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 Gene...

Author: M.S. Swaminathan

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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]

Printed at: Thomson Press

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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Prof. M. S. Swaminathan Chairman

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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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*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

17

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.

"This page is Intentionally Left Blank"

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

RPMP Vol. 29 - Drug Plants III

20

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.

Phytotherapeutic Approach to Alcohol Dependence

21

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 •

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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

22


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


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

24


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,


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

26


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)


27

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2.

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4. 5.

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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.


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]

32


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).

34


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


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


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

38


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


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).

40


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.

42


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

44


(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


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


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


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

48


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.


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


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)


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

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--

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

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~

300 0 700

750

800

850

900

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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


52

450

.....= ~

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300 7 3

761

150

0 700

750

800

850

900

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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

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Il

500 0 720

728

736

744

I, 752

760

768

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Fig 10. (Contd.)


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....=

~

53

70 60 50 40 30 20 10 0

600

-

700

800

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B

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...

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

600

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Fig 10. (Contd,)


54

2500 2000 ...l

.s

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758

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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


55

1600 1400 · 1200 '

~

:.:

1000 · 800 ' 600 . 400 ·

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200 •

o

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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

56


1250 1000 789.612

750

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500

I~J

250

.1 , .....11

o I"'" ",' 700

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.... 860

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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


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).

58


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.

59

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.


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]

62


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).

Screening ofNatural Products to Drug Discovery

63

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

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)=0

o 0

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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.

64


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).

65


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

,,

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.,

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I

Inactive fractions

,

Pharmacological evaluation and structure determination

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.,

,

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Known compounds

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,:

Validation oflead

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Synthesis of

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Fig 1. General approach to drug discovery from natural sources

I


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


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.

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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.,


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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.

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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

82


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


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


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


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.


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]


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.


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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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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)


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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



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

106


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.


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


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


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



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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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


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


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


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-

168


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' -


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.


170

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


171

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


176

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


177

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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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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Chemoprventive and Radioprotective Effects

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

Chemoprventiue and Radioprotective Effects

187

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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RPMP Vol. 29 - Drug Plants III Vitamins

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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.

Chinese Herbal Medicine for Coronary Artery Disease

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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).

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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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Non-commercial Plants of Medicinal Purposes

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


222

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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

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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.

232


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


233

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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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

The Treatment Period-Dependent Effects

239

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


240

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

241

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 0.05; 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


242

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:!

;: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

:)

00 ~

I>:)


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.


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 :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



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

;::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



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 9%) during the four


348 16

16 H3C

115

10 9

10

10

9

9

2

2

2

6-hydroxilycopodine

Sauroine

Lycopodine 16

16

H,C

. \15

10

10

10

9

9

9

2

2

Clavolonine

2

Lycodine

Sauroxine

16

16 16

10 10

9

o

H,C 2

N-metillycodine

Huperzine A

N-acetillycodine

Fig 1. Lycopodium alkaloids isolated from Argentinean specimens of Huperzia saururus

seasons: sauroine, sauroxine and 6-0H lycopodine, and the other alkaloid had a minority content «9% and> 1%) and in traces ( 30 mg/day) can cause an hypercarotenemia that is an abnormal yellowing of the skin (Pusztai et al., 2000; McGowan et al., 2004). This manifestation can also occur in individuals consuming large amounts of carrots or products containing finely grated carrots or carrot juice. It should be however noted that the rare cases of hypercarotenemia mentioned so far are entirely benign and have no adverse effects.

Conclusions In conclusion, this survey of the literature showed that carrot extracts from various parts (seeds, leaves, stems, umbels-flowers, roots) exhibited interesting biological properties that could be exploited in human or veterinary medicine. However, composition analysis demonstrated a tremendous chemical variability of extracts.

Daucus carota L.: A Common Plant

405

Thus, care should be taken to the species or species derivatives, maturation state, part of the plant used, and to the extraction procedures that are undertaken to produce bio-active extracts.

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23 Capsaicin: A Spice Derived Phytochemical that Modulates Calcium Homeostasis, Energy Inter-conversion and Cellular Metabolism

Abstract Capsaicin is the chemical compound that makes chili peppers taste hot. CPS is an agonist of the transient receptor potential Vanilloid subfamily 1 (TRPV1), a non-selective cation channel abundantly expressed in sensory neurons. CPS intake has been linked with the suppression of several malignant transformations through a mechanism that is not fully understood. In addition, many studies in the medical literature have linked the consumption of chili-containing meals with increased energy expenditure and fat oxidation. In this regard, a seeming effect of chili consumption is the rapid increase in body core temperature and subsequent increased heat loss. We have recently discovered a prominent target for the action of capsaicin, namely, the sarcoplasmic reticulum calcium ATPase. We showed that capsaicin uncouples this ion pump. Here I briefly review old and new information on capsaicin and account on the physiological effects of this drug in light of our new finding. 2

Key words: Capsaicin, Ca +-ATPase, Intracellular calcium, Thermogenesis, TPRV

Introduction For centuries, chili peppers is known to contain a component that causes irritation and burning effect when comes in contact with the skin. In 1816, the active molecule in chili peppers was first isolated in crystalline form by P.A. Bucholz. In 1846, L.T. Thresh introduced the name capsaicin. Despite 1. Institute of Physiology and Biophysics, University of Aarhus, Denmark.

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

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this early discovery, it was not until 1919 that the exact chemical structure of capsaicin (Fig 1) was determined, and was demonstrated to be identical with trans-8-methyl-N-vanillyl-6-nonenamid (Nelson, 1919). Capsaicin was first completely synthesized in 1930 (Spath & Darling, 1930). In 1961, similar substances were isolated from chili peppers by the Japanese chemists S. Kosuge and Y. Inagaki, who named them capsaicinoids. To date, six different capsaicinoids have been described, these include homocapsaicin, dihydrocapsaicin, homodihydrocapsaicin, and nordihydrocapsaicin. Capsaicinoids are also referred to as Vanilloids, as they contain a vanilloyl functional group (Davis et al., 2007). Due to its possible role in pain relieve capsaicin is indeed one of the best characterized drugs, retrieving around 10,000 articles when searched in Medline.

Fig 1. Chemical structure of capsaicin

Origin, nomenclature, distribution, and isolation Capsaicinoids are found in the fruits of plants belonging to the genus Capsicum (Paprika or Cayenne, botanical names: Capsicum frutescens, Capsicum annuum). These plants belong to the Solanaceae family (also termed nightshade- or potato family) (Fig 2). Capsicum is a native ofthe Americas, where it was cultivated for thousands of years by the people of the tropical Americas. Now, the plant is cultivated worldwide. The effectiveness of a given capsaicinoid, also called pungency or spiciness, is measured in Scoville units (Scoville, 1912). The more pungent a capsaicinoid the more stimulation it causes in the sensory neurons in the skin (and hence more burning effect). Capsaicin and dihydrocapsaicin have a pungency value of -16 million units. That is, the alcoholic extract ofthe compound has to be diluted 1:16,000,000 in order for the burning effect to be completely undetectable when sensed by

Fig 2. Left. Spanish pepper flowers (Capsicum annuum). Right. Capsicum with the green and red pepper fruits.

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the tip of the human tongue. The other capsaicinoids have a pungency value of about 8 million units. The fact that capsaicin constitutes about 70% of capsaicinoid mixtures (versus -18% for dihydrocapsaicin and less that 10% for the other capsaicinoids) puts capsaicin as the active component in chili peppers. It should be mentioned that the fruits of Capsicum have a variety of commercial names depending on the shape of the fruit, the place where they are grown, and on the degree of spiciness. The capsaicin content in red chilies varies depending on the variety and age ofthe plant (Mueller-Seitz et al., 2008). The content of capsaicin in Capsicum annuum is - 0.2% and in Capsicum frutescens is - 0.05% of the dry weight (Glinsukon et al., 1980). Isolation of capsaicin from its natural sources is a reliable process. The peppers are ground into a fine powder and further refined to the oleoresin which is a reddish-brown liquid. Capsaicinoids are then obtained by purification procedures employing thin layer chromatography and high performance liquid chromatography (Govindarajan, 1986; Carter, 1991). Products resulting from the hydrolysis of capsaicin have been identified as vanillylamine and an unsaturated fatty acid. Furthermore, the reaction of vanillylamine and the acid component from natural capsaicin results in the formation of a product with the same physical, chemical, and pungent properties as natural capsaicin (Cordell & Araujo, 1994).

