in vitro cytotoxicity activity and in vivo oral toxicity

IN VITRO CYTOTOXICITY ACTIVITY AND IN VIVO ORALTOXICITY OF Euphorbia hirta

KWAN YUET PING

UNIVERSITI SAINS MALAYSIA2014

IN VITRO CYTOTOXICITY ACTIVITY AND IN VIVO ORALTOXICITY OF Euphorbia hirta

by

KWAN YUET PING

Thesis submitted in fulfillment of the requirements for the degree ofDoctor of Philosophy

AUGUST 2014

ii

ACKNOWLEDGEMENTS

I wish to express my heartfelt gratitude to everyone who had helped me and made

my pursuit for the PhD degree a pleasant and fulfilling experience. First and

foremost, I would like to attribute my deepest gratitude to my supervisor, Dr.

Sasidharan Sreenivasan, for giving me the opportunity to learn and work under his

supervision. I would like to extend my appreciation for his expert guidance, advice

and encouragement, without of which my PhD project would not have been so

fruitful. Besides, my heartfelt thanks go to my co-supervisor Prof. Darah Ibrahim

and field-supervisor Prof. Umi Kalsom binti Yusuf for giving me invaluable advice

and encouragement throughout the duration of my study.

I would like to forward my appreciation to the laboratory staffs Mrs. Norhalida, Mrs.

Dyana and Mrs. Fatihah from Institute for Research in Molecular Medicine

(INFORMM) for their technical help and support in using cell culture and

microscope facilities. I am thankful to Dr. Faisal and Mrs. Nor Fadhilah from

Advanced Medical & Dental Institute (AMDI), for their technical help and assistance

in using flow cytometer. I am very grateful to Dr. Lim Sheh Hong, Mr. Shanmugam,

Mr. Soma, Mr. Letchimanan, Mrs. Shantini, Mr. Hamzah and Mr. Johari in School

of Biological Sciences, for their guidance and kind help in the process of laboratory

works. I would also like to thank Dr. Isma and Mr. Yusof for their kind support

during the animal studies in Animal Research and Service Centre (ARASC). I am

also grateful to the financial support of a research scholarship from the Ministry of

Higher Education, Malaysia. To my fellow colleagues, Jothy, Kavitha, Vijaya, Grace,

Boon Ping, Mooi Kwai, Wee Yee, Priscilla and Soo Ai, thank you for all the support

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and patience you have shown me through the years. Your encouragement and help

made my PhD journey a much sweeter one.

Last but not least, I would like to thank my mother and sister for their endless love,

faith and support. Thank you for always believing in me and encouraging me to go a

little further, dream a little bigger. Special thanks go to Sean for being my pillar of

support and for being there with me always, through the high and lows of my PhD

journey. May all individuals mentioned above be blessed with constant love, health

and happiness.

KWAN YUET PING

Institute for Research in Molecular Medicine

Universiti Sains Malaysia

August 2014

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iv

LIST OF FIGURES xii

LIST OF TABLES xxiii

LIST OF ABBREVIATIONS xxv

ABSTRAK xxvi

ABSTRACT xxx

CHAPTER 1.0: INTRODUCTION 1

1.1 Overview and rationale of the study 1

1.2 Research objectives 4

CHAPTER 2.0: LITERATURE REVIEW 5

2.1 Pharmaceuticals from plants 5

2.1.1 Background of herbal medicine in Malaysia 5

2.1.2 Safety concern on the use of pharmaceutical from plants 7

2.2 Euphorbia 8

2.2.1 Euphorbia hirta L. 8

2.2.1.1 Botanical description 8

2.2.1.2 Origin and geographical distribution 9

2.2.1.3 Phytochemistry 13

2.2.1.4 Pharmacological activities 15

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2.2.1.5 Ethnopharmacology 16

2.2.1.6 Plant derived anticancer agents 17

2.3 Cell death 19

2.3.1 Apoptosis and necrosis 20

2.3.2 Mechanism of apoptotic and necrotic cell death 23

2.3.2.1 Apoptosis pathways 23

2.3.2.1.a Intrinsic pathway 23

2.3.2.1.b Extrinsic pathway 24

2.3.2.2 Necrotic cell death 25

2.4 The cell cycle 27

2.4.1 The cell cycle regulation and checkpoints 29

2.4.2 Dysregulation of cell cycle in cancers 30

2.5 Genotoxicity 31

2.5.1 Overview of DNA damage and repair 31

2.5.2 Genotoxicity testing 32

2.5.3 Plant extracts as genotoxic agents 34

2.6 Toxicological studies 35

2.6.1 Three R’s alternative methods 36

2.6.2 Acute toxicity studies 37

2.6.3 Sub-chronic toxicity studies 39

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CHAPTER 3.0: CHROMATOGRAPHIC AND SPECTRALFINGERPRINTING STANDARDIZATION OFEuphorbia hirta

40

3.1 Introduction 40

3.2 Materials and methods 41

3.2.1 Chemicals and reagents 41

3.2.2 Plant sample collection 41

3.2.3 Plant material extraction 41

3.2.4 Qualitative investigation 42

3.2.5 Fourier Transform Infrared (FTIR) fingerprinting 42

3.2.6 Gas chromatography-mass spectrometry (GC-MS) analysis 43

3.2.7 Heavy metal analysis 43

3.2 8 DPPH free radical scavenging assay 44

3.3 Statistical analysis 45

3.4 Results 46

3.4.1 Extraction 46

3.4.2 Herbarium of E. hirta 46

3.4.3 Qualitative investigation of E. hirta plant parts 48

3.4.4 FTIR analysis 53

3.4.5 GC-MS analysis 55

3.4.6 Heavy metal analysis 59

3.4.7 DPPH assay 61

3.5 Discussion 63

3.6 Conclusion 72

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CHAPTER 4.0: IN VITRO CYTOTOXICITY OF E. hirta 73

4.1 Introduction 73



4.2.2 Cell culture 75

4.2.2.1 Cell culture media 75

4.2.2.1.1 Dulbecco’s Modifies Eagle’s Medium (DMEM) 75

4.2.2.1.2 Roswell Park Memorial Institute Medium 1640(RPMI)

75

4.2.2.2 Cell lines 76

4.2.3 Resuscitation of frozen cells 78

4.2.4 Subculturing of cells 78

4.2.5 Cell quantification 79

4.2.6 Brine shrimp lethality assay 81

4.2.7 MTT cytotoxicity assay 82

4.2.8 Morphological detection of apoptosis using light microscopy 82

4.2.9 Lactate Dehydrogenase (LDH) leakage assay 83

4.2.10 Annexin V/PI for apoptosis detection 84

4.2.11 DNA fragmentation 85

4.2.12 Morphological detection of apoptosis using AcridineOrange/Propidium Iodide (AO/PI) staining

86

4.2.13 Cell cycle analysis by flow cytometry 87

4.2.14 Reactive Oxygen Species (ROS) assay 88

4.2.15 Detection of caspase activity by colorimetric assay 89

4.2.16 Bradford protein assay 89

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4.4 Results 91

4.4.1 Brine shrimp lethality assay 91

4.4.2 Cytotoxic of E. hirta methanol extract on several cell lines 93

4.4.3 Morphology detection of apoptosis using light microscopy 100

4.4.4 LDH leakage assay 105

4.4.5 AO/PI staining 107

4.4.6 Annexin V/PI assay 111

4.4.7 DNA fragmentation 114

4.4.8 Cell cycle analysis 116

4.4.9 ROS analysis 119

4.4.10 Colorimetric assay of caspase 2, 3, 6, 8, 9 121

4.5 Discussion 123

4.6 Conclusion 135

CHAPTER 5.0: GENOTOXICITY OF E. hirta 136

5.1 Introduction 136


5.2.1 Cell lines 137


5.2.3 Allium cepa assay 137

5.2.3.1 Pre treatment 137

5.2.3.2 Slides preparation 138

5.2.3.3 Observation of specimen 138

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5.2.4 Comet assay 139


5.4 Results 141

5.4.1 Effect of E. hirta extract on mitotic index (MI) 141

5.4.2 Effect of E. hirta extract on chromosome aberrations 143

5.4.3 Effect of E. hirta extract on micronuclei 145

5.4.4 Comet assay 152

5.5 Discussion 155

5.6 Conclusion 161

CHAPTER 6.0: IN VIVO TOXICITY TESTING OF E. hirta 162



6.2.1 Preparation of plant extract 164

6.2.2 Animals 164

6.2.3 Acute oral toxicity study 165

6.2.4 Sub-chronic toxicity study 166

6.2.5 Relative organ weight 167

6.2.6 Blood analyses 167

6.2.7 Histopathological analyses 167


6.4 Results 172

6.4.1 Effect of acute oral administration of E. hirta on the general behaviourin rats

172

6.4.2 Effect of acute oral administration of E. hirta on body weight in rats 174

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6.4.3 Effect of acute oral administration of E. hirta on haematologicalindices in rats

