The Effects of Gynura Procumbens Extracts on Drug Metabolizing Enzymes

THE EFFECTS OF GYNURA PROCUMBENS EXTRACTS ON DRUG METABOLIZING ENZYMES

ATIQAH BINTI AFANDI

UNIVERSITI SAINS MALAYSIA

2015

THE EFFECTS OF GYNURA PROCUMBENS EXTRACTS ON DRUG METABOLIZING ENZYMES

ATIQAH BINTI AFANDI

Thesis submitted in fulfillment of the requirements for the degree of Master of Science

July 2015

ACKNOWLEDGEMENT

First and foremost, all praise be to Allah, the Almighty, the Benevolent for His blessing and guidance for giving me the patience and facilitate the completion of my thesis. I would like to express my gratitude to Prof. Dr. Sharif Mahsufi Mansor,

Director of Centre for Drug Research, for giving me the opportunity to continue my master study in this Centre as a full research master’s student and also providing me with facilities vital to the completion of my master study. I would like to extend my appreciation to my supervisor, Assoc. Prof. Dr. Sabariah Ismail for her constructive criticism, guidance, understanding and endless support during the completion of my study. I am thankful to all lab assistants and staffs of Centre for Drug Research for their assistance during the research, especially Nuraziah Hanapi, Nur Sabrina Mohd

Yusof and Aznorhaida Ramli for their continuous encouragement. I would like to express my special appreciation to all who have helped in one way or another, especially my dearest lab mates and friends, Nurul Afifah Mohd Salleh, Nor Liyana

Mohd Salleh, Zulhilmi Husni and Munirah Haron for their sound judgements and moral support during my study. My special gratitude to the USM Graduate Assistant

Scheme, My Brain 15 by the Ministry of Higher Education Malaysia and Short Term

Grant Scheme (Modulation of Drug Metabolizing Enzyme Activity by Gynura procumbens Standardized Extracts) for their financial support in these two years.

Finally, I owe deepest gratitude to my dear husband Mohd Halimhilmi Zulkiffli, who supports me, giving me strength to finish up my thesis and also to my lovely parents, brothers and sisters for their endless love, prayers and moral support. I am indebted and grateful to those who indirectly contributed to this research.

Last but not least, I would like to thank my son, Ahmad Luthfi Hakim bin Mohd

Halimhimi (3 months old), who being such a good son while I’m doing my thesis correction. Thank you very much.

Atiqah binti Afandi

USM, July, 2015

TABLE OF CONTENTS

Page

Acknowledgement ii

Table of Contents iii

List of Tables viii

List of Figures x

List of Symbols xviii

List of Abbreviations xix

List of Appendices xix

Abstrak xxiv

Abstract xxvi

CHAPTER ONE – INTRODUCTION 1

1.1 Background of the study 1

1.2 The Problem Statement of the Study 8

1.3 The purpose of the study 8

1.4 The objectives of the study 9

CHAPTER TWO - LITERATURE REVIEWS

2.1 Description of Gynura procumbens 10

2.2 Taxonomy of Gynura procumbens 11

2.3 Pharmacological potentials of Gynura procumbens 12

2.4 Phytochemical constituents of Gynura procumbens 13

2.5 Phytochemical analysis of Gynura procumbens 14

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2.6 Drug metabolism 15

2.7 Phase I drug metabolizing enzymes 17

2.8 CYP3A4 Isoform 19

2.9 CYP1A2 Isoform 23

2.10 P450-GloTM screening system 26

2.11 Phase II drug metabolizing enzymes 27

2.12 UDP-Glucuronosyltransferases (UGTs) 27

2.13 Glutathione S-transferases (GSTs) 30

2.14 In-vitro tools in herb-drug interaction studies 33

CHAPTER THREE - MATERIALS AND METHODS 36

3.1 Chemicals and reagents 36

3.2 Equipment and Instruments 37

3.3 Extraction process of Gynura procumbens 38

3.3.1 Gynura procumbens leaves collection 38

3.3.2 Preparation of Gynura procumbens Methanol Extract 38

3.3.3 Preparation of Gynura procumbens Ethanol Extract 38

3.3.4 Preparation of Gynura procumbens Aqueous Extract 39

3.4 HPLC profiling of Gynura procumbens extracts 39

3.4.1 Preparation of sample and standard solution 39

3.4.2 Chromatographic conditions 40

3.4.3 Quantification of kaempferol-3-O-rutinoside and 40 astragalin in Gynura procumbens extracts

3.5 Antioxidant studies on Gynura procumbens extracts 40

3.5.1 Determination of total phenolic content of Gynura 40 procumbens extracts

3.5.2 Determination of total flavonoid content of Gynura 41 procumbens extracts

3.5.3 Evaluation of antioxidant activity of Gynura procumbens 42 extracts

3.6 Phase I CYP450 enzymes inhibition assay 43

3.6.1 Preparation of plant extracts samples and positive controls 43

3.6.2 Generating a D-luciferin standard curve 44

3.6.3 Evaluation on the effect of Gynura procumbens extracts 45 and known inhibitors on CYP450 enzyme activity

3.7 Determination of protein content in RLM and RLCF 48

3.8 Preparation of plant extracts samples and positive controls 49

3.9 Phase II UGT enzymes inhibition assay 49

3.9.1 Preparation of p-nitrophenol (pNP) standard curve 49

3.9.2 Optimization of UGT enzyme assay parameters 50

3.9.2 (a) Linearity of incubation time 50

3.9.2 (b) Linearity of protein concentration 51

3.9.3 (c) Optimization of triton X-100 52

3.9.3 Determination of maximal velocity of reaction (Vmax) and 52

Michaelis constant (Km) of pNP glucuronidation

3.9.4 Evaluation on the effect of Gynura procumbens extracts 53 and known inhibitors on UGT enzyme activity

3.10 Phase II GST enzymes inhibition assay 55

3.10.1 Linearity of incubation time and protein concentration 55

3.10.2 Evaluation on the effect of Gynura procumbens extracts 56 and known inhibitor on GST enzyme activity

3.11 Statistical Analysis 57

CHAPTER FOUR - RESULT 58

4.1 Extraction process of dried leaves of Gynura procumbens 58

4.2 Quantification of Kaemperol-3-O-rutinoside and astragalin in 58 Gynura procumbens extracts

4.3 Antioxidant properties of Gynura procumbens extracts 64

4.3.1 Total phenolic content of Gynura procumbens extracts 64

4.3.2 Total flavonoid content of Gynura procumbens extracts 65

4.3.2 DPPH Free Radical Scavenging Capacity of Gynura 67 procumbens extracts

4.4 Phase I CYP450 enzymes inhibition assay 68

4.4.1 D-Luciferin Standard Curve 68

4.4.2 The effect of known CYP450 inhibitors on CYP450 70 isoforms activity

4.4.3 The effect of Gynura procumbens ethanol extract on 71 CYP450 isoforms

4.4.4 The effect of Gynura procumbens methanol extract 73 on CYP450 Isoforms

4.4.5 The effect of Gynura procumbens aqueous extract 75 on CYP450 isoforms

4.5 Determination of protein content in RLM and RLCF 77

4.6 Phase II UGT enzyme inhibition assay 79

4.6.1 p-nitrophenol (pNP) standard curve 79

4.6.2 Optimization of UGT enzyme assay parameters 80

4.6.2 (a) Incubation time 80

4.6.2 (b) Protein concentration 81

4.6.2 (c) Optimization of triton X-100 82

4.6.3 Determination of maximal reaction velocity (Vmax) and 83

Michaelis constant (Km) of UGT enzyme inhibition assay

4.6.4 The effect of known UGT inhibitor and Gynura 84 procumbens extracts on UGT enzyme activity

4.7 Phase II GST enzyme inhibition assay 87

4.7.1 Optimization of incubation time and protein concentration 87

4.7.2 The effect of known GST inhibitor and Gynura 89 procumbens extracts on GST enzyme activity

3.7.2 Inhibitory Effect of Known Inhibitor (Tannic Acid) on 75 GST Enzyme Assay

3.7.3 Inhibitory Effect of Gynura procumbens Extracts on 76 GST Enzyme Assay

CHAPTER FIVE - DISCUSSION 92

5.1 Extraction process of Gynura procumbens and quantification of 92 its marker compounds

5.2 Total phenolic content, total flavonoid content and antioxidant 94 activity of Gynura procumbens extracts

5.3 The effect of Gynura procumbens extracts on human CYP3A4 96 and CYP1A2 enzyme activity using recombinant enzymes

5.4 The effect of Gynura procumbens extracts on rat UGT enzyme 102 activity using RLM

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5.5 The effect of Gynura procumbens extracts on rat GST enzyme 105 activity using RLCF