Antimicrobial and mutagenic activity Capsicum annuum extracts have been found to have a strong effect against the cercaria of Schistosoma mansoni which is estimated to have infected over 200 million people world-wide. In terms of pharmacology, nevertheless, these results cannot rule out the possibility that this activity is ascribed to the presence of capsaicinoids or due to other compounds, as the toxicity was assayed with crude fruit extracts (Frischkorn et al., 1978). Molina-Torres et al. (1999) performed the first study evaluating the antimicrobial activity of isolated capsaicin. These authors followed the growth ofliquid bacterial/yeast cultures in the presence of varying concentrations of capsaicin. Inocula were obtained from cultures growing exponentially in the same medium, and growth was followed by determining turbidity at 650 nm in a spectrophotometer. Capsaicin, at a concentration of up to 0.3 mg/ml, inhibited the growth of Escherichia coli. On the other hand, the same concentration of the drug retarded the growth of Pseudomonas solanacearum by only 20%. In addition, inhibition of the growth of Bacillus subtilis was observed after treatment with 0.025 mg/ml capsaicin. The effect of capsaicin on Saccharomyces cerevisiae was not definitive. Short-term cellular growth was stimulated at concentrations as high as 0.15 and 0.3 mg/m!. However, Capsaicin had no effect on the long-term (24 h) growth of Saccharomyces cerevisiae at concentrations as high as 0.3 mg/ml (MolinaTorres et al., 1999).

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Capsaicin was also tested for mutagenic activity using different Salmonella typhimurium strains with and without 89 metabolic activation (Billings et al., 1985), and 20 min standard preincubation time (Azizan & Blevins, 1995). Indeed, there was no correlation between mutagenicity and the pungent properties of different molecules. However, pure capsaicin was found to be mutagenic to a single strain among three strained tested and only in the presence of the 89 metabolic activation system. In addition, the mutagenic activity of capsaicin was found to be partially relieved by components from the acetone extract of Capsicum annuum (Azizan & Blevins, 1995).

Uses of capsaicin The analgesic effect of Capsicum has been recognized for many centuries. Native Americans rubbed their gums with pepper pods to relieve toothache. The therapeutic potential of capsaicin was documented for the first time in 1850 where alcoholic hot pepper extract was used on sore teeth for instant relief(Turnbull, 1850). Currently, uses for individual products containing capsaicin vary widely. Capsaicinoids evoke a sensation of burning by interacting with and activating a nonselective cation channel (capsaicin receptor, see below) on the sensory nerve endings, releasing sensory neuropeptides that trigger a neurogenic inflammatory response, developing an unpleasant effect in animals. Consequently, capsaicin has been registered for the first time as a natural pesticide to repel vertebrate pests such as rabbits, cats and dogs. Interestingly, it was found that primary sensory neurons from birds are fully insensitive to hot chili peppers (capsaicin), although their sensory nerve endings express a similar cation channel ortholog (Geisthovel et al., 1986). This discovery represented a major economic advantage in the field of ecophysiology because it highlighted a role for birds in Capsicum seed dispersal and the insurance of plant existence (Tewksbury & Nabhan, 2001). This species-specific difference fact was also employed in the field of food industry. In fact, chicken feed can be "flavored" with dried pepper powder. Capsaicinoid feeding changes the color of the yolks of chicken eggs orangered and most importantly function as a feeding depressant for other animals that can have access to the food such as rats and squirrel (Rouhi, 1996). The insensitivity of birds to capsaicin has prompted scientists to investigate the molecular basis of this fascinating difference and it was shown that the avian cation channel is insensitive to capsaicin due to the dissimilarity of two highly conserved amino acids in the loop connecting transmembrane domains 2 and 3 in the channel (Jordt & Julius, 2002). Even before identification of the molecular targets of capsaicin, the medical significance of capsaicin has been studied intensively. The selective and reversible effects of capsaicin applied locally to the skin or nasal mucosa have allowed the use of the drug to treat certain neurological disorders involving sensory neurons. In some patients, topical application of capsaicin


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to the skin was found to relieve pain associated with post-herpetic neuralgia (Watson et al., 1988), diabetic neuropathy (Ross & Varipapa, 1989), local stump pain (Rayner et al., 1989), and rheumatoid arthritis (Deal et al., 1991). Several capsaicin-containing creams (e.g., Zostrix, Zostrix-HP, and Axsain) are commercially available for the treatment ofthe above mentioned painful conditions. Many studies in the medical literature have linked the consumption of chili-containing meals with increased energy expenditure and fat oxidation (Westerterp-Plantenga et al., 2006; Diepvens et al., 2007). Capsaicin capsules are being used by athletics and body builders to stimulate thermogenesis and thereby lose excess fat before competitions. Recently, interest has been directed into the role of capsaicin in treating chronic diseases such as cancer. Indeed, capsaicin intake has been linked with the suppression of several malignant transformations through a mechanism that is not fully understood. In particular, CPS treatment was reported to significantly slow down the proliferation of prostate cancer cells (Mori et al., 2006). Other types of malignant transformations have also been shown to be suppressed by capsaicin treatment (e.g., Morre et al., 1995; Zhang et al., 2003; Qiao et al., 2005; Chu-Chung et al., 2009). The possible role of capsaicin in cancer treatment is currently under investigation by several research groups.

Dosage The daily intake of the fruits of Capsicum annuum largely depends on cultural factors. In India, the average daily intake of red chili is around 0.08 g/kg body weight. Dosage of capsaicin is difficult to estimate because the content ofthe two major capsaicinoids vary significantly in the different fruits. Some health food companies in Europe describe a dose of 1-4 g/day. Thus, doses as much as 3-6 g/day can be tolerated by healthy humans. Since the content of capsaicin in chili peppers varies significantly, consumption should be individually evaluated in a reasonable manner (see below).

Precautions for usage It is apparent that moderate oral doses of capsaicin (0.02-0.06 mg/kg) are well tolerated by healthy humans. It must be mentioned that hot chili peppers are food additive and must not be considered as an ordinary meal. The oral toxicity of capsaicin has been evaluated using animal models and it was documented that capsaicin can indeed exhibit cytotoxicity at high doses (Saito & Yamamoto, 1996). In fact, eating chili peppers alone can be dangerous and in the worst case may strongly influence heart metabolism. Through its effects on calcium transporting proteins (see below) capsaicin may have a strong influence on calcium homeostasis and this may differentially affect different people, especially at extremely high doses and when the length and frequency of exposure varies. Indeed, capsaicin content in chili peppers vary significantly. In some types of chili peppers like Naga Jolokia, Dorset Naga and Red Savina Habanero the capsaicin content may be much higher

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than in other types. Thus the capsaicin content (degree of spiciness) may increase by as much as 20 fold when comparing two different types of peppers. Furthermore, the pharmacological properties of many compounds in the Capsicum fruits is not even firmly documented: although capsaicin is considered as the major active constitute in peppers it is known that Capsicum contains over 100 distinct volatile compounds that may function in many ways dissimilar to capsaicin. Capsaicin contact with the eyes as well as compound inhalation should be avoided (as in the case with black pepper and many other herbs and food additives). Inhalation of capsaicin may cause temporal bronchoconstriction, coughing, and nausea (Chanda et al., 2004). Furthermore, prolonged inhalation may cause lung inflammation and widespread damage to tracheal, bronchial, and alveolar cells (Reilly et al., 2003, 2005). A single case of an arterial hypertensive crisis was reported in a 19-year-old Italian man who admitted to have ingested large doses of chili peppers (Patane et al., 2009) a day before registration. This unusual and odd effect is probably due to a very high capsaicin dose. Major cases in which chili pepper intake associates with health problems occur almost exclusively as a result of challenges between teenagers on how many chili peppers a winner must eat. On the other hand, it is true that millions of people consume tons of Capsicum fruits without virtually no harmful effect, indicating the safety of the drug when used in a reasonable manner.

