PHYTOCHEMICAL AND ANTIDIABETIC ACTIVITY INVESTIGATIONS OF TETRACERA

INDICA MERR.

BY

BASHAR BELLO S. DOGARAI

A thesis submitted in fulfillment of the requirement for the Degree of Master of Pharmaceutical Chemistry

Kulliyah of Pharmacy

International Islamic University Malaysia

MAY 2011

ii

ABSTRACT

The work embodied in the present thesis relates to the studies on the biological activity and chemical constituents of the leaves of Tetracera indica Merr. (Dilleniaceae) (mempelas paya) and is presented in two parts. Part A involves studies on the antidiabetic activity of aqueous (AQ) and methanol (MeOH) extracts of the leaves of T. indica Merr. in vivo and isolation of the active principle (wogonin). Part B describes the isolation and characterization of chemical constituents from MeOH extracts of the leaves of T. indica Merr. Part A comprises of investigation of antidiabetic activity of the aqueous (AQ) and methanol (MeOH) extracts of the leaves of T. indica along with the active principle isolated from MeOH extract in vivo in alloxan induced diabetic and normal male albino rats (Sprague Dawley strain (SD)). Two doses of each extract 250 and 500 mg/kg b.w. and 0.5 and 1mg/kg b.w in the case of active principle (wogonin) were evaluated. Both AQ and MeOH extracts exhibited significant anti-hyperglycemic activity in alloxan induced diabetic rats, however in normal rats, no hypoglycemic activity was observed, when compared with both +ve and –ve controlled groups except that of AQ extract which exhibited hypoglycemic activity in normal rats at 250 mg/kg b.w. The antidiabetic activity was also found to be comparable to that of the effect produced by glibenclamide (GLBC) (0.25 mg/kg b.w.). The LD50 of both AQ and MeOH extracts was found to be more than 5000 mg/kg body weight and no lethal toxicity was observed within this range. Wogonin was isolated as an active principle from MeOH extract which revealed anti-hyperglycemic activity in vivo comparable to GLBC. Part B deals with the isolation and structure elucidation of the active principle (wogonin) along with other chemical constituents from MeOH extract of the leaves of T. indica Merr. MeOH extract upon repeated silica gel and sephadex LH-20 column chromatography afforded two terpenoids and six flavonoids viz., mixture of glycosides of β-sitosterol and stigmasterol, betulinic acid; 5,7-dihydroxy-8-methoxyflavone (wogonin); 5,7,8-trihydroxyflavone (norwogonin); 4’,5,7-trihydroxy-8-methoxyflavone (isoscutellarein methyl ether); 4’3,5,7,-tetrahydroxyflavone (kaempferol); 3’,4’3,5,7-pentahydroxyflavone (quercetin) and 5-hydroxy-7-methoxyflavone (techtochrysin). Occurrence of norwogonin, isoscutellarein methyl ether, kaempferol, quercetin, techtochrysin and isomeric mixture of β-sitosterol and stigmasterol is being reported for the first time from the leaves of T. indica Merr. Keywords: Tetracera indica Merr., Dillineceae, Antidiabetic activity in vivo, active principle, other phytoconstituents.

iii

البحث خلاصة

القسم أ يتضمن تحري )القسم أ والقسم ب(العمل الحالي تم إنجازه على قسمين المضادة للسكري للخلاصتين المائية والميتانولية لأوراق ؟؟؟ إضافة إلى الفعالية

المكون الفعال المعزول من الخلاصة الميتانولية في الجسم الحي في ذآور الجرذان Sprague Dawley(البيضاء المصابة بالداء السكري المحرض بالالوآسان

strain (SD) .( تم تقييم جرعتين من آل خلاصة)250 and 500 mg/kg b.w. (آلتا الخلاصتين المائية والميتانولية . في حالة المكون الفعال) and 1mg 0.5(و

أظهرت فعالية هامة مضادة لارتفاع سكر الدم في الجرذان المصابة بالداء السكري المحرض بالألوآسان، لكن في الجرذان الطبيعية، لم يلاحظ أية فعالية خافضة لسكر

باستثناء أنه بالنسبة ve and –ve+لمقارنة مع مجموعة المقارنة الدم، وذلك عند اللخلاصة المائية التي أظهرت فعالية خافضة لسكر الدم في الجرذان الطبيعية بجرعة

250 mg/kg b.w. . لقد وُجد أن الفعالية المضادة للسكري يمكن مقارنتها بتلكصتين المائية للخلا LD50الـ). .GLBC)0.25 mg/kg b.w المُحدثة بالـ