176

6.4.4 Effect of acute oral administration of E. hirta on biochemical indicesin rats

178

6.4.5 Effect of acute oral administration of E. hirta on organ weight in rats 180

6.4.6 Effect of acute oral administration of E. hirta on histologicalassessment of organs in rats

182

6.4.7 Effect of sub-chronic oral administration of E. hirta on the generalbehaviour in rats

143

6.4.8 Effect of sub-chronic oral administration of E. hirta on body weightin rats

195

6.4.9 Effect of sub-chronic oral administration of E. hirta on haematologicalindices in rats

198

6.4.10 Effect of sub-chronic oral administration of E. hirta on biochemicalindices in rats

201

6.4.11 Effect of sub-chronic oral administration of E. hirta on organ weightin rats

204

6.4.12 Effect of sub-chronic oral administration of E. hirta on histologicalassessment of organs in rats

206

6.5 Discussion 232

6.6 Conclusion 240

CHAPTER 7.0: FRACTIONATION AND IDENTIFICATION OFCYTOTOXIC FRACTION FROM E. hirta

241



7.2.1 Preparation of plant extract 242

7.2.2 Fractionation and isolation of cytotoxic fraction from E. hirta 242

7.2.2.1 Solvent partitioning method 242

7.2.2.2 Medium Pressure Liquid Chromatography (MPLC) 242

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7.2.2.3 Liquid Chromatography-Mass Spectrometry (LC-MS) 243

7.2.2.4 MTT cytotoxicity assay 244

7.3 Results 246

7.3.1 Fractionation of methanol extract of E. hirta 246

7.3.2 Cytotoxic effects of the subfractions on MCF-7 cells 246

7.3.3 Isolation and identification of compounds 253

7.4 Discussion 256

7.5 Conclusion 259

CHAPTER 8.0: SUMMARY 260

8.1 General conclusion 260

8.2 Suggestions for future studies 264

REFERENCES 265

APPENDICES 289

LIST OF PUBLICATIONS 311

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LIST OF FIGURES

Page

Figure 2.1 Taxonomic classification of E. hirta 10

Figure 2.2 Euphorbia hirta plant 11

Figure 2.3 E. hirta plant (1: plant habit; 2: young cyathium; 3:mature cyathium; 4: seed)

12

Figure 2.4 Chemical structure of some known compounds foundin E. hirta

14

Figure 2.5 Types of cell death 22

Figure 2.6 Cell cycle and DNA damage checkpoint 28

Figure 3.1 The herbarium voucher of Euphorbia hirta 47

Figure 3.2 Microscopy of E. hirta leaf transverse section 50

Figure 3.3 Microscopy of E. hirta root transverse section 51

Figure 3.4 Microscopy of E. hirta stem transverse section 52

Figure 3.5 FTIR spectrum of methanolic extract of E. hirta 54

Figure 3.6 GC-MS profile of 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid at retentiontime 11.7

57

Figure 3.7 Calibration curve of 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid by GC-MS

58

Figure 4.1 Haemacytometer chamber grid pattern showing foursquares to be used to count viable cells

80

Figure 4.2 Haemacytometer chamber grid pattern showing viablecells

80

Figure 4.3 Toxic effects of the E. hirta extract after 24 h usingbrine shrimp lethality assay

92

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Figure 4.4 Toxic effects of potassium dichromate after 24 h usingbrine shrimp lethality assay

92

Figure 4.5 Cell viability curve of Vero cells treated with E. hirtaextract by using MTT assay

96

Figure 4.6 Cell viability curve of MCF-7 cells treated withE. hirta extract by using MTT assay

96

Figure 4.7 Cell viability curve of HeLa cells treated with E. hirtaextract by using MTT assay

97

Figure 4.8 Cell viability curve of HT-29 cells treated withE. hirta extract by using MTT assay

97

Figure 4.9 Cell viability curve of Vero cells treated withdoxorubicin hydrochloride by using MTT assay

98

Figure 4.10 Cell viability curve of MCF-7 cells treated withdoxorubicin hydrochloride by using MTT assay

98

Figure 4.11 Cell viability curve of HeLa cells treated withdoxorubicin hydrochloride by using MTT assay

99

Figure 4.12 Cell viability curve of HT-29 cells treated withdoxorubicin hydrochloride by using MTT assay

99

Figure 4.13 Morphological changes of MCF-7 cells treated with0.5% (v/v) DMSO as negative control, viewed underan inverted light microscope

101

Figure 4.14 Morphological changes of MCF-7 cells treated withIC50 of E. hirta extract for 24 h, viewed under aninverted light microscope

101


102


102

Figure 4.17 Morphological changes of MCF-7 cells treated withIC50 of doxorubicin hydrochloride for 24 h, viewedunder an inverted light microscope

103

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103


104

Figure 4.20 The influence of E. hirta extract on the membraneintegrity of MCF-7 cells after 24 h of treatment

106

Figure 4.21 AO/PI staining of MCF-7 cells treated with 0.5% (v/v)DMSO as negative control, viewed under afluorescence microscope

108

Figure 4.22 AO/PI staining of MCF-7 cells treated with IC50 ofE. hirta extract for 24 h, viewed under a fluorescencemicroscope

108

Figure 4.23 AO/PI staining of MCF-7 cells treated with IC50 of E.hirta extract for 48 h, viewed under a fluorescencemicroscope

109

Figure 4.24 AO/PI staining of MCF-7 cells treated with IC50 of E.hirta extract for 72 h, viewed under a fluorescencemicroscope

109

Figure 4.25 Assessment of frequencies of viable, apoptotic andnecrotic cells in E. hirta extract (25 μg/mL) treatedMCF-7 cancer cells at various incubation times

110

Figure 4.26 Annexin V/PI double-staining assay of MCF-7 cellstreated with E. hirta extract (25 μg/mL)

112

Figure 4.27 Annexin V/PI double-staining assay of MCF-7 cellstreated with E. hirta extract (50 μg/mL)

113

Figure 4.28 DNA fragmentations in MCF-7 cells were treated for24 h with indicated concentrations of E. hirta.

115

Figure 4.29 Cell cycle distribution of the cells upon E. hirtatreatment

117

Figure 4.30 Effect of E. hirta on cell cycle distribution of MCF-7cells after 24 hour of treatment.

118

Figure 4.31 Effect of E. hirta extract on intracellular ROS 120

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production by MCF-7 cells

Figure 4.32 Measurement of ROS with DCFH-DA in MCF-7 cellstreated with H202 and E. hirta at various concentrationswith increasing incubation time

120

Figure 4.33 A standard curve of protein concentration usingBradford method

122

Figure 4.34 Caspases activity in MCF-7 cells was analyzed usingcaspase colorimetric protease assay kit

122

Figure 5.1 Effect of E. hirta extract and ethyl methanesulfonateon mitotic index

142

Figure 5.2 Effect of E. hirta extract and ethyl methanesulfonateon micronuclei

146

Figure 5.3 A. cepa root tips without treatment showing normalprophase

147

Figure 5.4 A. cepa root tips treated with E. hirta extract showingC-mitosis

147

Figure 5.5 A. cepa root tips without treatment showing normalmetaphase

148

Figure 5.6 A. cepa root tips treated with E. hirta extract showingsticky chromosome

148

Figure 5.7 A. cepa root tips without treatment showing normalanaphase

149

Figure 5.8 A. cepa root tips treated with E. hirta extract showingchromosome bridge

149

Figure 5.9 A. cepa root tips without treatment showing normaltelophase

150

Figure 5.10 A. cepa root tips treated with E. hirta extract showingvagrant chromosome

150

Figure 5.11 A. cepa root tips without treatment showing normalinterphase

151

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Figure 5.12 A. cepa root tips treated with E. hirta extract showingmicronuclei

151

Figure 5.13 Photomicrographs of stained DNA of untreatedMCF-7 cells showing no DNA damage

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Figure 5.14 Photomicrographs of stained DNA of MCF-7 cellstreated with 25 μg/mL E. hirta for 24 h showing DNAdamage in comet tail

153

Figure 5.15 Effect of E. hirta and doxorubicin on tail momentusing Comet assay DNA damage in MCF-7 cells