Molecular targets for capsaicin In fact, early attempts to identify a capsaicin target have been unsuccessful due to technical problems owing to the hydrophobic nature of the molecule. Experiments aiming at understanding the hot and painful action of several vanilloyl group containing compounds led to the identification of the capsaicin-related molecule resiniferatoxin (RTX) as a potent capsaicin analogue. In the beginning of the nineties, identification of a binding site for RTX has revolutionized research in this field: binding studies have indicated the presence of a single class of saturable binding sites for [3HlRTX on membranes isolated from dorsal root and trigeminal ganglia (Szallasi & Blumberg, 1990). In 1997, a Vanilloid receptor, termed VR1, was cloned and shown to be activated by capsaicin as well as heat and low pH (Caterina et al., 1997). VR1 was later proposed to function as a molecular integrator of pain signals in response to the aforementioned stimuli (Tominaga et al., 1998). The cloning of VR1 also enabled the discovery of other members of the transient receptor potential of the vanilloid type (TRPV). Similar to TRPV1, these receptors function as molecular detectors of physical and chemical stimuli, such as innocuous and noxious heat, as well as mechanical energy. Novel TRP channels sensitive to low temperatures also have been cloned, namely, TRPM8 and TRPA1 (see Patapoutian et al., 2009 for recent review). Now, these proteins belong to a growing family of noxious stimuli detectors in mammals (Levine &


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Alessandri-Haber, 2007). Currently, several research groups worldwide investigate the structure, mechanism, and physiology of TPRV using vanilloids as a tool. Until recently, TPRV was believed to be the only target for the action of capsaicin. On this basis, capsaicin is expected to mediate its action by activating passive ion fluxes across the membranes where TPRV are expressed. The physiological impact of capsaicin was thus expected merely to be the induction of action potential from sensory neurons which ends up with the activation of targets down the ~-adrenergic signaling pathway.

Capsaicin, sarcoplasmic reticulum Ca-ATPase, and heat production The sarcoplasmic reticulum is a highly specialized organelle that plays a key role in muscle physiology and pathophysiology. This organelle has developed an elaborate set of calcium-regulatory proteins that provide a near-instantaneous release of calcium upon excitation and a slower means of calcium reuptake and maintained calcium storage during muscle relaxation and inactivity. Specifically, the processes of calcium storage, release, and reuptake are balanced by the concerted action of three major classes of sarcoplasmic reticulum calcium-regulatory proteins: (1) luminal calcium-binding proteins (calsequestrin, histidine- rich calcium-binding protein, junctate, and sarcalumenin) for calcium storage; (2) sarcoplasmic reticulum calcium release channels (type 1 ryanodine receptor or RyR1 and IP3 receptors) for calcium release; and (3) sarcoplasmic reticulum Ca 2 +-ATPase pumps for calcium reuptake. In the working muscle, the sarcoplasmic reticulum Ca 2 +-ATPase, one of the major regulators of calcium transport, rapidly clears cytoplasmic calcium to ensure muscle relaxation. Sarcoplasmic reticulum Ca 2 +-ATPase uses energy from ATP hydrolysis to build up a calcium gradient across the sarcoplasmic reticulum membrane that can reach up to four orders of magnitude. This calcium gradient is pivotal for the operation of contraction-relaxation cycles. The sarcoplasmic reticulum Ca 2+-ATPase uses about 25% of the total energy to maintain calcium concentration gradient in the resting muscle cell. Worm-blooded animals are able to keep their body temperature at a roughly constant level, regardless ofthe ambient temperature. This implies the ability to produce more body heat in cold environments. An increase in heat production occurs merely through increasing the metabolic rate. Heat production can either be autonomic (shivering) or facultative (non-shivering). In human infants and hibernating mammals, non-shivering thermogenesis occurs mainly in the brown adipose tissue (Lean, 1989). In this process, free fatty acids remove purine (e.g. ADP and GDP) inhibition of uncoupling protein-1 which causes an influx of hydrogen into the matrix of the mitochondria and bypasses the ATP synthase channel (Argyropoulos &

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Harper, 2002). This uncouples oxidative phosphorylation and the energy from the proton motive force is dissipated as heat rather than producing ATP from ADP (Morrison et al., 2008). In the adult human body, which contains significantly less concentrations of brown adipose tissue (Lean et al., 1986), skeletal muscle is the main source of heat production. Mammalian cells spend an important fraction of their energy in maintaining steady state ion gradients, and there is evidence that such mechanisms may be used for the purpose of increasing ATP consumption in a futile manner. When ion gradients across the membrane are reduced either by leakage or by utilization of them to support trans-membrane transport, the cell is forced to spend more ATP to keep the steady state gradient. Calcium cycling is one of the major mechanisms whereby heat generation can take place across the sarcoplasmic reticulum membrane in the skeletal muscle cell. Heat production by muscle most likely involves different mechanisms such as increase in the passive calcium efflux from the lumen of the sarcoplasmic reticulum (thereby indirectly activating the calcium pump and enhances ATP hydrolysis) or directly activating ATP hydrolysis by the calcium pump without calcium mobilization. A direct link between capsaicin and a sarcoplasmic reticulum protein has not been observed earlier. In our laboratory, we found that capsaicin directly stimulates the hydrolytic activity of the sarcoplasmic reticulum Ca2+ATPase in vitro (Mahmmoud, 2008). As with all chemical processes, the Ca 2+ATPase works at a certain degree of efficiency, that is, it is impossible to convert all energy from ATP hydrolysis into dynamic calcium gradient across sarcoplasmic reticulum membrane (100% efficiency). A substantial part of the energy from ATP hydrolysis has to be given off as heat, especially when a calcium gradient is already established across the sarcoplasmic reticulum membrane. The balance between passive calcium entry to the cell cytoplasm and the energy required to empty calcium from the cytoplasm will represent how much heat energy is produced. Coupled Ca2+-ATPase is that mediating active calcium transport across the sarcoplasmic reticulum membrane, whereas uncoupled Ca2+-ATPase is that hydrolyzing ATP without net calcium transport, that is, generating heat. Capsaicin stabilizes Ca 2+-ATPase conformation mediating uncoupled transport (Mahmmoud, 2008).

