DRUG METABOLISM AND TOXICITY STUDIES OF

Orthosiphon stamineus, Benth (MISAI KUCING) IN RATS

by

CHIN JIN HAN

Thesis submitted in fulfillment

of the requirements for the degree of

Doctor of Philosophy

November 2006 Acknowledgements

I wish to extend a special note of 3ppreciation to those who helped me over the years to the development of this thesis. Without their assistance and support, this thesis could not be has been completed. First, I would like to acknowledge deep gratitude to Associate Professor Dr. Abas Hj Hussin, my main supervisor and

Dr. Sabariah Ismail, my co-supervisor for their cooperative and helpful guidance during the developmental process of this thesis. Many thanks also to their helpful suggestions and corrections until the completion of this thesis. They greatly improved my knowledge and gave most valuable help to me in assembling all the material into a final product. Second, I sincerely thank the non-academic staffs of the School of

Pharmaceutical Sciences and the Centre for DnJQ Research. They are Mr. Rosli

Hassan, Mr. Yusuf, Mr. Adnan, Madam Yeong, Mr. Arunchalam Ramachandran and

Mr. Rahim Ali Musa who have helped and supported me from inception of my study.

Third, writing this thesis has been significantly aided by the stimulating discussion and suggestion with my laboratory mates in the Department of Pharmacology. Fourth, the

School of Pharmaceutical Sciences and the Centre for Drug research have been most generous with infrastructural support. Besides that, I wish to thank the Ministry of

Science, Technology and Innovative, Malaysia for their encouragement and financial support for this study and Professor Zhari Ismail for supplying the standardised methanol extract of 0. stamineus. Finally, I owe the deepest gratitude to my lovely wife, my parent, sister and brother for their moral support during my study. I gratefully acknowledge their love and support, making this thesis possible.

Chin Jin Han, 2006

Universiti Sains Malaysia

II TABLE OF CONTENT

Page

TITLE

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iii

LIST OF TABLES xvi

LIST OF FIGURES xix

LIST OF APPENDICES XX

LIST OF ABBREVIATIONS xxi

ABSTRAK xxiv

ABSTRACT xxvi

CHAPTER ONE: GENERAL INTRODUCTION 1

1.0 INTRODUCTION 1

1.1 Use of herbal medicine worldwide 1

1 1 .1 The usage of herbs to treat diseases 3

1.2 Literature review of Misai Kucing ( stamineus, Benth) 5

1.2.1 Misai Kucing description 5

1.2.2 Classification of Misai Kucing 6

1.2.3 Pharmacologic effects of Misai Kucing 7

1.2.4 Herb-drug interaction and adverse effects of methanol 9 extract of Misai Kucing

1.2.5 Evaluation of Misai Kucing chemical constituents 11

1.3 Hepatic drug metabolism reactions 12

1.3.1 Introduction to drug metabolism 12

1.3.1.1 Phase I hepatic drug metabolism reaction 18

1.3.1.2 Phase II hepatic drug metabolism reaction 20

iii 1.3.1.3 Isolation and subcellular fraction in the liver 21

1.3.2 Herb-drug interaction 23

1.3.2.1 Pharmacokinetic interaction 28

1.3.2.2 Pharmacodynamic interaction 30

1.3.3 The interpretation of data from rat to human 31

1.4 Objectives of the study 34

CHAPTER TWO: THE EFFECT OF THE STANDARDISED METHANOL 36 EXTRACT OF MISAI KUCING (Orthosiphon stamineus, Benth) ON PHASE I HEPATIC DRUG METABOLISING ENZYME, AMINOPYRINE N-DEMETHYLASE ACTIVITY IN NORMAL AND DIABETIC SO RAT HEPATOCYTES

2.0 INTRODUCTION 36

2.1 Liver and drug metabolism 36

2.1.1 Liver perfusion technique and its application 37

2.2 Introduction to cytochrome P450 38

2.2.1 Nomenclature of cytochrome P450 38

2.2.2 Cytochrome P450 and its functions 38

2.2.3 Catalytic cycle of cytochrome P450 41

2.2.4 Induction mechanism for cytochrome P450 42

2.2.4.1 Induction of cytochrome P450 by 42 active compounds in herbs

2.3 Aminopyrine metabolism in liver 43

2.3.1 Aminopyrine N-demethylase assay 43

2.3.2 Literature review on aminopyrine metabolism 48 and N-demethylation pathway

2.4 Second messenger pathway involved in drug metabolism 49

2.4.1 G protein-coupled receptor (GPCR) signal transduction 49

2.4.2 Heterotrimetic G Protein in signal transduction 50

2.4.3 Cyclic adenosine monophosphate (cAMP) pathway 51

2.4.4 Cyclic guanosine monophosphate (cGMP) pathway 51

IV 2.4.5 Phosphatidyl inositol pathway 52

2.4.6 Phosphodiesterase (POE) in second messenger pathway 54

2.4.7 Protein kinases in second messenger pathway 55

2.4.8 Phosphorylation and dephosphorylation of target protein 56

2.5 Biological factors affecting phase I hepatic metabolism reaction 57

2.6 Objectives of the study 61

2.7 METHODS AND METERIALS 62

2. 7.1 Sources of chemicals used 62

2.7.2 Sources of lab equipments and instrumentals used 64

2. 7.3 Experimental animals 65

2.7.3.1 Source of animals used 65

2.7.3.2 Selection of experimental animal 65

2.7.3.3 Induction of type I diabetes mellitus 66 by streptozotocin (STZ)

2.7.3.4 Preparation of standardised methanol extract of 66 Misai Kucing (0. stamineus) solution

2.7.4 Preparation of buffer and others solutions 68

2. 7.4.1 Stock solutions for aminopyrine N-demethylase assay 68

2.7.4.2 Solutions prepared fresh daily 69

2.7.5 Preparation of hepatocytes 70

2.7.5.1 Counting total number of isolated hepatocytes 71

2. 7.6 Preparation of formaldehyde standard curve 71

2.7.7 Aminopyrine N-demethylase assay 72

2.7.7.1 In vitro study of the effect of standardised methanol 72 extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in normal and diabetic SD rat hepatocytes

2.7.7.2 Ex vivo study of the effect of standardised methanol 73 extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in normal and diabetic SD rat hepatocytes

2. 7. 7.3 Molecular mechanism elucidation of the effect of 74

v standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in SO rat hepatocytes

2.7.7.4 Statistical analysis 76

2.8 RESULTS 77

2.8.1 Preparation of formaldehyde standard curve 77

2.8.2 In vitro experiment of the effect of the standardised 77 methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in SO rat liver hepatocytes

2.8.2.1 In vitro experiment of the effect of the 77 standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in normal SO rat liver hepatocytes

2.8.2.2 In vitro experiment of the effect of the 78 standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in diabetic SO rat liver hepatocytes

2.8.3 Molecular mechanism elucidation of the effect of standardised 80 methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in SD rat hepatocytes

2.8.3.1 Molecular mechanism evaluation of the effect of 80 standardised methanol extract of Misai Kucing ( 0. stamineus)on aminopyrine N-demethylase activity in normal old male SO rat hepatocytes

2.8.3.2 Molecular mechanism evaluation of the effect of 84 standardised methanol extract of Misai Kucing (0. stamineus) on aminopyrine N-demethylase activity in normal young female SO rat hepatocytes

2.8.4 Ex vivo (acute-1 day treatment) effect of the standardised 88 methanol extract of Misai Kucing (0. stamineus) on aminopyrine N-demethylase activity in SO rat hepatocytes

2.8.4.1 Ex vivo (acute-1 day treatment) effect of the 88 standardised methanol extract of Misai Kucing (0. stamineus) on aminopyrine N-demethylase activity in normal SO rat hepatocytes

2.8.4.2 Ex vivo (acute-1 day treatment) effect of the 88 standardised methanol extract of Misai Kucing (0. stamineus) on aminopyrine N-demethylase activity in diabetic SO rat hepatocytes

2.8.5 Ex vivo (sub-chronic-14 days treatment) effect of the 91 standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in SO rat hepatocytes

VI 2.8.5.1 Ex vivo (sub-chronic-14 days treatment) effect of the 91 standardised methanol extract of Misai Kucing (0. stamineus) on aminopyrine N-demethylase activity in normal SO rat heratocytes

2.8.5.2 Ex vivo (sub-chronic-14 days treatment) effect of the 91 standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in diabetic SO rat hepatocytes

2.9 DISCUSSION 94

2.9.1 The standard curve for formaldehyde 94

2.9.2 The in vitro and ex vivo (acute or sub-chronic) effect of 94 standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in SO rat hepatocytes

2.9.2.1 The influence of age, gender and disease on 96 in vitro and ex vivo aminopyrine metabolism in SO rat hepatocytes

2.9.3 Molecular mechanisms elucidation of the effect of standardised 98 methanol extract of Misai Kucing (0. stamineus) on aminopyrine metabolism in SO rat hepatocytes

2.9.3.1 The elucidation of the effect of standardised methanol 99 extract of Misai Kucing ( 0. stamineus) on aminopyrine metabolism in normal old male SO rat hepatocytes

2.9.3.2 The elucidation of the effect of standardised methanol 103 extract of Misai Kucing (0. stamineus) on aminopyrine metabolism in normal young female SO rat hepatocytes

