, palpitations, an irregular heart rhythm and a darkened tongue (Jiang, 2005; Zhao et al., 2007). Approximately 250 Chinese medicinal herbs are prescribed in the TCM treatment of CAD. According to the literature, the 24 herbs listed below have been used most frequently by clinicians (Zhou et al., 2005; Wu et al., 2007) (Table 1). Based on a large body of chemical and pharmacological research (Shen, 2005; Gu et al., 2005), their mechanisms of action including: (1) Increase coronary blood flow, and lessening the preload and afterload of the heart; (2) Attenuate the procoagulant and prothrombotic state; (3) Antithrombotic effects; (4) Anti-oxidative stress and inhibiting lipid peroxidation; (5) Improve endothelial function; (6) Attenuate inflammatory reaction; (7) Anti-Apoptosis. Table 1. 24 herbs used most frequently in TCM for CAD Pharmacopeia name

Binomial name

Bulbus Allii Macrostemonis Flos Carthami Fructus Aurantii Fructus Crataegi Fructus Schisandrae Chinensis Fructus Triehosanthis Hirudo Lignum Dalbergiae Odoriferae Poria Radix Astragali Radix Angelieae Sinensis Radix Codonopsis Radix Cureumae Radix Glyeyrrhizae Radix N otoginseng Radix Ophiopogonis Radix Paeoniae Rubra Radix Puerariae Lobatae Radix Salviae Miltiorrhizea Ramulus Cinnamomi Rhizoma Aeori Tatarinowii Rhizoma Chuanxiong Rhizoma Pinelliae Semen Persieae

Allium macrostemon Carthamus tinctorius Citrus aurantium Crataegus pinnatifida Schisandra chinensis Trichosanthes kirilouii Whitmania pigra Dalbergia odorifera Poria cocos Astragalus membranaceus Angelica sinensis Codonopsis pilosula Curcuma wenyujin Glycyrrhiza uralensis Panax notoginseng Ophiopogon japonicus Paeonia lactifZora Pueraria labata Salvia miltiorrhiza Cinnamomum cassia Acorus tatarinowii Ligusticum chuanxwng Pinellia ternata Prunus persica

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TCM prescriptions, according to the comparability principle in TCM, are often used to strengthen herbal effectiveness and to mitigate toxic effects. Hundreds of combined formulae to treat CAD have been documented in various historical Material Medica and contemporary journals (Zhou et al, 2005). Currently 10 unique prescriptions for treating CAD are approved by the Chinese State Food and Drug Administration. The most frequently used, compositions and usage interpretations of TCM are listed in Table 2.

Pharmacological studies and clinical survey of 5 widely used TCM herbal treatments in CAD Current research suggests several mechanisms by which TCM herbal treatments effectively treat CAD including: anti-atherosclerotic; antioxidative stress; improving hemorrheology; anti-inflammation and so on. Combined herbal formulae would be expected to incorporate several mechanisms to treat symptoms of CAD. The following section describes the potential mode of action of five frequently used herbal prescriptions for CAD treatment.

Xuefu zhuyu decoction Wang et al. (2005c) reported treating angina pectoris of coronary heart disease in fifty patients with either Xuefu Zhuyu decoction or regular therapy, at the 456th Hospital of People's Liberation Army. Patients received Xuefu Zhuyu decoction or regular therapy for 8 weeks. The efficacy ofXuefu Zhuyu decoction was 93.75%, which was significantly higher than 66.67% in the regular therapy. The pharmacological study suggested that Xuefu Zhuyu decoction could improve the functions of vascular endothelium by lowering the levels of endothelium-derived contracting substances, enhancing the levels of endothelium-derived relaxing substances, and reducing the cell adhesions, and hence to raise the therapeutic effects on UAP (Huang et al., 2007). The other studies showed that the clinical effect ofXuefu Zhuyu Decoction was related with anti-platelet activating effect in vitro (Xue et al., 2008).

Shengmai san Clinical study shows that Shengmai San has a favorable effect for treating CAD (Ichikawa et al., 2003). The study of Wang et al. (2005a) was designed to ascertain whether the possible occurrence of overproduction ofinducible nitric oxide synthase-dependent nitric oxide in the brain and inflammatory cytokines in the peripheral blood exhibited during heat stroke can be reduced by prior administration of Shengmai San. The results suggest that the Shengmai San significantly attenuated the heat stress-induced arterial hypotension, cerebral ischemia, and increased levels of brain inducible nitric oxide synthase -dependent nitric oxide production and serum cytokines formation. Futher, they found that Shengmai San is effective for improving circulatory shock and oxidative damage in the brain during heatstroke (Wang et al., 2005b). And the other reports showed that Shengmai San effectively

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Table 2. TCM formulas and their traditional use in CAD treatments Chinese name

Composition

Xuefu Zhuyu decoction

The formula consists of 11 crude drugs: Semen Persicae; Flos Carthami; Radix Angelicae Sinensis; Rhizoma Chuanxiong; Radix Rehmanniae; Radix Paeoniae Rubra; Radix Cyathulae; Radix Platycodonis; Radix Bupleuri; Fructus Aurantii; Radix Glycyrrhizea; in a ratio of6:1:5:3:6:6:6:5:3:6:3 on the dry weight basis. The formula consists of 7 crude drugs: Radix Bupleuri; Paeonia; Fructus Aurantii; Pericarpium Citri Reticulatae; Rhizoma Chuanxiong; Rhizoma Cyperi; Radix Glycyrrhizae Preparata in a ratio of 2:3:2:2:2:2:1 on the dry weight basis. The formula consists of 5 crude drugs: Fructus Aurantii Immaturus; Cortex Magnoliae Officinalic; Bulbus Allii Macrostemonis; Ramulus Cinnamomi; Fructus Trichosanthis in a ratio of 2:2:2:1:2 on the dry weight basis. The formula consists of 3 crude drugs: Radix Ginseng; Radix Ophiopogonis; Fructus Schisandrae Chinen sis in a ratio of 3:3:2 on the dry weight basis. The formula consists of 14 crude drugs: Radix Ginseng; Radix Scrophulariae; Radix Salviae Miltiorrhizea; Poria; Fructus Schisandrae Chinen sis; Radix Polygalae; Radix Platycodonis; Radix Angelicae Sinensis; Radix Asparagi; Radix Ophiopogonis; Semen Platycladi; Semen Ziziphi Spinosae; Radix Rehmanniae; Cinnabaris in a ratio of 1:1:1:1:1:1:1:2:2:2:2:2:8:2 on the dry weight basis. The formula consists of 3 crude drugs: Fructus Trichosanthis; Bulbus Allii Macrostemonis; Rhizoma Pinelliae in a ratio of 2: 1: 1 on the dry weight basis. The formula consists of 11 crude drugs: Radix Astragali; Radix angelicae sinensis; Radix Paeoniae Rubra; Pheretima; Rhizoma Chuanxiong; Semen Persicae; Flos Carthami; in a ratio of 8:4:3:2:2:2:2 on the dry weight basis. The formula consists of 2 crude drugs: Rhizoma Chuanxiong and Broneolum Syntheticum (Lin et al.,1995). The formula consists of 4 crude drugs: Radix Ginseng; Radix Aconiti Lateralis Praeparata; Rhizoma Zingiberis Recens; Fructus Jujubae in a ratio of 3:1:1:1 on the dry weight basis. The formula consists of 5 crude drugs: Rhizoma Salviae Miltiorrhizea;Rhizoma Chuanxiong; Radix Paeoniae Rubra; Flos Carthami; Lignum Dalbergiae Odoriferae in a ratio of 2:1:1:1:1 on the dry weight basis (Footnotes).

Chaihu Sugan powder

Zhishi Xiebai Guizhi decoction

Shengmai San

Tianwang Buxin pill

Kuolou Feibai Banxia decoction

Buyang huanwu tang

Suxiao jiuxin wan Shengfu decotion

Guan-Xin-Er-Hao

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prevented cerebral oxidative injury in rats when it administered into the duodenum before cerebral ischemia-reperfusion (Ichikawa et al., 2003 and 2005; Wang et al., 2003).

Guan-Xin-Er-Hao Guan-Xin-Er-Hao was orally administered to 15 healthy volunteers at the dose ofO. 75,1.5,3,6 g/kg. It was demonstrated that the oral administration of Guan-Xin-Er-Hao increased coronary flow acutely in a dose-dependent manner without modification of systemic hemodynamic parameters, when compared with a placebo group (Zhao et al., 2007). Subsequently, a systematic studies were carried out (Qin et al., 2009; Zhao et al., 2008; Huang et al., 2009), The above results implied that the absorbed bioactive components of Guan-Xin-Er-Hao is ferulic acid, hydroxyl safflor yellow A, and tanshinol. And these three components are likely to contribute to the ability of GuanXin-Er-Hao in protecting the heart from ischemic injury by inhibiting myocardial apoptosis and caspase-3 activity(Huang et al., 2009). Further, ferulic acid, hydroxyl saffior yellow A, and tanshinol had been widely used in treating for CAD, the synergistical interactions among the 3 components have happened when absorbed into blood (Huang et al., 2009).

Suxiaojiuxin wan Randomised controlled trials of Suxiao Jiuxin Wan compared to standard treatment in people with angina. Studies with a treatment duration> 4 weeks were included. Fifteen trials involving 1776 people were included. There was weak evidence that Suxiao Jiuxin Wan compared with nitroglycerin improved ECG measurements, reduced symptoms, reduced the frequency of acute attacks of angina, reduced diastolic pressure and reduced the need for supplementary nitroglycerin. There was also weak evidence that Suxiao Jiuxin Wan compared with Salvia miltiorrhiza reduced symptoms and improved ECG measurements. There was no significant difference when comparing Suxiao Jiuxin Wan with isosorbide dinitrate both for ECG improvement and for symptom improvement (Duan et al., 2008). The results show that Suxiao Jiuxin Wan appears to be effective in the treatment of angina pectoris and no serious side effects were identified (Cao et al., 2007; Duan et al., 2008).

Gualou xiebai banxia decoction Liu and Zhang (2008) reported treating angina pectoris of coronary heart disease in 117 patients with either Gualou Xiebai Banxia decoction (60 patients) or regular therapy (57 patients). Patients received gualou xiebai Banxia decoction or regular therapy for 15 days. The clinical efficacy of Gualou Xiebai Banxia decoction was 95%, which was significantly higher than 78.9% in the regular therapy. While the electrocardiogram efficacy of Gualou Xiebai Banxia decoction was 81. 7%, which was 59.6% in the regular therapy. The other result also shows that Gualou Xiebai Banxia decoction

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appears to be effective in the treatment of CAD and no serious side effects (Hu, 2001).

Conclusions CAD is one of the most common ailments directly influencing a peoples' quality oflife. Revascularization and standard anti anginal therapy are usually employed rapidly to reduce the symptoms of CAD . However, many patients continue to experience symptoms of stable angina and progression of their disease. Therefore, the traditional Chinese medicine that works through other pathways that can be used in combination with standard therapy or as monotherapy for patients unable to tolerate other agents would be useful. TCM have been used to protect and treat conventional chronic diseases throughout China's long history. In other words, the effectiveness of TCM herbs was first verified in patients and then contemporary scientific technologies have gradually validated their effectiveness. Since Chinese herbal treatments (simple or combinatorial) are effective and often have reduced side effects, medicinal herbs are considered to be an alternative to conventional remedies for many diseases. Herbal prescriptions, containing unique herbal formulations, effectively treat CAD by regulate Spleen and Stomach functions. Herbal formulations improve the benefits of each constituent while minimizing the toxic effects of others, thereby promoting the best therapeutic benefit with minimal side-effects. In fact, most herbs can eliminate intrinsic causes of disease. The precise mechanisms of TCM herbal action need to be further investigated via phytochemical analysis, to identify the active compounds from herbs and herbal formulations and meet the growing demands for herbal medicine and phytotherapies in the coming decades.

Acknowledgements This project was supported by National Science Fund for Distinguished Young Scholars (No. 30325045) and was partly supported by the National Natural Science Foundation of China (No. 30572339).

References Abrams, J. 2005. Chronic stable angina. New England Journal of Medicine 352: 25242533. Cao, S., Yan, X., Zhang, J., Zhang, G., Zhang, Y., La, D. and Sun, F. 2007. Observation of short-term curative effect of Suxiaojiuxin Pill on coronary heart disease and angina pectoris. Chinese Traditional Patent Medicine 29(4): 486-488. Cheng, J.W.M. 2006. Ranolazine for the Management of Coronary Artery Disease. Clinical Therapeutics 28(12): 1996-2007. Duan, X., Zhou, L., Wu, T., Liu, G., Qiao, J., Wei, J., Ni, J., Zheng, J., Chen, X. and Wang, Q. 2008. Chinese herbal medicine suxiao jiuxin wan for angina pectoris. Cochrane Database of Systematic Reviews 1: CD004473.

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Gu, X.Q., Ren, P. and Huang, X. 2005. Traditional chinese medicine protects the heart induced by acute ischemic myocardial injury in rats: the treatment progress on hemodynamic function. Zhong Yao Cai 28(6): 527-529. Huang, Q., Qiao, X. and Xu, X. 2007. Potential synergism and inhibitors to multiple target enzymes ofXuefu Zhuyu Decoction in cardiac disease therapeutics: A computational approach. Bioorganic & Medicinal Chemistry Letters 17(6): 1779-1783. Huang, X., Qin, F., Zhang, H.M., Xiao, H.B., Wang, L.x., Zhang, X.Y. and Ren, P. 2009. Cardioprotection by Guanxin II in rats with acute myocardial infarction is related to its three compounds. Journal ofEthnopharmacology 121(2): 268-273 Holubkov, R., Laskey, W.K and Haviland, A 2002. For the NHLBI Dynamic Registry investigators. Angina 1 year after percutaneous coronary intervention: A report from the NHLBI Dynamic Registry. American Heart Journal 144:826-833. Hu, X. 2001. Clinical Observation on 36 Cases of Angina Pectoris of coronary heart disease treated by GUALOUXIEBAIBANXlA decoction. Hunan Guiding Journal of Traditional Chinese Medicine and Pharmacology 9: 454 Hueb, W., Soares, P.R. and Gersh, B.J. 2004. The medicine, angioplasty, or surgery study (MASS-II): A randomized, controlled clinical trial of three therapeutic strategies for multivessel coronary artery disease: One-year results.Journal of the American College of Cardiology 43: 1743-1751. Ichikawa, H., Wang, L. and Konishi, T. 2006. Prevention of cerebral oxidative injury by post-ischemic intravenous administration of Shengmai San. American Journal of Chinese Medicine 34(4): 591-600. Ichikawa, H., Wang, X. and Konishi, T. 2003. Role of component herbs in antioxidant activity of shengmai san - a traditional Chinese medicine formula preventing cerebral oxidative damage in rat. Am J Chin Med 31(4): 509-521. Jiang, W.Y. 2005. Therapeutic wisdom in traditional Chinese medicine: a perspective from modern science. Trends in Pharmacological Sciences 26: 558-563 Lin, H.W., Xu, Z.F. and Zhao, L. 1995. The kinetic effects on blood flow ofsu-xiao-jiu-xinwan andjiu-sin-tau. Zhong Guo Zhong Xi Yi Jie He Za Zhi 15: 46-47. !iu, Y.H. and Zhang, Y.J. 2008. Clinical Observation on 60 Cases of Angina Pectoris of Coronary Heart Disease Treated by Gualou-Xieba-Banxia Decoction. Zhong Xi Yi Jie He Xin Xue Guan Za Zhi. 7: 12-14. Miller, AL. 1998. Botanical influences on cardiovascular disease. Alternative Medicine Review 3(6): 422-431. Nabel, E.G. 2003. Cardiovascular Disease. New England Journal ofMedicine 349(1): 60-72. Qin, F., Liu, Y.X., Zhao, H.W., Huang, X., Ren, P. and Zhu, Z.Y. 2009. Chinese medicinal formula Guan-Xin-Er-Hao protects the heart against oxidative stress induced by acute ischemic myocardial injury in rats. Phytomedicine 16: 215-221. Simon, B., Bob, F. and Robert, C. 2003. The Treatment of Cardiovascular Diseases with Chinese Medicine. Blue Poppy Press, Boulder, Colorado USA, pp 110-111. Shen, S.L. 2005. The mechanism of action for chinese medicine in coronary heart disease. Chinese Traditional and Herbal Drugs 36(4): 634-636. Thorn, T., Haase, N. and Rosamond, W. 2006. Heart disease and stroke statistics - 2006 update. A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 113: 85-151. Venardos, KM., Perkins, A, Headrick, J. and Kaye, D.M. 2007. Myocardial ischemiareperfusion injury, antioxidant enzyme systems, and selenium: a review. Current Medicinal Chemistry 14: 1539-1549. Wang, L., Nishida, H., Ogawa, Y. and Konishi, T. 2003. Prevention of oxidative injury in PC12 cells by a traditional Chinese medicine, Shengmai San, as a model of an antioxidant-based composite formula. Biological & Pharmaceutical Bulletin 26(7): 1000-1004. Wang, L., Zhou, G.B. and Liu, P. 2008. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic

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leukemia. Proceedings of the National Academy of Sciences of the United States of America 105(12): 4826-4831. Wang, N.L., Chang, C.K, Liou, Y.L., Lin, C.L. and Lin, M.T. 2005a. Shengmai San, a Chinese herbal medicine protects against rat heat stroke by reducing inflammatory cytokines and nitric oxide formation. Journal of Pharmaceutical Sciences 98(1): 1-7. Wang, N.L., Liou, Y.L., Lin, M.T., Lin, C.L. and Chang, C.K 2005b. Chinese herbal medicine, Shengmai San, is effective for improving circulatory shock and oxidative damage in the brain during heatstroke. Journal of Pharmaceutical Sciences 97(2): 253-265. Wang, W.L., Su, Y.M., Yang, R.Y., Zhang, J. and Xu, Y. 2005c. Follow-up efficacy of integrative Chinese and Western drugs on localized scleroderma with vitamin B6 and Xuefu Zhuyu decoction. Chinese Journal of Integration Medicine 11(1): 34-36. Wu, R., Liu, X., Wang, J. and Zhou, X. 2007. Study on law using Chinese drug of famous old docter of traditional Chinese medicine to coronary heart disease based on association rules. Zhongguo Zhong Yao Za Zhi 32(17): 1786-1788. Xue, M., Chen, KJ., Ma, X.J., Liu, J.G., Jiang, Y.R., Miao, Y. and Yin, H.J. 2008. Effects ofXuefu Zhuyu Oral Liquid on hemorheology in patients with blood-stasis syndrome due to coronary disease and their relationship with human platelet antigen-3 polymorphism. Zhong Xi Yi Jie He Xue Bao 6(11): 1129-1135. Zhou, L., Huang, X., Fu, C. and Song, S. 2005. Study on regularity of using compound herbal formulae in treating coronary heart disease. Shanghai Journal of Traditional Chinese medicine 39(2): 65-66. Zhao, H.W., Qin, F., Liu, Y.X., Huang, X. and Ren, P. 2008. Antiapoptotic mechanisms of Chinese medicine formula Guan-Xin-Er-Hao in the rat ischemic heart. Tohoku Journal of Expenmen tal Medicine 216(4): 309-316. Zhao, J., Huang, X., Tang, W., Ren, P., Xing, Z., Tian, X., Zhu, Z. and Wang, Y. 2007. Effect of oriental herbal prescription Guan-Xin-Er-Hao on coronary flow in healthy volunteers and antiapoptosis on myocardial ischemia-reperfusion in rat models. Phytotherapy Research 21: 926-931.

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13 Non-commercial Plants of Medicinal Purposes from the Brazilian Biomes for the Treatment of Gastrointestinal Diseases CLAUDIA HELENA PELLIZZON!*, ARIANE LEITE ROZZAl, PAULO CESAR DE PAULA VASCONCELOS!, MARCIO ADRIANO ANnRE0 2 , WAGNER VILEGAS 3 AND CLELIA AKrKO HIRUMA-LIMA4

Abstract Medicinal plants are used, in many cases, with little or without knowledge regarding their pharmacological and toxicological properties. Researches involving natural products are normally guided by the ethnopharmacological knowledge and have been contributing for the drug improvement leading to the determination of new structures and action mechanisms. In this chapter, we will discuss three medicinal plants from Cerrado - Alchornea glandulosa Poepp. & Endl. (Euphorbiaceae), Byrsonima fagifolia Nied. (Malpighiaceae) and Mouriri pusa Gardn.(Melastomataceae) indicating by ethnopharmacological tools to gastrointestinal disturbs. Based on pre-clinical assays (cicatrisation and cytoprotective process in gastric models) that simulated the gastrointestinal pathogenesis in man these plants proved that chemical and pharmacological research can be of an effective therapies safe and efficient against gastroduodenal diseases. Key words: Alchornea glandulosa, Byrsonima fagifolia , Mouriri pusa, Gastric ulcer cicatrisation, Colitis 1. Sao Paulo State University, Depto. De Morfologia Instituto De Biociencias De Botucatu UNESP, Brazil. 2. Sao Paulo University, Depto. De Fisica e Quimica na F-aculdade De Ciencias Farmaceuticas De Ribeirao Preto USP, Brazil. 3. Sao Paulo State University, Depto. De Quimica Organica - Instituto De Quimica De Araraquara-UNESP, Brazil. 4. Sao Paulo State University, Depto. De Fisiologia Instituto De Biociencias De Botucatu UNESP, Brazil. * Corresponding author: E-mail: [email protected]

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Introduction Medicinal plants are used, in many cases, with little or without knowledge regarding their pharmacological and toxicological properties (Veiga Junior et al., 2005). The phytotherapy in Brazil exists mainly in the informal market, what represents a considerable danger to the population's health due to the lack of necessary controls of identification and/or purity (Andreo, 2008). In function to this problematic, it is necessary the accurate plant identification skills, as well as the knowledge concerning their chemical properties, pharmacological and toxicological mechanisms and the suitable concentration to guarantee the safe and efficacy of this alternative therapy. The plant's biome that will be studied in this chapter is the Cerrado - a region that occupies approximately 21% of the Brazilian territory, what is equivalent to 2 2,031,990 km at the equatorial zone (Fig 1) (Motta et al., 2002). The area of this biome is smaller than the Amazon biome; nevertheless, it is believed that there is a huge number of species (Table 1). The predominant weather in the Cerrado is the Seasonal Tropical, dry winter, with considerable amplitude of temperature, which varies from 10 to 40 ac. The flora of the Cerrado shows strong adaptation to resist along the dry period such thick bark, thick leaves, and high ability to regenerate (http:// www.biodiversityhotspots.org/xp/Hotspots/cerrado/Pages/biodiversity.aspx).

Fig 1. Area occupied by the Cerrado biome in the Brazilian territory. Source: http:// www.wwf.org.br/natureza_brasileira/biomas/bioma3errado/index.cfm

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Table 1. Distribution of the taxonomic group highlighting the number of species in general and endemic of the region ofthe Brazilian Cerrado Taxonomic group Plants Mammals Birds Reptiles Amphibious Fish

Number of species

Number of endemic species

10,000 195 607 225 186 800

4,400 14 17 33

28 200

Modified from http://www. biodiversity hotspots .org/xp/Hotspots/cerrado/Pages/ biodiversity.aspx

Methodology The study goals: GASTROINTESTINAL ALTERATIONS Gastric disturbs are important among the current diseases, being highly debilitating to population in general. In Brazil, in 2001, it was estimated that 10% of the population suffered this disturb (Eisig & Laudanna, 2001). Peptic ulcer is an injured area in the gastric mucosa or duodenal mucosa that may reach the submucosa and it is caused by the action of the gastric juice. The ulcer is the result ofthe unbalance between the protective and injurious elements in the stomach. Among the protective elements, we mentioned the formation of the mucus, bicarbonate, the proper blood flow, antioxidants and prostaglandins among others. The injurious factors include hydrochloric acid, refluxed bile salts, alcohol, foodstuffs , pepsin, H 2 0 2 OR among others (Maity et al., 2003; Wallace, 2008). Gastric and duodenal ulcers are chronic and recurring diseases (Makola et al., 2007) and the most common disturb in the gastrointestinal medical practice (Bafna & Balaraman, 2004). This disease is more common in adults, occurring in 5-10% of the world population. Although it presents a healing probability of up to 95%, the chance of relapse is between 65-80% one year after healing and almost 100% two years after (Fan et al., 2005). Therefore, peptic ulcer has been considered as a new epidemic of the 2pt century (O'Malley, 2003). Many pharmaceutical products have been used for the treatment against ulcer, resulting in the fall of the mortality and morbidity rates, but they are not fully effective and produce several side-effects (Rates, 2001). Anti-secretory drugs such as proton bomb-inhibitor (omeprazole and lansoprazole) and histamine-H 2 -receptor blocker (cimetidine and ranitidine) have been exhaustively used to control the acidic secretion increased and the development of gastric ulcers. However, such drugs result in adverse effects and reincidence at long term (Martelli et al., 1998; Wolfe & Sachs, 2000). Another important gastrointestinal disturbs are ulcerative colitis as well as the Crohn's disease that are intestinal inflammatory diseases characterized by the malfunction ofthe mucosal T cells, abnormal production

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of cytokines and cell inflammation, which lead to colon injury (Fiocchi, 1998). The etiology of the disease remains unknown, although affecting a huge part of the population. One of the hypotheses is that ulcerative colitis is caused by deregulation of the mucosal immune system and pathological responses of the T cells in genetically susceptible individuals (Neurath et al., 2002). Other hypothesis is that disturb in the mucosa barrier is an initial factor and subsequent attacks by colonial commensal bacteria cause the inflammation of the mucosa (Stremmel et al., 2005). Most of current therapies against ulcerative colitis include glucocorticosteroids , sulfasalazine and 5-aminosalicilic acid, immunosuppressive agents and anti-TNF-a monoclonal antibodies. These therapies present a limited efficiency; they are not specifics and generate important clinical side effects. The last remaining alternative is the colostomy (Cheng et al., 2007) that generates a huge discomfort to the patient. Thus, there are priorities of the development of new treatments and discovery of new drugs effective against ulcerative colitis. Thus, there is an increasing interest to alternative therapies such the use of natural products, especially the plant-derived ones (Rates, 2001; Schmeda-Hirschmann & Yesilada, 2005). The majority ofthe plantderived drugs reduce the aggressive factors to the mucosa and it is safe, clinically efficient, more tolerable by the patient, relatively cheap, and globally competitive (Goel & Sairam, 2002). According to the World Health Organization (WHO), almost 65% ofthe world population has incorporated plants as their option in the health care (Farnsworth et al., 1985). The search for new drugs from plant sources is a multidisciplinary study involving the routine biological screening, toxicological evaluation and the development of in vitro and in vivo bioassays that simulated the disease in human. The experimental gastric lesions are observed clearly using the chronic model first described by Takagi et al. (1969) and Okabe and Amagase (2005) and the several acute models such as acute lesions induced by ethanol described by Morimoto et al. (1991) (Fig 2). These methods are very

Fig 2. Stomachic lesions obtained by the models using ethanol (A) model Morimoto et al . (1991), and acetic a cid (B) model Takagi et al . (1969) and Okabe & Amagase (2005). In A, it is noticed the lesions indicated by the arrows and the asterisk shows the non-glandular region of the stomach of the rat. In B, it is observed the lesion indicated by the arrow by of the lesion (#) and the arrows show the healing area.

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functional experimental models to the understanding of the gastric cicatrisation and cytoprotective process in gastric mucosa, and to colon this experimental models are important to understands the cytoprotective process and reduction of lesion area and diarrhoea. Next, we will describe the experimental models.

Gastrointestinal models ofstudy Healing gastric ulcer model: Using the model described by Takagi et al. (1969) and Okabe and Amagase (2005), it is possible to analyse the gastric cicatricial process. Ulcers induced by absolute ethanol: This methodology is based on the modifications made from the work of Morimoto et al. (1991), where is possible to evaluate the gastroprotective effect ofthe tested substances. The aspect of the injury of the gastric mucosa observed in this type of experiment are numerous hemorrhagic points in parallel lesions and intensively red along the greater stomachic axis; this type of the lesion is possibly the consequence ofthe oxygen radical formation from the free radicals and from the neutrophil infiltrates in the gastric mucosa which is one of the strongest injurious factors to the mucosa (Chow et al., 1998). Experimental colitis: Intracolonial administration, in rats, of 10 mg trinitrobenzenesulfonic acid (TNBS) dissolved in a volume of 0.25 ml of 50% ethanol/water (Morris et al., 1989). This model generates an inflammatory process in the rat's large intestine which lasts at least six weeks.

Results Alchornea glandulosa The species Alchornea glandulosa Poepp. & Endl. (Euphorbiaceae) is a 20 meter-tall tree, commonly known as tapia, tanheiro de folha redonda, tanheiro or canela-de-raposa. In Brazil, the use of plants of this genus by the population for therapeutic purposes regarding the treatment of gastric alteration is considerably frequent (Osabede & Okoye, 2003; Calvo et al., 2007). From the fractionalization ofthe methanolic extract ofthe leaves ofA. glandulosa through GPC and purification by HPLC it was possible to observe the presence of gallic acid, methyl gallate, the flavonoids myricetin-3-0-a-Lrhamnopyranoside, quercetin -3-0-~- D-galactopyranoside, quercetin-3-0-aL-arabinopyranoside, quercetin, the biflavonoid amentoflavone, and the alkaloid pterogynidine, identified by comparison oftheir spectroscopic data with those reported in the literature by Calvo et al. (2007) (Fig 3) where their concentrations as a whole may be observed in the Table 2. Through another method of spectroscopic analysis of RMN1H and 13C, and DEPT 900 and 1350, COSY 1H-1H and HETCOR experiments performed by Conegero et al. (2003), they observed the presence of a mixture of steroids

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OH

CO:

OH HO

*

HO

I./.:

OH

OH

OH

R B (2) CBs

(3) (4) (5) (6)

(1)

o

0

R.

R1 a-L-rhamnopyranoside I3-D-galactopyranoside a-L-arabinopyranoside OB

OB B B B

OH ~CH,

OH OH HO HO

=<

NH~CH'

HN

NH~CH, CH,

(7)

(8)

Fig3. Chemical Structure of Alchornea glandulosa: I-gallic acid, 2-methyl gallate, 3amentoflavone, 4-amentoflavone derivative, 5-myricetin 3-0-a-L-rhamnopyranoside, 6-quercetin 3-0-~- D-galactopyranoside, 7-quercetin 3-0-a-L-arabinopyranoside, 8quercetin (Calvo et al., 2007). Table 2. Percentage of the components contained in the total extract of Alchornea glandulosa isolated through HPLC (modified from Calvo et al., 2007) No.

Components isolated through HPLC

1 2 3 4 5 6 7 8

Gallic acid Methyl gallate Amentoflavone Amentoflavone derivative Myricetin 3-0-a- L-rhamnopyranoside Quercetin 3-0-~ -D-galactopyranoside Quercetin 3-0-a -L-arabinopyranoside Quercetin

%

16.69 10.99 5.99 7.34 16.22 17.25 21.07 3.25

B-sitosterol and stigmasterol, loliolide, guanidic alkaloid N-l,N-2,N-3triisopentenylguanidine and corilagin. Such chemical variation may be due to the seasonality of the plant's sampling and plant's location.

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It carried the toxicity assay using the model of Souza-Brito (1994), where it was used an oral dose of 5 glkg from the methanolic extract of Alchornea glandulosa and mice were observed during 14 days. After this period, the animals were sacrificed and weighed and from them extracted the main organs target of toxicity. In the work of Calvo et al. (2007), there were not observed significant alterations in the body weight, kidneys, spleen and heart nor caused death. There was no alteration of the parameters related to the kidneys (urea and creatinine) or liver (AST - aspartate aminotransferase and ALT - alanine aminotransferase). Based on the absence of toxicological activity that the methanolic extract of Alchornea glandulosa was carried, the study of the effect of the different doses using the following models of ulcer induction by ethanol in rats and ulcer induction by HCliethanol and non-steroidal anti-inflammatory (NSAIDs) in mice was carried. The doses-response curve of250, 500 and 1000 mglkgwere selected for the antiulcerogenic activity test ofthis plant (Lima et al., 2004). Through these methods, it is possible to verifY the gastroprotective action of this extract in experimental assay (Lima, 2006) (Table 3). This species also has anti secretory effect by decreased total gastric acid content from gastric juice. This anti secretory action joined with gastroprotective effect increase the antiulcer effect of this medicinal species.

One of the possible cytoprotective action mechanisms of the methanolic extract of Alchornea glandulosa must be due to the presence of Table 3. The effect of different doses of the methanolic extract of Alchornea glandulosa in different models of gastric ulcer induction in rats and mice. Method Ethanol (Rats)

Treatment (p.o.) Salina Lanzoprazole

Aglandulosa Hey Ethanol (Mice)

Salina Lanzoprazole

Aglandulosa NSAID (Mice)

Salina Lanzoprazole

Aglandulosa

Dose (mg/kg) 30 250 500 100 30 250 500 100 30 250 500 100

Number of animals 5 5 4 4 4 8 6 5 5 5 5 5 5 5 5

Lesion index 89.0 35.40 * 79.25 37.75* 13.0* 71.0 30.0** 25.80** 38.40* 48.60 50.8 12.8** 11.20** 33.8** 58.0

Inhibition (%)

60 11 57 85

58 64 46 32 75 78 34 -14

Results expressed as means. Ethanol: Anova, Dunnett Test * P
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quercetin and gallic acid - efficient in the presence of ulcers due to its antioxidant property (Gonzalez et al., 2001). Kahraman et al. (2003) revealed that quercetin inhibits the development of gastric ulcers when administering ethanol orally. In the model using the NSAIDs, the piroxicam is the injurious drug to the gastric mucosa, since it is the COX-1 preferential inhibitor. The cyclooxygenase (COX) is constitutive secretory pathway in the gastrointestinal tract and it is important to the mucosal integrity maintenance, and the enzyme fundamental to the prostaglandin pathway (Halter et al., 2001). It was observed that the methanolic extract of A. glandulosa presents important gastroprotective effect considering the dose of 250 mg/kg presented in Table 3. But the gastroprotective effect was decreased in higher dose of A. glandulosa. This result shows that the compounds present in the extract has inflammatory property; the increase of the concentration of flavonoids may change the properties from antioxidant to pro-oxidant (Gracioso et al., 2002). Repetto and Llesuy (2002) describe that the low concentration of phenolic compounds stimulate the synthesis of prostaglandin, while high concentrations of such compounds inhibit the production of prostaglandins. This fact may justify the noneffectiveness of the high doses in the gastric protection (Table 3) and confirm the data of Banerjee et al. (2002), in which amentoflavone and quercetin suppressed the biosynthesis ofPGE2 through the downregulation of COX2/iNOS expression. It was also performed the analysis of the gastric ulcer cicatrisation process through the methanolic extract of Alchornea glandulosa 250 mg/ kg in chronic model of Takagi et al. (1969), model that is similar to a human chronic ulcer, where it was possible to observe macroscopically that the injured external area has not presented alteration when compared with the check groups; however, there is a decrease of the internal border area, where it is noticeable a cicatricial rate of 43%. Microscopically, at the region of the border of the ulcer, there is a complete alteration of the glandules of the gastric mucosa where it is observed a huge dilation and the presence of mucopolysaccharide in these glands, becoming similar to the content secreted by the glands of the animals treated with cimetidine (Fig 4). Another important element for the cicatrisation process is the stimulus to the cell proliferation (Tarnawski et al., 2001), process dependent of several factors such the blood platelet-derived growth factor, fibroblast growth factor and endothelium growth factor that stimulate the vascularization and the cell proliferation (Tatematsu et al., 2003). Using the methanolic extract ofAlchorneaglandulosa in the treatment of gastric ulcer, there was observed the presence of positive PCNA cells, which corroborate with the data of Kitajima et al. (1993) that demonstrated the increase of the proliferative activity in the cicatrisation of the ulcer by PCNA methods.

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Fig 4. Photomicrograph of stomach of rats treated with cimetidine (a) and methanolic extract of AZchornea gZanduZosa (b). Where it is possible to notice the healing process pointed by the arrow.

It is noticed a significative increase ofPCNA cells at the basal region ofthe gland_ It is important to highlight that the action ofthis plant in the healing process of the gastric ulcers promotes mucous secretion and cell proliferation, what differs from a drug of reference used for gastric ulcersthe cimetidine, which inhibits the cell proliferation in cell lineages under culture. According to Finn et al. (1996), there is evidence of dependency of the stimulus ofthe H2receptor for the cell proliferation. Thus, the Alchornea glandulosa is an important plant for future studies regarding the cicatricial effects in gastric ulcers, since the action mechanism is related to the increase ofthe production of mucus and the cell proliferation without causing visible toxic signals.