Mechanism of capsaicin action Activation ofTRPVl channels Primary sensory neurons represent the communication channels between the internal body organs and the environment. Sensory neurons are found in the skin as well as in tissues having terminals reacting to sensory stimuli. Noxious and thermal stimuli act directly on the peripheral terminals of a high-threshold primary sensory neurons, referred to as nociceptors, to elicit nociceptive pain. Many of the transduction channels that convert thermal,

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Capsaicin: A Spice Derived Phytochemical Action potential

~ /

Transmitter release

Signal transmission

Fig 3. The role of TRPV1 channel in the peripheral terminals of nociceptors. Agonist binding to the vanilloid receptor opens the channel pore and leads to cation influx (Wood et al ., 1988). This cation influx causes membrane depolarization, which if reaches a certain threshold, induces an action potential that propagates along the entire length of the vanilloid-sensitive neuron and causes a sense of pain (Holzer, 1991). Apparently the same occurs up on activation with thermal nociceptive stimuli. Vanilloid-sensitive neurons use glutamate, ATP and a variety ofneuropeptides as transmitters, see also Lundberg (1996).

mechanical or chemical stimuli into electrical activity are transient receptor potential (TRP) channels. Activation of the peripheral terminal leads to action potential signaling towards the central nervous system and a local release of vasoactive peptides that produce neurogenic inflammation (Fig 3). TRPV1 is largely expressed in nociceptors. The nociceptor presynaptic terminal contains excitatory amino acid and peptide transmitters, the release of which is also modulated by several TRP channels. The function ofTPRV1 has been extensively studied using the gain-of-function approach, i.e., to treat the channel by activating ligands and probe the physiological consequences of such activation. In this regard, capsaicin has been an invaluable tool in studies on the mechanism and function ofTPRVl. Analysis of TRPV1 knock-out mice revealed that the VR1-j- mice showed normal responses to noxious mechanical stimuli but exhibited no capsaicin-evoked pain behavior. This shows that the painful effect of capsaicin is entirely mediated by TRPV1 (Caterina et al., 2000; Davis et al., 2000). It should be mentioned that treatment of isolated neurons with capsaicin or RTX has been shown to disrupt vital cellular organelles due to a significant increase in cytoplasmic calcium concentrations (Olah et al., 2001). The same likely happens following epidural administration ofRTX to animal models. Capsaicin has been reported to increase thermogenesis by enhancing catecholamine secretion from the adrenal medulla in rats, mainly through activation ofthe central nervous system (Watanabe et al., 1988). Activation of beta-adrenergic receptors raises cAMP levels and increase the expression of genes responsible for mitochondrial biogenesis and function. This process seems to be dependent on the thyroid hormone levels (Silva, 2006; Bianco et al., 2005). It is not clear how gene expression of mitochondrial protein

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leads to increased heat generation. In addition, the stimulation of uncoupled sarcoplasmic reticulum Ca 2+-ATPase seems to be a significant mechanism through which heat generation is induced (see below).

Activation of sarcoplasmic reticulum Ca 2 +-ATPase Hypothetically, hydrolysis of one ATP molecule leads to transport of two calcium ions from the cytoplasm to the sarcoplasmic reticulum lumen (Inesi et al., 1980). However, sub-stoichiometric efficiencies of SERCA are commonly observed in the presence of a calcium gradient across the sarcoplasmic reticulum membrane. The energy from ATP hydrolysis not coupled to calcium transport is presumably dissipated as heat. Definite information on whether the Ca 2+-ATPase hydrolyzes/synthesizes ATP, transports calcium, or generates heat has been obtained by measuring calcium transport, ATP hydrolysis, together with pump mediated heat exchange (de Meis, 2001). Subsequent investigations in this direction accentuated the presence of regulatory mechanisms that would allow Ca2+ATPase to generate heat at the disbursement of calcium transport (Ketzer & de Meis, 2008; Kjelstrup et al., 2008). Indeed, the amount of heat released during the ATP hydrolysis and calcium transport was found to vary between 7 and 32 kcallmol. This finding indicated that SERCA are able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel which is used for calcium transport and the parcel of energy that is used for the heat production. Heat generation and burning calories are implicated in the regulation of several physiological processes including body rectal temperature, cellular metabolism, obesity, energy balance and cold acclimation (Block, 1994; Jansky, 1995). The heat derived from Ca2+-ATPase maneuver may therefore play an important role in the regulation of non-shivering thermogenesis and obesity control (Lowell & Spiegelman, 2000; Belza et al. , 2007). Earlier studies have shown that adrenaline infusion, which causes a 25% increase in whole body energy expenditure in humans, stimulates forearm muscle oxygen consumption by as much as 90% (Simonsen et al., 1992). Assuming that forearm muscle is representative of total body musculature, skeletal muscle then accounts for 40% of adrenaline infusion-induced thermogenesis in humans. Studies on other species have identified muscle tissues specifically involved in heat production (Carey, 1982; Block & FranziniArmstrong, 1988; Block et al., 1994). Indeed, recent studies on rabbits have shown that these animals (which in contrast to rats lacks brown adipose tissue) can regulate their body temperature in response to cold exposure through muscle mediated heat generation. Gross anatomy ofthe gastrocnemius muscle in the control and in cold exposed rabbits indicated a dark red colour in the cold exposed rabbits, characteristic of oxidative muscle fibers. Interestingly, cold tolerance was also found to occur in hypothyroid rabbits, indicating that the muscle mediated heat generation occurs independent on thryroid


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of< o o

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o. o· o· I~ o· oj o o

o o o

t

:

1Pyruvic acid. ,..---------_.

glucose

Fig 4. Simple illustration of calcium mobilization and energy inter-conversion in a muscle cell. Reduction in the ATP levels affects the equilibrium between ADP and phosphocreatine (PCr) to restore ATP levels. This equilibrium is extremely efficient at maintaining constant ATP levels at varying demands. In particular, it has been experimentally determined that the concentration ofPCr can decrease by 90% of its initial level before the ATP concentration begins to decrease by only 10%. Breakdown of muscle glycogen releases glucose, which, under anaerobic condition is converted to lactic acid (energy from this reaction is limited, giving 2 ATP molecule per glucose molecule). Under aerobic conditions, pyruvic acid is enrolled in the Krebs cycle in mitochondria, giving extra 36 molecules of ATP. Cellular energy is used by three main ways; 1) Actomyosin ATPases (contraction), 2) Sarcoplasmic reticulum Ca 2+-ATPase (relaxation) and other metabolic functions . RyR. Ryanodine receptor, SR. Sarcoplasmic reticulum.

hormones (Arruda et al., 2008). Fig 4 provides a simple illustration of how capsaicin can affect calcium homeostasis and energy inter conversion in the muscle cell.

Physiological effects of capsaicin: Ca 2 + homeostasis, cellular energy and metabolism. The above information strongly indicate that the sarcoplasmic reticulum is a system that is involved in heat generation beside its role in calcium cycling. Indeed, it is known that a significant portion of the variation in metabolic rate between humans can be accounted for by differences in the energy expenditure of skeletal muscle at rest. The importance of the sarcoplasmic reticulum proteins in regulating tissue thermal balance can be emphasized by looking into the heater organ offish and the pathological condition known as malignant hyperthermia. The 'heater organ' is a derivative of muscle that is relatively devoid of contractile elements. These specialized cells make up most ofthe superior rectus muscle in the orbit and generate heat for the brain and eyes during cold-water dives, providing heat to maintain the function of the eye and adjacent brain at temperatures as high as 14°C over the water temperature. Like typical muscle cells, heater cells possess abundant acetylcholine receptors and have an extensive network of sarcoplasmic reticulum and

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T-tubules. Mitochondria are also extremely abundant in heater cells, comprising over 60% of total cell volume. Interestingly, the ryanodine receptor channel expressed in this organ is similar to that in slow-twitch muscle, which may be more important for thermogenesis than fast-twitch muscle. Thermogenesis in heater cells is initiated by depolarization, which causes calcium release by the sarcoplasmic reticulum. ATP is then consumed by Ca2+-ATPase, which returns calcium to the sarcoplasmic reticulum. The increased demand for ATP required to sustain this futile cycle drives fuel oxidation. Thus, depolarization-induced calcium entry into the cytoplasm causes ion cycling mediated thermogenesis. A drastic illustration ofthe potential of heat regulation in mammals is malignant hyperthermia, wherein in genetically predisposed individuals or animals (it may result from a mutation in the skeletal muscle ryanodine receptor), certain environmental factors such as some anesthetics or stress can make the sarcoplasmic reticulum leaky, with an ensuing life-threatening hyperthermia. Furthermore, it is possible that some leakage occurs in the normal resting muscle contributing to obligatory thermogenesis. Based on observations made in isolated sarcoplasmic vesicles it was estimated that the calcium recycling across the sarcoplasmic membrane could account for 3070% (depending on the calcium pool size in the muscle) of the resting muscle energy expenditure. As mentioned, muscle is a major site of non-shivering thermogenesis in birds, and it has been found that SERCAI and RyR channels increase in muscle of ducklings during cold adaptation. Such observations altogether support to the hypothesis that sarcoplasmic reticulum calcium leak, coupled to rapid recapture by the sarco/endoplasmic reticulum Ca 2+ATPase could sub-serve a thermogenic role. The effect of ingested capsaicin on heat generation in the muscle can thus be accounted for (at least in part) by its direct effect on SERCA.