154

Figure 6.1 Flow chart for acute toxicity study in accordance to

OECD guideline 420 (OECD, 2001a)

169

Figure 6.2 Flow chart for subchronic toxicity study in accordance

to OECD guideline 408 (OECD, 1998)

170

Figure 6.3 2-3 rats of same sex were placed in the cages 171

Figure 6.4 Mean body weight of male rats in acute toxicity study 175

Figure 6.5 Mean body weight of female rats in acute toxicitystudy

175

Figure 6.6 Photomicrograph showing section of heart of malecontrol rats in acute toxicity study

183

Figure 6.7 Photomicrograph showing section of heart of male ratstreated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

183

Figure 6.8 Photomicrograph showing section of heart of femalecontrol rats in acute toxicity study

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Figure 6.9 Photomicrograph showing section of heart of femalerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

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Figure 6.10 Photomicrograph showing section of lung of malecontrol rats in acute toxicity study

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Figure 6.11 Photomicrograph showing section of lung of male ratstreated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

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Figure 6.12 Photomicrograph showing section of lung of femalecontrol rats in acute toxicity study

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Figure 6.13 Photomicrograph showing section of lung of femalerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

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Figure 6.14 Photomicrograph showing section of kidney of malecontrol rats in acute toxicity study

187

Figure 6.15 Photomicrograph showing section of kidney of malerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

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Figure 6.16 Photomicrograph showing section of kidney of femalecontrol rats in acute toxicity study

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Figure 6.17 Photomicrograph showing section of kidney of femalerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

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Figure 6.18 Photomicrograph showing section of liver of malecontrol rats in acute toxicity study

189

Figure 6.19 Photomicrograph showing section of liver of male ratstreated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

189

Figure 6.20 Photomicrograph showing section of liver of femalecontrol rats in acute toxicity study

190

Figure 6.21 Photomicrograph showing section of liver of femalerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

190

Figure 6.22 Photomicrograph showing section of spleen of malecontrol rats in acute toxicity study

191

Figure 6.23 Photomicrograph showing section of spleen of malerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

191

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Figure 6.24 Photomicrograph showing section of spleen of femalecontrol rats in acute toxicity study

192

Figure 6.25 Photomicrograph showing section of spleen of malerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study

192

Figure 6.26 Mean body weight of male rats in sub-chronic toxicitystudy

196

Figure 6.27 Mean body weight of female rats in sub-chronictoxicity study

196

Figure 6.28 Mean body weight of rats in satellite group in sub-chronic toxicity study

197

Figure 6.29 Photomicrograph showing section of heart of malecontrol rats in sub-chronic toxicity study

207

Figure 6.30 Photomicrograph showing section of heart of male ratstreated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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Figure 6.33 Photomicrograph showing section of heart of male ratsas the satellite group in sub-chronic toxicity study

209

Figure 6.34 Photomicrograph showing section of heart of femalecontrol rats in sub-chronic toxicity study

209

Figure 6.35 Photomicrograph showing section of heart of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

210

Figure 6.36 Photomicrograph showing section of heart of femalerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study

210

Figure 6.37 Photomicrograph showing section of heart of femalerats treated with 1000 mg/kg/day of E. hirta extract in

211

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sub-chronic toxicity study

Figure 6.38 Photomicrograph showing section of heart of femalerats as the satellite group in sub-chronic toxicity study

211

Figure 6.39 Photomicrograph showing section of lung of malecontrol rats in sub-chronic toxicity study

212

Figure 6.40 Photomicrograph showing section of lung of male ratstreated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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Figure 6.43 Photomicrograph showing section of lung of male ratsas the satellite group in sub-chronic toxicity study

214

Figure 6.44 Photomicrograph showing section of lung of femalecontrol rats in sub-chronic toxicity study

214

Figure 6.45 Photomicrograph showing section of lung of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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Figure 6.48 Photomicrograph showing section of lung of femalerats as the satellite group in sub-chronic toxicity study

216

Figure 6.49 Photomicrograph showing section of kidney of malecontrol rats in sub-chronic toxicity study

217

Figure 6.50 Photomicrograph showing section of kidney of malerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

217

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Figure 6.53 Photomicrograph showing section of kidney of malerats as the satellite group in sub-chronic toxicity study

219

Figure 6.54 Photomicrograph showing section of kidney of femalecontrol rats in sub-chronic toxicity study

219

Figure 6.55 Photomicrograph showing section of kidney of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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Figure 6.58 Photomicrograph showing section of kidney of malerats as the satellite group in sub-chronic toxicity study

221

Figure 6.59 Photomicrograph showing section of liver of malecontrol rats in sub-chronic toxicity study

222

Figure 6.60 Photomicrograph showing section of liver of male ratstreated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

222


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223

Figure 6.63 Photomicrograph showing section of liver of male ratsas the satellite group in sub-chronic toxicity study

224

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Figure 6.64 Photomicrograph showing section of liver of femalecontrol rats in sub-chronic toxicity study

224

Figure 6.65 Photomicrograph showing section of liver of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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Figure 6.68 Photomicrograph showing section of liver of femalerats as the satellite group in sub-chronic toxicity study

226

Figure 6.69 Photomicrograph showing section of spleen of malecontrol rats in sub-chronic toxicity study

227

Figure 6.70 Photomicrograph showing section of spleen of malerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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Figure 6.73 Photomicrograph showing section of spleen of malerats as the satellite group in sub-chronic toxicity study

229

Figure 6.74 Photomicrograph showing section of spleen of femalecontrol rats in sub-chronic toxicity study

229

Figure 6.75 Photomicrograph showing section of spleen of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study

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231

Figure 6.78 Photomicrograph showing section of spleen of femalerats as the satellite group in sub-chronic toxicity study

231

Figure 7.1 Flow chart for fractionation of E. hirta methanolextract

245

Figure 7.2 Cell viability curve of MCF-7 cells treated withE. hirta hexane fraction by using MTT assay

248

Figure 7.3 Cell viability curve of MCF-7 cells treated withE. hirta EH Hex 4 subfraction by using MTT assay

248

Figure 7.4 Cell viability curve of MCF-7 cells treated withE. hirta EH Hex 7 subfraction by using MTT assay

249

Figure 7.5 Cell viability curve of MCF-7 cells treated withE. hirta EH EtOAc 1 subfraction by using MTT assay

249


250


250


251

Figure 7.9 Cell viability curve of MCF-7 cells treated withE. hirta EH BuOH 8 subfraction by using MTT assay

251

Figure 7.10 Cell viability curve of MCF-7 cells treated withE. hirta EH BuOH 9 subfraction by using MTT assay

252

Figure 7.11 LC chromatogram of E. hirta EH Hex 4 subfraction 254

Figure 7.12 ESI total ion chromatogram of E. hirta hexane extract 254

Figure 7.13 MS spectra for E. hirta EH Hex 4 subfraction 254

Figure 7.14 3,3’,4’,5,7-Pentahydroxy-8-(5-oxo-2-pyrrolidinyl)flavone isolated from the E. hirta

255

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LIST OF TABLES

Page

Table 3.1 The main compounds identified in methanol extract ofE. hirta crude extract

56

Table 3.2 Biological activity of major phyto-componentsidentified in E. hirta crude extract by GC-MS

56

Table 3.3 Heavy metal concentration in E. hirta extract 60

Table 3.4 DPPH antioxidant activity of 1 mg/mL E. hirta extractwithin duration of 3 months

62

Table 4.1 Types of cell lines used on the in vitro screening andtheir complete growth medium requirements

77

Table 4.2 Cytotoxic effects of methanol extract of E. hirta onseveral cancer cells and normal cells at their respectiveIC50

95

Table 4.3 Cytotoxic effects of doxorubicin hydrochloride onseveral cancer cells and normal cells at their respectiveIC50

95

Table 5.1 Mitotic index in control and E. hirta treatmentconcentrations on A. cepa root cells

142

Table 5.2 Chromosomal aberrations in control and E. hirtatreatment concentrations on A. cepa root cells

144

Table 5.3 Micronuclei in control and E. hirta treatmentconcentrations on A. cepa root cells

146

Table 5.4 Mean percentage of DNA damage by the comet assayin MCF-7 cells treated with E. hirta and doxorubicinhydrochloride

154

Table 6.1 Cage side observations on general appearance andbehavioural observations for control and treatedgroups

173

Table 6.2 Haematology values of rats treated with single dose ofE. hirta methanol extract in acute oral toxicity study

177

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Table 6.3 Biochemistry values of rats treated with single dose ofEuphorbia hirta methanol extract in acute oral toxicitystudy