Byrsonima fagifolia Byrsonima fagifolia Nied. (Malpighiaceae) is an important Brazilian herb known as murici or murici-do-mato whose fruits are used as food. The leaves and bark infusions are used in folk medicine as anti-emetics and diuretics, and as treatments for ulcers, gastritis and diarrhoea (Silva et al., 2000). Lima et al. (2008) investigated the gastroprotective, healing activities against gastric ulcer, anti-diarrhoeal, antimicrobial and mutagenic activities of the methanolic extract from Byrsonima fagifolia leaves. Through HPLC, there were identified in the extract the following important phenolic compounds: gallic acid, and quercetin (Fig 5) and another compounds methyl gallate, quercetin-3-0-~-D-glycopyranoside,quercetin-3-0-~-D xylopyOH COOH 6 5

HO

OH 8

HO

6' 6

Gallic acid MW170

OR OH

0 Quercetin

Fig 5. Chemical structure of some compounds present in the extract ofleaves of Byrsonima fagifoZia (Sannomiya et aZ., 2007)

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ranoside, quercetin-3-0-(2"-galloyl)-p-D-galactopyranoside and quercetin -3O-(2"-galloyl)-p-D-glycopyranoside. The extract presented antioxidant activity in the model using TLC silica-gel plates sprayed with p-carotene and DPPH reagents. All the compounds identified from Byrsonima fagifolia have a catecholic group in their structures, which may explain the antioxidant properties observed during the TLC test (Azuma et al., 2000). Byrsonima fagifolia has presented gastroprotective effect against ulcers induced by ethanol at the doses of 500 or 1000 mglkg and induced by HCIethanol in the doses of 250, 500 or 1000 mglkg in rodent. This effect could be mediated by the ability offlavonoids to scavenge free radicals. Byrsonima fagifolia did not exert anti-ulcer action by an anti-secretory mechanism like cimetidine, as it has not shown significative gastroprotection in the hypothermic-restraint stress model. Byrsonima fagifolia has not shown gastroprotetive activity in the NSAIDs model in none of the three doses previously mentioned, what suggest that the gastroprotective mechanism also does not involve endogenous prostaglandins. The administration of the NO-sintase inhibitor attenuate the gastroprotection suggesting that the endogenous NO participates in the gastroprotective effect of Byrsonima fagifolia. There are also involvement of sulphydryl compounds (SH) in gastroprotective effect that strengthened the gastric mucosa barrier against injurious agents (Lima et al., 2008). The extract is effective not only in the gastroprotection but also in the cicatrisation of gastric ulcers induced by acetic acid in rats, where Byrsonima fagifolia produced lesion healing of 45% (external area) and 42% (internal area) compared to the vehicle treated control group (Fig 6). These values are similar to those obtained using cimetidine, a standard drug (58 and 60%, respectively). In 14 days of treatment, there were not observed visible signs oftoxicity, there was no alteration in the organ's weight (heart, lung, liver, kidneys and spleen) neither in biochemical renal or hepatic parameters oftoxicity (Lima et al., 2008). There was observed that the compounds responsible for the gastroprotective effects are concentrated in the ethyl acetate fraction and not in the aqueous fraction, which has not presented gastroprotection in the dose of 100 mglkg, while the ethyl acetate fraction exerted significative

• Fig 6. Histological analyses of rat's stomach treated with (A) vehicle or (B) Byrsonima fagifolia (500 mg/kg) stained with hematoxylin and eosin. The arrow in (A) indicates the lesion area, and arrowhead in (B) the regeneration area.

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inhibitions in the lesion inductions at respective doses of 50, 100 or 200 mg/ kg. Phytochemical analyses ofthe acetate fraction yielded gallic acid, methyl gallate, quercetin-3-0- ~-D-glycopyranoside, quercetin-3-0-~-D­ xylopyranoside, quercetin-3-0-(2"-galloyl)-~- D-glycopyranoside and quercetin3-0-(2"-galloyl)-~-D-galactopyranoside. Thus, it is probable that the presence of these phenolic compounds in Byrsonima fagifolia may be involved in ulcer-preventing activity, since flavonoids possess important anti-ulcer activity in several experimental models of gastric ulceration (Deshpande et al., 2003). Byrsonima fagifolia also displayed significant activity against castor oil-induced diarrhoea, which was comparable to the anti-diarrhoeal agent as loperamide. Castor oil increases peristaltic activity and alters the permeability ofthe intestinal mucosa (Palombo, 2006). The gastroprotective, healing and antidiarrhoeal effects of B. fagifolia was also followed by antimicrobial action. The minimal inhibitory concentration (MIC) of ethyl acetate fraction against Helicobacter pylori or Staphylococcus aureus was 0.25 mglmL and this result represents important antimicrobial action of B. fagifolia. This species has not presented mutagenic effects against the bacterium Salmonella typhimurium strains in the Ames test for methanolic extract, aqueous and acetate fractions usingTA98, TA97a (frameshift mutation), TA100 and TA102 (base-pair substitution) strains, with and without microsomal activation (Lima et al., 2008). Finally, Lima et al. (2008) proved the following effects: anti-oxidant, gastroprotective and healing against gastric ulcers (involving NO and SH, but not involving the prostaglandin and its mechanism is not anti-secretive), anti-diarrhoeal, non-toxic and not-mutagenic from this important medicinal species from Cerrado Bioma.

Mouriri pusa The species of Mouriri pusa Gardn. (Melastomataceae) is found frequently in the Cerrado (in the States of Piaui, Ceara, Pernambuco, Bahia, and Minas Gerais) (Fig 7). It is commonly known as pu<;a,jabuticaba do mato in Goias State, manapu<;a or mandapu<;a in Minas Gerais State, and in Mato Grosso State asjabuticaba do cerrado and moroso cigano in Ceara State. This species is a 5-7 meter-tall tree that presents: small leaves, ovate, hard and petiolate; pentameric flowers, white or pink with round apex and dry berrylike fruit, black, smooth, with 1 or 2 seeds. It presents a comestible fruit that is produced from September to October, which is very appreciated by the natives ofthe Northern Brazil (Fig 8). Its wood is used to build stake (Correa, 1984). Through a ethnopharmacological survey species of the Cerrado of To cantins State performed by Silva et al., 2000, the Mouriri pusa was cited several times by the local population as useful in the treatment against digestive tract disturbs, ulcers, gastritis when using its aerial parts to make tea.

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Fig 7. Photo ofthe flower of Mouriri sp.

Fig 8. "Pw;:a", comestible fruit of Mouriri pusa

Phytochemical studies of the methanolic extract (polar) of Mouriri pusa have shown that the main constituents are condensed tannins (70%).

There are present the traces ofhydrolysable tannins, saponins and terpenes (6%). More, there was detected a huge variety of flavonoids (13%), whose phytochemical study resulted in the isolation and or identification of a flavone, two flavonoids, three flavonols, twelve flavonol monoglycosides and three flavonol di-glycosylated (Fig 9). The remarkable presence of phenolic substances, overall flavonoids and tannins, gives to the species a potential anti-oxidant. Data point that the catechins, condensed tannins and hydrolysable tannins have the property of capturing the free radicals of oxygen, which are important in the antiinflammatory process (Hatano el al., 1989). In experiences with animals, it was observed that there was no significative alteration in the behavior, in the weights of the organs or in the body weight of the animals treated in comparison to the check group. N either alterations in serological biochemical parameters such as creatinine, urea, AST, ALT and gamma-GT was observed (Vasconcelos et at., 2008 A). With regard to the mutagenicity, the extract has in vitro mutagenic effect for the TA98, TA100 and TA97a cell lineages, with and without metabolic activation. On the other hand, it was not mutagenic for the TA102lineage (Santos et at., 2008). These results support a more deepened study to better establish parameters for the safe utilization ofthe medicinal plants.

Flavonoid

Content 0.39

H

H

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OH OH

H

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1.96

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0.49

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Kaempferol-3-0-a-arabinopyranose

0.29

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Quercetin-3-0-a-arabinopyranose

0.39

H

H

Kaempferol-3-0-J3-xylopyranose

0.20

OH

H

Quercetin-3-0-J3-xylopyranose

0.73

H

H

Kaempferol-3-0-J3-g1ucopyranose

0.20

Myricetin -3-0-J3-g1 ucopyranose

0.88

H

Quercetin -3-0-a-arabinofuranose

0.39

~

Quercetin-3-0-J3-galactopyranose

4.41

Myricetina-3-0-J3-galactopyranose

0.88

H H

HO~O\ H \ =T~H HO

H

,OH

H

HO~< H HO

O H \

OH

OH

OH

H

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1-

OH H

OH

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(%)

H 0

H

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.

Fig 9 Contd. ..

RJ

R2

OH

OH

H

H

Flavonoid

Ra

oo¥Z'y" H

m J#:" 0 H

H

o

Myricetin-3-0-a-rhamnopyranose

0.49

Kaempferol-3-0-a- L-rhamnopyranosy1(6 l)-~-D-galactopyranose

0.49

Kaempferol-3-0-a-L-rhamnopyranosy1(6 l)-~-D-glucopyranose

0.49

.

H

o

t>:)

c..-

o

HH C

OH

Content (%)

H

H

H

H

.

H

H

OH

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m

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o

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.

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~

~

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0

OH

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. -9" "-.,.....__..oH

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I

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I

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II'

Quercetin -3-0-a-L-rhamnopyranosyl(6 ll-~-D-glucopyranose 4' ,5,6-trihydroxi-7 -metoxyflavone 6,8-dihydroxikaem pferol-3-0-~gal acto pyranose (- )-epicatequin (+ l-epicatequin

0.98 0.24 0.24

~

!""

~

I

~

'"1

0.49 0.49

Fig 9. Flavonoids from the polar extract of Mouriri pusa and its content in the flavonoid fraction (Source: Andreo, 2008)

~

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'"

~

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231

Confirming the efficacy of the popular use of the plant, the polar extract of Mouriri pusa at the doses above 250 mg/kg exerts a significative gastroprotection against the ethanol, acidic ethanol, non-steroidal antiinflammatory drug and stress, with protections varying from 51 until 100%. Such protection as demonstrated experimentally is due to the local action of the extract, mainly, not being observed by systemic action (Andreo et al., 2006). It was noticed that Nitric Oxide (NO) participates in the gastroprotective action ofthis extract (Andreo et al., 2006). The NO participates in the gastric defense mechanisms regulating the mucosal blood flow. It promotes vasodilatation in the gastric microcirculation during the acid secretion (Pique et al., 1992). The endogenous NO contributes also in the inhibition ofthe acid secretion. Furthermore, the NO regulates the secretion of the mucous ofthe gastric epithelial cells (Esplugues et al., 1993).

The extract of Mouriri pusa possibly acts through the sulphydryl compound that strengthened the gastric mucosa barrier (Andreo et al., 2006). Gastric injuries induced by ethanol come from multifactorial agents, including those associated to the depletion of endogenous sulphydryl groups, which besides acting as anti-oxidant protect the gastric mucus when uniting its subunits by disulfide linkage. If these linkages are reduced, the mucus becomes more soluble, making the mucosa more susceptible to harmful agents (Avila et al., 1996). The treatment with the polar extract of Mouriri pusa shows efficacy also in the healing of experimental gastric ulcer induced by acetic acid. In prolonged treatments (14 or 30 consecutive days), the extract (250 mg/kg) exhibits better results than the cimetidine (standard drug against gastric ulcer, blocker of the H2 receptors of histamine). This healing promotion action is involved with the increase of the gastric mucus, angiogenesis, induction of the cell proliferation and neutrophil and mast cell mobilization (Vasconcelos et al., 2008 A). These beneficial effects against the gastric ulcers regarding the extract, although in high dose (250 mg/kg), were reproduced faithfully also on the part of its fractions oftannins and flavonoids with doses from 5 to 10 times smaller, showing that the active constituents are predominantly in these fractions (Vasconcelos et al., 2007). The condensed tannins constitute a class of polyphenol extensively distributed in the entire plants. Although the antioxidant activity of these substances is higher than the vitamin C or E, its functional properties are little understood. The mechanisms of this activity involve the inactivation of radicals and inhibitory actions upon enzymes. Yoshida et al. (2000) associated the activity against H. pylori, bacterium that is the main cause of gastric ulcer, to the occurrence of tannins.

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The condensed tannins also inhibit the stomachic acid secretion through G cells, besides having the ability to precipitate microproteins at the place of ulceration forming a protective pellicle that avoids the absorption oftoxic substances and resists to the attacks of proteolytic enzymes (John & Onabanjo, 1990; Nwafor et al., 1996). The catechins and flavonoids show anti-oxidant activity similar to vitamins C and E, which also reduce the risk to certain types of cancer (Manfredini et al., 2004). They are considered important cytoprotectors because of a powerful anti-oxidant activity, and cell apoptosis prevention performed by some of them (Spencer et al., 2001). They promote also important vascular benefits (Schroeter et al., 2006). The (-)-epicatechin present in the extract was also useful in either acute or chronic experimental ulcerative colitis, being preventive against the lesion recurrence. It is due, mainly, to its powerful anti-oxidant action of glutathione in colon (Vasconcelos et al., 2008 B), a tripeptide that exerts an important role as antioxidant protecting against the oxidative stress, acting in the detoxification of several electro phi Is and regulating the transcription activity of the genes (Meister, 1985).

Conclusions Global expansion of consumption of alcohol, smokes and non-steroidal anti-inflammatory drugs (NSAID) and inappropriate diets have contributed to growing gastrointestinal etiopathology. In this way, the peptic ulcer is considered a disease of modern times, related to the addictions that are increasingly frequent in the society and to its stressful lifestyle. Treatment with natural products presents promise of a cure. Plants have been raw material for the synthesis of many drugs and they remain an important source of neW therapeutic agents. Cerrado Bioma is one of the major biogeographic regions of the world and also the most threatened. Many of these plants are used as natural medicines by people living in the Cerrado area to treat several diseases. An ethnopharmacological inventory made in the Cerrado of central Brazil showed a high number of medicinal plants used to treat gastrointestinal disturbs. This research is based on ethnopharmacological investigation, followed by the chemical and pharmacological investigation ofthree medicinal plants. The determination of the antiulcerogenic mechanisms we investigated through the effect of the isolated substances (or enriched fractions) on specific receptors, enzymes and substances produced in response to the gastric lesion. Simultaneously, the antioxidant activity of extracts/substance were evaluated mainly those related to the mechanisms of the anti ulcerogenic activity. Additionally, assays for the detection of mucus, prostaglandins, sulphydryl compounds and antimicrobial action against Helicobacter pylori were also evaluated. Our studies shown that the apparent incompatibility between chemical and pharmacological research of a plant species can be

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solved with the strong determination of dealing rationally with the problem - the search of phytomedicine with efficacy and safety of use from gastroduodenal diseases.

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Finn, P. E., Purnell, P. and Pilkington, G.J. 1996. Effect of histamine and the H2 antagonist cimetidine on the growth and migration of human neoplastic glia. Neuropathol. Appl. Neurobiol. 22: 559. Fiocchi, C. 1998. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 115: 182-205. Goel, R.K and Sairam, K 2002.Antiulcer drugs from indigenous sources with emphasis on Musa sapientum, Tamrabhasna, Asparagus racemosus and Zingiber officinale. IndianJ. Pharmacol. 34: 100-110. Gonzalez, E., Montenegro, M.A., Nazareno, M.A. and Lopez, M.B. 2001.Carotenoid composition and vitamin A value of an Argentinian squash (Cucurbita moschata). Arch. Latinoam. Nutr. 51: 395-399. Gracioso, J.S., Vilegas, W., Hiruma-Lima, C.A. and Souza-Brito, A.R.M. 2002. Effects of tea from Turnera ulmifolia L. on mouse gastric mucosa support the Turneraceae as a new source of antiulcerogenic drugs. BioI. Pharm. Bull. 25: 470-491. Halter, F., Tarnawski, A.S., Schmassmann, A. and Peskar, B.M. 2001.Cyclooxygenase 2implications on maintenance of gastric mucosal integrity and ulcer healing: controversial issues and perspectives Gut 49: 443-453. Hatano, T., Edamatsu,R.,Hiramtsu, M., Mori,A.,Fujita, Y, Yasuhara, T., Yoshida, T. and Okuda, T. 1989. Effects ofthe interaction of tannins with co-existing substances. 6. Effects of tannins and related polyphenols on superoxide anion radical, and on 1,1diphenyl-2-picrylhydrazyl radical. Chem. Pharm. Bull. pp. 2016-2021. http://www.bdc.ib.unicamp.br/gamaivisualizarMaterial.php?idMaterial=574 http://www. biodiversityhotspots.orglxplHotspotsicerradolPageslbiodiversity.aspx John, T.A. and Onabanjo, A.O. 1990. Gastroprotective effects of an aqueous extract of Entandrophragma utile bark in experimental ethanol-induced peptic ulceration in mice and rats. J. Ethnopharmacol. 29: 87-93. Kahraman, A., Erkasap, N., Koken, T., Serteser, M., Aktepe, F. and Erkasa, P. 2003. The antioxidative and antihistaminic properties of quercetin in ethanol-induced gastric lesions.Toxicol. 183: 133-142. Kitajima, T., Okuhira, M., Tani, K., Nakano, T., Hiramatsu, A., Mizuno, T. and Inoue, K 1993. Cell proliferation kinetics in acetic acid-induced gastric ulcer evaluated by immunohistochemical staining of proliferating cell nuclear antigen. Clin. Gastroenterol. 17: Sl16-S120. Lima, Z. P., Ballesteros, KV.R., Silva, J.S., Hiruma-Lima, C.A., Rocha, L.R.M., Calvo, T.R., Vilegas, W. and Brito, A.R.M.S. 2004. Efeitos antiulcerogenicos daAlchornea glandulosa. In: Congresso brasileiro de farmacologia e terapeutica experimental, 2004, Aguas de Lind6ia. XXXVI Congresso Brasileiro de Farmacologia e Terapeutica Experimental, Conference Proceeding 299. Lima, Z.P. 2006. Avaliac;ao da atividade antiulcerogenica dos extratos e frac;oes de Alchornea triplinervia e Alchornea glandulosa. Dissertac;ao de Mestrado UNESP/ Botucatu, 200p. Lima, Z.P. 2006.Avaliac;ao da atividade antiulcerogencia dos extratos e frac;oes de Alchornea triplinervia eAlchorneaglandulosa. Dissertac;ao de mestrado IBBJUNESP: 157. Lima, Z.P., Dos Santos, R.D., Torres, T.U., Sannomiya, M., Rodrigues, C.M., Dos Santos, L.C., Pellizzon, C.H., Rocha, L.R., Vilegas, W., Souza Brito, A.R., Cardoso, C.R., Varanda, E.A., de Moraes, H.P., Bauab, T.M., Carli, C., Carlos, I.Z. and HirumaLima, C.A. 2008. Byrsonima fagifolia: An integrative study to validate the gastroprotective, healing, antidiarrhoeal, antimicrobial and mutagenic action. J. Ethnopharmacology 120: 149-160. Lorenzi, H. 1992. Arvores Brasileiras Manual de indentificac;ao e cultivo de plantas arb6reas Nativas do Brasil. Editora Plantarium. Maity, P., Biswas, K, Roy, S., Banerjee, R.K and Bandyopadhyay, U. 2003. Smoking and the pathogenesis of gastroduodenal ulcer-recent mechanism update. Molecular and Cellular Biochemistry 253: 329-338.

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Makola, D., Peura, D.A. and Crowe, S.E. 2007. HellCobacter pylori infection and related gastrointestinal diseases. J. Clin. Gastroenterol. 41: 548-558. Manfredini, V., Martins, V.D. and Benfato, M.S. 2004. Chit verde: beneficios para a saude hum ana. Infarma, pp. 9-10. Martelli, A, Mattlioli, F., Mereto, E., Brambilla, C.G., Sini, D., Bergamaschi, R. and Brabilla, G. 1998. Evaluation of omeprazole genotoxicity in a battery of m vitro and in vivo assays. Toxicol. 30: 29-41. Meister, A 1985. Glutathione synthetase from rat kidney. Methods. Enzimol. 113: 393399. Morimoto, Y., Shimohara, K, Oshima, S. and Sukamoto, K 1991. Effects of the new antiulcer agent Kb-5492 on experimental gastric mucosal lesions and gastric mucosal defensive factors, as compared to those of terpenone and cimetidine. Japan J. Pharmacol. 57: 495-505. Morris, G.P., Beck, P.L., Herridge, M.S., Depew, W.T., Szewczuk, M.R. and Wallace, J.L. 1989. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795-803. Motta, P.E.F., Curi, N. and Franzmeier, D.P. 2002. In the Cerrados of Brazil. Ecology and natural history ofa Neotropical Savanna. Oliveira & Marquis Editor 398 p. Neurath, M.F., Finotto, S. and Glimcher, L.H. 2002. The role of Th1ffh2 polarization in mucosal immunity. Nat. Med. 8: 567-573. Nwafor, P.A, Effraim, KD. and Jacks, T.W. 1996. Gastroprotective effect of aqueous extract of Khaia senegalensls bark on indomethacin-induced ulceration in rats. West African Journal of Pharmacology and Drug Research 12: 45-50. Okabe, S. and Amagase, K 2005. An overview of acetic acid ulcer models - The History and state of the art of peptic ulcer research. Biol. Pharm. Bull. 28(8): 1321-1341. O'Malley, P. 2003. Gastric ulcers and GERD: the new "plagues" of the 21st century update for the clinical nurse specialist. Clin. Nurse Spec. 17(6): 286-289. Osabede, P.O. and Okoye, F.B.C. 2003. Anti-inflammatory effects of crude methanolic extract and fractions ofAlchornea cordlfolia leaves. J. Ethnopharmacol. 89(1): 19-24. Palombo, E.A 2006. Phytochemicals from traditional medicinal plants used in the treatment of diarrhoea: modes of action and effects on intestinal function. Phytotherapy Research 20: 717-724. Pique, J.M., Esplugues, J.v. and Whittle, B.J. 1992. Endogenous nitric oxide as a mediator of gastric mucosal vasodilatation during acid secretion. Gastroenterology 102: 168-174. Rates, S.M.K 2001. Plants as source of drugs. Toxlcon 39: 603-613. Repetto, M.G. and Llesuy, S.F. 2002.Antioxidant properties of natural compounds used in popular medicine for gastric ulcers. Braz. J. Med. Biol. Res. 35: 523-534. Sannomiya, M., dos Santos, L.C., Carbone, V., Napolitano, A, Piacente, S., Pizza, C., Souza-Brito, A.R.M. and Vilegas, W. 2007. Liquid chromatography/electrospray ionization tandem mass spectrometry profiling of compounds from the infusion of Byrsonima fagifolia Niedenzu. Rapid Commun. Mass Spectrom. 21: 1393-1400. Santos, F.V., Tubaldini, F.R., Colus, I.M., Andreo, M.A, Bauab, T.M., Leite, C.Q., Vilegas, W. and Varanda, E.A, 2008. Mutagenicity of Mouriri pusa Gardner and Mouriri elliptlca Martius. Food Chem. Toxicol. 46: 2721-2727. Schmeda-Hirschmann, G. and Yesilada, E. 2005. Traditional medicine and gastroprotective crude drugs. J. Ethnopharmacol. 100: 61-66. Schroeter, H., Heiss, C., Balzer, J., Kleinbongard, P., Keen, C.L., Hollenberg, N.K, Sies, H., Kwik-Uribe, C., Schmitz, H.H. and KeIrn, M. 2006. (-)-Epicatechin mediates beneficial effects offlavanol-rich cocoa on vascular function in humans. Proc. Nat!. Acad. Sci. U.S.A. 103: 1024-1029. Silva, E.M., Hiruma-Lima, C.A and Lolis, S.F. 2000. Levantamento etnofarmacologico no municipio de Porto Nacional, Tocantins. XVI Simposio de Plantas Medicinais do Brasil. Recife, PE, Brazil. Conference Proceeding, 106.

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Souza Brito, AR.M. 1994.Manual de ensaios toxicol6gicos in vivo. Editora da Vniversidade Estadual De Campinas, VNICAMP. Spencer, J.P., Schroeter, H., Kuhnle, G., Srai, S.K., Tyrrell, R.M., Hahn, V. and RiceEvans, C. 2001. Epicatechin and its in vivo metabolite, 3'-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase3 activation. Biochem. J. 354: 493-500. Stremmel, W., Merle, V., Zahn, A, Autschbach, F., Hinz, V. and Ehehalt, R. 2005. Retarded release phosphatidylcholine benefits patients with chronic active ulcerative colitis. Gut 54: 966-971. Takagi, K., Okabe, S. and Saziki, R. A 1969.New method for the production of chronic gastric ulcer in rats and the effect of several drugs on its healing. Jap. J. Pharmacol. 19: 418-426. Tarnawski, A, Szabo, I.L., Husain, S.S. and Soreghan, B. 2001. Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways. J. Physiol. (Paris) 95: 337-344. Tatematsu, M., Tsukamoto, T. and Inada, K. 2003. Stem cells and gastric cancer: Role of gastric and intestinal mixed intestinal metaplasia. Cancer Sci. 94: 135-141. Vasconcelos, P.C.P., Andreo, M., Hiruma-Lima, C.A, Vilegas, W. and Pellizzon, C.H. 2007. Avalia~ao do efeito gastroprotetor das fra~oes de flavon6ides e de taninos do extrato metan6lico das folhas de Mouriri pusa Gardn. 39° Congresso Brasileiro de Farmacologia e Terapeutica Experimental. Ribeirao Preto. Ref Type: Conference Proceeding Vasconcelos, P.C.P., Kushima, H., Andreo, M., Hiruma-Lima, C.A, Vilegas, W., Takahira, R.K. and Pellizzon, C.H. 2008. A Studies of gastric mucosa regeneration and safety promoted by Mouriri pusa treatment in acetic acid ulcer model. J. Ethnopharmacol. 115: 293-301. Vasconcelos, P.C.P., Seito, L.N., Di Stasi, L.C., Hiruma-Lima, C.A and Pellizzon, C.H., 2008 B. Effect of (-)-epicatechin from Mouriri pusa against acute ulcerative colitis in rats. XX Simp6sio de Plantas Medicinais do Brasil & X Congresso Internacional de Etnofarmacologia. Sao Paulo. Ref Type: Conference Proceeding. Veiga Junior, V.F., Pino, AC. and Maciel, M.AM. 2005. Plantas medicinais: cura segura. Quim. Nova 28: 519-528. Wallace, J.L. 2008. Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn't the stomach digest itself? Physiol. Res. 88: 1547-1565. Wolfe, M.M. and Sachs, G. 2000. Acid suppression: optimizing therapy for gastroduodenal ulcer healing, gastroesophageal reflux disease, and stress-related erosive syndrome. Gastroenterol. 118: S9-S31. Yoshida, T., Hatano, T. and Ito, H. 2000. Chemistry and function of vegetable polyphenols with high molecular weights. Biofactors 13: 121-125.

14 The Treatment Period-Dependent Effects of Ginger Extract (Zingiber officinale) and Ibuprofen in Patients with Osteoarthritis MAsOUD HAGHIGHI 1 *, ALI KHALVAT2 AND TAYEBEH TOLIYAT3

Abstract To evaluate the treatment period-dependent effects of ginger extract and ibuprofen on patients suffering from osteoarthritis (GA). Eighty outpatients (61 men, and 19 women) with symptomatic osteoarthritis, in range of 52-64 years, were included after randomization in a double blind study for two months of treatment. These patients were randomized into two groups of 40, including ginger extract (GE), and ibuprofen aBP) groups. After a washout period of one week (week 0), patients received either 30 mg GE in two 500 mg capsules, or 400 mg IBP three times daily for 2 months. Acetaminophen tablet (325 mg) was prescribed as a rescue analgesic during the study. The clinical assessments included a visual analogue scale (VAS), gelling pain, joint swelling measurement and joint motion slope measurement. Results were evaluated by a 100 mm VAS of pain on movement. Joint motion slope measured by goniometry (normal=130°, limited=120°, and very limited=1100). The results showed that the improvement of symptoms (defined as reduction in the mean of score) was superior on pain relief at the end of two than one month of treatment in VAS, both in GE group (p < 0.0001) and in IBP group (p < 0.0001); and also, in gelling pain scores, both in GE group (p =0.02) and in IBP group (p = 0.0002). However, there was no a significant difference between the ginger extract and the ibuprofen groups in VAS and in gelling pain scores, neither at the end of one nor two month of treatment. Also, 1. Department of Pharmacology, Agriculture Research and Education Organization,

Tonekabon, Iran. 2. Department of Rheumatology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran. 3. Faculty of Pharmacy, Tehran University, Tehran, Iran. * Corresponding author: E-mail: [email protected]

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there was no significant difference between groups in the remaining outcome parameters including: joint swelling measurement and joint motion slope measurement at the end of one or two months of treatment. These results showed a statistically significant effect of GE which was comparable with IBP effect on reducing pain of patients with OA. Key words : Ginger, Zingiber officinale, Ethanol extract, Osteoarthritis, Pain, Alternative medicine

Introduction Osteoarthritis (also known as OA) is the non-inflammatory degenerative joint disease occurring chiefly in older persons, characterized by degeneration of the articular cartilage, hypertrophy of bone at the margin, and changes in the synovial membrane. It is accompanied by pain, swelling and loss of motion of the joint. OA can range from very mild to very severe. It typically affects hands and weight-bearing joints such as knees, hips, feet and back. There is an increasing awareness, both in the medical community and in the public, ofthe use of unconventional or alternative treatment modalities by patients (Eisenberg et al., 1993; Murray & Rubel, 1992). It is understandable that patient suffering from chronic painful disease, in particular osteoarthritis disease, for which there is no cure, will attempt to seek any additional help or treatment modality which might give them some symptomatic relief. Alternative therapies used for the treatment of osteoarthritis include herbs, supplements, and non-drug modalities such as exercise, physical therapy, acupuncture, and electromagnets. Ginger is the rhizome of Zingiber officinale Roscoe (Zingiberaceae), a plant cultivated in many tropical and subtropical countries. The herbal remedy Zingiber officinale (ginger rhizome) has been used for perhaps thousands of years in the Far East to treat diseases, including osteoarthritis. However, no controlled study had been performed by last decade. Ginger is one of the most popular herbal medications for rheumatic diseases (Visser et al., 1992). It has been an important plant for the traditional Chinese and Indian pharmacopoeia. Although one of its indications has been historically to treat rheumatic disorders and although ginger extracts have shown the ability to inhibit arachidonic acid metabolism and anti- inflammatory and/or antirheumatic properties (Srivastava & Mustafa, 1992; Sharma et al., 1994), however, beneficial effects of ginger have been reported casuistically (Srivastava & Mustafa, 1989). Now, there are a few controlled studies on beneficial effects of ginger extract in patients with osteoarthritis (Biddal et al., 2000; Altman & Marcussen, 2001). The treatment currently available for OA affords only palliative care. The prescription of simple analgesics such as acetaminophen to reduce pain generally precedes treatment with non-steroidal anti-inflammatory drugs (NSAIDs). Although the use of NSAIDs in osteoarthritis is highly controversial (Doherty & Jones, 1995), the fact is that many physicians and patients do favor these agents for short

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and long-term use. However, the therapeutic utility of these agents is frequently limited by the development of side effects, especially renal toxicity, gastrointestinal ulceration and ulcer complications. Ulcer complications, such as bleeding and perforation, associated with NSAID therapy often occur without warning and may be life threatening. The aim of the present study was to evaluate the treatment perioddependent effects of ginger extract on pain relief and improvement of functional disability of patients with osteoarthritis.

Materials and Methods Plant material and extract preparation Fresh rhizome of ginger (Zingiber officinale Rosce) was purchased from a local market in India and authenticated by a botanist from Institute of Medicinal Plants, Jehad-e-Daneshgahi. The plant was dried in the shade. The dried rhizome was powdered mechanically and extracted by cold percolation with 95% ethanol for 24 h. The extract was recovered and 95% ethanol was further added to the plant material and the extraction continued. The process was repeated three times. The three extracts were pooled together and the combined extract was concentrated under reduced pressure (22-26 mmHg) at 45-60°C. Solvent free extract (30 g) was equivalent to 1 kg of dried powder of ginger (WIW). The concentrate was weighed and combined with excipients, and also formulated in capsules of 500 mg which each was contained 15 mg of ginger extract (all of the above-mentioned procedures were undertaken in industrial pharmacy department of the Faculty of Pharmacy, Tehran University of Medical Sciences).

Patients' selection and study design This study was approved by the local committee for medical ethics and prior written informed consent was obtained from all patients. Eighty outpatients with OA (61 men, 19 women, aged 52 to 64 years old, mean: 58.5 year) were recruited for this study, which was carried out in the rheumatology clinic of Imam Khomeini hospital in Iran. All patients had complaints of clinical dysfunction and pain due to OA. Radiologically, it was verified that they had OA in the hip or Knee with pain on movement of more than 30 mm on a 100 mm visual analogue scale (VAS) (Huskisson, 1982) (mean 71 mm) on their first visit for this study. The study was a double-blind randomized clinical trial. Exclusion criteria were rheumatoid arthritis, metabolic disorders (diabetes), gastrointestinal disorders (gastritis and duodenum ulcer), neurological disorders, and dementia. The patients were then randomized into two treatment groups of 40, receiving either 30 mg ginger extract divided in two 500 mg capsules daily, or three 400 mg ibuprofen tablets daily for two months. Acetaminophen was used as a rescue medication throughout the study (1 to 3 tablets of325 mg, daily). Treatment with analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) was

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discontinued during the one-week wash-out period before the initiation of the study. The following measurements were taken from the above-mentioned subjects: •

100 mm VAS for assessing the severity of pain;

• • •

Gelling pain; Joint swelling measurements; and Joint motion slope measurements.

Data analysis

The data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to compare group means. A comparison between the groups was carried out by t-test. The differences were considered significant, when p < 0.05. Calculations were performed on a personal computer using the Instat program before breaking of the code.

Results Characteristics A total of 80 patients with OA were enrolled in two treatment groups: ginger extract, and ibuprofen. Table 1 shows a brief characteristic comparison ofthe study groups before the start ofthe treatment (baseline). There were no significant difference between the two groups for mean age, pain, joint swelling measurement, joint motion slope measurement (t-test) and sex (Chi-square). Table 1. Baseline characteristics of patients evaluated at the end of the washout period. Characteristic Mean age (years) Range Sex (man: woman) VAS Gelling pain after rising scores Joint swelling scores Joint motion slope scores

Ginger extract groupN=40

Ibuprofen groupN=40

Pvalue

58.3 (55-64) 29: 11 71.7 ± 3.5 3.65 ± 0.18

53.8 (52--62) 32 :8 71.2 ± 2.4 3.0 ± 0.20

>0.05 >0.05 > 0.05 >0.05

1.25 ± 0.06 1.62 ± 0.07

1.15 ± 0.05 1.45 ± 0.07

>0.05 >0.05

Efficacy During the treatment period, no patient was excluded from this study. At the end of first month of treatment, VAS and gelling or regressive pain after rising changed in comparison to the baseline values (before

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The Treatment Period-Dependent Effects

treatment), both in the ginger extract group and the ibuprofen group, but not in the remaining outcome parameters including joint swelling measurements and joint motion slope measurements (Tables 2 and 3). At the end of first month of treatment, VAS changed from the entry median value of 71. 7 ± 3.5 mm to 30 ± 3.7 mm in the ginger extract group (***p < 0.0001; Table 2) and from 71.2 ± 2.4 mm to 28 ± 3.4 mm in the ibuprofen group (***p < 0.0001; Table 3); which was no significant difference between the two groups (p > 0.05; Fig 1). At the end of second month of treatment, these values were 3.7 ± 1.0 mm both in the ginger extract group and in the ibuprofen group; which were significant differences in comparison to identical at the end of first month of treatment (***p < 0.0001 in both; Tables 2 and 3); but there was no significant difference between the two groups (p > 0.05; Fig 1). Also, gelling or regressive pain after rising scores changed from entry median values of3.65 ± 0.18 to 1.3 ± 0.13 in the ginger extract group (p <0.0001; Table 2) and 3 ± 0.20 to 0.97 ± 0.11 in the ibuprofen group at the end of first month of treatment (* **p < 0.0001; Tables 3). At the end of second month of treatment, these values were 0.27 ± 0.07 in the ginger extract group, and 0.32 ± 0.08 in the ibuprofen group, which were significant differences. in comparison to identical at the end of first month of treatment (*p = 0.02 and ***p = 0.0002 respectively; Tables 2 and 3); but there was no significant difference between the two groups (p > 0.05; Fig 2). Although, the remaining outcome parameters including: joint swelling measurement and joint motion slope measurement were lower both at the end of the first and second month of treatment in comparison to before the start of treatment, however, there were no statistically Table 2. The change in outcome pa rameters at the end of first and second month of treatment with ginger extract Characteristic

Baseline (Before treatment)

Mter one month of treatment N= 40

P value

Mter two months of treatment N = 40

P value

VAS

71.7 ± 3.5 3.65 ± 0.18

30 ± 3.7 1.30 ± 0.13

< 0.0001 * < 0.0001 *

3.7 ± 1.0 0.27 ± 0.07

< 0.0001-

1.25 ± 0.06

1.12 ± 0.05

> 0.05

1 ± 0.01

> 0.05

1.62 ± 0.07

1.55 ± 0.07

> 0.05

1.22 ± 0.06

> 0.05

Gelling pain after rising scor es Joint swelling scores Joint motion slope scores

= 0.02-

* A sterisks indicate sig nificant differences between baseline and the end offirst month

-

of treatment Squares indicate sig nificant differences between the end offirst and second month of treatment

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Table 3. The change in outcome parameters at the end of first and second month of treatment with ibuprofen Characteristic

Baseline (Before treatment)

Mter one month of treatment

P value

P value

N= 40

N =40

VAS Gelling pain after rising scores Joint swelling scores Joint motion slope scores

Mter two months of treatment 3.7 ± 1.0 0.32 ± 0.08

< 0.0001-

71.2±2.4 3.0 ± 0.20

28 ± 3.4 0.97 ± 0.11

< 0.0001 * < 0.0001 '"

1.15 ± 0.05

1.10 ± 0.04

>0.05

1 ± 0.04

>0.05

1.45 ± 0.07

1.40 ± 0.07

>0.05

1.25 ± 0.06

> 0.05

= 0.0002-

* Asterisks indicate significant differences between baseline and the end offirst month of treatment Squares indicate significant differences between the end of first and second month of treatment

-

~

100

E

80

E

.t-

• Before the start of treatment C At the end of one month of treatment o At the end of two months of treatment

'iii

c: Q)

] c:

60

"§

40

'" ~.S

20

'@ p...

