Recent Progress in Medicinal Plants 29 - Drug Plants III

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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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(¥r ~ ~ f'lmTT) NATIONAL COMMISSION ON FARMERS GOVERNMENT OF INDIA MINISTRY OF AGRICULTURE (DEPARTMENT OF AGRICULTURE & COOPERATION)

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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ATIII concentration (UlIml)

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

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

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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

RPMP Vol. 29 - Drug Plants III

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

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

Phytotherapeutic Approach to Alcohol Dependence

23

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

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

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RPMP Vol. 29 - Drug Plants III

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

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

Phytotherapeutic Approach to Alcohol Dependence

25

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

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

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

26

RPMP Vol. 29 - Drug Plants III

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

OH 0 OH HO HO

OH A

o

B

c

E

F

~

CH3

1

f?

""I

H,C

CH 3

OH

D

HO

H,C

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

Phytotherapeutic Approach to Alcohol Dependence

27

References 1.

2.

3.

4. 5.

6. 7.

8.

9.

10.

11. 12.

13. 14.

15.

16.

Abenavoli, L., Bardazzi, G., Cracolici, F., Quaranta, C., Santini, G., Graziosi, S., Polero, L., Leggio, L. and Addolorato, G. 2008. Complementary therapies for treating alcoholism. First Annual meeting by Complementary Medicine Research Group ofthe Italian Society for Alcohol Studies - May 5, 2006, Florence, Italy. Fitoterapia 79: 142-147. Addolorato, G., Abenavoli, L., Leggio, L. and Gasbarrini, G. 2005. Alcoholism treatment study group. How many craving? Pharmacological aspects of craving treatment in alcohol addiction: A review. Neuropsychobiology 51: 59-66. Addolorato, G., Leggio, L., Abenavoli, L. and Gasbarrini, G. 2005. Neurobiochemical and clinical aspects of craving in alcohol addiction: A review. Addict. Behav. 30: 1209-1224. Ernst, E. 1996. Complementary medicine: from quackery to science? J. Lab. Clin. Med. 127: 244-245. Overstreet, D.H., Keung, W.M., Rezvani, AH., Massi, M. and Lee, D.Y. 2003. Herbal remedies for alcoholism: Promises and possible pitfalls. Alcohol Clin. Exp. Res. 27: 177-185. Rezvani, AH., Overstreet, D.H., Perfumi, M. and Massi, M. 2003. Plant derivatives in the treatment of alcohol dependency. Pharmacol. Biochem. Behav. 75: 593-606 Bardazzi, G., Merluzzi, J.A, Voller, F., Fontana, A, Abenavoli, L., Leggio, L. and Addolorato, G. 2006. Complementary medicine for alcohol dependence in Italian services: A mail questionnaire. Complement. Ther. Clm. Pract. 12: 216-221. Nahrstedt, A. and Butterweck, V. 1997. Biologically active and other chemical constituents of the herb of Hypericum perforatum L. Pharmacopsychiatry 30(SuppI2): 129-134. Barnes, J., Anderson, L.A and Phillipson, J.D. 2001. St. John's wort (Hypencum perforatum L.): A review of its chemistry, pharmacology and clinical properties. J. Pharm. Pharmacol. 53: 583-600. Kumar, V., Mdzinarishvili, A, Kiewert, C., Abbruscato, T., Bickel, D., van der Schyf, C.J. and Klein, J. 2006. NMDA receptor-antagonistic properties ofhyperforin, a constituent of St. John's Wort. J. Pharmacol. Sci. 102: 47-54. Treiber, K., Singer, A., Henke, B. and Milller, W.E. 2005. Hyperforin activates nonselective cation channels (NSCCs). Br. J. Pharmacol. 145: 75-83. Fisunov, A., Lozovaya, N., Tsintsadze, T., Chatterjee, S., Niildner, M. and Krishtal, 0.2000. Hyperforin modulates gating ofP-type Ca 2+ current in cerebellar Purkinje neurons. Pflugers Arch. Eur. J. Physiol. 440: 427 -434. Dzbay, T.1. 2008. Hypericum perforatum and substance dependence: A review. Phytother. Res. 22: 578-582. Wright, C.W., Gott, M., Grayson, B., Hanna, M., Smith, AG., Sunter, A. and Neill, J.C. 2003. Correlation ofhyperforin content of Hypericum perforatum (St. John's wort) extracts with their effects on alcohol drinking in C57BLl6J mice: A preliminary study. J. Psychopharmacol. 17: 403-408. Chatterjee, S.S., Biber, A. and Weibezahn, C. 2001. Stimulation of glutamate, aspartate and gamma-amino butyric acid release from synaptosomes by hyperforin. Pharmacopsychiatry 34(Suppll): 11-19. Panocka, I., Perfumi, M., Angeletti, S., Ciccocioppo, R. and Massi, M. 2000. Effects of Hypericum perforatum extract on alcohol intake and on behavioral despair: A search for the neurochemical systems involved. Pharmacol. Biochem. Behav. 66: 105-111.