179

Table 6.4 Relative organ weights of rats treated with single doseof E. hirta methanol extract in acute oral toxicity study

181

Table 6.5 Cage side observations on general appearance andbehavioural observations for control and treatedgroups

194

Table 6.6 Haematological values of male rats treated with dailydose of E. hirta methanol extract in sub-chronictoxicity study

199

Table 6.7 Haematological values of male rats treated with dailydose of E. hirta methanol extract in sub-chronictoxicity study

200

Table 6.8 Biochemical values of male rats treated with dailydose of E. hirta methanol extract in sub-chronictoxicity study

202

Table 6.9 Biochemical values of female rats treated with dailydose of E. hirta methanol extract in sub-chronictoxicity study

203

Table 6.10 Relative organ weights of rats treated with daily doseof E. hirta methanol extract in sub-chronic toxicitystudy

205

Table 7.1 IC50 values of E. hirta extract various isolated fractionstowards MCF-7 at 24 hour as determined by usingMTT assay

247

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LIST OF ABBREVIATIONS

AAS Atomic Absorption Spectroscopy

ALT Alanine aminotransferase

ANOVA Analysis of variance

AST Aspartate aminotransferase

ATP Adenosine triphosphate

DCFH-DA 2’, 7’-Dichlorodihydrofluorescin diacetate

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO Dimethyl Sulfoxide

E. hirta Euphorbia hirta

EDTA Ethylenediaminetetraacetic acid

FBS Fetal Bovine Serum

FITC Fluorescein isothiocyanate

FTIR Fourier Transform Infrared

GC-MS Gas Chromatography-Mass Spectrometry

HPLC High Performance Liquid Chromatography

IC50 Inhibitory Concentration 50%

LC-MS Liquid Chromatography-Mass Spectrometry

LD50 Lethal Dose 50%

MPLC Medium Pressure Liquid Chromatography

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NOAEL No Observed Adverse Effect Level

OECD Organisation for Economic Cooperation and Development

PBS Phosphate Buffered Saline

PI Propidium Iodide

RPMI 1640 Rosswell Park Memorial Institute

RNAse Ribonuclease

ROS Reactive Oxygen Species

SEM Standard Error of the Mean

SPSS Statistical Package for the Social Sciences

WBC White Blood Cell count

WHO World Health Organisation

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AKTIVITI SITOTOKSIKAN SECARA IN VITRO DAN UJIAN

KETOKSIKAN ORAL SECARA IN VIVO DARIPADA Euphorbia hirta

ABSTRAK

Euphorbia hirta adalah sejenis tumbuhan yang digunakan dalam perubatan

tradisional secara meluas untuk mengubati pelbagai jenis penyakit. Sitotoksik,

genotoksik secara in vitro dan penilaian ketoksikan secara in vivo ekstrak metanol

daripada E. hirta telah diselidik dalam kajian ini. Penilaian pelbagai parameter

farmakognosi telah dijalankan mengikut garis panduan Pertubuhan Kesihatan

Sedunia (WHO) untuk pempiawaian ekstrak E. hirta. Pempiawaian kandungan kimia

melibatkan kuantifikasi sebatian kimia utama dalam ekstrak E. hirta. Kaedah GC-

MS yang telah digunakan untuk kuantifikasi asid 1,3,4,5

tetrahidroksisikloheksanakarbosilik dalam ekstrak adalah cepat, tepat, dan linear.

Enam puncak utama dalam lingkungan 600 - 1500 dan 2800 - 3400 cm-1

diperhatikan dalam spektrum FTIR. Kepekatan logam berat dalam ekstrak E. hirta

adalah didapati berada di bawah had yang dibenarkan. Dalam asai antioksidan

terhadap radikal bebas DPPH, ekstrak E. hirta menunjukkan aktiviti antioksidan

50% ke atas, tidak bergantung pada masa pengekstrakan. Kajian potensi ketoksikan

ekstrak E. hirta telah dilakukan dengan menggunakan kaedah kematian anak udang

dan ujian sitotoksisiti terhadap sel-sel kanser. Ekstrak E. hirta menunjukkan kesan

toksik yang ketara terhadap anak udang dengan nilai (kepekatan maut) LC50

sebanyak 620.38 µg/mL (24 jam). Perbandingan keputusan ini dengan kesan toksik

kawalan positif iaitu kalium dikromat didapati bahawa kesan aktiviti ketoksikan

yang ditunjukkan oleh ekstrak metanol adalah sederhana. Ujian sitotoksisiti MTT

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menunjukkan bahawa ekstrak E. hirta merencatkan pertumbuhan sel MCF-7 dengan

nilai (kepekatan perencatan) IC50 sebanyak 25.26 µg/mL yang semakin meningkat

seiring dengan peningkatan kepekatan ekstrak dan masa pengeraman. Kajian

mikroskopik menunjukkan bahawa sel-sel yang dirawat dengan ekstrak E. hirta

menandakan ciri-ciri morfologi apoptosis. Ekstrak E. hirta juga dikesan tidak

mempunyai pengaruh terhadap kebocoran laktat dehidrogenase (LDH) dan

penghasilan species oksigen reaktif (ROS) secara intrasel. Oleh itu, E. hirta mungkin

merangsang kesan sitotoksisiti melalui mekanisma yang tidak bersandarkan kepada

ROS terhadap sel-sel MCF-7. Analisis sitometri aliran dengan pewarnaan Annesin

V/Propidium Iodida mengesahkan bahawa ekstrak E. hirta merangsang apoptosis

dalam sel-sel MCF-7. Rawatan ekstrak E. hirta juga menyebabkan fragmentasi DNA

dalam sel-sel MCF-7. Tambahan pula, rawatan E. hirta menyebabkan pengumpulan

sel-sel di fasa S and G2/M kitaran sel dengan menginduksi apoptosis. Kajian aktiviti

kaspase mendedahkan bahawa ekstrak E. hirta mengaruhi apoptosis tanpa

bersandarkan laluan kaspase-3 tetapi melalui pengaktifan kaspase-2, 6, 8 dan 9.

Untuk mengenal pasti sebatian kimia yang bersifat sitotoksiti, penyisihan ekstrak

E. hirta telah dilakukan berpandukan bioasai. Subfraksi heksan E. hirta iaitu EH Hex

4 menunjukkan aktiviti sitotoksiti yang signifikan di kalangan semua fraksi yang

diuji. Analisis selanjutnya dengan menggunakan kaedah LC-MS terhadap fraksi EH

Hex 4 membawa kepada identifikasi sebatian 3,3’,4’,5,7-Pentahydroxy-8-(5-oxo-2-

pyrrolidinyl) flavone sebagai agen cenderung kepada sitotoksik di dalam ekstrak

E. hirta. Data yang diperolehi daripada kajian ini menunjukkan bahawa E. hirta

menyebabkan kematian sel secara apoptotik dan mencadangkan bahawa E. hirta

boleh digunakan sebagai agen anti-kanser yang mengaruhi apoptosis untuk rawatan

kanser payudara dengan kajian lanjutan yang terperinci. Kesan potensi genotoksik

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E. hirta telah dikaji dengan menggunakan ujian Allium cepa and asai komet. Dalam

ujian A. cepa, ekstrak E. hirta menunjukkan kesan genotoksik dan mitodepresif yang

signifikan pada kepekatan sebanyak 1000 µg/mL.. Peningkatan dalam nilai aberasi

kromosom yang bersandarkan kepada kepekatan ekstrak telah diperhatikan seperti

kromosom kelekitan, kromosom c-mitosis, kromosom jambatan dan kromosom

vagrant. Sel mikronukleus juga telah diperhatikan pada interfasa. Dalam ujian komet,

rawatan dengan 25 µg/mL ekstrak E. hirta selama 72 jam pada sel-sel MCF-7

menyebabkan peningkatan kerosakan DNA sebanyak 48.16% berbanding dengan

kawalan yang tidak dirawat. Data ini mengesahkan kesan genotoksik ekstrak E. hirta.

Ujian ketoksikan oral akut dan sub-kronik ekstrak E. hirta telah dilakukan dengan

menggunakan tikus Sprague Dawley. Ekstrak pada dos tunggal iaitu sebanyak 5000

mg/kg berat badan tidak menunjukkan kematian atau kesan toksik yang berkaitan

dengan rawatan tersebut dalam tempoh pemerhatian selama 14 hari. Oleh itu, dos

LD50 (dos membunuh) tumbuhan ini dianggarkan melebihi 5000 mg/kg. Dalam

kajian ketoksikan sub-kronik, pemberian ekstrak E. hirta sebanyak 50 mg/kg, 250

mg/kg, 1000 mg/kg/hari berat badan menunjukkan tiada sebarang perbezaan yang

signifikan dalam makanan dan penggunaan air, perubahan berat badan, parameter

hematologi dan biokimia, berat relatif organ dan pemerhatian secara kasar

berbanding dengan kumpulan kawalan. Pemeriksaan makropatologi dan

histopatologi kesemua organ tidak menunjukkan sebarang perubahan morfologi.