من وزن الجسم ولم يلاحظ أية سُمّية mg/kg 5000والميتانولية وُجدت أآثر من آمكون فعال من الخلاصة wogonin لقد تم عزل الـ. قاتلة ضمن هذا المجال

.GLBCالميتانولية التي أبدت فعالية مضادة لارتفاع سكر الدم يمكن مقارنتها بالـلمكون الفعال إضافة إلى المكونات الكيميائية الجزء ب تم فيه فصل وتوصيف ا

من خلال تكرار آروماتوغرافيا . الأخرى من الخلاصة الميتانولية لأوراق ؟؟؟الخالصة الميتانولية مرآبين أعطتsephadex LH-20 العمود على السيليكا جل والـ

-mixture of glycosides of β من التربينوئيدات وستة فلافونوئيدات وهي؛ sitosterol and stigmasterol; Betulinic acid; 5,7-dihydroxy-8-methoxyflavone (wogonin); 5,7,8-trihydroxyflavone (norwogonin); 4’,5,7-trihydroxy-8-methoxyflavone (isoscutellarein methyl ether); 4’,3,5,7-tetrahydroxyflavone (kaempferol); 3’,4’,3,5,7-pentahydroxyflavone (quercetin) and

5-hydroxy-7-methoxyflavone (techtochrysin).

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APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion; it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Pharmaceutical Chemistry.

…………………………………... Supervisor

I certify that I have read this study and that in my opinion; it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Pharmaceutical Chemistry.

…………..………………………. Examiner

This thesis was submitted to the Department of Pharmaceutical Chemistry and is accepted as a fulfilment of the requirement for the degree of Master of Pharmaceutical Chemistry.

…………..………………………. Head of Department Pharmaceutical Chemistry

This thesis was submitted to the Kulliyyah of Pharmacy and is accepted as a fulfilment of the requirement for the degree of Master of Pharmaceutical Chemistry.

…………..………………………. Dean, Kulliyyah of Pharmacy

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DECLARATION

I here by declare that this thesis is the result of my own investigations, except where

otherwise stated. I also declare it has not been previously or concurrently submitted as

a whole for any other degrees at IIM or other institutions.

Bashar Bello S. Dogarai.

Signature:……………………………… Date……………………………

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INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION

OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright © 2011 by International Islamic University Malaysia. All rights reserved.

PHYTOCHEMICAL AND ANTIDIABETIC ACTIVITY INVESTIGATIONS OF TETRACERA INDICA MERR.

I hereby affirm that The International Islamic University Malaysia (IIUM) hold all rights in

the copyright of this work and henceforth any reproduction or use in any form or by means

whatsoever is prohibited without the written consent of IIUM. No part of this unpublished

research may be reproduced, stored in a retrieval system, or transmitted, in any form or by

means, electronic, mechanical, photocopying, recording or otherwise without prior written

permission of the copyright holder.

Affirmed by Bashar Bello S. Dogarai

.................................... .....................................

Signature Date

vii

I dedicate this work to Almighty Allah, the sustainer, Who gave me patient to go along with this research work, my beloved grandmother (Aishatu Shehu Adili S.Dogarai

(Kabo)) for her loving and caring throughout my childhood. May Allah forgive her shortcomings and her soul rest in peace, ameen.

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ACKNOWLEDGEMENTS

All praises are due to Allah for his guidance and bounteous mercy upon us to see this thesis writing completed with a great success. May He (S.W.A) give us the maximum benefit attached to this work, ameen. I would like to express my sincere gratitude to my supervisor (Dr. Qamar Uddin Ahmed) for his patience, motivation; enthusiasm and persistent support in my M.Sc. research. May Allah grant him and his family Jannatul firdaus, ameen. I also want to extend my appreciation to the staff of Kulliyah of Pharmacy and most especially to the Department of Pharmaceutical Chemistry for their assistance and being patient with me through out the period of my research.

My sincere gratitude goes to the Prof. Dr. Jalifa Latif, Prof. Dr. Ambar, Br. Zahir and Br. Alifi, from the Department of Chemistry and Food Technology, Universty Kebangsaan Malaysia for their help in providing us with necessary spectra. May Allah S.W.T reward them, ameen.