2.9.4 The significance of aminopyrine assay to drug development 104

2.9.5 Suggestions for further study 106

2.10 CONCLUSION 107

CHAPTER THREE: THE EFFECT OF STANDARDISED METHANOL 109 EXTRACT OF MISAI KUCING (Orthosiphon stamineus, Benth) ON PHASE II HEPATIC DRUG METABOLISING ENZYMES, UDP-GLUCURONOSYLTRANSFERASE(UGT)AND GLUTATHIONE-S-TRANSFERASE (GST) ACTIVITY IN NORMAL AND DIABETIC SD RATS

3.0 INTRODUCTION 109

3.1 Introduction to UDP-glucuronosyltransferase (UGT) reaction 109

3.1.1 Localisation and tissue distribution of UGT enzyme 111

3.1.2 Nomenclature of UGT multigene family 111

VII 3.1.3 The roles of UGT and glucuronidation in drug metabolism 112

3.1.4 Induction of UGT isoforms 113

3.1.5 p-nitrophenol (pNP) - UGT assay 114

3.2 Introduction to Glutathione-S-transferase (GST) assay 116

3.2.1 Localisation and tissue distribution of GST 117

3.2.2 The nomenclature of GST enzyme 117

3.2.3 Functions of GST enzyme 118

3.2.4 Induction of glutathione (GSH) and GST 120

3.2.5 Glutathione conjugation towards 121 1-chloro-2,4-dinitrobenzene (CDNB)

3.3 Biological factors affecting phase II liver metabolising enzymes 123

3.4 Objectives of the study 125

3.5 METHODS AND MATERIALS 126

3.5.1 Sources of chemicals used 126

3.5.2 Sources of laboratory equipments and instruments used 127

3.5.3 Experimental animals 128

3.5.3.1 Selection of experimental animal 128

3.5.3.2 Induction of type I diabetes mellitus 128 by streptozotocin (STZ)

3.5.4 Preparation of standardised methanol extract of 128 Misai Kucing ( 0. stamineus) solution

3.5.5 Preparation of buffer and other solutions 129

3.5.5.1 Solutions used for rat liver fraction 129 and microsomes preparation

3.5.5.2 Solutions used for determination of protein content 129 by Lowry method

3.5.5.3 Solutions used for UDP-glucuronosyltransferase 130 (UGT) assay

3.5.5.4 Solutions used for glutathione-S-transferase 130 (GST) assay

3.5.6 Preparation of liver cytosolic fraction and microsomes 131

viii 3.5.6.1 Preparation for liver cytosolic fraction 131

3.5.6.2. Preparation for liver microsomes 131

3.5.7 Determination of the protein contents in liver samples 132

3.5.8 Preparation of p-nitrophenol (pNP) standard curve 133

3.5.9 Optimization of UGT assay in rat liver microsomes 134

3.5.1 0 Optimization of GST assay in rat liver cytosolic fraction 135

3.5.11 pNP-UGT assay 137

3.5.11.1 In vitro study of the effect of standardised methanol 137 extract of Misai Kucing (0. stamineus) on UGT activity in normal and diabetic SO rat liver microsomes

3.5.11.2 Ex vivo study of the effect of standardised methanol 138 extract of Misai Kucing ( 0. stamineus) on UGT activity in normal and diabetic SO rat liver microsomes

3.5.11.3 Calculation of the UGT activity 140

3.5.12 GST assay 141

3.5.12.1 In vitro study of the effect of standardised methanol 141 extract of Misai Kucing (0. stamineus) on GST activity in normal and diabetic SO rat liver cytosolic fraction

3.5.12.2 Ex vivo study of the effect of standardised methanol 142 extract of Misai Kucing (0. stamineus) on GST activity in normal and diabetic SO rat liver cytosolic fraction

3.5.12.3 Calculation of the GST activity 143

3.5.13 Statistical analysis 144

3.6 RESULTS 145

3.6.1 Optimization for the UGT assay 145

3.6.2 Optimization for the GST assay 145

3.6.3 In vitro effect of standardised methanol extract of Misai Kucing 146 ( 0. stamineus) on UGT activity in rat liver microsomes

3.6.3.1 In vitro effect of the standardised methanol extract of 146 Misai Kucing (0. stamineus) on UGT activity in normal SO rat liver microsomes

3.6.3.2 In vitro effect of the standardised methanol extract of 14 7 Misai Kucing (0. stamineus) on UGT activity in diabetic rat liver microsomes

IX 3.6.4 In vitro effect of standardised methanol extract of Misai Kucing 151 (0. stamineus) on GST activity in SD rat liver cytosolic fraction

3.6.4.1 In vitro effect of the standardised r.1ethanol extract of 151 Misai Kucing (0. stamineus) on GST activity in normal SD rat liver cytosolic fraction

3.6.4.2 In vitro effect of the standardised methanol extract of 153 Misai Kucing (0. stamineus) on GST activity in diabetic SD rat liver cytosolic fraction

3.6.5 Ex vivo (acute-1 day treatment) effect of standardised methanol 156 extract of Misai Kucing (0. stamineus) on UGT activity in SD rat liver microsomes

3.6.5.1 Ex vivo (acute-1 day treatment) effect of standardised 156 methanol extract of Misai Kucing (0. stamineus) on UGT activity in normal SD rat liver microsomes

3.6.5.2 Ex vivo (acute-1 day treatment) effect of standardised 157 methanol extract of Misai Kucing (0. stamineus) on UGT activity in diabetic SD rat liver microsomes

3.6.6 Ex vivo (acute-1 day treatment) effect of the standardised 160 methanol extract of Misai Kucing (0. stamineus) on GST activity in SD rat liver cytosolic fraction

3.6.6.1 Ex vivo (acute-1 day treatment) effect of the standardised 160 methanol extract of Misai Kucing ( 0. stamineus) on GST activity in normal SD rat liver cytosolic fraction

3.6.6.2 Ex vivo (acute-1 day treatment) effect of the standardised 161 methanol extract of Misai Kucing (0. stamineus) on GST activity in diabetic SD rat liver cytosolic fraction

3.6.7 Ex vivo (sub-chronic-14 days treatment) effect of standardised 164 methanol extract of Misai Kucing (0. stamineus) on UGT activity in SD rat liver microsomes

3.6.7.1 Ex vivo (sub-chronic-14 days treatment) effect of 164 standardised methanol extract of Misai Kucing (0. stamineus) on UGT activity in normal SD rat liver microsomes

3.6.7.2 Ex vivo (sub-chronic-14 days treatment) effect of 164 standardised methanol extract of Misai Kucing (0. stamineus) on UGT activity in diabetic SD rat liver microsomes

3.6.8 Ex vivo (sub-chronic-14 days treatment) effect of standardised 168 methanol extract of Misai Kucing (0. stamineus) on GST activity in SD rat liver cytosolic fraction

X 3.6.8.1 Ex vivo (sub-chronic-14 days treatment) 168 effect of standardised methanol extract of Misai Kucing (0. stamineus) on GST activity in normal SO rat liver cytosolic fraction

3.6.8.2 Ex vivo (sub-chronic-14 days treatment) 169 effect of standardised methanol extract of Misai Kucing (0. stamineus) on GST activity in diabetic SO rat liver cytosolic fraction

3.7 DISCUSSION 172

3. 7.1 The in vitro and ex vivo effect of standardised methanol extract of 172 Misai Kucing (0. stamineus) on UGT and GST activity in SO rat liver

3.7.2 The influence of age, gender and disease factor 174 on UGT and GST activity in SO rat liver

3. 7.3 The significance of UGT and GST assay to herb-drug interaction 175 study

3. 7.4 Suggestion for future study 176

3.8 CONCLUSION 176

CHAPTER FOUR: TOXICITY AND ANTI-HEPATOTOXICITY STUDIES OF 178 THE STANDARDISED METHANOL EXTRACT OF MISAI KUCING (Orthosiphon stamineus, Benth) IN SD RATS

