IN VITRO AND IN VIVO RADIOPROTECTIVE ACTIVITIES...

IN VITRO AND IN VIVO RADIOPROTECTIVE

ACTIVITIES OF Polyalthia longifolia AGAINST LETHAL

IRRADIATION

JO THY LACHUMY A/P SUBRAMANION

UNIVERSITI SAINS MALAYSIA

2015

IN VITRO AND IN VIVO RADIOPROTECTIVE ACTIVITIES OF

Polyalthia longifolia AGAINST LETHAL IRRADIATION

by

JO THY LACHUMY A/P SUBRAMANION

Thesis submitted in fulfillment of the requirements for the degree of

Doctor of Philosophy

July 2015

ii

ACKNOWLEDGEMENT

This thesis is the end of my long journey in obtaining my PhD degree in

Universiti Sains Malaysia. In the name of God foremost, I would like to express my

heartfelt thanks to almighty for giving me the most gracious and the most merciful

strength to complete my PhD of Science. This dissertation would not have been

possible to complete without His grace. Moreover, there are some people who made

this journey easier with words of encouragement and more intellectually satisfying

by offering different places to look to expand my theories and ideas. I take this

opportunity to express my profound gratitude and deep regards to all of them.

Foremost, I would like to express my sincere gratitude to my supervisor

Associate Professor Dr. Sasidharan Sreenivasan for the continuous support of my

PhD research study for his patience, motivation, immense knowledge,

encouragement and providing an excellent atmosphere to complete the research

work successfully. I would like to thank him for his exemplary assistance on editing

my thesis; also his comments and suggestions throughout my project were very

helpful to build an excellent research work. The blessing, help and guidance given

by him time to time shall carry me a long way in the journey of life on which I am

about to embark. Secondly, I would like to express my deepest sense of gratitude to

my Co-supervisor Professor Dr. Azlan Abdul Aziz for his cordial support and

valuable information, guidance which help me in completing this project through

various stages.

I am truly indebted and thankful to Dr Cheah and Dr Lim from Oncologist

and Radiotherapist departments of LohGuanLye Specialist Center, Pulau Pinang for

helping me in irradiating the animal with highly recommended lethal irradiation

iii

doses. I also thankful to all staffs from Animal House of Universiti Sains Malaysia

specially Dr Hismah and Encik Yussof for providing me space and animals during in

vivo study. I am thankful to Ms. Shantini for her assistance in the histology study. I

would like to express my deepest gratitude to Dr. Tamio Saito and lab colleagues

from chemical biology department of RIKEN, Tokyo Japan for assisting and

providing me with appropriate facilities for isolation and identification of bioactive

compounds during my short internship. I acknowledge my gratitude to Institute for

Research in Molecular Medicine, Universiti Sains Malaysia, Pulau Pinang for

providing me proper lab facilities with appropriate materials and technical support

for my research.

I would like to forward my appreciation to Dr Chen Yeng from Universiti

Malaya for her guidance and knowledge. To my fellow colleagues Dr Kwan Yuet

Ping, thank you for all the support and patience you have shown me through the

years.

Finally, I take this great opportunity to express my profound gratitude to

Mybrain15 (MyPhD) Fellowship from Ministry of Higher Education, Malaysia

which support me financially and help me to complete my research project without

financial constrain. I would like to thank Universiti Sains Malaysia who funded my

research project by providing Research University Post Graduate Research Grant

Scheme (RU-PRGS).

JO THY LACHUMY SUBRAMANION

INFORMM, Universiti Sains Malaysia,

Pulau Pinang,

July 2015

iv

TABLE CONTENTS

PAGE

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iv

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVATIONS xxi

ABSTRAK xxiii

ABSTRACT xxvi

CHAPTER 1.0: GENERAL INTRODUCTION 1

1.1 Overview and rationale of the study 1

1.1.1 Research objectives 6

CHAPTER 2.0: LITERATURE REVIEW 7

2.1 Radiation 7

2.2 Ionization and formation of free radicals 10

2.2.1 Free radicals and cell death 12

2.2.2 Free radicals and cancer 13

2.3 Radioprotection mechanisms by plant extract or compounds 14

2.4 Plant as anti-radiation sources 16

2.4.1 Traditional usage of medicinal plant as radioprotective agent 16

2.4.2 Medicinal plant with radioprotective effects 17

2.5 Antiradiation compounds 18

2.5.1 Modern technique for evaluation of radioprotective activity of

medicinal plants

23

2.5.2 Extraction 23

v

2.5.3 In vitro test 25

2.5.3.1 Antioxidant 25

2.5.3.2 Comet assay 28

2.5.3.3 Plasmid relaxation assay 32

2.5.3.4 Allium cepa Assay 35

2.6 In vivo screening assays 38

2.6.1 Acute toxicity and Maximum Tolerable Dose (MTD) (LD50) 40

2.6.2 Whole- body survival, body weight and dose reduction factor

(DRF)