I am grateful to the traditional practitioners and field supervisors who helped me selfishlessly to collect the plant material for my research. My warm thank goes to the botanists of FRIM (Kuala Lumpur, Malaysia) for botanical determination of the plant. My profound thanks and appreciations go to my parents, and the rest of Ahmad Magayaki’s and Yari Husaini’s families for their prayers. They provided me with every opportunity and encouragement I needed to succeed in life. May Allah bless them immensely.

May I seize this opportunity to extend my zillion thanks to our father Alhaji Umaru Ibrahim Gidan Kanawa for his tireless efforts and encouragements for us to excel and realize our dream. May Allah reward him with jannatul firdaus, ameen. My sincere gratitude also goes to my Mentors and guardians, Assoc. Prof. Dr. Bala Yauri Muhammad and Assistant Prof. Dr. Abubakar Abdullahi from Kulliyyah of Pharmacy for their motivational guidance and constructive advice to realize my set objectives.

I also want to extend my gratitude to my friends and colleagues for whom I

have great regard such as Abdulrashid Umar, Abubakar Malami, Na’em Balogun, Abubakar Sule, Nafiu Abdurazak Bodemi, Dr. Aminu Ishaka, Dr. Hassan Yankuzo, Abdulateef Raji, Moshood Yusuf, Mansur Asabur, Akbar John, Dr. Osama Jodan, Dr. Haitham Qaribullah, Dr. Marwan, Suhaib Ibrahim, Hassan Sheikh, Dr. Muhammad Ludfi, Br. AbdulMunim Syria, Br. Harith Akram, Br. Muhammad Fahmi, Br. Hafiz Zarul Bin Abdulhalim, Br. Ashraf, Br. Habibi Baharudeen, Br. Hafiz Huzazi, Br. Abdurrahman Abdu, Br. Muhammad Syria, Br. Aiman, Br. Mulham, Sis. Aiza, Sis. Hayati, Sis. Laina, Sis. Zafira Liyana, and to all those who have contributed directly or indirectly for the completion of this study.

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TABLE OF CONTENTS

Abstract ................................................................................................................... ii Arabic Abstract ........................................................................................................ iii Approval page .......................................................................................................... iv Declaration ............................................................................................................... v Declaration of copy right ......................................................................................... vi Dedication ................................................................................................................ vii Acknowledgements .................................................................................................. viii Table of content ...................................................................................................... ix List of Tables ........................................................................................................... xii List of Figures .......................................................................................................... xiii List of Abbreviation ................................................................................................ xv CHAPTER ONE ................................................................................................... 1

1.0 Introduction .......................................................................................... 1 1.1 Medicinal Plant .................................................................................... 1 1.2 Diabetes ................................................................................................ 3 1.3 Tetracera indica, Merr ......................................................................... 5 1.4 Objectives of the research .................................................................... 6 1.5 Specific objectives ............................................................................... 6 1.6 Hypothesis of the research ................................................................... 6

CHAPTER TWO: LITERATURE REVIEW ..................................................... 7

2.1 Medicinal Plant .................................................................................... 7 2.2 Drug discovery from medicinal plants ................................................. 9 2.3 Synthesis and role of plant secondary metabolite ................................ 11 2.4 Phenolic compounds ............................................................................ 13

2.4.1 Flavonoids ............................................................................... 15 2.4.2 Tannins .................................................................................... 18 2.4.3 Terpenes .................................................................................. 20

2.5 Diabetes mellitus .................................................................................. 21 2.5.1 Type-1 diabetes ....................................................................... 21 2.5.2 Type-2 diabetes ....................................................................... 22 2.5.3 Gestational diabetes mellitus .................................................. 23 2.5.4 Other types of diabetes ............................................................ 23 2.5.5 Diabetes diagnosis ................................................................... 24 2.5.6 Management of diabetes ........................................................ 24

2.6 Dilleniaceae background ..................................................................... 27 2.6.1 Tetracera ................................................................................... 27 2.6.2 Tetracera alfolia wild ................................................................ 27 2.6.3 Tetracera volubilis .................................................................... 29 2.6.4 Tetracera asperula .................................................................... 29

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2.6.5 Tetracera costata mart eitchler ................................................. 29 2.6.6 Tetracera tigare ...................................................................... 30 2.6.7 Tetracera macrophylla vall ...................................................... 30 2.6.8 Tetracera potaoria ................................................................... 30 2.6.9 Tetracera scanderns ................................................................ 31 2.6.10 Tetracera indica ...................................................................... 32

2.6.10.1 Scientific classification ..................................................... 32 2.6.10.2 Traditional use .................................................................. 33 2.6.10.3 Phytochemical examination ............................................. 34