Penganalisaan keputusan ini bersama-sama dengan informasi mengenai tanda-tanda

penyakit, tingkah laku dan pemantauan kesihatan boleh membawa kepada

kesimpulan bahawa pengambilan jangka panjang ekstrak E. hirta selama 90 hari

tidak menyebabkan ketoksikan sub-kronik. Kesimpulannya, keputusan ujian

ketoksikan secara in vitro dan in vivo yang diperolehi dalam kajian ini membuktikan

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tentang kepentingan kedua-dua kaedah tersebut untuk memperolehi data penilaian

toksikologi yang komprehensif untuk agen terapeutik semula jadi.

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IN VITRO CYTOTOXICITY ACTIVITY AND IN VIVO ACUTE ORAL

TOXICITY OF Euphorbia hirta

ABSTRACT

Euphorbia hirta is an annual plant that has been widely used in traditional medicine

to treat various diseases. In vitro cytotoxicity, genotoxicity properties and in vivo

toxicological evaluation of E. hirta methanol extract were investigated in this study.

Various pharmacognostical parameters evaluation was carried out as per World

Health Organization (WHO) guidelines procedure for the standardization of E. hirta

extract. The chemical constituent aspect of standardization involves quantification

of the main chemical components in E. hirta. The GC-MS analysis was used for

quantification of 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid in the extract was

rapid, accurate and linear. Six major peaks in the range of 600 - 1500 and 2800 -

3400 cm-1 were observed in the FTIR spectra. The concentrations of heavy metals

determined in E. hirta extract were well below the permissible limit. In 2,2-

Diphenyl-1-picylhydrazyl radical (DPPH) assay, the extract showed a general

consistency on antioxidant activity above 50%, independent of the extraction time.

The potential cytotoxicity effect of E. hirta was investigated using brine shrimp

lethality assay and the cytotoxicity assay against cancer cells. The extract of E. hirta

showed significant toxicity against brine shrimp with (Lethal Concentration) LC50

value of 620.38µg/mL (24 h). Comparison with positive control potassium

dichromate signifies that cytotoxicity exhibited by the methanol extract have

moderate activity. The 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyltetrazolium

bromide (MTT) cytotoxicity assay showed that E. hirta inhibited MCF-7 cell

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viability with the (Inhibitory Concentration) IC50 values 25.26 µg/ml in a dose and

time-dependent manner. Microscopic studies showed that E. hirta treated cells

exhibited marked morphological features characteristic of apoptosis. E. hirta extract

also had an ignorable influence on the lactate dehydrogenase (LDH) leakage and

generating intracellular reactive oxygen species (ROS). Therefore, E. hirta might

induce the cytotoxicity via a ROS-independent mechanism in MCF-7 cells. The

Annexin V/Propidium Iodide flow cytometry study confirmed that E. hirta extract

induced apoptosis in MCF-7 cells. E. hirta extract treatment also resulted in DNA

fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the

accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase

activity study revealed that E. hirta extract induced apoptosis through the caspase-3

independent pathway by the activation of caspase-2, 6, 8 and 9. To identify the

cytotoxic compound, E. hirta extract was subjected to bioassay-guided fractionation.

E. hirta hexane subfraction, namely EH Hex 4 demonstrated highest activity among

all the fractions tested. Further Liquid Chromatography–Mass Spectrometry (LC-MS)

analysis of EH Hex 4 led to identification of 3,3’,4’,5,7-Pentahydroxy-8-(5-oxo-2-

pyrrolidinyl) flavone as the likely cytotoxic agent in E. hirta extract. The data

obtained from this study revealed that E. hirta induced apoptotic cell death and

suggests that E. hirta could be used as an apoptosis-inducing anti-cancer agent for

breast cancer treatment with further detailed studies. The potential genotoxic effect

of E. hirta was investigated using Allium cepa and comet assay. In A. cepa assay, the

result showed that the methanol extract of E. hirta exerted significant genotoxic and

mitodepressive effects at 1000 µg/mL. A dose-dependent increase of chromosome

aberrations was also observed such as stickiness, c-mitosis, bridges and vagrant

chromosomes. Micronucleated cells were also observed at interphase. In the comet

xxxii

assay, the treatment of 25 µg/mL of E. hirta extract for 72 h on MCF-7 cells caused

an increase in DNA damage by approximately 48.16% compared to the unchallenged

control which confirmed the genotoxic effect of E. hirta extract. The acute and sub-

chronic oral toxicity of E. hirta was evaluated in Sprague Dawley rats. The extract at

a single dose of 5000 mg/kg did not produce treatment related signs of toxicity or

mortality in the animals tested during the 14-day observation period. Therefore, the

(Lethal Dose) LD50 of this plant was estimated to be more than 5000 mg/kg. In the

subchronic toxicity study, the administration of 50 mg/kg, 250 mg/kg, 1000

mg/kg/day of E. hirta extract per body weight revealed no significant difference in

the body weight change, haematological and biochemical parameters, relative organ

weights and gross findings compared to the control group. Macropathology and

histopathology examination of all organs did not reveal morphological alteration.

Analyses of these results with the information of signs, behaviour and health

monitoring could lead to the conclusion that the long-term oral administration of

E. hirta extract for 90 days does not cause toxicity. In conclusion, in vitro and in vivo

toxicity results obtained in this study proved the importance of both methods to

obtain comprehensive toxicological evaluation of natural product agents.

1

CHAPTER 1.0: INTRODUCTION

1.1 Overview and rationale of the study

Natural products, especially medicinal plants, have played a significant role in drug

discovery and development of therapeutic agents. Plants contain many biologically

active compound(s) which have potential for development as therapeutic agents.

More than 35,000 plant species have been reported to be used in various human

cultures around the world for medical purposes (Lewington, 1993; Faleyimu and

Oso, 2012). There is a growing scientific consensus about the impending loss of

tropical species as some of these species may not be available to future generations

of natural product drug discovery scientists (Gurib-Fakim, 2006). Plants of the

tropical rainforests especially in Malaysia serve as a potential source of

pharmaceutical leads due to the high chemical diversity. Hence, toxicity studies play

an important role in the identification and isolation of pharmaceutical leads from

medicinal plants.

Although Euphorbia hirta (E. hirta) has been used for generations as a

medicinal herb, however little is known of the toxicological potential of the herb.

Based on their long-term use by humans, one might expect E. hirta used in

traditional medicine to have low toxicity. However, recent research have revealed

that many plants used as food or in traditional medicine showed toxic effects in in

vitro assays (Cardoso et al., 2006; Mohd-Fuat et al., 2007). This raises concern about

the potential toxic hazards resulting from the short-term and long-term use of

E. hirta. Therefore, evaluating the toxicological effects of E. hirta extract intended to

be used in humans is a crucial part of its assessment for potential hazards. Regarding

drug discovery and development, there are different weights of interest of all

2

concerned groups, which need to be taken into consideration (Olejniczak et al.,

2001). The general public, patients and consumers are mostly interested in the fast

access to safe and efficient medicines, as well as in animal welfare. Health

authorities are obliged to make sure that laws and regulations are followed, i.e., both

regulatory laws on medicines and on animal protection.

Moreover, the pharmaceutical industry plans to produce safe and ethical

medicine in the most economic way. The different interests of the public, the

pharmaceutical industry and health authorities may lead to potential conflicts.