40

2.6.3 Endogenous Spleen Colony Forming Unit (CFU) assay 42

2.6.4 Haematological assays 43

2.6.5 Gastrointestinal damage assays 44

2.7 Polyalthia longifolia 45

2.7.1 Botany 46

2.7.1.1 Distribution 46

2.7.1.2 Botanical Description 46

2.7.1.3 Propagation 48

2.7.2 Ethnomedicinal Uses 48

2.7.3 Phytochemistry 50

2.7.4 Pharmacological Activities of Polyalthia longifolia 54

2.7.4.1 Antibacterial activity 54

2.7.4.2 Antioxidant activity 56

2.7.4.3 Anti-inflammatory activity 57

2.7.4.4 Anticancer activity 58

2.7.4.5 Hepatoprotective activity 59

vi

2.7.5 Toxicological Assessment 59

2.7.6 Precautions/Safety for Usage 60

CHAPTER 3.0: CHROMATOGRAPHIC AND SPECTRAL

FINGERPRINTING OF Polyalthia longifolia A SOURCE OF

PHYTOCHEMICALS

61

3.1 INTRODUCTION 61

3.2 MATERIALS AND METHODS 64

3.2.1 Chemicals and Reagents 64

3.2.2 Plant Sample Collection 64

3.2.3 Microscopic Studies and Powder Analysis 64

3.2.4 Solvent Extraction 65

3.2.5 Phytochemical Analysis 65

3.2.5.1 Saponins (Frothing/ Foam test) 65

3.2.5.2 Tannins (Braemer’s test) 66

3.2.5.3 Alkaloids (Wagner test) 66

3.2.5.4 Terpenoids (Salkowski test) 66

3.2.5.5 Anthraquinones (Borntrager’s test) 66

3.2.5.6 Carbohydrates (Barfoed's test) 66

3.2.5.7 Steroid (Liebermann-Burchard test) 67

3.2.5.8 Glycoside (Keller -Killiani test) 67

3.2.6 Standardization of P. longifolia leaf methanolic extract 67

3.2.6.1 Quantification of Rutin 67

3.2.7 Fourier Transform Infrared (FTIR) Analysis 69

3.2.8 HPTLC Finger Print profiles for P. longifolia Leaf Extract 69

3.2.8.1 TLC condition 69

vii

3.2.8.2 Procedures 69

3.2.9 Heavy Metal Analysis 70

3.3 RESULTS 71

3.3.1 Plants extract yield percentage 71

3.3.2 Microscopy of Leaf 71

3.3.2.1 Transverse section 71

3.3.2.2 Leaf powder 71

3.3.3 Phytochemicals Analysis 74


3.3.4.1 Quantification of rutin 74


3.3.6 High-Performance Thin Layer Chromatography (HPTLC)

Fingerprinting

80

3.3.7 Fourier Transform Infrared (FTIR) Spectral Fingerprinting 80

3.4 DISCUSSION 84

3.4.1 Light microscopy of Leaf 84

3.4.1.1 Light microscopy Leaf powder 85

3.4.2 Phytochemicals Analysis 85



3.4.5 High-Performance Thin Layer Chromatography (HPTLC)

Fingerprinting

87

3.4.6 Fourier Transform Infrared (FTIR) Spectral Fingerprinting 88

3.5 CONCLUSION 91

viii

CHAPTER 4.0: ANTIOXIDANT ACTIVITY AND

HEPATOPROTECTIVE POTENTIAL OF Polyalthia longifolia LEAF

AGAINST PARACETAMOL-INDUCED LIVER INJURY

92

4.1 INTRODUCTION 92


4.2.1 Plant collection and plant extract preparation 95

4.2.2 Determination of total phenolic and flavonoid contents 95

4.2.3 In vitro antioxidant Assays 96

4.2.3.1 DPPH radical-scavenging assay 96

4.2.3.2 Reducing power assay 97

4.2.3.3 Hydroxyl radical scavenging assay 97

4.2.3.4 Nitric oxide scavenging assay 98

4.2.3.5 Ferrous ion chelating assay 99

4.2.3.6 Anti-lipidperoxidation (ALP) assays 99

4.2.4 In vivo Hepatoprotective activity of P. longifolia leaf extract 100

4.2.4.1 Animals 100

4.2.4.2 Paracetamol Dose Regimen 101

4.2.4.3 Grouping of Mice and Treatments 101

4.2.4.4 Sacrifice and Organ Harvesting 101

4.2.4.5 Biochemical Parameters 102

4.2.4.6 Statistical Analysis 102

4.3 RESULTS 103

4.3.1 Total phenolic and flavonoid contents 103

4.3.2 DPPH radical scavenging assay 103

4.3.3 Reducing Power assay 107

ix

4.3.4 Hydroxyl radical scavenging assay 107

4.3.5 Nitric oxide scavenging assay 107

4.3.6 Ferrous ion chelating assay 111

4.3.7 Anti lipid peroxidation activity 111

4.3.8 Biochemical Parameters 111

4.3.9 Histopathological Analysis 115

4.4 DISCUSSION 117

4.4.1 Antioxidant activity 117

4.4.2 Total phenolic and flavonoid contents 117

4.4.3 DPPH radical scavenging assay 119

4.4.4 Reducing Power assay 119

4.4.5 Hydroxyl radical scavenging assay 120

4.4.6 Nitric oxide scavenging assay 121

4.4.7 Ferrous ion chelating assay 122

4.4.8 Anti lipid peroxidation activity 122

4.4.9 Biochemical Parameters 123


4.5 CONCLUSION 125

CHAPTER 5.0: EVALUATION OF THE GENOTOXIC POTENTIAL

AGAINST H2O2-RADICAL MEDIATED DNA DAMAGE AND ACUTE

ORAL TOXICITY OF STANDARDIZED EXTRACT OF Polyalthia

longifolia LEAF

126

5.1 INTRODUCTION 126



x

5.2.2 Acute Oral Toxicity Study 129

5.2.2.1 Target Animal 129

5.2.2.2 Acute Toxicity Assays 129

5.2.2.3 Organs and Body Weight Analysis 130

5.2.2.4 Histopathology of Heart, Kidney, Liver, Lung and

Spleen

130

5.2.2.5 Blood Biomarker Assays 131

5.2.2.6 Statistical Analysis 131

5.2.3 Plasmid relaxation assay 131

5.2.4 Cytotoxicity screening 131

5.2.4.1 Vero Cell line 131

5.2.4.2 Cytotoxicity assay 132

5.2.5 Comet assay 133

5.2.5.1 Cell culture and treatment 133