CHAPTER THREE: METHODOLOGY ......................................................... 36

3.1 General experimental procedures ....................................................... 36 3.2 Chemical ........................................................................................... 36 3.3 Collection of plant material ................................................................ 37

3.3.1 Preparation of polar extracts .................................................... 37 3.3.2 Aqueous extract (AQ) of the leaves of T.indica Merr. ........... 37 3.3.3 Methanol extract (MeOH) of the leaves of T.indica Merr ....... 38

3.4 Isolation of the active principle compound along with other biological active compounds from the methanol extract .............................................. 38 3.5 Experimental animals ......................................................................... 47 3.6 Acute toxicity evaluation of AQ and MeOTH extracts of T.indica Merr ...................................................................................................................... 48 3.7 Induction of diabetes .......................................................................... 48 3.8 Experimental design .......................................................................... 49

CHAPTER FOUR .................................................................................................. 51

4.1 Preparation of extract ............................................................................ 51 4.2 Isolation and purification of Methanol extract of the leaves of T.indica merr .............................................................................................................. 51 4.3 Statistic analysis .................................................................................... 55 4.4 Acute toxicity evaluation of AQ and MeOTH extracts of T.indica Merr ...................................................................................................................... 55 4.5 Induction of diabetes ............................................................................. 55 4.6 Antidiabetic activity of Aqueous extract (AQ) ..................................... 56 4.7 Antidiabetic activity of Methanol extract (MeOH) ............................... 59 4.8 Antidiabetic activity of active principle ................................................ 62

CHAPTER FIVE ................................................................................................... 66

5.1 Antidiabetic activity of AQ and MeOH extracts of the leaves of T. indica Merr. in vivo .................................................................................. 66 5.2 Isolation and Structure elucidation (Discussion) ................................. 68

CONCLUSION ..................................................................................................... 90

SUGGESTION ............................................................................................. 91

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BIBLIOGHRAPHY................................................................................................93 CONFERENCES....................................................................................................103 PUBLICATIONS....................................................................................................103 AWARD/HONOURS..............................................................................................103 APPENDICES ........................................................................................................ 104

Appendix A1: BQ2 1H-NMR spectrum ...................................................... 104 Appendix A2: BQ-2 1H-NMR spectrum (Expanded 1) ............................... 105 Appendix A3: BQ-2 1H-NMR spectrum (Expanded 2) .............................. 106 Appendix A4: BQ-2 1H-NMR spectrum (Expanded 3) ............................... 107 Appendix A5: BQ-2 13C-NMR spectrum .................................................... 108 Appendix A6: BQ-2 13C-NMR spectrum (Expanded 1) .............................. 109 Appendix A7: BQ-2 13C-NMR spectrum (Expanded 2) .............................. 110 Appendix A8: BQ-2 Mass spectrum ........................................................... 111 Appendix A9: BQ-2 FT-IR spectrum ......................................................... 112 Appendix B1: BQ-3 1H-NMR spectrum ..................................................... 113 Appendix B2: BQ-3 1H-NMR spentrum (Expanded) .................................. 114 Appendix B3: BQ-3 13C-NMR spentrum .................................................... 115 Appendix B4: BQ-3 Mass spectrum ............................................................ 116 Appendix B5: BQ-3 FT-IR spectrum ........................................................... 117 Appendix C1: BQ-5 1H-NMR spectrum ...................................................... 118 Appendix C2: BQ-5 13C-NMR spectrum ..................................................... 119 Appendix C3: BQ5 Mass spectrum…………………………………… ...... 120 Appendix C4: BQ5 FT-IR spectrum ............................................................ 121 Appendix D1: BQ6 1-HNMR spectrum ....................................................... 122 Appendix D2: BQ-6 13C-NMR spectrum ..................................................... 123 Appendix D3: BQ-6 Mass spectrum ............................................................ 124 Appendix D4: BQ-6 FT-IR spectrum .......................................................... 125 Appendix E1: BQ-7 1H-NMR spectrum ...................................................... 126 Appendix E2: BQ-7 13C-NMR spectrum ..................................................... 127 Appendix E3: BQ-7 Mass spectrum ............................................................ 128 Appendix E4: BQ-7 FT-IR spectrum ........................................................... 129 Appendix F1: BQ-8 1H-NMR spectrum ...................................................... 130 Appendix F2: BQ-8 13C spectrum ................................................................ 131 Appendix F3: BQ-8 Mass spectrum ............................................................. 132 Appendix F4: BQ-8 FT-IR spectrum ........................................................... 133 Appendix G1: BQ-9 1-HNMR spectrum ...................................................... 134 Appendix G2: BQ-9 13C-NMR spectrum ..................................................... 135 Appendix G3: BQ-9 Mass spectrum ............................................................ 136 Appendix G4: BQ-9 FT-IR spectrum .......................................................... 137 Appendix H1: BQ-10 1-HNMR spectrum .................................................... 138 Appendix H2: BQ-10 13C-NMR spectrum ................................................... 139 Appendix H3: BQ-10 Mass spectrum .......................................................... 140 Appendix H4: BQ-10 FT-IR spectrum ........................................................ 141