Therefore, the ICH (International Conference on Harmonisation of Technical

Requirements for Registration of Pharmaceuticals for Human Use), the common

regulatory initiative of the EU, USA and Japan has addressed these topics in their

mission and goals, which are: to provide and assure safe and ethical medicines; to

maintain regulatory compliance; to achieve fast development and market

authorization; to economize costs and to consider animal protection (ICH, 2007). In

light of the given situation of therapeutic agents development and because of the

above described interests, new approaches are progressing in different phases of drug

development that allow for more efficient, yet safe procedures. One possible way to

economize time and costs, as well as to address animal protection, is to introduce

alternative methods into non-clinical safety testing. Currently, animal tests are

mandatory for the evaluation of acute toxicity of chemicals and new drugs. The

replacement of the in vivo tests by alternative in vitro assays would offer the

opportunity to screen and assess numerous plants extract at the same time, to predict

acute oral toxicity and thus accelerate drug development processes. Furthermore, the

substitution of in vivo tests by in vitro methods shows a proactive pursuit of ethical

3

and animal welfare issues. There is little toxicological data available on E. hirta

although the plant has been used as herbal medicine since ancient time. Hence, the

current study was designed to compare the in vivo and in vitro toxicity of E. hirta

extract besides evaluating its potential as anticancer agents and its mechanism of

actions. The conflict between in vitro and in vivo studies persists as the results

obtained from in vitro studies are often not directly interpretable in the context of

potential in vivo exposures (Yoon et al., 2012). In vitro data from cell-based assays

should be viewed as indicators of potential toxicity, but not as absolute markers. On

the other hand, in vivo data from animal studies are more indicative of toxicity and

may be considered to be safety markers (WHO, 2000).

Cancer is a growing health problem around the world characterised by

uncontrolled cell growth. Breast cancer is the one of the most common cancer in

females. Global breast cancer case has increased from 1.38 million women in 2008

to more than 1.6 million in 2010 (Ferlay et al., 2012). There were 3242 female breast

cancer cases diagnosed in 2007, accounted for 18.1% of all cancer cases reported

and 32.1% of all female cases (Zainal and Nor Saleha, 2011). It is estimated over

60% of anticancer drugs available in the market are of natural origin and majority of

these compounds are obtained from higher plants (Newmann et al., 2003). The

development of anticancer drugs, especially cytotoxic agents from medicinal plants,

differs significantly from the drug development process for any other indication.

Major research efforts are aimed at discovery of molecular targets that are specific

for cancer cells, development of agents that are toxic to cancer cells, and at devising

their synergistic combinations. Hence, the identification toxicity of medicinal plants

may contributes for the identification of medicinal plants that are selectively induces

4

cytotoxicity in cancer cells but not in normal cells, which is an unusual property that

is not shared by conventional chemotherapeutic agents (Blagosklonny and Pardee,

2001; Li et al., 2003). The development of anticancer drugs from natural sources

requires toxicity information on the constituent of interest. It should be emphasized

that toxic effects of the anticancer agent on the host cells must be considered, as a

substance may exhibit an apparent anticancer activity by virtue of its toxic effect on

the cells. Therefore, in this study besides various toxicity evaluation of E. hirta

extract, the anticancer potential of this plant on cancer cells was also evaluated to

develop therapeutic agents which selectively toxic (toxicity to cancer cells but not to

normal cells) on cancer cells.

1.2 Research objectives

The current study was undertaken with the following objectives:

1. To standardize the methanolic extract of E. hirta

2. To determine the in vitro cytotoxicity of methanolic extract of E. hirta on

several cell lines and Artemia salina

3. To determine the mode and mechanism of cell death in MCF-7 cells treated

with methanolic extract of E. hirta

4. To determine the genotoxicity of methanolic extract of E. hirta

5. To evaluate in vivo acute and sub-chronic oral toxicity of E. hirta extract

administration in albino rats

6. To identify cytotoxic fraction/compound (s) from E. hirta

5

CHAPTER 2.0: LITERATURE REVIEW

2.1 Pharmaceuticals from plants

2.1.1 Background of herbal medicine in Malaysia

From prehistoric times, plants have been a valuable source of natural products for

treatment of various diseases. This plant-based, traditional medicine system

continues to play an essential role in health care, with about 80% of the world’s

population relying mainly on traditional medicines for their primary modality of

health care (Farnsworth et al., 1985; Jyothi et al., 2011).

Malaysia is among the world’s top 12 rich biodiversity countries where

Ayurveda, Siddha, traditional Chinese, traditional Malay, Unani and other traditional

systems of medicine are commonly practised. Therefore, Malaysia is to become a

significant global player given that the global trade of herbal products, which

amounted to RM777 billion in 2006 and is projected to triple by 2020 (Anonymous,

2011). Malaysia’s herbal industry has been identified as one of the agriculture Entry

Point Projects (EPPs) under the National Key Economic Areas (NKEAs) in the

Economic Transformation Programme (ETP). Several popular Malaysian herbs have

been identified as the focus, including Eurycoma longifolia (Tongkat Ali), Labisia

pumila (Kacip Fatimah), Orthosiphon stamineus (Misai Kucing), Andrographis

paniculata (Hempedu Bumi) and Phyllanthus niruri (Dukung Anak) (Anonymous,

2011).

Herbal medicine or phytomedicine refers to the use of a plant's seeds, berries,

roots, leaves, bark or flowers for medicinal purposes. Historically, herbal drugs were

used as tinctures, poultices, powders and teas followed by formulations, and lastly as

6

pure compounds. There are 119 pure chemical compounds extracted from higher

plants used in medicine and 74% of these compounds came from folklore claims

(Gurib-Fakim, 2006). WHO recognizes herbal medicines as valuable resources and

states it is necessary to develop a systematic inventory of medicinal plants to

introduce regulatory measures, apply good manufacturing practices and to include

herbal medicines in the conventional pharmacopoeia of each nation (WHO, 1998a).

Well-known examples of plant-derived prescription drugs include the

antimalarial drug quinine obtained from the bark of Cinchona officinalis; the

analgesics, codeine and morphine from Papaver somniferum; the antihypertensive

reserpine from Rauwolfia serpentina; and the cardiac glycoside, digoxin, from

Digitalis purpurea (Cragg and Newman, 1999). Initially, quinine from cinchona bark

was used to manage the symptoms of malaria long before the disease was identified

and the raw ingredients of a common aspirin tablet have been a popular painkiller for

far longer than we have had the access to tablet-making machinery (Ansari and

Inamdar, 2010).

Tropical rain forests continue to support a vast reservoir of potential drug from

plant species due to their ability to synthesize various chemicals as defense agents

against pests, diseases and predators. They are an excellent reservoir of medicines

and chemical leads with which researchers can design and synthesize new drugs

(Zakrzewski, 2002). The potential for finding more compounds is enormous as to

date only less than 1% of tropical forest plant species have been tested for chemical

compounds and medicinal value, yet at least 25 % of all modern drugs originally

came from rainforests (Rates, 2001). Due to the disappearance of tropical plant

7

species, there is a sense of urgency to increase screening efforts and investigate

commercial potential of tropical plant chemicals.

2.1.2 Safety concern on the use of pharmaceutical from plants

Safety is a fundamental principle in the provision of herbal medicines and a critical

component of quality control. Herbal medicines are generally considered as safe

based on their long-standing use in various cultures. However, there were case

reports of serious adverse effects after administration of herbal products. High

profile issues such as adverse reactions associated with Ephedra and Aristolochia

have shown that herbal medicine can produce toxicity after consumption in humans

(Jordan et al., 2010). A number of other plant-derived agents were entered into

clinical trials and were terminated due to the lack of efficacy or unacceptable

toxicity. Some examples are acronycine, bruceantin, maytansine and thalicarpine

(Cragg et al., 1993). Some adverse effects reported in association with herbal

products are attributable to quality issue. Toxicity associated with herbal medicines

may result from misidentification of plant species, contamination with toxic metals

and pesticide, adulteration with pharmacologically active synthetic compounds and

herb-drug interactions (Ernst, 2004; Van Breemen et al., 2008).

This issue has led to the regulation of traditional medicines through legally

enforced product registration in Malaysia in January 1992. However, herbal

medicines are not rigorously regulated by the Drug Control Authority (DCA) of

Malaysia. The evaluation of the quality and safety of herbal medicines by the DCA

is limited to control on the content of specified adulterants and contaminants such as

heavy metals and micro-organisms (Aziz, 2009). Given that herbal medicines are

often assumed as being harmless, the public may be at risk of adverse effects from

8

the use of herbal medicines. The scientific evaluation of safety and efficacy of herbal

products and medicinal preparation is thus of vital importance from both medicinal

and economic perspectives.

2.2 Euphorbia

Euphorbia is a genus of flowering plants belonging to the family Euphorbiaceae,

comprises about 300 genera and 5,000 species, common to tropical countries (Figure

2.1). The genus Euphorbia is the largest in spurge family, comprising more than

2000 species (Aslam et al., 2014). It is characterized by the presence of white milky

latex which is toxic to a certain extent (Kumar et al., 2010).