'2

..c:

il'c:o

'"

..c: Q

Fig 1. The effects of ginger extract and ibuprofen on the change in the visual analogue scale (VAS). Values are mean ± SEM. There were significant differences between before treatment (baseline) and the end of one or two months of treatment with ginger extract (*** p
significant differences (Tables 2 and 3).

Discussion The effect of ginger extract on reducing pain of patients with osteoarthritis has been shown previously CHaghighi et ai., 2005). The aim of the present study was that to evaluate the treatment period-dependent effects of ginger extract on pain relief and improvement of functional disability of patients with OA. The results of the present study showed that ginger extract was

The Treatment Period-Dependent Effects

243

6

• Before the start of treatment o At the end of one month of treatment 0 At the end of two months of treatment

t>

'in

.,

a"

-

"

4

'@ 0..

bJl

;.§ 0;

o

::

2

Ginger Extract group

I buprofen group

Fig 2. The effects of ginger extract and ibuprofen on the change in the gelling pain intensity. Values are mean ± SEM. There were significant differences between before treatment (baseline) and the end of one or two months of treatment with ginger extract (***p < 0.0001, and *p =0.02, respectively) or ibuprofen (***p < 0.0001 , ***p = 0.0002; respectively).

significantly superior on pain relief and improvement offunctional disability of patients with OA at the end of second than first month of treatment. The active components of ginger are not known certainly, but studies of the lipophilic rhizome extracts have yielded the potentially active components gingerols and shogaols (Bisset, 1994). Ginger contains chemical substances with an anti-inflammatory potential, and the effect might be attributed to the actions of gingerols, shogaols, diarylheptanoids and dialdehyde diterpenes which may inhibit inflammatory prostaglandins (Kiuchi et at., 1992; Kawakishi et at., 1994; Suekawa et at., 1986). These agents are dual inhibitors of eicosanoid synthesis which makes the substances even more interesting in the field of rheumatology (Backon, 1986; Weidner, 1997; Srivastava, 1984). Thus, antiinflammatory effect of ginger may be due to a decrease in the formation of prostaglandins and leukotrienes (Mustafa et at., 1993). It has been suggested that eugenol and ginger oil have potent anti-inflammatory and/or antirheumatic properties on severe chronic adjuvant arthritis in rats (Mascolo et at., 1989; Sharma et at., 1994). Recently, it has been demonstrated the anti-arthritic effects of ginger root extract (GRE) on decreasing the production of inflammatory mediators in sow osteoarthritis cartilage explants (Shen et al., 2003). Also, it has been investigated the comparative effects of GRE on the production of inflammatory mediators, including nitric oxide (NO) and prostaglandian E2(PGE 2), in normal chondrocytes and osteoarthritis chondrocytes isolated from sow cartilage explants (Shen et al., 2005). In this study, it has been shown that GRE decreased both NO and PGE 2linearly in both the normal chondrocytes and osteoarthritis chondrocytes. The

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inhibitory effects of GRE on NO and PGE 2 production by sow osteoarthritis chondrocytes observed in this study suggest an important role for GER as an anti-arthritic agent. It has been discovered that ginger extract blocks activation of proinflamatory mediators, in vitro human synoviocyte assay (Frondoza et al., 2004). Symptomatic effect on pan relief and improvement of functional disability was also reported with the use of ginger extract and other nutriceuticals, including avocado/soybean extracts in the treatment of osteoarthritis CReginster et al. , 2000). These observations indicate that ginger extract offers a complementary and alternative approach to relieve the pain and to modulate the inflammatory process. However, ginger extract should be more extensively investigated. Not long ago, in various randomized, double-blind, placebo controlled trials ginger was shown to reduce symptoms of osteoarthritis (Biddal et al., 2000; Altman & Marcussen, 2001). In one ofthese trials, it has been evaluated the efficacy and safety of a highly purified and standardized ginger extract on reducing knee pain in patients with OA (Biddal et al., 2000). Ginger extract effect was moderate accompanying a good safety profile, with mostly mild gastrointestinal adverse events . A randomized, placebo-controlled, crossover study comparing ginger extract and ibuprofen was performed on 75 individuals with osteoarthritis ofthe hip or knee (Altman & Marcussen, 2001). Patients received either 170 mg ginger extract, 400 mg ibuprofen, or placebo three times per day and were followed for three weeks. The study revealed significant improvement in symptoms for both the ginger and ibuprofen groups before crossover; however, at the study's end there was no difference between ginger and placebo. No side effects were noted in the ginger group; however, side effects prompting removal the study occurred in the ibuprofen group. The finding of our study also indicated efficacy of the ginger extract on reducing pain symptoms in patients with OA without being gastrointestinal adverse events. These observations indicate that ginger extract could be effective on pain relief and improvement of functional disability of patients with OA. In the present study, it has been demonstrated that the two months than one month period oftherapy with ginger extract was superior on reducing pain of patients with ~A . These finding suggest the treatment perioddependent effects of ginger extract on pain relief of patients with OA. The two month periods of therapy with only one dose of ginger extract applied might not have been adequate for all the effects of ginger extract to be detected. More studies are recommended using different doses and duration oftreatment to assess the efficacy of ginger extract for this condition.

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Conclusions The results of our study shows efficacy of ginger extract on reducing pain symptoms to a degree similar to that of NSAID drugs but with fewer side effects.

References Altman, RD. and Marcussen, KC. 2001. Effects of a ginger extract on knee pain in patients with osteoarthritis. Arthritis Rheum 44(11): 2531-2538. Backon, J. 1986. Ginger: inhibition of thromboxane synthetase and stimulation of prostacyclin: relevance for medicine and psychiatry. Medical Hypothesis 20: 271-278. Biddal, H., Rosetzsky, A., Schlichting, P., Weidner, M.S., Andersen, L.A., Ibfelt, H.H., Christensen, K, Jensen, O.N. and Barslev, J. 2000. A randomized, placebo-controlled, cross-over study of ginger extracts and ibuprofen in osteoarthritis. Osteoarthritis and Cartilage 8: 9-12. Bisset, N.G. 1994. Herbal drugs and phytopharmaceuticals: a handbook for practice on a scientific basis. Boca Raton, FL: CRC Press. Doherty, M. and Jones, A. 1995. Indometacin hastens large joint osteoarthritis in humanshow strong is the evidence. J Rheumatol 22: 2013-2016. Eisenberg, D.M., Kessler, RC., Foster, C., Norlock, F.E., Calkins, D.R and Delbanco, T.L. 1993. Unconventional medicine in the United States. Prevalence, costs, and patterns of use. N Engl J Med 328: 246-252. Frondoza, C.G., Sohrabi, A., Polotsky, A., Phan, p.v., Hungerford, D.s. and Lindmark, L. 2004, An In vitro screening assay for inhibitors of proinfalmmatory mediators in herbal extracts using human synoviocyte cultures. In vitro Cell Dev BioI Anim 40(3-4): 95-101. Haghighi, M., Khalvat, A., Toliat, T. and Jallaei, S.H. 2005. Comparing the effects of ginger (Zingiber officinale) extract and ibuprofen on patients with osteoarthritis. Arch Iranian Med 4: 267-271. Huskisson, E.C. 1982. Measurment of pain. J Rheum 9: 768-769 Kawakishi, S., Morimitsu, Y. and Osawa, T. 1994. Chemistry of ginger componds and inhibitory of arachidonic acid cascade. American Chemical Society Symposium Series 547: 244-250. Kiuchi, F., Iwakami, S., Shibuya, M., Hanaoka, F. and Sankawa, U. 1992. Inhibition of prostaglandin and leukotriene bio-synthesis by gingerol and diaryheptanoids. Chem PharmBull40: 387-391. Mascolo, N., Jain, R, Jain, S. and Capasso, F. 1989. Ethnopharmacologic investigation of ginger (Zingiber officinale). J Ethnopharmacol27: 129-140. Murray, RH. and Rubel, A.J. 1992. Physicians and healers - unwitting partners in health care. N Engl J Med 326(1): 61-64. Mustafa, T., Srivastava, KC. and Jeusen, KB. 1993. Drug development reports. Pharmacology of ginger, Zingiberofficinale. Journal of Drug Development 6: 25-39. Reginster, J.Y., Gillot, V., Bruyere, O. and Henrotin, Y. 2000. Evidence of nutrice utica I effectiveness in the treatment of osteoarthritis. Curr Rheumatol Rep 2(6): 472-477. Sharma, J.N., Srivastava, KC. and Gan, E.K 1994. Suppressive effects of eugenol and ginger oil on arthritic rats. Pharmacology 49(5): 314-318. Shen, C.L., Hong, KJ. and Kim, S.W. 2003. Effects of ginger (Zingiber officinale Rosc.) on decreasing the production ofinflammatoty mediators in sow osteoarthritis cartilage explants. J Med Food 6(4): 323-328.

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Shen, C.L., Hong, KJ. and Kim, S.W. 2005. Comparative effects of ginger root (Zingiber officinale Rose.) on the production of inflammatoty mediators in normal and osteoarthrotic sow chondrocytes. J Med Food 8(2): 149-153. Srivastava, KC. and Mustafa, T. 1992. Ginger (Zingiber officinale) in rheumatism and musculoskeletal disorders. Medical Hypotheses 39: 342-348. Srivastava, KC. and Mustafa, T. 1989. Ginger (Zingiber officinale) and rheumatic disorders. Medical Hypotheses 29: 25-28. Srivastava, KC. 1984. Aqueous extracts of onion, garlic and ginger inhibit platelet aggregation and alter arachidonic acid metabolism. Biomed Biochem Acta 43: S335-S346. Suekawa, M., Yuasa, K and Isono, M. 1986. Pharmacological studies on ginger: IV. Effects of (6)-shogaol on the arachidonic cascade. Folia Pharmacologia Japan 88: 239-270. Visser, G.J., Peters, L. and Rasker, J.J. 1992. Rheumatologists and their patients who seek alternative care: an agreement to disagree. Br J Rheumatol 31: 485-489. Weidner, M.S. 1997. HMP-33 ginger extract-a new anti-inflammatory compound. Osteoarthntis Cartilage 5(suppIA): 42.

15 Exploring the Anti-diabetic Effect of Terminalia arjuna in In vivo Animal Model MANONMANI GANAPATHYl*,

K.

BALAKRISHNA2 AND

C.S. SHYAMALA DEVIl

Abstract Since ancient times, plants have been an exemplary source of medicine. A number of Indian medicinal plants have been used from time immemorial for treating various human ailments by the traditional system of medicine. Ayurvedic practitioners used many plants for management and treatment of diabetes, cancer and various other diseases. The medicinal properties of plants have been investigated in the light of recent scientific developments around the world, due to their potent pharmacological activities, low toxicity and economic viability. Diabetes is a chronic metabolic disorder and the incidence rate of diabetes and its complications have increased by several fold throughout the world. Many diabetic patients choose alternative therapeutic approaches such as herbal or traditional medicine, thus making alternative therapy for diabetes a popular remedy. Terminalia arjuna, a reputed heart tonic and a component of several popular drugs of the ayurvedic systems of medicine has been reported to have hypoglycemic activity without troublesome side effects. We have shown that T. arjuna was found to upregulates antioxidant enzymes and antioxidants and concordant downregulation of lipid peroxidation products in alloxan- induced diabetic rats. The precise mechanism of action ofT. arjuna is not completely studied yet. The main objective of the present study was to explore the anti-diabetic effect ofT. arjuna in preclinical animal models. Experimental animals (Male Wistar rats: 180-250 g) were divided into the following groups: control (2% 1. Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 02[;. India. Present address: Medical Research Division, Edinburg Regional Academic Health Center, University of Texas Health Science Center at San Antonio, 1214, West Schunior, Edinburg, Texas-78541, USA. 2. Central Research Institute for Siddha, Arumbakkam, Chennai-600 106, India. * Corresponding author: E-mail: [email protected]

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carboxy methyl cellulose as drug vehicle), extract treated control, diabetic (alloxan-120 mg / kg body weight, i.p.) and extract treated diabetic and each group has six animals. Effective dose of chloroform extract of T. arjuna (350, 400 and 450 mg / kg body weight) were assessed by analyzing the body weight changes, glycosylated hemoglobin (HbAic), fasting blood glucose, glucose tolerance test in experimental animals. Efficacy was evaluated by determining the levels of urea, serum uric acid, alanine transaminase (ALT), aspartate transaminase AST and alkaline phosphatase (ALP), liver carbohydrate metabolic changes, serum and liver lipid profile, glomerular filtration rate (GFR) and creatinine levels. We have found 400 mg / kg bodyweight of the extract showed strong anti-diabetic effect without showing toxic side effects. The extract treated control and control rats showed no significant changes in urea, serum ALT, AST and ALP, thereby suggesting the non-toxic nature of the drug. The significantly (p
Introduction Diabetes is defined as a state in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by the pancreatic hormone, insulin; resulting in an increased blood glucose level. Diabetes is a progressive disease and is one of the major killers in recent times. World Health Organization (WHO) suggests that the global population is in the midst ofa diabetes epidemic with people in Southeast Asia and Western Pacific being mostly at risk. The number of cases for diabetes that is currently at 171 million is predicted to reach 366 million by the year 2030 (WHO, 2006). Although, oral hypoglycemic agents/insulin are the mainstay of treatment for diabetes and are effective in controlling hyperglycemia, but they have a prominent side effects and fail to significantly alter the course of diabetic complications (Rang & Dale, 1991). As the knowledge of heterogeneity of this disorder increases, there is need to look for more efficacious agents

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with lesser side effects. Ayurveda, the traditional Indian herbal medicinal system practiced for over thousands of years have reported to possess several medicinal properties with no known side effects. Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them. A number of medicinal plants have been used for treatment of various human ailments by the traditional system of medicine for the management and treatment of diabetes since the time of Charaka and Sushruta (6 th century BC) (Grover & Vats, 2001; Manonmani et al., 2002; Manonmani et al. , 2005), cancer (Kumar et al., 2007; Ghosh et al., 2008) and various diseases (Ilavarasan et al., 2001; Jainu et al., 2006; Jainu & Mohan, 2008; Qin et al., 2008). More than 400 medicinal plants have been recommended for the treatment of diabetes mellitus by the traditional health care centers (lvorra et al. , 1989; Baily & Day, 1989). Moreover, only a limited number of medicinal plants have received detailed scientific scrutiny thereby prompting the World Health Organisation to recommend that this area be comprehensively investigated.

T. arjuna is a deciduous tree found throughout India (sub-Himalayan tracts of Utter Pradesh, south Bihar, Madhya Pradesh), Deccan region, Myanmar, Srilanka and Southestern countries and grows to a height of6090 feet. The wood is reddish white and heart wood is brown variegated with darker coloured streaks, flowers, sessile in short axillary spikes or interminal panicles, bracteoles are linear and lanceolate shorter than flowers, caducocus with glabrous calyx. It has spreading branches and the leaves are subopposite, ovate, coriaceous and bitter in taste. The thick and white to pinkish gray bark has been used as India's native ayurvedic medicine (Vagbhatta, 1963) for more than 3000 years. T. arjuna belongs to the family Combretaceae (Roxb) Wight and Arn (Nadkarni & Nadkarni, 1976) and is commonly called Marudham in Tamil (Fig 1a). Botanical name (Latin) Sanskrit Common name (English) Hindi Tamil Marathi

Terminalia arjuna Arjuna, N adisarjja Arjuna myrobalan Arjun, Kabu Marudham Arjun Sadada

Fig 1. a . Terminalia arjuna (Combreta ceae) Wight and Am; b. Stem bark

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The plant T. arjuna has been used in Ayurveda for cardiac ailments in ancient times (Nadkarni & Nadkarni, 1976). It is useful in dysentery, haemorrhages, leucorrhoea, urethrorrhea, tumors, inflammations, cirrhosis ofliver, cough, asthma, bronchitis, diabetes, piles, febrifuge, anthelmintic and useful in wound healing and fractures. The juice of fresh leaves is a good remedy for ear ache (Kirthikar & Basu, 1935; Chopra et al., 1956; Warrier et al., 1974; The Wealth of India, 1976; Nadkarni & Nadkarni, 1976; Kurup et al., 1979). Its active constituents include tannins, triterpenoid saponins (arjunic acid, arjunolic acid (King et al., 1954; Honda et al., 1976), arjungenin, arjunglycosides), flavanoids (arjunone, arjunolone, leuteolin), gallic acid, ellagic acid, oligomeric proanthocyanidins, phytosterols, calcium, magnesium, essential oil, zinc and copper (Bone, 1996; Kapoor, 1990) have been reported to possess variety of medicinal properties (Sumitra et al., 2001). T. arjuna is an essential ingredient of many commercially available ayurvedic formulations and are sold as cardiotonics (Nesamony & Oushadyha, 1988). They also show the beneficial effects on hepatic, venereal and viral disease (arjunin - used to treat cardiac necrosis and palpitations). Abana, a polyherbal formulation in which T. arjuna is one of the major ingredients and is reported to possess cardioprotective effect in rats (Sheela Sasikumar & Shyamala Devi, 2000). Recently, both experimental and clinical studies have claimed that the dried bark powder ofT. arjuna has significant beneficial effects in ischemic heart disease (Tripathi, 1993; Dwivedi & Agarwal, 1994; Sumitra et al., 2001). Apart from cardio protective effect, the plant T. arjuna has also been demonstrated to have several medicinal properties such as hypoglycemic (Shaila et al., 1997b), antioxidant (Sheela Sasikumar & Shyamala Devi, 2000; Gupta et al., 2001; Gauthaman et al., 2001; Manonmani et al., 2002; Raghavan & Kumari, 2006), renal and liver disorder (Manna et al., 2006), lowering lipid peroxidation products in diabetic rats (Manonmani et al., 2002; Raghavan & Kumari, 2006) antibacterial (Samy & Ignacimuthu, 2001), antineoplastic (Pettit et al., 1996), antigenotoxic (Scassellatic et al., 1999), hypolipidemic (Shaila et al., 1997a), hypocholesterolemic (Shaila et al., 2000), antimutagenic (Kaur et al., 1997), antianginal activity (Dwivedi & Agarwal, 1994) and stimulate the tumor suppressor gene p53 in human transformed osteosarcoma (UPS) cells in vitro (Avinash et al., 2000). Bhavapriya et al. (2001) has reported that T. arjuna, as one of the components of Aaviraikudineer (a poly herbal formulation), which showed hypoglycemic activity in alloxan-induced diabetic rats. The present study was, therefore, undertaken to investigate the protective role of chloroform extract of T. arjuna against alloxan-induced diabetes using in vivo rat model.

Materials and Methods Chemicals Alloxan was obtained from Sigma Chemical Company, St. Louis, MO, U.S.A. Glycine and cholesterol were obtained from SD'S Lab Chemical Industry,

Exploring the Anti-diabetic effect of Terminalia arjuna

251

Mumbai, India. Acids, bases, solvents and salts used for the investigation were of analytical grade (AR) and were obtained from Glaxo Laboratories, SRL, Mumbai, India and Fischer Inorganics and Aromatics, Chennai.

Plant collection Stem bark of Terminalia arjuna (Fig Ib) was obtained from the local herbal market and duly authenticated by Dr. P. Brindha, Botanist, Central Research Institute for Siddha, (CRIS), Arumbakkam, Chennai, India.

Preparation of the plant extract Shade dried and coarsely powdered stem bark (2 kg) was extracted with chloroform in the cold (48 h). The extract was filtered and distilled on a water bath. A reddish brown syrupy mass was obtained and it was further concentrated in a rotary vacuum evaporator to remove the last traces of the solvent (Yield 10.12 g). The extract thus obtained were used for further studies. The extraction procedure was repeated to get more yield.

Animals Male Wistar rats weighing 180-250 g, procured from the Tamil Nadu University of Veterinary and Animal Sciences (TANUVAS), Chennai, India, were used for the study. Rats were housed in an air-conditioned room with a 12 h lightJdark cycle, received a standard rat chow (Amrut Laboratory Animal Feed, Bangalore) and drank tap water. All procedures complied with the standards for the care and use of animal subjects as stated in the guidelines laid by Institutional Animal Ethical Committee (IAEC), University of Madras, Guindy Campus, Chennai-600 025.

Experimental induction ofdiabetes Male Wistar rats weighing 180-250 g were made diabetic by alloxanisation procedure (Hammarstrom & Ullberg, 1966). The animals were allowed to fast for 16 h and were injected, intraperitoneally, with freshly prepared alloxan (5,6-dioxyuracil monohydrate, USA, 120 mglkg body weight) in 0.9% saline. Alloxan treated animals were allowed to drink 5% glucose solution overnight to overcome drug induced hypoglycemia. Four days later, diabetes was confirmed by estimating blood glucose levels. Rats with basal glycemia ranging between 250-300 mg/dl were considered as diabetic. The blood glucose levels were checked periodically for 45 days to confirm the presence of uncontrolled hyperglycemic condition.

Dose assessment ofT. aTjuna extract The biochemical effects of the plant extract, at different concentrations (350,400 and 450 mglkg b.wt.) on control and experimental diabetic rats were studied to assess the optimum dosage of the extract, which produced normoglycemic effects in the diabetic rats, without any side effects. The body weight changes, fasting blood glucose, oral glucose tolerance test and

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glycosylated Hb levels were analyzed to fix the effective dosage (400 mg/kg body weight) and treatment duration (25 days) (data not shown). Since 400 mg/kg body weight/day of chloroform extract of T. arjuna for 25 days was found to be the most effective dosage and treatment duration, therefore, further studies were carried out with the same dosage for 25 days.

Experimental design The experimental rats were divided into four major experimental groups of six rats in each group. Group I (control) - rats received 2% CMC by orally; Group II (extract treated control) - rats treated with chloroform extract ofT. arjuna (400 mg/kg body weight/day; 2% CMC as drug vehicle) by oral adminstration; Group III (diabetic) - Alloxan (120 mg/kg body weight dissolved in 0.9% saline, single i.p.injection) induced diabetic rats; Group IV(extract treated diabetic) - Alloxan induced diabetic rats treated with chloroform extract of T. arjuna (400 mg/kg body weight/day; 2% CMC as drug vehicle) through oral adminstration for 25 days. The animals were weighed and drug was given through oral administration everyday for 25 days. They were kept under good environmental conditions and food and water were given ad libitum. At the end of the experimental period, the rats were fasted overnight and anaesthetized with anesthetic ether and the blood was collected through cardiac puncture. Blood collected without anticoagulant was used for serum separation. The liver and kidney tissues were dissected out and washed in ice cold saline. Known amount ofthe tissues were homogenized in 0.1 M Tris - HCI buffer, pH 7.4 at 4°C, in a Potter - Elvehjem homogenizer with a Teflon pestle at 600 rpm for 3 min. The homogenate was centrifuged at 3,000 x g for 10 min at 4°C using Sorva1l5 B refrigerated centrifuge. The supernatant was used to measure the biochemical parameters such as carbohydrate metabolizing enzymes and lipid contents .

Biochemical parameters Basic biochemical parameters The blood samples were used to measure the glucose levels (Sasaki & Matsui, 1972). The plasma protein (Lowry et al., 1951) and urea (Natelson et al., 1951) levels were also determined. Serum from the experimental animals were used to measure uric acid (Caraway, 1963), ALT (Mohur & Cook, 1957), AST (Mohur & Cook, 1957), ALP (Kind & King, 1954) levels and to measure lipid profiles.

Carbohydrate metabolizing enzymes The supernatant from the liver homogenate were used to assay the carbohydrate metabolizing enzymes such as hexokinase (Branstrup et al., 1957), pyruvate kinase (Pogsun & Denton, 1967), glucose-6-phosphatase (King, 1965), fructose-1-6-di phosphatase (Gancedo & Gancedo, 1971), glucose6-phosphate dehydrogenase (Ells & Kirkman, 1961) and lactate

Exploring the Anti-diabetic effect of Terminalia arjuna

253

dehydrogenase (King, 1965).

Extraction and estimation of tissue lipids Total lipids were extracted by the method of Folch et al. (1957) using chloroform: methanol mixture (2:1 VN). The extracted lipid were used to measure the total cholesterol (Parekh & Jung, 1970), phospholipids (Bartlett, 1959) by digestion with perchloric acid and estimating the amount of the PI liberated (Fiske & Subbarow, 1925), triacyl glycerol (Rice, 1970), and free fatty acids (Hron & Menahan, 1981) with the color reagent ofItaya, (1977).

Renal function tests The levels of plasma creatinine was estimated according to the method of Broad and Sirota (1948). GFR was estimated based on creatinine clearance test as assessed by 24 h urinary excretion rates of creatinine in relation to plasma concentration. Plasma creatinine (Pcreatmm) and urine creatinine clearance was estimated by calculating the amount of creatinine excreted in 24 h urine sample (U). GFR was calculated from the urine to plasma creatinine ratio: GFR = UIPcreatmme x Vu where Vu is the urine flow rate. Urinary glucose and urinary albumin were estimated by using uristrix and albustrix (Mis. Boerinhger Mannheim, India Ltd.).

Statistical analysis Results are expressed as mean ± S.D. and Student's t-test was performed to assess the statistical significance.

Results and Discussion Effect ofT. arjuna extract on basic biochemical parameters The levels of protein, urea and other basic biochemical parameters were investigated in order to test the toxicity of the drug. As shown in Table 1, we measured blood glucose, plasma protein, urea and serum uric acid in control and experimental rats. There was a significant decrease (p < 0.001) in the levels oftotal protein in alloxan-induced diabetic rats (group III) when compared to control rats (group I) and the levels of protein were brought back to near normal upon treatment with T. arjuna extract in group IV rats (p
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Table 1. Levels of blood glucose, plasma protein, urea, serum uric acid and activities of serum AST, ALT and ALP of control and experimental groups of rats. Values are expressed as mean ± S.D. of six rats in each group. Parameters

Blood glucose (mg/dl) Plasma protein (g/dl) Plasma urea (mg/dl) Serum uric acid (mg/dl) Serum AST (lUll) Serum ALT (lUll) Serum alkaline phosphatase (KA UnitsIL)

Control

112.5±3.2

Extract treated control

Diabetic

Extract treated diabetic

113.8 ± 3.9NS

270.3 ± 4.8'"

169±3.T

7.02 ± 0.11

7.14 ± 0.75 NS

16.73 ± 1.99

16.50 ± 1.29NS

1.81 ±0.03

1.79 ± 0.06 NS

258 ± 9.4B 52.1 ± 5.1 7.51 ±2.31

262.9 ± 1O.49NS 54.3± 5.9 NS 8.61 ± 1.50NS

5.63±0.22 . 41.56 ± 3.76" 3.98 ± 0.20'"

7.0±0.1O'· 27.97 ± 2.97'" 2.99±0.25" .

370.8±19.08'" 286.5±15.80'" 94.5±8.0· " 55.33 ± 5.4<" 22.2±3.0r 11.08 ± 1.49""

Statistical comparison: control vs extract treated control; control vs diabetic; diabetic vs extract treated diabetic, p values: "·p
glucose levels are maintained mainly by insulin that facilitates the uptake, utilization and storage of glucose. During diabetes, the blood glucose levels are drastically increased which results from reduced glucose utilization by various tissues (Soling & Kleineke, 1976). In the present study, the alloxaninduced diabetes as shown by persistant increase in blood glucose level was almost normalized upon extract treatment which may be due to stimulation of glucose utilization by the peripheral tissues or through increased transport of glucose into the cells. Hypoproteinemia is a common problem in diabetic animals and is generally attributed to diabetic nephropathy. Protein serves as a source of nutrition for the tissues and its synthesis and regulation determines normal function. Insulin plays a pivotal role in protein synthesis. Diabetes mellitus shows profound changes in circulating aminoacids and hepatic aminoacid uptake (Felig et al., 1977). The significant decrease of plasma protein in alloxan-induced diabetic rats could be attributed to suppressed protein synthesis. Urea is a non-protein nitrogenous waste product whose level reflects a normal and continued protein metabolism. The increase in synthesis of urea in diabetic rats may be due to the enhanced catabolism of both liver and plasma proteins. Hyperuremia in blood reflects either over synthesis of urea or its decreased excretion. During diabetes, there is an increased protein catabolism with inflow of amino acid to the liver, which facilitates gluconeogenesis and accelerates urea synthesis thereby resulting in hyperuremia. This was observed in the present study where the alloxaninduced diabetic rats showed lowered levels of plasma protein and increased

Exploring the Anti-diabetic effect of Terminalia arjuna

255

levels of urea (Table 1). Such aberrated values were brought back to near normal level upon extract treatment, probably by decreasing proteolysis. Uric acid is the biomarker for the development of diabetic complications. Chou et al. (1998) and Costa et al. (2002) suggested increased uric acid concentration to be a risk factor for cardiovascular diseases. In the present investigation, the levels of serum uric acid in diabetes induced rats were found to be increased (p
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Effect ofT. arujna extract on carbohydrate metabolism Liver is the prime organ for glucose metabolism. Glycolysis and gluconeogenesis are the two complementary events that are involved in glucose homeostasis. In Type I diabetes, simultaneous decrease in the rate of glycolysis and increased rate of gluconeogenesis in hepatic tissues produces more glucose. Glucose phosphorylation is the first step in glucose metabolism which is activated by hexokinase, an inducible enzyme and its activity is regulated by insulin (Scohor et al., 1979). Activities of liver carbohydrate metabolizing enzymes in control and experimental groups of rats are presented in Table 2. The activities of hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase were decreased significantly (p
257

Exploring the Anti-diabetic effect of Terminalia arjuna

Table 2. Activities of hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6phosphatase, fructose-1,6-bisphosphatase and glucose-6-phosphate dehydrogenase in liver of control and experimental groups of rats. Values are expressed as mean ± S.D. of six rats in each group Parameters

Hexokinase (Ilmoles of G6P formed/minlmg protein) Pyruvate kinase (Ilmoles of pyruvate formed! minlmg protein) Lactate dehydrogenase (Ilmoles of pyruvate formed! minlmg protein) Glucose-6phosphatase (Ilmoles of Pi liberated! minlmg protein) Fructose-1,6bis phosphatase (nmoles of Pi liberated!minl mg protein) Glucose-6phosphate dehydrogenase (Ilmoles ofNADPH formed!minlg tissue)

Control

Extract treated control

Diabetic

Extract treated diabetic

263.5 ± 7.3

264.6 ± 7.12NS

120 ± 2.58'"

231.6 ± 6.35'"

10.95 ± 0.48

11.06 ± 0.36 NS

2.29 ± 0.35"--

7.42 ± 0.37'"

174.7±4.85

174.9 ± 4.7NS

280.6±11.62"· 214.2 ± 7.60'"

12.7 ± 1.0

12.61 ± 0.93 NS

35.3 ± 2.6'"

15.07 ± 1.7'"

4.50±0.24

4.55±0.26 NS

11.48 ± 0.17'"

7.42 ± 0.16'"

7.32 ± 0.21

7.38 ± 0.17NS

2.36 ± 0.14'"

4.57 ± 0.12'"

Statistical comparison: control us extract treated control; control us diabetic; diabetic us extract treated diabetic. p values: "'p<0.001 ; NS - Non significant.

concentration. Noguchi et al. (1982) have reported that, the mRNA for PK was diminished in STZ-induced diabetic rats. Lactate dehydrogenase is the enzyme which is involved in the final step of anaerobic glycolysis. LDH oxidizes NADH to form NAD+, which is essentially utilized by glyceraldehyde3-phosphate dehydrogenase as cofactor. During diabetes, the activity ofLDH is significantly elevated (Table 2). Belfiore et al. (1970) have reported that the activity of LDH was drastically increased in diabetic condition by facilitating the continuous supply of NAD+ to glyceraldehyde-3-phosphate dehydrogenase and was concordant with our results. T. arjuna extract treatment for 25 days showed significant elevation (p<0.001) of hexokinase and pyruvate kinase enzyme activities in diabetic rats. Enhancement ofthese enzyme activities by the plant extract suggests

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an increase of glycolytic flux, which stimulates proper oxidation of glucose. The plant extract reduces the significant enhancement of hepatic LDH that may be due to regulation of coenzyme NAD+ required for the enzymic activity or removal ofthe substrate pyruvate for further oxidation in the TCA cycle: Glucose-6-phosphatase is one of the important enzymes in gluconeogenesis and glycogenolysis, which is involved in the regulation of blood glucose level. Fructose-1,6-bisphosphatase catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate which is one of the irreversible steps in gluconeogenesis. In the present investigation, the activities ofthese enzymes are highly elevated (p
Effect ofT. arjuna extract on lipid profiles We have investigated whether the T. arjuna extract induces hypolipidaemic effect through the modulation oflipid moieties. As shown in Fig 2 and Table 3, we observed significantly elevated (p
Exploring the Anti-d iabetic effect of Terminalia arjuna

259

50.00 45.00 40.00 J.

~

35. 00

~

25.00

tL

20.00

E

15.00

1: .",

ao.oo

r-

10.00 5.00 0.00

r'

1" " - . I, i

C o t) trol

Fig 2. Levels ofFFA, TG, PL and TC in liver of control and experimenta l groups of rats. Values are expressed as mea n ± S.D. of six rats in each group

hyperlipidemia. The elevated levels oflipid is either due to defective removal or overproduction of one or more lipoproteins (New et al., 1963; Nikilla & Hormila, 1978) and presents a risk factor for developing coronary heart disease (AL-Shamaony et al., 1994). Diabetes is associated with modified blood lipoprotein pattern and premature atherosclerosis. The abnormal high concentration of serum lipids in diabetes is mainly due to an increased activation ofthe hormone sensitive lipase (McGarry & Foster, 1977; Stanely et al., 1999). The increased level of TG in serum was reported by Zeenat et al. (1997), which causes cardiac depression. Hence, the levels ofTG and FFA are drastically increased during diabetes (Ghosh & Suryawanshi, 2001; Monica et al., 2002) and these levels were consistent with our present data (Fig 2 and Table 3). Hypertriglyceridemia occurs in diabetic conditions resulting in either over production of TG rich lipoprotein or its decreased clearance (Ebara et al. , 1994), which may be due to drop in LPL activity (Howard & Howard, 1994) and thus blocks the catabolism of TG rich lipoprotein and reduces the removal of TG from the circulation (Agarth Table 3. Levels of serum free fatty acid, triacylglycerol, phospholipid and total cholesterol in control and experimental groups of rats (mg/dl). Values are expressed as mean ± S.D. of six rats in each group Parameters

Free fatty a cids Triacylglycerol Phospholipid Total cholesterol

Control

Extract treated control

48 .13 ± 4.39 61.56 ± 1.25 109.3 ± 3.45 65.9 ± 4.3

47 .85±4.19 NS 61.83 ± 1.28 NS 111.0 ± 4.13 NS 66.3 ± 4.0NS

Diabetic

97.99 ± 3.19'" 191.0 ± 4.5'" 157.1 ± 5.16" > 130.1 ± 5.9'"

Extract treated diabetic 61.8 157.0 137.2 109.0

± 3.81 '" ± 3.5'" ± 3.84'" ± 3.2'"

Statistical comparison : control us extract treat ed control ; control us diabetic; diabetic us extract treated diabetic. p values : ·" p
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et al., 1982), whose activity depends only on insulin levels (Nakai et al., 1979; Ohno et al., 2000). Liver plays a pivotal role in lipid homeostasis and is severely affected during diabetes. During diabetes the hormone sensitive lipase (HSL) gets activated and increases the removal of free fatty acids from adipose tissues, thereby providing substrates for TG synthesis, which causes fatty liver (Murthy & Shipp, 1979; Chauhan et al., 1984). Earlier reports suggested that the lipid profile in diabetic liver tissues were severely altered due to the diminished activity of the lipogenic enzyme (West, 1982) G6PD, which substantially reduces the oxidation of the hydrogen shuttle system and the redox state of the cell (Gupta et al., 1999). The elevated levels of hepatic lipid content are observed in the present study (Fig 2) which is consistent with the previous reports (Sekar & Govindasamy, 1991; Suresh Kumar & Menon, 1992; Stan ely, 1999). Keelan et al. (1985) and Kanzaki et al. (1987) have reported sphingomyelin and glycosphingolipid levels are highly elevated during diabetes. The above findings implicate a potential role of hypolipidaemic activity of T. arjuna extract in alloxan induced diabetes.