4.0 INTRODUCTION 178

4.1 History of toxicology 178

4.1.1 Principles of acute oral toxicity studies 179

4.1.2 Definition of LD 50 182

4.1.3 Possible mechanism of hepatotoxicity 183

4.1.4 Mechanisms of P450-mediated liver injury 186

4.2 Anti-Hepatotoxicity effect of the herbs 188

4.2.1 Acetaminophen-induced hepatotoxicity 188

4.2.1.1 Types of liver injuries induced by acetaminophen 189

4.2.1.2 Cytochrome P450, UGT and GST enzymes in the 190 prevention of acetaminophen-induced liver injury

4.3 The design of experimental model 192

4.3.1 Cage-side observations 192

XI 4.3.2 Blood serum biochemical analyses 193

4.3.2.1 Blood serum biochemical parameters of 195 Liver function test

4.3.2.2 Blood serum biochemical parameters of 199 Kidney function test

4.3.2.3 Blood serum Lipid profile 200

4.4 Factors that may affect the experimental results 201

4.4.1 Biological factors in experimental animals 201

4.4.2 Environment of experimental animals 202

4.4.3 Route of administration 203

4.5 Objectives of the study 203

4.6 METHODS AND MATERIALS 204

4.6.1 Sources of chemicals used 204

4.6.2 Sources of laboratory equipments and instruments used 205

4.6.3 Preparation of the buffer and other solutions 206

4.6.3.1 Preparation of gum tragacanth 206

4.6.4 Experimental design for toxicity study 206

4.6.4.1 Experimental animals 206

4.6.4.1.1 Specification of animals 206

4.6.4.1.2 The procedure for the selection of rats 206

4.6.4.2 Dose selection for toxicity studies 207

4.6.4.2.1 Dose preparation and administration 207

4.6.4.3 The protocol of toxicity study 207

4.6.4.3.1 Acute toxicity study (1 day treatment) 208

4.6.4.3.2 Sub-chronic toxicity study (14 days treatment) 208

4.6.4.3.3 Cage-side observation 209

4.6.4.3.4 Blood collection and preparation of blood serum 210

4.6.4.3.5 Recovery period 210

4.6.4.3.6 Statistical analysis 211

xii 4.6.5 The experimental design for anti-hepatotoxicity study 211

4.6.5.1 Experimental animals 211

4.6.5.1.1 Specification of animals 211

4.6.5.1.2 Procedure for the selection of SO rats 211

4.6.5.2 Dose selection for anti-hepatoxicity study 211

4.6.5.2.1 Dose selection for standardised methanol 211 of Misai Kucing ( 0. stamineus)

4.6.5.2.2 Dose selection of acetaminophen 212

4.6.5.3 The protocol of anti-hepatoxicity study 213

4.6.5.3.1 Acute (1 day treatment) anti-hepatotoxicity study 213

4.6.5.3.2 Sub-chronic (14 days treatment) 214 anti-hepatotoxicity study

4.6.5.3.3 Blood sample collection and 214 serum preparation

4.6.5.3.4 Percentage hepatocytes viability 215

4.6.5.3.5 Statistical analysis 215

4.7 RESULTS 216

4. 7.1 Acute ( 1 day treatment) toxicity effect of the 216 standardised methanol extract of Misai Kucing (0. stamineus) in SO rats

4.7.1.1 Blood serum biochemical analyses 216

4.7.1.1.1 Acute (single dose-1 day treatment) 216 effect of standardised methanol extract of Misai Kucing (0. stamineus) on serum AST, ALT and ALP level in normal young male and female SO rats

4.7.1.1.2 Acute(singledose-1 daytreatment) 218 effect of standardised methanol extract of Misai Kucing (0. stamineus) on serum urea and creatinine level in normal young male and female SO rats

4.7.1.1.3 Acute (single dose-1 day treatment) 220 effect of standardised methanol extract of Misai Kucing (0. stamineus) on serum total cholesterol and triacylglycerollevel in normal young male and female SO rats

XIII 4.7.1.2 Cage-side observation 222

4.7.1.3 Body weight and water intake 222

4.7.1.4 Relative organ weight 224

4. 7 .1.5 Recovery period-acute toxicity study 225

4. 7.2 Sub-chronic (repeated dose-14 days treatment) effect of the 229 standardised methanol extract of Misai Kucing ( 0. stamineus) in SD rats

4.7.2.1 Blood serum biochemical analyses 229

4. 7 .2.1.1 Sub-chronic (repeated dose-14 days treatment) 229 effect of standardised methanol extract of Misai Kucing (0. stamineus) on serum AST, ALT and ALP level in normal young male and female SD rats

4.7.2.1.2 Sub-chronic (repeated dose-14 days treatment) 230 effect of standardised methanol extract of Misai Kucing ( 0. stamineus) on serum urea and creatinine level in normal young male and female SD rats

4.7.2.1.3 Sub-chronic (repeated dose-14 days treatment) 233 effect of standardised methanol extract of Misai Kucing (0. stamineus) on serum total cholesterol and triacylglycerol level in normal young male and female SD rats

4.7.2.2 Cage-side Observations 233

4.7.2.3 Body weight, food consumption and water intake 234

4.7.2.4 Relative organ weight 234

4.7.2.5 Recovery period 240

4.7.3 Acute (1 day treatment) anti-hepatotoxicity studies of the 243 standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminophen-induced liver injury in SD rats

4.7.3.1 Acute (1 day treatment) anti-hepatotoxicity studies of the 243 standardised methanol extract of Misai Kucing (0. stamineus) on acetaminophen-induced liver injury in adult male SD rats

4.7.3.2 Acute (1 day treatment) anti-hepatotoxicity studies of the 244 standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminophen-induced liver injury in adult female SD rats

4.7.4 Sub-chronic (14 days treatment) anti-hepatotoxicity studies 248

XlV 4.7.4.1 Sub-chronic (14 days treatment) anti-hepatotoxicity 248 effect of standardised methanol extract of Misai Kucing (0. stamineus) on acetaminophen-induced liver injury in adult male SD rats

4.7.4.2 Sub-chronic (14 days treatment) anti-hepatotoxicity 249 effect of standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminophen-induced liver injury in adult female SD rats

4.8 DISCUSSION 253

4.8.1 Toxicity studies of standardised methanol extract of Misai Kucing 254

4.8.1.1 Acute (1 day treatment) toxicity study of the 254 standardised methanol extract of Misai Kucing ( 0. stamineus) in normal young male and female SD rats

4.8.1.2 Sub-chronic (14 days treatment) toxicity study of 256 the standardised methanol extract of Misai Kucing ( 0. stamineus) in normal young male and female SD rats

4.8.1.3 The significance of toxicity study to drug development 258

4.8.1.4 Suggestions for further study 260

4.8.2 Anti-hepatotoxicity effect of the standardised methanol extract of 260 Misai Kucing ( 0. stamineus) on acetaminophen-induced liver damage in SD rats

4.8.2.1 The acute (1 day treatment) anti-hepatotoxicity effect of 261 standardised methanol extract of Misai Kucing ( 0. stamineus) acetaminophen-induced liver damage in adult male and female SD rats

4.8.2.2 The sub-chronic (14 days treatment) anti-hepatotoxicity 263 effect of standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminophen-induced liver damage in adult male and female SD rats

4.8.2.3 The significance of the anti-hepatotoxicity study 264 to drug development

4.8.2.4 Suggestions for further study 266

4.9 CONCLUSION 266

CHAPTER FIVE: GENERAL CONCLUSION 267

BIBLIOGRAPHY 276

APPENDICES 297

PUBLICATION 310

XV LIST OF TABLES

Page

1.1 List of chemical constituents of Orthosiphon stamineus aerial part extract 13

1.2 List of chemical constituents of Orthosiphon stamineus leaves extract 14

1.3 Major biotransformation reaction 24

1.4 Characteristics of Phase I enzymes 25

1.5 Characteristics of Phase II enzymes 26

1.6 Examples of the herb-drug interactions documented in human 32

1. 7 Equivalent surface area dosage conversion factors 33

2.1 Cytochrome P450 oxidative system and substrate specificity 40

2.2 Induction mechanisms for cytochrome P450 44

2.3 Potent inhibitors of eDNA-derived enzymes 45

2.4 In vitro effect of standardised methanol extract of Misai Kucing 79 (0. stamineus) on aminopyrine N-demethylase activity in normal and diabetic SO rat hepatocytes

2.5 The effect of the standardised methanol extract of Misai Kucing 83 (0. stamineus) on aminopyrine N-demethylase activity in normal old male SO rat hepatocytes in the presence or absence of cellular inhibitors/stimulants

2.6 The effect of the standardised methanol extract of Misai Kucing 87 (0. stamineus) on aminopyrine N-demethylase activity in normal young female SO rat hepatocytes in the presence or absence of cellular inhibitors/stimulants

2.7 Ex vivo (acute- 1 day treatment) effect of the standardised methanol 90 extract of Misai Kucing (0. stamineus) on aminopyrine N-demethylase activity in normal and diabetic SO rat hepatocytes

2.8 Ex vivo (sub-chronic-14 days treatment) effect of the standardised 93 methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in normal and diabetic SO rat hepatocytes

3.1 In vitro effect of the standardised methanol extract of Misai Kucing 150 (0. stamineus) on UGT activity in normal and diabetic SO rat liver microsomes

xvi 3.2 In vitro effect of the standardised methanol extract of Misai Kucing 155 (0. stamineus) on GST activity in normal and diabetic SD rat liver cytosolic fraction

3.3 Ex vivo (acute-1 dav treatment) effect of the standardised methanol 159 extract of Misai Kucing (0. stamineus) on UGT activity in normal and diabetic SD rat liver microsomes

3.4 Ex vivo (acute-1 day treatment) effect of the standardised 163 methanol extract of Misai Kucing ( 0. stamineus) on GST activity in normal and diabetic SO rat liver cytosolic fraction

3.5 Ex vivo (sub-chronic-14 days treatment) effect of the standardised 167 methanol extract of Misai Kucing (0. stamineus) on UGT activity in normal and diabetic SO rat liver microsomes

3.6 Ex vivo (sub-chronic-14 days treatment) effect of the standardised 171 methanol extract of Misai Kucing ( 0. stamineus) on GST activity in normal and diabetic SO rat liver cytosolic fraction

4.1 Some potential fatal and their toxicity profiles 180

4.2 Classification of chemicals according to relative toxicity 185

4.3 Acute (1 day treatment) effect of the standardised methanol extract of 217 Misai Kucing (0. stamineus) on serum AST, ALT and ALP level in normal young male and female SD rats

4.4 Acute (1 day treatment) effect of the standardised methanol extract of 219 Misai Kucing (0. stamineus) on serum urea and creatinine level in normal young male and female SD rats

4.5 Acute (1 day treatment) toxicity study of the standardised methanol 221 extract of Misai Kucing (0. stamineus) on serum total cholesterol and triacylglycerollevel in normal young male and female SO rats