CHAPTER SIX - CONCLUSION 109

REFERENCES 111

APPENDICES 126

LIST OF PUBLICATIONS 131

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LIST OF TABLES

Page Table 1.1 Interactions between herbal medicines and drug 3 metabolizing enzymes.

Table 2.1 List of factors that affect the activity of drug metabolizing 16 enzymes

Table 2.2 List of CYP3A4 substrates 22

Table 2.3 List of CYP1A2 substrates 25

Table 4.1 Type of extract and percentages of yield obtained for each 58 extraction method.

Table 4.2 Retention time of standard marker compound in Gynura 59 procumbens extracts.

Table 4.3 The amount of kaempferol-3-O-rutinoside and astragalin in 63 Gynura procumbens extracts.

Table 4.4 Total phenolic content of Gynura procumbens extracts. 65

Table 4.5 Total flavonoid content of Gynura procumbens extracts. 66

Table 4.6 IC50 values for DPPH scavenging activity of Gynura 68 procumbens extracts. Values are expressed as mean in microgram per milliliter of Gynura procumbens extracts ± SEM for four replicates (n=4).

Table 4.7 IC50 values of positive controls of CY1A2 and CYP3A4 70 enzymes.

Table 4.8 The half maximal inhibitory concentration (IC50) of Gynura 77 procumbens extracts on the metabolism mediated by CYP3A4 and CYP1A2.

Table 4.9 Concentration of protein in rat liver microsome (RLM) and 78 rat liver cytosolic fraction (RLCF) for male Sprague Dawley rats.

Table 4.10 Enzyme kinetic parameters for UGT enzyme-mediated pNP 84 glucuronidation in RLM.

Table 4.11 Relative activity of UGT enzyme after incubated with 86 Gynura procumbens extracts and diclofenac at three different concentrations of 10, 100 and 1000µg/mL.

Table 4.12 IC50 values for Gynura procumbens extracts and tannic acid 91 showing inhibition towards 1-chloro-dinitrobenzene (CDNB) conjugation reaction catalyzed by GST enzyme.

Table 5.1 The structure of flavones. 99

LIST OF FIGURES

Page Figure 1.1 The experimental design for the effects of Gynura 7 procumbens extracts on drug metabolizing enzymes study

Figure 2.1 Gynura procumbens 10

Figure 2.2 Capsule of Gynura procumbens sold in the market. Pictures 11 are adopted from panels (a) www.sambungnyawa.com; (b) www.etalasemuslim.com; and (c) www.istanaherbal.com.

Figure 2.3 Conversion of derivatives of D-luciferin to D-luciferin by 26 CYP450 enzymes. * Indicates the site of modification by CYP450 enzymes.

Figure 2.4 Glucuronidation reaction of nucleophilic substrate p- 29 nitrophenol (pNP) with UDPGA catalyzed by UDP- Glucuronosyltransferases (UGT) enzymes.

Figure 2.5 Conjugation reaction of electrophilic substrate 1-chloro-2,4- 31 dinitrobenzene with glutathione (GSH) catalyzed by glutathione S-transferase (GST) enzymes

Figure 2.6 Pathway of paracetamol metabolism 32

Figure 4.1 HPLC chromatograms of the reference standards (A) and 60 methanol extract of Gynura procumbens (B)

Figure 4.2 HPLC chromatograms of the ethanol extract of Gynura 61 procumbens (A) and aqueous extract of Gynura procumbens (B) Figure 4.3 Calibration curve of reference standard of kaempferol-3-O- 62 rutinoside.

Figure 4.4 Calibration curve of reference standard of astragalin. 63

Figure 4.5 Standard curve of gallic acid. A range of gallic acid 64 concentrations (0.02 – 0.64mg/mL) were incubated with Folin-Ciocalteu phenol reagent for 3 minutes, mixed with

Na2CO3 (1N) and the absorbance were measured at 725nm. Each point represent the mean absorbance values of gallic acid detected for various gallic acid concentrations ± SD for four replicates (n=4).

Figure 4.6 Standard curve of quercetin. A range of quercetin 66 concentrations (0.02 – 0.64mg/mL) were incubated with

NaNO2 (5%) solution for 6 minutes; mixed with AlCl3 (10%) solution and the absorbance were measured at 510nm. Each point represent the mean absorbance values of quercetin detected for various quercetin concentrations ± SD for four replicates (n=4).

Figure 4.7 DPPH scavenging effect of test samples which are Gynura 67 procumbens extracts (methanol, ethanol and aqueous extracts) and ascorbic acid (positive control) with concentration ranged from 0.00625µg/mL to 0.8µg/mL for all Gynura procumbens extracts and ascorbic acid. Each point represent the mean of percentages of DPPH scavenging effect ± SEM for four replicates (n=4).

Figure 4.8 Standard curves of D-luciferin attained from the incubation 69 of standard D-luciferin (concentration ranged from 0.016µM - 2µM) with non-CYP1A2 enzyme and luciferin methyl ether (substrate for CYP1A2).

Figure 4.9 Standard curves of D-luciferin attained from the incubation 70 of standard D-luciferin (concentration ranged from 0.016µM - 2µM) with non-CYP3A4 enzyme and luciferin benzyl ether (substrate for CYP3A4).

Figure 4.10 Inhibition of concentrations (0.1 - 250µg/mL) of ethanol 71 extract of Gynura procumbens leaves in formation of D- luciferin generated by dealkylation reaction of CYP3A4 and CYP1A2 substrates in the presence of human CYP3A4 (1.0pmole) and CYP1A2 (0.5pmole) recombinant enzymes during 30 minute and 10 minute at 37ºC respectively. The values were expressed as the mean percentage activity relative to control ± SD for three replicates (n=3). Statistical analysis was conducted using one way ANOVA followed by Dunnet’s test. * indicates significant difference from control (without Gynura procumbens extracts) (p < 0.05).

Figure 4.11 Inhibitory effect of ethanol extract of Gynura procumbens 72 on D-luciferin formation by CYP450 isoforms. Concentration of ethanol extract ranged from 0.1µg/mL – 250µg/mL. Data are expressed as the mean percentage activity relative to control ± SEM for three replicates (n = 3). Goodness of fit values were greater than 0.9.

Figure 4.12 Inhibition of concentrations (0.1 - 250µg/mL) of methanol 73 extract of Gynura procumbens leaves in formation of D- luciferin generated by dealkylation reaction of CYP3A4 and CYP1A2 substrates in the presence of human CYP3A4 (1.0pmole) and CYP1A2 (0.5pmole) recombinant enzymes during 30 minute and 10 minute at 37ºC respectively. The values were expressed as the mean percentage activity relative to control ± SD for three replicates (n=3). Statistical

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analysis was conducted using one way ANOVA followed by Dunnet’s test. * indicates significant difference from control (without Gynura procumbens extracts) (p < 0.05).