Effect ofT. arjuna extract on renal function To determine the effect of T. arjuna on renal funtion, we performed GFR using creatinine clearance, urinary glucose, albumin, fluid intake, food intake, urine volume and kidney mass . As shown in Table 4, these levels were significantly elevated (p
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Exploring the Anti-diabetic effect of Terminalia arjuna

Table 4. Fluid intake, food intake, urine volume and levels of urinary glucose and albumin, plasma creatinine and GFR in control and experimental groups of rats. Values are expressed as mean ± S.D. of six rats in each group Parameters

Fluid intake (mliday) Food intake (g/kg/day) Urine volume (mliday) Glucose in urine Urinary albumin Plasma creatinine (mg/dl) GFR (mlimin) Kidney weight(g)

Control

Extract treated control

Diabetic

Extract treated diabetic

211.5 ± 2.62

212.0 ± 3.67 NS

353.8 ± 3.75'"

252.0

114.4 ± 2.15

115.1 ± 2.07 NS

176.8

±

3.85 '"

169.8 ± 1.41"

10.6 ± 0.67

10.7 ± 0.69 NS

37.3

±

1.79'"

ND ND 1.05 ± 0.07 NS

ND ND 1.03 ± 0.03 NS

+++ +++ 4.0 ± 0.09'"

0.73 0.79

0.74 ± 0.03 NS 0.8 NS

±

0.04

1.77 ± 0.03'" 1.45'"

19.4

±

±

1.34'"

1.30'"

ND ND 1.55 ± 0.01'" 0.86

±

0.04'*,

0.91'"'

Statistical comparison: control us extract treated control; control us diabetic; diabetic us extract treated diabetic. p values: 'd p
permeability defect, inhibiting elevated GFR and proteinuria. The T. arjuna extract reduces microalbuminuria is concordant with lowered blood glucose by the extract in diabetic rats. Glomerular filtration rate is a fundamental parameter of renal function. So creatinine clearance test was performed to assess GFR (Table 4) in experimental rats. The increased GFR in diabetic condition is associated with an increased kidney size (Mogensen & Anderson, 1973) and is consistent with our findings (Table 4). Yamada et at. (1992) has reported that increased kidney size is accompanied with elevation of glomerular volume and capillary surface area. In the diabetic rats, impaired renal function led to elevated levels of plasma and urine creatinine (Becker et at., 1996; Hwang et at., 1997; Murali & Goyal, 2002) which was correlated with our laboratory findings (Table 4) and GFR levels were found to be lowered upon treatment. These results suggest that T. arjuna, may be responsible for the observed biological activities and induce glycolysis and Ii polysis in preclinical animal model by targeting critical carbohydrate and lipid metabolizing enzymes.

Acknowledgements The financial assistance provided to Manonmani Ganapathy by the University of Madras in the form ofTamilnadu Junior Research Fellowship is gratefully acknowledged. The author MG also would like to acknowledge Dr. Usman Ali, Central Research Institute for Siddha for suggesting the problem and lively discussions throughout the investigation.

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References Agardh, C.D., Nilsson-Ehle, P. and Schersten, B. 1982. Improvement of the plasma lipoprotein pattern after institution of insulin treatment in diabetes mellitus. Diabetes Care 5(3): 322-325. AI-Shamaony, L., AI-Khazraji, S.M. and Twaij, H.A. 1994. Hypoglycemic effect of Artenmisia herba alba 11. Effect of a valuable extract on some blood parameters in diabetic animals. J. Ethnopharmacol43: 167-171. Avinash, N., Laxhman, S.M., Kaur, S., Grover, 1.S., Renu, W. and Sunil, C.K 2000. Growth suppression of human transformed cells by treatment with bark extracts from a medicinal plant Terminalia arjuna. In vitro Cell. Dev. Biol. Animal 36: 544-547. Bailey, C.J. and Day, C. 1989. Traditional plant medicines as treatment for diabetes, Diabetes Care 12: 553 - 564. Bartlett, G.R. 1959. J. Biol. Chem 234: 466-468. Barzilai, N. and Rossetti, L. 1993. Role of glucokinase and glucose-6-phosphatase in the acute and chronic regulation of hepatic glucose fluxes by insulin. J. Biol. Chem. 268: 25019-25025. Becker, D.J., Reul, B., Ozcelikay, A.T., Buchet, J.P., Henquin, J.C. and Brichard, S.M. 1996. Oral selenate improves glucose homeostasis and partly reverses abnormal expression of liver glycolytic and gluconeogenic enzymes in diabetic rats. Diabetologia 39: 3-11. Begum, N. and Shanmugasundaram, KR. 1978. Transaminases in experimental diabetes. Arogya 4: 112-116. Belfiore, F., Lovecchio, L. and Napoli, E. 1970. Serum enzymes in diabetic coma, Boll. Soc. Ital. Biol. Spero 46: 376-379. Bhavapriya, V., Kalpana, S., Govindasamy, S. and Apparanantham, T. 2001. Biochemical studies on hypoglycemic effect of Aavirai kudineer: A herbal formulation in alloxan diabetic rats. Ind. J. Exp. Biol. 39: 925-928. Bone, K 1996. Clinical applications of ayurvedic and Chinese herbs. Phytotherapy Press, Warwick, Queensland, Australia. pp. 131-133. Branstrup, N., Krik, J.E. and Bruni, C. 1957. The hexokinase and phosphogluco isomerase activities of aorta and pulmonary artery tissue in individuals of various ages. J. Gerontol. 12: 166-171. Broad, J. and Sirota, J.H. 1948. Renal clearance of endogenous creatinine in man. J. Clm. Invest. 27: 645. Caraway, W.I. 1963. Uric acid. In: Seligson, D. ed., Standard methods of clinical chemistry. Academic Press, New York, 4: 239-247. Chauhan, U.P.S., Jaggi, C.B., Ahiya, H.C. and Singh, V.N. 1984. Metabolism of hepatic lipids in alloxan diabetes. Ind. J. Exp. Biol. 22: 386-390. Chopra, R.N., Nayar, S.L. and Chopra, I.C. 1956. Glossary of Indian Medicinal Plants, CSIR, New Delhi, India, pp. 251. Chou, P., Li, C.L., Wu, G.S. et al., 1998. Progression to Type II diabetes among high-risk groups in Kin-Chen. Kinmen. Diabetes Care 21: 1183-1187. Costa, A., Igula, I., Bedin, J., Quinto, L. and Conget, 1. 2002. Uric acid concentration in subjects at risk of Type 2 diabetes mellitus. Relationship to components of the metabolic syndrome. Metabolism 51(3): 372-375. Dutt, P. and Sarkar, A.K. 1993. Alterations in rat intestinal sucrase and alkaline phosphatase activities in alloxan induced experimental diabetes. Ind. J. Biochem and Biophys, 30: 177-180. Dwivedi, S. and Agarwal, M.P. 1994. Antianginal and cardioprotective effects ofT. arjuna. an indignous drug in coronary artery disease. J. Assoc. Physicians India 42(4): 287289. Ebara, T., Hirano, Namo, J.C.L., Sakamaki, R., Furukawa, S., Nagano, S. and Takahashi, T. 1994. Hyperlipidemia in STZ diabetic hamsters as a model for human insulin

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dependent diabetes mellitus: comparison to STZ diabetic rats. Metabolism 43(3): 299-305. Ells, H.A and Kirkman, H.M. 1961. A colorimetric method for assay of erythrocytic glucose-6-phosphate dehydrogenase. Proc. Soc. Exp. Bwl. Med, 106: 607-609. EI-Maghrabi, M.R, Market, P.J. et aZ. 1988. cDNA sequence of rat liver fructose-1,6bisphosphatase that evidence for down regulation of its mRNA by insulin. Proc. Natl. Acad. Sci. USA 85: 8430-8434. Felig, P., Wahren, J., Sherwin, RS. and Palaigos, G. 1977. Protein and amino acid metabolism in diabetes mellitus. Arch. Intern. Med. 137: 507. Ferre, T., Riu, E., Bosch, F. et al. 1996. Evidence from transgenic mice that glucokinase is rate limiting for glucose utilization by liver. FASEB J. 10: 1213-1218. Fiske, C.H. and Subbarow, Y. 1925. The colorimetric determination of phosphorus. J. BioI. Chem 66: 375-400. Folch, J., Lees, M. and Slone Stanley, G.H. 1957. A simple method for the isolation and purification oftotallipids from animal tissues. J. Bioi. Chem., 226: 497-509. Gancedo, J.M. and Gancedo, C. 1971. Fructose-1,6 bisphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non-fermenting yeasts. Arch. Mikrobiol. 76: 132-138. Gauthaman, K., Maulik, M., Kumari, R, Manchanda, S.C., Dinda, A.K. and Maulik, S.K. 2001. Effect of chronic treatment with bark of T. arJuna extract a study on the isolated ischemic- reperfused rat heart. J. Ethnopharmacol 75: 197-201. Ghosh, R, Ganapathy, M., Alworth, W.L., Chan, D.C. and Kumar, AP. 2008. Combination of2-methoxyestradiol (2-ME(2» and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells. J. Steroid Biochem. Mol. Bwl. Nov 21st (Article in Press: doi:1O.1016/j.jsbmb.2008.11.002) Ghosh, S. and Suryawanshi, S.A. 2001. Effect of Vinca rosea extracts in treatment of alloxan diabetes in male albino rats, Ind. J. Exp. Bioi, 39: 748-759. Grover, J.K. and Vats, V. 2001. Shifting Paradigm "from conventional to alternate medicine." An introduction on traditional Indian medicine. Asia Pacific Bwtechnology News 5(1): 28-32. Gupta, D., Jayadev, Rand Baquer, N.Z. 1999. Modulation of some gluconeogenic enzyme activities in diabetic rat liver and kidney: Effect of antidiabetic compounds. Ind. J. Exp. Bioi. 37: 196-199. Gupta, D., Raju, J., Jayaprakash, R. and Baquer, N.Z. 1999. Change in the lipid profile, lipogenic and related enzymes in the livers of experimental diabetic rats: Effect of insulin and vanadate. Diabetes Res. and Clin. Practice 46: 1-7. Gupta, R., Singhal, S., Goyle, A. and Sharma, V.N. 2001. Antioxidant and hypocholesterolaemic effects of TerminaZia arJuna tree - bark powder: a randomised placebo-controlled trial. J. Assoc. Physicians. India 49: 231-235. Guy, L., Manuel, RA, Pierrette, F. and Christiane, L. 1984. Renal enzymes during experimental diabetes mellitus in the rat. Role of insulin- carbohydrate metabolism and ketoacidosis. Can. J. Physiol. Pharmacol 62: 70-75. Hammarstrom, L. and Ullberg, S. 1966. Nature 212: 708-714. Harris, H. 1959. Human Biochemical genetics, Cambridge University Press, pp.121. Honda, T., Murae, T., Tsuyuki, T., Takahashi, T. and Sawai, M. 1976. Bull. Chem. Soc. Japan 49: 3213. Howard, B.V. and Howard, W.J. 1994. Joslin's diabetes mellitus (l3 th edition). Khan, C.R and Weir, G.C. eds., Lea & Febiger, Philadelphia, pp. 372-396. Howard, B.W. 1987. Lipoprotein metabolism in diabetes. J. Lipid Res. 28: 613-628. Hron, W.T. and Menahan, L.A 1981. A sensitive method for the determination offree fatty acids in plasma. J. Lipid Res. 22: 377-381. Hwang, D.F., Lai, YS. and Chiang, M.T. 1997. Toxic effects of grass crap, snake and chicken bile juices in rats. Toxicol. Lett. 85(2): 85-92.

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llavarasan, R., Mohideen, S., Vijayalakshmi, M. and Manonmani, G. 2001. Hepatoprotective effect of Cassia angustifolia, Vahl. Indian J. Pharm. Sci. 63: 504-507. Itaya, K1977. A more sensitive and stable calorimetric determination offree fatty acids in plasma. J. Lip. Res. 18(5): 663-665. Ivorra, M.D., Paya, M. and Villar, A 1988. Hypoglycemic and insulin release effect of tormentic acid: a new hypoglycemic natural product. Planta Medica 54: 282-286. Jainu, M. and Mohan, KV. 2008. Protective role of ascorbic acid isolated from Cissus quadrangularis on NSAID induced toxicity through immunomodulating response and growth factors expression. Int. Immuno. Pharmacol. 8(13-14): 1721-7. Jainu, M., Vijai Mohan, K and Shyamala Devi, C.S. 2006. Gastroprotective effect of Cissus quadrangularis extract in rats with experimentally induced ulcer. Indian J. Med. Res. 123(6): 799-806. Kanthasamy, A, Sekar, N. and Govindasamy, S. 1988. Vanadate substitutes insulin role in chronic experimental diabetes. Ind. J. Exp. Bioi. 26: 778-780. Kanzaki, T., Ishikawa, Y., Morisaki, N., Shirai, K, Saito, Y. and Yoshida, S. 1987. Abnormal metabolism ofpolyunsaturatal fatty acids and phospholipids in diabetic glomeruli. Lipids 22: 704-710. Kapoor, L.D. 1990. Handbook of Ayurvedic Medicinal plants, CRC Press, Boca Raton, FL. pp.319-320. Kartz, R.N., Nauck, AM. and Wilson, T.P. 1979. Induction of glucokinase by insulin under the permissive action of decamethasone in primary rat hepatocyte cultures. Biochem. Biophys. Res. Commun. 88: 23-29. Keelan, M., Walker, K and Thomson, AB. 1985. Intestinal brush border membrane marker enzymes, lipid composition and villus morphology, Effect offasting diabetes mellitus in rats. Comp. Biochem. Physiol. 82: 83-89. Kind, P.R.N. and King, E.J. 1954. Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrine. J. Clin. Pathol. 7: 322-330. King, F.E., King, T.J. and Ross, J.M. 1954. The chemistry of extractives from hardwoods. Part XVIII. The constitution of aIjunolic acid, a triterpene from Terminalia arjuna. J. Chem. Soc. 23: 3995-4003. King, J. 1965. Practical clinical enzymology. Philadelphia, Dvan Nostrand Co., London. Kirthikar, KR. and Basu, B.D. 1935. Indian Medicinal Plants, International Book Distributors, Dehradun, India. 2: 1023. Kumar, A.P., Bhaskaran, S., Ganapathy, M., Crosby, K., Davis, M.D., Kochunov, P., Schoolfield, J., Yeh, LT., Troyer, D.A and Ghosh, R. 2007. AkticAMP-responsive element binding proteinlcyclin D 1 network: a novel target for prostate cancer inhibition in transgenic adenocarcinoma of mouse prostate model mediated by Nexrutine, a Phellodendron amurense bark extract. Clin. Cancer Res. 13(9): 2784-94. Kurup, P.N.V., Ramadas, V.N.K and Joshi, P. 1979. Hand book of medicinal plants. Central Council for Research in Ayurveda and Siddha, New Delhi, India, pp. 13. Lowry, O.H., Rusenbrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with folin-phenol reagent. J. Bioi. Chem. 193: 265-275. Manna, P., Sinha, M. and Sil, P.C. 2006. Aqueous extract of Terminalia arjuna prevents carbon tetrachloride induced hepatic and renal disorders, BMC Complement. Altern. Med.6:33. Manonmani, G., Anbarasi, K, Balakrishna, K, Veluchamy, G. and Shyamala Devi, C.S. Effect of Terminalia arjuna on the antioxidant defense system in alloxan induced diabetes in rats. Biomed. 22(52-61): 2002. Manonmani, G., Bhavapriya, V., Kalpana, S., Govindasamy, S. and Apparanantham, T. 2005. Antioxidant activity of Cassia fistula (Linn.) flowers in alloxan induced diabetic rats. J. Ethnopharmacol. 97: 39-42. Mathiux, G., Vidal, H., Zitoun, C., Bruni, N., Daniele, N. and Minassian, C. 1996. Glucose6-phosphatase mRNA and activity are increased to the same extent in kidney and liver of diabetic rats. Diabetes 45(7): 891-896.

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McGarry, J.D. and Foster, D.W. 1977. Hormonal control of ketogenesis, Arch. Intern. Med. 137: 445-450. Mogensen, C.E. and Anderson, M.J.F. 1973. Increased kidney size and glomerular filtration rate in early juvenile diabetes, Diabetes 22: 706-712. Mohur, AF. and Cook, I.J.Y. 1957. J. Clin. Pathol.l0: 394-396. Monica, G., Ayuso, E., Casellas, A, Costa, C., Devedjian, J.C. and Bosch, F. 2002. Bcell expression of IGF -I leads to recovery from type I diabetes, The Journal of Clin. Invest. 109(4): 1153-1163. Murali, B. and Goyal, R.K 2002. Effect of chronic treatment with losartan on STZ induced diabetic rats, Ind. J. Exp. Bio!. 40: 31-34. Murthy, V.K and Shipp, J.G. 1979. Synthesis and accumulation oftriglycerides in liver of diabetic rats effects of insulin treatment, Diabetes 28: 472-478. Nadkarni, KM. and Nadkarni, AK 1976. Indian Materia Medica, Popular Prakashan Pvt. Ltd., Bombay, India. VoLl pp. 1198-1202. Nakai, T., Yarnada, S., Tarnai, T., Kobayashi, T., Hayashi, T. and Takeda, R. 1979. Metabolism 28: 30-40. N atelson, S., Scott, M.L. and Begga, E. 1951. A rapid method for the estimation of urea in biological fluids by means of the reaction between diacetyl and urea, Am. J. Chem. Pathol. 21: 275-281. Nesamony, S. and Oushadyha, S. 1988. Medicinal plants. State Institute for Language, Kerala, India. pp.314-316. New, M.I., Roberts, T.N., Bierman, E.L. and Reader, G.G. 1963. The significance of blood lipid alteration in diabetes mellitus, Diabetes 12: 208-212. Nikkila, E.A. and Hormila, P. 1978. Serum lipids lipoproteins in insulin - treated diabetes demonstration of increased high density lipoprotein concentrations. Diabetes 27: 1078-1086. Noguchi, T., Inoue, H. and Tanaka, T. 1982. Regulation of rat liver L-type pyruvate kinase mRNA by insulin and by fructose. Eur. J. Biochem. 128(2-3): 583-588. Noguchi, T., Inoue, H. and Tanaka, T. 1985. Transcriptional and post-transcriptional regulation of L-type pyruvate kinase in diabetic rat liver by insulin and dietary fructose. J. BioI. Chem. 260(26): 14393-14397. Ohno, T., Horio, F., Tanaka, S., Terada, M., Namikawa, T. and Kitoh, J. 2000. Fatty liver and hyperlipidemia in IDDM ofSTZ treated shrews. Life Sciences 66(2): 125. Parekh, AC. and Jung, D.H. 1970. Cholesterol determination with ferric acetate - uranyl acetate and sulphuric acid - ferrous sulphate reagents, Anal. Chem. 42: 1423-1427. Pettit, R.P., Hoard, M.S., Doubek, D.L., Schmitt, J.M., Pettit, R.K, Tackett, L.P. and Chapuis, J.C. 1996. Antineoplastic agents 338: The cancer cell growth inhibitory constituents of Terminalia arjuna (combretaceae). J. ofEthnopharmacol. 53(2): 5763. Ploa, G.L. and Hewitt, W.R. 1989. Detection and evaluation of chemically induced liver injury. In: Hayes, W.A ed., Principles and Methods of Toxicology, 2nd edition. Raven Press Ltd., New York, pp. 599-628. Pogliaro, L. and Notarbartola, A 1961. LDH in liver of diabetic subjects. Boll. del. Soc. Ital. di. BioI. Spero 37: 334. Pogsun, C.L. and Denton, R.M. 1967. Nature London 212: 1053. Qin, L., Han, T., Zhang, Q., Cao, D., Nian, H., Rahman, K and Zheng, H. 2008. Antiosteoporotic chemical constituents from Er-Xian Decoction, a traditional Chinese herbal formula. J Ethnopharmacol. 118(2): 271-279. Raghavan, B. and Kumari, S.K 2006. Effect ofTerminalia arjuna stem bark on antioxidant status in liver and kidney of alloxan diabetic rats. Indian J. Physiol. Pharmacol. 50(2): 133-42. Rang, H.P. and Dale, M.M. 1991. In the endocrine system pharmacology. 2 nd edition, Longman group, London. pp. 504-508

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Rasch, 1979. Prevention of diabetic glomerulopathy in STZ diabetic rats by insulin treatment. The messengial region. Diabetologia, 17: 243-248. Rice, E.W. 1970. Triglycerides in serum. In: Roderick P and McDonald eds., Standard method for clinical chemistry, Academic Press, New York, 6: 215-222. Samy, RP. and Ignacimuthu, S. 2001. Antibacterial effects of the bark of Terminalia arjuna: Justification of Folklore beliefs. Pharmaceutical Biology, 39(6): 417-420. Saraswathi, P.R and Govindasamy, S. 2002. Effect of sodium molybdate on carbohydrate metabolizing enzymes in alloxan - induced diabetic rats. J. Nutritional Biochemistry 13: 21-26. Sasaki, T. and Matsui 1972. Effect of acetic acid concentration on the colour reaction is the toluidine-boric acid method for blood glucose determination. Rmsho Kagaku 1: 346-353. Saxena, A, Bhatnagar, M. and Garg, N.K. 1984. Enzyme changes in rat tissues during hyperglycemia Arogya. J. Hlth. Sci. 10: 33-37. Scassellatic, S.G., Villarini, M. et al. 1999. Antigenotoxic properties of Terminalia arjuna bark extracts. Journal of Environ. Pathol. Toxicol. and Oncol. 18(2): 119-125. Sekar, N. and Govindasamy, S. 1991. Insulin mimetic role of vanadate on plasma membrane insulin receptors. Biochem. Inti. 23(3): 461-466. Shalla, H.P., Udupa, S.L. and Udupa, A.L. 1997a. Hypolipidemic effect of Terminalia arjuna in cholesterol fed rabbits. Fitoterapia 68(5): 405-409. Shaila, H.P., Udupa, S.L. and Udupa, AL. 2000. Hypocholesterolemic activity in rats of different fractions from Terminalia arjuna. Pharm. Pharmacol. Commun. 6: 327330. Shaila, H.P., Udupa, S.L., Udupa, AL. and Nair, N.S. 1997b. Effect of Terminalia arjuna on experimental hyperlipidemia in rabbits. Int. J Pharmacogn. 35: 1-4. Sheela, C.G. and Augusti, K. T. 1992. Antidiabetic effects of S-allyl cysteine sulphoxide isolated from garlic Allium sativum Linn. Ind. J. Exp. Bioi. 30: 523-526. Sheela Sasikumar, C. and Shyamala Devi, C.S. 2000. Effect of Abana an ayurvedic formulation, on lipid peroxidation in experimental myocardial infarction in rats. Ind. J. Exp. Bio!. 38: 827-830. Sheela Sasikumar, C. and Shyamala Devi, C.S. 2000. Protective effect of Abana, a polyherbal formulation, on isoproterenol-induced myocardial infarction in rats. Ind. J. Pharmacol. 32: 198-201. Sochor, M., Baquer, N.Z. and McLean, P. 1979. Glucose over utilization in diabetes: Evidence from studies on the changes in hexo kinase, the pentose phosphate pathway and glucuronate-xylulose pathway in rat kidney cortex in diabetes. Biochem. Biophys. Res. Commun. 86(1): 32-39. Soling, D. and Kleineke, J. 1976. Gluconeogenesis. In: Hanson, RW., Mehlman, M.A eds., New York, John Wiley, pp. 369-462. Spence, T.J. 1983. Levels oftranslatable mRNA coding for rat liver glucokinase. J. Bioi. Chem. 258: 9143-9146. Stanely, M.P.P., Menon, V.P. and Gunasekaran, G. 1999. Hypolipidaemic action of Tinospora cordifolia roots in alloxan diabetic rats. J. Ethnopharmacol. 64: 53-57. Stanely, M.P.P., Menon, V.P. and Pari, L. 1997. Effect of Syzigium cumini extracts on hepatic hexokinase and glucose-6-phosphatase in experimental diabetes. Phyto Res. 11: 529-531. Sumitra, M., Manikandan, P., Kumar, D.A., Arutselvan, N., Balakrishna, K., Manohar, B.M. and Puvanakrishnan, R 2001. Experimental myocardial necrosis in rats: role of arjunolic acid on platelet aggregation, coagulation and antioxidant status. Mol. Cell Biochem. 224(1-2): 135-42. Suresh Kumar, J.S. and Menon, V.P. 1992. Peroxidative changes in experimental diabetes mellitus, Ind. J. Med. Res. 96: 176-181. The Wealth ofIndia, Raw Material. 1976. CSIR, New Delhi, India. 10: 161.

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Tripathi, Y.B. 1993. T. arjuna extract modulates the contraction of rat aorta induced by KCI and norepinephrine. Phyto. Res. 7: 320-322. Vagbhatta, 1963. Astanga Hridayam (Commentaries by Pandit Lalchandra Vaidya), 1't edition. Motilal Benarasi Das, Varanasi, India. pp. 414. Vestergoard, H. 1999. Studies of gene expression and activity of hexokinase, phosphorfructokinase and glycogen synthetase in human skeletal muscle in states of altered insulin stimulated glucose metabolism. Dan Med Bull. 46: 13-34. Viberti, G.C., Jarrett, RJ., Mahmud, V., Hill, RD., Argyropoulos, A. and Keen, H. 1982. Microalbuminuria as a predictor of clinical nephropathy in IDDM. Lancet I: 14301432. Warrier, P.K, Nambiar, V.P.K and Raman Kutty, C. 1974. Indian Medicinal Plants. Orient Longman Ltd., Hyderabad, India. 5: 253. West, KM. 1982. Hyperglycemia as a cause oflongterm complications. In: Keen, H. and J arret, J. eds., Complication of diabetes, 2 nd edition. Edward Arnold, London, pp. 318. World Health Organization, (WHO). Diabetes Programme. 2006. Available at http// www.who.Intidiabetes/eni. Accessed October 15, 2007. Yamada, H., Hishida, A., Kumagai, H. and Nishi, S. 1992. Effects of age, renal diseases and diabetes mellitus on renal size reduction accompanied by the decrease on renal function. Nippon Jinzo Gakkai Shi. 34(10): 1071-1075. Yudkin, J.S., Forrest, RD. and Jacson, C.A. 1988. Microalbuminuria as a predictor of vascular disease in non-diabetic subjects. Lancet II: 530-533. Zeenat, S.H., Patel, B.K and Goyal, RK 1997. Effects of chronic Ramipril treatment in STZ-induced diabetic rats. Ind. J. Physial. Pharmacal. 41(4): 353-360.

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16 Inhibition of Angiotensin Converting Enzyme (ACE) by Medicinal Plants Exhibiting Antihypertensive Activity JALAHALLI M. SIDDESHAl, CLETUS J.M. D'SOUZAl AND BANNlKUPPE

S. VISHWANATH l *

Abstract This chapter summarizes the screening of plants traditionally used against hypertension for ACE inhibitory activity. The maximum number ofplant species (18) exhibiting ACE inhibition belongs to the family Fabaceae. Following Fabaceae, the plant species (6-7) that inhibit ACE activity belongs to Euphorbiaceae, Araliaceae, Lamiaceae, Oleaceae, Asteraceae and Malvaceae. While, only a few species belonging to other diversified families Amaranthaceae, Combretaceae, Rosaceae, Anacardiaceae, Apiaceae, Aristolochiaceae, Crassulaceae, Cucurbitaceae, Lauraceae, Liliaceae, Rubiaceae, Sapindaceae, Acanthaceae, Berberidaceae, Ericaceae, Myrsinaceae, Onagaraceae, Polygonaceae, Rutaceae, Solanaceae, Theaceae etc. showed ACE inhibition. The major class ofcompounds showing ACE inhibition is found to be flavonoids followed by pep tides, alkaloids, phenylpropanoid glycosides, terpenes, iridoids, lipids, polyphenols, tannins and xanthones. In our ongoing research, methanolic and ethanolic leafextracts ofArtocarpus altilis exhibited potent ACE inhibition compared to aqueous and acetone extracts. The methanolic, ethanolic and aqueous leaf extracts of Catharanthus roseus, Pongamia pinnata and Azadirachta indica as well as that of Tamarindus indicus seed coat showed good ACE inhibition compared to acetone extracts, suggesting the benefit of polar compounds as potent ACE inhibitors. In conclusion, the scientific investigations ofplants that are used in traditional antihypertensive medicine and the isolation ofbioactive polar compounds will be helpful to develop safe and effective antihypertensive drugs. 1. Department of Studies in Biochemistry, University of Mysore, Manasagangotri,

*

Mysore-570 006, India. Corresponding author: E-mail: [email protected]

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Key words: Angiotensin Converting Enzyme Antihypertensive, Artocarpus altilis

inhibitor,

ACE,

Introduction Plants are the basis of life on earth and are central to people's livelihoods. Over many years, the focus on plant research has increased all over the world and a plenty of evidences show immense potential of medicinal plants in the management of hypertension (Barbosa-Filho et al., 2006; Kwan et al., 2002; Wright et al., 2007 ). The concept of medicinal application of plants is based on the traditional medicine systems, which utilize plants as a therapeutic approach for the treatment of cardiovascular diseases and high blood pressure. Hypertension is a growing undesired symptom that damages health and threatens mostly the developed societies and is a significant health problem worldwide. It is one among the major independent risk factors for atherosclerosis, stroke, myocardial infarction and end stage renal disease (Lam Ie et al., 2007; Frantz, 2003). The main objective of hypertensive treatment is to reduce the blood pressure and to control other cardiovascular risk factors. Since, the angiotensin converting enzyme (ACE) catalyzes conversion of inactive angiotensin I to active angiotensin II and concomitant inactivation of bradykinin; suppression of its enzymatic activity is an ideal target in the treatment of hypertension. ACE inhibitors constitute an established therapy and play an important role as first-line therapy in hypertensive patients with cardiovascular complications, diabetic nephropathy and type II diabetes mellitus (Dostal et al., 1996; Lam Ie et ai, 2007). ACE inhibitors have shown protection against stroke, coronary events, heart failure, progression of renal disease, progression to more severe hypertension and all-cause mortality (Psaty et al., 1997; Moser & Hebert, 1996). Despite this advantage, pharmaceutically designed ACE inhibitors have exhibited adverse effects such as cough, angioedema, taste disturbances, skin rashes and allergic reactions (de Lima, 1999). Therefore, worldwide the medicinal plants have gain more importance because oftheir better cultural acceptability and compatibility with the human body and lesser side effects. In this chapter, we are presenting the review data regarding the utility of plant extracts and plant-derived products as ACE inhibitors. We are also presenting the initial results of our ongoing phytoceutical research on angiotensin converting enzyme inhibitory activity of medicinal plants exhibiting antihypertensive effects.

Role of angiotensin converting enzyme in hypertension Angiotensin converting enzyme (EC 3.4.15.1) is a zinc-containing dipeptidyl carboxy-peptidase associated with the renin-angiotensin system. It is found

Inhibition of Angiotensin Converting Enzyme (ACE)

271

in a wide variety of mammalian tissues, principally as a membrane-bound ectoenzyme (Erdos, 1990). ACE plays a critical role in the control of blood pressure by virtue of its participation in the renin-angiotensin-aldosterone system. It removes C-terminal dipeptide from prohormone angiotensin I to generate the powerful, active vasoconstrictor octapeptide, angiotensin II. The same enzyme also degrades and inactivates the vasodilatory peptide, bradykinin by the sequential removal of dipeptides from the C-terminus (Ondetti & Cushman, 1982; Soffer, 1976). Thus produced, angiotensin II acts directly on vascular smooth muscle cells and causes the contraction of blood vessels and thereby raising blood pressure (Folkow et al., 1961). The angiotensin II interacts with the sympathetic nervous system both peripherally and centrally to increase vascular tone (Corvol, 1995). It is also known to stimulate both the synthesis and release of aldosterone from the adrenal cortex and this event increases blood pressure via sodium retention. The volume expansion takes place due to sodium retention via aldosterone and renal vasoconstriction (Zimmerman, 1984) as well as due to fluid retention via antidiuretic hormone (Biron, 1961). Apart from elevating the blood pressure, angiotensin II promotes migration, proliferation and hypertrophy at the cellular level (Padfield et al., 1977; Bell et al., 1990; Itoh et al., 1993). The mechanism of action of ACE and angiotensin II in elevating the blood pressure in human beings and animals is shown in Fig 1.

1

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Fig 1. Mechanism of action of Angiotensin Converting Enzyme

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Importance of ACE inhibition in hypertension Among the six major classes of antihypertensive drugs; diuretics, ACE inhibitors, adrenergic receptor blockers, calcium channel blockers, a and ~ blockers, ACE inhibitors constitute an established therapy in the management of high blood pressure (Cushman & Ondetti, 1991). This class of drugs effectively lowers the mean, systolic and diastolic pressures in hypertensive patients as well as in salt-depleted normotensive subjects (Vidt et al., 1982; Todd et al., 1986; Pool et al., 1987). ACE inhibitors alter the balance between the vasodilatory and natriuretic properties of bradykinin and the vasoconstrictive and salt-retentive properties of angiotensin II, by decreasing the formation of angiotensin II and the degradation of bradykinin. Since the original discovery of ACE inhibitors in snake venom, pharmacologically active ACE inhibitors captopril, enalapril, lisinopril, benazepril, fosinopril, ramipril, perindropil, quinapril and many more compounds have been developed and are currently in use (Brown & Vaughan, 1998). These synthetic ACE inhibitors have established themselves in the therapy of hypertension and congestive heart failure (Cheung, 1973). Synthetic ACE inhibitors are remarkably effective, but they cause adverse side effects such as cough, angioedema, taste disturbances, skin rashes and allergic reactions (de Lima, 1999). Therefore, in recent times, the trend has been set towards the development of natural, safe and effective ACE inhibitors with minimized adverse effects.

Utilization of plants as source of ACE inhibitors The screening for antihypertensive activity in traditional medicines has been performed over many years. The antihypertensive activity ofthe majority of the plants is found to be through the inhibition of ACE. From the different parts of the world, plants with antihypertensive activity have been reported. In this chapter we have reviewed the literature on ACE inhibitory activity of about 200 plant extracts and presented in Table 1. Many plant species belonging to diversified families shown to inhibit ACE activity. A total number of18 plant species belonging to Fabaceae showed ACE inhibitory activity. Families like Euphorbiaceae, Araliaceae, Lamiaceae, Oleaceae, Asteraceae and Malvaceae, each included 6-7 species with ACE inhibitory activity. Whereas, only a few species belonging to other diversified families Amaranthaceae, Combretaceae, Rosaceae, Anacardiaceae, Apiaceae, Aristolochiaceae, Crassulaceae, Cucurbitaceae, Lauraceae, Liliaceae, Rubiaceae, Sapindaceae Acanthaceae, Berberidaceae, Ericaceae, Myrsinaceae, Onagaraceae, Polygonaceae, Rutaceae, Solanaceae, Theaceae etc. showed ACE inhibition. The inhibition ofACE activity has been shown from the different parts ofthe plants including leaves, stem, stem bark, root, fruit, aerial parts and even from the whole plant. The selection of the plants and their parts is made based on the previous knowledge about the usefulness of traditional medicine in combating the high blood pressure and other complications.