28 17.

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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.

29

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

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

*

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

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RPMP Vol. 29 - Drug Plants III

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

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

Effects of Chinese Herbal Medicines on Bone Loss

33

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

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

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RPMP Vol. 29 - Drug Plants III

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

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

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

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

35

Effects of Chinese Herbal Medicines on Bone Loss

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

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

(n)

Adrenals

1. OVX-Control 2. OVX-EED

(8) (8)

16.2 ± 0.8 26.4 ± 1.5**

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

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

16.4 16.3 17.7 26 .5

± 0.9 ± 0.6 ± 0.8 ± 1.0**

Uterus 20.0 187.6 20.0 20.0 20.0 20.0

± ± ± ± ± ±

0.9 16.5** 0.6 0.9 1.0 14.6*

Spleen 157.1 163.0 157.1 157.1 157.1 157.1

± ± ± ± ± ±

6.3 9.1 7.1 8.1 11.6 8.7

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

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

RPMP Vol. 29 - Drug Plants III

36

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

Groups

(n)

Ca(mg/dL)

Eg

1. OVX-Control

(B)

9.B4±0.11

ND

2. 0VX-EED

(B)

10.6± 0.3**

35.3 ±3.5**

3. 0VX-NYT

(B)

9.91±0.11

2.1B±0.lB

4. 0VX-OKT

(B)

9.94 ± 0.1O

2.0B±0.21

5.0VX-HET

(B)

9.93 ± 0.16

2.00±0.22

6. Normal-Control

(B)

10.4±0.1**

27 .B±7.7**

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

i!

•• ••

8

rn I

< ~

•

~

Ei

e

4

~

rn

o OVX

E,

NYT

- Control

OKT

HET

Normal - Control

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

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

37

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

Whole

150

100

50

0

0

Metaphysis

100

200

OVX -Control

**

E.

*

NYT

OKT

*

HET

Normal -Control

* **

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

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

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

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

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

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

Effects of Chinese Herbal Medicines on Bone Loss

39

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

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

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

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

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

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

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

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

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

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

Production of ET743, Bryostatin, and Taxol

43

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

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

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

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

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

Production of ET743, Bryostatin, and Taxol

45

Bacteria

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

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

o

HO

of;"r0° H .f O''"

~OH

~O'''',,-

00NH ~ ~

o

0

0);-

"-

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

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

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

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

Production of ET743, Bryostatin, and Taxol

47

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

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

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

(2)

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

(3)

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

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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

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50

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

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

40000000

927.4

20000000

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

500550600650700750 SOO 850 900 9501000

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

Production of ET743, Bryostatin, and Taxol

8.0 E + 07

51

j

...

=

/

'; 4.0 E + 07

"il ~

_IL 0.0 E + 00 800 825 850 875 900

..AM.. 925 950 975 1000

m/z

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

3750

... .s

3000 2250

~ 1500 ~

750 0 750

J•.