Figure 4.13 Inhibitory effect of methanol extract of Gynura procumbens 74 on D-luciferin formation by CYP450 isoforms. Concentration of methanol extract ranged from 0.1µg/mL – 500µg/mL. Data are expressed as the mean percentage activity relative to control ± SEM for three replicates (n = 3). Goodness of fit values were greater than 0.9.

Figure 4.14 Inhibition of concentrations (0.1 - 250µg/mL) of aqueous 75 extract of Gynura procumbens leaves in formation of D- luciferin generated by dealkylation reaction of CYP3A4 and CYP1A2 substrates in the presence of human CYP3A4 (1.0pmole) and CYP1A2 (0.5pmole) recombinant enzymes during 30 minute and 10 minute at 37ºC respectively. The values were expressed as the mean percentage activity relative to control ± SD for three replicates (n=3). Statistical analysis was conducted using one way ANOVA followed by Dunnet’s test. * indicates significant difference from control (without Gynura procumbens extracts) (p < 0.05).

Figure 4.15 Inhibitory effect of aqueous extract of Gynura procumbens 76 on D-luciferin formation by CYP450 isoforms. Concentration of aqueous extract ranged from 0.1µg/mL – 500µg/mL. Data are expressed as the mean percentage activity relative to control ± SEM for three replicates (n = 3). Goodness of fit values were greater than 0.9.

Figure 4.16 Standard curves of bovine serum albumin (BSA) attained 78 from one of the incubation of standard BSA (concentration ranged from 25µg/mL - 400µg/mL) with Lowry’s reagent and Folin-Ciocalteu reagent. Absorbance was measured at

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750nm. Each point represent the mean of absorbance value from the protein ± SD for four replicates (n=4).

Figure 4.17 Standard curve of p-nitrophenol (pNP). Incubation mixtures 79 for constructing the standard curve of p-nitrophenol (pNP) (200µL) consist of standard pNP (concentration ranged from 0.2mM - 1.0mM), 100mM of Tris-HCl, and 50mM

MgCl2. Each point represent the mean absorbance value of p-nitrophenol (pNP) detected for various p-nitrophenol (pNP) concentration ± SD for four replicates (n=4).

Figure 4.18 Optimization of time incubation. The conjugation reaction 80 of p-nitrophenol (pNP) catalyzed by UGT enzyme was performed in the total incubation volume of 200µL in the presence of pNP (500µM) and rat liver microsomes (0.1 mg) for 0 – 60 at 37ºC. Each point represent the mean in nanomole per milligram of p-nitrophenol (pNP) glucuronide formed in various time incubation ± SD for four replicates (n=4).

Figure 4.19 Optimization of protein concentration. The conjugation 81 reaction of p-nitrophenol (pNP) catalyzed by UGT enzyme was performed in the total incubation volume of 200µL in the presence of pNP (500µM) and rat liver microsomes (0 to 0.4mg) for 30 minutes at 37ºC. Each point represent the mean in nanomole per milligram of p-nitrophenol (pNP) glucuronide formed in various protein concentration ± SD for four replicates (n=4).

Figure 4.20 Optimization of triton X-100 concentration. The 82 conjugation reaction of p-nitrophenol (pNP) catalyzed by UGT enzyme was performed in the total incubation volume of 200µL in the presence of pNP (500µM) and the rat liver

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microsomes (0.1mg) was activated by triton X-100 (0.005 to and 0.04% (%v/v)) for 30 minutes at 37ºC. Each point represent the mean in nanomole per milligram of p- nitrophenol (pNP) glucuronide formed in various protein concentration ± SD for four replicates (n=4).

Figure 4.21 Hyperbolic relationship between the rate of formation of 83 pNP glucuronide (nmole/min/mg) and the concentration of pNP (µM). The reaction (200µL) was performed in the presence of RLM (0.5mg/mL) and pNP (concentration ranged from 50 – 3000µM). Each point represent the mean of nmol per minute per miligram of pNP glucuronide formed ± SEM of three replicates (n=3).

Figure 4.22 Effect of Gynura procumbens extracts on pNP 85 glucuronidation. The reaction was performed in the presence of pNP (0.5mM), RLM (0.5mg/mL) at three different concentrations of the extracts and diclofenac (10, 100, 1000µg/mL). Each bar represents the mean percentage activity relative to control ± SD for five replicates (n=5). Statistical analysis was conducted using one way ANOVA followed by Dunnet’s test. * indicates significant difference from control (without inhibitor) (p < 0.05).

Figure 4.23 Optimization of time incubation. The conjugation reaction 87 of 1-chloro-dinitrobenzene (CDNB) catalyzed by GST enzyme was performed in the total incubation volume of 300µL in the presence of 1-chloro-dinitrobenzene (CDNB) (1mM) substrate for GST enzyme and the rat liver cytosolic fraction (RLCF) (0.125mg/mL) for 0 – 5 minutes reaction time. Each point represent the absorbance of dinitrobenzene-glutathione conjugate formed in various time incubation ± SD for five replicates (n=5).

Figure 4.24 Optimization of protein concentration. The conjugation 88 reaction of 1-chloro-dinitrobenzene (CDNB) catalyzed by GST enzyme was performed in the total incubation volume of 300µL in the presence of 1-chloro-dinitrobenzene (CDNB) (1mM) substrate for GST enzyme and the rat liver cytosolic fraction (RLCF) (0.0625 to 4mg/mL) for 5 minutes reaction time. Each point represent the absorbance of dinitrobenzene-glutathione conjugate formed in various time incubation ± SD for five replicates (n=5).

Figure 4.25 Effect of Gynura procumbens extracts on CDNB 89 conjugation reaction. The conjugation reaction was performed in the presence of 1-chloro-dinitrobenzene (CDNB) substrate of GST enzyme (1mM) and rat liver cytosolic fraction (RLCF) (0.125mg/mL) at five different concentrations of Gynura procumbens extracts (0.01 to 250µg/mL). Each bar represents the mean percentage of activity relative to control ± SD for five replicates (n=5). Statistical analysis was conducted using one way ANOVA followed by Dunnet’s test. * indicates significant difference from control (without Gynura procumbens) (p < 0.05).

Figure 4.26 Inhibitory effect of the tannic acid and Gynura procumbens 91 extracts on CDNB conjugation reaction catalyzed by the GST enzyme from rat liver cytosolic fraction. Concentration of all extracts and tannic acid ranged from 0.01 – 250 µg/mL. Data are expressed as the mean percentage activity relative to control ± SEM for five replicates (n=5). Goodness of fit values were greater than 0.9.

Figure 5.1 The structure of quercetin 96

Figure 5.2 Structure of flavone, chalcone and coumarins 107

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LIST OF SYMBOLS

% Percentage sign

°C Degree Celsius

µg Microgram

μg/mL Microgram per milliliter

μL Microliter

μM Micromolar g Grams mg Milligram min Minute mM Milimolar pmol Picomole nmole Nanomole