S<

Table 1. Antihypertensive plants exhibiting angiotensin converting enzyme inhibition

~

Botanical names of plant Abrus precatorius Acacia nilotica Achyranthes aspera Actinida deZiciosa Actinostemma Zobatum Adenopodia sprcata Agapanthus africanus Agave americana AZisma orientaZe Allium ursinum AllophyZus edulis Amaranthus dubius Amaranthus hybridus Andrographis echioides AngeZica acutiloba AngeZica gigas AngeZica keiskei Antidesma madagascariense Antirrhea borbonzca AphZoia thezformzs

Common name

Family

Part used

Extract

Reference

§: ....

o·

;:s

Water Ethanol, Acetone Ethanol (95%) Ethanol (70%), Water Methanol-Water (1:1)

Nymun et aZ., 1998 Nymun et aZ., 1998 Hansen et aZ., 1995 Jung et aZ., 2005 Inokuchi et aZ., 1984

~

Water, Ethanol Water, Ethanol Ethanol, Water Water Lyophilized Butanol, Ethanol (70%) Methanol Water Acetone

Duncan et aZ., 1999 Duncan et aZ., 1999 Duncan et aZ., 1999 Han, 1991 Sendl et aZ., 1992 Arisawa et aZ., 1989

;:s

Dried root Dried leaf Leaf

Ethanol (95%) Decoction Ethanol (95%) Ethanol (80%) Acetone

Ham et at., 1996 Kanetoshi et aZ., 1993 Ham et aI., 1996 Shimizu et aZ., 1999 Adsersen & Adsersen, 1997

Leaf Leaf

Acetone Acetone

Adsersen & Adsersen, 1997 Adsersen & Adsersen, 1997

Crab's eye Scented-pod acacia Devil's horsewhip Kiwifruit Goki-zuru

Fabaceae Fabaceae Amaranthaceae Actinidiaceae Cucurbitaceae

Spiny splinter-bean Agapanthus Century plant, Agave Alisma, Ze xie Bear's garlic COCll., Chal chal

Mimosaceae Agapanthaceae Agavaceae Alismataceae Liliaceae Sapindaceae

Aerial parts Thorn Aerial parts Fruit Dried entire plant Leaf Leaf Leaf Dried rhizome Fresh leaf Branches

Spleen amaranth Smooth amaranth False waterwillow

Amaranthaceae Amaranthaceae Acanthaceae

Leaf Leaf Aerial parts

Japanese Angelica

Apiaceae

Dried root

Korean Angelica Ashitaba Antidesma

Apiaceae Apiaceae Euphorbiaceae

Not found Albino-berry

Rubiaceae Flacourtiaceae

Ramesar et aZ., 2008 Ramesar et aZ., 2008 Somanadhan et aZ., 1999

~

~

o· ~

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<:!

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k

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Table 1. (Contd.) Botanical names of plant

Common name

Family

Part used

Extract Chromatog. Fraction Methanol-Water (1:1) Tannin fraction Water

Inokuchi et al., 1984 Inokuchi et al., 1984 Inokuchi et al., 1996a Han, 1991

Water Acetone, Ethanol (95%) Water Ethanol (95%) Ethanol Water

Han, 1991 Hansen et ai., 1995

Water

Han et al., 1991

Methanol Methanol Acetone, Ethanol, Water Ethanol Water Methanol Ethanol Acetone, Ethanol Ethanol

Alasbahi & Melzig, 2008 Ramesar et al., 2008 Adsersen & Adsersen, 1997 Adsersen & Adsersen, 1997 Hansen et al., 1995 Oleski et al., 2006 Nymun et al., 1998 Nymun et al., 1998 Braga et al., 2007

Acetone, Ethanol, Water

Adsersen & Adsersen, 1997

Areca catechu

Betel nut palm

Arecaceae

Dried seed

Arisaema consanguineum Aristolochia debilis Aristolochia manshuriensis Aristotelia chilensis Artemisia pallens Asarum heterotropoides Asarum sieboldii

Himalayan cobra lily Pipe vine Birthwort

Arecaceae

Dried rizhome Dried root Stem

Macqui Davana Ginger

Elaeocarpaceae Asteraceae Aristolochiaceae

Siebold wild ginger Not found Chinese violet Not found

Aristolochiaceae

Bark Aerial parts Dried aerial parts Leaf

Asteraceae Acanthaceae Myrsinaceae

Leaf Leaf Bark

Aspilia helianthoides Asystasia gangetica Badula barthesia Boerhauia diffusa Boswellia elongate Butea frondosa Butea parui/Zora Calophyllum brasiliense Calophyllum tacamahaca

..,.

....;J

Aristolochiaceae Aristolochiaceae

Spreading Hogweed Not found Forest flame Mountain ebony Santa maria

Nyctaginaceae Burseraceae Fabaceae Fabaceae Clusiaceae

Root Bark Pulvinus Bark Stem

Poon tree

Clusiaceae

Leaf

Reference

Hansen et al., 1995 Somanadhan et al., 1999 Han et al., 1991

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~

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~

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-;

~

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'"

E::1

~

Table 1. (Contd.) Botanical names of plant Camellia smensis

Canarium euphyllum Cardiospermum halicacabum Carica papaya Cassia fistula

Common name Tea, Green tea, Japanese Sencha Black tea, Indian Assam Broken Orange Pekoe Indian white mahogany Balloonvine heartseed Papaya Golden shower tree Sickle pod Devil's gut, Seashore dodder Australian Pine

Family

Part used

Extract

Theaceae

Green leaf, Leaf

Acetone, Phosphatebuffered saline Phosphatebuffered saline

Reference Cho et al., 1993 Persson et al., 2006 Persson et al., 2006

.Q., ~

~

....c·

'" OJ

Burseraceae

Bark

Acetone, Ethanol

Somanadhan et al., 1999

Sapindaceae

Stem

Water

Somanadhan et al., 1999

Caricaceae Fabaceae

Leaf Bark

Adsersen & Adsersen, 1997 Somanadhan et al., 1999

S· Q ;:!

Fabaceae Lauraceae

Root Herb

Casuarinaceae

Fruit

Ethanol, Water

Adsersen & Adsersen, 1997

Red cecropia Ambay pumpwood Black-oil tree

Cecropiaceae Cecropiaceae

Leaf, Stipule Leaf

Methanol Methanol

Dubois et al., 2001 Dubois et al. ,2001

Celastraceae

Gotu kola

Apiaceae

Chrysanthemum lavandulaefolium

Pepperback

Asteraceae

Dried flowers

Acetone, Ethanol, Water Acetone, Ethanol, Water Water

Somanadhan et al., 1999

Centella asiatIca

Bark, Fruit, Seed Aerial parts

Casuarma equisetifolia Cecropia glaziovii Cecropia pachystachya Celastrus paniculatus

....§: c· ;:!

;:!

Water Acetone, Ethanol, Water Acetone, Ethanol Acetone, Ethanol

Cassia tora Cassytha filiformis

;::,-

Somanadhan et al., 1999 Adsersen & Adsersen, 1997

<:::

'"

;:t ~. t:>::l

;:!

~ ;:l

S'"

C1

E:J

Hansen et al., 1995 Han et al., 1991 t-.:)

-1 01

Table 1. (Contd.) Botanical names of plant

t-:l ...;J

Common name

Family

Part used

Extract

Reference

m

Cinnamomum cassia Cinnamomum zeylanicum

Chinese cinnamon Cinnamon

Lauraceae

Dried bark

Tannin fraction

Inokuchi et al., 1984

Lauraceae

Dried bark

Inokuchi et al., 1984

Cissus hamaderohensis Citrus limon Clausena anisata Clerodendrum infortunatum Coffea mauritanica Combretum fruticosum Cordemoya integrifolia Crataegus pinnatifida Crataegus sp. Cuphea cartagenesis Cupressus sempervirens Cynostemma pentaphylla Dalbergia odorifera

Achira, African aloe Lemon Horsewood Bhantaka

Vitaceae

Leaf

Chromatog. Fraction, Water, MethanolWater (1:1) Methanol

Oleski et al., 2006

Rutaceae Rutaceae Verbenaceae

Leaf Leaf Leaf

Water Water Water

Adsersen & Adsersen, 1997 Duncan et al., 1999 Somanadhan et al., 1999

Maroon coffee Chameleon vine

Rubiaceae Combretaceae

Leaf Leaf

Water Ethanol

Adsersen & Adsersen, 1997 Braga et al., 2007

Rushfoil, Croton

Euphorbiaceae

Bark

Adsersen & Adsersen, 1997

Chinese haw

Rosaceae

Dried fruit

Acetone, Ethanol, Water Water

Han et al., 1991

~

Hawthorn Tenuissimum cigar flower Italian cypress

Rosaceae Lytharaceae

Fresh fruit Leaf

Methanol-Water (1:1)

Inokuchi et al., 1984 Castro Braga et al., 2000

I""

Cupressaceae

Dried fruit

Flavonoid fraction

~ ~

1.>0

Ie

Sweet tea vine

Cucurbitaceae

Fragrant rosewood

Fabaceae

Dried aerial parts Dried wood

Meunier et al., 1987

Infusion

Chen et al., 1996

Water

Han et al., 1991

I

..,t::::;

~

~ ~

;:s 1;l"

~

;;-

Table 1. (Contd.) Family

Part used

Extract

Trefle gros

Fabaceae

Root

Famola kantsy

Fabaceae

Root

Ethanol, Acetone, Water Ethanol (95%), Water

Coin·leaf desmodium Sickle bush

Fabaceae

Leaf, Stem

Fabaceae

Leaf

Botanical names of plant

Common name

Desmodium triquetrum Desmodium gangeticum Desmodium styracifolium Dichrostachys cinerea Dwtes iridioides Diospyros melanoxylon Dodonea viscosa

Reference

~

§: .....

o·

Acetone, Ethanol (95%) Water Water

;:l

Nymun et al., 1998

~

Hansen et al., 1995

~

Hansen et al., 1995

;:l

~

Nymun et al., 1998

o·.... ~

;:;. til

~

;:l

African iris, Fortnight lily Ebony

lridaceae

Leaf

Water

Duncan et al., 1999

Ebenaceae

Leaf, Root

Water, Ethanol

Nymun et al., 1998

~ ~

;:t.

Jg' t.:t.l

;:l

Native hop, Hop bush Oni ukogi

Sapindaceae

Leaf

Acetone

Adsersen & Adsersen, 1997

~

Araliaceae

Butanol

Leem, 1990

S

Chloroform

Takashari et al., 1993

'-"

~ ~

Eleutherococcus divaricatus Eleutherococcus senticosus Embelia angustifolia Embelia basal Entada pursaetha Ephedra sinica

Siberian ginseng Liane soap

Araliaceae

Dried stem, Bark Dried stem

Myrsinaceae

Leaf

Acetone, Ethanol

Adsersen & Adsersen, 1997

Coat buttons Sea bean Ma huang

Myrsinaceae Fabaceae Ephedraceae

Ethanol, Acetone Ethanol (95%), Water Tannin fraction

Somanadhan et al., 1999 Hansen et al., 1995 Inokuchi et al., 1985

Ephedra sp.

Ephedra

Ephedraceae

Fruit stem Seed Dried aerial parts Dried entire plant

Chroma tog fraction

Inokuchi et al., 1984

R

t>:l -l -l

Table 1. (Contd.) Botanical names of plant Epilobium angustifolium Epimedium alpinum Epimedium brevicornum Epimedium macranthum Equisetum hyemale Erythroxylum laurifolium Eugenia heyneana Euodia simplex Euphorbia hirta Euphorbia humifusa Fritillaria sp. Fritillaria ussuriensis Fuchsia magellanica Galinsoga parviflora Geranium core-core Gunnera tinctoria

t-.:J

Common name Willow herb, Fire weed Alpine epimedium Horny goat weed Barrenwort

Family

Part used

Extract

Onagraceae

Herb

Ethylacetate

Kiss et al., 2004

Berberidaceae

Dried aerial parts Not specified

Methanol-Water (1:1) Tannin fraction Water

Inokuchi et al., 1985

Dried entire plant Dried stem Dried leaf

Chroma tog. Fraction Methanol-Water (1:1) Water Ethanol (100%) Ethanol, Acetone Ethanol, Water Ethanol, Acetone Methanol Water

Berberidaceae Berberidaceae

Horsetail Bois de ronde

Equisetaceae Erythroxylaceae

Katjamun Not found Cats hair Trailing spurge Fritillaria Pingbei mu

Myrtaceae Rutaceae Euphorbiaceae Euphorbiaceae Liliaceae Liliaceae

Root Leaf Dried leaf Dried aerial parts Dried bulb Bulb

Fuchsia

Onagraceae

Gallant soldier, Potato weed Geranium core-core Gunnera

Asteraceae

Reference

-l C/J

Han et al., 1991 Inokuchi et al., 1984 Han et al., 1991 Hansen et al., 1996a Adsersen & Adsersen, 1997 Nymun et al., 1998 Adsersen & Adsersen, 1997 Williams et al., 1997 Han et al., 1991

:== Inokuchi et al., 1984 Kang et al., 2002

Aerial parts

Methanol-Water (1:1) Butanol, Ethylacetate, Water Water

Leaf

Methanol

Ramesar et al., 2008

Hansen et al., 1995

~

"= ~

I"'"

~

I

tl

Geraniaceae

Aerial parts

Water

Hansen et al., 1995

Gunneraceae

Leaf

Butanol, Ethanol (95%), Ethyl acetate

Hansen et at., 1995

'1

~

~ ~

;::I

<-;.

'"

~

Table 1. (Contd.) Botanical names of plant Gynura procumbens Hedysarum polybotrys Hexachlamys edulis Hibiscus sabdariffa Houttuynza cordata Humboldtia vahliana Jasminun azoricum

Jasminun grandi{lorum Jasminum multi{lorum Jasminum sambac Jatropha curcas Justicia {lava Kalanchoe farinacea Leea guinenis Leea rubra

Common name

Family

Part used

Reference

~

0;.

Sambung nyawa Not found

Asteraceae

Leaf

Fabaceae

Dried root

Pessegueirodo-mato Roselle

Myrtaceae

Leaf

Malvaceae

Calyx

Chameleon plant

Extract

;;;;::-

Saururaceae

o· ;:l

Aqueous fraction (FA-I) Water

Hoe et al., 2007

.s;.,

Han et al., 1991

~

Acetone-Water (1:1), Ethanol (95%) Water

Hansen et al., 1995

;:l

Entire plant

Jelavedesa Jasmine

Fabaceae Oleaceae

Spanish jasmine

Oleaceae

Downy jasmine, Star jasmine Arabian jasmine, Mogra Barbados nut, Physic nut Yellow justicea Mealy kalanchoe Leia-alaranjada Hawaiian Holly, Red Leea

Oleaceae

Acetone, Water, Ethanol (95%) Bark Ethanol, Acetone Dried aerial parts Dichloromethane, Chromatog. Fraction, Ethyl acetate, Water Aerial part Ethanol, Acetone, Water Fresh leaf Acetone

Oleaceae

Leaf

Water

Euphorbiaceae

Leaf

Water, Ethanol

Acanthaceae Crassulaceae Leeaceae Leeaceae

Leaf Aerial parts Leaf Aerial parts

Water Methanol Ethanol, Acetone Ethanol

~

o· ~

Of)

S· Herrera-Arellano et al., 2007 Hansen et at., 1995

~

;:l c:!

"".., ....

~.

Somanadhan et al., 1999 Somanadhan et al., 1998

t:r::l

;:l

~ ;3

""

Somanadhan et al., 1999

~

Somanadhan et al., 1999

"-'

&S

Somanadhan et al., 1999 Adsersen & Adsersen, 1997 Ramesar et al., 2008 Oleski et al., 2006 Adsersen & Adsersen, 1997 Braga et al., 2007 t-:l -l ~

~

Table 1. (Contd.) Botanical names of plant Lespedeza capitata Lycium chinese Lygodium japonicum Machilus thunbergii Mangifera indica Mansoa hirsuta Merremia tridentate Marrubium radiatum Mesembruanthemum spp Molinaea alternifolia Momordica balsam ina Monimia ovalifolia Monimia rotundifolia Moringa oleifera Morus alba Musanga cecropioides

Oenothera biennis

Family

Part used

Extract

Roundhead lespedeza Chinese boxthorn

Fabaceae

Dried leaf

Flavonoid fraction

Wagner & Elbl, 1992

Solanaceae

Dried root bark

Japanese climbing fern Tabu-no-ki tree Indian mango Cip6-de-alho Mogra Hoarhound Ice plant

Schizaeaceae

Morota et al., 1987 Yahara et al., 1993 Han et al., 1991

Lauraceae Anacardiaceae Bignoniaceae Convolvulaceae Lamiaceae Aizoaceae

Dried aerial parts Bark Bark Leaf Aerial parts Aerial parts Leaf

Chloroform Not stated Water

Methanol, Chloroform Oh et al., 1997 Acetone, Ethanol Somanadhan et al., 1999 Castro Braga et al., 2000 Ethanol (95%), Water Hansen et al., 1995 Decoction Loizzo et al., 2008 Water, Ethanol Duncan et al., 1999

Sapindaceae Cucurbitaceae

Leaf Leaf

Ethanol, Water Water

Adsersen & Adsersen, 1997 Ramesar et al., 2008

Monimiaceae Monimiaceae

Leaf Leaf

Ethanol, Water Ethanol, Acetone

Adsersen & Adsersen, 1997 Adsersen & Adsersen, 1997

Moringaceae

Leaf fruit

Water

Moraceae Moraceae

Dried leaf Leaf, Bark Leaf Seed

Methanol-Water (1:1) Water, Methanol

Somanadhan et al., 1999 Adsersen & Adsersen, 1997 Inokuchi et al., 1984 Dongmo et al., 2002 Adeneye et at., 2006 Dubois et al., 2001 Scholkens et al., 1982

Common name

White wood gaulettes Balsam apple, Bitter melon Mapou Mapou with large sheets Drumstick, Horseradish tree White mulberry Mrican Corkwood, Umbrella Tree Common evening primrose

Reference

00 0

~

Onagraceae

Seed oil

~

"ti

~

I"'" t\Q

Ie

I

0 ~ "j

~

>:!

;:s @"

::::

;;-

Table 1. (Contd.) Botanical names of plant

~

Family

Part used

Extract

Wild olive

Oleaceae

Leaf

Water

Adsersen & Adsersen, 1997

~

Bois malaya Oregano Not found Mbigili, Song'e Chinese peony Moutan

Oleaceae Lamiaceae Ochnaceae Polygonaceae Paeoniaceae Paeoniaceae

Leaf

Water, Ethanol Water

Adsersen & Adsersen, 1997 Apostolidis et al., 2006 Castro Braga et al., 2000 Ramesar et al., 2008 Han et al., 1991 Inokuchi et al., 1984 Inokuchi et al., 1984 Inokuchi et al., 1985 Persson et al., 2006a Adsersen & Adsersen, 1997 Okamot et al., 1994

~

Common name

Reference

§: ,...

o·

;:l

Olea europaea ssp. africana Olea lancea Origanum vulgare Ouratea semiserrata Oxygonum sinuatum Paeonia albiflora Paeonia moutan

Panax ginseng Passiflora edulis Passiflora quadrangularis Pavonia odorata Philippia montana Phyllanthus niruri Phyllanthus phillyretfolius Physalis VLscosa Phoenix roebelinii Pinellia ternata Piper betle Piper futokadsura

Asian ginseng Passion fruit Giant granadilla

Araliaceae Passifloraceae Passifloraceae

Stem Leaf Dried root Dried bark

Pavonia Branle white Stone breaker

Malvaceae Ericaceae Euphorbiaceae

Wood negresse

Euphorbiaceae

Root Leaf Dried entire plant Entire plant Leaf Dried entire plant Bark

Starhair groundc herry Pigmy date palm Crowdipper Betel Kasura stem

Solanaceae

Leaf

Arecaceae Araceae Piperaceae Piperaceae

Leaf Dried rhizome Leaf Dried aerial parts

Water Water Chromatog. Fraction Methanol-Water (1:1) Tannin fraction Water Water, Acetone Water Ethanol Ethanol, Water Chromatog. Fraction

Somanadhan et al., 1999 Adsersen & Adsersen, 1997 Veno et al., 1988

Ethanol, Acetone, Water Methanol

Adsersen & Adsersen, 1997 Ramesar et al., 2008

Ethanol Water Water, Ethanol Water

Braga et al., 2007 Han et al., 1991 Somanadhan et al., 1999 Han et al., 1991

~

o· 1? ;:l C/)

S·

g ;:l

<::

'",... ""j

~.

ttl

;:l

~

~

'" ~ &3

'-"

t>:)

00 ~

I>:)

Table 1. (Contd.) Botanical names of plant

00

Common name

Family

Part used

Extract

Dried aerial parts Entire plant

Flavonoid fraction

Sanz et al., 1993

Acetone, Ethanol (95%), Water Not stated Tannin fraction

Hansen et al., 1995 Ikemizu et al., 1995 Inokuchi et al., 1985

Methanol-Water (1:1)

Inokuchi et al., 1984

Water Tannin fraction

Han et al., 1991 Inokuchi et al., 1985

Chromatog. Fraction, Methanol-Water (1:1) Ethanol, Water, Acetone Ethanol, Water Acetone, Ethanol (95%) Ethanol (95%), Water

Inokuchi et al., 1984

Pistacia lentiscus

Mastic tree

Anacardiaceae

Plantago asiatica

Chinese plantain

Plantaginaceae

Pleurotus sajor-caju Polygonatum aviculare

Oyster mushroom Polyporaceae Birdgrass, Doorweed Liliaceae

Polygonum multiflorum Tuber fleece flower Chinese cinquefoil Potentilla chinensis

Polygalaceae Rosaceae

Potentilla sp.

Cinquefoil

Rosaceae

Poupartia borbonica

Bios de poupart

Anacardiaceae

Fruit body Dried aerial parts Dried entire plant Dried root Dried aerial parts Dried entire plant Bark

Psathura borbonica Pseudarthria hookeri

Brittle wood Velvet bean

Rubiaceae Fabaceae

Leaf Root

Pseudarthria viscida

Moovila, Long pepper Kudzu vine Japanese felt fern

Fabaceae

Root

Fabaceae Polypodiaceae

Root Entire plant

Quinchamali

Santalaceae

Aerial parts

Pueraria lobata Pyrrosia lingua Quinchamalium chilense

Ethanol (95%) Acetone, Ethanol (95%), Water Ethanol (95%)

Reference

I>:)

Adsersen & Adsersen, 1997 ~

~

Adsersen & Adsersen, 1997 Hansen et al., 1995

"ti

Hansen et al., 1995

I"'"

Hansen et al., 1995 Hansen et al., 1995 Hansen et al., 1995

~

~

I

...,t! ~ ~ ~

;:,

....

'"

~

;;;:r.

Table 1. (Contd.) Botanical names of plant

Common name

Family

Part used

Extract

Reference

§: ""'" o·

;:s

Rabdosia coetsa Rhodiola crenulata Rhodiola rosea Rheum palma tum Rheum sp. Rosmarinus officinalis Salsola oppositifolia Salsola soda Salvadora persica Salvia acetabulosa Salvia miltiorrhiza Sanguisorba officinalis Saussurea lappa Schinus latifolius

Duo mao bian zhong Da hua hong jingtian Golden root, Roseroot Turkey rhubarb Rhubarb Rosemary Tumbleweed Tumbleweed Salt bush, Toothbrush tree Not found Danshen Burnet bloodworl Costus, Kuth Chilean pepper tree

Lamiaceae

Ethylacetate

Li et al., 2008

~

Crassulaceae

Water

Kwon et al., 2006

~

Crassulaceae

Ethanol, Water

Kwon et al., 2006

1;i ;:s

Tannin fraction Chromatog. Fraction, Methanol-Water (1:1) Water Ethylacetate Ethylacetate Water

Inokuchi et aI., 1985 Inokuchi et al., 1984

~

Polygonaceae Polygonaceae Lamiaceae Amaranthaceae Amaranthaceae Salvadoraceae

Dried rhizome Dried rhizome

Aerial parts Aerial parts Unripe seed

Lamiaceae Lamiaceae Rosaceae Asteraceae Anacardiaceae

Aerial parts Dried root Dried root Bark

Scleropyrum pentandrum Sedum sarmentosum

Not found

Santalaceae

Nut shell

Stringy stonecrop

Crassulaceae

Entire plant

Sida acuta

Common wireweed

Malvaceae

Root

Decoction Water Methanol-Water (1:1) Methanol-Water (1:1) Butanol, Ethyl acetate, Ethanol (95%), Ethanol- Water (7:3) Acetone, Ethanol, Water Acetone, Water, Ethanol (95%) Acetone, Water

o·

'" S· ~

;:s <::: eo ""i

Apostolidis et al., 2006 Loizzo et al., 2007 Loizzo et al., 2007 Nymun et al., 1998 Loizzo et al., 2008 Kang et al., 2002a Inokuchi et al., 1984 Inokuchi et al., 1984 Hansen et al., 1995

""'" ~. t:>:l

;:s

~ ;3 eo

S &3 '-"

Nymun et al., 1998 Hansen et al., 1995 Hansen et al., 1995 t-:l 00 C.:>

Table 1. (Contd.) Botanical names of plant Sida cordifolia

I>.?

00

Common name

Family

Part used

Extract

Reference

Malvaceae

Root

Acetone, Water

Hansen et al., 1995

Malvaceae

Root

Acetone, Water

Hansen et al., 1995

Sinomenium acutum Solanum nigrum

Heart-leaf sida, Indian hemp Paddy's lucern, Jelly leaf Chinese moon seed Black nightshade

Menispermaceae Solanaceae

Water Water

Han et al., 1991 Han et al., 1991

Stange ria eriopus Terminalia bentzoe Terminalia bialata Terminalia catappa

Natal grass cycad Catappa benzoin Silver greywood Indian almond

Stangeriaceae Combretaceae Combretaceae Combretaceae

Water Water Ethanol Ethanol, Water

Terminalia chebula Tribulus terrestris

Myrobalan, Hardad Combretaceae Bullhead, Gokshura Zygophyllaceae

Acetone Water

Duncan et al., 1999 Adsersen & Adsersen, 1997 Somanadhan et al., 1999 Braga et al., 2007 Adsersen & Adsersen, 1997 Somanadhan et al., 1999 Somanadhan et al., 1999

Trichosanthes kirilowii

Cucurbitaceae

Water

Han et al., 1991

Tiliaceae

Root

Ethanol (95%), Water

Hansen et al., 1995

Tulbaghia violacea

Snakegourd, Chinese cucumber Burr bush, Diamond burrbark Wild garlic

Dried root Dried aerial parts Leaf Leaf Bark Aerial parts, Leaf Fruit Aerial parts, Fruit Dried fruit

Alliaceae

Leaf, Leaf root

Uncaria rhynchophylla

Cat's Claw

Rubiaceae

Sida retusa

Triumfetta rhomboidea

Vaccinium ashei reade Blueberry Vaccinium macrocarpon Cranberry

Ericaceae Ericaceae

Methanol, Water Water, Ethanol Dried branches, Methanol-Water (1:1) Acetone, Ethanol Root (95%) Water Leaf Water Water

Ramesar et al., 2008 Duncan et al., 1999 Inokuchi et al., 1984 Hansen et al., 1995 Sakaida et al., 2007 Apostolidis et al., 2006

""-

~

~ ~

I""

t-:I

I;.C

I

..,t:::I J% ~

Q

;:s ....

'"

~

Table 1. (Contd.) Botanical names of plant

Viburnum opuZus

Viscum trifZorum Vitis uimfera Weinmannia tinctoria Wrightia tinctoria

Common name European cranberrybush viburnum, Crampbark Mistletoe fruit Wine grape Arbre mouche a miel Pala indigo, Indrajao

Family

Part used

Extract

Reference

Caprifoliaceae

Dried bark

Ethanol-Water (1:1)

Jonader et aZ., 1989

Loranthaceae Vitaceae Cunoniaceae Apocynaceae

Leaf Dried fruit Leaf Seed

Water, Acetone Flavonoid fraction Ethanol, Acetone Water

Adsersen & Adsersen, 1997 Meunier et aZ., 1987 Adsersen & Adsersen, 1997 Nymun et aZ., 1998

Table 2. List of plant-derived ACE inhibitory compounds. Chemical name

Class of the chemical compound

Botanical name (Source)

Common name

Acteoside

CZerodendron trichotomum Abeliophyllum distichum CZerodendron trichotomum

Harlequin glorybower Verbenaceae White forsythia Oleaceae Harlequin glorybower Verbenaceae

Afzelin Ala-Tyr Apigenin Arrivacin A & B Asperoside

Phenyl propanoid glycoside Phenylpropanoid glycoside Flavonoid Peptide Flavonoid Sesquiterpene Cardenolide

ErythroxyZum ZaurifoZium Zea mays AiZanthus exceZsa Ambrosia psiZostachya Eucommia uZmoides

Bois de ronde Corn Ailanthus, Ardu Cuman ragweed Hardy rubber tree

Astragalin Atractylodinol Butein

Flavonoid Oxygen heterocycle Polyphenol

Diospyros kaki AtractyZoides japonica PZant compound

Japanese Persimmon Chinese Atractylodes

Acteoside isomer

Family

Reference

Kang et aZ., 2003 Oh et at., 2003a Kang et aZ., 2003

Erythroxylaceae Hansen et aZ., 1995 Poaceae Yang et aZ., 2007 Simaroubaceae Loizzo et at., 2007 Asteraceae Chen et aZ., 1991 Eucommiaceae Yamadaki et aZ., 1992 Ebenaceae Kameda et aZ., 1987 Sakurai et at., 1993 Asteraceae Kang et aZ., 2003b

Table 2. (Contd.) Chemical name

t--:l 00

Class of the chemical compound

Botanical name (Source)

Common name

Family

Reference

~

Caffeoylquinates Capsianoside A Capsianoside C

Tannin Diterpene Diterpene

Capsicum sp. Capsicum sp.

Chinese herbs Chilli pepper Chilli pepper

Solanaceae Solanaceae

Catechin, (+ 1 Catechin-3-0gallate, epi (-1 Corilagin Coriolic acid, (DLl Crenatoside

Flavonoid Flavonoid

Allophytus edulzs Camellia sinensis

Cocti, Chal chal Korean green tea

Sapindaceae Theaceae

Tannin Lipid Phenylpropanoid glycoside Flavonoid Lipid

Phyllanthus niruri Fritillaria vertic illata Microtoena prainiana

Stone breaker Fritillary Prain microtoena

Euphorbiaceae Liliaceae Lamiaceae

Cho et at., 1993 Cho et at., 1993 Uchida et at., 1987 Ueno et at., 1988 Niitsu et at., 1987 Li et at., 2004

Daphne odora Lycium chinense

Winter daphne Chinese boxthorn

Thymelaeaceae Solanaceae

Takai et at., 1999 Morota et at., 1987

Lipid

Frittitaria vertic illata

Fritillary

Liliaceae

Niitsu et at., 1987

Lipid

Frittilaria verticillata

Fritillary

Liliaceae

Niitsu et at., 1987

~

Phenolic acid

Cuscuta japonica

Japanese dodder

Cuscutaceae

Dh et at., 2002

!"'"

Phenolic acid Coumarin Flavonoid

Cuscuta japonica Phyllanthus niruri Camellia sinensis Camellia sinensis Camellia sinensis Camellia sinensis Oenothera paradoxa

Japanese dodder Stone breaker Green tea Black tea Green tea Black tea Evening prime rose

Cuscutaceae Euphorbiaceae Theaceae Theaceae Theaceae Theaceae Dnagraceae

Dh et at., 2002 Ueno et at., 1988 Persson et at., 2006 Persson et at., 2006 Persson et at., 2006 Persson et at., 2006 Kiss et at., 2008

Liu et at., 2003 Yahara et at., 1990 Izumitani et at.,

1990

Daphnodorin A & B Dimorphecolic acid, alpha Dimorphecolic acid, alpha (DLl Dimorphecolic (DLl acid, beta 3,4-Di-Ocaffeoylquinic acid 3,5-Di-OEllagic acid (-l-Epicatechin (-l-Epicatechin gallate

~ ~

l'IO Ie

Flavonoid

I ..,t:::l ~

~ >:l ,....

;:l

'"

~

~

Table 2. (Contd.) Chemical name

Class of the chemical compound

Gallotannins Geraniin

Tannin Tannin

Gossypol

Botanical name (Source)

Common name

Family

Reference

o·

;:::I

Sesquiterpene

Phyllanthus niruri Allophylus edulis Phyllanthus urinaria Gossypium sp.

Chinese herbs Stone breaker COCll, Chal chal Gripeweed Cotton

Euphorbiaceae Sapindaceae Euphorbiaceae Malvaceae

Liu et al., 2003 Ueno et al., 1988 Arisawa et al., 1989 Lin et al., 2008 Krassnigg et al.,

HHL 2"-hydroxy -nicotianamine IAP

Peptide Alkoloid

Glycine max Fagopyrum esculentum

Korean soybean Buckwheat

Fabaceae Polygonaceae

Shin et al., 2001 Aoyagi, 2006

Peptide

Triticum spp.

Wheat

Poaceae

IAYKPAG IFL Isoacteoside

Peptide Peptide Phenylethanoid glycoside Phenylethanoid glycoside Phenylpropanoid glycoside Flavonoid

Spinacia oleracea Glycine max Abeliophyllum distichum

Spinach Soybean White forsythia

Amaranthaceae Fabaceae Oleaceae

Motoi & Kodama, 2003 Yang et al., 2003 Kuba et al., 2003 Oh et al., 2003a

1984

Isocrenatoside Isomartynoside Isoorientin

Isorhamnetin3-beta-y lucopyranoside Isoquercitrin

Phenylpropanoid glycoside Flavonoid

~

§: .,...

Microtoena prainiana

Prain microtoena

Clerodendron trichotomum

Harlequin glorybower Verbenaceae

Kang et al., 2003

Cecropia glaziovll Cecropia hololeuca Musanga cecropioide Sedum sarmentosum

Red cecropia Silver cecropia Mrican Corkwood Stringy Stonecrop

Cecropiaceae Cecropiaceae Moraceae Crassulaceae

Dubois et al., 2001 Dubois et al., 2001 Dubois et at., 2001 Oh et al., 2004

Diospyros kaki

Japanese persimmon

Ebenaceae

Kameda et al., 1987

Lamiaceae

Li et al., 2004

.!;, ~

~

o· .,...

'";:::I til

S·

bl ;:::I <:::

;:; '"

ktz:J ;:::I

~

~

s:'" C""::l

~

I\:)

00 -:]

I>:l

Table 2. (Contd.) Chemical name

Class of the chemical compound

Botanical name (Source)

Common name

Family

(-)-Epigallocatechin

Flavonoid

Epigallocatechin gallate (-)-Epigallocatechin gallate Ethyl caffeate Evocarpine Fagopyrum tripeptide Fangchinolium hydroxide Fenfangjine F, H and I Ficus oligopeptide FLP-1, -2 & -3 Ficus peptide FLP-1, -2 & -3 FVNPQAGS Gallic acid Gallocatechin, (+ 1 Gallocatechin, epi (-l Gallocatechin, epi, 3-0-gallate (- 1 Gallocatechin -3-0gallate (-l, epi (-)

Flavonoid

Camellia sinensis Camellia sinensis Camellia sinensis

Green tea Black tea Green tea

Theaceae Theaceae Theaceae

Persson et al., 2006 Persson et al., 2006 Persson et al., 2007

Phenolic Alkaloid Peptide

Camellia sinensis Camellia sinensis Rabdosia coetsa Evodia rutaecarpa Fagopyrum sp.

Green tea Black tea Duo mao bian zhong Evodia, Wu-Zhu-Yu Buckwheat

Theaceae Theaceae Lamiaceae Rutaceae Polygonaceae

Persson et al., 2006 Persson et al., 2006 Li et al., 2008 Lee et al., 1998 Koyama et al., 1993

Alkaloid

Stephania tetrandra

Han fang ji

Menispermaceae

Ogino et al., 1986

Alkaloid

Stephania tetrandra

Han fangji

Menispermaceae

Ogino et al., 1998

Peptide

Ficus carica

Common fig

Moraceae

Peptide

Ficus carica

Common fig

Moraceae

Flavonoid

Reference

Peptide Benzenoid Flavonoid Flavonoid Flavonoid

Helianthus annuus Phyllanthus niruri Camellia sinensis Camellia sinensis Camellia sinensis

Sunflower Stone breaker Korean green tea Korean green tea Korean green tea

Asteraceae Euphorbaceae Theaceae Theaceae Theaceae

Maruyama et al., 1990 Maruyama et al., 1989 Megias et al., 2004 Ueno et al., 1988 Cho et al., 1993 Cho et al., 1993 Uchida et al., 1987

Flavonoid

Camellia sinensis

Korean green tea

Theaceae

Cho et al., 1993

oa oa

~

~

~ t'" ~

I ..,t:l

~

~ '"

;:l ....

