THE EFFECTS OF GYNURA PROCUMBENS EXTRACTS ON …eprints.usm.my/31622/1/ATIQAH_AFANDI.pdfiv 2.6 Drug metabolism 15 2.7 Phase I drug metabolizing enzymes 17 2.8 CYP3A4 Isoform 19 2.9

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

by

ATIQAH BINTI AFANDI

Thesis submitted in fulfillment of the requirements for the

degree of

Master of Science

July 2015

ii

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

iii

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

iv

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

astragalin in Gynura procumbens extracts

40

3.5 Antioxidant studies on Gynura procumbens extracts 40

v

3.5.1 Determination of total phenolic content of Gynura

procumbens extracts

40

3.5.2 Determination of total flavonoid content of Gynura

procumbens extracts

41

3.5.3 Evaluation of antioxidant activity of Gynura procumbens

extracts

42

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

and known inhibitors on CYP450 enzyme activity

45

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

Michaelis constant (Km) of pNP glucuronidation

52


and known inhibitors on UGT enzyme activity

53

3.10 Phase II GST enzymes inhibition assay 55

3.10.1 Linearity of incubation time and protein concentration 55

vi


and known inhibitor on GST enzyme activity

56

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

Gynura procumbens extracts

58

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

procumbens extracts

67

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

isoforms activity

70

4.4.3 The effect of Gynura procumbens ethanol extract on

CYP450 isoforms

71

4.4.4 The effect of Gynura procumbens methanol extract

on CYP450 Isoforms

73

4.4.5 The effect of Gynura procumbens aqueous extract

on CYP450 isoforms

75

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

vii

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

Michaelis constant (Km) of UGT enzyme inhibition assay

83

4.6.4 The effect of known UGT inhibitor and Gynura

procumbens extracts on UGT enzyme activity

84

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

procumbens extracts on GST enzyme activity

89

3.7.2 Inhibitory Effect of Known Inhibitor (Tannic Acid) on

GST Enzyme Assay

75

3.7.3 Inhibitory Effect of Gynura procumbens Extracts on

GST Enzyme Assay

76

CHAPTER FIVE - DISCUSSION 92

5.1 Extraction process of Gynura procumbens and quantification of

its marker compounds

92

5.2 Total phenolic content, total flavonoid content and antioxidant

activity of Gynura procumbens extracts

94

5.3 The effect of Gynura procumbens extracts on human CYP3A4

and CYP1A2 enzyme activity using recombinant enzymes

96

5.4 The effect of Gynura procumbens extracts on rat UGT enzyme

activity using RLM

102

viii

5.5 The effect of Gynura procumbens extracts on rat GST enzyme

activity using RLCF

105

CHAPTER SIX - CONCLUSION 109

REFERENCES 111

APPENDICES 126

LIST OF PUBLICATIONS 131

ix

LIST OF TABLES

Page

Table 1.1 Interactions between herbal medicines and drug

metabolizing enzymes.

3

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

enzymes

16

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

extraction method.

58

Table 4.2 Retention time of standard marker compound in Gynura

procumbens extracts.

59

Table 4.3 The amount of kaempferol-3-O-rutinoside and astragalin in

Gynura procumbens extracts.

63

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

procumbens extracts. Values are expressed as mean in

microgram per milliliter of Gynura procumbens extracts ±

SEM for four replicates (n=4).

68

Table 4.7 IC50 values of positive controls of CY1A2 and CYP3A4

enzymes.

70

x

Table 4.8 The half maximal inhibitory concentration (IC50) of Gynura

procumbens extracts on the metabolism mediated by

CYP3A4 and CYP1A2.

77

Table 4.9 Concentration of protein in rat liver microsome (RLM) and

rat liver cytosolic fraction (RLCF) for male Sprague

Dawley rats.

78

Table 4.10 Enzyme kinetic parameters for UGT enzyme-mediated pNP

glucuronidation in RLM.

84

Table 4.11 Relative activity of UGT enzyme after incubated with

Gynura procumbens extracts and diclofenac at three

different concentrations of 10, 100 and 1000µg/mL.

86

Table 4.12 IC50 values for Gynura procumbens extracts and tannic acid

showing inhibition towards 1-chloro-dinitrobenzene

(CDNB) conjugation reaction catalyzed by GST enzyme.

91

Table 5.1 The structure of flavones. 99

x

LIST OF FIGURES

Page

Figure 1.1 The experimental design for the effects of Gynura

procumbens extracts on drug metabolizing enzymes study

7

Figure 2.1 Gynura procumbens 10

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.

11

Figure 2.3 Conversion of derivatives of D-luciferin to D-luciferin by

CYP450 enzymes. * Indicates the site of modification by

CYP450 enzymes.

26

Figure 2.4 Glucuronidation reaction of nucleophilic substrate p-

nitrophenol (pNP) with UDPGA catalyzed by UDP-

Glucuronosyltransferases (UGT) enzymes.

29

Figure 2.5 Conjugation reaction of electrophilic substrate 1-chloro-2,4-

dinitrobenzene with glutathione (GSH) catalyzed by

glutathione S-transferase (GST) enzymes

31

Figure 2.6 Pathway of paracetamol metabolism 32

Figure 4.1 HPLC chromatograms of the reference standards (A) and

methanol extract of Gynura procumbens (B)

60

Figure 4.2 HPLC chromatograms of the ethanol extract of Gynura

procumbens (A) and aqueous extract of Gynura procumbens

(B)

61

Figure 4.3 Calibration curve of reference standard of kaempferol-3-O-

rutinoside.