R2 Coefficient of determination v/v Volume over volume

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LIST OF ABBREVIATIONS

AICI3 Aluminium chloride

ANOVA Analysis of variance

BSA Bovine serum albumin

CDNB 1-chloro-2,4-dinitrobenzene

CuSO4.5H2O Copper(II) sulfat pentahydrate

CYP450: Cytochrome P450

DPPH 2,2-diphenyl-1-picrylhydrazyl

DSHEA Dietary Supplement Health and Education Act

DOX Doxorubicin

EGCG Epigallocatechin-3-gallate

FDA Food and Drug Administration

FMO Flavin monooxygenase

GAE Gallic acid equivalent

GSH Glutathione

GST Glutathione S-transferases

HPLC High Perfomance Liquid Chromatography

HPTLC High Perfomance Thin Layer Chromatography

IC50 Half maximal inhibitory concentration

Ki Inhibitor constant

Km Michaelis-Menten constant

Luciferin-BE Luciferin benzyl ether

Luciferin-ME Luciferin methyl ether

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MgCl2 Magnesium chloride

MMP-1 Matrix metalloproteinase-1

MMP-9 Matrix metallopeptidase 9

MS Mass spectrophotometry

Na2CO3 Sodium carbonate

NADPH Nicotinamide adenine dinucleotide phosphate hydrogenase

NADP+ Nicotinamide adenine dinucleotide phosphate

NaK Tartrate Sodium potassium tartrate

NaNO2 Sodium nitrite

NaOH Sodium hydroxide

NAPQI N-acetyl-p-benzoquinone imine

NATs N-acetyltransferases

PAH Polycyclic aromatic hydrocarbon pNP Para-nitrophenol

QE Quercetin equivalent

RLM Rat liver microsome

RLU Relative Light Unit

ROS Reactive oxygen species

SEM Standard error mean

SULTs Sulfotransferases

TCA Trichloroacetic acid

TIM Traditional Indian Medicine

Tris-HCl Tris-hydrochloride

UDPGA Uridine 5’-diphospho-glucuronic acid

UGT Uridine-diphospho-glucuronosyl transferases

US United States

Vmax Maximal reaction velocity

WHO World Health Organization

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LIST OF APPENDICES

Appendix A Animal Ethical Clearance Letter

Appendix B Concentration of Reaction Components in the P450-GloTM Screening Systems.

Appendix C Preparation of CYP3A4 and CYP1A2 Reaction Mixture

Appendix D Preparation of Control Reaction Mixture for CYP3A4 and CYP1A2

Appendix E Preparation of CYP3A4 and CYP1A2 NADPH Regeneration System

Appendix F Percentage of CYP3A4 Specific Activity when Ketoconazole was added into the Reaction Mixture.

Appendix G Percentage of CYP1A2 Specific Activity when α- naphthoflavone was added into the Reaction Mixture.

Appendix H Percentage of UGT Specific Activity when Diclofenac was added into the Reaction Mixture

Appendix I Non-linear regression plot for diclofenac, positive inhibitor of UGT enzyme

Appendix J Percentage of GST Specific Activity when Diclofenac was added into the Reaction Mixture.

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KESAN EKSTRAK GYNURA PROCUMBENS TERHADAP ENZIM

METABOLISME DRUG

ABSTRAK

Kebangkitan penggunaan ubat-ubatan herba di seluruh dunia, dan penggunaan bersama drug konvensional dan terapi tradisional telah menjadi kebiasaan. Gynura procumbens ialah herba malar hijau yang lazimnya dimakan secara mentah atau diminum sebagai teh di Malaysia. Walaupun pelbagai kajian mengenai aktiviti farmakologi Gynura procumbens telah dijalankan, interaksi di antara herba ini dengan enzim metabolisme drug masih tidak diketahui. Ini adalah kajian pertama berkaitan dengan modulasi ekstrak Gynura procumbens terhadap enzim metabolisme drug. Pengekstrakan daun Gynura procumbens dengan air, etanol dan metanol masing-masing menghasilkan peratusan hasil sebanyak 27.50%,

7.80% dan 4.20%. Kuantifikasi dua sebatian penanda kaempferol-3-O-rutinosida dan astragalin di dalam setiap ekstrak adalah berjaya kecuali ekstrak akueus Gynura procumbens. Setiap ekstrak etanol dan metanol masing-masing mengandungi kaempferol-3-O-rutinosida dan astagalin sebanyak 1.60% dan 1.79% dan 2.33% dan

3.83%. Bagaimanapun, kaempferol-3-O-rutinosida dan astragalin tidak dapat dikenal pasti di dalam ekstrak akueus. Kandungan sebatian fenolik di dalam ekstrak Gynura procumbens boleh disenaraikan dalam turutan menurun seperti berikut: metanol > etanol > akueus, manakala kandungan sebatian flavonoid di dalam ekstrak Gynura procumbens boleh disenaraikan dalam turutan menurun seperti etanol >metanol > akueus. Ekstrak metanol menunjukkan penghapus radikal bebas yang paling aktif berbanding ekstrak lain.

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Ekstrak etanol menunjukkan kesan perencatan yang kuat terhadap enzim CYP3A4,

CYP1A2 dan GST masing-masing dengan nilai IC50 32.01 ± 1.11 µg/mL, 7.87 ± 1.22

µg/mL dan 44.62 ± 1.12 µg/mL. Bagaimanapun, ekstrak etanol tidak menunjukan perencatan yang ketara terhadap enzim UGT. Ekstrak methanol menunjukkan kesan perencatan yang lemah terhadap enzim CYP3A4, CYP1A2 dan GST masing-masing dengan nilai IC50 lebih daripada 100 µg/mL. Ekstrak metanol juga tidak menjejaskan enzim UGT. Ekstrak akueus sebaliknya, tidak menunjukkan kesan perencatan terhadap semua enzim yang dikaji. Kesimpulannya, perencatan enzim metabolisme drug mengikut turutan kandungan sebatian flavonoid (ekstrak etanol > ekstrak metanol > ekstrak akueus) iaitu semakin tinggi kandungan sebatian flavonoid, semakin kuat perencatan enzim metabolisme drug.

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THE EFFECTS OF GYNURA PROCUMBENS EXTRACTS ON DRUG

METABOLIZING ENZYMES

ABSTRACT

Resurgence in the use of herbal medicines worldwide and the co-use of conventional drug and traditional therapies is becoming more common. Gynura procumbens is an evergreen herb that has been commonly eaten raw or drink as tea in Malaysia. Despite various studies conducted on the pharmacological activities of

Gynura procumbens, the interaction between this herb with drug metabolizing enzymes is still unknown. This is the first study regarding the modulation of Gynura procumbens extracts towards drug metabolizing enzymes. Extraction of Gynura procumbens leaves with water, ethanol and methanol produced a percentage of yields of 27.50%, 7.80% and 4.20% respectively. Quantification of two marker compounds kaempferol-3-O-rutinoside and astragalin in each extracts is successful except for the aqueous extract of Gynura procumbens. Each ethanol and methanol extracts contain

1.60% and 1.79% and 2.33% and 3.83% of kaempferol-3-O-rutinoside and astragalin respectively. Kaempferol-3-O-rutinoside and astragalin, however, could not be identified in aqueous extract. The content of phenolic compounds in Gynura procumbens extracts can be ranked in decreasing order as methanol extract > ethanol extract > aqueous extract, whereas the content of flavonoid compounds in Gynura procumbens extracts can be ranked in decreasing order as follows: ethanol extract > methanol extract > aqueous extract.

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The methanol extract of Gynura procumbens exhibited the most active free radical scavenger compared to the other extracts. Ethanol extract exhibited strong inhibitory effect on CYP3A4, CYP1A2 and GST enzyme with IC50 values of 32.01 ±

1.11µg/mL, 7.87 ± 1.22µg/mL and 44.62 ± 1.12µg/mL respectively. However, ethanol extract did not show significant inhibition on UGT enzymes. Methanol extract exhibited weak inhibitory effect on CYP3A4, CYP1A2 and GST enzymes with IC50 values more than 100 µg/mL. Similar to ethanol extract, methanol extract also did not affect UGT enzymes. Aqueous extract, on the other hand, demonstrated no inhibitory effect on all enzymes studied. In conclusion, the inhibition of drug metabolizing enzymes in this study follows the rank order of total flavonoid content

(ethanol extract > methanol extract > aqueous extract) in which the higher the total flavonoid content, the stronger the inhibition of drug metabolizing enzymes studied.