5.2.5.2 Assessment of cellular DNA damage 133

5.2.6 Allium cepa assay 134

5.2.6.1. Pre-treatment 134

5.2.6.2 Preparation of slides 134

5.3 RESULTS 136

5.3.1 General signs and behavioural observation 136

5.3.2 Organ and body weight analysis 136


5.3.4 Haematology and Biochemical Analysis 141


5.3.6 Determinations of CC50 concentration 148

xi



5.4 DISCUSSION 156

5.4.1 General signs and behavioural observation 156

5.4.2 Organ and body weight analysis 156

5.4.3 Histopathological analysis 157

5.4.4 Haematology and biochemical analysis 157


5.4.6 Determinations of CC50 concentration 160



5.5 CONCLUSION 163

CHAPTER 6.0: RADIO-MODULATORY EFFECTS OF Polyalthia

longifolia AGAINST X-RAY IRRADIATION INDUCED

HEMATOPOIETIC, ENDOGENOUS ANTIOXIDANT, LIVER AND

GASTROINTESTINAL DAMAGE IN SWISS ALBINO MICE

164




6.2.2 Administration of plant extract 167

6.2.3 Animals 167

6.2.4 Irradiation 169

6.2.5 Experimental design 169

6.2.5.1 Whole body survival studies 169

6.2.5.2 Clinical Signs observations 170

xii

6.2.5.3 Hematological Study 170

6.2.5.4 Spleen colony-forming units (CFU-S) assay 170

6.2.5.5 Biochemical Estimations of Endogenous antioxidant 170

6.2.5.5.1 Lipid peroxidation (LPx) assay 171

6.2.5.5.2 Superoxide dismutase (SOD) assay 171

6.2.5.5.3 Catalase assay 172

6.2.5.6 Macropathology and Histopathology 172

6.2.6 Statistical Analysis 173

6.3 RESULTS 174

6.3.1 Whole body survival studies 174

6.3.2 Clinical Signs observations 174

6.3.3 Hematological Study 177

6.3.3.1 Hemoglobin (Hb) 177

6.3.3.2 Red blood cell (RBC) 177

6.3.3.3 White blood cell (WBC) 179

6.3.3.4 Platelets 179

6.3.4 Spleen colony-forming units (CFU-S) assay 179

6.3.5 Biochemical Estimation of Endogenous antioxidant 181

6.3.5.1 Lipid peroxidation (LPx) 181

6.3.5.2 Superoxide dismutase (SOD) 181

6.3.5.3 Catalase activity 184

6.3.6 Macropathology and Histopathology 184

6.3.6.1 Spleen 184

6.3.6.2 Small intestine (Ileum) 188

xiii

6.3.6.3 Protective effects of P. longifolia leaf extract on X-

ray irradiation-induced liver damage in mice

190

6.4 DISCUSSION 194

6.5 CONCLUSION 206

CHAPTER 7.0: BIOASSAY GUIDED ISOLATION OF ANTIOXIDANT

COMPOUND FROM Polyalthia longifolia

207




7.2.2 Bioassay Guided Isolation of MPLC fractions from the

solvents partitions

209

7.2.3 Purification of sub fraction EtOAc_F007 by using preparative

HPLC

212

7.2.4 Identification of antioxidant compound using mass

spectrometry analysis

213

7.3 RESULTS 216

7.3.1 Bioassay guided fractionations of P. longifolia crude extract 216

7.3.2 Evaluation of antioxidant activity of sub fractions 216

7.3.3 Purification of sub fraction EtOAc_F007 222

7.3.4 Identification of antioxidant compound using mass

spectrometry analysis

222

7.4 DISCUSSION 230

7.5 CONCLUSION 233

CHAPTER 8.0 GENERAL CONCLUSIONS AND SUGGESTIONS FOR

FUTURE STUDIES

234

8.1 Future work 239

xiv

REFERENCES 241

APPENDICES

LIST OF PUBLICATIONS

xv

LIST OF TABLES

PAGE

Table 2.1 Plant with radioprotective activity or antioxidant activity 19

Table 2.2 In Vitro antioxidant assays 27

Table 3.1 Extraction yields in percentage for Polyalthia longifolia leaf 72

Table 3.2 Phytochemical Analysis of the methanolic extract of Polyalthia

longifolia leaf

75

Table 3.3 Quantification of rutin in P. longifolia leaf methanolic extract 78

Table 3.4 Heavy Metal Concentrations in Polyalthia longifolia Leaf Extract 79

Table 4.1 Total phenolic contents of leaf extract Polyalthia longifolia 105

Table 4.2 Total flavonoid contents of leaf extract of Polyalthia longifolia 105

Table 4.3 Effect of Polyalthia longifolia leaf extract on liver marker

enzymes and serum bilirubin content

114

Table 5.1 General appearance and behavioral observations for control and

treated groups

137

Table 5.2 Effect of single oral administration of Polyalthia longifolia leaf

extract on organ-to-body weight index in rat

139

Table 5.3 Effect of single oral administration of the extract on

hematological parameters in Sprague Dawley (SD) rat

145

Table 5.4 Effect of single oral administration of the extract on biochemical

parameter in Sprague Dawley (SD) Rat

146

Table 5.5 Cytogenetic analysis of Allium cepa root tips exposed to different

concentrations of Polyalthia longifolia leaf and Fenton reagents

153

Table 6.1 Effect of Polyalthia longifolia leaf extract on liver marker

enzymes and serum bilirubin content

193

Table 7.1 The yield of the each partition obtained 217

Table 7.2 The yield of Hexane fractions (F1-F8) 218

Table 7.3 The yield of Ethyl acetate fractions (F1-F8) 219

Table 7.4 The yield of Butanol fractions (F1-F8) 220