 

xii

    

LIST OF TABLES

Table 2.1 Page No. 2.1 Some major plant drugs for which no synthetic one is Available 7 4.1 Effect of different doses of AQ extract of the leaves of T. indica on blood glucose levels (mmol/L) in normal and diabetic rats at different intervals (h). 57 4.2 Effect of different doses of MeOH extract of the leaves of T. indica on blood glucose levels (mmol/L) in normal and diabetic rats at different intervals (h). 60 4.3 Effect of different doses of active principle (wogonin) obtained from MeOH extract of the leaves of T. indica on blood glucose levels (mmol/L) in normal and diabetic rats different interval 63 5.1 1H and 13 C –NMR of Betulinic acid, (3β)-3-Hydroxy-lup-(20) (29)en-28-oic acid (BQ-02) 71

5.2 1H and 13 C –NMR of Wogonin (5, 7-dihydroxy-8- methoxyflavone); BQ-5 75

5.3 1H and 13 C –NMR of Norwogonin (5, 7, 8-trihydroxy flavone); BQ-6 78 5.4 1H and 13 C –NMR of Isoscutellarein -8-methylether (5, 7- dihydroxy-8-methoxy flavone); BQ- 81 5.5 1H and 13 C –NMR of Kaempferol (3, 5, 7, 4’-tetrahydroxy flavone); BQ-8 84 5.6 1H and 13 C –NMR of Quercetin (3, 5, 7, 3’, 4’-pentahydroxy- flavone); BQ-9 86 5.7 1H and 13 C –NMR of Techtochrysin (5-hydroxy-7-methoxy flavone); BQ-10 89

xiii

LIST OF FIGURES Figure No. Page No. 2.1 Structures of Major plant drugs for which no synthetic one is Currently available 9 2.2 Generic structure of major classes of flavonoid 16 2.3 Structures of some tannin compounds 19 2.4 Example of some terpenes strucutures 20 2.5 Picture of Tetracera indica (plant) 35 3.1 Systematic diagrams of Methanolic extract extraction and purification Processes 43 4.1 Effect of different doses of AQ extract of the leaves of T.indica Merr. On blood glucose levels (mmol/L) in normal and diabetic rats at different interval 58 4.2 Graph percentage falls of blood glucose of different doses of AQ extract of the leaves of T.indica in diabetic rats in mmol/L 59 4.3 Effect of different doses of MeOH extract of the leaves T.indica Merr. on blood glucose levels (mmol/L) in normal and diabetic rats in mmol/L 61 4.4 Graph percentage falls of blood glucose of different doses of MeOH

extract of the leaves of T. indica in diabetic rats in mmol/L 62 4.5 Effect of different doses of active principle (wogonin) from MeOH

extract of the leaves T.indica Merr. on blood glucose levels (mmol/L) in normal and diabetic rats in mmol/L 64

4.6 Graph percentage falls of blood glucose of different doses of active

principle (wogonin) in diabetic rats in mmol/L 65

xiv

5.1 Structure of betulinic acid (3β)-3-Hydroxy-lup-(20) (29) en-28-0ic acid (BQ-.2) 72 5.2 Structure of Wogonin (5, 7-dihydroxy flavones-8-methoxyflavone); BQ-5 76 5.3 Structure of Norwogonin (5, 7, 8-trihydroxyflavone); BQ-6 79 5.4 Structure of isoscutellarein -8-methyl ether (4’, 5, 7-trihydroxy-8-

methoxyflavone); BQ-7 82 5.5 Structure of Kaempferol (4’, 3, 5, 7-tetrahydroxyflavone); BQ-8 84 5.6 Structure of Quercetin (3’, 4’, 3, 5, 7-pentahydroxyflavone); BQ-9 87 5.7 Structure of Techtochrysin (5-hydroxy-7-methoxyflavone); BQ-10 89

xv

LIST OF ABBREVIATIONS

Ac Acetone AQ Aqueous b.w Body weight C Carbon CC Column Chromatography C6D6 Hexadeuteriobenzene CDCl3 Deuteriochloroform COSY Correlation Spectroscopy CHCl3 Chloroform d oublet dd Doublet of the doublet dL Deci litre DCM Dichlomethane EtOH Ethanol EtOAc Ethyl acetate FeCl3 Ferric chloride Fig. Figure GLBC Glibenclamide GC Gas Chromatography HPLC High Performance Liquid