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CHAPTER 1

INTRODUCTION

1.1 Background of the study

Herbal medicine has gained great attention throughout the world. Herbal medicine is defined as a medicine which is made from plants, including seeds, berries, roots, leaves, bark. Herb plants have been used for medicinal treatment as early as 3,000

BC by the ancients Chinese and Egyptians. Africans and native Americans have also used herbs in their healing rituals while the Indians practiced the use of herbs in

Ayurveda treatment, which is a system of traditional Indian medicine (TIM)

(Patwardhan et al., 2005). In the latest issue of Herbal Gram, American Botanical

Council reported herbal supplement sales in all channels reached up to $5.3 billion in the United States in 2011, which is an increase of 4.3% of the total sales in 2010

(Schultz, 2012). Herbal medicine has been receiving continuous overwhelming response from all over the world because it is safe, sustainable, readily available (can be eaten raw) and it is an alternative medicine to conventional drug which is usually expensive. In the year 1994, U.S. Dietary Supplement Health and Education Act

(DSHEA) have classified herbal medicine as ‘dietary supplement’. As a result, herbal medicines, unlike prescription drug, can be sold without prior safety and effectiveness tests and if the U.S Food and Drug Administration (FDA) prove a particular herbal medicine as harmful and dangerous for human consumption, only then it will be removed from the market (Brent and Bauer, 2003).

There is huge demand for herbal medicinal plants from pharmaceutical companies, phytopharmaceutical companies, health product companies, traditional and alternative practitioners due to their great medical and health benefits. Currently, there are 40 top-selling herbal dietary supplements in the United States and cranberry

(Vaccinium macrocarpon) is the best-selling herbal product in the year of 2011, followed by, saw palmetto (Serenoa repens), soy (Glycine max), ginkgo (Ginkgo biloba), garlic (Allium sativum), milk thistle (Silybum marianum) and so forth

(Blumenthal et al., 2012). Ginkgo (Ginkgo biloba) is believed to heal memory impairment, stroke, edema, inflammation, Alzheimer’s disease and vasso oclusive crisis (Diamond et al., 2000) while garlic (Allium sativum) is suggested to have antilipidemic, antihypertensive, antiglycemic and antithrombotic properties

(Ackermann et al., 2001). Echinacea or purple coneflower (Echinacea purpurea) is commonly used to treat influenza and common cold in the United States. Besides that, ginseng (Panax ginseng), another traditional herbal plant widely used in the

United States is known to enhanced human immune system, increase physical stamina and decrease fatigueness (Foti and Wahlstrom, 2008). Although herbal medicine promises a great deal of benefits health care, a large number of investigations have reported its adverse side effects and possible fatality in some cases. Herbal medicine usually contains a mixture of more than one active ingredient. Interactions between herbal medicine and its corresponding active constituents towards drug metabolizing enzyme are summarized in Table 1.1.

Table 1.1: Interactions between herbal medicines and drug metabolizing enzymes.

Herbal medicines Scientific Name Active constituent Interaction References

Black cohosh Actaea racemosa Triterpene glycoside Inhibit CYP2D6 Gurley et al., 2005

Inhibit CYP2C, 2D1 and Angelica root Angelica dahurica Furanocoumarin derivatives Ishihara et al., 2000 3A

Black pepper Piper nigrum Alkaloid piperine Inhibit CYP3A4 Bhardwaj et al., 2002

Bergamottin, 6’7’- Inhibit 1A2, 2A6, 2C9, Grapefruit juice Citrus paradisi He et al., 1998 dihydroxybergamottin 2C19, 2D6, 2E1 and 3A4 Inhibit CYP1A, 2C9, Kava Piper methysticum Kavalactones Foster et al., 2003 2C19, 3A4 and 2D6 Decrease bilirubin Milk thistle Silybum marianum Silymarin Salmi and Sarna, 1982 conjugation Inhibit CYP1A2, 2C9, St. John’s wort Hypericum perforatum Hypericin, hyperforin Wang et al., 2001 2C19, 2D6 and 3A4

Soy Glycine max Daidzein, genistein CYP3A4 Foster et al., 2003

Fatty acid, plant sterols, Inhibit CYP3A4, Saw palmetto Serenoa repens Yale et al., 2005 flavonoids 2D6,2C9

Table 1-1. Continued.

Herbal medicines Scientific Name Active constituent Interaction References

Schisandra fruit Schisandra chinensis Schisandrin, gomisin Inhibit CYP3A4 Iwata et al., 2004

Valerenic acid, valepotriates, Inhibit 2C19, 2D6 and Valerian Valeriana officinalis alkaloids, furanofuran lignans, Strandell et al., 2004 3A4 free amino acids

Licorice Glycyrrhiza glabra Glycyrrhizin Inhibit CYP3A4 Budzinski et al., 2000

Hempedu bumi Andrographis paniculata Andrographalide Inhibit UGT2B7 Zainal Abidin et al., 2014

Sinensetin, eupatorin, rosmarinic Misai Kucing Orthosiphon stamineus Inhibit GST Tan et al., 2011 acid Curcumin, curcumene, Temu lawak Curcuma xanthorrhiza Inhibit UGT1A1 and 2B7 Mohd Salleh., 2015 xanthorrhizol

Kratom Mitragyna speciosa Mitragynine Weakly inhibit GST Azizi et al., 2010

Anthocyanins, flavonols, Cranberry Vaccinium oxycoccos Inhibit CYP3A Uesawa et al., 2006 quercetin

For example, grapefruit juice contains two of the most abundant furanocoumarins, namely bergamottin and 6’,7’-dihydroxybergamottin, which are known to be responsible for herb-drug interaction (Zhou et al., 2004). Milk thistle (Silybum marianum) contains flavonolignans, which are present as multiple structural isomers including silymarin, and flavonolignans have been evalueted to inhibit Phase I drug metabolizing enzyme at low concentration (Foti and Wahlstrom, 2008). Piperine, the active chemical constituent that can be found in Piper nigrum Linn and Piper

Longum Linn, is responsible for raising the concentration of several drugs in blood plasma such as phenytoin (antiepileptic drug), propanolol (drug used to treat high blood pressure) and theophylline (drug used to treat respiratory disease). It is also increased the plasma concentration of rifampicin in patients with pulmonary tuberculosis (Hu et al., 2005). Garlic (Allium sativum) is reported to have several of compounds such as allicin and alliin, flavonoids, polysaccharides, prostaglandins, saponins and terpenes. Some reports have stated the co-administration of warfarin with garlic extract increases the clotting time and international normalised ratio and result in spontaneous spinal epidural haematoma and postoperative bleeding (Hu et al., 2005).

Gynura procumbens which is known as Sambung Nyawa in Malaysia is widely distributed in South East Asian countries. Gynura procumbens leaves have been used traditionally to treat various diseases. Many researchers reported that this herbal plant exhibited pharmacological potential as anti-diabetic, anti-inflammatory, antihypertensive and more (Hassan et al., 2010; Lee et al., 2012; Iskander et al., 2002;

Kim et al., 2006).

Despite numerous studies on the various pharmacological effects of Gynura procumbens, its inhibitory effects on drug metabolizing enzymes have not been investigated to date. Hence, the present study was carried out to expand the study on the effects of varying Gynura procumbens extracts on drug metabolizing enzymes activities. Quantification of two marker compounds using a high performance liquid chromatography (HPLC) method and standardization of Gynura procumbens extracts by biological profiling (determination of total phenolic and flavonoid content, and antioxidant activity) were also carried out. The experiments of the present study were summarized in Figure 1.1.

Extraction of dried leaves of Gynura procumbens

Water Ethanol Methanol

Standardization of Gynura procumbens extracts by identification and quantification of the marker compounds kaempferol-3-O-rutinoside and astragalin

Antioxidant Properties of Gynura procumbens Drug Metabolizing Enzymes Activity Assay

Phase I Inhibition Assay Phase II Inhibition Assay Total Total DPPH Phenolic Flavonoid Scavenging UGT Content Content Activity CYP450

GST CYP3A4 CYP1A2

Figure 1.1: The experimental design for the effects of Gynura procumbens extracts on drug metabolizing enzymes study.