xvi

LIST OF FIGURES

PAGE

Figure 2.1 Production of free radical and related damages by lethal

irradiation

8

Figure 2.2: Chemical structure of Cysteine 11

Figure 2.3 Various steps involved in the development and evaluation of

radioprotective property of medicinal plants

24

Figure 2.4 Principle of comet assay in alkaline micro-gel electrophoresis

to cellular DNA damage.

30

Figure 2.5 Comet images with different level of DNA damage (A)

normal cell; (B) irradiated cell

31

Figure 2.6 (a): Genoprotective effect of the irradiated pUC18 plasmid

DNA in the presence of plant extract (b): various form of

plasmid

33

Figure 2.7 Stages of mitotic division in cells of Allium cepa exposed to

radiation and treated with plant extract

37

Figure 2.8 In vivo screening assays 39

Figure 2.9 Polyalthia longifolia 47

Figure 2.10 Various phytochemicals isolated from Polyalthia longifolia 51

Figure 3.1 Transverse section of the leaf of Polyalthia longifolia 72

Figure 3.2 Powder microscopic features of leaves of Polyalthia

longifolia

73

Figure 3.3 Extracted Ion Chromatogram of (A) P. longifolia leaf

methanolic extract and (B) standard rutin

76

Figure 3.4 A mass spectra of rutin in positive ion mode 77

Figure 3.5 Calibration curve of standard rutin 77

Figure 3.6 HPTLC profile of Polyathia longifolia leaf extract under

visible light (a) and 365 nm UV light (b)

81

Figure 3.7 HPTLC Chromatogram of Polyalthia longifolia leaf extract

showing the present of 10 peaks

82

Figure 3.8 FT-MIR spectrum of methanolic leaf extract of Polyalthia

longifolia

83

xvii

Figure 4.1 Standard curve for determination of Gallic acid Equivalents

for total phenolic content of leaf extract of P. longifolia

104

Figure 4.2 Standard curve for determination of catechin Equivalents for

total flavonoids content of leaf extract of P. longifolia

104

Figure 4.3 Scavenging effect of methanolic leaf extract of Polyalthia

longifolia on DPPH free radicals compared with butylated

hydroxytoluene (BHT).

106

Figure 4.4 Regression analyses for total phenolic content and 2-

diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

determined in Polyalthia longifolia methanolic leaf extract

106

Figure 4.5 Reducing power of methanolic leaf extract of Polyalthia

longifolia compared to butylated hydroxytoluene (BHT)

108


longifolia on hydroxyl radicals compared to ascorbic acid

109


longifolia on nitric oxide radicals compared with Quercetin

110


longifolia on ferrous ions compared to ascorbic acid

112

Figure 4.9 Anti-lipidperoxidation activity of methanolic leaf extract of

Polyalthia longifolia compared to ascorbic acid

113

Figure 4.10 Light microphotographs of liver cell of control (a), mice

exposed to paracetamol (b) and treated with Polyathia

longifolia extract (c)

116

Figure 5.1 Effect of single dose (5000 mg/kg) administration of the

Polyalthia longifolia leaf extract in rats

140

Figure 5.2 Representative histological photomicrographs of (A) spleen,

(B) lung, (C) liver of control and Polyalthia longifolia leaf

extract treated groups (at dose 5000 mg/kg).

143

Figure 5.2 Continued. (D) Kidney and (E) heart of control and

Polyalthia longifolia leaf extract treated groups (at dose 5000

mg/kg)

144

xviii

Figure 5.3 Agarose gel electrophoretic analysis of fenton-mediated DNA

oxidation

147

Figure 5.4 Effect of concentration on cytotoxicity of Polyalthia

longifolia leaf extract on Vero cells

149

Figure 5.5 The quantitation of DNA damage and repair in Vero cell line

represent the comet tail length

150

Figure 5.6 Protective effect of Polyalthia longifolia leaf extract against

H2O2-induced DNA damage and migration

151

Figure 5.7 Chromosome aberrations observed in Allium cepa

meristematic cells exposed to extracts of Polyalthia longifolia

leaf

154

Figure 6.1 (a) Survival rate of the X-ray irradiated mice treated with or

without Polyalthia longifolia (500 mg/kg and 250 mg/kg

b.w.). (b) Body weight response of mice pretreated with or

without Polyalthia longifolia for 30 days at exposure of 10

Gy whole body irradiation

175

Figure 6.2 General radiation Sickness and behavioral appearance of the

mice from control and experimental groups

176

Figure 6.3 Haematological alteration in blood of the mice post whole

body exposure to 10 Gy irradiation at with or without

Polyalthia longifolia at dose 500 mg/kg b.w. and 250 mg/kg

b.w. (a) Haemoglobin; (b) red blood cell; (c) white blood cell;

(d) platelets

178

Figure 6.4 Colony forming units (CFU) in spleen treated with Polyalthia

longifolia at dose 500 mg/kg b.w. and 250 mg/kg b.w.