4.6 Acute (1 day treatment) effect of the standardised methanol extract of 223 Misai Kucing ( 0. stamineus) on body weight and water intake and percentage of lethality in normal young male and female SO rats

4.7 Acute (1 day treatment) effect of the standardised methanol extract of 226 Misai Kucing ( 0. stamineus) on relative organ weight in normal young male and female SO rats

4.8 Acute (1 day treatment) effect of the standardised methanol extract of 227 Misai Kucing (0. stamineus) on body weight, water intake and food consumption in normal young male and female SO rats after fourteen days recovery period

4.9 Acute (1 day treatment) effect of the standardised methanol extract of 228 Misai Kucing ( 0. stamineus) on relative organ weight in normal young male and female SO rats after fourteen days recovery period

4.10 Sub-chronic (repeated dose-14 days treatment) effect of the 231 standardised methanol extract of Misai Kucing ( 0. stamineus) on serum

xvii AST, AL T and ALP level in normal young male and female SD rats

4.11 Sub-chronic (repeated dose-14 days treatment) effect of the 232 standardised methanol extract of Misai Kucing ( 0. stamineus) on serum urea and creatinine level in normal young male and female SD rats

4.12 Sub-chronic (repeated dose-14 days treatment) effect of the 235 standardised methanol extract of Misai Kucing ( 0. stamineus) on serum total cholesterol and triacylglycerollevel in normal young male and female SD rats

4.13 Sub-chronic (repeated dose-14 days treatment) effect of the 236 standardised methanol extract of Misai Kucing ( 0. stamineus) on body weight and percentage of lethality in normal young male and female SD rats

4.14 Sub-chronic (repeated dose-14 days treatment) effect of the 237 standardised methanol extract of Misai Kucing ( 0. stamineus) on food consumption in normal young male and female SO rats

4.15 Sub-chronic (repeated dose-14 days treatment) effect of the 238 standardised methanol extract of Misai Kucing ( 0. stamineus) on water intake in normal young male and female SO rats

4.16 Sub-chronic (repeated dose-14 days treatment) effect of the 239 standardised methanol extract of Misai Kucing ( 0. stamineus) on relative organ weight in normal young male and female SO rats

4.17 Sub-chronic (repeated dose-14 days treatment) effect of the 241 standardised methanol extract of Misai Kucing ( 0. stamineus) on body weight, water intake and food consumption in normal young male and female SD rats after 14 days recovery period

4.18 Sub-chronic (repeated dose-14 days treatment) effect of the 242 standardised methanol extract of Misai Kucing (0. stamineus) on relative organ weight in normal young male and female SO rats after 14 days recovery period

4.19 In vivo (acute-1 day treatment) anti-hepatotoxicity effect of 246 standardised methanol extract of Misai Kucing (0. stamineus) on acetaminophen-induced liver injury in adult male SO rats

4.20 In vivo (acute-1 day treatment) anti-hepatotoxicity effect of the 247 standardised methanol extract of Misai Kucing (0. stamineus) on acetaminophen-induced liver injury in adult female SO rats

4.21 In vivo (sub-chronic-14 days treatment) anti-hepatotoxicity effect 251 of the standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminohen-induced liver injury in adult male SO rats

4.22 In vivo (sub-chronic-14 days treatment) anti-hepatotoxicity effect 252 of the standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminophen-induced liver injury in adult female SO rats

xviii LIST OF FIGURES

Page

1.1 Misai Kucing (Orthosiphon stamineus, Benth) 10

1.2 Schematic diagram of the preparation of subcellular fraction 27

2.1 Aminopyrine N-demethylase reactions 47

3.1 The general role of UGT in conjugation with foreign compounds 110

3.2 Glucuronidation of p-nitrophenol (pNP) catalysed by UGT enzyme 115

3.3 Conjugation of glutathione (GSH) and 122 1-chloro-2,4-dinitrobenzene (CONS) as catalysed by glutathione-S-transferases (GST) enzyme

4.1 Metabolism pathway of acetaminophen through conjugation reaction 195

XIX LIST OF APPENDICES

Page

1.1 Overview of the experimental design in the study 297

1.2 Experiment design of the effect of standardised 298 methanol extract of Misai Kucing (Orthosiphon stamineus) on aminopyrine N-demethylase, glutathione-S-transferase (GST) and UDP-glucuronosyltransferase (UGT) activity in rat liver

2.1 The standard curve for formaldehyde 299

3.1 Optimization of incubation time and protein concentration for 300 the UGT enzyme assay

3.2 Optimization of the UDPGA concentration and triton X-100 ratio 301 for UGT assay

3.3 Optimization of glutathione (GSH) and 1-chloro-2,4-dinitrobenzene 302 (CDNB) concentration for the GST assay

3.4 Optimization of liver cytosolic fraction concentration and 303 incubation time for the GST assay

3.5 The standard curve of bovine serum albumin (BSA) 304

3.6 The standard curve of p-nitrophenol (pNP) 305

4.1 Mean control ranges of serum clinical biochemistry measurements 306 in normal male and female SD rats

4.2 The effect of single dose administration of 650 mg/kg and 2g/kg of 307 acetaminophen on liver damage in normal adult male and female SD rats

4.3 Overview of the experimental design for the acute (1 day treatment) 308 and sub-chronic (14 days treatment) effect of 0.5, 1.0, 3.0 and 5.0 g/kg of standardised methanol extract of Misai Kucing ( 0. stamineus) on normal young male and female SD rats

4.4 Overview of the experimental design for the acute (1 day treatment) 309 and sub-chronic (14 days treatment) effect of 5, 31.25, 125 and 500 mg/kg of standardised methanol extract of Misai Kucing ( 0. stamineus) on acetaminophen-induced liver injury in adult male and female SD rats

XX LIST OF ABBREVIATIONS

ADP Adenosine-5'-diphosphate

Ad libitium To be taken as wanted

AGC adenyl guanyl cyclase

ALD Approximate lethal dose

ALT Alanine aminotransferase

ALP Alkaline phosphatase

AST Aspartate aminotransferase

ATP Adenosine-5'-triphosphate

BSA Bovine serum albumin

Ca/CaM Calcium calmodulin complex

Ca2+ Calcium ion cAMP cyclic adenosine-3', 5'-monophosphate cGMP cyclic guanosine-3', 5'-monophosphate

CDNB 1-chloro-2,4-dinitrobenzene

DAG Diacylglycerol

DMSO Dimethylsulphoxide eta/ Else where or and others

FAD Flavin adenine dinucleotide g Gram

!Jg/ml microgram per milliliter ng/ml nanogram per milliliter mg/kg Miligram per kilogram mg/ml Miligram per mililiter

Go Alpha sub-unit of guanine nucleotide regulatory protein

GAP Good Agriculture Practice

XXI GGT Gamma-glutamyltransferase

g/kg Gram per kilogram

GLP Good Laboratory Practice

GMP Good Manufacture Practice

G-protein A guanine nucleotide regulatory protein

5' -Guanylylimidodiphosphate

GSH Glutathione

GST Glutathione-S-transferase

GTP Guanosine triphosphate

HBSS Hank's Balanced Salt Solution

IBMX 3-isobutyl-1-methylxanthi ne

lnositol-1 ,4,5-triphosphate

Inhibition constant

Michaelis Menten constant

KT5720 cAMP-dependent protein kinase inhibitor

KT5823 cGMP-dependent protein kinase inhibitor

LDso Dose caused 50% lethality population ml Mililiter mmoi/L Milimol per liter nM nanomolar

NADPH Nicotinamide adenine dinucleotide phosphate (reduced)

NAPQI N-acetyl-p-benzoquinone imine pNP p-nitrophenol

NOAEL No observable Adverse effect level

NOEL No observable effect level

OECD Organization for Economic Cooperation and Development

OA Okadaic acid

xxii OTC Over-the-counter

POE Phosphodiesterase enzyme

PKA Protein kinase A

PKc Protein kinase C

PKG Protein kinase G

PMA 4~-phorbol-12~-myristate-13a-acetate

PPA2 Phosphatase A2 r Coefficient factor

S.D Standard deviation

STZ Streptozotocin

TPA Trifluoperazine

U/L Unit per liter

UDP Uridine diphosphate

UDPGA UDP-glucuronic acid

UGT UDP-glucuronosyltransferase

USA United States of America

US$ United States dollar

Vmax Maximal velocity

WHO World Health Organization

XXIII KAJIAN METABOLISME DRUG DAN TOKSISITI Orthosiphon stamineus, Benth

(MISAI KUCING) DALAM TIKUS

ABSTRAK

Orthosiphon stamineus, Benth (Famili: Lamiceae) atau dikenali dengan nama

tempatan sebagai Misai Kucing, digunakan secara meluas di Malaysia untuk merawat

hipertensi, masalah sistem urinasi dan penyakit batu karang ginjal. Objektif kajian ini

ialah untuk mengkaji kesan ekstrak metanol Misai Kucing piawai ke atas enzim

penghadarnan drug hepatik fasa I dan II di dalam tikus Sprague-Oawley (SO).