Phytochemicals in spice as a potential cancer drugs Spices have been known for their flavor, taste, and importance in food processing. However, scientific interest in medicinal plants has increased strongly during the last decade , and many efforts have been made to understand and explain the beneficial effects of many plant-derived chemicals. Extensive research in several laboratories have uncovered the pharmacological significance of many spice-derived phytochemical (Calixto et al ., 2005; Aggarwal et al., 2008). Other phytochemical-derived drugs include curcumin (from Curcuma Zonga), thymoquinone (from Nigella sativa), ursolic acid (from Rosmarinus officinaZis). These molecules seem to have the ability to prevent chronic diseases such as cancer, diabetes, as well as pulmonary, cardiovascular and neurological disorders. Curcumin has been shown to repress tumor initiation, promotion, and metastasis by inhibiting transcription factors, enzymes, and molecules (see Aggarwal et al. , 2003 for review). This broad specificity is understandable, when looked at in light of the fact that curcumin has several targets in the cell. Thus, this molecule directly modulates the activity of house-keeping enzymes


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such as ATPases (Logan-Smith et al., 2001; Mahmmoud, 2005) and protein kinases (Liu et al., 1993; Mahmmoud, 2007). Many other targets have been identified in intact cell systems (Aggarwal et al., 2007; Kunnumakkara et al., 2008), however, the possibility that a given target may be modulated indirectly following curcumin treatment cannot be ruled out. We have proposed that calcium ions may modulate the interaction of curcumin with its targets (Mahmmoud, 2007), indicating that the effect of the drug may depend on cytoplasmic calcium concentration. We also have evidence that curcumin may interact at the lipid/protein interface, pointing out to the conclusion that curcumin may not have a specific interaction site on one or several of its protein targets (Mahmmoud, unpublished observations). Several other drugs are also known to exert their effects via changing membrane events. The beneficial effects of curcumin on intact cells is likely a result of integration of several mechanisms modulated by this drug. It can be indicated from the above considerations that the effect of most phytochemicals is rather related to their role as chemopreventive agents. This is in contrast to other drugs that have unique target enzymes such as ouabain, a highly specific inhibitor of the sodium potassium pump (Manunta et al., 2009), or thapsigargin, a highly specific inhibitor of the sarcoplasmic reticulum Ca2+-ATPase (Treiman et al., 1998). However, we believe that specific cellular effects may be feasible by optimizing the treatment conditions. This is possible through performing further experiments to understand the role of yet unknown cellular components (e.g. calcium, lipids, and/or regulatory proteins) in modulating the effect of a given phytochemical.

Since calcium handling proteins is involved in several pathological conditions, selective modulators of these proteins would be substances of potential interest to treat such diseases. Once again, natural products seem to be also interesting sources of compounds that may function as prototype TRPV1 ligands. With the possible existence of TRPV1 in skeletal muscle (Xin et al., 2005), it would be of special pharmacological significance to find a drug that differentiates between TRPV1 and Ca2+-ATPase in order to better understand how calcium mobilization through a single pathway modulates thermogenesis.

Concluding remarks Adaptive thermogenesis is an increasingly attractive target for the development of several disorders including hypothermia and antiobesity. As the key molecular components in the pathway of heat production become defined, screening for drugs that increase energy dissipation is becoming a more attainable goal. The mechanism of action of capsaicin and other related molecules is expected to be very complicated, as it involves modulation of at least two major targets. We are currently testing more vanilloids on the function of sarcoplasmic reticulum Ca2+-ATPase. This may shade light on whether the uncoupling effect is mediated on a single (or perhaps more) step(s) in the calcium pump cycle.

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It is clear that capsaicin and other related molecules will continue to be an important research tool in the future. Plants have developed over thousands of years. They have interacted with climate, surrounding environmental factors as well as unwelcome enemies. What plants have synthesized over the course of evolution deserves in my opinion a great deal of attention.

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Subject Index A Abeliophyllum distichum 285, 287, 293 Abrus precatorius 85, 273 Acacia nilotica 273 Acanthaceae 87 ACE 270 ACE inhibitors 272 Achyranthes aspera 273 Acnistus arborescens 71, 72, 69 Acorus tatarinowii 209 Actinida deliciosa 273 Actinostemma lobatum 273 Addiction 19 Adenopodia spicata 273 Aedes aegypti 397 Aegle marmelos 118, 120, 136 Aeromonas hydrophila 105 A sob ria 105 Afromomum latifolium 85 Afzelia quanzensis 85 Agapanthus africanus 273 Agave americana 273 Aglycons 2 Ailanthus excels 285, 289, 290, 292 Alchorneaglandulosa 217, 221, 223 Alcohol dependence 19 Aldose reductase 1 Aldose reductase (AR) inhibitors 1, 3 Alisma orientale 273 Allium 183 A cepa 85 A hirtifolium 201 A macrostemon 209 A sativum 120, 190 A ursinum 273 Allophylus edulis 273, 286, 287, 293, 294 Alloxan 131 Aloe buettneri

Alternaria alternata 397 Alternative medicine 238 Alzheimer's disease 93, 96, 330 Amaranthus dubius 273 A hybridus 273 Ambrosia psilostachya 285 Amenorrhea 93, 96 Anacardiaceae 72, 87 Andrographis echioides 273 A paniculata 118 Angelica acutiloba 273 A gigas 273 A keiskei 273 A sinensis 209 Angina 207,208 Angina pectoris 192 Angiotensin 270 Angiotensin Converting Enzyme (ACE) 269, 270 Annona muricata 71, 72 A senegalensis 85 A squamosa 120 A stenophylla 365, 368 Annonaceae 72, 85 Anogeissus leiocarpa 85 Anti-allergic action 106 Antianginal agent 207, 208 Antianginal therapy 207, 208 Antibacterial 98, 100, 385 Antibiotic 93, 96 Anticancer 98, 402 Anticholinesterase activity 345 Anticoagulant 1 Antidesma madagascariense 273 Anti-diabetic effect 113, 247 Antifebrile 1 Anti-fertility 385, 399 Antifungal 93, 97, 385, 395 Antifungal compounds 396 Anti-HIV activity 337 Antihydrotic 93, 96


432 Antihyperglycemic therapies 113 Antihypertensive 269, 270, 300 Anti-inflammatory 99, 385,400,402 Antimicrobial 97, 98 Antimutagenic 97 Antioxidant 93, 98, 99, 247, 300, 363, 385,402 Antioxidant enzymes 247 Antioxidative 100 Anti-parasitic 385 Antiperspiration 93 Antiplatelet activity 1 Antiretroviral drugs 329 Antirrhea borbonica 273 Antischistosomal agents 79 Anti-secretory drugs 219 Antiseptic 1, 96 Antispasmodic 93, 96, 192 Anti-steroidogenic 385, 399 Anti-thrombotic 385, 400 Antitumour activity 309 Antiviral 93, 100, 337 Aphloia theiformis 273 Apigenin 333 Apocynaceae 88 Araliaceae 25 Areca catechu 274 Arecaceae 72 Arisaema consanguineum 274 Aristolochia debilis 274 A. manshuriensis 274 A. rugosa 69, 72 Aristolochiaceae 72 Aristotelia chilensis 274 Artemisia absinthium 120 A. annua 84 A. capillaries 138 A. pallens 274 Artocarpus altilis 269, 270, 296, 299 Asarum heterotropoides 274 A. sieboldii 274 Asclepiadaceae 85, 87 Aspergillus niger 396 Aspilia helianthoides 274 Asteraceae 72, 85, 86 Astragalus membranaceus 209 Astringent 93, 96 Asystasiagangetica 274 Atherosclerosis 192 Atractyloides japonica 285