~

~

Table 2. (Contd.) Chemical name Isovitexin

Class of the chemical compound

Botanical name (Source)

Flavonoid

Musanga cecropioides Cecropia pachystachya Sesamum indicum Triticum spp. Brassica napus

African Corkwood Ambay pumpwood Sesame wheat Rapeseed Greens

Moraceae Cecropiaceae Pedaliaceae Poaceae Brassicaceae

Common name

Family

Reference

§: ,.,. 0·

;:l

IVY

Peptide

IY Kaempferol

Peptide Flavonoid

Kaempferol-3-al phaarabinopyranoside Kaempferol-3-0alpha-ara binopyranoside Kaempferol-3-0betagalactopyranoside Kaempferol-3O-galloyl-glucose KDYRL KLPAGTLF Lanosten (20-R) Lanosten (20-S) Leucosceptoside

Flavonoid

Sedum sarmentosum

Stringy Stonecrop

Crassulaceae

Dubois et al., 2001 Dubois et al., 2001 Hong et al., 2008 Matsui et al., 1999 Marczak et al., 2003 Olszanecki et al., 2008 Oh et al., 2004

Flavonoid

Ailanthus excelsa

Ailanthus, Ardu

Simaroubaceae

Loizzo et al., 2007

Ligstroside aglycones Liriodendrin

;:::-

~ ~

~

0· ctl" ;:l

'" S· ~ ;:l <:::

'" ;:;.

~. ~

;:l

Flavonoid

Ailanthus excelsa

Ailanthus, Ardu

Simaroubaceae

Loizzo et al., 2007

Flavonoid

Diospyros kaki

Japanese persimmon

Ebenaceae

Kameda et al., 1987

Peptide Peptide Triterpene Triterpene Phenylpropanoid glycoside Iridoid Lignan

Vigna radiata Vigna radiata Schinus moUe Schinus moUe Clerodendron trichotomum

Mung bean Mung bean Peruvian pepper Peruvian pepper Wild privet, European privet Danshen, Red sage Hardy rubber tree

Fabaceae Fabaceae Anacardiaceae Anacardiaceae Oleaceae

Li et al., 2006 Li et al., 2006 Olafsson et al., 1997 Olafsson et al., 1997 Kang et al., 2003

Lamiaceae Eucommiaceae

Kiss et al., 2008 Yamadaki et al., 1992

Ligustrum vulgare Eucommia ulmoides

~ ;3

S'" ~ "-"

l\:)

00 <:0

~

Table 2. (Contd.)

~

Chemical name

Class of the chemical compound

Botanical name (Source)

Common name

Family

Reference

Lithospermic acid B Luteolin Luteolin-7-0-

Polyphenol Flavonoid Flavonoid

Salviae miltiorrhiza Ailanthus excelsa Ailanthus excelsa

Red sage, Danshen Ailanthus, Ardu Ailanthus, Ardu

Lamiaceae Simaroubaceae Simaroubaceae

Kang et al., 2003a Loizzo et al., 2007 Loizzo et al., 2007

Lyciumin A

Peptide

Lycium chinense

Wolfberry, goji berry

Solanaceae

Lyciumin B

Peptide

Lycium chinense

Wolfberry, goji berry

Solanaceae

LRIPVA LRP, LSP, LQP, LPP LVY, LSA, LQP, LKY Martynoside

Peptide Peptide Peptide Phenylpropanoid glycoside Tannin

Spinacia oleracea Zea mays Sesamum indicum Clerodendron trichotomum Cuscuta japonica

Spinach Maize Sesame Harlequin glorybower

Amaranthaceae Poaceae Pedaliaceae Verbenaceae

Yahara et ai., 1989 Morita et al., 1996 Yahara et al., 1989 Yahara et al., 1993 Yang et al., 2003 Hong et ai., 2008 Hong et al., 2008 Kang et al., 2003

Japanese dodder

Cuscutaceae

Oh et al., 2002

Tannin

Cuscuta japonica

Japanese dodder

Cuscutaceae

Oh et al., 2002

Polyphenol lridoid

Rabdosia coetsa Jasminum azoricum

Duo mao bian zhong Jasmine

Lamiaceae Oleaceae

Li et ai., 2008 Somanadhan et al.,

Alkaloid Peptide Pepide Peptide Xanthone

Crotalaria sp. Spinacia oleracea Spinacia oleracea Corchorus olitorius Trypterospermum lanceolatum

Rattle pod Spinach Spinach Jute

Fabaceae Amaranthaceae Amaranthaceae Tiiaceae

Molteni et al., 1984 Yang et al., 2003 Yang et al., 2003 Kimoto et al., 1998 Sutter & Wang,

0

~-glucopyranoside

Methyl 3,4-Di-Ocaffeoylquinate Methyl 3,5-Di-Ocaffeoylquinate Methyl rosmarinate Molihuaside A

~

1998 Monocrotaline MRW MRWRD Nicotinamide Norathyriol

1993

~"tj ~

r~

I t? ""1

~

~

>=l

;::I

0;.

'"

~

Table 2. (Contd.) Chemical name Octadeca -10trans-12-cis-15cis-trienoic acid, 9-hydroxy Octadeca-9trans-ll-transdienoic acid, 13-hydroxy Oenothein B Oleacein Oleuropein 6"-0-malonyldaidzin 6" -0-malony1genistin 3'''-0methylcrenatoside Peimisine Penta-O-g alloy I-beta -D-gI ucose Procyanidins (dimer and hexamerl Proanthocyanidin B3 Pro cyanidin B-1 Pro cyanidin B-2, 3,3'-di-0-gallate Pro cyanidin B-3

Class of the chemical compound

Botanical name (Source)

Lipid

Lycium chinensis

Common name

Family

Reference

S' ;::,§:

.....

c· .s;, ;:s

Wolfberry, Goji berry

Solanaceae

Morota et al., 1987

>

Jg

c· .....

Lipid

Fnttllana vertic illata

Fritillary

Liliaceae

Niitsu et al., 1987

'";:s OJ>

S·

g ;:s <:::

Ellagitannin Iridoid Iridoid

Oenothera paradoxa Olea europaea Ligustrum vulgare

Isoflavone Isoflavone Glycoside

;::; '"

Onagraceae Oleaceae Oleaceae

Kiss et al., 2008 Hansen et al., 1996 Kiss et al., 2008

~.

Glycine max Glycine max Microtoena prainiana

Evening primerose Olive Wild privet, European privet Soybean Soybean Prain microtoena

Fabaceae Fabaceae Lamiaceae

Wu & Muir, 2008 Wu & Muir, 2008 Li et al., 2004

S'"

Alkoloid Flavonoid

Fritillaria ussuriensis Oenothera paradoxa

Ping bei mu Evening primerose

Liliaceae Onagraceae

Oh et al., 2003 Kiss et al., 2008

Flavonoid

Rhubarb

Polygonaceae

Uchida et al., 1987

Flavanol Flavonoid Flavonoid

Rhet rhizoma / Rheum palmatum Oenothera paradoxa Lespedeza capitata Camellia sinensis

Evening primerose Roundheadlespedeza Korean green tea

Onagraceae Fabaceae Theaceae

Kiss et al., 2008 Wagner et al., 1992 Uchida et al., 1987

Flavonoid

Lespedeza capitata

Roundheadlespedeza

Fabaceae

Wagner et al., 1992

I;tj

;:s

~ ;3 ~

~

I:\:) ~

f-'

Table 2. (Contd.)

I>:)

~

Chemical name

Class of the chemical compound

Botanical name (Source)

Procyanidin B-3, 3-0-gallate Procyanidin B-5, 3,3'-di-O-gallate

Flavonoid

Camellia sinensis

Flavonoid

Procyanidin B-6 Procyanidin C 1

Flavonoid Flavonoid

Procyanidin C-1, 3,3',3"-tri-O-gallate

Flavonoid

Procyanidin C-2 Procyanidin glycoside Pycnogenol Quercetin -3-0-al phaarabinopyranoside Quercetin-3-0-alpha(6"'-caffeoylglucosylbeta-1,2-rhamnosidel, Quercetin 3-0-alpha(6'" -p-coumaroyl glucosyl-beta-1, 2-rhamnosidel Quercetin-3-0(2"-O-galloyDglucoside

Flavonoid Flavonoid Flavonoid Flavonoid

Camellia sinensis Rhei rhizoma I Rheum palmatum Lespedeza capitata Cecropia glaziovii Cecropia hololeuca Camellia sinensis Rhei rhizoma I Rheum palmatum Lespedeza capitata Rhei rhizomal Rheum palma tum Ailanthus excelsa Pinus maritima

Flavonoid

Common name

Family

Reference

Korean green tea

Theaceae

Cho et al., 1993

Korean green tea Rhubarb

Theaceae Polygonaceae

Uchida et al., 1987 Uchida et al., 1987

Roundheadlespedeza Red cecropia Silver cecropia Korean green tea Rhubarb

Fabaceae Cecropiaceae Cecropiaceae Theaceae Polygonaceae

Wagner et al., 1992 Dubois et al., 2001 Dubois et al., 2001 Uchida et al., 1987 Uchida et al., 1987

Roundheadlespedeza Rhubarb

Fabaceae Polygonaceae

Wagner et al., 1992 Uchida et al., 1987

Simaroubaceae Pinaceae

Loizzo et at., 2007 Packer et al., 1999

Sedum sarmentosum

Ailanthus, Ardu Cluster pine, Maritime pine Stringy Stonecrop

Crassulaceae

Oh et al., 2004

Flavonoid

Sedum sarmentosum

Stringy Stonecrop

Crassulaceae

Oh et al., 2004

Flavonoid

Diospyros kaki

Japanese Persimmon

Ebenaceae

Kameda et al., 1987 Arisawa et at., 1989

I>:)

~

~

"ti

~ !"'< l>O

~

I ..,t::::! ~

~

t:l

;:s

.....

'"

~

Table 2. (Contd.) Chemical name Quercetin -3-0beta-D-glucoside Quercetin-3beta-glucopyranoside Quercitrin 1996a Quercitrin, iso Quinolone, 1methyl-2- [(cis-4-cis7l-4,7-tridecadienyll Quinolone, 1-methyl2-[pentadeca-cis6-cis-9-dienyl] RDHP Rosmarinic acid Rutin RIY Salvianolic acid Salvianolic acid B Sambacein I, II and III Sambacoside A

Class of the chemical compound

Botanical name (Source)

Flavonoid

Allophylus edulis

Common name

Family

Reference

S' ;::,~ ....

o· .s;, ;:l

Cocli, Chal chal

Sapindaceae

Arisawa et al., 1989

Flavonoid

Sedum sarmentosum

Stringy Stonecrop

Crassulaceae

Oh et al., 2004

Flavonoid

Erythroxylum laurifolium

Bois de ronde

Erythroxylaceae

Hansen et al.,

Flavonoid

Dwspyros kaki

Japanese Persimmon

Ebenaceae

Kameda et al., 1987

Alkaloid

Evodia rutaecarpa

Evodia, Wu-Zhu-Yu

Rutaceae

Lee et al., 1998

~

~

o· ....

'"

;:l

en

S·

2? ..,....'"<::: ;:l

k

t:>:J

;:l

~

Alkaloid

Evodia rutaecarpa

Evodia, Wu-Zhu-Yu

Rutaceae

Lee et al., 1998

Peptide Polyphenol Flavonoid glycoside Peptide Polyphenol Polyphenol Iridoid

Oryza sativus Rabdosia coetsa Abeliophyllum distichum Brassica napus Salvia miltwrrhiza Salvia miltiorrh!za Jasminum azoricum

Asian rice Duo mao bian zhong White forsythia Rapeseed Red sage, Danshen Red sage, Danshen Jasmine

Poaceae Lamiaceae Oleaece Brassicaceae Lamiaceae Lamiaceae Oleaceae

lridoid

Jasmmum azoricum

Jasmine

Oleaceae

Hong et al., 2008 Li et ai., 2008 Oh et ai., 2003a Marczak et al., 2003 Gao et al., 2004 Gao et al., 2004 Somanadhan et al., 1998 Somanadhan et al., 1998

~

'" ~ &3

'-.:

t-,J

'"'"

I).? (0

Table 2. (Contd.) Chemical name

Class of the chemical compound

Botanical name (Source)

Common name

Family

Syringin

Phenylpropanoid

Eucommia ulmoides

Hardy rubber tree

Eucommiaceae

Taxol

Diterpene

Taxus brevifolia

Taxaceae

TQVY Veratridine Verticine Verticinone Vicenin 2 Vitexin Vitexin, iso Vitexin-2"-Oalpha-L-rhamnoside VHLPP VHLPPP VIY VLIVP VTPALR VW VWIS WL Xanthone, 1,3,5, 6-tetrahydroxy Xanthone, 3,4, 6,7 -tetrahydroxy

Peptide Alkaloid Alkoloid Alkoloid Flavonoid Flavonoid Flavonoid Flavonoid

Oryza sativus Veratrum sp. Fritillaria ussuriensis Fritillaria ussuriensis Allophylus edulis Allophylus edulis Allophylus edulis Allophylus edulis

Pacific yew, Western yew Asian rice Corn lily Ping bei mu Ping bei mu COCll, Chal chal COCll, Chal chal COCll, Chal chal COCll, Chal chal

Yamadaki et al., 1992 Sauru et al., 1995

Poaceae Liliaceae Liliaceae Liliaceae Sapindaceae Sapindaceae Sapindaceae Sapindaceae

Hong et al., 2008 Ball et al., 1986 Oh et al., 2003 Oh et ai., 2003 Arisawa et ai., 1989 Okamot et al., 1994 Arisawa et al., 1989 Arisawa et al., 1989

Peptide Peptide Peptide Peptide Peptide Peptide Peptide Peptide Xanthone

Zea mays Sesamum indicum Glycine max Glycine max Vigna radiata Brassica napus Brassica napus Glycine max Tripterospermum lanceolatum Tripterospermum lanceolatum

Maize Sesame Soybean Soybean Mung bean Rapeseed Rapeseed Soybean

Poaceae Pedaliaceae Fabaceae Fabaceae Fabaceae Brassicaceae Brassicaceae Fabaceae

Arisawa et al., 1989 Hong et al., 2008 Hong et al., 2008 Hong et al., 2008 Li et al., 2006 Marczak et al., 2003 Marczak et al., 2003 Kuba Met al., 2003 Chen et al., 1992

Xanthone

Reference

Chen et al., 1992

*'"

::tl

~

"ti

~

!""

~

I tJ

"1

Jg ~

!;:l

;:s

'""'"

~

Inhibition of Angiotensin Converting Enzyme (ACE)

295

The ACE inhibitory activity ofthe plants or plant extracts is attributed to their diversified chemical composition. Since, the plants contain a number of different chemical compounds, isolation and characterization of the bioactive compounds responsible for antihypertensive cum ACE inhibitory activity is essential. In this regard, a number of chemical compounds with ACE inhibitory activity have been isolated and characterized from different plant species. The detailed phytochemical studies of various plant species have reported more than 150 chemical compounds as ACE inhibitors. The major class of compounds showing ACE inhibition is found to be flavonoids followed by peptides, alkaloids, phenylpropanoid glycosides, terpenes, iridoids, lipids, polyphenols, tannins and xanthones. Majority of them are characterized to be polar in nature. Table 2 summarizes the information on plant-derived ACE-inhibitory compounds. Although, most ofthe isolated compounds possess fairly high IC 50 values in comparison with the commercial ACE inhibitors, we still consider plantderived inhibitors to be of value in developing both traditional and modern medicine (Nyman et al., 1998). The utilization of plants as a source of ACE inhibitors is worth due to their tolerability and minimized side effects compared to the western medicine in humans. The results obtained from the earlier studies signify the possible usefulness of isolation and purification of potent Table 3a. Description of Azadirachta indica A. Juss Botanical name Common name Family Origin and distribution

Parts used Description of the plant

Chemical composition

Azadirachta indica A. Juss. Neem, Bevu, Nimba, DogonYaro, Vempu etc Meliaceae Native to India, Mayanmar, Bangladesh and Pakistan. Distributed throughout India, deciduous forests , tropical and semi-tropical regions. Bark, leaves, flowers, seeds, oil. A medium to large sized tree, 15-20 m in height with a clear bole of7 m having grayish to dark grey tubercled bark; leaved compound, imparipinnate, leaflets , subopposite, serrate, very oblique at base; flowers cream or yellowish white in axillary panicles, staminal tubes conspicuous, cylindric, widening above, 9-10 lobed at the apex; fruits one-seeded drupes with woody endocarp greeninsh yellow when ripe, seeds ellipsoid, cotyledons thick, fleshy and oily. Tannin, red dye, nimbin, nimbinin, nimbidin, 6-desacetylnimbinene, n-hexacosanol, noncosane, nimbicidin e, nimbinol , nimbandiol , quercetin, beta-sitosterol, azadirachtin, salannin, gedunin, azadirone etc.

RPMP Vol. 29 - Drug Plants III

296 Table 3a. (Contd.) Reference

A Handbook of Medicinal plants: A complete Source Book by Narayan Das Prajapati et at., Agrobios India publications.Mahesware, J.K. 1963; The flora of Delhi. Council of Scientific and Industrial Research, New Delhi. http://www.ayurhelp.com

Table 3b. Description of Artocarpus altilis Fosb Botanical name Common name Family Origin and distribution

Parts used

Chemical composition

Reference

Artocarpus altilis Fosb Breadfruit, Seemapanasa, Seema pila etc Moraceae Native to the region including Southeast Asia, New Guinea and the South Pacific, although the exact location is uncertain. Widely cultivated throughout the humid tropics. Bark, leaves and root. It is a large tree, 10-35 m tall with sticky, white latex and large spirally or alternately arranged lobed leaves. Flower monoecious. Mature fruits (Syncarps) relatively large, yellow-green to yellow-brown, fleshy, with numerous moderate sized seeds, exuding latex where damaged. Ripe fruit are often available throughout the year.

Pectins, starch, artocarpin, hydrocynic acid, beta amyrin acetate, alpha amyrin, flavonoids, lectin, oleic, linoleic and linolenic acids (seed oil), cycloratenol, cycloartenone, cycloartenyl acetate, folic acid, cycloaltilisin, cyclomorusin. A Handbook of Medicinal plants: A complete Source Book by Narayan Das Prajapati et at., Agrobios India publications.

Table 3c. Description of Catharanthus rose us Botanical name Common name

Family Origin and distribution

Parts used

(L.)

G.Don

Catharanthus roseus (L.) G.Don Madagascar periwinkle, Rosy-flowered Indian periwinkle, Cape periwinkle, Old maid etc. Apocynaceae Native to the Indian Ocean island of Madagascar. This herb is common in many tropical and subtropical regions worldwide, including the southern United States . Common wild plant in coastal areas and is cultivated as an ornamental plant. Leaves and root.

Inhibition of Angiotensin Converting Enzyme (ACE) Table 3c. (Contd.) Description of the plant

Chemical composition

Reference

297

It is a fleshy perennial herb growing to 30-80 cm height. Stems pinkish-red , muchbranched. Leaves opposite, obovate, glabrous on both sides, dark shining above. Flowers pink or white in the axil of the leaves. Follicle cylindrical, narrow, slightly arched-recurved in pairs ; seeds numerous, tiny, blackishbrown.

Alkoloids; serpentine, ajmaline, ajmalicine, catharanthine, catharanthinole, vindoline, vindolinine, vincaleucoblastine. Leurosidine, vincristine. A Handbook of Medicinal plants: A complete Source Book by Narayan Das Prajapati et al., Agrobios India publications.

Table 3d. Description of Morus alba L. Botanical name Common name Family Origin and distribution

Parts used Description ofthe plant

Chemical compounds

Morus alba L. Mulberry, White mulberry, Hipnerle etc. Moraceae Native of China, cultivated throughout the world wherever silkworms are raised, and is occasionally cultivated elsewhere in Europe, North America, and Mrica. Having escaped, trees often appear on roadsides, along fencerows, and as ornamentals. Root bark and leaves It is small to medium-sized monoecious or dioecious shrub or tree, up to 15 m tall, widespreading, round-topped, trunk attaining 60 cm in diameter; leaves alternate, stipulate, variable in shape, lobed or unlobed, cordate, dentate, acuminate, long-petiolate, 12 x 8 cm on fruiting branches, up to 25 x 20 cm on vigorous nonfruiting branches, usually smooth above, glabrous or pubescent along veins beneath, thin, light green; flowers small, greenishyellow, in dense spikes to 2 cm long; sepals 4; stamens 4; pistils with two styles; staminate spikes soon deciduous; pistillate spikes maturing into an aggregate fruit (syncarp) of drupelets; syncarp ovoid to oblong-cylindric, 1-5 cm long, white, pinkish or purplish to nearly black, edible long before ripe, sweet, but insipid; seeds brown, 1-1.2 mm long. Citral, linalyl acetate, linalol, terpinyl acetate, hexenol, ~-sitosterol, sterols, pipecolic acid, 5-hydroxy pipecolic acid.

298 Table 3d. (Contd.) Reference

RPMP Vol. 29 - Drug Plants III

Council of Scientific and Industrial Research. 1948-1976. The wealth ofIndia. 11 vols. New Delhi.http://www .hort.purdue.eduA Handbook of Medicinal plants: A complete Source Book by Narayan Das Prajapati et al., Agrobios India publications.

Table 3e. Description of Pongamia pinnata (L.) Pierre Botanical name Common name Family Origin and distribution

Parts used Description ofthe plant

Chemical compounds

Pongamia pinnata (L.) Pierre

Indian beech, Poongam oil tree, Karanj, Honge, Ponge etc. Fabaceae An Indomalaysian species, a medium-sized subevergreen tree, common throughout India, in tidal and beach forests, cultivated often as avenue trees. Now found in Australia, Florida, Hawaii, India, Malaysia, Oceania, Philippines, and Seychelles. Root, bark, leaves, flowers, seeds oil. It is a fast growing, medium-sized semievergreen glabrous, deciduous tree with a short bole and spreading crown upto 18m or more in height, bark grayish green or brown, very often mottled with bark brown dots, specks, lines or streaks; leaves compound, leaflets 5-9, ovate acuminate or elliptic, the terminal leaflet larger than the others; stipules caducous. Flowers fragrant, white to pinkish, paired along rachis in axillary, pendent, long racemes or panicles; calyx campanulate or cup-shaped, truncate, shortdentate, lowermost lobe sometimes longer; standard suborbicular, broad, outside; wings oblique, long, somewhat adherent to the obtuse keel; keel petals coherent at apex; stamens monadelphous, vexillary stamen free at the base but joined with others into a closed tube; ovary subsessile to short-stalked, pubescent; ovules 2, rarely 3; style filiform, upper halfincurved, glabrous; stigma small, terminal. Pod short stalked, oblique-oblong, flat, smooth, thickly leathery to subwoody, indehiscent, 1- seeded; seed thick, reniform to nearly round, smooth or wrinkled. alkaloids, pongaglabrone, glabrin, karangin, pongapin, diketonepongamol, pongamol, demethoxy-kanugin, gamatay, glabrosaponin, kaempferol, kanjone , kanugin, neoglabrin, pinnatin, pongapin, quercitin, saponin, ~-sitosterol, and tannin

Inhibition of Angiotensin Converting Enzyme (ACE) Table 3e. (Contd.) Reference

299

http://www.hort.purdue.edu, Council of Scientific and Industrial Research. 1948-1976. The wealth of India. 11 vols. New Delhi.A Handbook of Medicinal plants: A complete Source Book by Narayan Das Prajapati et al., Agrobios India publications.

Table 3f. Description of Tamarindus indicus L. Botanical name Common name Family Origin and distribution

Parts used Description ofthe plant

Chemical composition

Reference

Tamarindus indicus L. Tamarind tree, Imli, Hunase etc Fabaceae Possibly native to western Madagascar, tropical Africa. Today it has a worldwide distribution and has been adopted in several countries due to its culinary properties, as an ornamental tree and for its environmental characteristics. It is found in Asia, Mrica, the Pacific and America. It is distributed throughout India, particularly in the South India, often cultivated. Roots, fruits, seeds It is a large to very large evergreen, droughtresistant tree upto 30 m in height with dark grey bark having longitudinal fissures and deep cracks; leaves pari pinnate up to 15 cm long, rachis slender, channeled, leaflets 10-20 pairs, subsessile, oblong; flowers yellow , striped with red in laxz, few flowered racemes at the ends of the banchlets; fruit pods, brownish ash coloured, slightly curved , subcompressed, with a shallow oblong pit on each side ofthe flat faces; seeds enveloped by a toughy leathery membrane (the endocarp) and pulpy mesocarp, tests shining, hard. Tartaric acid, citric maleic acid, potassium bitartarate, oxalic acid, flavonoid glycosides saponaretin, vitexin, orientin, homoorientin, hordenine. A Handbook of Medicinal plants: A complete Source Book by Narayan Das Prajapati et al., Agrobios India publications. California Rare Fruit Growers www Site.

ACE inhibitory molecule in combating the cardiovascular complications associated with the hypertension. The isolation of pure plant compound might be a lead molecule for developing a newer, safe and effective antihypertensive drug. In our ongoing phytoceutical research, as an initial step to isolate ACE inhibitory molecules, we have selected Artocarpus altilis, Azadirachta

300

RPMP Vol. 29 - Drug Plants III

indica, Catharanthus roseus, Morus alba, Pongamia pinnata and Tamarindus indicus, based on their use in traditional medicine as antioxidant as well as antihypertensive. The information regarding these plants with botanical and common names, family, origin and distribution, description of different parts of the plants, parts used for the medicinal purpose and chemical composition is summarized in Table 3a-f. The leaf extracts of A. altilis, A. indica, C. roseus, M. alba, P. pinnata and seed coat extract of T. indicus were screened for their ACE inhibitory activity in vitro. The successive solvent extracts of plant materials (acetone, ethanol, methanol and water) exhibited differential ACE inhibitory activity differential in vitro. The methanolic and ethanolic leaf extract of A. altilis exhibited potent ACE inhibition while the aqueous and acetone extracts poorly inhibited ACE activity. The methanolic, ethanolic and aqueous leaf extracts of A. indica, C. roseus, and P. pinnata as well as that of T. indicus seed coat extract showed fairly good ACE inhibition compared to acetone extracts (Table 4). Thus, in our study the ACE inhibition was found to be higher in polar solvents, suggesting the benefit of polar compounds as potent ACE-inhibitors compared to non-polar compounds. Among, all the plant extracts studied, M. alba extracts did not show ACE-inhibitory activity.

Conclusions Determination of the ACE-inhibitory activity of plants and their derived compounds that are used in traditional medicine is helpful for the development of modern medicine. Although, many plant extracts have been characterized for their ACE inhibitory activity, further scientific investigations and isolation of bioactive polar compounds from plants will be supportive to develop safe and effective antihypertensive drugs.

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Shin, Z.I., Yu, R, Park, S.A, Chung, D.K, Ahn, C.W., Nam, H.S., Kim, KS. and Lee, H.J. 2001. His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. Journal of Agricultural and Food Chemistry 49(6): 3004-3009. Singh, P.D. and Johnson, J.H. 1984. Muraceins-muramyl peptides produced by Nocardia orientalis as angiotensi-converting enzyme inhibitors. II. Isolation and structure determination. The Journal of Antibiotics 37: 336-343. Soffer, RL. 1976. Angiotensin-converting enzyme and the regulation of vasoactive peptides. Annual Review of Biochemistry 45: 73-94. Somanadhan, B., Varughese, G., Palpu, P., Sreedharan, R, Gudiksen, L., Smitt, U.W. and Nyman, U. 1999. An ethnopharmacological survey for potential angiotensin converting enzyme inhibitors from Indian medicinal plants. Journal of Ethnopharmacology 65: 103-112. Sutter, M.C. and Wang, Y.X. 1993. Recent cardiovascular drugs from Chinese medicinal plants. Cardiovascular Research 27: 1891-1901. Takai, S., Sakaguchi, M., Jin, D., Baba, K and Miyazaki, M. 1999. Effects of daphnodorin A, daphnodorin B and daphnodorin C on human chymase-dependent angiotensin II formation. Life Sciences 64: 1889-1896. Takashari, T., Sato, T. and Kaneshima, H. 1993. Inhibitor of angiotensin converting enzyme from Acathopanax senticosus. Hokkaidoritsu Eisei Kenkyusho Ho 43: 63-64. Todd, P.A and Heel, RC. 1986. Enalapril: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in hypertension and congestive heart failure. Drugs 31: 198-248. Uchida, S., Ikari, N., Ohta, H., Niwa, M., Nonaka, G.I., Nishioka, I. and Ozaki, M. 1987. Inhibitory effects of condensed tannins on angiotensin converting enzyme. Japanese Journal of Pharmacology 43: 242-246. Ueno, H., Horie, S., Nishi, Y., Shogawa, H., Kawasaki, M. et al., 1988. Chemical and pharmaceutical studies on medicinal plants in Paraguay. Geraniin, an angiotensinconverting enzyme inhibitor from 'Paraparai mi', Phyllanthus niruri. Journal of Natural Products 51: 357-359. Vidt, D.G., Bravo, E.L. and Fouad, F.M. 1982. Captopril. The New England Journal of Medicine 306: 214-219. Wagner, H. and Elbl, G. 1992. ACE-inhibitory procyanidins from Lespedeza capitata. Planta Medica 58: 297-298. Wagner, H., Elbl, G., Lotter, H. and Guinea, M. 1991. Evaluation of natural products as inhibitors of angiotensin-I converting enzyme (ACE). Pharmaceutical and Pharmacological Letters 1: 15-18. Williams, LAD., Gossell-Williams, M., Barton, E.N. and Fleischhacker, R 1997. Angiotensin converting enzyme inhibiting and anti-dipsogenic activities of Euphorbia hirta extracts. Phytotherapy Research 11: 401-402. Wright, C.I., Van-Buren, L., Kroner, C.1. and Koning, M.M. 2007. Herbal medicines as diuretics: A review of the scientific evidence. Journal ofEthnopharmacology 114(1): 1-31. Wu, J. and Muir, AD., 2008. Isoflavone content and its potential contribution to the antihypertensive activity in soybean Angiotensin I converting enzyme inhibitory peptides. Journal ofAgricultural and Food Chemistry 56(21): 9899-9904. Yahara, S., Shigeyama, C., Nohara, T., Okuda, H., Wakamatsu, K and Yasuhara, T.1989. Structures of anti-ACE and renin peptides from Lycii radicis cortex. Tetrahedron Letters 30: 6041-6042. Yahara, S., Shigeyama, C., Ura, T., Wakamatsu, K, Yasuhara, T. and Nohara, T. 1993. Ciclic peptides, acyclic diterpene glycosides and other compounds from Lycium chinense Mill. Chemical & Pharmaceutical Bulletin 41: 703-709. Yamadaki, M. and Shimoyama, A 1992. Angiotensin-converting enzyme I inhibitor extraction from the Eucommia ulmoides leaves. Patent-Japan Kokai Tokkya Koho04,368,336,5PP.

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Yang, Y., Marczak, E.D., Yokoo, M., Vsui, H. and Yoshikawa, M. 2003. Isolation and antihypertensive effect of angiotensin I-converting enzyme (ACE) inhibitory peptides from spinach Rubisco. Journal ofAgricultural and Food Chemistry. 51(17): 48974902. Yang, Y., Tao, G., Liu, P. and Liu, J. 2007. Peptide with angiotensin I-converting enzyme inhibitory activity from hydrolyzed corn gluten meal. Journal ofAgricultural and Food Chemistry 55(19): 7891-7895. Ye, R.D. 2005. VIC Department of Pharmacology 312-996-5087 CMW 406. Pharmacology of antihypertensive drugs. www.uic.edu/classes/pcol/pcol331/dentalhandouts2005/ dentlecture23. Zimmerman, B.G., Sybertz, E.J. and Wong, P.C. 1984. Interaction between sympathetic and renin-angiotensin system. Journal of Hypertension 2: 581-587.

17 Pesticidal Activities of Some Important Chinese Medicinal Plant

Abstract Chinese traditional medicine has a long history which dates back thousands of years. To date, Chinese people still believe in traditional medicine and herbal medications are very popular in Chinese societies around the world. The latest encyclopedia of Chinese traditional medicine lists over 5500 natural sources (82.8% of which are plants). Of these, some provide compounds with significant antitumour activity and some have been demonstrated to cure tough disease. To improve whole understanding of the benefits of Chinese medicinal plants, more scientific information on their pesticidal activities should be made available to researchers. In this part, the focus will be on such efforts made in the study of pesticidal activities of some important Chinese medicinal plants. Key words: Pesticidal activities, Chinese medicinal plants CTM, Anti-tumor

Azadirachta indica A. Juss Common name: Indian-lilac, margosa, neem, nimtree, margosier Botanical name: Azadirachta indica A. Juss Family: Meliaceae Neem (Azadirachta indica A. Juss) is a evergreen tree, up to 15 m high. It grows on dry, sandstone, shrubland, grassland, sand, clay, disturbed pindan and shallow soils having a pH range of 5.0 to 8.5. Leaves are alternate, spiral, compound, pinnate, and petiolate, with a petiole ranging from 70 cm 1. The Key Lab. of Natural Pesticide and Chemical Biology (South China Agricultural University), Ministry of Education, P. R. China * Corresponding author: E-mail: [email protected]

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to 90 mm long. Flowers are arranged in panicles; predominantly white that are 0.3 to 0.4 cm across. Flowering occurs January through May. Fruit are smooth, green, indehiscent, ellipsoidal drupes, 1.2 to 1.8 cm long, and 1.0 cm wide that turn yellow to brown when ripe. The brown seeds are 1 cm in length and 4 to 5 mm wide, ovoid or spherically pointed apically with a thin testa. The hilum is not well marked. Seeds generally fall during rainy season and lose viability within 2 to 3 weeks.

Origin, distribution, commercially cultivated or wild Neem is a subtropical tree native to Asia-Tropical and cultivated & naturalized in tropical Asia, exact native range obscure.

Chemical constituents of medicinal value, their structures, formula and properties Triterpenoids, fatty acids, triacylglycerols, and sterols in neem oil have been determined (Momchilova, et al., 2007). In China, Wu in 1997 reported a new way of isolating azadirachtin: neem kernels were degreased with petroleum ether and extracted with methanol. Then azadirachtin was extracted into ethyl acetate, after clearup by column chromatography, and the azadirachtin was obtained. The method of mass and high-speed manufacturing azadirachtin-based insecticide formulation was found by Lab. ofInsect Toxicology, South China Agricultural University, which is applicable in industrialized production. In this method, neem seeds were extracted with methanol and degreased with petroleum ether and the azadirachtin was obtained by two times of silica gel column chromatography. Recently, surpercritical-fluid chromatography and micro-wave extraction method were used in extraction and isolation of natural production. Now, experiments aiming to carry these new techniques into azadirachtin production are in their way. The following are some formula structures of azadirachtin:

Pesticidal activities Insecticidal activities of neem extract and azadirachtin have been broadly studied. It was found that neem extract and azadirachtin showed high antifeedant property, growth inhibition, stomach poison, deterrant, respiration inhibition, contact poison and sterbility activities against the larvae of Pieris rapae, Spodoptera litura, Plutella xylostella, Oxya chinesis, Ostrinia furnacalis, Locusta migratoria manilensis, Tryporyza incertulas, Nilaparvata lugens,Cnaphalocrocis medinalis,Orseolia oryzae,Sitophilus zeamais, Sitophilus oryzae, Tesseratoma papillosa, Panonychus citri, Lyonetia citri, Dendrolimus punotatus, Ceraoris kiangsu,Ceraoris nigricornis ,Hemiberlesia pitysophila, Scelio uvarovi, Lycosa pseudoannulata, Coptotermes formosan us, Mythimna separata and mosquitoes. The

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o

o

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AcO AcO azadirachtin - A

azadirachtin - B

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The chemical structures of azadirachtins

antifeedant property, growth inhibition and deteration activity were the most prominent. It was reported that, at the dosage of 0.5 ~ml, azadirachtin could affect ovary development of Oxya chinensis and induce ovary shrink, which resulted in the oviposition failure. But an interesting phenomenon was found as owning to the different content of azadirachtin that activities ofneem samples gathered from various

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areas were different. The antifeedant property against the larvae of Pieris rapae and Spodoptera litura of methanol extract of the neem seeds gathered from Yuanjiang, Yuanmo and Mandalay were obviously different (He, 1999). Neem products have also displayed antibacterial activity against a wide variety of pathogenic bacteria. Both the MIC and MBC results obtained in this study indicate that neem (A. indica) oil displayed a greater antibacterial effect on Gram positive bacteria than Gram negative bacteria(Chiu, 1989; National Research Council., 1992).

Mechanism ofaction The main targets of azadirachtin were the brain neurosecretory system, the corpus paracar and the prothoracic glands. Azadirachtin can interfere with the internal secretion, influence synthesis and release of PTTH, decrease susceptivity of prothoracic glands to PTTH, induce deficiency of 20-hydroxyecdysone, and inhibit the development of pests (Li, 1995). Moreover, azadirachtin could stimulate the neurone of the sensillum basiconicum of the maxillae of the larvae of Pieris rapae, and inhibited the transmission of neural signal (Zhong, 1995). Qiu reported that the larval period of Ostrinia furnacalis was prolonged and the larvae couldn't pupate and died when fed with artificial diet that contained 20 Jlg/ml azadirachtin for 2 days (Qiu, 1984). When the larvae of Spodoptera litura were treated with azadirachtin, the content of haemolymph and the activity of carboxylesterase were inhibited. The thoracic legs became black, brown spot appeared on the thorax, the cerebrum atrophied, and the genitalia and the prothoracic glands became tumorous (Tian, 1993).