Aminopirin, p-nitrofenol (pNP) dan 1-kloro, 2,4-dinitrobenzena (CONS) telah digunakan

sebagai substrat untuk menentukan aktiviti aminopirin N-demetilase,

UOP-glukuronosiltransferase (UGT) dan glutatione-S-transferase (GST) masing­

masing di dalam hati tikus SO. Pengasingan hepatosit, fraksi sitosolik hati dan

mikrosom telah disediakan dengan menggunakan teknik perfusi kolagenase dan

pengemparan berperingkat. Kesan ketoksikan serta anti-hepatotoksik oleh ekstrak metanol Misai Kucing piawai ke atas tikus SO juga diuji. Oaripada keputusan yang diperolehi, kebanyakan aktiviti UGT dan GST di dalam hati tikus SO normal dan diabetik adalah dipengaruhi secara signifikan oleh ekstrak metanol Misai Kucing piawai. Hanya beberapa kumpulan tikus SO normal dan diabetic menunjukkan kesan yang signifikan terhadap metabolisme aminopirin oleh ekstrak methanol Misai Kucing piawai. Kemungkinan mekanisme tindakan in vitro 1 mg/ml ekstrak metanol Misai

Kucing ke atas metabolisme aminopirin di dalam hepatosit tikus jantan tua normal adalah melalui pengaktifan G-protein dan protein kinase C manakala kesan in vitro

xxiv 0.001 mg/ml ekstrak metanol Misai Kucing piawai ke atas metabolisme aminopirin di

dalam hepatosit tikus SD betina muda adalah berkemungkinan melalui pengaktifan

protein kinase A. Aras LD50 untuk ekUrak metanol Misai Kucing piawai tidak dapat

ditentukan dalam ujikaji ini. Tiada kematian dan kesan toksik diperhatikan pada tikus jantan dan betina muda normal setelah dirawat dengan 0.5, 1.0, 3.0 dan 5.0 g/kg ekstrak metanol Misai Kucing piawai. Pra-rawatan dengan ekstrak metanol Misa!

Kucing piawai dapat meningkatkan peratusan viabiliti hepatosit serta menurunkan setengah dari aras AST, ALT, ALP dan GGT serum di dalam tikus SD jantan dan betina dewasa yang dirangsang kecederaan hati dengan 2 g/kg acetaminofen. Secara kesimpulan, ekstrak metanol Misai Kucing piawai mempunyai kesan yang lebih besar ke atas aktiviti UGT dan GST jika dibandingkan dengan aktiviti aminopirin

N-demetilase di dalam hati tikus SD. Ekstrak metanol Misai Kucing piawai pada

0.5 g/kg adalah diklasifikasikan sebagai paras tidak menyebabkan kesan (NOEL) dan ia mempamerkan kesan anti-hepatotoksisiti ke atas tikus SD jantan dan betina dewasa yang dirangsang kerosakan hati dengan acetaminofen.

XXV DRUG METABOLISM AND TOXICITY STUDIES OF Orthosiphon stamineus, Benth

(MISAI KUCING) IN RATS

ABSTRACT

Orthosiphon stamineus, Benth (Family: ) or locally known as Misai

Kucing, is widely used in Malaysia for treating hypertension, urinary system ailments and kidney stone disease. The objective of this study is to investigate the effect of standardised methanol extract of Misai Kucing on phase I and phase II hepatic drug metabolising enzymes in Sprague-Oawley (SO) rat. Aminopyrine, p-nitrophenol (pNP) and 1-chloro-2,4-dinitro benzene (CONB) was used as a substrate to determine the aminopyrine N-demethylase, UOP-glucuronosyltransferase (UGT) and glutathione-S­ transferase (GST) activity respectively in SD rat liver. Isolated hepatocytes, liver cytosolic fraction and liver microsomes from SO rats were prepared using the collagenase perfusion technique and differential centrifugation technique. The possible toxic and anti-hepatotoxicity effects of standardised of methanol extract of Misai Kucing on SO rats were also examined. Results obtained showed that most of the UGT and

GST activity in normal and diabetic SO rat liver were significantly affected by standardised methanol extract of Misai Kucing. Only a few groups of normal and diabetic SO rats showed significant effect on aminopyrine metabolism in hepatocytes in the presence of standardised methanol extract of Misai Kucing. The possible mechanism of action ofthe in vitro effect of 1 mg/ml of standardised methanol extract of Misai Kucing on aminopyrine metabolism in normal old male SD rat hepatocytes was probably mediated through the activation of G-protein and protein kinase C while

xxvi the in vitro effect of 0.001 mg/ml of Misai Kucing extract on normal young female SO rat hepatocytes was probably mediated through the activation of protein kinase A. The

LOse of standardised of methanol extract of Misai Kucing could not be determined in this study. No lethality incident and any toxic effects were observed in normal young male and female SO rats after being treated with 0.5, 1.0, 3.0 and 5 g/kg of standardised methanol extract of Misai Kucing. Pre-treatment with standardised methanol extract of Misai Kucing increased the percentage of hepatocytes viability and decreased some of the serum AST, ALT, ALP and GGT level in 2g/kg of acetaminophen induced liver damage in adult male and female SO rats. In conclusion, standardised methanol extract of Misai Kucing had a greater influence on UGT and

GST activity than the aminopyrine N-demethylase activity in SO rat liver. Standardised methanol extract of Misai Kucing at 0.5 g/kg is classified as not-observable effect level

(NOEL) and it exhibited anti-hepatotoxicity effect on acetaminophen-induced liver injury in adult male and female SO rats.

XXVII CHAPTER ONE

GENERAL INTRODUCTION

1.0 INTRODUCTION

1.1 Use of herbal medicine worldwide

Herbal medicine systems vary from one country to another country. Many of the

herbal medicine practices are originated from their culture and region. Herbal medicine

practices have been handed down from one generation to another generation and

some countries had practiced it for hundreds and even thousand of years. For

example, conventional United States and European herbal practices are based on

ancient Greek-Roman experiences by using single herbs. In contrast, Chinese

traditional herbal practice is based on formulas using multiple herbs

(Rotblatt & Ziment, 2002). Old Chinese herbal medication especially is based on the

experiences and absence of scientific evidence based products may not be acceptable

by the European herbal medicine practitioners although both systems are giving same therapeutic effects. Many countries are still practicing herbal medicine which remains the backbone of medicine.

In recent years, herbal medicine has been gaining more acceptance and attention around the world (Ernst, 2002). Herbal medicine has been practiced in many countries including Europe to Asia. Today, the World Health Organization (WHO) estimates that 80 % of the world's population use herbal medicine

(Fetrow & Avila, 2000). Herbal medicine or phytomedicine is a common element in the practice of ayurvedic, homeopathy and traditional oriented medicine system by using plants for healing purposes. Botanically, herbs are defined as a plant, plant part or extract for medicinal usage and can also be used as foods, fragrance, spices and essential oils (Blumenthal & lsraelsen, 1998). There are great movements in herbal medicine around the world by conventionally using herbal remedies in the form of herbal tea or crude tablets to extracted and standardised form of herbal remedies in today's modern herbal medicine. The most popular botanical medications in the USA in

1996 are echinacea, garlic, ginseng, gingko, goldenseal, mahuang, psyllium, siberian ginseng, saw palmetto and Cascara sagrada (Dennehy & Tsourounis, 2001 ).

Many consumers feel more comfortable with natural herbal products because it is commonly believed that herbal products have fewer side effects as compared to modern drugs especially after chronic use. Combination of herbs with certain drugs may alter pharmacological action of the drugs or even produce unwanted side effects such as toxicity. Therefore, scientific-based evidence of herbal medicine on the possible herb-drug interaction and toxicity is important to ensure the efficacy, safety and quality of the herbal remedies-based products.

In Malaysia, herbs are very common among the Malaysian community for medicinal purpose, foods and supplements. They used different part of the plant such as leaf, stem, root, flower or even seeds for medicinal purposes. A variety of herbal products are increasingly available at the Malaysian local market and many of these herbal products are sold as over-the-counter (OTC) medicine. One can easily find traditional medicine practitioners in Malay villages such as "nujum" (clairvoyant),

"bomoh" or "mak bidan" to help in diagnosing illnesses and diseases. Normally they use certain type of plants like limau nipis or limau purut or even pap rice for medicinal purposes (Muhammad & Mustafa, 1994). Recently, numerous studies on local herbals have been studied extensively in Malaysia. Many scientific studies have proven that local traditional herbs such as Tongkat Ali (Eurycoma longifolia), Hempedu Bumi

(Andrographis panicu/ata), Kacip Fatimah (Labisia pumila), Lempoyang (Zingiber

2 zerumbet) exhibit medicinal value for treatment of various diseases. Many other research on its use of herbs for medication are currently underway.

1.1.1 The usage of herbs to treat diseases

Worldwide, there are at least 250,000 species of flowering plants. In the

Southeast Asia alone, there are about 35,000 species of flowering plants of which

8,000 species are found in Malaysia (Muhammad & Mustafa, 1994). Malaysia is endowed with a large biodiversity of flora and fauna. Traditionally, unprocessed herbs are used for medicinal purposes. For instance, herbs are prepared as tea for internal use by boiling the parts of plant with water. Currently, herbal preparation has changed to extraction of the herbs with different organic solvents to increase the effectiveness and quality of the preparation for therapeutic purposes.

Plants contain many types of chemical compounds which help to protect it from predators or attract pollinators. These secondary metabolites actually are of therapeutic values to the human. Phytomedicinal chemicals are generally assigned into nine important categories namely alkaloids, bioflavonoids, essential oils, glycosides, resins, saponins, sterols, tannins and terpenes (Rotblatt & Ziment, 2002).

i) Alkaloids are defined as basic amine group with no volatile character and their name always end with '-ine'. Briefly, there are 13 or more subclasses of alkaloids.