180

Figure 6.5 Effect of Polyalthia longifolia administration on lipid

peroxidation (LPx) in Swiss albino mice

182

Figure 6.6 Effect of Polyalthia longifolia administration on superoxide

dismutase (SOD) activity in Swiss albino mice

183

Figure 6.7 Effect of Polyalthia longifolia administration on catalase

activity in Swiss albino mice

185

xix

Figure 6.8 Macropathology of (A) spleen; (B) Small intestine (ileum). 186

Figure 6.9 Histopathological demonstration of protective effect of

Polyalthia longifolia in the spleen of irradiated mice.

187


Polyalthia longifolia in the small intestine (Ileum) of

irradiated mice

189


Polyalthia longifolia in the Liver of irradiated mice

191

Figure 7.1 Schematic illustration of the solvent ratio for the partitions

obtained

210

Figure 7.2 Schematic diagram of solvent-solvent extraction 211

Figure 7.3 Flow chart of the isolation and identification of the bioactive

compound(s) from Polyalthia longifolia leaf extract

215

Figure 7.4 Four partitions were obtained from solvent-solvent extraction

method

217

Figure 7.5 Hexane fractions (Hex1-8) obtained from Medium Pressure

Liquid Chromatography (MPLC)

218

Figure 7.6 Ethyl acetate fractionS (EtOA1-8) obtained from Medium

Pressure Liquid Chromatography (MPLC)

219

Figure 7.7 Butanol fractionS (BuOH1-8) obtained from Medium

Pressure Liquid Chromatography (MPLC)

220

Figure 7.8 DPPH radical scavenging activity of (a) hexane, (b) ethyl

acetate and (c) butanol sub fractions of Polyalthia longifolia

obtained from MPLC

221

Figure 7.9 Pure compound (yellow) isolated from EtOAc_F007 using

HPLC

223

Figure 7.10 HPLC chromatograms of antioxidant compound isolated from

EtOAc_F007 fraction of Polyalthia longifolia leaf at 11.862

retention time

224

Figure 7.11 Positive full scan of EtOAc_F007 isolated from P. longifolia

leaf using LC-QTOF-MS

225

xx

Figure 7.12 MS/MS spectra and proposed fragmentation pathways for the

EtOAc_F007 isolated antioxidant compound from P.

longifolia leaf at the collision energy of 10 eV

226

Figure 7.13 Typical patterns and the percentage of abundance of rutin at

higher collision energy of 20 and 40 eV in ESI positive mode

228

Figure 7.14 Chemical structure of rutin 229

xxi

LIST ABBREVIATIONS

ANOVA BHT DMSO DPPH GAE LC-MS IC50

LD50 Rf HPTLC v/v w/v TLC SD ABS ROS AST ALP ALT CFU M.I. DRF P. longifolia

TBIL

Analysis of varians Buthylated hydroxytoluene Dimethyl sulfoxide 2, 2-diphenyl-1-picrylhydrazyl Gallic acid equivalents Liquid Chromatography-Mass Spectrometry Inhibitory Concentration at 50% Lethality Dosage at 50% Retention Factor High Performance Thin Layer Chromatography Volume per volume Weight per volume Thin Layer Chromatography Standard Deviation Absorbance Reactive Oxygen Species Aspartate aminotransferase Alkaline phosphate Alanine aminotransferase Colony Forming Units Mitotic Index Dose Reduction Factor

Polyalthia longifolia Total billirubin

xxii

MTT CV TBE MTD SOD LPX

HB WBC RBC HPLC MPLC DAD UV RRLC QTOF-MS ESI OECD

3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide Central vein Tris-borate-EDTA Maximum Tolerable Dose Superoxide Dismutase Lipid Peroxidation Heamoglobin White Blood Cell Red Blood Cell High Performance Liquid Chromatography Medium Pressure Liquid Chromatography Diode Array Detector Ultraviolet Rapid Resolution liquid chromatography Accurate-Mass quadrupole time of flight mass spectrometer Electron spray ionization

Organization for Economic Co-operation and Development

xxiii

AKTIVITI RADIOPROTEKTIF OLEH Polyalthia longifolia

TERHADAP SINARAN RADIASI MAUT SECARA IN VITRO

DAN IN VIVO

ABSTRAK

Tumbuhan ubatan yang kaya dengan pelbagai bahan fitokimia dengan sifat

antioksidan boleh bertindak sebagai agen pelindung sinaran radiasi maut alternatif.

Kajian ini telah dijalankan untuk menilai ekstrak daun P. longifolia sebagai

pelindung sinar radiasi maut yang berpotensi. Kuantifikasi rutin telah dilakukan

untuk tujuan pemiawaian dan didapati ianya adalah 8.96 μg (0.896%) di dalam 1000

μg ekstrak daun P. longifolia. Kajian mikroskopi cahaya terhadap keratan melintang

daun P. longifolia memperlihatkan kehadiran pelbagai jenis sel tumbuhan.

Keputusan analisa fitokimia ekstrak menunjukkan kehadiran alkaloid, triterpenoid,

tanin, saponin, antrakuinon dan glikosida. Kepekatan logam berat yang ditentukan di

dalam ekstrak adalah di bawah had yang dibenarkan. Analisis HPTLC ekstrak daun

metanol P. longifolia menunjukkan sepuluh puncak spesifik. Dua belas puncak

utama dalam lingkungan 4,000 hingga 500 cm-1 diperhatikan dalam spektrum FTIR,

yang mewakili pelbagai kumpulan berfungsi khusus. Kajian antioksidan in vitro

menunjukkan aktiviti antioksidan yang memuaskan yang bergantung kepada dos

yang digunakan berbanding dengan agen antioksidan piawai. Kandungan jumlah

sebatian fenolik dan flavonoid dalam ekstrak yang diuji berkorelasi rapat dengan

keupayaan antioksidan ekstrak tumbuhan yang diuji. Terapi menggunakan ekstrak P.

longifolia menunjukkan kesan perlindungan hati terhadap perubahan biokimia dan

histopatologi. Dalam ujian komet, rawatan dengan 23.88 µg/mL kepekatan ekstrak

xxiv

P. longifolia selama 24 jam pada sel-sel Vero menyebabkan pengurangan kerosakan

DNA sebanyak 50.94% berbanding dengan kawalan yang tidak dirawat. Dalam ujian

relaksasi plasmid and komet ekstrak daun P. longifolia mempamerkan aktiviti

perencatan yang kuat terhadap kesan kerosakan yang diakibatkan oleh H2O2.

