in vitro cytotoxicity activity and in vivo oral toxicity
TRANSCRIPT
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
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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 Materials and methods 74
4.2.1 Chemicals and reagents 74
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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4.3 Statistical analysis 90
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 Materials and methods 137
5.2.1 Cell lines 137
5.2.2 Chemicals and reagents 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
5.3 Statistical analysis 140
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
6.1 Introduction 162
6.2 Materials and methods 164
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
6.3 Statistical analysis 168
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
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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
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6.4.8 Effect of sub-chronic oral administration of E. hirta on body weightin rats
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6.4.9 Effect of sub-chronic oral administration of E. hirta on haematologicalindices in rats
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6.4.10 Effect of sub-chronic oral administration of E. hirta on biochemicalindices in rats
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6.4.11 Effect of sub-chronic oral administration of E. hirta on organ weightin rats
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6.4.12 Effect of sub-chronic oral administration of E. hirta on histologicalassessment of organs in rats
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6.5 Discussion 232
6.6 Conclusion 240
CHAPTER 7.0: FRACTIONATION AND IDENTIFICATION OFCYTOTOXIC FRACTION FROM E. hirta
241
7.1 Introduction 241
7.2 Materials and methods 242
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
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Figure 3.7 Calibration curve of 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid by GC-MS
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Figure 4.1 Haemacytometer chamber grid pattern showing foursquares to be used to count viable cells
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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
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Figure 4.4 Toxic effects of potassium dichromate after 24 h usingbrine shrimp lethality assay
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Figure 4.5 Cell viability curve of Vero cells treated with E. hirtaextract by using MTT assay
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Figure 4.6 Cell viability curve of MCF-7 cells treated withE. hirta extract by using MTT assay
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Figure 4.7 Cell viability curve of HeLa cells treated with E. hirtaextract by using MTT assay
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Figure 4.8 Cell viability curve of HT-29 cells treated withE. hirta extract by using MTT assay
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Figure 4.9 Cell viability curve of Vero cells treated withdoxorubicin hydrochloride by using MTT assay
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Figure 4.10 Cell viability curve of MCF-7 cells treated withdoxorubicin hydrochloride by using MTT assay
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Figure 4.11 Cell viability curve of HeLa cells treated withdoxorubicin hydrochloride by using MTT assay
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Figure 4.12 Cell viability curve of HT-29 cells treated withdoxorubicin hydrochloride by using MTT assay
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Figure 4.13 Morphological changes of MCF-7 cells treated with0.5% (v/v) DMSO as negative control, viewed underan inverted light microscope
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Figure 4.14 Morphological changes of MCF-7 cells treated withIC50 of E. hirta extract for 24 h, viewed under aninverted light microscope
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Figure 4.15 Morphological changes of MCF-7 cells treated withIC50 of E. hirta extract for 48 h, viewed under aninverted light microscope
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Figure 4.16 Morphological changes of MCF-7 cells treated withIC50 of E. hirta extract for 72 h, viewed under aninverted light microscope
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Figure 4.17 Morphological changes of MCF-7 cells treated withIC50 of doxorubicin hydrochloride for 24 h, viewedunder an inverted light microscope
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Figure 4.18 Morphological changes of MCF-7 cells treated withIC50 of doxorubicin hydrochloride for 48 h, viewedunder an inverted light microscope
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Figure 4.19 Morphological changes of MCF-7 cells treated withIC50 of doxorubicin hydrochloride for 72 h, viewedunder an inverted light microscope
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Figure 4.20 The influence of E. hirta extract on the membraneintegrity of MCF-7 cells after 24 h of treatment
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Figure 4.21 AO/PI staining of MCF-7 cells treated with 0.5% (v/v)DMSO as negative control, viewed under afluorescence microscope
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Figure 4.22 AO/PI staining of MCF-7 cells treated with IC50 ofE. hirta extract for 24 h, viewed under a fluorescencemicroscope
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Figure 4.23 AO/PI staining of MCF-7 cells treated with IC50 of E.hirta extract for 48 h, viewed under a fluorescencemicroscope
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Figure 4.24 AO/PI staining of MCF-7 cells treated with IC50 of E.hirta extract for 72 h, viewed under a fluorescencemicroscope
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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
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Figure 4.26 Annexin V/PI double-staining assay of MCF-7 cellstreated with E. hirta extract (25 μg/mL)
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Figure 4.27 Annexin V/PI double-staining assay of MCF-7 cellstreated with E. hirta extract (50 μg/mL)
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Figure 4.28 DNA fragmentations in MCF-7 cells were treated for24 h with indicated concentrations of E. hirta.
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Figure 4.29 Cell cycle distribution of the cells upon E. hirtatreatment
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Figure 4.30 Effect of E. hirta on cell cycle distribution of MCF-7cells after 24 hour of treatment.