1.2 The Problem Statement of the Study

Gynura Procumbens have been used traditionally to treat various types of diseases. It is also have been studied extensively by researchers to investigate the pharmacology properties of Gynura procumbens so that this plant can be a natural and non-toxic medication for diseases for example in treating diabetes patient. Since Gynura procumbens has been eaten or drank for medicinal purposes, it is crucial to investigate the effect of this herbal plant on drug metabolizing enzymes to predict any changes in drug metabolizing enzymes activity which may lead to serious side effects.

1.3 The Purpose of the Study

The aim of the study is to investigate the effects of Gynura procumbens extracts on

Phase I and Phase II drug metabolizing enzymes.

1.4 The Objectives of the Study

The objectives of the present study are:

1. To quantify the amount of two marker compounds (kaempferol-3-O-

rutinoside and astragalin) in each extract of Gynura procumbens.

2. To study the total phenolic content, total flavonoid content and DPPH

scavenging activity of each extracts of Gynura procumbens.

3. To evaluate the effect of Gynura procumbens extracts on human recombinant

CYP3A4 and CYP1A2 enzymes isoforms respectively by using luciferin

derivatives as the marker reaction for CYP3A4 and CYP1A2 enzymes

activity.

4. To study the effect of Gynura procumbens extract on rat liver microsomes

(RLM) UDP-glucuronosyltransferases (UGT) enzyme by employing p-

nitrophenol (p-NP) as the marker reaction for UGT enzyme activity.

5. To study the effect of Gynura procumbens extract on rat liver cytosolic

fraction glutathione S-transferases (GST) enzyme by employing 1-chloro-2,4-

dinitrobenzene (CDNB) as the marker reaction for GST enzyme activity.

CHAPTER TWO

LITERATURE REVIEWS

2.1 Description of Gynura procumbens

Gynura procumbens (Figure 2.1) is an annual evergreen shrub of the Compositae family which can be found in Indonesia, Thailand, and Malaysia. Gynura procumbens is locally known in Malaysia as ‘Akar Sebiak’, ‘Kecam Akar’ or

‘Sambung Nyawa’ (Bhore and Vaishana, 2010). It is factual that the leaves of

Gynura procumbens are not bitter at all and it can be eaten raw as salad. Gynura procumbens is mostly used by the Malays in Malaysia as salad. They also can be sun-dried, preserved as tea. The tea can be prepared by steeping the leaves of Gynura procumbens in boiled water for about 5 minutes and it is ready to be consumed either hot or cold. Honey or sugar can be added to reduce the bitterness of Gynura procumbens tea. Gynura procumbens also sold over-the-counter in capsule form especially in Indonesia (Figure 2.1).

Figure 2.1: Gynura procumbens

A B C

Figure 2.2: Capsule of Gynura procumbens sold in the market. Pictures are adopted from panels (a) www.sambungnyawa.com; (b) www.etalasemuslim.com; and (c) www.istanaherbal.com.

2.2 Taxonomy of Gynura procumbens

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Asterales

Family : Asteraceae

Genus : Gynura

Species : Gynura procumbens

2.3 Pharmacological Potentials of Gynura procumbens

Gynura procumbens is traditionally used to treat various types of illnesses such as fever, rash inflammation, kidney disease, hemorrhoids and diabetes mellitus (Kim et al., 2006; Saiman et al., 2012).

The leaves of Gynura procumbens are proven to be non-toxic (Yam et al., 2009) and they exhibited anti-diabetic (Hassan et al., 2010; Lee et al., 2012), anti-oxidative

(Puangpronpitag et al., 2010), anti-inflammatory (Iskander et al., 2002), and antihypertensive characteristics (Kim et al., 2006). Ethanol extract of Gynura procumbens may also have biguanide-like activity since it is shown to reduced serum cholesterol and triglyceride levels when an optimum dose was given over a period of a group of diabetic rats (Zhang and Tan, 2000). Gynura procumbens has been demonstrated to decrease blood pressure in spontaneously hypertensive rats via the inhibition of the angiotensin-converting enzyme (Hoe et al., 2007).

However, the same author has proposed butanolic fraction of Gynura procumbens may contribute to hypotensive effect in rats via other mechanism. The investigation has reported butanolic fraction of Gynura procumbens resulted in lowered blood pressure (hypotensive effect) in rats as a result of vasodilation due to inhibition of

Ca2+ influx via receptor-operated and/or voltage-dependent calcium channel (Hoe et al., 2007). In addition, ethanol extract of Gynura procumbens inhibited MMP-1 and

MMP-9 expressions which are induced by UVB irradiation via the inhibition of pro- inflammatory cytokine mediator release and reactive oxygen species (ROS) production (Kim et al., 2011).

Ethanol extract of Gynura procumbens is also shown to have antiproliferative activity on male rat’s livers induced by 7,12-dimethylbenz[α]anthracene (Nisa et al.,

2012). According to Nurulita (2012) in her recent study, ethyl acetate fraction of

Gynura procumbens in combination with doxorubicin (DOX) potentiate DOX effect on breast cancer cell growth inhibition and hence this fraction could be developed as co-chemotherapy agent in reversing multidrug resistance.

2.4 Phytochemical constituents of Gynura procumbens

Numerous studies have exposed that Gynura procumbens leaves extract contains various active compounds such as flavanoids, saponins, sterol glycoside, terpenoids and tannins (Zahra et al., 2011). Previous studies have demonstrated that Gynura procumbens which extracted using various concentration of ethanol as extraction solvent (95%, 75%, 50%, 25%, and 0% of ethanol (% v/v)) with different extraction methods (Soxhlet, maceration and ultra-sonication) showed vary in phenolic content

(Algariri et al., 2013).

The total content of phenolic compounds in various extracts of Gynura procumbens is ranked in decreasing order as 50% ethanol > 75% ethanol > 95% ethanol > 25% ethanol > 0% ethanol. However, total flavonoid content of Gynura procumbens was found to decrease with the increase in polarity of the extraction solvent (Algariri et al., 2013). Crude methanol extract of Gynura procumbens and its fractions

(chloroform, ethyl acetate, n-butanol, and aqueous) had showed different level of antioxidant potential. Ethyl acetate fraction exhibited the highest antioxidant properties compared to the other extracts and fractions.

2.5 Phytochemical Analysis of Gynura procumbens

The chemical constituents of Gynura procumbens that is responsible for its antioxidant action was determined using HPTLC and it is found that the content of astragalin and kaempferol-3-O-rutinoside were relatively high in the ethyl acetate fraction (Yam et al., 2008). Three components from Gynura procumbens ethanol extract (quercetin 3-O-rutinoside, isobioquercetin and kaempferol 3-O-rutinoside) have also been identified by direct comparison of their respective molecular weights and retention time using mass spectrophotometer and high performance liquid chromatography (HPLC) (Kim et al., 2011). Akowuah et al. (2001, 2002) conducted preliminary phytochemical analysis on Gynura procumbens methanol extract, and this analysis led to isolation of flavonol and flavonol glycoside including rutin, quercetin, kaempferol, and quercetin-3-O-rhamnosyl (1-6) glucoside, quercetin-3-O- rhamnosyl (1-6) galactoside, kaempferol-3-O-rhamnosyl 1-6) glucoside and kaempferol-3-O-glucoside.

In addition, kaempferol-3-O-rutinoside and kaempferol-3-O-glucoside in the Gynura procumbens methanol extract both have been determined using high performance thin layer chromatography (Yam et al., 2009). Hassan et al. (2010) performed the same phytochemical analysis as Yam et al. (2009) towards Gynura procumbens aqueous extract and revealed that this plant extract contains 0.76% and 2.65% of kaempferol-3-O-rutinoside and kaempferol-3-O-glucoside respectively.