Peningkatan dalam nilai aberasi kromosom yang bersandarkan kepada kepekatan

ekstrak juga telah diperhatikan dalam ujian Allium cepa. Keabnormalan yang

diperhatikan adalah seperti kelekitan, c-mitosis, jambatan dan kromosom vagrant.

Sel mikronukleus juga telah diperhatikan dalam interfasa. Eksperimen ini adalah

laporan pertama tentang kesan perlindungan P. longifolia terhadap kerosakan DNA

yang disebabkan oleh radikal hidroksil. Tambahan pula, dalam ujian ketoksikan oral

akut, tikus betina telah dirawat dengan kepekatan ekstrak daun P. longifolia

sebanyak 5000 mg/kg berat badan tikus dan diperhatikan untuk tanda-tanda

ketoksikan selama 14 hari. Ekstrak daun P. longifolia tidak menunjukkan sebarang

tanda-tanda kesan toksik yang menunjukkan bahawa daun tersebut tidak toksik

berkaitan dengan rawatan tersebut. Seterusnya, kesan aktiviti radioprotektif P.

longifolia telah dikaji dengan menggunakan tikus. Rawatan P. longifolia pada tikus

menunjukkan peningkatan yang signifikan dalam jumlah hari tikus hidup (27 hari),

berbanding dengan 100% kematian dalam kumpulan tikus yang didedahkan pada

radiasi dalam tempoh 14 hari. Peningkatan ketara dalam kepekatan hemoglobin, sel

darah merah, sel darah putih dan jumlah platelet diperhatikan pada haiwan yang

menerima prarawatan ekstrak daun P. longifolia berbanding dengan kumpulan yang

didedahkan radiasi sahaja. Pemberian ekstrak daun P. longifolia sebelum

pendedahan radiasi juga telah meningkatkan jumlah CFU limpa dan saiz limpa

relatif. Penurunan yang bergantung kepada dos rawatan dalam nilai pengoksidaan

lipid diperhatikan dalam haiwan yang menerima prarawatan dengan P. longifolia.

xxv

Walau bagaimanapun, haiwan yang menerima prarawatan dengan P. longifolia

mempamerkan peningkatan yang ketara dalam aktiviti superoksida dismutase dan

katalase, tetapi nilai-nilai ini kekal di bawah nilai biasa di dalam hati dan usus.

Prarawatan dengan P. longifolia sebelum pendedahan pada radiasi juga

menyebabkan regenerasi semula krip mukosa dan vili usus. Tambahan pula, rawatan

awal ekstrak daun P. longifolia juga menunjukkan kesan perlindungan ke atas

kerosakan hati yang disebabkan oleh sinaran radiasi-X pada tikus dengan pemulihan

struktur sel hati yang normal dan pengurangan ketara aras ALT, AST dan tahap

bilirubin berbanding dengan tikus yang didedahkan dengan sinaran radiasi-X.

Keputusan ini menunjukkan keupayaan radioprotektif ekstrak daun P. longifolia

yang dimanifestasikan dalam beberapa sistem haiwan ujian. Untuk mengenal pasti

sebatian antioksidan, ekstrak daun P. longifolia telah dikenakan fraksinasi

berpandukan bioasai. Fraksi P. longifolia etil asetat, iaitu EtOAc_F007 menunjukkan

aktiviti antioksidan tertinggi di kalangan semua fraksi yang diuji. Analisis

selanjutnya dengan menggunakan kaedah LC-MS terhadap fraksi EtOAc_F007

membawa kepada pengenalan sebatian rutin sebagai agen antioksidan dalam ekstrak

P. longifolia. Kesimpulannya, hasil kajian ini menyokong potensi penggunaan

ekstrak daun P. longifolia sebagai produk semula jadi untuk diaplikasikan di masa

hadapan sebagai pembangunan agen pelindung radiasi maut baru yang bersifat

semula jadi.

xxvi

IN VITRO AND IN VIVO RADIOPROTECTIVE ACTIVITIES OF

Polyalthia longifolia AGAINST LETHAL IRRADIATION

ABSTRACT

Medicinal plants rich with various phytochemicals with antioxidant properties could

serve as an alternative radioprotective agent. The current study was designed to

evaluate the P. longifolia leaf extract as a potential radioprotector. Rutin

quantification was performed for standardization and was found to be 8.96 µg

(0.896%) in 1000 µg of P. longifolia leaf extract. Light microscopy of a transverse

section of the leaf of P. longifolia revealed the presence of various plant cells.