Chromatography HSC Heteronuclear Shift Correlation Hz Hertz H2SO4 Glacial sulphuric acid IDDM Insulin dependent diabetic mellitus IR Infrared I2 Iodine J Coupling constant Kg Kilogram LD50 Lethal dose (50% of the total no.

animal dies) LADA Latent Autoimmune Diabetes in Adults m/z Mass-to-charge ratio MeOH Methanol MS Mass Spectrometry m Multiplet

xvi

mg Milligram MODY Maturity –Onset Diabetes of the

Young NDM Neonatal Diabetic Mellitus NMR Nuclear Magnetic Resonance NOESY Nuclear Overhauser Enhancement

Spectroscopy NIDDM Non-insulin dependent diabetic

mellitus OGTT Oral glucose tolerance test prep. Preparative

ppm Parts per million PTLC Preparative Thin Layer

Chromatography P Petrol P.B.F. Pyridine: Benzene: Formic acid s Singlet t Triplet T.E.F. Toluene: Ethyl formate: Formic acid TLC Thin Layer Chromatography UV Ultraviolet 1H-NMR Proton Nuclear Magnetic Resonance 2D Two Dimensional 13C-NMR 13Carbon Nuclear Magnetic Resonance

 

1

CHAPTER ONE

INTRODUCTION

1.1 MEDICINAL PLANTS

Interest in medicinal plants as a re-emerging health aid has been fuelled by the rising

costs of prescription drugs in the maintenance of personal health and well-being, and

the bioprospecting of new plant-derived drugs. Several issues as well as a range of

interests and activities in a number of countries are dealt with. Based on current

research and financial investments, medicinal plants will, seemingly, continue to play

an important role as a health aid. Medicinal plants, since times immemorial, have been

used virtually in all cultures as a source of medicine. The widespread use of herbal

remedies and healthcare preparations, as those described in ancient texts such as the

Vedas and the Bible, and obtained from commonly used traditional herbs and

medicinal plants, has been traced to the occurrence of natural products with medicinal

properties. The use of traditional medicine and medicinal plants in most developing

countries, as a normative basis for the maintenance of good health, has been widely

observed (UNESCO, 1996).

Furthermore, an increasing reliance on the use of medicinal plants in the

industrialized societies has been traced to the extraction and development of several

drugs and chemotherapeutics from these plants as well as from traditionally used rural

herbal remedies (UNESCO, 1998). Moreover, in these societies, herbal remedies have

become more popular in the treatment of minor ailments, and also on account of the

increasing costs of personal health maintenance. Indeed, the market and public

2

demand has been so great that there is a great risk that many medicinal plants today,

face either extinction or loss of genetic diversity (Lucy and Edger, 1998).

Malaysia has been classified as one of the 12 megadiversity countries of the

world. Altogether, these twelve megadiversity countries comprise at least 60% of the

world’s known species (Latiff, 2005). Plant has been used for ages for food, shelter,

treat human disorders and disease. Malaysia has about 15,000 species of flowering

plants of which about 10% are said to be medicinal (Faridah et. al., 2001). Medicinal

plant is one of the valuable non-timbers in the forest. Traditional medicine is an

important part in Malaysian culture and is practiced by ancestors long before the

introduction of modern medicine.

The complete reports on the Malay traditional medicinal plants were reported

by (Burkill, 1935), provided the first comprehensive knowledge about the medicinal

plants of Peninsular Malaysia, and that became the starting point for the

phytochemists and ethnobotanist to do some studies and research relating to the

medicinal plants. All of these works add a comprehensive knowledge to the account of

the Malaysian medicinal plants. In conjunction with phytochemical screenings and

chemical studies by the chemists, the above compilation attempted to introduce the

diversity of medicinal plants to the Malaysian public and this serves as a key for

future bioprospecting in Malaysia.