There are imidazolesatropine, indoles, isoquinolines, amides, piperidines, pyridines, pyrrolidines, pyrrolizidines, quinolines, quinolizidines, steroidal, purines, and terpenoids

(Rotblatt & Ziment, 2002). Alkaloids are found to exert multiple types of therapeutic effects including atropine, emetine, capsaicin, cocaine, morphine, quinine, methylxanthines (such as caffeine and theophylline), strychnine and nicotine

(Fetrow & Avila, 2000). However, some alkaloids are harmful to human.

3 ii) Bioflavonoids lack nitrogen and usually contain two 6-carbon rings joined by three carbon atoms. About half of the 8,000 plant phenolic compounds are made up of flavonoids. They are found in high concentrations in many flowers, fruits ana vegetables (Moridani et a/., 2001 ). Flavans, flavanones, isoflavanones, flavones, isoflavones, chalcones, anthocyanidines and flavonolignans are the subclasses for flavonoids (Hodek eta/., 2002). Flavonoids are commonly believed to be antioxidants and antiiflammatory and protective against various cancers (Challis & Barlett, 1975).

Rutin, quercetin, kaempferol, genistein, licoricidin, cyanidin, isoliquiritigenin, hispidol, coumestiol and silymarin are common example of bioflavonoid compounds that exhibit therapeutic effects on human and animals (Badger eta/., 2001).

iii) Essential oils are isoprene derivatives. The important essential oils are found in

10 classes which are assigned as alcohols, aldehydes, esters, ethers, furans, hydrocarbons, ketones, phenols, sesquiterpinoids and sulfur compounds

(Rotblatt & Ziment, 2002). Essential oils are used therapeutically and also in aromatherapy (Sadler, 2001 ). Essential oils from mint family are used as flavors and drugs.

iv) Glycosides are sugar derivaties attached to aglycones {Fetrow & Avila, 2000).

Glycosides are subdivided into cardiac glycosides, saponins, anthraquinones, cyanogenic glycosides, isothiocyanates, aldehydes, phenolics, alcohols, lactones and coumarins. Examples of glycosides used therapeutically are digoxin in allopathic medicine and anthraquinones which is used as laxative {Rotblatt & Ziment, 2002).

v) Another important compound is called terpene. Terpenes are derived from 5-carbon isoprene units. These terpene groups are subdivided into monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyterpene and

4 meroterpenes. Terpenoid compounds are mainly found in green vegetable, soy and

grains and many have antioxidant properties (Rotblatt & Ziment, 2002).

vi) Other compound like sterols group may benefit human; stigmasterol and

13-sitosterol may reduce low-density lipoproteins and lower serum cholesterol level

(Rotblatt & Ziment, 2002).

vii) Triterpenes glycosides have widespread distribution in plants and are sometimes

referred to as saponins as they have soap-like properties and readily form foams

(Heinrich eta/., 2004).

viii) Triterpenes are also components of resins and resinous exudates from plants (e.g frankincense and myrrh). Resins are produced following damage to the tree as a physical barrier to attack by fungi and bacteria. Additionally, many of the terpenoid components of these resins have high antimicrobial activity (Heinrich eta/., 2004).

ix) Tannin comprises of water-soluble polyphenolic compounds, which may have high molecular weight. They are broadly divided into two groups i.e. hydrolysable tannins, which are formed by the esterification of sugars with the simple phenolic acids that are shikimate-derived (e.g gallic acid) and non-hydrolysable tannins which occur due to polymerization reactions between flavonoids (Heinrich eta/., 2004).

1.2 Literature review of Misai Kucing (Orthosiphon stamineus, Benth)

1.2.1 Misai Kucing plant description

In general, Misai Kucing is a medicinal herb easily found in Southeast Asia. The leaves are arranged in opposite pairs. The petiole is relatively short, about 0.3 em in length and reddish purple in colour. The flowers are borne on verticals about 16 em length, white to bluish in colour with long far-exerted filaments, making it look like eat's

5 whispers (Wiart, 2002). However, there are many different types of Misai Kucing which

can be easily distinguished by its flower, these including with

white flower and Orthosiphon grandifolis with red colour flower. Identification of the

species of Misai Kucing is very important to control its therapeutic effects because

each species of Misai Kucing may have its own active ingredients and are not found in

other species of Misai Kucing.

Misai Kucing is also found in other countries such as Thailand, Indonesia and

Europe. In these countries, Misai Kucing is also known as yaa Nuat Maeo, Rau Meo or

Cay Bac (Thailand), Kumis Kucing or Remujung (Indonesia), moustaches de chat

(French) and Java Tea and Kidney Tea (European). In Malaysia, Misai Kucing can be

seen in the garden, roadsides and wastelands. Misai Kucing is easily propagated

through 3 or 4 noded stems cuttings from a mother plant of more than 5 months of age

(lndubala & Ng, 2000). Misai Kucing has been cultivated for a long time. However,

there are many important factors in determining the growth and yield of Misai Kucing.

These factors include population density, fertilizer, soil type and weather. Recently,

researchers from Malaysia Agriculture Research and Development Institute (MARDI)

showed that population density of 29 333 plants/hectare with spacing of 120 em between row and 45 em between plants within the row for Misai Kucing on bris soil significantly gave highest fresh and dry yield compared to population density of

21 333 plants/hectare or lower (Zaharah & Salbiah, 2004).

1.2.2 Classification of Misai Kucing

The features of Misai Kucing as described by Wiart (2002) is a beautiful , flower conspicuous, white and arranged in a terminal raceme. Stamens of Misai Kucing are long, expanding and shaped like eat's whiskers and its leaves are simple, without stipules and secussate, diamond-shaped, dark green above and paler below and secondary nerves 5-6. The Misai Kucing's stems are quadrangular with

6 purple lines at each corner and pithy (Figure 1.1 ). Based on the features described by

Wiart (2002), the of Misai Kucing is:

Class: Magnoliopsida.

Subclass: Asteridae

Order:

Family: Lamiaceae (or Labiate)

Genus: Orthosiphon

Species: stamineus

Scientific name: Orthosiphon stamineus, Benth

1.2.3 Pharmacologic effects of Misai Kucing (0. stamineus)

Misai Kucing became a popular herbal tea among the community of Southeast

Asia and European countries (lndubala & Ng, 2002). The Misai Kucing products are appearing in the forms of tea sachets, capsules and dried leaves. This plant is believed to have several pharmacological and therapeutic effects. The evaluations of Misai

Kucing extract for medicinal purposes are as below:

a) Kidney stone elimination effect: Traditionally, Misai Kucing has been widely used by Malaysian community as a diuretic and for treating catarrh of the bladder. Misai

Kucing has been used to eliminate stones in the bladder (lndubala & Ng, 2002). In

Malaysia, the leaves are used as diuretic and for treating catarrh of the bladder. A decoction prepared from the plants is used to eliminate stones in the bladder

(lndubala & Ng, 2002).

b) Diuretic effect Diuretic effect of Misai Kucing has been proven through scientific experiments. Hydroalcohol extract of 0. stamineus has been tested for their diuretic activities in rats and it revealed that they led to an increase in urine flow and urinary sodium excretion (Beaux eta/., 1999).

7 c) Cytotoxic effect: Some flavonoids and diterpenes isolated from Misai Kucing which

included 7,3',4'-tri-0-methylluteolin, eupatorin and ladanein showed cytotoxic activity

towards highly liver metastatic murine colon 26-LS carcinoma cells with the EDso value

between 10 ~g/ml to 90 ~g/ml (Tezuka et at., 2000). A mechanism which may be

responsible for the anticarcinogenic potency of the extract may be related to the

modulation of drug metabolising enzymes, for example, glutathione-S-transferase

(GST) involved in carcinogen detoxification.

d) Cardiovascular effect Methylripariochromene A, another flavonoid isolated from

aqueous extract of Misai Kucing exhibits hypotensive and vasodilating properties and

decreases the cardiac output in animals (Matsubara, 1999).

e) Anti-inflammatory effect: Researchers from Japan reported that two novel highly

oxygenated pimarane diterpenes known as orthosiphol A and B isolated from dry

leaves of Orthosiphon stamineus, Benth exert potent inhibitory activity against the inflammation induced by tumor promoters, TPA ( 12-0-tetradecanoylphorbol-13- acetate) on mouse ears (Masuda eta/., 1992).

f) Antioxidant effect Shantanova (1998) had reported that the administration of dry extract of Orthosiphon stamineus, Benth., Desmodium canadensis D.S., Poligonum aviculare L., Arctostaphylos uva-ursi L., to post-ischemic acute renal insufficiency white rats decreased the concentration of lipid peroxidase products in rat kidneys and markedly inhibit haemolysis. The inhibition of haemolysis was due to inactivation of free radical particles by binding it with phenolic compounds in the preparations. The leaves extract of herb are believed to have highest antioxidant property as compared to other part of herb studied (Chung eta/., 1998).

8 g) Nitric oxide inhibition effect: Three highly oxygenated isopimarane-type

diterpenes, (siphonol A-C) and a norisopimarane-type diterpene named siphonol E

were isolated from the methanolic extract of 0. stamineus sh0wed more potent

inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS) activated

macrophage-like J774.1 cells than NG-monomethyh-arginine (L-NMMA)

(Awale eta/., 2002).