Phytochemical screening results of the extract revealed the presence of alkaloids,

triterpenoids, tannins, saponin, anthraquinones, and glycosides. The concentrations

of heavy metals determined in the extract were well below the permissible limit. The

HPTLC analysis of the methanolic extract of P. longifolia leaf showed ten specific

peaks. Twelve major peaks in the range of 4,000 to 500 cm-1 were observed in the

FTIR spectra, which represented various specific functional groups. The in

vitro antioxidant assays showed considerable in vitro antioxidant activities in a dose-

dependent manner when compared to the standard antioxidant Phenolic and

flavonoid content of these extracts is significantly correlated with antioxidant

capacity. Therapy of P. longifolia showed the liver protective effect on biochemical

and histopathological alterations. Moreover, histological studies also supported the

biochemical finding, that is, the maximum improvement in the histoarchitecture of

the liver. In the comet assay, the treatment of 23.88 µg/mL of P. longifolia extract

for 24 h on Vero cells caused decrease in DNA damage by approximately 50.94%

xxvii

compared to the unchallenged control. In the plasmid relation and comet assay,

the P. longifolia leaf extract exhibited strong inhibitory effects against H2O2-

mediated DNA damage. A dose-dependent increase of chromosome aberrations was

also observed in the Allium cepa assay. The abnormalities scored were stickiness, c-

mitosis, bridges, and vagrant chromosomes. Micronucleated cells were also observed

at the interphase. This experiment is the first report for the protective effect of P.

longifolia on DNA damage-induced by hydroxyl radicals. Additionally in an acute

oral toxicity study, female rats were treated at 5000 mg/kg body weight of P.

longifolia leaf extract and observed for signs of toxicity for 14 days. P.

longifolia leaf extract did not produce any signs of toxicity which indicates that the

P. longifolia was not toxic. Subsequently, the radioprotective effect of P. longifolia

was studied in mice. P. longifolia treatment rendered remarkable improvement in

mice survival (27 days), compared to 100% mortality in irradiated groups mice

within 14 days. Significant increases in hemoglobin concentration, red blood cell,

white blood cells and platelets counts were observed in the animals pretreated with

P. longifolia leaf extract in comparison to the irradiation-alone group. Pre-irradiation

administration of P. longifolia leaf extract also increased the CFU counts of spleen

colony and increased spleen relative size. A dose-dependent decrease in lipid

peroxidation levels was observed in animals pretreated with P. longifolia. However,

the animals pretreated with P. longifolia exhibited a significant increase in

superoxide dismutase and catalase activity, but the values remained below normal

both in liver and intestine. Pre-irradiation administration of P. longifolia also

resulted in regeneration of mucosal crypts and villi of intestine. Moreover,

pretreatment of P. longifolia leaf extract also shows protective effects on X-ray

irradiation-induced liver damage in mice by restoration of normal liver cell structure

xxviii

and significant reduction in the elevated levels of ALT, AST and bilirubin level

compared with the X-ray irradiated mice. These results suggest that the

radioprotective ability of P. longifolia leaf extract which was manifested in several

systems in experimental animal. To identify the antioxidant compound, P. longifolia

leaf extract was subjected to bioassay-guided fractionation. P. longifolia ethyl

acetate fraction, namely EtOAc_F007 demonstrated highest antioxidant activity

among all the fractions tested. Further LC-MS analysis of EtOAc_F007 led to

identification of rutin as the antioxidant agent in P. longifolia extract. In conclusion,

the results from this study strongly imply the potential use of P. longifolia leaf

extracts from natural product in future application for the development of natural

products based radioprotection agents.

1

CHAPTER 1.0: GENERAL INTRODUCTION

1.1 Overview and rationale of the study

The adverse effects of radiation has begun to realize immediately after the discovery

of X-ray by Roentgen (1896) and radioactivity by Becquerel (1896) and it was

considered as a remarkable turning point of human health care. Basically, humans

are constantly exposed to radiation either from planned included diagnostic,

therapeutic uses and industrial sources or unplanned included the nuclear explosion

such as atomic bomb blast, which brought tremendous damage at Hiroshima and

Nagasaki, Japan in 1945 and the natural background radiation emanating from the

earth or other radioactive sources (Jagetia, 2007). In general, the chances of radiation

exposure have been increased extensively and this further enhance by the rapid

advancement in technology which also leads to the additional radiation stresses.

Broadly, the radiation sources can be categorised into ionizing and non-

ionizing radiation. Ionizing radiation can be defined as any types of electromagnetic

or particles radiation with sufficient energy to knock electrons off of atoms or

molecules and such phenomenon known as ionization. Ionized molecules are

unstable and quickly undergo chemical changes. The amount of damage in the cell is

related to the dose of radiation it receives. The types of ionizing radiation included

X-ray and Gamma ray which widely used in cancer treatment. Meanwhile, non-

ionizing radiation has low energy that does not directly damage in molecules.

Common types of non-ionizing radiation include ultraviolet (UV) rays, visible light,

infrared rays, microwaves, and radiofrequency rays (radio waves), and cell phones

all emit (send out) non-ionizing radiation.

Ionizing radiation is an important modality in cancer treatment and almost

80% of cancer patients required radiotherapy during some point of their clinical

2

management either for curative or palliative purpose (Piya Paul et al., 2011). The

basic principle of radiotherapy is to destroy the cancer cells. However, the radiation

also induced damage to the normal tissues which results in adverse side effects after

months or years of therapy and this restricts the therapeutic doses of radiation, hence

limits the effectiveness of the treatments. Consequently, when the ionizing radiation

passing through living tissues it transfuses deleterious effects in biological system

through direct deposition of energy into crucial bio-macromolecules or by radiolysis

of water which leads to generation of reactive free radicals such as hydroxyl radicals

and hydrogen radicals. Thus the overproduction of these free radicals tend to become

reactive oxygen species and toxic which can interact with the critical bio-

macromolecules such as DNA, proteins, or membranes and induce cell damage

eventually leads to cell dysfunction and death (Hosseinimehr, 2007). In general, the

amount of reactive oxygen species increases in the biological system following

exposure to irradiation with sufficient dose and this directly correspond to the

intensity of cell damage.