Commercial products available already Early in 4 century BC, neem leaves were burned for driving out mosquitoes or applied in barns or folded clothes for driving out pests by ancient Hindoo (Sigh et al., 1996), and people of a few countries still used this simple method to control pests. Suspension concentrateygranule, ultra low volume agent (Mordue et al., 1993) and wettable powder (Xu, 1999) of azadirachtin have been developed successively. There were a few of reports about effect of azadirachtin application in field in China. The field trial ofazadirachtin 0.3%EC against the resistant Plutella xylostella and Phyllotreta vittata was performed in Guangdong province, Guangxi province, Hebei province and Shan dong province (Xu Hanhong et al., 2001), and results showed that azadirachtin 0.3%EC was an outstanding insecticide against the two pests. More than 40 kinds of commercial insecticides that using azadirachtin as raw and processed material, such as Neenmix 4.5 (USA), Neem Gold (India), Fortune AZA (India), NeemAzal-F (Germany), Green Gold (Australia), Safer'ENI(Canada) and azadirachtin 0.3% EC, have been exploited all

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over the world since the first azadirachtin insecticide-Margosan-O was permitted to be registered in 19S5.0thers are Azatin, T urlex, Align, Neemrich, Neemgaurd, Nemidin, Nemol, Nemicidine, Margocideckok, Ackook, RD-9 (Repelin), Neemark, etc. In China, 0.3% neem EC has been successfully developed and is widely used now. Because azadirachtin comprises some unstable groups, such as ROOC-, epoxy, it is unstable and decomposed easily in the condition of ultraviolet radiation, illumination and high temperature. It is reported that azadirachtin decompounded completely and became invalid after the neem extract had been applied in field for 5-S days. How to regulate stabilities of azadirachtin-based insecticide formulation is a difficult problem and a factor which limits azadirachtin application in market (Rong, 2000).

Melia azedarach Common name: China berry tree; Botanical name: Melia azedarach. Family: Meliaceae. Trees to 10 m tall, deciduous. Bark brownish gray, longitudinally exfoliating. Branches spreading; branchlets with leaf scars. Leaves oddpinnate, 2-pinnate or 3-pinnate, 20-40 cm; leaflets opposite; leaflet blades ovate, elliptic, or lanceolate, 3-7 x 2-3 cm but terminal one usually slightly larger, both surfaces with stellate trichomes when young but glabrescent, secondary veins 12-16 on each side of midvein, outspread and ascending, base ± oblique and cuneate to broadly cuneate, margin crenate or sometimes entire, apex shortly acuminate. Flowers fragrant. Calyx 5parted; sepals ovate to oblong-ovate, outside puberulent, apex acute. Petals lilac-colored, obovate-spatulate, 0.9-1.3 cm, both surfaces puberulent but usually outside more densely so. Ovary spherical, glabrous, 5-S-locular, with 2 ovules per locule; style acerose; stigma capitate, not included within filament tube, apex 5-dentate. Drupe globose to ellipsoid, 1-3 x 0.S-1.5 cm; endocarp ligneous. Seed ellipsoid. Fl. Mar-May, fro Oct-Dec.

Origin, distribution, commercially cultivated or wild This species is cultivated and sometimes naturalized in many warmtemperate and tropical parts ofthe world. Because ofits extensive cultivation and tendency to become naturalized in disturbed habitats, its original wild distribution is uncertain. It is distributed in Laos, Nepal, Papua New Guinea, Philippines, Sri Lanka, Thailand, Vietnam; tropical Australia, Pacific islands (Solomon Islands)

Chemical constituents of medicinal value, their structures, formula and properties Melia azedarach distributes widely in China and contains toosendanin. Toosendanin (TSN) is a triterpenoid extracted from Melia toosendan Sieb

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o II -c I

V

HO

H

The chemical structure of Toosendanin

et Zucc, which was used as a digestive tract-parasiticide and agricultural insecticide in ancient China. Toosendanin (C30H3s011' FW = 574), a triterpenoid derivative, was extracted from the bark of Melia toosendan Seib et Zucc by Chinese scientists in the 1950s and used as an ascarifuge in China instead of imported sendanin in the following 20 years (Shi, 2007).

Pesticidal activities Toosendanin is a kind of druggery that can repel bellyworm and its pharmacological activities were studied systematically in China. Toosendanin possesses strong antifeedant activity at high concentration against the larvae of Tryporyza incertulas, Spodoptera venalba, Plutella xylostella and Ostrinia furnacalis and stomach poison at low concentration against the larvae of T. incertulas, Pieris rapae. It also can inhibit the growth process of insects. Methanol extracts of the seed kernels of M. azedarach and M. toosendan, as well as toosendanin (the most important active ingredient of M. azedarach and M. toosendan. which was first isolated and identified in China and mainly used as anthelmintic against ascariasis) were very effective against S. litura as an antifeedant. The 5th instar larvae were so sensitive to these materials that the AFC 50 was found to be as low as 0.0027%, 0.124% and 0.0027%, respectively in the leaf disc choice test. The 2 nd instar larvae had a reduction of feeding by 99.6% in choice test when the leaves were treated with 1% methanol extract of M. azedarach. The acetone extracts of the leaves, barks and stems of the two species of chinaberry also were very effective at a concentration of 1-2% (weight of plant tissue/volume ofthe solvent) as feeding deterrents. Both the methanol and ethanol extracts showed promising antifeedant activity against the larvae of P. rapae in both choice and no-choice tests. However, it was interesting to find that the larvae almost stopped eating either the treated or untreated leaves after feeding on the bioactive materials for several hours. The larvae were killed when treated with high concentrations of the plant extracts. Growth inhibition, oviposition deterrency and egg hatching disruption from treatments with lower concentrations were observed. The results of field plot trials demonstrated that the vegetables were effectively protected from the injury of the larvae of P. rapae by spraying these plant extracts.

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Toosendanin at a concentration of 0.1% gave an effectiveness approximate to that of 0.01%. Therefore, commercial product of toosendanin, 0.5% Toosedarin EC has been developed recently (Chiu, 1988).

Mechanism ofaction TSN was demonstrated to be a selective presynaptic blocker and an effective antibotulismic agent. By interfering with neurotransmitter release through an initial facilitation followed by a subsequent depression, TSN eventually blocks synaptic transmission at both the neuro-muscular junction and central synapses. Despite sharing some similar actions with botulinum neurotoxin (BoNT), TSN has a marked antibotulismic effect in vivo and in vitro. Studies suggest that the antibotulismic effect of TSN is achieved by preventing BoNT from approaching its enzymatic substrate, the SNARE protein. It is also found that TSN can induce differentiation and apoptosis in several cell lines, and suppress proliferation of various human cancer cells. TSN inhibits various K+-channels, selectively facilitates Ca2 +-influx via L-type Ca2 + channels and increases intracellular Ca2 +concentration ([Ca 2+]). The TSNinduced [Ca2+] increase and overload could be responsible for the TSNinduced bipha~ic effect on transmitter release, cell differentiation, apoptosis as well as the cytoxicity of TSN(Shi, 2007). Because TSN inhibits feeding and development in insects (Chiu, 1989 and Carpinella et al., 2003), its use as a botanical insecticide in China has become popular. The experiments with radioactive isotopes indicated that the 3H-toosendanin eaten by the imported cabbage worm was found in the blood and then transported to various organs. The phenomenon of antifeeding occurred only after 3H-toosendanin reached the blood. There seemed to be a correlation between the fluctuations of 3H-toosendanin in blood and the response of the insects (Zhang Yeguang, 1984). By feeding or injecting the 5th ins tar larvae at the dosage of 1-3 ].lg/larva of toosendanin, the pathological changes in histology could be observed, which indicated that the tissues of midgut was destroyed seriously. The pertrophic membrane disappeared, so that the food in the gut contacted the tissue of ventriculus directly and damaged it.

Chrysanthemum coronarium Common name: Garland chrysanthemum Botanical name: Chrysanthemum coronarium Family; Compositae A leafy annual herb growing to 1.2 m and has yellow florets grouped in small rayed flower heads and aromatic, bipinnately lobed leaves. It is in flower from July to September, and the seeds ripen from August to October. The flowers are hermaphrodite (have both male and female organs) and are pollinated by Bees, flies, beetles and Lepidoptera (Moths & Butterflies). The plant is self-fertile.

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Origin, distribution, commercially cultivated or wild It grows very well in mild or slightly cold climates, but will go quickly into premature flowering in warm summer conditions. Seeds are sown in early spring and falL The plant prefers light (sandy), medium (loamy) and heavy (clay) soils and requires well-drained soiL The plant prefers acid, neutral and basic (alkaline) soils. It can grow in semi-shade (light woodland) or no shade. It requires moist soiL It is popular in Cantonese cuisine, especially in the cuisine of Hong Kong.

Chemical constituents of medicinal value, their structures, formula and properties The leaves are expectorant and stomachic. In conjunction with black pepper it is used in the treatment of gonorrhoea. The flowers are aromatic, bitter and stomachic. They are used as a substitute for camomile. The bark is purgative, it is used in the treatment of syphilis (Duke et al., 1985). The active chemical of pescticidal activities is spiro enol ether:

The chemical structure of spiro enol ether

Pesticidal activities As spiro enol ether, extracted from Chrysanthemum coronarium, exhibits antifeedant activity against some insects, therefore, a series of analogues, named after the sequence, of spiro enol ether were synthesized accordingly at Shanghai Institute of Organic Chemistry, Chinese Academy. When treated with leaves that had dipped into the solution, the LC 50 of compounds No. 12 and No. 20 against the 2rd instar larva of the diamondback moth, Plutella xylostella after 5 days were 498.74 p.glmL and 299.82 p.glmL, while against the 3rd instar larva of Pieris rapae after 6 days were 655.79 p.glmL and 465.74 p.glmL and after 8 days 382.12 p.glmL and 278.86 p.glmL (Zhang, 2001). Against the 3rd instar larvae of S. Iitura, the LC 50 of compound No. 12 and No. 20 were 1295.76 p.glmL and 945.25 p.gI mL. Injected with compound No. 12 and No. 20 at the concentration of 10 p.g per larvae, the, mortality rate within 24 h of the 4rd instar larvae of S. Iitura were 66.67 and 87.50%. At the concentration of500 p.glmL, these two compounds could affect the weight oflarvae obviously (Xu, 2000a; Xu, 2000b). As to the antifeedant activities of these two compounds, it is reported that the AFC 50 of No. 12 and No. 20 against the 4rd instar larva of the diamondback moth were 205.06 glmL and 405.97 p.glmL 24h after the

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treatment, and 235.08 Jlg/mL and 261.97 Jlg/mL 48h after the treatment, whereas against the 3rd instar larva of Pieris rapae were 370.00 Jlg/mL and 226.93 Jlg/mL 24 h after the treatment (Cheng, 2002). The AFC 50 of No. 12 against the 3rd instar larvae of S. litura in choice test and the 4rd instar larvae of S. litura in no-choice test were 403.83 Jlg/mL and 340.39 Jlg/mL, respectively (Xu, 2000a). In addition, compounds No.4, No.8, No. 12 and No. 20 also showed oviposition deterrent and hatch delay activities against the 2rd ins tar larvae of the diamondback moth. At the concentration of 1000 Jlg/mL, the oviposition deterrent rate were 39.90, 57.90, 25.56 and 47.17%, respectively (Xu, 2000b).

Stellera chamaejasme L. Botanical name: Stellera chamaejasme Family: Thymelaeaceae It is in flower in June, and the seeds ripen from August to October. The flowers are hermaphrodite.

Origin, distribution, commercially cultivated or wild Stellera chamaejasme L. is widespread in the north of China. The plant prefers light (sandy) and medium (loamy) soils and requires well-drained soil. The plant prefers acid, neutral and basic (alkaline) soils. It cannot grow in the shade. It requires moist soil.

Chemical constituents of medicinal value, their structures, formula and properties S. chamaejasme L. has been known to contain biflavonoids. Tsai and Lin have isolated an active principle possessing selective fungistatic and bacteriostatic activity from the rootstock ofthis plant, which they tentatively named as "stellerin". Natural stellerin is soluble in ether, ethanol, acetone, benzene, glycerol and clove oil, soluble in water to the extent of 100 mg/L, insoluble in light petroleum, carbon tetrachloride and chloroform. Thirteen compounds were isolated from roots of Stellera chamaejasme L. (Thymelaeaceae). They are 3-sitosterol, simplexin, pimelea factor P2, daucosterol, (+ )-3-hydroxy-1,5-diphenyl-1-pentanone, 4-ethoxy-benzoic acid, 2,4,6-trimethoxy-benzoic acid, (+)-afzelechin, fumaric acid, N,N-dimethylL-aspartic acid, umbelliferone, daphniretin) and a novel bicoumarin named bicoumastechamin. In vitro bioassays showed that pimelea factor P2 inhibited cancer cell growth, daphniretin exhibited immunomodulatory activity, and (+ )-3-hydroxy-1,5-diphenyl-1-pentanone exhibited both immunomodulatory and anti-tumor activity (Xu et al., 2001). The daphnane-type diterpene gnidimacrin, isolated from the root, was found to strongly inhibit cell growth of human leukemias, stomach cancers and non-small cell lung cancers in vitro at concentrations of 10(-9) to 10(-10) M. On the other hand, even at 10(-6) to 10(-5) M, the small cell lung cancer

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cell line H69 and the hepatoma cell line HLE were refractory to gnidimacrin. The agent showed significant antitumor activity against murine leukemias and solid tumors in an in vivo system (Yoshida et al., 1996). Gnidimacrin, a diterpene compound, isolated from the methanol extract of Stellera chamaejasme L, showed significant antitumor activities against mouse leukemia P-388 and L-1210 in vivo. At the dosages of 0.020.03 mg/kg i.p., the in increase in life span (ILS) was 70 and 80%, respectively (Feng et al., 1995).

Pesticidal activities Stellera chamaejasme L. is an interesting poisonous plant, which is reputed to have medicinal and insecticidal value. It is a repellant and a contactpoison and possibly acts as a stomach poison. The LD50 of the extract of root of Stellera chamaejasme L. with ethanol against the 5th instar larvae of imported cabbage worm were 12.32 flg/ larvae 48 h after the treatment. While the antifeedant rate of no-choice was 56.07% by topical application 24 h after at the dosage of 25 flg/larvae (Zhang, 2000a). Furthermore, the extract of root of Stellera chamaejasme L. with ethanol also exhibited oviposition deterrent activity and ovicidal activity against imported cabbage worm (Zhang, 2000b). In addition to stellerin, the other pesticidal active ingredients are 7hydro-coumarin, daphnoritin and chamaechromone. It was found that they possessed strong antifeedant and growth inhibition activities. The AFC 50 of daphnoritin and chamaechromone to the 5th instar larvae of imported cabbage worm were 160.57 pg/mL and 229.49 pg/mL 24 h after the treatment and 76.94 pg/mL and 131.30 pg/mL 48 h after the treatment respectively (Zhang, 2000c).

Mechanism of action Physio-biochemical studies revealed that daphnoritin increased the content of sugar, while decreased the content of protein in haemolymph significantly. In contrast, chamaechromone decreased the content of sugar, while increased the esterase activity in midgut significantly (Zhang, 2000).

Daphne tangutica Maxim. Botanical name: Daphne tangutica Maxim Family: Thymelaeaceae Shrubs evergreen, 0.5-2.5 m tall. Branches yellowish green, turning grayish yellow or purplish red to grayish brown, sparsely pubescent, glabrescent. Leaves alternate; petiole ca. 1 mm, glabrous; leaf blade lustrous dark green, lanceolate to oblanceolate, 2-10 x 0.5-2.2(-3) cm, leathery, abaxially glabrous or sparsely puberulous, adaxially glabrous, base cuneate or decurrent, margin sometimes revolute, apex obtuse, rarely retuse or

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acute to acuminate. Inflorescences terminal, capitate, 3-12-flowered; peduncle 1-3 mm, pubescent; bracts caducous, ovate or ovate-Ianceolate, 5-9 x 3-4 mm, glabrous, ciliate on margin, apex obtuse. Pedicel to ca. 1 mm, densely pubescent. Calyx pink to white flushed purplish red abaxially; tube cylindric, 9-13(-15) mm, exterior glabrous; lobes 4, ovate or ovateelliptic, 5-8(-10) x (3-)4-5(-6) mm, apex obtuse. Stamens 8, lower whorl inserted slightly above middle of calyx tube, upper whorl in throat; filaments short; anthers oblong, 1-1.2 mm; upper ones slightly exserted from calyx tube. Disk annular, ca. 0.5 mm wide, irregularly lobed. Ovary oblong-obovoid, 2-3(-4) mm, glabrous; style short; stigma discoid-capitate, 4-lobed. Drupe red, subglobose or ovoid, 6-8(-10) mm; flowering Apr-Jun, fruit May-Jul.

Origin, distribution, commercially, cultivated or wild Daphne tangutica Maxim., a shrub ofThymelaeaceae family, mainly grows in northwest area of China at altitude of 1500-4000 m. It is an evergreen shrub that grows in the semi-tropical area such as Bhutan and northern Burma.

Chemical constituents of medicinal value, their structures, formula and properties The roots of D. tangutica have been used to treat wounds, bruises and faucitis as a folk medicine in China (Song, 1999). The root barks of this plant are utilized as a traditional Tibetan medicine for releasing pain, curing rheumatism and as an abortifacient (Qinghai Provincial Institute of Tibetan Medicine, 1996). Different classes of natural products have been isolated from this plant, including flavonoids, coumarins and diterpenoids. It is widely used to treat cancer in folk medicine, and various compounds isolated from these plants showed antileukemic activity against P-388 lymphocytic leukemia in mice (Torrance et al., 1979; Wang, 1980; Hall et al., 1982; Taniguchi, 1996; Li et al., 2002; Zhang, 2007). According to previous research, daphnane diterpenes were found in the families ofEuphorbiaceae and Thymelaeaceae, and were considered to be the major toxic and active constituents ofThymelaeaceae plants (Zhuang, 1982; Pan, et al., 2006).

Pesticidal activities Insecticidal activity of Daphne tangutica Maxim.was firstly reported by Chen in 2000. The air-dried and powdered plant was extracted with petroleum ester, acetone, methanol, ethyl acetate, chloroform and benzene. The crude extracts of the five solvents were proved to possess strongly antifeedant activity against the 3rd instar larva of tobacco cutworm, S. litura, through the preliminary bioassay with AFC 50 of 108.72, 111.69, 102.14, 71.15 and 65.51 g dried powderlL (Chen, 2000). Further bioassay revealed there existed strong stomach toxicities along with antifeedant activity of these extracts

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against the 5th instar larvae of imported cabbage worm at the concentration of 100 mg fried powder/mL (Chen, 2000). Moreover, the methanol extract exhibited high effect of antifeedant and stomach poison. Fraction 9 of silica gel column chromatography proved to be the most active fraction among those partitioned from the methanol extract. The larvae fed on leaf discs treated with fraction 9 grew slowly, and their bodies were definitely smaller than those of the control. Histological study indicated that the tissue of midgut was destroyed and fat-body became to fade out by autolysis. The preliminary physiological reaction studies showed that fraction 9 could reduce the content of protein in haemolymph and inhibited the activity of esterase in midgut significantly (Xu, 2000). The benzene extract possessed the highest insecticidal activity against the 5 th instar larvae of imported cabbage worm, chloroform the second. Among root bark, xylem of root, stem bark, xylem of stem and leaf, possessed strong antifeedant activity and stomach poison. The residues of different parts extracted by benzene were re-extracted with methanol, and the leaf extracts gave insecticidal activity to some extent. Therefore, it suggested the content of active ingredients in the leaf should be the highest (Chen, 2000).

Xanthium sibiricum Patrin Botanical name: Xanthium sibiricum Patrin Family: Compositae An annual spring weed. Stem 40-60 cm tall, straight, rigid, simple or somewhat branchy, rounded below, striated upwardly. Leaves are triangularovoid or cordate, 5-9 cm long, almost entire or indistinctly irregularly serrate-dentate, with fine thin decumbent hairs or bristly pubescent on both sides; petioles are 11 cm long. Involucre embracing hemicarp, oval or ellipsoid, narrowing based, swollen, 12-15 mm long and 4-7 mm wide, with glandular pubescence; regularly covered with short (1-2 mm long) and thin, hardly thickened at base and hooked at apex, yellowish or greenish thorns. Involucral apical beaks are mostly straight, seldom crescent, sharp, frequently unequal, parallel, less often converging, pubescent, 1.5-2.5 mm long. Blossoming and fructifying occurs from July until September (Korchagina et al., 1972; Li, 1998; Wang, 1990; Zhang, 2000). Origin, distribution, commercially, cultivated or wild It distributes at the Caucasus, Western and Eastern Siberia, the Far East, Central Asia, northern Iran, Kashmir, China, Japan.

Chemical constituents of medicinal value, their structures, formula and properties Xanthium is an invaluable herb for the treatment of acute and chronic rhinitis and chronic paranasal sinusitis. It is relied upon to open the nasal

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passages and clear out nasal discharges. It also helps clear sinus headaches. Xanthium is commonly added to formulations that soothe skin conditions with itching, including atopic dermatitis and chronic eczema. Its broad scope of anti-inflammatory and anti-allergic action also makes it useful for chronic arthritis with generalized stiffness and pain (Jiangsu Medical College, 1971; Huang, 1993). Previous research indicated that this plant contained xanthatin, xanthiazinone, xanthiazone, xanthiside, xanthienopyran and 5hydroxypyrrolidin-2-one along with eremophilanolides, sibiriolides A and B (Zhang et al., 2006).

Pesticidal activities Early in 1000 years ago, it had been used to control some insects. It was also recorded as an effective pesticide against the imported cabbage worm in "Chinese Indigenous Pesticides" in 1959. Recent studies showed that it contained many a chemical, such as, xanthostrumarin, carboxyatractyloside, xanthatin, atractyloside, xanthin, xanthunin, xanthanodiene, strumaroside and so on. Bioassay indicated that xanthatin and atractyloside exhibited potent stomach-poison and growth inhibition activities as well as antifeedant activity. The LD50 of xanthatin against the 5th instar of the imported cabbage worm was 2.07 Ilg per larva 24 h after the treatment. When treated at the dosage of 50 Ilg/mL of xanthatin, the pupation rate of the imported cabbage worm was only 26.67% 4 days after the treatment (He, 2002).

Mechanism of action Mechanism studies indicated that the contents of sugar and protein in haemolymph of the imported cabbage worm were both decreased significantly, and the activity of esterase in the mid-gut was inhibited obviously as well (He, 2002).

Bidens pilosa Linn. Common name: Hairy beggarticks Botanical name: Bidens pilosa Linn. Family: Compositae It is a weak annual herb usually a metre or less in height, with spreading branches. The leaves are opposite and are divided pinnately into 3-5 leaflets with toothed margins (edge). The terminal and lateral leaflets are ovate to lanceolate shaped. The petiole (the stalk of a leaf) is very slightly winged. The flowers (summer-autumn), technically heads of tiny flowers, terminate all branches and branchlets. The flowerheads (capitula) are white and yellow and 5-15 mm in diameter. They are borne on long slender peduncles (stalks) at the end ofthe stems. Each flower head

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has 4 or 5 short, broad, white petals (ray florets) with numerous, yellow disc florets (tubular florets in centre offlowerhead). The outer involucral bracts (a whorl or several whorls of a more or less modified leaves surrounding a flower or an inflorescence) have finely hairy margins (edges) and are shorter than the inner bracts. The seeds are slender, linear, curved, black and rigid, they are 4 angled 6-12 mm long with 2 or 3 barbed awns (stiff bristle). Flowering occurs throughout the year but primarily summerautumn (Harden, 2002).

Chemical constituents of medicinal value, their structures, formula and properties It is used for coughs, conjunctivitis, dysentry, haematuria, urethritis, custitis, cloudy urine, benign prostratic hypertrophy, kidney stones, etc. in China.

Phenylheptrine (PHT), isolated from Bidens pilosa, possessed obviously photoactivity to the larvae of mosquitoes and flies.

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Mechanism of action The main active ingredient in this plant is a photoactivated chemical, a-To Two photo-oxidation mechanisms were proposed for the mechanism of photo activated pesticides, namely photo-induced toxicity and photo-kinetic reaction (Heitz, 1987). Researches on mechanisms of action of photo active insecticides have been done in our group, mainly focusing on polyacetylenes, such as, a-T and some synthetic polyacetylenes. In 1975, Callaham reported the inhibition of the acetylcholinesterase from the imported fire ant, Sole nopsis richteri (Forel) by dye-sensitized photooxidation (Callaham et al., 1975a; Callaham et al., 1975b). Recently, ESR analysis on active oxygen radicals and endogenous enzyme activity in the protective enzyme system indicated that a-T could enhance the amount of active oxygen radicals in

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the 4th instar larvae of Culex pipiens pallens, and the effect of a-T could also be increased by exposure to near ultraviolet radiation. The activity of superoxidedismutase (SOD), peroxidase (POD) and catalase (CAT) could be inhibited by a -Tin vivo (Jiang et al., 2000; Jiang et al., 2003a). Experiments on Na+-K+-ATPase from the larval heads of Helicoverpa armigera and Ostrinia furnacalis suggested that Na+-K+-ATPase might be also involved in the mechanism of action of a-T (Jiang et al., 2003b). Additionally, a-T had effect on GSTs. High concentration of a-T inhibited the GSTs in vitro in Helicoverpa armigera. Following topical administration, high doses of a-T increased the GSTs activities in vivo both in Helicoverpa armigera and Ostrinia furnacalis (Jiang et al., 2003c). Enzyme experiment ofthe 11 synthetic polyacetylenes (the molecular structures of which were mentioned above in part 2) showed that 1-t-butyl4-hydroxymethyl diacetylene and di-ethyl-2-propargyl thiophosphate could inhibit the activities of AChE and Na+-K+-ATPase of adult Periplaneta americana (Wan et al., 2005). The mechanisms of programmed cell death caused by ± T analogues have been studied recently. The oxidative damage to Spodoptera litura (SL) cells by hematoporphyrin mono methyl ether (HMME), the second generation photosensitizer for PDT, was investigated. IC 50 value, the output of MDA and the content of GSH, and the alteration of cell organelles were assayed or observed to explore the feasibility of applying HMME and its derivatives to control agricultural pest insects. IC values obtained with MTT at 24h and 48h post HMME treatment with irradiation were 8.35 J.lg/mL and 7.66 J.lg/mL, respectively. The results obtained with TBA method showed that photo activated HMME could induce MDA increasing in dose dependent manner. When the treatment concentration of HMME was 50.000 J.lg/mL, the output of MDA at 48 h post treatment was 173.08 ± 3.51 nmollL. The GSH level, however, exhibited a contrary tendency. When the treatment concentration of HMME was 50.000 J.lg/ mL, the GSH content decreased by 39.59% compared with the same concentration treatment without irradiation. The SEM photographs showed that distinct pores in SL cells treated with 6.250 J.lg/mL HMME and irradiation, and sunken and plicate membrane appeared in the treated SL cells. All the evidence indicated that HMME had induced oxidative damage to SL cells (Wang et al., 2007). The oxidative damage of a-terthienyl (a-T) to Spodoptera Iitura (SL) cell and its mechanism were investigated also. MTT was used to compare the toxicity of a-T and rotenone to SL cell. The output of malondialdehyde and relative content of glutathione were determined with 2-thiobarbituric acid and 5,5'-dithio-bis(2-nitrobenzoic acid) respectively. Transmission electron microscope (TEM) was employed to observe the influence of a-T on the membrane and organelle of SL cell. The IC 50 value of a-T to SL cell was 0.21 J.lg/mL, whereas the corresponding

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dose of rotenone was 12.25 J.1g/mL. The output ofMDA positively responded to the concentration of a-T, whereas the content of GSH negatively correlated with the concentration of a-To According to TEM, cell membrane and karyotheca swelled and couldn't retain integrity. The intracellular substances leaked out. Unidentified granule appeared in SL cell. The mitochondria expanded, and the membrane and subcellular organelle were damaged severely. Mter oxidative damage induced by a-T occurring, the output of MDA increased notably, whereas the relative content of GSH decreased. This indicated that the antioxidant ability of cell weakened. The result of TEM implied that SL cell suffered from oxidative damage under the tested dose (Wang et al., 2007).

Commercial products available already, composition, recipes etc. Efforts have also been made to improve the quality of formulations of photo activated insecticides. For example, an effort has been made to improve the stability and effectiveness of a-T by some new formulations such as nano-suspension formulation and beta-cyclodextrin inclusion complex formulation. Contrasted with a-T, degradation rate of beta-cyclodextrin inclusion complex and nano-suspension were only half, whereas the bioactivity of a-T nano-suspension to Tribolium castaneum had been improved 5.66 times (Hu et al, 2003). Now, the nano-suspension preparation of a-T "5% Guangwei EC", with a prolonged effective period and enhanced stability, has been widely used in the Pearl River Delta in China. Additionally, to minimize the dependence of the produce of a-T on the changeable nature condition which thereby caused some unanticipated influence on the formulation production, hairy root systems of Tagetes erecta has been established by use of young and health leaves as explants and cocultured with Agrobacterium rhizogene R1601 (Hou et al., 1999).

Xanthopappus subacaulis C. Winkle Botanical name: Xanthopappus subacaulis C. WinkL Family: Compositae It is a coarse herb ca. 50 cm in diam.; involucral bracts green; pappus white or yellow. It distributes on dry, sun-baked slope.

Chemical constituents and pesticidal activities Three new photo activated insecticidal thiophene derivatives, xanthopappins A-C, were isolated from Xanthopappus subacaulis, along with three known thiophene acetylenes, 5-hydroxymethyl-2-(E)-hept-5-ene1,3-diynylthiophene, 5-{1,2-dihydroxyethyl)- 2-(E)-hept -5-ene-1,3diynylthiophene, and 5-( 1,2-diacetoxyethyl)-2-(E)-hept-5-ene-1,3diynylthiophene. Xanthopappins A-F exhibited significant photoactivated and insecticidal activity against the 4th instar larvae of the Asian tiger mosquito (Tian, 2005; Tian et al., 2006).

Pesticidal Activities of Some Important Chinese Medicinal Plant HO

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References Callaham, M.F., Broome, J.R., Lindig, O.H. and Heitz, J.R. 1975. Dye-sensitized photooxidation reactions in the boll weevil, Anthonomous grandis, Environmental Entomology 4(5): 837-841. Callaham, M.F., Lewis, L.A., Holloman, M.E., Broome, J.R. and Heitz, J.R. 1975. Inhibition of the acetylcholinesterase from the imported fire ant, Solenopsis richteri (Forel) by dye-sensitized photooxidation, Compo Biochem. Physiol. 51(1): 123-128. Carpinella, M.C., Carpinella, M.T., Defago, G. and Valladares, S.M. 2003. Palacios, Antifeedant and insecticide properties of a limonoid from Melia azedarach (Meliaceae) with potential use for pest management, J. Agric. Food Chem. 51: 369-374. Chen Li, Xu Hanhong and Chiu Shinfoon. 2000. Antifeedant activity and stomach-poison effects of Daphne tangutica Maxim against Pieris rapae. Natural Product Research and Development 12(6): 22-26. Chen Li, Xu Hanhong and Chiu Shinfoon. 2000. Studies on antifeedant activity of Daphne tangutica Maxim against tobacoo cutworm, Spodoptera litura. Journal of South China Agricultural University 20(1): 44-46.

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Cheng Dongmei, Zhang Zhixiang, Xu Hanhong, et al., 2002. Antifeedant activity of spirol enol ether analogues against vegetable insects. Journal of Huazhong Agricultural University 21(4):343-346. Chiu Shinfoon and Zhang Yeguang. 1989. Successful introduction of neem tree (Azadirachta indica A. Juss) in China, Neem Newsletter 6(2): 16-18 Chiu Shinfoon. 1988. Experiments on the practical application of chinaberry, Melia azedarach, and other naturally occurring insecticides in China, Proc.3 rd Int .Neem Conf., Nairobi, Kenya, pp.661-668. Chiu Shinfoon. 1989. Chiu, Recent advances in research on botanical insecticides in China. In: J.T. Amason, B.J.R. Philogene and P. Morand, Eds, Insecticides of Plant Origin. Am. Chern. Soc. Symp. Ser. vol. 387, American Chemical Society, Washington, DC. pp. 69-77. Duke, J.A. and Ayensu, E.S. 1985. Medicinal Plants of China. Reference Publications, Inc. ISBN 0-917256-20-4. Ermel, K and Kleeberg, H. 1995. Commercial products, their standardization and problems of quality control in the neem tree (Schmutterer, H., ed.), Verlagsgesellschaft, Wienham, GDR. Feng, W., Tetsuro, I. and Mitsuzi, Y. 1995. The antitumor activities of gnidimacrin isolated from Stellera chamaejasme L. Zhonghua Zhong Liu Za Zhi. 17(1): 24-26. Hall, I.H., Tagahara, K and Lee, KH. 1982. Antitumor agents LIII: The effects of daphnoretin on nucleic acid and protein synthesis of Ehrlich ascites tumor cells. J Pharm Sci 71: 741-744. Harden, G.J. (ed) 2002. 'Flora of New South Wales.' (University of New South Wales Press Ltd: Sydney, Australia). He Daohang. 1999. Bioactivity and application of neem. Master Dissertation of South China Agricultural University. Heitz, J.R. 1987. Development of photo activated compounds as pesticides, light activated pesticides, Heitz, J.R. and Downum, KR. (eds.), American Chemical Society Symposium Series 339, American Chemical Society, Washington, D.C. pp. 1-21. Hou Xuewen, Xu Hanhong and Chiu Shinfoon. 1999. Preliminary studies of the establishment of hairy root system producing a-terthienyl. Guangzhou Chemical Industry 27: 28-29. Huang, KC. 1993. The Pharmacology of Chinese Herbs Drugs p. 160. CRC Press, Boca Raton. Jiang Zhisheng and Yan Zengguang. 2003c. Inhibition of a-terthienyl on Na +-K +-ATPases activity in Helicoverpa armigera and Ostrinia furnacalis larvae. Chinese Journal of Pesticide Science 5(2): 47-52. Jiang Zhisheng, Shang Zhizhen, Wan Shuqing, Xu Hanhong and Zhao Shanhuan. 2003a. ESR analysis of a photoactivated insecticide and its effects on superoxide dismutase, peroxidase and catalase activity in Culex pipiens pallens. Acta Entomologica Sinica 46(1): 22-26. Jiang Zhisheng, Shang Zhizhen, Wan Shuqing, Xu Hanhong and Zhao Shanhuan. 2000. The effect of photo activated insectsicide on the protective enzyme system of Spodoptera litura. Zherjiang Chemical Industry 31(suppl): 100-102. Jiang Zhisheng, Yan Zengguang and Shang Zhizhen. 2003b Effect of a-terthienyl on glutathione S-transferases in Helicoverpa armigera and Ostrinia furnacalis larvae. Chinese Journal of Pesticide Science 5(3): 76-79. Jiangsu Medical College. 1977. Encyclopedia of Chinese Materia Medica p. 1071. Korchagina, V.A., Penchukov, V.M., Morozov, N.A., Smashevskaya, G.A., Kolomiitsev, F.B., Trubeeva, A.1. and Baranova, M.M. 1972. Control of weeds in the Far East. Khabarovsk: Khabarovsk Publishing House. 160 p. (In Russian). Li Xiaodong and Chiu Shinfoon. 1995. Mechanism of neem. Chinese Journal of South China Agricultural University. Li Yanghan. 1998. Weeds of China. Beijing: Agriculture Press.