.tIL 770

790

810

830

mJz

--

850

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

1200 817.816 900

... = 600

.....

~

300 0 700

750

800

850

900

950

1000

mJz

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

RPMP Vol. 29 - Drug Plants III

52

450

.....= ~

789 817

300 7 3

761

150

0 700

750

800

850

900

950

1000

mJz

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

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

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Production of ET743, Bryostatin, and Taxol

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RPMP Vol. 29 - Drug Plants III

54

2500 2000 ...l

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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

Production of ET743, Bryostatin, and Taxol

55

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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

RPMP Vol. 29 - Drug Plants III

1250 1000 789.612

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

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

Production of ET743, Bryostatin, and Taxol

57

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

References 1.

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

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

Production of ET743, Bryostatin, and Taxol 25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

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.

"This page is Intentionally Left Blank"

5 Screening of Natural Products to Drug Discovery

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

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

*

de la Habana, Cuba.

Corresponding autlwr : E-mail: [email protected]

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

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

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).

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

1.

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

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2.

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

3. 4.

5.

6. 7.

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

2.

3. 4.

5.

6.

7. 8.

9.

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

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

65

Screening ofNatural Products to Drug Discovery

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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Non-commercial Plants of Medicinal Purposes

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

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

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

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

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

References Andreo, M.A. 2008. Prospecc;ao quimico-farmaco16gica em plantas superiores: Atividade de Mouriri SSP sobre 0 sistema gastrointestinal. Tese de Doutoramento IQ UNESP l04p. Andreo, M.A., Ballesteros, KV., Hiruma-Lima, C.A., Machado da Rocha, L.R., Souza Brito, A.R., Vilegas, W., 2006. Effect of Mouriri pusa extracts on experimentally induced gastric lesions in rodents: Role of endogenous sulfhydryls compounds and nitric oxide in gastroprotection. J. Ethnopharmacol. 107(3): 431-441. Avila, J.R., de La Lastra, C.A., Martin, M.J., Motilva, V., Luque, I., Delgado, D., Esteban, J., Herrerias, J., 1996. Role of endogenous sulphydryls and neutrophil infiltration in the pathogenesis of gastric mucosal injury induced by piroxicam in rats. Inflamm. Res. 45: 83-88. Azuma, K, Ippoushi, K, Nakayama, M., Ito, H., Higashio, H. and Terao, J. 2000. Absorption of chlorogenic acid and caffeic acid in rats after oral administration. J. Agriculture Food Chemistry 48: 5496-5500. Bafna, P.A., Balaraman, R. 2004.Anti-ulcer and antioxidant activity ofDHC-l, a herbal formulation. J. Ethnopharmacol. 90: 123-127. Banerjee, T., Van Der Vilet, A. and Ziboh, A. 2002. Downregulation ofCOX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostaglandins Leukot. Essent.Fatty Acids 66: 485-492. Calvo, T.R., Lima, Z.P., Silva, J.S., Ballesteros, KV.R., Pellizzon,C.H., Hiruma-Lima, CA., Tamashiro, J., Brito, A.R.M.S, Takahira, R.K and Vilegas, W. 2007. Constituents and antiulcer effect of Alchornea glandulosa: activation of cell proliferation in gastric mucosa during the healing process. Bioi. Pharm. Bull. 30(3): 451-459. Cheng, H., Xia, B., Guo, Q., Zhang, L., Wang, F., Jiang, L., Wang, Z., Zhang, Y. and Li, C. 2007. Sinomenine attenuates 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice. Int. Immunopharmacol. 7: 604-611. Chow, J.y', Ma, L. and Cho, C.H.1998.Effect of cigarette smoke on ethanol-induced gastric mucosal lesions: the role of nitric oxide and neutrophils. Eur. J. Pharmacol. 342: 253-260. Conegero, L.S., Ide, R.M., Nazari, A.S., Sarragiotto, M.H., Dias Filho, B.P., Nakamura, C. V. 2003. Constituintes quimicos de Alchornea glandulosa (Euphorbiaceae), Quim. Nova. 26(6): 825-827. Correa, M.P. 1984. Dicionario das plantas uteis do Brasil e das ex6ticas cultivadas. Editora Imprensa Nacional, Rio de Janeiro. Deshpande, S.S., Shah, G.B. and Parmar, N.S. 2003. Antiulcer activity of Tephrosia purpurea in rats, Indian Journal Pharmacology 35: 168-172. Eisig, J. N. and Laudanna, A.A. 2007.(Jlcera peptica. Programa de Saude da Familia. Disponivel em: Acess 25jan. Esplugues, J.V., Barrachina, M.D., Calatayud, S., Pique, J.M. and Whittle, B.J. 1993. Nitric oxide mediates the inhibition by interleukin-l beta of pentagastrin-stimulated rat gastric acid secretion. Br. J. Pharmacol. 108: 9-10. Fan, T.Y., Feng, Q.Q., Jia, C.R., Fan, Q., Li, C.A. and Bai, X.L. 2005.Protective effect of Weikang decoction and partial ingredients on model rat with gastric mucosa ulcer. World J. Gastroenterol. 11(8): 1204-1209. Farnsworth, N.R., Akerele, 0., Bingel, A.S., Soejarto, D.D. and Guo, Z. 1985.Medicinal plants in therapy. Bull WHO 63: 965-981.