Azadirachta indica 120, 269, 295, 299, 309 Azadirachtin 312

B Bacillus 160 R. cereus 105, 393, R. megatherium 105 B. subtilis 105, 393, 415 Bacterial amplification chamber 42 Bactericidal activities 105 Bacteriostatic 105, 93 Badula barthesia 274 Baicalein 329, 330 Baicalin 329 Balanites aegyptiaca 85 Berkheya speciosa 85 Bidens pilosa 321 Bignoniaceae 72 Bioactive compounds 61 Biological activity 93, 385 Biomaterials 159 Boerhavia diffusa 274 Bone loss 31 Bone mineral density (BMD) 32, 36 Boswellia elongate 274 Bougainvillea spectabilis 118, 135 Brassicajuncea 120, 137 B. napus 289, 293, 294 Bryostatin 41, 42 Bugula neritina 42, 44, 47 Burseraceae 86 Butea frondosa 274 B. parviflora 274 Byrsonima fagifolia 217, 225, 226

C Cadaba farinose 85 Caenorhabditis elegans 397 Caesalpiniaceae 72 Calophyllum brasiliense 274 C. tacamahaca 274 Calotropis procera 85 Camellia sinensis 120, 275, 286, 291 Campylobacter jejuni 394 Canarium euphyllum 275 Cancer 247, 385 Candidatus endobugula sertula 44 Capparidaceae 85 Capparis tomentosa 85 Capraria biflora 69, 71, 72

433

Subject Index Capsaicin 413, 420 Capsicum sp. 286 C. annuum414,415,416,417 C. frutescens 414 Carcinogenesis 183, 187 Cardiac complications 192 Cardiospermum halicacabum 275 Carica papaya 275 Carminative 96 Carthamus tinctorius 209 Cassia fistula 275 C. italic 85 C. nigricans 86 C. sieberiana 86 C. tora 275 Cassytha filiformis 275 Casuarina equisetifolia 275 Catharanthus roseus 120, 269, 296, 300 Cecropia glaziouii 275, 287, 292 C. hololeuca 287, 292 C. pachystachya 275, 289 Celastrus paniculatus 275 Centella asiatica 275 Ceraoris kiangsu 310 C. nigricornis 310 Ceriops tagal 139 Chemical composition of S. officinalis 93 Chemical constituents of D. Carota 385 Chemopreventive 183, 402 Chemotherapy 79 Chinese Herbal Medicine 31, 97, 207, 209 Chronic stable angina 207, 208 Chrysanthemum coronarium 315, 316 C. lauandulaefolium 275 Chrysin 334 Chrysobalanaceae 72 Chrysobalanus icaco 72, 69 Cichorium intybus 119, 139 Cinnamomum cassia 209, 276 C. zeylanicum 139, 276 Cissus hamaderohensis 276 C. quadrangularis 86 C. sicyoides 118 Citrobacter freundi 393 Citrus aurantifolia 86 C. aurantium 183, 195, 196, 209 C. limon 276 C. nobilis 71, 72

Clausena anisata 276, 367, 368 Clerodendron trichotomum 285, 287, 289, 290 C. infortunatum 276 Cnaphalocrocis medinalis 310 Cochlospermaceae 86 Cochlospermum tinctorium 86 Cocos nucifera 69, 72 Codonopsis pilosula 209 Coffea mauritanica 276 Colitis 217 Combretaceae 85, 86 Combretum fruticosum 276 C. micranthumi 86 Commiphora mol mol 86 Complementary medicine 19 Compositae 315 Condalia microphylla 191 Congestive heart failure 192 Conventional chronic diseases 213 Conventional medicine 375 Coptotermes formosanus 310 Corchorus olitorius 290 Cordemoya integrifolia 276 Coronary artery disease (CAD) 207 Coronary revascularization 207 Cortex phellodendri 337 Cortidis rhizome 140 Crassulaceae 72 Crataegus sp. 276 C. laeuigata 191 C. monogyna, 191 C. oxyacantha 191 C. pinnatifida 209, 276 Creatinine 248 Crescentia cujete 71, 72 Crohn's disease 219 Crotalaria sp. 290 Cuphea cartagenesis 276 Cupressus semperuirens 276 Curcuma longa 86, 424 C. wenyujin 209 Cuscuta japonica 286, 290 Cyclic oligosaccharides 159 Cyclodextrins 159, 160 Cynostemma pentaphylla 276

D Dalbergia odorifera 209, 276 Daphne odora 286 D. tangutica 318, 319


434

Datura stramonium 69, 71, 72 Daucus carota 385 Dehydro-epiandrosterone sulfate 32 Dendrolimus punotatus 310 Desmodium gangeticum 277 D. styracifolium 277 D. triquetrum 277 Diabetes 247 Diabetes mellitus 113 Dichrostachys cinerea 277 Dicoma anomala 86, 366, 368 Dietes iridioides 277 Digestive stimulants 1 Digestive tract disturbs 227 Diospyros kaki 285, 287, 289, 292, 293 D. melanoxylon 277 Discodermia dissolute 63 Dodonea viscose 277 Drug development 329 Dysmenorrheal 93, 96

E Ebenaceae 86 Ecteinascidia turbinate 42, 46 Elephantorrhiza goetzei 86 Eleutherococcus divaricatus 277 E. senticosus 277 Embelia angustifolia 277 E. basal 277 Emmenagogues 1 Entada Africana 86 E. pursaetha 277 Enterobacter gergoviae 105 Enterohepatic recirculation 329 Eomecon sp. 277 Eomecon chionantha 82 Ephedra sinica 277 Epidemiology studies 183 Epilobium angustifolium 278 Epimedium alpinum 278 E. brevicornum 278 E. macranthum 278 Equisetum hyemale 278 Erythroxylum laurifolium 278, 285, 293 Escherichia coli 105, 393, 415 Essential oil 385 Estrogenic 93, 96 ET743 41, 42 Ethnomedicinal plants 69 Ethnomedicines 69

Ethnopharmacological know ledge 217 Ethnopharmacology 79 Ethnoveterinary medicine 69 Euclea natalensis 86 Eucommia ulmoides 120, 285, 289, 294 Eugenia heyneana 278 E. jambolana 118 Euodia simplex 278 Euphorbia hirta 87, 278 E. humifusa 278 Euphorbiaceae 87, 88, 217 Evidence based medicine (EBM) 339 Evodia rutaecarpa 288, 293 Eye and dental problems 69

F Flaveria haumanii 1 F. bidentis 1, 2 Fabaceae 22,79,85,86,87,88 Fagopyrum sp. 288 F. esculentum 287 Ficus carica 288 F. thonningii 87 Flatulent dyspepsia 96 Folk medicine 386 Fritillaria sp. 278 F. ussuriensis 278, 291, 294 F. verticillata 286, 291, 286 Fructus gardeniae 337 Fuchsia magellanica 278 Fusarium oxysporum 105, 396

G Galactorrhoea 96 Galinsoga parviflora 278 Gastric ulcer cicatrisation 217 Gastritis 227 Gastroduodenal diseases 217 Gastrointestinal alterations 219 Gastrointestinal disturbs 219 Genetic diabetic models 123 Gepeduculanta 88 Geranium core-core 278 Ginger 237, 238, 243 Gingivitis 96 Glomerular filtration rate 248, 261 Glucose tolerance test 248 Glycine max 287, 291, 294 Glycosides 2

435

Subject Index Glycyrrhiza uralensis 209 Gossypium sp. 287 Grifola frondosa 120 Guazuma ulmifolia 139 Gunnera tinctoria 278 Gynura procumbens 279