2.2.1 Euphorbia hirta L.

2.2.1.1 Botanical description

E. hirta is a small annual, branched herb prostrate to ascending with branches

reaching 70 cm in height (Figure 2.2). It is reddish or purplish in color, with

abundant latex and is covered with short hairs. Its leaves are opposite, distichous and

simple (not lobed or divided). The leaf blades are lanceolate-oblong, long elliptic or

ovate-lanceolate. The inflorescence of E. hirta has a terminal or axillary cluster of

flowers, called a ‘cyathium’, with several cyathia densely clustered into a cyme

(Figure 2.3). The flowers of E. hirta are unisexual; the male flowers are sessile, the

bracteoles are linear, fringed, the perianth is absent and possesses one stamen,

whereas the female flowers have short pedicel, the perianth is rimmed, the ovary is

superior, covered with short hairs, three-celled, possesses three styles, minute and

the apex is two-fid. The fruit of E. hirta is exerted, acutely three-lobed, base

truncate, covered in short hairs and three-seeded. The seeds are oblong, four-sided

9

prismatic, slightly wrinkled, pinkish brown and caruncle absent. Flowering duration

of individual plant is usually throughout the year. E. hirta often grows in cultivated

areas in lowland, paddy fields, gardens, roadsides and waste places. They prefer dry

condition, from sea-level up to 2000 m altitude. E. hirta has many synonyms in

different countries. The common names for E. hirta are snakeweed, asthmaweed,

hairy spurge and milkweed. In Malaysia, it is called ambin jantan, kelusan, gelang

susu or keremak susu (Huang et al., 2012). Figure 2.1 shows the taxonomic

classification of E. hirta plant.

2.2.1.2 Origin and geographical distribution

E. hirta is native to Central America and a very common weed of the tropics and

subtropics; it occurs throughout tropical Africa and also in South Africa (PROSEA,

2014)

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Figure 2.1: Taxonomic classification of E. hirta (Mamun-Or-Rashid et al., 2013)

11

Figure 2.2: Euphorbia hirta plant

12

Figure 2.3: E. hirta plant (1: plant habit; 2: young cyathium; 3: mature cyathium; 4:seed) (PROSEA, 2014)

13

2.2.1.3 Phytochemistry

Phytochemical analysis of E. hirta showed the presence of reducing sugar, alkaloids,

flavonoids, sterols, tannins and triterpenes in the whole plant. Sterols isolated from

E. hirta are cycloarternol, 24- methylene-cycloarternol, β-sitosterol, euphorbol

hexacozonate, 1-hexacosanol, tinyaloxin, campesterol and stigmasterol (Shih and

Cherng, 2012). E. hirta contains three hydrolysable tannins, namely, dimeric

hydrolysable tannin, euphorbin E and the dimeric dehydroellagitannins, euphorbin A

and euphorbin B (Yoshida et al., 1990). Polyphenols from the leaves of E. hirta were

also isolated by using physicochemical and spectroscopic methods: gallic acid, 2,4,

6-tri-O-galloyl-D-glucose, quinic acid ester, 1,2,3,4, 6-penta-Ogalloyl- β-D-glucose

and 3,4-di-O-galloylquinic acid (Chen, 1991). The triterpenes β-amyrin, 24-

methylencycloartenol and β-sitosterol have been identified from E. hirta (Martinez et

al., 1999). Flavonol glycosides were isolated from methanolic extract of E. hirta and

chemically characterized as afzelin, quercitrin, and myricitrin (Liu et al., 2007). The

chemical structure of 2,4, 6-tri-O-galloyl-D-glucose, β-sitosterol, myricitrin, gallic

acid and quercitrin found in E. hirta are showed in Figure 2.4.

14

2, 4, 6-tri-O-galloyl-D-glucose β-sitosterol

Myricitrin Gallic acid

Quercitrin

Figure 2.4: Chemical structure of some known compounds found in E. hirta(Source: http://www.chemspider.com/Chemical-Structure)

15

2.2.1.4 Pharmacological activities

E. hirta exhibited several pharmacological activities as reported in literature. The

antibacterial activity of E. hirta has been comprehensively investigated and proven.

The ethanolic extract of E. hirta inhibited the growth of the Escherichia coli,

Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtili (Ogbulie et

al., 2007). Aqueous and chloroform leaf extracts of E. hirta possess an antibacterial

activity against Klebsiella pneumonia (Suresh et al., 2008).

The antidiarrheal effect of the herb decoction was studied in mice. The

lyophilised decoction of E. hirta was found to possess antidiarrheal activity induced

by castor oil, arachidonic acid and prostaglandin E2 (Galvez et al., 1993a).

Quercitrin isolated from E. hirta showed an antidiarrheal activity at a dose of 50

mg/kg against castor oil and prostaglandin E2-induced diarrhea in mice (Galvez et

al., 1993b).

The antioxidant activity of E. hirta was comparable with that of ascorbic acid

and found to be dose dependent (Basma et al., 2011). The aqueous extract of E. hirta

showed an antioxidant effect and a free radical scavenging activity in various in vitro

models like total antioxidant and total ferric reducing power determination, assay for

free radical-scavenging activity using ABTS, DPPH and hydroxyl radical scavenging

assays. It showed maximum antioxidants and free radical scavenging activities at

0.25 mg/mL (Sharma et al., 2007).

E. hirta was reported to possess antimalarial activity. The bioassay-guided

fractionation of methanol extracts of the aerial parts of E. hirta resulted in isolation

of flavonol glycosides afzelin, quercetrin and myricitrin. The compounds showed

16

inhibition of the proliferation of Plasmodium falciparum with IC50 values of 1.1, 4.1,

5.4 µg/mL, repectively. Furthermore, they showed little cytotoxic activity against

human epidermoid carcinoma KB3-1 cells (Liu et al., 2007).

Antiinflammatory effects of E. hirta were exhibited in 12-o-tetradecanoyl

phorbol acetate-induced ear inflammation in mice whereby the result showed a dose-

dependent effect (Martinez et al., 1999; Lanhers et al., 1991). In some recent studies,

E. hirta produced a remarkable antiinflammatory effect via its active component of

beta-amyrin and showed a dose-related inhibition against LPS-induced NO

production (Camuesco et al., 2004; Shih et al., 2010).

E. hirta was found to have an antiasthmatic activity due to the relaxation effect

on the bronchial tubes and a depressant action on respiration (Kumar et al., 2010).

E. hirta reduced asthma attack has been shown as effective as corticosteroid did in

the BALB/c asthmatic mouse mode (Ekpo and Pretorius, 2008). The possible active

component of E. hirta is thought to be quercitrin. E. hirta ethanol extract

significantly prevented eosinophil accumulation and eosinophil peroxidase activity

and reduced the protein content in bronchoalveolar lavage fluid in a 'mild' model of

asthma (Singh et al., 2006).

2.2.1.5 Ethnopharmacology

E. hirta plays a very important role in traditional use due to its wide range of

biological and pharmacological properties. The plant part used are the whole plant,

latex, aerial part, root and leaf. Different formulations are used, including crude

drug, decoction, infusion, lotion and powders. The latex and fluid extract of the

tincture are used to treat gastrointestinal disorders (diarrhea, dysentery, intestinal

17

parasitosis), bronchial and respiratory diseases (asthma, bronchitis, hav fever)

(Kumar et al., 2010). The decoction of the root is used to alleviate vomiting.

Decoction of dry herbs is used for skin diseases. Root decoction is used for snake

bites, sores, wounds, boils and is beneficial for nursing mothers with deficient milk.

The entire plant is prescribed as an antidote; it is considered hemostatic, sedative and

narcotic. In Australia, the most common use of E. hirta is to treat hypertension,

asthma, edema and pectoral complaints. In the Philippines, leaves are mixed with

datura metel leaves and flowers in the preparation of "asthma-cigarettes" (Huang et

al., 2012).

2.2.1.6 Plant derived anticancer agents

Plants have enormous contribution to modern medicine with the origin of numerous

drugs in use today are derived from plants. Most of these plant-derived drugs were

originally discovered through the study of herbal cures and folk knowledge of

traditional people. Some of these could not be synthesised economically and are still

obtained from wild or cultivated plants despite the enormous advancement in

synthetic chemistry. Natural product secondary metabolites from plants and

microbes in particular play a very important role in the amelioration of cancer. In the

field of anticancer activity, a correlation between biological activity and plants used

in folklore has been validated (Farnsworth and Kaas, 1981). More than 60% of all

cancer drugs are of natural origin and a majority of these compounds are obtained

from higher plants (Newmann et al., 2003).