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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
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Figure 4.33 A standard curve of protein concentration usingBradford method
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Figure 4.34 Caspases activity in MCF-7 cells was analyzed usingcaspase colorimetric protease assay kit
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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
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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
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Figure 5.5 A. cepa root tips without treatment showing normalmetaphase
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Figure 5.6 A. cepa root tips treated with E. hirta extract showingsticky chromosome
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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
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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
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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
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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
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Figure 6.6 Photomicrograph showing section of heart of malecontrol rats in acute toxicity study
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Figure 6.7 Photomicrograph showing section of heart of male ratstreated with 5000 mg/kg/day of E. hirta extract inacute toxicity study
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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
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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
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Figure 6.19 Photomicrograph showing section of liver of male ratstreated with 5000 mg/kg/day of E. hirta extract inacute toxicity study
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Figure 6.20 Photomicrograph showing section of liver of femalecontrol rats in acute toxicity study
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Figure 6.21 Photomicrograph showing section of liver of femalerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study
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Figure 6.22 Photomicrograph showing section of spleen of malecontrol rats in acute toxicity study
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Figure 6.23 Photomicrograph showing section of spleen of malerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study
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Figure 6.24 Photomicrograph showing section of spleen of femalecontrol rats in acute toxicity study
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Figure 6.25 Photomicrograph showing section of spleen of malerats treated with 5000 mg/kg/day of E. hirta extract inacute toxicity study
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Figure 6.26 Mean body weight of male rats in sub-chronic toxicitystudy
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Figure 6.27 Mean body weight of female rats in sub-chronictoxicity study
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Figure 6.28 Mean body weight of rats in satellite group in sub-chronic toxicity study
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Figure 6.29 Photomicrograph showing section of heart of malecontrol rats in sub-chronic toxicity study
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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 6.31 Photomicrograph showing section of heart of male ratstreated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.32 Photomicrograph showing section of heart of male ratstreated with 1000 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
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Figure 6.34 Photomicrograph showing section of heart of femalecontrol rats in sub-chronic toxicity study
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Figure 6.35 Photomicrograph showing section of heart of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.36 Photomicrograph showing section of heart of femalerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.37 Photomicrograph showing section of heart of femalerats treated with 1000 mg/kg/day of E. hirta extract in
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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
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Figure 6.39 Photomicrograph showing section of lung of malecontrol rats in sub-chronic toxicity study
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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 6.41 Photomicrograph showing section of lung of male ratstreated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.42 Photomicrograph showing section of lung of male ratstreated with 1000 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
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Figure 6.44 Photomicrograph showing section of lung of femalecontrol rats in sub-chronic toxicity study
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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 6.46 Photomicrograph showing section of lung of femalerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.47 Photomicrograph showing section of lung of femalerats treated with 1000 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
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Figure 6.49 Photomicrograph showing section of kidney of malecontrol rats in sub-chronic toxicity study
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Figure 6.50 Photomicrograph showing section of kidney of malerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.51 Photomicrograph showing section of kidney of malerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.52 Photomicrograph showing section of kidney of malerats treated with 1000 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.53 Photomicrograph showing section of kidney of malerats as the satellite group in sub-chronic toxicity study
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Figure 6.54 Photomicrograph showing section of kidney of femalecontrol rats in sub-chronic toxicity study
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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 6.56 Photomicrograph showing section of kidney of femalerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.57 Photomicrograph showing section of kidney of femalerats treated with 1000 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
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Figure 6.59 Photomicrograph showing section of liver of malecontrol rats in sub-chronic toxicity study
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Figure 6.60 Photomicrograph showing section of liver of male ratstreated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.61 Photomicrograph showing section of liver of male ratstreated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.62 Photomicrograph showing section of liver of male ratstreated with 1000 mg/kg/day of E. hirta extract insub-chronic toxicity study
223
Figure 6.63 Photomicrograph showing section of liver of male ratsas the satellite group in sub-chronic toxicity study
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Figure 6.64 Photomicrograph showing section of liver of femalecontrol rats in sub-chronic toxicity study
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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 6.66 Photomicrograph showing section of liver of femalerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.67 Photomicrograph showing section of liver of femalerats treated with 1000 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
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Figure 6.69 Photomicrograph showing section of spleen of malecontrol rats in sub-chronic toxicity study
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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 6.71 Photomicrograph showing section of spleen of malerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
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Figure 6.72 Photomicrograph showing section of spleen of malerats treated with 1000 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
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Figure 6.74 Photomicrograph showing section of spleen of femalecontrol rats in sub-chronic toxicity study
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Figure 6.75 Photomicrograph showing section of spleen of femalerats treated with 50 mg/kg/day of E. hirta extract insub-chronic toxicity study
230
Figure 6.76 Photomicrograph showing section of spleen of femalerats treated with 250 mg/kg/day of E. hirta extract insub-chronic toxicity study
230
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Figure 6.77 Photomicrograph showing section of spleen of femalerats treated with 1000 mg/kg/day of E. hirta extract insub-chronic toxicity study
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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
Figure 7.6 Cell viability curve of MCF-7 cells treated withE. hirta EH EtOAc 6 subfraction by using MTT assay
250
Figure 7.7 Cell viability curve of MCF-7 cells treated withE. hirta EH EtOAc 7 subfraction by using MTT assay
250
Figure 7.8 Cell viability curve of MCF-7 cells treated withE. hirta EH EtOAc 8 subfraction by using MTT assay
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
xxiv
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
xxv
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
xxvi
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
xxvii
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
xxviii
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
xxix
tentang kepentingan kedua-dua kaedah tersebut untuk memperolehi data penilaian
toksikologi yang komprehensif untuk agen terapeutik semula jadi.
xxx
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
xxxi
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)
10
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
18
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).
24
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