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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.
II
Chin Jin Han, 2006
Universiti Sains Malaysia
TABLE OF CONTENT
TITLE
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDICES
LIST OF ABBREVIATIONS
ABSTRAK
ABSTRACT
CHAPTER ONE: GENERAL INTRODUCTION
1.0
1.1
1.2
1.3
INTRODUCTION
Use of herbal medicine worldwide
1 1 .1 The usage of herbs to treat diseases
Literature review of Misai Kucing (Orthosiphon stamineus, Benth)
1.2.1 Misai Kucing plant description
1.2.2 Classification of Misai Kucing
1.2.3 Pharmacologic effects of Misai Kucing
1.2.4 Herb-drug interaction and adverse effects of methanol extract of Misai Kucing
1.2.5 Evaluation of Misai Kucing chemical constituents
Hepatic drug metabolism reactions
1.3.1 Introduction to drug metabolism
1.3.1.1 Phase I hepatic drug metabolism reaction
1.3.1.2 Phase II hepatic drug metabolism reaction
iii
Page
ii
iii
xvi
xix
XX
xxi
xxiv
xxvi
1
1
1
3
5
5
6
7
9
11
12
12
18
20
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
2.1
2.2
INTRODUCTION
Liver and drug metabolism
2.1.1 Liver perfusion technique and its application
Introduction to cytochrome P450
2.2.1 Nomenclature of cytochrome P450
2.2.2 Cytochrome P450 and its functions
2.2.3 Catalytic cycle of cytochrome P450
2.2.4 Induction mechanism for cytochrome P450
2.2.4.1 Induction of cytochrome P450 by active compounds in herbs
2.3 Aminopyrine metabolism in liver
2.4
2.3.1 Aminopyrine N-demethylase assay
2.3.2 Literature review on aminopyrine metabolism and N-demethylation pathway
Second messenger pathway involved in drug metabolism
2.4.1 G protein-coupled receptor (GPCR) signal transduction
2.4.2 Heterotrimetic G Protein in signal transduction
2.4.3 Cyclic adenosine monophosphate (cAMP) pathway
2.4.4 Cyclic guanosine monophosphate (cGMP) pathway
IV
36
36
37
38
38
38
41
42
42
43
43
48
49
49
50
51
51
2.5
2.6
2.7
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
Biological factors affecting phase I hepatic metabolism reaction 57
Objectives of the study 61
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 7 4
v
2.8
standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in SO rat hepatocytes
2.7.7.4 Statistical analysis
RESULTS
76
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
2.9
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
DISCUSSION
standardised methanol extract of Misai Kucing ( 0. stamineus) on aminopyrine N-demethylase activity in diabetic SO rat hepatocytes
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
3.1 Introduction to UDP-glucuronosyltransferase (UGT) reaction
3.1.1 Localisation and tissue distribution of UGT enzyme
3.1.2 Nomenclature of UGT multigene family
VII
109
109
111
111
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
3.6
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
3.5.12 GST assay
140
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
3.5.13 Statistical analysis
RESULTS
143
144
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
3.7
3.8
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
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
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 LD50 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
4.4
4.5
4.6
4.3.2 Blood serum biochemical analyses
4.3.2.1 Blood serum biochemical parameters of Liver function test
4.3.2.2 Blood serum biochemical parameters of Kidney function test
4.3.2.3 Blood serum Lipid profile
Factors that may affect the experimental results
4.4.1 Biological factors in experimental animals
4.4.2 Environment of experimental animals
4.4.3 Route of administration
Objectives of the study
METHODS AND MATERIALS
193
195
199
200
201
201
202
203
203
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
4.7
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
RESULTS
4.6.5.3.1 Acute (1 day treatment) anti-hepatotoxicity study 213
4.6.5.3.2 Sub-chronic (14 days treatment) anti-hepatotoxicity study
4.6.5.3.3 Blood sample collection and serum preparation
4.6.5.3.4 Percentage hepatocytes viability
4.6.5.3.5 Statistical analysis
214
214
215
215
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
4.8
4.9
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
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
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 plants 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