62

xi

Figure 4.4 Calibration curve of reference standard of astragalin. 63

Figure 4.5 Standard curve of gallic acid. A range of gallic acid

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).

64

Figure 4.6 Standard curve of quercetin. A range of quercetin

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).

66

Figure 4.7 DPPH scavenging effect of test samples which are Gynura

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).

67

Figure 4.8 Standard curves of D-luciferin attained from the incubation

of standard D-luciferin (concentration ranged from

0.016µM - 2µM) with non-CYP1A2 enzyme and luciferin

methyl ether (substrate for CYP1A2).

69

xii

Figure 4.9 Standard curves of D-luciferin attained from the incubation

of standard D-luciferin (concentration ranged from

0.016µM - 2µM) with non-CYP3A4 enzyme and luciferin

benzyl ether (substrate for CYP3A4).

70

Figure 4.10 Inhibition of concentrations (0.1 - 250µg/mL) of ethanol

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).

71

Figure 4.11 Inhibitory effect of ethanol extract of Gynura procumbens

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.

72

Figure 4.12 Inhibition of concentrations (0.1 - 250µg/mL) of methanol








73

xiii




Figure 4.13 Inhibitory effect of methanol extract of Gynura procumbens


Concentration of methanol extract ranged from 0.1µg/mL –




74

Figure 4.14 Inhibition of concentrations (0.1 - 250µg/mL) of aqueous











75

Figure 4.15 Inhibitory effect of aqueous extract of Gynura procumbens


Concentration of aqueous extract ranged from 0.1µg/mL –




76

Figure 4.16 Standard curves of bovine serum albumin (BSA) attained

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

78

xiv

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

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).

79

Figure 4.18 Optimization of time incubation. The 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 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).

80

Figure 4.19 Optimization of protein concentration. The 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 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).

81

Figure 4.20 Optimization of triton X-100 concentration. The

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

82

xv

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

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).

83

Figure 4.22 Effect of Gynura procumbens extracts on pNP

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).

85

Figure 4.23 Optimization of time incubation. The conjugation 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) (0.125mg/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).

87

xvi

Figure 4.24 Optimization of protein concentration. The conjugation

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).

88

Figure 4.25 Effect of Gynura procumbens extracts on CDNB

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).

89

Figure 4.26 Inhibitory effect of the tannic acid and Gynura procumbens

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.

91

Figure 5.1 The structure of quercetin 96

Figure 5.2 Structure of flavone, chalcone and coumarins 107

xviii

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

xix

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

xx

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

xxi

UGT Uridine-diphospho-glucuronosyl transferases

US United States

Vmax Maximal reaction velocity

WHO World Health Organization

xxii

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.

xxiv

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.

xxv

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.

xxvi

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.

xxvii

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.

1

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).

2

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.

3

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

Angelica root Angelica dahurica Furanocoumarin derivatives Inhibit CYP2C, 2D1 and

3A Ishihara et al., 2000

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

Grapefruit juice Citrus paradisi Bergamottin, 6’7’-

dihydroxybergamottin

Inhibit 1A2, 2A6, 2C9,

2C19, 2D6, 2E1 and 3A4 He et al., 1998

Kava Piper methysticum Kavalactones Inhibit CYP1A, 2C9,

2C19, 3A4 and 2D6 Foster et al., 2003

Milk thistle Silybum marianum Silymarin Decrease bilirubin

conjugation Salmi and Sarna, 1982

St. John’s wort Hypericum perforatum Hypericin, hyperforin Inhibit CYP1A2, 2C9,

2C19, 2D6 and 3A4 Wang et al., 2001

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

Saw palmetto Serenoa repens Fatty acid, plant sterols,

flavonoids

Inhibit CYP3A4,

2D6,2C9 Yale et al., 2005

4

Table 1-1. Continued.

Herbal medicines Scientific Name Active constituent Interaction References

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

Valerian Valeriana officinalis

Valerenic acid, valepotriates,

alkaloids, furanofuran lignans,

free amino acids

Inhibit 2C19, 2D6 and

3A4 Strandell et al., 2004

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

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

Misai Kucing Orthosiphon stamineus Sinensetin, eupatorin, rosmarinic

acid Inhibit GST Tan et al., 2011

Temu lawak Curcuma xanthorrhiza Curcumin, curcumene,

xanthorrhizol Inhibit UGT1A1 and 2B7 Mohd Salleh., 2015

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

Cranberry Vaccinium oxycoccos Anthocyanins, flavonols,

quercetin Inhibit CYP3A Uesawa et al., 2006

5

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, anti-

hypertensive and more (Hassan et al., 2010; Lee et al., 2012; Iskander et al., 2002;

Kim et al., 2006).

6

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.

7

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

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

Total

Phenolic

Content

Total

Flavonoid

Content

DPPH

Scavenging

Activity

Drug Metabolizing Enzymes Activity Assay

Phase I Inhibition Assay Phase II Inhibition Assay

CYP450

CYP1A2 CYP3A4

UGT

GST

Antioxidant Properties of Gynura procumbens

8

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.

9

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.

10

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

11

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

A B C

12

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 anti-

hypertensive 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).

13

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.

14

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.

15

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.

16

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).

17

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).

18

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.

19

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).

20

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).

21

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.

22

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)

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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).

24

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.

Documents

THE EFFECTS OF GYNURA PROCUMBENS EXTRACTS ON …eprints.usm.my/31622/1/ATIQAH_AFANDI.pdfiv 2.6 Drug metabolism 15 2.7 Phase I drug metabolizing enzymes 17 2.8 CYP3A4 Isoform 19 2.9