In the 1970s, one of the significant breakthroughs in the field of anti-cancer

drugs comes from the Madagascan Periwinkle. The Periwinkle has a long history of

treating a wide variety of diseases and has also been used for centuries against

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diabetes. This traditional use as a cure for diabetes resulted in preliminary laboratory

investigations. Laboratory animals developed significantly low counts of white blood

cells leaving them defenseless against infections caused by bacteria. The result

showed that one or more of the Periwinkle alkaloids might slow or halt white blood

cell production and this is probably the mechanism that evolved in nature to

discourage herbivorous predators from eating the Madagascan Periwinkle. Hence,

further bioassay-guided isolation of the plant led to the characterization or the active

complex alkaloidal compounds namely vincristine and vinblastine. The level of

vincristine in the plant is extremely low (0.0002%) and thus making it a very

expensive antitumour agent. They have been proven to be effective agents against

childhood leukaemia, breast cancer and Hodgkin’s disease. Vincristine and

vinblastine exert their anticancer properties by inhibiting mitosis by binding to

tubulin, thus preventing the cell from making spindles it needs to be able to move its

chromosomes around as it divides (Gurib-Fakim, 2006).

Other examples of plant-derived anticancer drug are etoposide and teniposide,

which are semisynthetic derivatives of the natural product epipodophyllotoxin.

Epipodophyllotoxin is an isomer of podophyllotoxin that was isolated as the active

antitumor agent from the roots of various species of the genus Podophyllum. These

plants have a long history of medicinal use by early American and Asian cultures,

including the treatment of skin cancers and warts. Etoposide is now marketed as

Vepesid for small cell lung cancer, testicular cancer and lymphomas while

Teniposide is used in treating brain tumours (Cragg and Newman, 1999). Recent

plant-derived anticancer drug are homoharringtonine isolated from the Chinese tree,

Cephalotaxus harringtonia (Cephalotaxaceae) and elliptinium, a derivative of

19

ellipticine isolated from species of plant family Apocynaceae including Bleekeria

vitensis, a Fijian medicinal plant with reputed anti-cancer properties. Combretastatin

isolated from the bark of the South African tree Combretum caffrum Kuntze is active

against colon, lung and leukemia cancers. It is considered to be the most cytotoxic

phytomolecule isolated so far (Unnati et al., 2013).

2.3 Cell death

Cell death is a general biological phenomenon, which continues throughout the lives

of organisms and is important to maintain normal cellular homeostasis. Cell deaths

during embryonic development are vital for successful organogenesis and the

crafting of complex multicellular tissues (Nika and Stanley, 2004). Defects in

apoptotic cell death regulation may lead to many diseases. Acquired

Immunodeficiency Syndrome (AIDS), neurodegenerative disorders, insulin-

dependent diabetes, hepatitis C infection, myocardial infarct and atherosclerosis are

examples of disorders with inappropriate apoptosis or increased rate of apoptosis,

whereas autoimmune diseases and cancer are disorders with decreased rate of

apoptosis (Ulukaya et al., 2011).

During cell death, activation of distinct biochemical cascades follows and

manifest with different morphological features. Cell death can occur by either of

two distinct mechanisms, namely, apoptosis or necrosis. Apoptosis or programmed

cell death is the physiological process by which unwanted or useless cells are

eliminated during development and other normal biological processes. The

biochemical and morphological events that lead to apoptosis usually have a highly

regulated series of events. Necrosis or accidental cell death is a pathological process

20

in response to trauma generated by external factors or overwhelming cellular injury

(Figure 2.5) (Fa´tima and Pedro, 2010).

2.3.1 Apoptosis and necrosis

The term ‘apoptosis’ was first coined by Kerr et al. (1972) to describe the specific

morphological features leading to the programmed cell death. Apoptosis is of Greek

origin, having the meaning "falling off or dropping off", in analogy to leaves falling

off trees or petals dropping off flowers (Sankari et al., 2012). This analogy

emphasizes that the death of living matter is an integral and necessary part of the life

cycle of organisms.

Apoptosis is a form of cell death characterized by cell shrinkage, membrane

blebbing, chromatin condensation and looses contact to its neighbouring cells.

Apoptotic cell is fragmented into compact membrane-enclosed structures, called

'apoptotic bodies' which contain cytosol, the condensed chromatin and organelles.

Apoptosis is an active process as the apoptotic bodies are engulfed by macrophages

and thus are removed from the tissue without causing an inflammatory response.

These morphological changes are a result of characteristic molecular and

biochemical events leading to the activation of proteolytic enzymes which eventually

mediate the cleavage of DNA into oligonucleosomal and cleavage of a multitude of

specific protein substrates which usually determine the integrity and shape of the

cytoplasm or organelles (Saraste and Pulkki, 2000).

The term necrosis was originally used to define all types of cell death until

Kerr (1971); Kerr et al. (1972) introduced the concept of apoptosis. Necrosis is of

Greek origin, having the meaning "corpse” (Zong and Thompson, 2006). For a long

21

time, necrosis has been considered an accidental and uncontrolled form of cell death

lacking underlying signaling events. Morphologically, necrosis is characterized by

cytoplasmic and organelle swelling, followed by the loss of cell membrane integrity

and loss of intracellular contents (Duprez et al., 2009). During necrosis, the

intracellular contents are released uncontrolled into the cell's environment which

results in damage of surrounding cells and a strong inflammatory reaction in the

corresponding tissue (Leist and Jaattela, 2001).

22

Figure 2.5: Types of cell death [Source: Walker et al., (1988)].

23

2.3.2 Mechanism of apoptotic and necrotic cell death

2.3.2.1 Apoptosis pathways

Occurrence of apoptosis is controlled by a number of complex proteins, which are

activated by various triggers and arranged in sequential signaling modules. There are

two main pathways that lead to apoptosis. The first, referred to as the intrinsic or

mitochondrial pathway that when stimulated leads to the release of cytochrome-c

from the mitochondria and activation of the death signal. The second pathway is the

extrinsic or cytoplasmic pathway, is triggered through the Fas death receptor, a

member of the tumor necrosis factor (TNF) receptor superfamily. Both pathways

converge to a final common pathway involving the activation of caspases that cleave

regulatory and structural molecules resulting in the death of the cell (Ghobrial et al.,

2005).

2.3.2.1.a Intrinsic pathway

In the intrinsic or mitochondrial pathway of apoptosis, caspase activation is closely

linked to permeabilization of the outer mitochondrial membrane (Green and

Kroemer, 2004). This permeabilization is regulated by proteins from the Bcl-2

family. The Bcl-2 family includes proapoptotic members such as Bax, Bak, Bad,

Bcl-Xs, Bid, Bik, Bim, and Hrk, and antiapoptotic members such Bcl-2, Bcl-XL,

Bcl-W, Bfl-1, and Mcl-1. Antiapoptotic Bcl-2 members act as supressors of

apoptosis by blocking the release of cytochrome-c, whereas proapoptotic members

act as promoters (Reed, 1997).

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Upon triggered by a death signal, proapoptotic proteins undergo

posttranslational modifications that include dephosphorylation and cleavage

resulting in their activation and translocation to the mitochondria leading to

apoptosis. The activation of Bcl-2 members such as Bax and Bak may cause an

increase of mitochondrial membrane permeability, leading to the release of

cytochrome-c and second mitochondria-derived activator of caspase (SMAC) or

inihibitor of apoptosis proteins (IAPs) into cytosol. The release of cytochrome c then

binds and activate with Apaf-1, as well as procaspase-9, forming an “apoptosome”.

Activation of caspase-9 then activates caspase-3, which subsequently activates the

rest of the downstream caspases and leads to apoptosis (Ghobrial et al., 2005).

2.3.2.1.b Extrinsic pathway

The extrinsic pathway or death receptor pathway initiates apoptosis via

transmembrane receptor-mediated interactions. These involve death receptors that

are members of the TNF receptor gene superfamily (Susan, 2007). These proteins

comprise death receptors, the membrane-bound Fas ligand, the Fas complexes, the

Fas-associated death domain, and caspases 8 and 10 (Ghobrial et al., 2005).

Activation of the extrinsic pathway is initiated with the ligation of cell surface

receptors called death receptors (DRs). Fas is a member of the tumor necrosis factor

receptor superfamily, also known as Apo-1 or CD95. Other TNF receptors include

TNF R1, DR3 (Apo 2), DR4 (TRAIL R1), DR5 (TRAIL R2), and DR6 (Zapata et

al., 2001).

Following a death stimulus, the FasL interacts with the inactive Fas

complexes and forms the death-inducing signaling complex which contains the

adaptor protein Fas-associated death domain protein and caspases 8 and 10. This

in vitro cytotoxicity activity and in vivo oral toxicity

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