ADP
Ad libitium
AGC
ALD
ALT
ALP
AST
ATP
BSA
Ca/CaM
Ca2+
cAMP
cGMP
CDNB
DAG
DMSO
eta/
FAD
g
!Jg/ml
ng/ml
mg/kg
mg/ml
Go
GAP
LIST OF ABBREVIATIONS
Adenosine-5'-diphosphate
To be taken as wanted
adenyl guanyl cyclase
Approximate lethal dose
Alanine aminotransferase
Alkaline phosphatase
Aspartate aminotransferase
Adenosine-5'-triphosphate
Bovine serum albumin
Calcium calmodulin complex
Calcium ion
cyclic adenosine-3', 5'-monophosphate
cyclic guanosine-3', 5'-monophosphate
1-chloro-2,4-dinitrobenzene
Diacylglycerol
Dimethylsulphoxide
Else where or and others
Flavin adenine dinucleotide
Gram
microgram per milliliter
nanogram per milliliter
Miligram per kilogram
Miligram per mililiter
Alpha sub-unit of guanine nucleotide regulatory protein
Good Agriculture Practice
XXI
GGT
g/kg
GLP
GMP
G-protein
GSH
GST
GTP
HBSS
IBMX
KT5720
KT5823
LDso
ml
mmoi/L
nM
NADPH
NAPQI
pNP
NOAEL
NOEL
OECD
OA
Gamma-glutamyltransferase
Gram per kilogram
Good Laboratory Practice
Good Manufacture Practice
A guanine nucleotide regulatory protein
5' -Guanylylimidodiphosphate
Glutathione
Glutathione-S-transferase
Guanosine triphosphate
Hank's Balanced Salt Solution
3-isobutyl-1-methylxanthi ne
lnositol-1 ,4,5-triphosphate
Inhibition constant
Michaelis Menten constant
cAMP-dependent protein kinase inhibitor
cGMP-dependent protein kinase inhibitor
Dose caused 50% lethality population
Mililiter
Milimol per liter
nanomolar
Nicotinamide adenine dinucleotide phosphate (reduced)
N-acetyl-p-benzoquinone imine
p-nitrophenol
No observable Adverse effect level
No observable effect level
Organization for Economic Cooperation and Development
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, AL T, 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: Lamiaceae) 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
1.0 INTRODUCTION
CHAPTER ONE
GENERAL 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 phytochemical 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 Orthosiphon aristatus 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
flowering plant, 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 taxonomy of Misai Kucing is:
Class: Magnoliopsida.
Subclass: Asteridae
Order: Lamiales
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 1 0 g dried leaves in
1 00 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 polyphenols 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
Vl
R30 PhCOO '·· ...
~ 20
CH3COO ... ...
R1o······ 19
........ /! 13 15
141~ 0
OH ·· .. ,,OR2
R' = COCH3. R" = H, R'" = CoPh, Orthosiphol F [1]
PhC09
CH3coo ... '•,
CH3coo······
R' = COCH3, R" = R"' = H, Orthosiphol G [2] R = H, Orthosipholl [4]
y?'
R' = R"= COCH3. R"' = COPh Orthosiphol H [3] R = COCH3 , Orthosiphol J [5]
.. ..... OH
( ~ 15 16
FtilX) FtlXX) .......
CI¥IX? j . \ :
~
Staminolactone A [8]
0 17
HaD. : n.£Y'V'\ ...
~j\ PhCOO,, 12
Phcoq ···· ...
CH3COO\.
CH3co6
Staminolactone 8 [9] Norstaminol A [1 0]
PhCOQ PhCOO ···· ..
CH3COO .. , '•.
CH3coo·····
R = COCH3 , Staminol A [6]
R = H, Staminol 8 [7]
OBz ~
OBz .
Orthosiphol S [11]
. ..... /
........ /!
Oleanolic acid [12]
! 0\
HO
13-sitosterol [15]
COOH
::
Ursolic acid [13]
OCH3
H3CO R4
R2
R1 0
) ···· ...
Betulinic acid [14]
R, = R2 = R3 = OCH3 ,R4 = H
Sinensetin [18]
COOH
R1 = OH, R2 = R3 = OCH3, R4 = H,
3'-hydroxy-5 ,6, 7 ,4'-tetramethoxyflavone
[19]
R, = R4 = H, R2 = OCH3, salvigenin [20]
R1 = R4 = H, R2 = OH ladanein [21]
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
Eupatorin [17]
R, = CH3, R2 = OH, R4 = H, 6-hydroxy-
5,7,4'-trimethoxyflavone [23]
OH
0 OH
~OH .00
OH 0 OH
Kaempferol-3-0-~-glucoside [24]
0 -...)
Jo HO
OH
Rosmarinic acid [27]
PhCO-Phe-PheoJ---COCH3
Aurantiamide acetate [28]
OH
HO
OH 0
OH
~roH ~ OH
~OH OH
Quercetin-3-0-~-glucoside [25]
0
OH OH
bH Caffeic acid [30]
Vomifoliol [29]
0 ~0
q -0 OH
OH ,f/ 0
OH
OH
OH
Caffeoyl tartrate [26]
•I 0 H 0-:F C -o =o . 0-C
-:;:?' O 0 H /
OH OH OH
2,3-Dicaffeoyl tartrate [31]
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
N ~
Table 1.3 Major biotransformation reaction (Timbrel!, 2000).
PHASE I PHASE II
Oxidation Glucuronidation
Reduction Glutathione conjugation
Hydrolysis Sulphation
Hydration Acetylation
Amino acid conjugation
Methylation
PHASE Ill
Further metabolism of
glutathione conjugate
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