H Hawthorn 183, 191 Headaches 69 Heater organ 423 Hedysarum polybotrys 279 Helianthus annuus 288 Helicobacter pylori 394 Helicoverpa armigera 323 H. zea 397 Heliothis virescens 397 Heliotropium zeylanicum 118 Hemiberlesia pitysophila 310 Hemostat 96 Hepatic injuries 385 Hepatoprotective 337, 402 Herb 207 Herbal and combination formulae 207 Herbal drugs 113, 213, 309 Hesperidin 200 Hexachlamys edulis 279 Hibiscus sabdariffa 279 Hippocampal plasticity 345 Hippocampus synaptic plasticity 345 HN 329 Hochuekkito (RET) 31 Houttuynia cordata 279 Huang-qin 329, 337 Humboldtia vahliana 279 Huperzia 345, 354 H. saururus 345, 346, 346, 353, 354, 358 Hyacinthaceae 87, 88 Hyperglycemia 248 Hyperhydrosis 96 Hypericum perforatum 20 Hypertension 192 Hypertriglyceridemia 259

Hyperuremia 254 Hypnotic 98, 99, 100 Hypoglycemic 96,113, 114,247 Hypoproteinemia 254

I Immune-enhancing 402 Indian medicinal plants 247 Ipomoea aquatic 119, 135

J Jasminum azoricum 279, 290, 293 J. multiflorum 279 J. sambac 279 J. grandiflorum 279 Jatropha curcas 279 Justicia flava 279

K Kalanchoe farinacea 279 K pinnata 69, 71, 72 Klebsiella oxytoca 105, 160 K pneumonia 160

L Lycopodium carinatum 352 L. casuarinoides 352 L. clavatum 346 L. hamiltonii 353 L. sieboldii 353 Lactobacillus curvatus 105 L. plantarum 393 L. sakei 105 Lamiaceae 23, 72, 87, 93 Laxative 96 Ledebouria ovatifolia 87 Leea guinenis 279 L. rubra 279 Lepianthes peltata 69, 71, 72 Leptadenia hastate 87 Lespedeza capitata 280, 291, 292 Leucas martiniensis 87 Leucorrheal 96 Ligusticum chuanxiong 209 L. vulgare 289, 291 Liliaceae 85 Limitations 61 Locusta migratoria 310 Lonchocarpus laxiflorus 87


436

Long-Dan-Xie-Gan-Tang 337 Lycium chinense 280, 286, 290, 291 Lycopodiaceae 345 Lycopodiella 345 Lycopodium 345 L. alkaloids 346 L. complanatum 346 L. saururus 346 Lycosa pseudoannulata 310 Lygodium japonicum 280 Lymnaea luteola 80 Lyonetia citri 310

M Machilus thunbergii 280 Malpighiaceae 217 Manduca sexta 397 Mangifera indica 280 Manilensis 310 Mansoa hirsute 280 Marrubium radiatum 119, 280 Mass spectrometry 42 Matricaria chamomilla 119, 120, 142 Medicinal plant 183, 363 Medicinal use 93 Melastomataceae 217 Melia azedarach 313, 314 M. toosendan 313, 314 Meliaceae 88 Memory retention 345 Mentha piperita 120 Merremia tridentate 280 Mesembruanthemum ssp. 280 Microbial Amplification System 41 Micrococcus luteus 160 Microtoena prainiana 286, 287, 291 Molinaea alternifolia 280 Momordica balsamina 280 M. charantia 119, 120, 136, 137, 139 Monimia ovalifolia 280 M. rotundifolia 280 Moraceae 87 Moringa oleifera 280 Morus alba L. 280, 297, 300 Mosquitoes 310 Mouriripusa 217, 227, 231 Murraya koenigii 118, 120, 135, 136, 137 Musa sp. 69, 71, 72 Musaceae 72 Musanga cecropioide 280, 287, 289

Myocardial infarction 208 Myrcia uniflora 119 Mythimna separate 310

N Natural product 42, 61, 79, 375 Natural product prototype 375 Natural sources 61 Nervine 96 Neuroprotective effects 339 Nigella sativa 84, 87, 424 Nilaparvata lugens 310 Ninjin'yoeito (NYT) 31 Non-commercial plants 217 NSAID drugs 245 NSAID therapy 239 Nymphea micrantha 87 Nympheaceae 87

o Ocimum gratissimum 69, 71, 72 O. sanctum 118, 120, 136 O. tenuiflorum 118, 135 Oenothera biennis 20, 280 O. paradoxa 286, 291 Ogikenchuto (OKT) 31 Olacaceae 88 Olea europaea 281, 291 O. lancea 281 Ophiopogon japonicas 209 Origanum vulgare 281 Orseolia oryzae 310 Oryza sativus 293, 294 Osteoarthritis 237, 238 Osteopenia 32 Ostrinia furnacalis 310, 312, 314, 323 Ouratea semiserrata 281 Ovariectomy 32 Oxidative stress 183 Oxya chinesis 310 Oxygonum sinuatum 281 Ozoroa insignis 87

p Paeonia albiflora 281 P. lactiflora 209 P. moutan 281 Panagrellus redivivus 397 Panax ginseng 20, 25, 281 P. notoginseng 209

Subject Index P. quinquefolius 118 Panonychus citri 310 Parasitic infections 79 Parkia biglobosa 118 Parkinson's disease 330 Parkinsonia aculeate 118 Passiflora edulis 281 P. quadrangularis 281 Pavonia odorata 281 Peristrophe bicalyculata 87 Pesticidal activities 309 Pharmaceutical industry 61 Pharmacodynamics 329 Pharmacokinetics 329, 340 Pharmacological activities 89, 217, 247 Pharyngitis 96 Philippia montana 281 Phlegmariurus squarrosus 352 Phlomis anisodonta 120, 142 Phoenix roebelinii 281 Phyllanthus niruri 281, 286, 287, 288 P. phillyreifolius 281 P. urinaria 287 Phyllotreta vittata 312 Phyloglossum 345 Physalis viscose 281 Phytochemical 79, 213, 389 Phytolacca dodecandra 87 Phytolaccaceae 87 Phytotherapies 213 Pieris rapae 310, 312, 314 Pinellia ternate 209, 281 Pinus densiflora 119 P. maritime 292 Piper betle 119, 136,281 P. futokadsura 281 P. hispidum 71, 72 Piperaceae 72 Pistacia lentiscus 282 Plantago asiatica 282 P. ovate 119 Pleurotus sajor-caju 282 Plutella xylostella 310, 312, 314, 316 Poaceae 88 Polygalaceae 88 Polygonatum aviculare 282 Polygonum hydropiper 2 P. multiflorum 282 Pongamia pinnata 269, 298, 300 Poria cocos 209

437 Potential biomolecular targets 79 Potentilla sp. 282 Potentilla chinensis 282 Poupartia borbonica 282 Prostate carcinoma 330 Prunus persica 209 Psathura borbonica 282 Pseudarthria hookeri 282 P. viscid 282 Pseudomonas aeruginosa 393, 105 P. solanacearum 415 Pterocarpus angolensis 87, 268, 366 Pterodon emarginatus 89 Pueraria labata 20, 22, 209, 282 Pyrrosia lingua 282

Q Quillaja saponaria 120 Quinchamalium chilense 282

R Rabdosia coetsa 283, 288, 290, 293 Radioprotective 183 Radix Scutellariae 329, 330, 333, 335, 336, 337, 339 Ranunculaceae 87 Revascularization 213 Revitalizing body systems 159, 172 Rhei rhizoma 291, 292 Rheum sp. 283 Rheum palmatum 283, 291, 292 Rhizoma coptidis 337 Rhodiola crenulata 283 R. rosea 283 Ricinus communis 69, 71, 72 Rosa rugosa 121 Rosmarinus officinalis 69, 71, 72, 121, 283, 424 Rutaceae 72, 86

s Saba senegalensis 88 Saccharomyces cerevisiae 415 Saffron 183, 200 Salacia reticulate 172 Salmonella typhimurium 227, 416 Salsola oppositifolia 283 S. soda 283 Salvadora persica 283 Salvia acetabulosa 119,283 S. candelabrum 96