1.2.4 Herb-drug interaction and adverse effects of methanol extract of Misai

Kucing

Induction of hepatic enzyme activities in animals following the treatment with many natural compounds or traditional medicines which include Gingko bi/oba

(Fessenden et a/., 2001), grapefruit juice (Lilja eta/., 2000; Kane & Lipsky, 2000),

St. John's Wort (Hypericum perforatum) (Durr et a/., 2000) and Kava

(Piper methysticum) (Almeida & Grimsley, 1996) had previously been reported.

However, there is no previous scientific study reported on the effect of standardised methanol extract of Misai Kucing on herb-drug interaction and toxicity in laboratory animals and in man. Therefore, we describe for the first time the effect of standardised methanol extract of Misai Kucing on phase I and phase II hepatic drug metabolising enzymes activity in rat liver and toxicity studies in rat following the Misai Kucing methanolic extract treatment. Therefore, our research provides very important information about safety and efficacy of the standardised methanol extract of Misai

Kucing.

9 Figure 1.1 Misai Kucing (Orthosiphon stamineus, Benth)

10 1.2.5 Evaluation of Misai Kucing ( 0. stamineus) chemical constituents

There are numerous factors which affect the quality, efficacy and safety of the extract obtained. These factors include growing conditions. method of drying and grinding, different processing methods and solvents used for extraction and also storage conditions (Rotblatt & Ziment, 2002). Good Agriculture Practice (GAP), Good

Processing Practice (GPP) and Good Manufacturing Practice (GMP) are the few followed guidelines in order to maintain the quality and standard of the Misai Kucing extract obtained.

In order to maintain the quality of the extract. leaves used for Misai Kucing extraction are collected in the late afternoon, from 30-45 days old white flowered plants. The leaves are chopped and dried at approximately 40°C for 3 days. Methanol extracts of Misai Kucing are prepared by using a proportion of 10 g dried leaves in

100 ml of methanol by warming for 4 hours at 40°C. The solution is filtered through filter paper (Whatman No.1). concentrated and spray-dried to obtain the crude methanol extract of Misai Kucing (Akowuah et a/.. 2004 ). Recent researchers from

Universiti Sains Malaysia had reported that rosmarinic acid is the main component in the standardised methanol extract of Misai Kucing with concentration ranging from

5.1 % to 29.9% of the total dry leaf weight. Concentrations of 3'-hydroxy-5,6,7,4'­ tetramethoxyflavone (TMF), eupatorin (EUP) and sinensetin (SEN) ranged from 0.05% to 0.69%, 0.34% to 3.37% and 0.22% to 1.76% respectively (Akowuah eta/., 2004).

Sixteen known compounds from methanol extract of Misai Kucing have been isolated and identified by Tezuka eta/ (2000). The identified compounds were five new isopimarane-type diterpenes (orthosiphols F-J] and two diterpenes [staminols A and B] with a novel carbon framework and three new highly-oxygenated stamina-type diterpenes [staminolactones A and B and norstaminol A]. Other compounds such as

II 7,3' ,4'-tri-0-methylluteolin, 5-hydroxy-6, 7 ,3' ,4'-tetramethoxyflavone, eupatorin,

sinensetin, salvigenin, ladanein, tetramethylscutellarein, 6-hydroxy-5, 7,4'-

trimethoxyflavone, vomifoliol, aurantiamide acetate, rosmarinic acid, caffeic acid,

oleanolic acid, ursolic acid, betulinic acid and ~-sitosterol are also identified in the

same extract (refer to table 1.1 ). Six phenolic compounds from leaves extract of Misai

Kucing were isolated and identified by Sumaryono et at (1991 ). These identified

phenolic compounds were caffeoyl tartrate, rosmarinic acid, aurantiamide acetate,

vomifoliol, caffeic acid and 2, 3-dicaffeoyl tartrate (refer table 1.2). Sinensitine,

eupatorin, rosmarinic-, cichoric- and caffeic-acids were the main identified

in two tinctures (A: 50 % v/v ethanol and B: 70% v/v ethanol) of Orthosiphon stamineus

(Oiah eta/., 2003).

1.3 Hepatic drug metabolism reactions

1.3.1 Introduction to drug metabolism

The liver is one of the hardest working organs in the body and works closely with other organs system in the body. It is the most abundant organ in the body for xenobiotic and drug metabolism and detoxification process (de Ia Iglesia eta/., 1999).

In general, the term "metabolism" refers to the sum of biochemical reactions in the body, including anabolism (building of complex) and catabolism (breakdown of a complex). However, drug metabolism or biotransformation is used to describe the conversion of drugs or other toxins into metabolites. These reactions are generally called biochemical breakdown of the drug. Biotransformation whether completed by the

P450 system or other enzyme system is rarely a simple process. These reactions involve several different processes concurrently and a single metabolic transformation does not usually occur.

12 Table 1.1 List of chemical constituents of Orthosiphon stamineus aerial part extract (Tezuka eta/., 2000)

Diterpenes Triterpenes Flavones orthosiphols F [1] Oleanolic acid [12] 7,3 ,4-tri- 0-methylluteolin [16] orthosiphols G [2] ursolic acid [13] eupatorin [17], orthosiphols H [3] betulinic acid [14] sinensetin [18], orthosiphols I [4] f3-sitosterol [15], 3'-hydroxy-5,6, 7 ,4·- tetramethoxyflavone [19] orthosiphols J [5] salvigenin [20], staminols A [6] ladanein [21], staminols 8 [7], Tetramethylscutallarein [22], staminolactones A [8] 6-hydroxy-5, 7,4- trimethoxyflavone [23], staminolactones 8 [9] kaempferoi-3-0-f3- glucoside [24], norstaminol A [1 0] quercetin-3-0-f3-glucoside [25], orthosipholl S [11]

The number in bracket are chemical compounds found in 0. stamineus and their individual chemical structures can be found in pages 15-17.

13 Table 1.2 List of chemical constituents of Orthosiphon stamineus leaves extract (Sumaryono eta/., 1991}

Phenolic acids caffeoyl tartrate [26] rosmarinic acid [27] aurantiamide acetate [28], vomifoliol [29], caffeic acid [30], 2, 3-dicaffeoyl tartrate [31]

The number in bracket are chemical compounds found in 0. stamineus and their individual chemical structures can be found in pages 17.

14 R30 y?' PhCOQ PhCOO '·· ...... /! PhCOO ····...... /! ~ 20 13 15 CH3COO ...... 141~ PhC09 ...... OH CH3COO .. , 0 '•. CH coo ... OH 3 '•, R o······ 1 ·· ..,,OR2 CH3coo·····

19 CH3coo······

R' = COCH3. R" = H, R'" = CoPh, Orthosiphol F [1] R = COCH3 , Staminol A [6]

R' = COCH3, R" = R"' = H, Orthosiphol G [2] R = H, Orthosipholl [4] R = H, Staminol 8 [7]

R' = R"= COCH3. R"' = COPh Orthosiphol H [3] R = COCH3 , Orthosiphol J [5]

Vl

15 16 ~ OBz ~ PhCOO,, ( HaD. : 12 OBz . FtilX) . FtlXX) ...... n.£Y'V'\ ... Phcoq ···· ...... / 0 CI¥IX? j . ~j\ CH3COO\. 17 \ :

CH co6 ~ 3 Orthosiphol S [11] Norstaminol A [1 0] Staminolactone A [8] Staminolactone 8 [9] ) ···· ... COOH COOH

::

Ursolic acid [13] Betulinic acid [14]

Oleanolic acid [12]

R, =R2 = R3 =OCH3 ,R4 =H ! Sinensetin [18] 0\ OCH3 R1 =OH, R2 =R3 =OCH3, R4 =H, H3CO R4 3'-hydroxy-5 ,6, 7 ,4'-tetramethoxyflavone [19] R2 R, R4 H, R2 OCH3, salvigenin [20] HO R1 0 = = = R1 =R4 =H, R2 =OH ladanein [21] 13-sitosterol [15] R1 = R2 = H, R4 = OCH3 , 7,3',4'-tri-0- R1 = CH3, R2 = OCH3, R4 = H methylluteolin [16] tetramethylscutellarein [22] R1 =R3 =OH , R2 =OCH3 , R4 =H R, = CH3, R2 = OH, R4 = H, 6-hydroxy- Eupatorin [17] 5,7,4'-trimethoxyflavone [23] OH OH 0 q .00 -0 OH OH ~roH 0 0 ~OH HO ~OH ~ OH ,f/ 0 ~OH OH OH 0 OH OH 0 OH OH Kaempferol-3-0-~-glucoside [24] OH Quercetin-3-0-~-glucoside [25]

Caffeoyl tartrate [26]

0 OH 0 •I 0 H -...) C -o =o Jo 0-:F. 0-C -:;:?' O 0 H / HO OH OH OH OH OH Rosmarinic acid [27] OH bH 2,3-Dicaffeoyl tartrate [31] PhCO-Phe-PheoJ---COCH3 Caffeic acid [30] Vomifoliol [29] Aurantiamide acetate [28] As shown in table 1.3, biotransformation reactions have been generally termed as phase I (functionalisation reaction), phase II (conjugation) and phase Ill (export of the conjugate from cells) (Timbrel!, 2001 ). In phase I reactions, xenobiutics are generally converted to more polar, hydroxylated derivatives and these derivatives are conjugated with molecules such as glucuronic acid, sulphate or glutathione in phase II reaction later and eventually excreted in the urine or bile (Murray, 1998). Xenobiotics that already possess a functional group can bypass phase I metabolism and directly participate in the conjugation reaction (loannides, 2002). In mammals, a majority of the enzyme systems that contribute to phase I and phase II metabolisms are encountered in every tissue but particularly in the endoplasmic recticulum and cytosolic fraction of the liver (loannides, 2002).