The Malaysian medicinal plants cover 135 families and more than 1000

species of medicinal plants have been discovered. Among these, only about 103

families and 768 dicotyledonous species documented to be commonly used in

traditional practices, and hence would definitely impede the country’s efforts to better

and judiciously utilize them for the benefits of the populations’ (Latiff, 2007).

According to Tuan’ Marina (2007), there was high species richness, abundance and

3

economic value of medicinal plants at Tranum Forest Reserved, Raub, and Pahang.

This forest categorized as hill forest. The most popular and high demanding medicinal

plants within this area are Phyllagathis rotundifolia, Labisia pumila, Mapania

cuspidata, Homalomena sagittifolia, Peliosanthes teta and Tacca integrifolia. It

proved that, hill forest also have abundance of medicinal plants to look at (Ali, 2010).

1.2 DIABETES

Diabetes mellitus is no longer an epidemic that can be ignored. It is a complex

disorder characterized by a relative or absolute insufficient secretion independent

diabetes mellitus (IDDM) or concomitance resistance of the metabolic action of

insulin on target tissue non insulin diabetes mellitus (NIDDM) (Alberto and Swapnil,

2001). The prevalence of diabetes for all age-groups worldwide has been estimated to

be 2.8% in 2000 and reached up to 4.4% in 2030 (Sarah et al., 2004). The

International Diabetes Federation (IDF) has estimated that 246 million people

worldwide suffer from diabetes and this figure is projected to increase to 380 million

by the year of 2025 (International Diabetes Federation, Diabetes Atlas, Third Edition,

2006).

The prevalence of diabetes in Asian populations has increased rapidly in recent

decades. In 2007, more than 110 million individuals in Asia were living with

diabetes, with a disproportionate burden among the young and middle aged. Similarly,

rates of overweight and obesity are increasing sharply, driven by economic

development, nutrition transition, and increasingly sedentary lifestyles (Lee et al.,

2007).

The number of diabetics in Malaysia has increased by almost 80% in the last

10 years from 1996-2006 to 1.4 million adults above the age of 30. Ninety five

4

percent of diabetics suffer from type-2 diabetes, of which 50 percent will develop

cardiovascular disease within 10 years

(http://thestar.com.my/news/story.asp?file=/2010/1/11/nation).

In the management of diabetes mellitus, several approaches are often

employed which include dietary intervention, use of different classes of oral but safe

hypoglycemic agents, insulin injection, aerobic exercise and food supplements (Davis

and Granner, 2001). However, in developing countries and in some other developed

countries, these therapeutic options are expensive, and not readily accessible,

especially to the poor. Beside these, the therapeutic regimen are considered rigid,

multi-pharmaceutical and often associated with intolerable side effects. These factors

militate against effective management/treatment of the patients. In view of these

shortcomings, herbal pharmacotherapy is often explored by these patients. The

treatment goals for patients with diabetes have evolved significantly over the last 80

years, from preventing imminent mortality, to alleviating symptoms, to the now

recognized objective of normalization or near normalization of glucose levels with the

intent of forestalling diabetic complications. Although there are several drug

treatments currently available, the need for new herbal agents for treatment of diabetes

are required. Plants are frequently considered to be less toxic and free from side

effects than synthetic ones (Jung et al., 2006).

Recently, the search for appropriate antidiabetic agents has been focused on

plants used in traditional medicine partly because of leads provided by traditional

medicine to natural products that may be better treatments than currently used drugs

responsible for unrevealing serious side effects among diabetics (Rates, 2001; Prince

et al., 2004).

5

1.3 TETRACERA INDICA MERR.

(DILLENIACEAE) (T. indica Merr.), (synonyms: Assa indica Christm & Panz)

(Malay name: Mempelas minyak or Memepelas paya), is a large, woody, rain forest

climber of Malaysia and Indonesia. It has a medium sized and simple leaves that

arrange alternate with each other. The texture of its stem is rough to such an extent

that it can hurt if touched by the skin. Its flowers are white in color with small pink

calyxes. The flowers also have a nice fragrance. It has fruits that taste kind of sour and

look like berries (Christophe, 2002).

T. indica Merr. is also one of the active ingredients in a local herbal medicine

in Malaysia viz, Plantisol, which is widely prescribed to treat diabetes in Malaysia by

the herbalists. Other active ingredients of Plantisol are Barrintoga racemosa,

Pithecellobium jiringa, Tinospora crispa and Andrographis paniculata which

collectively make ‘Plantisol’ worthy for the treatement of diabetes efficaciously

(http://www. klik4sihat.com/kencingmanis).