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Li, S.H., Wu, L.J. and Yin, H.Y. 2002. Chemical and pharmacological advances of the study on Zushima. China J Chin Mater Med 27: 401-403. Momchilova Svetlana, Antonova Daniela and Marekov Ilko. 2007. Fatty acids, triacylglycerols, and sterols in neem oil (Azadirachta indica A. Juss) as determined by a combination of chromatographic and spectral techniques. Journal of Liquid Chromatography & Related Technologies 30(1): 11-25. Mordue, A.J. and Blackwell, A. 1993. Azadirachtin: an update, J. Insect Physiol. 39(11): 903-924. National Research Council. 1992 Neem: a tree for solving global problems, National Academy Press,Washington D.C. National Research Council. 1992. Neem: A tree for solving global problems, National Academy Press,Washington D.C. Pan Li, Zhang Xiaofeng, Wu Haifeng and Ding Lisheng. 2006. A New Daphnane Diterpene from Daphne tangutica. Chinese Chemical Letters 17(1): 38-40. Qinghai Provincial Institute for Drug Control, Qinghai Provincial Institute of Tibetan Medicine, Tibetan Medicine of China, Vol. 1, Shanghai Science and Technology Press, Shanghai, 1996,545. Qiu Yutong. 1984. The resistance of Dimondback moth and the control by using of botanical pesticides. Ph. D. Dessertation of South China Agricultural University. Rong Xiaodong. 2000. Application of neem on control of the diamondback moth. Ph. D. Dessertation of South China Agricultural University. Singh, R.P. and Singh, S. 1996 Neem for management of insect pest: advantages and disadvantages, Recent Advance in India Entomology, ed. Lal, 0 P., APC Publications, pp.67-82. Song. 1999. China Herbal. China Herbal Shanghai Science and Technology Press: Shanghai, 5: 407-415. Taniguchi, M., Fujiwara, A. and Baba, K. 1996. Three flavonoids from Daphne odora. Phytochemistry 42: 1447-1453. Tian Shiyao. 1993. The bioactivities and mechanism of botanical pesticides on Spodoptera litura. Ph. D. Dessertation of South China Agricultural University. 17(1): 118-122. Tian Yongqing, Wei Xiaoyi and Xu Hanhong. 2006. Photoactivated insecticidal thiophene derivatives fromXanthopappus subacaulls. Journal ofNatural Product 69(8): 12411244. Tian Yongqing. 2005. Studies on photoactivated insecticidal activity and active ingredients of Xanthopappus subacaulis C. Wink!. Ph. D. Dessertation of South China Agricultural University. Torrance, S.J., Hoffmann, J.J. and Cole, J.R. 1979. Wikstromol, antitumor lignan from Wikstroemia foetida var. oahuensis Gray and Wikstroemia uua-ursi Gray (Thymelaeaceae). J Pharm Sci 68: 664-665. Wan Shuqing, Xu Hanhong, Zhao Shanhuan, Jiang Zhisheng, Shang Zhizhen and Liu Zhun. 2005. Contact toxicity of polyacetylenes to Periplaneta americana and their effects on AChE and ATPase. Acta Entomologica Sinica 48(4): 526-530. Wang Yujian, Hu Lin, Zhang Zhixiang, Xu Hanhong, Liao Meide and Liao Shaoyu. 2007. Oxidative damage to Spodoptera litura cell induced by a-Terthienyl. Scientia Agricultura Sinica 40(7): 1403-1409. Wang, M.S.H. 1980. Studies on the chemical constituents ofZu Shi Ma (the third report). Chin Tradit Herb Drugs 11: 389-390. Wang, Z., Xin, M., Ma, D., Song, S., Wang, X., Van, C., Zhang, D., Feng, W., Ma, E. and Chen, J. 1990. Farmland Weeds in China. A collection of coloured illustrative plates. Agricultural Publishing House. China. Xu Hanhong and Chinese Patent. 1999. CN 1235766. Xu Hanhong and He Daohang. 2001. Botanical pesticide: azadirachtin. Abstract of Symposium on entomology of the mainland and Taiwan.

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Xu Hanhong, Chen Li, Chiu Shinfoon, et al., 2000. Insecticidal activities of Daphne tangutica Maxim against the larvae of Pieris rapae. Acta Entomologia Sinica 43(4): 364-372. Xu Hanhong, Zhang Zhixiang, Cheng Dongmei, et al., 2000a. Studies on bioactives of derivates of spirol enol ether against Spodoptera litura Fabricius. Journal ofHuazhong Agricultural University 19(6): 543-546. Xu Hanhong, Zhang Zhixiang, Cheng Dongmei, et al., 2000b. Studies on bioactives of spirol enol ether analogues against Plutella xylostella. Natural Product Research and Development 21(5):17-22. Xu, Z.H., Qin, G.W. and Xu, R.S. 2001. A new bicoumarin from Stellera chamaejasme L. J Asian Nat Prod Res. 3(4): 335-340. Yoshida, M., Feng, W., Saijo, N. and Ikekawa, T. 1996. Antitumor activity of daphnanetype diterpene gnidimacrin isolated from Stellera chamaejasme L. Int J Cancer. 66(2): 268-273. Yu Liang Shi and MuFeng Li. 2007. Biological effects of toosendanin, a triterpenoid extracted from Chinese traditional medicine. Progress in Neurobiology 82(1): 1-10. Yu Liang Shi. 2007. Toosendanin modifies K+-and Ca 2+-channel activity and intracellular Ca 2+concentration, Prog. Biochem. Biophys. 34(2): 132-137. Zhang Guozhou, Wang Yawei, Xu Hanhong, et al., 2000b. Oviposition deterrent activity and ovicidal activity ofthe extract of root of Stellera chamaejasme L. with ethanol against imported cabbage worm. Journal ofAnhuiAgricultural Sciences 28(5): 623628. Zhang Guozhou, Wang Yawei, Xu Hanhong, et al., 2000c. Physio-biochemical effects on insects by 2-sitosterol, daphnoritin and chamaechromone. Joural of Hunan Agricultural University (Natural sciences). 26(5): 366-367. Zhang Guozhou, Xu Hanhong and Wu Zhenting. 2000a. Antifeedant activity ofthe extract of root of Stellera chamaejasme L. with ethanol against imported cabbage worm. Journal of AnhuiAgricultural Sciences 28(4): 464-465. Zhang Wei, Zhang weidong and Zhang chuan. 2007. Antitumor activities of extracts and compounds from the roots of Daphne tangutica Maxim. Phytotherapy Research 21(11): 1113-1115. Zhang, X.Q., Ye, W.C., Jiang, R.W., Yin, Z.Q., Zhao, S.x., Mak, T.C. and Yao, X.S. 2006. Two new eremophilanolides from Xanthium sibiricum. Nat Prod Res. 20(13): 12651270. Zhang, Z.P. and Hirota, S. (edsl 2000. Chinese Colored Weed Illustrated Book. Institute for the Control of Agrochemicals, Ministry of Agriculture, P.R. China, and the Japan Association For Advancement ofPhyto-Regulators. Zhang Zhixiang, Xu Hanhong, Cheng Dongmei, et al., 2001. Studies on bioactives of spirol enol ether analogues. Journal of Northeast Agricultural University 32(2): 146-150 Zhongping. 1995. Insecticidal activities and mechanism of neem, Chinese Journal of Plant Protection 21(5): 30-32. Zhuang, L.G., Seligmann, 0., Jurcic, K. and Wagner, H. 1982. Inhaltsstoffe Von Daphne tangutica. Planta Med 45: 172-176.

18 The Pharmacokinetics and Pharmacodynamics of the Active Ingredients in Radix Scutellariae CHRISTOF KARRICK ARNOLD!, CHAO-FENG CHIEN!, JEN-CHIH CHANG\ Y U-TSE Wu! AND TuNG-HU TSAI! ,2*

Abstract This chapter reviews Radix Scutellariae - the root of Scutellaria baicalensis Georgi, We focus on its molecular components and the effort to isolate them, pharmacokinetic research, the original uses from traditional Chinese medicine (TCM), pharmacodynamic applications, and drug development, A particular focus is placed on baicalin and its aglycone, baicalein, due to the extensive amount of research done on these compounds, In the case of baicalin and baicalein, the pairing of a glycoside and its aglycone is a good example of enterohepatic recirculation, These two compounds have put Radix Scutellariae in the forefront of research on combining antiretroviral drugs and herbal treatments for HN. Key words : Baicalein, Baicalin, Pharmacokinetics, Scutellaria baicalensis Georgi, Traditional Chinese medicine

Introduction Scutellaria baicalensis Georgi, or Huang-qin as it is known in Chinese, has been used in traditional Chinese medicine since before the Western Han Dynasty (206 BC-24 CE); a fact that attests to the herb's reliable clinical efficacy. The root of the plant is the portion used for medicinal purposes; it is known by the pharmaceutical name Radix Scutellariae. The entire root is used in traditional Chinese herbal formulas, but the active compounds are processed, and they are involved in drug development. 1. Institute of Traditional Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. 2. Department of Education and Research, Taipei City Hospital, Taipei, Taiwan. * Corresponding author: E-mail: [email protected]

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Almost as numerous as the indications for this herb (ranging from HIV and cancer to headache, bloody nose, and anticonvulsant), are the active compounds, or molecular components. Baicalein is a model prodrug for the development of HIV reverse transcriptase inhibitors, due to its innate capacity to inhibit HIV reverse transcriptase in vitro. Radix Scutellariae also has medicinal uses related to melatonin. Melatonin is implicated in the regulation of circadian rhythms, sleep cycle, mood, and tumor growth. This herb contains a rich suite of useful flavonoid compounds, and they have a large number of existing of clinical applications that range from inflammation and prostate carcinoma to Alzheimer's disease and Parkinson's disease.

Fig 1. Radix Scutellariae sample from Yu-Sheng Chinese Medicine Clinic Taipei, Taiwan

Herbal analysis Isolating chemical compounds within plants is a powerful tool. The process of isolating chemical compounds has developed greatly in recent years due to a greater understanding of organic chemistry, specifically in the areas of molecular properties, bonding and behavior. Developments in our understanding of basic principles related to pH and the Henderson-Hassebach equation have also contributed to advancement in herbal analysis and separation science. In Radix Scutellariae, the most concentrated ingredient is baicalin. Baicalin was first isolated and described in the 1970's in Japan. By the mid 80's many other flavonoids were being isolated from Radix Scutellariae (i.e. baicalein, baicalin, wogonin, skullcapflavone II, (2 8),2',5,6' ,7tetrahydroxyflavanone, (2 R,3 R),2' ,3,5,6',7 -pentahydroxyflavanone, and 2',5,5',7-tetrahydroxy-6',8-dimethoxyflavone), as reported by Kimura Y. et al. in 1985. Baicalin did not gather much interest from researchers, however, until its application to human immunodeficiency virus (HIV) in 1989. By 1993, Li et al. began discussing the possibility of using this xenobiotic for the treatment of HIV-1 in humans. The research impetus and possibility of drug development arising from baicalin spurred on modern chemists to find better and more efficient methods of isolation and identification.

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The problem of separating baicalin from its aglycone baicalein proved especially significant in the area of separation analysis, specifically high pressure liquid chromatography (HPLC). In practice, the identification of a compound must be rapid and sensitive. HPLC when coupled with a UV detector is not specific enough to provide a clear separation of the two molecules in a reasonable time. Rather, techniques have been developed using electrochemical detection, which uses redox chemistry to measure the compounds. The nature of baicalin and baicalein to undergo oxidation reactions makes this detector well suited to achieve the level of specificity necessary to separate baicalin from baicalein (Kotani et al., 2006). Recent investment in both China (e.g., Chang et al., 2007) and Japan (e.g., Ohkoshi et al., 2008) attest to the pharmaceutical potential of Radix Scutellariae. Current efforts focus on developing cost efficient isolation methods.

Pharmacokinetics of Radix Scutellariae Absorption Absorption describes the process where pharmaceuticals move from the site of administration into systemic circulation. During the absorption phase, pharmaceuticals may simultaneously undergo metabolism or excretion. This is the case for the main flavone compounds of Radix Scutellariae, namely baicalin, wogonoside, and their aglycones, baicalein and wogonin. Glycosides are more soluble in water than the aglycones due to their structure containing a glucuronic acid group, thus the absorption of the two compounds are quite different. Baicalin is simply baicalien with a glucuronic acid attached to create a glycoside (Fig 2), and these two molecules are a good example ofthe role a glucuronic acid can play. The intestinal lining is a major sight of absorption for xenobiotics, partially due to the large surface area created by the myriad of microvilli on the apical surface of the epithelial cells found there. This environment is a higher pH than the stomach, and the pH continually rises as the distance from the stomach increases (pH = 13 in the stomach, 5-7 in the duodenum, and 7-8 in the ileum) (Laurence, 2006). In opposition with the large surface area and the higher pH of the intestines, the body has a defense mechanism in place, P-glycoprotein transporters function to pump baicalin out of intestinal goblet cells back into the intestinal lumen. Baicalin is a likely substrate for P-glycoprotein transport (Tsai et al., 2002; Meijer et al., 1997). Thus, it does not readily penetrate the epithelial cell wall of the gastrointestinal tract. Teruaki Akao et al. (2000) demonstrated that baicalin can first be processed by native bacteria in the GI-tract into the aglycone form, and then baicalein is absorbed into the blood stream. Once in the blood stream, baicalein passes through the portal vein into the liver where a portion is reconstituted to the original glycoside form, baicalin (Akao et al., 2000).

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332 o

o

II

H:o~: HO

OH

*: o

II

H:o

HO

OH

Wogonoside

Baicalin

HO

H

HO

HO

Baicalein

Melatonin

o

Wogonin

Scutellarin

Fig 2. Chemical structures for 6 major active ingredients of Radix Scutellariae

When the same concentration (224 }lmol/kg) ofbaicalin and baicalein were administered orally, baicalin was absorbed more slowly than baicalein (Lai et al., 2003b). Compared with baicalin, baicalein was well absorbed in the stomach and small intestine (Taiming & Xuehua, 2006). However, when rats had baicalin orally administered, the same metabolites appeared in the bile as were found in the orally administered baicalein group (Abe et al., 1990). Therefore, the bacteria located in the gastrointestinal tract play an important role in baicalin absorption through the process of hydrolysis, or the removal of the glucuronic acid. A study conducted by Akao et al. (2000) revealed that baicalein was readily created from baicalin in the gastrointestinal tract when baicalin was orally administered to conventional rats, and baicalin could be detected in the plasma for 24 h after administration. In contrast, when baicalin was orally administered to the germ-free rats, only small amounts of baicalin could be detected in the plasma within 2 h of administration. That study indicated that baicalin was poorly absorbed in the gut ofthe germ-free rats, but baicalin became well absorbed after it was transformed to baicalein by the native bacteria in the gut. Since Radix Scutellariae is widely used for treating inflammation, fever and allergies, it may be prescribed alongside conventional antibiotics. When baicalin (224 }lmol/kg) was orally co-administered with a mixture of

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neomycin and streptomycin, the absolute bioavailability decreased from 2.2 ± 0.2% to 1.5 ± 0.2% (Xing et al., 2005a; Taiming & Xuehua, 2006). Therefore, the aminoglycosides decreased the transformation ofbaicalin to baicalein by inhibiting the gastrointestinal bacteria and then influenced the pharmacokinetics and pharmacodynamics of the baicalin. In relation to biliary excretion, Xing et al. (2005b) assessed the absorption and enterohepatic circulation ofbaicalin in rats by a linked-rat model. Since baicalin and baicalein were detected in the bile-recipient rats (which were not directly given any baicalin or baicalein), this phenomenon confirmed the possibility of enterohepatic circulation and that the enterohepatic circulation ratio would be 18.7 and 19.3% for baicalin and baicalein, respectively. Mter intravenous administration of baicalein (37 llmollkg), 75.7% baicalein became conjugated (Lai et al., 2003b). The values for absolute bioavailability of baicalin were found to be 5.4% (Akao et al., 2000), and 2.2% (Xing et al., 2005a); whereas the value was much higher, 27.8%, for baicalein (Xing et al., 2005b). Compared with the aglycones, baicalin and wogonoside show relative polarity and they don't penetrate the enterocytes easily. Because of this, Lai et al. speculated that baicalein and wogonin were immediate-released and rapidly absorbed through the small intestine (Lai et al., 2003a). In contrast, baicalin and wogonoside were gradually transformed to the aglycones and absorbed later in the colon. Thus, they can be regarded as natural slow-released pharmaceuticals. When Radix Scutellariae is prescribed with other herbs, herb-herb interactions may take place. Huang-Lian-Jie-Du-Tang is the traditional decoction used for "heat-clearing" in TCM theory, and Radix Scutellariae is one of its herbal ingredients. When pure baicalin (188 mglkg) and wogonoside (47 mglkg) were orally administered to Sprague-Dawley rats, their plasma concentrations were higher than the rats which received the decoction of the herbal formula Huang-Lian-Jie-Du-Tang (doses of baicalin and wogonoside were 193 and 43 mg/kg in the Huang-Lian-Jie-Du-Tang, respectively)(Lu et al., 2007). The result revealed that some ingredients in the other herbs of Huang-Lian-Jie-Du-Tang will interact with baicalin and wogonoside influencing their pharmacokinetic profiles. This interaction may be due to the inhibition of bacterial-glucuronidase activities, which will decrease oral absorption ofbaicalin and wogonoside. Hence, the absorption mechanism of baicalin and baicalein likely involves all three processes of bacterial hydrolysis, first-pass effect, and enterohepatic circulation.

Distribution Baicalin, baicalein, wogonin, and wogonoside are four flavones with antioxidant activity and are considered major components, while other flavones found in Radix Scutellariae with bioactivities include apigenin,

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chrysin, and scutellarein (Horvath et al., 2005). This section will review the in vivo distribution of baicalin, baicalein, wogonin, and scutellarein. The apparent volume of distribution (V D)' a pharmacokinetic parameter, does not have a true physiologic meaning in terms of an anatomic space. Rather, VD represents a volume that must be considered in estimating the amount of drug in the body from the level of drug found in the sampling compartment (Shargel & Yu, 1999). Drug molecules that are highly proteinbound usually have a low VD' suggesting the restricted distribution mainly in the blood circulation. The VD offree baicalin was 16.981 ± 12.499 Ukg (Huang et al., 2008), and the volume of distribution at steady state (V ) of baicalein was approximately 15.3 Ukg in male Sprague-Dawley rats ~fter an intravenous administration of Radix Scutellariae extract (10 mglkg), which was equivalent to 4.4 mg/kg ofbaicalein (Kim et al., 2006). VD values for baicalin or baicalein greater than the total body water suggests that distribution of these compounds may not be limited to the extracellular fluid compartment, and that these molecules are extensively distributed to most organ tissues. Clinical studies have demonstrated that baicalin and baicalein have protective effects on neurodegenerative diseases including Alzheimer's disease (Heo et al., 2004) and Parkinson's disease (Zhu et al., 2004). Therefore, ifbaicalin and baicalein cross the blood-brain barrier then their brain distribution is of great importance. In our lab, at 20 min after intravenous administration of baicalein (60 mg/kg) we have observed mean brain regional concentrations ofbaicalein throughout the brain, including: rat brain stem (4.05 JIg/g), cerebellum (3.13 JIg/g), cerebral cortex (2.72 JIg/g), hippocampus (3.17 JIg/g), midbrain (4.09 JIg/g), and striatum (3.00 JIg/g). In addition, the treatment of cyclosporin A, which is a P-glycoprotein inhibitor, results in a significant increase in elimination half-life, mean residence time and area under the concentration versus time curve offree-form baicalein within the blood brain barrier (BBB), suggesting the accumulation and distribution of baicalein in the rat brain might be regulated by P-glycoprotein transporters (Tsai et al., 2002). A study conducted by Zhang et al. (2006) has revealed that baicalin could penetrate into brain and distribute to the cerebral nuclei, such that mean peak concentrations after intravenous injection of Radix Scutellariae extract (90 mg/kg ofbaicalin) were: cortex (0.373 mg/g), hippocampus (0.925 mg/g), striatum (2.039 mg/g), thalamus (1.300 mg/g), and brain stem (0.668 mg/g). Another study also suggests the BBB penetration offree baicalin, which is revealed by microdialysis technique and a sensitive UPLC-MS/MS detective method. The results show that the peak level in blood was 1250.8 JIg/L and the concentration in CSF was 344.2 JIg/L after intravenous administration ofbaicalein (24 mg/kg) (Huang et al., 2008). The VD ofwogonin is 0.83 ± 0.081 Ukg in male Sprague-Dawley rats after an intravenous injection of pure wogonin (5 mg/kg) (Tsai et al., 1996),

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and the VD of scutellarin is 0.09 ± 0.03 Llkg in male and female Wistar rats after a single intravenous dose of scutellarin (10 mglkg) (Hao et al., 2005). The evidence shows that VD values for wogonin and scutellarin are much smaller than that of baicalin and baicalein, so they will only partially distribute to peripheral tissues. One study might help to prove this conjecture. The distribution of scutellarin in Kunming mice was determined to be 0.34 ± 0.08 pg/mL in plasma, 0.06 ± 0.02 pg/g in heart, 1.38 ± 0.36 pg/g in liver, 0.82 ± 0.24 pg/g in spleen, 0.22 ± 0.05 pg/g in lung, and 0.11 ± 0.01 pg/g in kidney, at 30 min after intravenous administration of scutellarin (25 mglkg) (Xiong et al., 2006), these amounts are lower than those of baicalin and baicalein found in brain regions as described above.

Elimination ofRadix Scutelleriae Drug elimination refers to two processes, namely, metabolism and excretion. Drug metabolism is a process of drug biochemical modification or degradation, usually through specialized enzymatic systems. This mechanism often converts hydrophobic chemical compounds into more readily excreted polar products. Its rate is an important determinant of the duration and intensity of the pharmacological action of drugs. The second part of elimination is excretion - the process of eliminating the drug and its waste products created through metabolism. Although every tissue has some ability to metabolize drugs, the liver is the major organ for drug metabolism. Other tissues that have specific activity metabolizing drugs include the gastrointestinal tract, the lungs, the skin, and the kidneys. In addition to our native tissues, the microorganisms that live in intestines are also capable of many biotransformation reactions. On a cellular level, the glucuronic acid group acts as a marker for the body to recognize the molecule as a xenobiotic. The liver is an important site for xenobiotics to be identified by UDPglucuronosyltransferase, the enzyme generally responsible for adding the glucuronic acid to baicalein (Karl Walter et al., 2005). When baicalin was orally administered to conventional rats, baicalin was detected in plasma for 24 h after administration, but baicalein - the aglycone of baicalin - was not detected (Wakui et al., 1992). However, when baicalin was given to germ-free rats, only a small amount ofbaicalin was detected in their plasma after the administration indicating that a large amount of baicalin remained in the intestinal tract (Akao et al., 2000). This report indicated that the baicalin is indeed poorly absorbed and baicalin is converted to baicalein after biotransformation (hydrolysis) by intestinal bacteria. By a linked-rat model, Xing et al. (2005) demonstrate that baicalin undergoes extensive first-pass glucuronidation and enterohepatic circulation. This is a route for baicalin to be absorbed into the body and then excreted. When baicalein was administered orally to germ-free rats, high levels of baicalin, but not baicalein, were recovered from their intestinal tracts. These results indicate that baicalein is

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absorbed, but then a large proportion is secreted into intestinal lumen as baicalin (Akao et ai., 2000). According to Akao et ai., a large proportion of baicalein is transformed into baicalin within the intestinal mucosal cells and excreted through multidrug resistance-associated protein 2 (MRP2) into the intestinal lumen (Akao et ai., 2004). In summary, baicalin is converted to baicalein through hydrolysis by intestinal bacteria, and then baicalein can enter the blood vessels eventually reaching the portal vein. Once in the liver baicalein undergoes glycosylation by UDP-glycuronosyltransferases labeling the molecule for transport, and as baicalin it travels out the bile duct into the duodenum. This entire loop is a process known as enterohepatic recirculation (Fig 3). This enterohepatic recirculation of western physiological function relates to the traditional perception that Radix Scutellariae resolves damp heat in the liver and spleen systems of traditional Chinese medicine.

LIver

balcahn~=~:::::=$=;:' baicalin Glucuro-

1

nosyl

transf,,-

~!~~~'"-I

transferase

lbwc:ilinolH--brucalin Portal

IntestInal epithelIal

veIn

cell

baicalm

I

Intestinal baclena

balcalm

Fig 3. Enterohepatic circulation and intestinal recycling ofbaicalin and baicalein

Although glucuronidation is the main metabolic pathway for baicalin, two other minor metabolic pathways exist: methylation and sulfation result in a small amount of methylated and sulfate conjugated metabolites (Abe et ai., 1990; Muto et ai., 1998; Xing et ai., 2005; Feng et ai., 2005). Major metabolites ofbaicalin include baicalin-6-0-~-glucopyranuronoside-7-0-~­ gl ucopyran uronoside, baicalin -6- ~-gl ucopyran uronoside, 6-0- methy1baicalin -7 -O-~-gl ucopyranuronoside, baicalein -6-0-~-gl ucose-7 -O-~­ glucopyranuronoside and baicalin-6-0-sulfate-7 -O-~-glucopyranuronoside (Feng et ai., 2005). Another major constituent of Radix Scutellariae, wogonoside, shares the same property as baicalin because they are both glycosides. Wogonoside is hydrolyzed by intestinal bacteria and converted to the aglycone form, wogonin, then absorbed through the intestinal wall. It is circulated and excreted in conjugated form when rats were administered Radix Scutellariae in the same manner as is seen with baicalin (Lai et ai., 2003; Zuo et ai., 2003).

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Pharmacodynamics of Radix Scutellariae Historical perspective and traditional therapeutics In China's Ben Cao Jing, attributed to the demigod Shen Nong, Huang-qin is listed among the zhong-pin, or "central products". This herb is of bitter flavor, cool nature, enters the gallbladder, large intestine, lung, and stomach channels, good for clearing heat and detoxifying, indicated for jaundice, regulator of the bowels and urination, quenches burns and extinguishes skin inflammation, antimicrobial. Huang-qin makes its way into a large portion of clinically prescribed formulas. A famous formula, Huang-Lian-Jie-Du-Tang, first appeared in the Wai Tai Mi Yao Fang published in 752 CE, during the Tang Dynasty (618-907 CE). In this formula Huang-qin has the role of the official, within the traditional four positions - captain (Rhizoma Coptidis), official (Radix Scutellariae), assistant (Cortex Phellodendri), and envoy (Fructus Gardeniae Jasminoidis). From a strategic standpoint, the official could be considered the cavalry; however a more literal connotation would be imperial eunuch. Another example of Huang-qin as the official in an important traditional formula is Long-Dan-Xie-Gan-Tang. These formulae highlight the antiviral and hepatoprotective properties of Huang-qin, respectively, but this multifaceted herb cannot be limited to these categories alone. Huang-qin also acts on the middle and upper burners of the traditional three burner system in Chinese physiology. In the middle burner, Huangqin clears damp heat from the spleen system (not correlated to the spleen organ of western physiology). However, Huang-qin is more renowned for clearing wind heat and damp heat from the upper burner; Huang-qin can protect the lungs and reinforce the heart by quenching wind heat and extinguishing damp heat in the upper burner (Wang, 2004).

Potent anti-HIV activity ofRadix Scutellariae Research into traditional Chinese herbs for treatment of human immunodeficiency virus (HIV) is extensive including countries such as China, Taiwan, Japan, and the United States. Radix Scutellariae is proving to be one of the best herbal choices for drug development and treatment ofHIV. The first reports ofbaicalein's inhibitory effect on HIV-1 reverse transcriptase originate in Japan (Ono et al., 1989). The authors found that 2 J.lg/mL of baicalein caused 90% inhibition by binding to the template primer binding site and subsequently interfered with the reaction. Almost ten years later they were still providing good evidence for this mechanism of action, yet they did not claim that it was the sole mechanism of action for baicalein's inhibition ofHIV growth in cell culture (Kitamura et al., 1998). In 2000, research supported by the National Cancer Institute, in the United States, produced interesting evidence ofthe possibility that baicalin inhibits the ability ofHIV to bind to host cells (Li et al., 2000). The mechanism

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was not competitive binding ofthe CD4 receptor, rather the authors proposed that baicalin conjugates with chemokines and it is this complex that binds the CCR5, or CXCR4 coreceptors preventing HIV viral envelope fusion. This shows that baicalin is a strong candidate for first line treatment in the early stages of infection, and the authors concluded it is a good candidate for further development. In light of similar findings, a group researching HIV-l reverse transcriptase inhibitory potential have been studying baicalin and baicalein for further drug development (Yuan et al., 2001). In 2007, Yuan et al. demonstrated antiviral activity and attenuation of gastrointestinal side effects of the HIV protease inhibitor ritonavir following treatment with Radix Scutellariae and baicalein in an in vivo experiment (Yuan et al., 2007). Thus, Radix Scutellariae, and its active ingredients baicalin/baicalein, appears to have a multi touch inhibition of the HIV-l virus as well as moderating the severe side effects of a protease inhibitor. Research into the concurrent use of Radix Scutellariae may provide useful therapeutic regimens for patients desperately in need of help. In developing countries such as those in Mrica, or the Tibetan Autonomous Region of China, herbs can be utilized as a way to lower the cost of treatment, while providing a high quality of care to patients, and a possible benefit to quality oflife due to reducing the often unbearable side effects experienced with HAART pharmaceuticals alone. In fact the concurrent use of herbs in the community of HIV positive patients in the US is already a common occurrence (Liu, 2007). In our lab we are currently investigating the pharmacokinetic profile of an HIV protease inhibitor during concurrent use of Radix Scutellariae and indinavir to establish a safety profile for this combinatory treatment.

Other applications ofRadix Scutellariae In addition to showing good promise for concurrent treatment in antiretroviral HIV regimens, Radix Scutellariae has promise to treat other conditions. For example, there have been studies into the effect of Radix Scutellariae compounds on neural tissues, and previous work has demonstrated that some molecular components can pass through the BBB and enter the CNS (Huang et al., 2008; Tsai et al., 2002; Zhang et al., 2006). In 2002, Watanabe et al. reported the effects of taking this herb as part of an herbal formula on melatonin levels. Melatonin is a native hormonal product of the pineal gland with cellular receptors in multiple tissues including the brain, gut, ovaries, and blood vessels. It is partially responsible for regulation of such physiological functions as circadian rhythms, sleep cycle, mood, immunoregulation and tumor growth. An herbal formula containing Radix Scutellariae (Qing-Xin-Lian-Zi-Yin) is commonly used for Qi and blood deficiency with lingering heat in the heart, lung, and kidneys a syndrome often accompanying alcoholic behavior. Qing-Xin-Lian-Zi-Yin

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(Seishin-renshi-in, in Japanese) was given to patients suffering from a combination of diabetic neuropathy (this formula has been shown to improve insulin resistance in rats by Tominaga et al., 1995) with a chronic urogenital disorder (Radix Scutellariae showed good results as an anti-proliferative for prostate cancer both in vitro and in vivo by Bonham et al., 2005), who were suffering from CNS symptoms commonly associated with high diurnal melatonin (Wantanabe et al., 2002). Two weeks after beginning treatment with Qing-Xin-Lian-Zi-Yin the level of melatonin in these patients decreased. The neuroprotective effects of Radix Scutellariae have also been investigated in light of the common use of this herb in TCM for the treatment of stroke patients. Kim et al. made a convincing report of this phenomenon in CAl hippocampal neurons when they gave an intraperitoneal injection of 1 mg/kg of a 70% methanol extract (dried under vacuum and reconstituted with 0.89% saline) of Radix Scutellariae to rats that had under-gone a surgery to induce global cerebral ischemia. They found that the CAl hippocampal neuron cell density 7 days after ischemia was significantly increased compared to saline treated controls, even showing a significant difference when 0.1 mg/kg of the extract was administered (Kim et al., 2001). Although the exact mechanism of protection has yet to be delineated, it is proposed that a substantial inhibition of NO production is involved. They further demonstrated that pretreatment ofPC12 cells with the Radix Scutellariae extract significantly protected them from Hp2-induced injury. The NO radical-scavaging activity and inhibition of NO production by Radix Scutellariae has been reported elsewhere (Tezuka et al., 2001). Finally, researchers in Beijing have shown that intravenous administration to rats caused a release of the dopamine stored in the striatum, and dopamine levels in the hippocampus and cortex increased (Chen et al., 2006). This demonstrates the ability of Radix Scutellariae to mobilize the latent stores of dopamine in the brain, and stimulate the brain's own dopaminergic system.

Future considerations Radix Scutellariae may be one of the most versatile herbs in the pharmacopeia ofTCM. New evidence coming from researchers around the world on the herb's active ingredients supports the claim. Clinicians of TCM do not use this herb as a remedy by itself; rather it is combined with other medicinal herbs to create formulas and prepared in a decoction. This aspect of clinical treatment, noting that the proportions of the herb are adjusted to meet a patient's constitution as well as geographic area, make it difficult to directly apply laboratory results in the clinic. The move towards evidence based medicine (EBM) has put into focus a need for well done ethical clinical trials in international traditional herbal medicine research. A recent report outlining a framework for implementing

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an ethical standard for researchers includes requirements for collaborative partnership, social value, scientific validity, independent review, and respect for subjects (Tilburt et ai., 2008). All these requirements are important considerations for the further development of clinical studies on therapeutic efficacy in traditional medicine. The push for reliable, sound, well designed trials that are convenient to implement can be acknowledged by researchers globally to provide a higher level of care to the patients these trials are designed for. Yet, the basic building blocks of these herbal remedies are best studied in a laboratory - not a clinical setting. This underscores the importance of laboratory research in pharmaceutical development. The two-way communication from benchside research to bedside treatment and vice versa plays a central role in the movement towards an evidence based medical system. This approach not only makes good use oftechnology in the laboratory, but also helps to safeguard both patients and doctors from unnecessary litigation and overregulation of medical practices. The key to such a relationship lies in fidelity of scientific research methods and application of scientific method to the field of medicine only when appropriate. Pharmacokinetic research brings significant benefit to the clinical practitioner of herbal medicine. The ability to measure which tissues botanics are distributed to and in what concentration they are found there can be a powerful tool in designing treatment protocol, as well as providing insight into possible causes of adverse affects and interaction with conventional pharmaceuticals. There are obvious cases where limitations arise in using human subjects for such studies, however there are also times when it is necessary to take certain measurements only within a human subject pool. Liver enzymes used for metabolizing active ingredients of herbs can differ between individuals based on the person's genetic profile (Fan et ai., 2008). The large influence, these enzymes can have, makes measuring the plasma concentration of target molecules the safest way of administering herbs in combination with conventional pharmaceuticals. The global community relies on medicinal herbs in both developing countries and developed countries as a means of primary health care as well as complimentary health care. The significant portion of the world's population using medicinal herbs cannot be ignored. Research to define the pharmacokinetics and pharmacodynamics of active ingredients is a major building block for countries establishing proper regulatory systems as recommended by the World Health Organization (WHO traditional medicine strategy 2002-2005).

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Acknowledgements The authors would like to thank Benjamin Ford Arnold for his meticulous editing of the manuscript. Funding for this study was provided in part by research Grants: NSC96-2113-M-OIO-003-MY3 and NSC96-2628-B-OIO-006MY3 from the National Science Council, Taiwan; TCH 97002-62-037 from Taipei City Hospital.

References Abe, KI., Inoue, O. and Yumioka, E., 1990. Biliary excretion of metabolites ofbaicalin in rats, Chemical and Pharmaceutical Bulletin 38(1): 208-211. Akao, T., Kawabata, K., Yanagisawa, E., Ishihara, K, Mizuhara, Y., Wakui, Y., Sakashita, Y. and Kobashi, K 2000. Baicalin, the predominant flavone glucuronide of Scutellariae Radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form, Journal of Pharmacy and Pharmacology 52: 1563-1568. Akao, T., Sakashita, Y., Hanada, M., Goto, H., Shimada, Y. and Terasawa, K 2004. Enteric excretion ofbaicalein, a flavone of Scutellariae Radix, via glucuronidation in rat: involvement of multidrug resistance-associated protein 2, Pharmaceutical Research 21(11): 2120-2126. Bonham, M., Posakony, J., Coleman, I., Montgomery, B., Simon, J. and Nelson, P.S. 2005. Characterization of chemical constituents in Scutellaria baicalensis with antiandrogenic and growth-inhibitory activities toward prostate carcinoma, Clinical Cancer Research 11(10): 3905-3914. Chang, Y.L., Liu, B. and Shen, B. 2007. Orthogonal array design for the optimization of supercritical fluid extraction ofbaicalin from roots of Scutellaria baicalensis Georgi, Journal of Separation Sciences 30: 1568-1574. Fan, L., Zhang, W., Guo, D., Tan, Z.R., Xu, P., Li, Q., Liu, Y.Z., Zhang, L., He, T.Y., Hu, D.L., Wang, D. and Zhou, H.H. 2008. The effect of herbal medicine baicalin on pharmacokinetics of rosuvastatin, substrate of organic anion-transporting polypeptide 1B1, Clinical Pharmacology and Therapeutics 83(3): 471-476. Feng, N.P., Di, B. and Liu, W.Y. 2005. Comparison of the metabolism ofbaicalin in rats orally administered with Radix Scutellariae extract and Shuang-Huang-Lian extract, Chemical and Pharmaceutical Bulletin 53(8): 978-983. Hao, X., Cheng, G., Sun, J., Zou, M., Yu, J., Zhang, S. and Cui, F. 2005. Validation of an HPLC method for the determination of scutellarin in rat plasma and its pharmacokinetics, Journal ofPharmaceutical and Biomedical Ana