2.6 Drug Metabolism

Preclinical drug metabolism and pharmacokinetics is important in drug discovery and development (Gunaratna, 2000). Drug metabolism is defined as a process of elimination of foreign compound from the body. This process occurs mainly in the liver with the aid of enzymes. Enzymes which are responsible for converting or transforming or detoxifying foreign compound are called drug metabolizing enzymes. Drug metabolizing enzymes are separated into two groups including oxidative drug metabolizing enzymes and conjugative drug metabolizing enzymes.

Oxidative drug metabolizing enzymes are also known as Phase I drug metabolizing enzymes includes cytochrome P450 (CYP450) and flavin monooxygenase (FMO), in which both catalyze the introduction of an oxygen atom into substrate molecules resulting in hydroxylation and demethylation. Phase I reaction involves the process of converting non-polar parent compound into a more polar or hydrophilic compound by the addition of functional groups such as –OH, -SH, -NH2, -COOH.

The conjugative drug metabolizing enzymes or Phase II drug metabolizing enzymes include UDP-glyconosyltransferases (UGTs), glutathione transferases (GSTs), sulfotransferases (SULTs), and N-acetyltransferases (NATs). Phase II drug metabolism reaction involves the conjugation or the attachment of an ionized group to the substrate which then further increases aqueous solubility and decreases pharmacological activity of the substrate. This group comprises of glutathione, methyl group and acetyl group.

Phase I reaction occurs in liver microsome and usually precede Phase II reaction which takes place in the liver cells as the parent compound or the metabolite from

Phase I becomes conjugated. Numerous factors are knowns to affect drug metabolism reaction including enzyme induction and enzyme inhibition. Enzyme induction results in acceleration of drug biotransformation and as a result loses its therapeutic effects due to rapid metabolism. In worst cases, certain drugs with active metabolites can exhibit increased adverse drug effects and/or toxicity. On the other hand, enzyme inhibition occurs when two drugs are metabolized via the same pathway and if one of the resulting drug products is a potent inhibitor, it can decrease the metabolism of the other drug thus leading to adverse toxicity (Ogu and Maxa,

2000). There are several factors that influence the activity of drug metabolism

(Gibson and Skett, 1986) and these factors are summarized in Table 2.1 below.

Table 2.1: List of factors that affect the activity of drug metabolizing enzymes

Internal External Species Diet Genetic Environment Sex Age Hormonal control Disease *Adapted from Gibson and Skett, (1986)

To date, drug metabolism is one of the most intensely studied aspects in herb-drug interaction. Grapefruit juice has been found to disrupt the oral bioavailability of various CYP3A4 substrates including cyclosporin A, felodipine, midazolam, terfenadine, verapamil, saquinavir, and ethinyl estradiol (Schmiedlin-Ren et al.,

1997).

In addition, Bhardwaj et al. (2002) reported that, piperine, a chemical constituent found in black pepper inhibited P-glycoprotein-mediated, polarized transport of digoxin and cyclosporin A transport in monolayers of Caco-2 cells (Bhardwaj et al.,

2002). The same author also claimed that piperine inhibited CYP3A4-mediated formation of the verapamil metabolites D-617 and norverapamil (Bhardwaj et al.,

2002). Since P-glycoprotein and CYP3A4 are important in determination of bioavailability of many drugs such as digoxin, verapamil and cyclosporin A, inhibition of both proteins by concomitantly administered substance such as piperine or grapefruit juice may lead to elevation of plasma concentration of a drug due to a dual effect on drug transport and metabolism (Schmiedlin-Ren et al., 1997; Bhardwaj et al., 2002).

2.7 Phase I Drug Metabolizing Enzymes

Cytochrome P450 or CYP450 isoenzymes are superfamilies of hemoproteins. The term P450 is derived from spectrophotometric absorption peak of the enzyme at a wavelength 450 nm when it is bounded and reduced by carbon monoxide (Chang and

Kam, 1999). CYP450 isoenzymes are popular for their role in the metabolism of diverse exogenous materials such as drugs, environmental pollutants, and chemicals

(Guengerich, 1999). Fifty seven CYP450 genes have been found in human and only

CYP1, CYP2, and CYP3 families are known to mainly contribute to drug metabolism. Among CYP450 isoenzymes, only five accounts for major drug metabolism namely CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2

(Guengerich, 2003). CYP3A4 is the most important enzyme as it metabolizes almost all CYP450-mediated reactions (Guengerich, 1996).

Generally, xenobiotic compounds are removed from our body by oxidation reaction of CYP450 which then results in the formation of more water-soluble and less toxic metabolite. However, metabolic activation of carcinogens may also occur during the oxidative mechanism mediated by CYP enzymes (Gonzalez and Gelboin, 1994). For instance, CYP1 family is responsible for the metabolic activation of carcinogens such as benzo[α]pyrene and 7,12-dimethylben[α]anthracene (Slaga et al., 1979).

Therefore, inhibitor of CYP1A2 may possess chemo preventive properties (Kim et al., 2013). Several studies have been conducted regarding herbal and natural constituents that have inhibitory effects on CYP1A family and these constituents may play as chemopreventive agents in carcinogenesis due to exposure to polycyclic aromatic hydrocarbon (PAH) (Hwang et al., 2008; Pekthong et al., 2008).

Pharmacological studies have reported kava extract and/or kavalactones, potent inhibitors of CYP3A4, may decrease elimination of alprazolam upon co- administeration of kava and alprazolam (Zhou et al., 2004). Other studies have reported that tanshinones, an active constituent in Danshen, (Salvia miltiorrhiza) inhibited various CYP probe substrates in both human liver microsomes and specific human isoforms in vitro (Wang et al., 2010). In addition, drug interaction has also been observed for the two known chemical constituents in Schisandra fruit extract

(gomisin B and C) that showed a potent inhibitory effect on CYP3A4 activity comparable with that of ketoconazole, a known inhibitor for CYP3A4 enzyme (Iwata et al., 2004). Natural product has been consumed for different reasons. The leaves of

Mitragyna speciosa or commonly known as ketum in Malaysia, have been used tradionally to treat various diseases.

However, recent studies revealed that alkaloid extract of Mitragyna speciosa is a potent inhibitor of CYP3A4 and CYP2D6 and moderate inhibitor of CYP1A2 (Kong et al., 2011).

2.8 CYP3A4 Isoform

The literature data on CYP3A4 inhibition by herbal medicines has increased over the years. Goldenseal (Hydrastis canadensis), St John’s wort (Hypericum perforatum), cat’s claw (Uncaria tomentosa), Black samson (Echinacea angustifolia), wild cherry

(Trifolium pratense), chamomile (Matricaria chamomilla) and licorice (Glycyrrhiza glabra) significantly inhibited CYP3A4 metabolite with IC50 values lower than 1% to

2% of full strength (highest concentration) measured using fluorometric microtitre plate assay (Budzinski et al., 2000). Herbal components namely ginsenosides Rb1,

Rb2, Rc, and Rd (from ginseng quercetin) and ginkgolides A and B (from Ginkgo biloba) were investigated for their inhibitory effect on CYP2C9 and CYP3A4 and all these herbal components showed different potencies in inhibiting both the CYPs (He and Edeki, 2004). Hyperforin, a putative active antidepressant constituent from St

John’s wort (Hypericum perforatum) extract was a potent competitive inhibitor of

CYP3A4 activities with the inhibitor constant (Ki) value of 0.48µM (Obach, 2000).

Ethanol extract of kava and three purified kava lactones including methysticin, desmethoxyyangonin, and yangonin were previously investigated for their inhibitory effects on CYP450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6,

CYP2E1 and CYP3A4) expressed in a baculovirus / insect cell system and in cryopreserved human hepatocytes and it is found that they have IC50 values approximately 10µM (Zou et al., 2004).