In folk remedies, different parts of T. indica Merr. have been found to act

effectively in the treatment of fever, flue, sinus symptoms, skin rashes, itching, piles,

mouth ulcer, diarrhea, insects bites and diabetes. In Machang, Kelantan, Malaysia,

decoction of the climbing stems and roots is drunk to reduce high blood pressure and

the leaves, crushed and mixed with water, are applied on the whole body to treat fever

(Ong and Nordiana, 1999). Temuan tribe in Selangor, Malaysia uses its roots

concoction to treat high blood pressure and high fever, while leaves and root pounded

together is used to treat skin itching (Faridah and Nurulhuda, 1999). Moreover, its

shoot ground, wrapped in banana leaves are heated then applied to treat headache

(Latiff and Zakri, 1996).

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1.4 OBJECTIVES OF THE RESEARCH

The prime objective of this research is to investigate the antidiabetic effect of the

leaves of Tetracera indica Merr. (Dilleniaceae). This research is intended to prove

traditional claims of T. indica Merr. with regard to treat diabetes in Malaysia

scientifically. This research is also intended to provide a chemical lead for the

synthesis of new derivatives, which might be potential antidiabetic agents.

1.5 SPECIFIC OBJECTIVES

1. To prepare aqueous and methanolic extracts of the leaves of T. indica

Merr.

2. To evaluate the antidiabetic efficacy of aqueous and methanolic extracts in

vivo.

3. To isolate biologically active compounds through analytical techniques.

4. To characterize the structures of active principles through spectroscopy.

1.6 RESEARCH HYPOTHESIS

T. indica Merr. may provide biologically active agents particularly towards the

management of diabetes.

7

CHAPTER TWO

LITERATURE REVIEW

2.1 MEDICINAL PLANT

Natural products as sources of novel therapeutic agents experienced a steady increase

from around the turn of the twentieth century until it peaked in the 1970s and 1980s.

However since this time pharmaceutical research in natural products has experienced

a decline. Despite this trend the natural products industry now seems to be

experiencing a revival of sorts. The World Health Organization estimates that

approximately 80% of the world’s inhabitants rely on traditional medicine for their

primary health care (Farnsworth et al., 1985).

Malaysia has been endowed with flora of about 12,000 species of flowering

plants of which 1,300 species were said to be medicinal and only about hundred have

been fully investigated for their medicinal potential. Huge diversity of the Malaysian

flora is assumed to have well diverse chemical structures from their secondary

metabolites which serve as a factor for natural product excellent venture for the

screening program (Isma’il, 2001). However, 1,200 species of higher plant species

and 2000 species in Sabah and Sarawak, reported to have medicinal values and have

been used for generation in various traditional health care systems (Jantan, 1998).

Natural product preparations have historically been the major source of

pharmaceutical agents. Analysis of FDA new-drug approvals from 1981 to 2002

revealed that natural products continued to play a pivotal role during that time, even if

the industry had turned to other discovery strategies (Newman et al., 2003). Indeed,

more than 90% of current therapeutic classes derive from a natural product prototype.

8

Those ‘‘medicinal plants’’ are either preparations of or natural product substances

from plants that have potential utility as pharmaceutical agents (Balunas and

Kinghorn, 2005).

Table 2.1 Some Major plant drugs for which no synthetic one is currently available

(Kumar et al., 1997)

Drugs Plants Uses

Morphine Papaver somniferum Painkiller

Codeine Papaver somniferum Anticough

Artemisinin Artemesia annua Antimalarial

Barberine Berberis For leishmaniasis

Allicin Allium sativum Antifungal, amoebabiasis

Catechin Acacia catechu antiulcer

Vinblastine Catharanthus roseus Anticancer,hypotensive

Reserpine Rauvolfia serpentine Tranquilizer

Cocaine Erythroxylum coca Topic anesthetic

Pilocarpine Pilocarpus jaborandi Antiglucoma

Plants synthesize an extensive array of natural products or secondary

metabolites, and many of these possess pharmacological properties. These bioactive

compounds can be found in medicinal plants. Extracts, infusions and other

preparations from medicinal plants were the sole alternative to human healthcare

needs until the nineteenth century (Gurib-Fakim, 2006) The isolation of morphine in

1806 is regarded by many as the beginning of phytochemistry (Balunas and Kinghorn,

2005 ; Hartmann, 2007). From then on, the development of organic chemistry

provided standardized medications through synthethic drugs and active compounds

isolated from biological material.