438

S. hians 96 S.lyrala 96 S. miltiorrhiza 20, 23, 209, 283, 290, 293 S. officinal is 93, 97, 105, 106 Sanguisorba officinalis 283 Sapotaceae 88 Sarcoplasmic reticulum calcium Atpase 413 Saussurea lappa 283 Scelio uvarovi 310 Schinus latifolius 283 S. molle 289 Schisandra chinensis 209 Schistosoma cercariae 81 S. haematobium 80 S. intercalatum 80 S. japonicum 82, 80 S. mansoni 80, 397, 415 S. mekongi 80 Schistosomiasis 79 Scilla natalensis 88 Scleropyrum pentandrum 283 Scopulariopsis brevicaulis 105 Scrophularia 72, 334 Scutellaria baicalensis 121, 329 S. laterifolia20 Securidaca lon 88 Securinega virosa 88 Sedative 96, 98, 99, 100, 192 Sedum sarmentosum 283, 287, 289, 292,293 Senna occidentalis 69, 71, 72 S. petersiana 88 Sesamum indicum 289, 290, 294 Sida acuta 283 S. cordifolia 284 S. ret usa 284 Silybum marianum 20 Sinomenium acutum 284 Siraitiagrosvenori 118 Sitophilus oryzae 310 S. zeamais 310 Skin diseases 1 Smallanthus sonchifoliu 118, 119 Snake poisoning 1 Solanaceae 72 Solanum nigrum 284 Spasmolytic 96 Spinacia oleracea 287, 290 Spodoptera litura 310, 312, 314, 323 S. venalba 314

Spondias mombin 69, 72 Stachytarpheta urticifolia 96 Standard antianginal therapy 213 Stange ria eriopus 284 Staphyloccocus aureus 105,227,393 S. carnosus 105 S. xylosus 105 Stellera chamaejasme 317, 318 Stephania tetrandra 288 Stimulant 96 Stomatitis 96 Streptococcus pneumonia 395 Streptomyces scabies 393 Stylosanthes erecta 88 Sulphated flavonoids 1 Syzygium aromaticum 119 S. cumi 118 S. cumini 119, 135, 139

T Tabernanthe iboga 24, 20 Tagetes patula 69, 71, 72 Tamarindus indica 118, 269, 299, 300 Taxo141,42 Taxus brevifolia 92, 294 T. floridana 52 Terminalia arjuna 247, 248, 249 T. bentzoe 284 T. bialata 284 T. catappa 284 T. chebula 284 Tesseratoma papillosa 310 Theobroma cacao 121, 142 Therapeutic agents 375 Thermoanaerobacter thermosulfurigenes 160 Thermococcus 160 Thymus vulgaris 120 Tonic 96 Total phenolic content 363 Toxicological properties 217 Traditional Chinese medicinal 207, 329 Traditional herbal medicine 32 Tribulus terrestris 284 Trichilia emetic 88 Trichosanthes kirilowii 184, 209 Triclisia sacleuxii 82 Trigonella foenum-graecum 120, 136, 140 Tripterospermum lanceolatum 294

439

Subject Index Triticum spp. 287, 289 Triumfetta rhomboidea 284 Tryporyza incertulas 310, 314 Trypterospermum lanceolatum 290 Tulbaghia violacea 284 Turraea nilotica 367, 368

u Ulcerative colitis 219 Ulcers 221, 227 Umbelliferae 385 Umbelliprenin 183, 201 Uncaria rhynchophylla 284 Uvulitis 96

v Vaccinium angustifolium 139 V. ashei reade 284 V. macrocarpon 284 Validation of ethnomedicinal practices 69 Vangueria infausta 365, 368, 369 Vasodilator 192 Vatairea macrocarpa 118 Vermifuge 1, 96 Viburnum opulus 285 Vigna radiate 289, 294 Virola surinamensis 84

Viscum album 137 V. triflorum 285 Vitaceae 86 Vitellaria paradoxa 88 Vitis vinifera 285

w Weinmannia tinctoria 285 Western blotting 140 Whitmania pigra 209 Wrightia tinctoria 285

x Xanthium sibiricum 320 Xanthopappus subacaulis 324 Ximenia amaricana 88 X. caffra 365, 368, 369

Y Yucca schidigera 120

z Zea mays 88, 285, 290, 294 Zingiber officinale 237, 238, 239 Zingiberaceae 85, 86, 238 Ziziphus mucronata 367, 368 Zygophillaceae 85

Recent Progress in Medicinal Plants 29 - Drug Plants III

Medicinal Plants

Advances In Medicinal Plants

Medicinal Plants in Viet Nam

Bioactive medicinal plants

Endangered Medicinal Plants

Conservation of Medicinal Plants

Edible And Medicinal Plants

The constituents of medicinal plants

Bioactive Molecules and Medicinal Plants

Medicinal Plants: Utilisation & Conservation

Horticultural, medicinal and aromatic plants

Medicinal Plants of Myanmar Burma

Medicinal Plants and their Utilization

Traditional Medicinal Plants and Malaria

Bioactive Molecules and Medicinal Plants

Progress in Medicinal Chemistry : Volume 29

Medicinal Plants in Tropical West Africa

Modern Phytomedicine: Turning Medicinal Plants into Drugs

Medicinal Plants of Asia and the Pacific

Modern Phytomedicine - Turning Medicinal Plants into Drugs

The Constituents of Medicinal Plants (2nd Edn)

Medicinal Plants of Asia and Pacific

WHO monographs on selected medicinal plants

Medicinal Plants - Classification, Biosynthesis and Pharmacology

Who Monographs on Selected Medicinal Plants

Medicinal Plants: Utilisation and Conservation, Second Edition

Medicinal Plants of the Mountain West

Indian Medicinal Plants: An Illustrated Dictionary

Medicinal Plants of the World: Chemical Constituents, Traditional and Modern Medicinal Uses (Medicinal Plants of the World (Humana)) (Medicinal Plants of the World (Humana))

Progress in Medicinal Chemistry, Volume 45 (Progress in Medicinal Chemistry) (Progress in Medicinal Chemistry)

Recent Progress in Medicinal Plants 29 - Drug Plants III

Medicinal Plants

Advances In Medicinal Plants

Medicinal Plants in Viet Nam

Bioactive medicinal plants

Endangered Medicinal Plants

Conservation of Medicinal Plants

Edible And Medicinal Plants

The constituents of medicinal plants

Bioactive Molecules and Medicinal Plants

Medicinal Plants: Utilisation & Conservation

Horticultural, medicinal and aromatic plants

Medicinal Plants of Myanmar Burma

Medicinal Plants and their Utilization

Traditional Medicinal Plants and Malaria

Bioactive Molecules and Medicinal Plants

Progress in Medicinal Chemistry : Volume 29

Medicinal Plants in Tropical West Africa

Modern Phytomedicine: Turning Medicinal Plants into Drugs

Medicinal Plants of Asia and the Pacific

Modern Phytomedicine - Turning Medicinal Plants into Drugs

The Constituents of Medicinal Plants (2nd Edn)

Medicinal Plants of Asia and Pacific

WHO monographs on selected medicinal plants

Medicinal Plants - Classification, Biosynthesis and Pharmacology

Who Monographs on Selected Medicinal Plants

Medicinal Plants: Utilisation and Conservation, Second Edition

Medicinal Plants of the Mountain West

Indian Medicinal Plants: An Illustrated Dictionary

Medicinal Plants of the World: Chemical Constituents, Traditional and Modern Medicinal Uses (Medicinal Plants of the World (Humana)) (Medicinal Plants of the World (Humana))

Progress in Medicinal Chemistry, Volume 45 (Progress in Medicinal Chemistry) (Progress in Medicinal Chemistry)

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