By employing P450-GloTM Screening System, Alpine lovage (Mutellina purpurea

L.) methanol extract showed about 6 fold significant inhibition towards CYP3A4 compared to its aqueous extract (Sieniawska et al., 2012). In a cell-free system, schisandrol A (SCH) and gomisin A (GOM), two main dibenzocyclooctadiene lignants isolated from the fruit of Schisandra chinensis, inhibited CYP3A4 activity with IC50 value 32.02µM and 1.39µM respectively (Wan et al., 2010).

Four Malaysian medicinal plants namely Andrographis paniculata, Orthosiphon staminues, Mitragyna speciosa and Curcuma xanthorrhiza have been found to have the ability to inhibit CYP3A4 with IC50 value in the range from 28µg/mL to

300µg/mL (Hanapi et al., 2010). Earlier studies reported that, Orthosiphon stamineus dichloromethane and petroleum ether extract moderately inhibited CYP3A4 than aqueous and methanol, but eupatorin, the active constituent of Orthosiphon staminues was found potently inhibited CYP3A4 (Pan et al., 2011). In addition, the same author suggested that the inhibitory effect of dichloromethane and petroleum ether extract on CYP3A4 was probably because of the high content of eupatorin in dichloromethane and petroleum ether extracts and therefore, drug-herb interaction are likely to occur for CYP3A4 substrates (Pan et al., 2011). It is worth noting that, among 30 Indonesian medicinal plants, 4 medicinal plants (ethyl acetate soluble fraction), namely Pi. cuceba, Pi. nigrum fruit, Pi nigrum leaf, and Z. aromaticum showed inhibitory activity of more than 70% towards CYP3A4 (Usia et al., 2006).

Interestingly, (-) -hinokinin, a compound that contain two methylenedioxyphenyl in its chemical structure, isolated from Pi. Cuceba, is a potent inhibitor towards

CYP3A4 and therefore, the inhibitory effect of Pi. Cuceba on CYP3A4 may be due to this compound (Usia et al., 2006).

In contrast to Usia et al. (2006), schisandria fruit component which is schizandrin, a compound with no methylenedioxy group in its chemical structure, weakly inhibited

CYP3A4 with an IC50 value more than 100µM (Iwata et al., 2004).

It is noteworthy that, perturbation in CYP450 enzyme activities may lead to treatment failure or worse, clinically fatal due to toxicity (Gomez-Lechon et al.,

2008). Inhibition or induction of CYP450 enzymes may increase the drug plasma concentration level in the body. This situation may lead to toxicity or decrease the drug plasma concentration therefore, loses its therapeutic effect which may lead to treatment failure (Li et al., 1997). Based on the recent studies on Elephantopus scaber L. major constituent, a series of CYP induction and CYP inhibition have been done using P450 GloTM CYP3A4 assay. Deoxyelephantopin (major germacranolide sesquiterpene lactone isolated from E. Saber) showed induction of CYP3A4 enzyme activity at a concentration range of 0.01 – 0.1µM. Oddly, when the concentration was further increased, CYP3A4 enzyme activity starts to decrease and thus, deoxyelephantopin was found to be a weak inducer and a weak inhibitor and unlikely to stimulate negative effect in human (Koe et al., 2013). It can be conclude that, if a drug/herb inhibits CYP3A4 that catalyzes the metabolism of a concomitant drug, plasma concentration of the concomitant drug will increase and thus, this will lead to toxicity. Table 2.2 shows the list of CYP3A4 substrates that may cause herb-drug interaction or drug-drug interaction when CYP3A4 inhibitors are taken concurrently with herbal medicines.

Table 2.2: List of CYP3A4 substrates

CYP3A4 substrates Alfentanil (Alfenta) Carbamazepine (eg, Tegretol) Erythromycin Solifenacin (Vesicare) Alfuzosin (Uroxatral) Clonazepam (Klonopin) Estazolam (ProSom) Tinidazole (Tindamax) Atorvastatin (Lipitor) Cyclosporine (Neoral) Ethosuximide (Zarontin) Tipranavir (Aptivus) Amlodipine (Norvasc) Darunavir (Prezista) Felodipine (Plendil) Triazolam (Halcion) Bexarotene (Targretin) Dexamethasone (Decadron) Flurazepam (Dalmane) Verapamil (Calan) Budesonide (Entocort) Docetaxel (Taxotere) Galantamine (Reminyl) Vinblastine (Velbane) Buprenorphine (Subutex) Ergotamine (Ergomar) Gefitinib (Iressa) Vincristine (Oncovin) Halofantrine (Halfan) Indinavir (Crixivan) Irinotecan (Camptosar) Ziprasidone (Geodon) Itraconazole (Sporanox) Lopinavir (Kaletra) Levomethadyl (Orlaam) Zolpidem (Ambien) Lovastatin (Mevacor) Midazolam (Versed) Nefazodone Zonisamide (Zonegran) Paclitaxel (Taxol) Pioglitazone Ranolazine (Ranexa) Zopiclone (Imovane) Repaglinide (Prandin) Ritonavir (Norvir) Simvastatin (Zocor) Sildenafil (Viagra) Adapted from Horn and Hansten (2008)

2.9 CYP1A2 Isoform

CYP1A2 is one of the members of CYP450 enzymatic group and its expression is induced by certain polycyclic aromatic hydrocarbons (PAHs). It has the ability to metabolize some PAHs into carcinogenic intermediates. The main CYP450 enzymes involved in carcinogen activation are CYP1A1, CYP1A2, CYP1B1, CYP2A6,

CYP2E1 and CP3A4. The most common enzymes from group CYP450 involved in cancer induction are both CYP1A2 and CYP2A6 (Guengerich, 2003). Several studies have been conducted exclusively regarding the issue that some herbal or natural constituents have the capability to inactivate CYP1A family members thus playing an important role in preventing the effects in carcinogenesis due to exposure to PAHs (Hwang et al., 2008; Pekthong et al., 2008). Standardized Asian ginseng

(Panax ginseng) extract and standardized North American ginseng (Panax quinquefolius) extract inhibited human recombinant enzymes CYP1A1, CYP1A2, and CYP1B1 activities in a concentration-dependent manner (Chang et al., 2002).

Mollugin, a pharmacological compound isolated from Rubia cordifolia, inhibited recombinant enzyme CYP1A2 competitively thus changing the pharmacokinetic properties of other drugs such as caffeine and theophylline inevitably (Kim et al.,

2013). Tanshinones (tanshinones I, tanshinone IIA, and cryptotanshinone), major constituents of Salvia miltiorrhiza, competitively inhibited CYP1A2 with a Ki value of 1.5 – 2.5µM thus suggesting the occurrence of herb-drug interaction, given that

CYP1A2 is solely responsible for the metabolism and disposition of almost all drugs currently used (Wang et al., 2010).

Therefore, in recent time, modulation of the activity of CYP1A2 by dietary phytochemicals such as flavonoids and elucidation of the structure of flavonoids that are responsible in herb-drug interaction have gain worldwide attention. Zhai et al.

(1998) reported flavone and five hydroxylated derivatives of flavone showed different potencies and selectivities on inhibition of CYP1A enzymes. Mitragyna speciosa alkaloid extract showed moderate inhibition towards CYP1A2 with an IC50 value of 39µg/mL (Kong et al., 2011). According to Appiah-Opong et al. (2007), curcumin, a polyphenolic component of tumeric, inhibited CYP1A2 competitively with an IC50 value of 40.0µM. Kuo et al., (2004) stated that flavonols inhibited

CYP1A enzyme in the decreasing order of aglycone >> monoglycoside > diglycoside. CYP1A2 metabolizes important medicines as listed in Table 2.3, therefore, suggesting that interaction between medicines and herbal extract that metabolize by the same enzyme may increase the plasma concentration of those particular medicines that if it is taken concurrently.