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

Non-commercial Plants of Medicinal Purposes

235

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

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

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

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

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

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

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

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

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

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

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

100 mm VAS for assessing the severity of pain;

• • •

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

Data analysis

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

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

Ginger extract groupN=40

Ibuprofen groupN=40

Pvalue

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

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

>0.05 >0.05 > 0.05 >0.05

1.25 ± 0.06 1.62 ± 0.07

1.15 ± 0.05 1.45 ± 0.07

>0.05 >0.05

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

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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

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

Baseline (Before treatment)

Mter one month of treatment

P value

P value

N= 40

N =40

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

Mter two months of treatment 3.7 ± 1.0 0.32 ± 0.08

< 0.0001-

71.2±2.4 3.0 ± 0.20

28 ± 3.4 0.97 ± 0.11

< 0.0001 * < 0.0001 '"

1.15 ± 0.05

1.10 ± 0.04

>0.05

1 ± 0.04

>0.05

1.45 ± 0.07

1.40 ± 0.07

>0.05

1.25 ± 0.06

> 0.05

= 0.0002-

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

-

~

100

E

80

E

.t-

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

'iii

c: Q)

] c:

60

"§

40

'" ~.S

20

'@ p...

'2

..c:

il'c:o

'"

..c: Q

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

Table 2. (Contd.) Chemical name

Class of the chemical compound

Botanical name (Source)

Common name

Family

(-)-Epigallocatechin

Flavonoid

Epigallocatechin gallate (-)-Epigallocatechin gallate Ethyl caffeate Evocarpine Fagopyrum tripeptide Fangchinolium hydroxide Fenfangjine F, H and I Ficus oligopeptide FLP-1, -2 & -3 Ficus peptide FLP-1, -2 & -3 FVNPQAGS Gallic acid Gallocatechin, (+ 1 Gallocatechin, epi (-l Gallocatechin, epi, 3-0-gallate (- 1 Gallocatechin -3-0gallate (-l, epi (-)

Flavonoid

Camellia sinensis Camellia sinensis Camellia sinensis

Green tea Black tea Green tea

Theaceae Theaceae Theaceae

Persson et al., 2006 Persson et al., 2006 Persson et al., 2007

Phenolic Alkaloid Peptide

Camellia sinensis Camellia sinensis Rabdosia coetsa Evodia rutaecarpa Fagopyrum sp.