1.3.1.1 Phase I hepatic drug metabolism reaction

Main reactions of Phase I (functionalisation) liver metabolising process are oxidation, reduction and hydrolysis. The major organ that carries out phase I reaction is the liver and the major site within the liver is the endoplasmic reticulum of the liver cell

(Vainio & Hietanen, 1980). Some phase I hepatic enzymes are found in several subcellular compartments. Hepatic enzymes involved in the phase I reactions are distributed in many different compartment of the liver and have overlapping substrate specificities (refer table 1.4). a) Reduction

Reductase enzymes are found in gut flora and some mammalian tissues.

Nicotinamide adenine dinucleotide phosphate (NADPH) and Flavin adenine dinucleotide (FAD) are required in this reaction as electron donor but the function of

NADPH is inhibited by oxygen. The major reactions of reduction are azo and nitro reduction. Examples of reduction reactions are:

18 i) The conversion of prontosil to sulphanilamide (antibacterial drug) is a well-known

example of azo reduction reaction which is catalysed by the cytochrome P450 and

carried out by the reductase enzyme in the gut bacteria (Hodgson & Goldstein, 2001 ).

ii) Tertiary amide oxides are reduced by cytochrome P450.

iii) Hydroxylamines is reduced by cytochrome P450 and also reduced by a

flavoprotein which is part of a system which requires NADH and includes NADH

cytochrome bs reductase and cytochrome b5 (Timbrel!, 2000).

b) Oxidation

The cytochrome P-450s system is involved in many oxidation reaction including

xenobiotic activation and detoxification (Ortiz de Montellano, 1999). Monooxygenations

of xenobiotics are the major types of Phase I reaction (Nebbia, 2003). They are also

known as mixed-function oxidations. In general, the sequences of oxidation reactions

involve at least four distinct steps: (i) addition of substrate to the enzyme, (ii) donation of an electron, (iii) addition of oxygen and rearrangement and (iv) donation of a second electron and loss of water. Briefly, mixed-function oxidations involve reactions of one atom of the oxygen molecule is being accepted by the substrate while the other oxygen atom is reduced to water (Guengerich, 2002).

Oxidation reactions could be catalysed by enzymes known as microsomal or non microsomal oxidation enzymes. Non-microsomal oxidations are subdivided into amine oxidation, alcohol and aldehyde oxidation, dehalogenation, purine oxidation and aromatization. On the other hand, microsomal oxidation reactions include N-, S- and

0-dealkylation, N-hydroxylation, aliphatic hydroxylation, alicyclic hydroxylation,

N-oxidation and deamination (Timbrel!, 2000).

c) Hydrolysis

Major hydrolysis reactions are ester and amide hydrolysis

(Hodgson & Goldstein, 2001). Enzymes involved in catalysing hydrolysis reactions are carboxylesterases and amidases. Carboxylesterases and amidases are usually found

19 in the cytosol and some have been described in the microsomes. Hydrazines and carbamates may undergo hydrolysis reaction. The insecticide, carbaryl is hydrolysed to 1-naphthol by hydrolysis reaction (Timbrel~. 2000). d) Hydration

Epoxide may undergo addition of water upon hydration reaction. Hydration reaction involves addition of water to the epoxide catalysed by epoxide hydrolase, probably by nucleophilic attack by OH- on one of the carbon atom of the oxirane ring to yield a dihydrodiol (Gibson & Skett, 1994).

1.3.1.2 Phase II hepatic drug metabolism reaction

The phase II hepatic metabolising reactions include glucuronidation, glutathione conjugation, sulphate conjugation, amino acid conjugation, hydration, methylation and acetylation. Enzymes which are responsible for the phase II conjugation are located within various subcellular fraction of the liver (Table 1.5). Phase II reaction generally conjugates with a specific substitute group such as glucuronic acid, sulphate, amino acid or glutathione to the substrate and thereby increase its size and molecular weight.

However, methylation and acetylation are exceptions and they may decrease both the substrate polarity and its solubility in water (Murray, 1998).

Enzymes that catalyse phase II reactions similar to the cytochrome P450 system, are subjected to induction. Induction of phase II hepatic metabolising enzymes could be beneficial to the liver itself because it enhances the clearance of toxic or non toxic intermediate metabolites. Below are a few of common phase II reactions: i) Glucuronidation is the major metabolism pathway. In these reactions,

UDP-glucuronosyltransferase (UGT) or glucuronidase enzyme adds glucuronic acid to a substrate. The conjugation product has a relatively high molecular weight and is prone to excretion through bile and urine (Orzechowski eta/., 1994). The most readily conjugated functional groups are phenols, alcohols and carboxylic acids.

20 ii) Glutathione conjugation is catalysed by glutathione-S-transferase (GST) which is localized primarily in the cytosol and to much lesser extent in the endoplasmic reticulum. The major purpose of the glutathione conjugation appears to be the control of reactive electrophiles which are present naturally or form metabolism and have a deleterious effect on the body (Wilce & Parker, 1994). iii) During sulphate conjugation, inorganic sulphate, in the form of

3'-phosphoadenosine-5'-phosphosulphate (PAPS) is added to the molecule and this reaction is catalysed by sulphotransferase (Pacifici & Giuliani, 1997). The phenols and amino groups are among the groups to be sulphated to yield readily excretable sulphamates. iv) Acetylation is another important phase II reaction. An amide bond is formed between the amino group of the chemical and acetate. Aromatic and heterocyclic amines, hydrazines and sulphonamides may be undergoing acetylation reaction

(Murray, 1998). v) Glycine being the most common amino acid group is found to conjugate with xenobiotics. Other amino acids such as glutamine, taurine and ornithine are also important in the amino acid conjugation. Amino acid conjugation is formed by peptide bond between carboxyl groups of the xenobiotics with the a-amino group of the amino acid (Caldwell, 1982). vi) Methylation reaction plays an important role in the metabolism of endogenous substrates such as noradrenaline, neurotransmitters (Hirata, 1982) and polyphenolics such as the tea flavonoid, epicatechin. Methyltransferase are primarily cytosolic enzymes but are also found in the endoplasmic reticulum.

1.3.1.3 Isolation and subcellular fraction of the liver

Hepatic cells contain a multitude of metabolising enzymes that are usually localised in the specific organelles within the cells. Some of these enzymes may also be expressed in other specific organs. This study used freshly isolated perfused

21 hepatocytes and subcellular fraction of the liver as indicator of hepatic phase I and

phase II drug metabolism respectively.

Isolated perfused hepatocytes are prepared by using collagenase perfusion

technique and this technique is widely used by pharmacologists to study drug

metabolism (Chazouilleres et a/., 1989). Perfusions are normally performed in the

anterograde direction (from portal vein to cava vein) (Miller, 1973). Isolated perfused

hepatocytes preserve cells integrity and completely contain cell architecture such as

the membrane cell, surface receptor, nucleus and hepatic metabolic systems

(Axelsson eta/., 2003). Viable isolated hepatocytes are seen to undergo active DNA

synthesis, mitosis, cell proliferation and are more physiological as compared to

subcellular fraction preparation. In addition, all the cofactors and biochemical

substances needed for reactions are contained within the hepatocytes (Sinz, 1999).

Subcellular fractions of the liver consist of two different fractions known as liver

microsomes and the S-9 cytosolic liver fraction (Ekins et a/., 1999) (Figure 1.2). For

example, UDP-glucuronosyltransferases (UGT) enzymes are predominantly located

within the endoplasmic reticulum. Consequently, liver microsomes isolated from the

liver are most suitable to be used in studying glucuronidation reaction of the drug. The

advantages of using subcellular fractions in drug metabolism studies are the ease to

prepare, flexibility of incubation conditions such as cofactors, buffer, pH and

temperature and their stability in long-term storage under specific temperature and

condition. The disadvantages of hepatic subcellular preparation are the disruption of

the hepatic cellular heterogeneity by homogenisation and the limitations of sequential

metabolism that requires multiple cofactors to be completed. However, this limitation

becomes an advantage when the mechanism of the reactions can be controlled by adding suitable cofactor at a specific time and eases the detection at each single step of the drug metabolism reaction (Ekins eta/., 1999).

22 1.3.2 Herb-drug interaction

There is an increasing use of alternative medicine by the public with a variety of

plant-derived drugs or plant-based supplements which contain active compounds over the counter. Moreover, there are possibilities where patients seek herbalist treatment while taking prescribed medication. It is possible that one substance may alter the

bioavailability of another substance by inducing the phase I and phase II hepatic enzymes system and subsequently affect drug therapeutic effect or even generate toxicity when two or more substances are given concurrently. There are many documented reports about herb-drug interactions in human (Roblatt & Ziment, 2002;

Table 1.6). Herb-drug interaction can be defined as any alteration either in the pharmacokinetic or pharmacodynamic effect of the drug caused by concurrent treatment with herb and drug. Herb-drug interaction can be generally divided into pharmacokinetic and pharmacodynamic interactions (Sugiyama eta/., 2004).

23 Table 1.3 Major biotransformation reaction (Timbrel!, 2000).

PHASE I PHASE II PHASE Ill

Oxidation Glucuronidation Further metabolism of

Reduction Glutathione conjugation glutathione conjugate

Hydrolysis Sulphation

Hydration Acetylation

N Amino acid conjugation ~ Methylation