Green tea Black tea Duo mao bian zhong Evodia, Wu-Zhu-Yu Buckwheat

Theaceae Theaceae Lamiaceae Rutaceae Polygonaceae

Persson et al., 2006 Persson et al., 2006 Li et al., 2008 Lee et al., 1998 Koyama et al., 1993

Alkaloid

Stephania tetrandra

Han fang ji

Menispermaceae

Ogino et al., 1986

Alkaloid

Stephania tetrandra

Han fangji

Menispermaceae

Ogino et al., 1998

Peptide

Ficus carica

Common fig

Moraceae

Peptide

Ficus carica

Common fig

Moraceae

Flavonoid

Reference

Peptide Benzenoid Flavonoid Flavonoid Flavonoid

Helianthus annuus Phyllanthus niruri Camellia sinensis Camellia sinensis Camellia sinensis

Sunflower Stone breaker Korean green tea Korean green tea Korean green tea

Asteraceae Euphorbaceae Theaceae Theaceae Theaceae

Maruyama et al., 1990 Maruyama et al., 1989 Megias et al., 2004 Ueno et al., 1988 Cho et al., 1993 Cho et al., 1993 Uchida et al., 1987

Flavonoid

Camellia sinensis

Korean green tea

Theaceae

Cho et al., 1993

oa oa

~

~

~ t'" ~

I ..,t:l

~

~ '"

;:l ....

~

~

Table 2. (Contd.) Chemical name Isovitexin

Class of the chemical compound

Botanical name (Source)

Flavonoid

Musanga cecropioides Cecropia pachystachya Sesamum indicum Triticum spp. Brassica napus

African Corkwood Ambay pumpwood Sesame wheat Rapeseed Greens

Moraceae Cecropiaceae Pedaliaceae Poaceae Brassicaceae

Common name

Family

Reference

§: ,.,. 0·

;:l

IVY

Peptide

IY Kaempferol

Peptide Flavonoid

Kaempferol-3-al phaarabinopyranoside Kaempferol-3-0alpha-ara binopyranoside Kaempferol-3-0betagalactopyranoside Kaempferol-3O-galloyl-glucose KDYRL KLPAGTLF Lanosten (20-R) Lanosten (20-S) Leucosceptoside

Flavonoid

Sedum sarmentosum

Stringy Stonecrop

Crassulaceae

Dubois et al., 2001 Dubois et al., 2001 Hong et al., 2008 Matsui et al., 1999 Marczak et al., 2003 Olszanecki et al., 2008 Oh et al., 2004

Flavonoid

Ailanthus excelsa

Ailanthus, Ardu

Simaroubaceae

Loizzo et al., 2007

Ligstroside aglycones Liriodendrin

;:::-

~ ~

~

0· ctl" ;:l

'" S· ~ ;:l =l

;::I

0;.

'"

~

Table 2. (Contd.) Chemical name Octadeca -10trans-12-cis-15cis-trienoic acid, 9-hydroxy Octadeca-9trans-ll-transdienoic acid, 13-hydroxy Oenothein B Oleacein Oleuropein 6"-0-malonyldaidzin 6" -0-malony1genistin 3'''-0methylcrenatoside Peimisine Penta-O-g alloy I-beta -D-gI ucose Procyanidins (dimer and hexamerl Proanthocyanidin B3 Pro cyanidin B-1 Pro cyanidin B-2, 3,3'-di-0-gallate Pro cyanidin B-3

Class of the chemical compound

Botanical name (Source)

Lipid

Lycium chinensis

Common name

Family

Reference

S' ;::,§:

.....

c· .s;, ;:s

Wolfberry, Goji berry

Solanaceae

Morota et al., 1987

>

Jg

c· .....

Lipid

Fnttllana vertic illata

Fritillary

Liliaceae

Niitsu et al., 1987

'";:s OJ>

S·

g ;:s 9%) during the four

RPMP Vol. 29 - Drug Plants III

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 (