The radiation has been considered an enigma to the general public and the

use of radiation for therapeutic purpose as well as spectacular advances made further

increases awareness of human health and such phenomenon been always associated

with some skepticism. The use of radioprotectors represents an obvious strategy to

improve the therapeutic index in radiotherapy. Therefore, the development of

effective radioprotectors is an area of great significance due to its wide applications

in planned as well as unplanned radiation exposure to reduce the risk of radiation

injury or severity of damage to normal tissues (Jagetia, 2007; Piya Paul et al., 2011).

Research in the development of radioprotectors world wide has focused on screening

a variety of chemical and biological compounds. Among the molecular

3

radioprotectors, WR-2721 [S-2-(3-aminopropyl-amino)] ethyl phosphorothioic acid

also known as amifostine, ethiophos (USA) or gammaphos (former USSR), is the

most thoroughly investigated radioprotective drug, developed at Walter Reed Army

Research Institute, under the Antiradiation Drug Development Program USA

(Schuchter and Glick, 1993; Sweeney, 1979). However, the strategy becomes

jeopardized when it comes to using synthetic chemical compound during

radiotherapy as it associated with undesirable side effects at clinically effective doses

and exorbitant cost the limitation greatly restricted in clinical use. In addition,

conflicting preclinical and clinical reports formulate it convoluted to accept the use

of synthetic compounds during radiotherapy in an unequivocal manner (Jena et al.,

2010). Therefore, the side effects profile of these compounds necessitated the search

for alternative drugs, which could be less toxic and highly effective at optimum dose

levels. Hence, such phenomenon diverts many researches attention towards the

plants and natural products focus on new drug discovery which would be safer than

available synthetic drugs.

Plant products have various pharmacological properties and have been used

to treat various diseases since ancient time based on traditional medicinal system.

The problem of safety of the pharmaceutical products with modern system of

medicine triggers increase in global interest in medicinal plants. Therefore,

medicinal plants have gained importance in the international market. Moreover,

more than 50% of drugs in markets are still based on natural products. In recent

years, herbal market mainly in the United States, Germany, France, India, Japan and

others have become huge potential market and has great room for scientific research

and technology. According to WHO (World Health Organization), more than 80% of

world population relies on traditional medicines, largely plant based for their

4

primary health care needs. Medicinal plants are currently in demand and their

popularity is increasing day by day in many parts of the world and has made a great

contribution towards maintaining human health. In addition, plant extracts eliciting

radioprotective efficacy contain a plethora of compounds including antioxidants,

immunostimulants, and cell proliferation stimulators, antiinflammatory and

antimicrobial agents, some will act in single compound as well as in combination

with other compounds from the same plant. Most studies using natural plant products

have focused on evaluation of radioprotective efficacy of whole extracts or

polyherbal formulations, and in some cases fractionated extracts and isolated

constituents (Arora et al., 2005). In general, biologically active compounds isolated

from plant largely contribute to medicinal field compare to the available synthetic

products. This may be due to the variability in chemical structures of secondary

metabolites which increasing the potential of new defense mechanisms against

various radiations induce damage.

A good radioprotector should be able to protect against the deleterious effects

of ionizing radiation either during therapeutic procedures as well as during nuclear

accidents or background irradiation. Apart from that, the agents should meets all the

prerequisites of an ideal radioprotectors including should be cheap, no cumulative or

irreversible toxicity in a wide dose range, provides effective long-term protection,

remains stable for a number of years without losing shelf life, and can be easily or

orally administered (Arora et al., 2006a; Arora et al., 2006b). Apart from that,

radioprotective activity of plant mediated through several mechanisms, including

radicals scavenging potential, detoxifying the radiation induced reactive species,

inducing cellular radioprotector such as superoxide dismutase (SOD) and

glutathione, enhancing the DNA repair by triggering one or more cellular DNA

5

pathways and also able to delay cell division and inducting hypoxia in the tissues

(Nair et al., 2001). Since, plant products possess complex mixture of active chemical

ingredients, therefore, it is able to contribute more on novel approaches of

radioprotection and mechanistic aspects as mention above, hence, it would be safe

and effective paradigm for radioprotection.

Malaysia being one of the 12 mega-biodiversity centers of the world is rich in

all three levels of biodiversity, namely, species diversity, genetic diversity and

habitat diversity with many plants used for medicinal and nutritional purposes. There

are more than 35,000 plant species being used in various medicinal purposes around

the world. In Peninsular Malaysia 1,200 species of higher plants and 2,000 species in

Sabah and Sarawak are reported to have medicinal value and have been used for

many generation in traditional health care system (Yoga Latha et al., 2011).

Therefore scientific investigation may be utilized to develop drugs for various

diseases which is easily accessible, available and affordable for the poor community

of bottom billion society.

Polyalthia longifolia leaf has been used as potential medicinal plants since

ancient time based on traditional medicine systems. The P. longifolia was widely

used in traditional medicine as febrifuge and tonic (The Wealth of India, 1969).

Fundamentally, the selected plant should be rich in antioxidants to minimize free

radical generation, a savor for macromolecules like lipids, proteins and DNA, should

be able to enhance internal defense mechanism, possess properties of potential

disinfectant and also a good immune rejuvenator. Hence, current study was designed

to evaluate the Polyalthia longifolia methanolic leaf extract as a potential

radioprotector.

6

1.1.1 Research objectives

The current study was undertaken with the following objectives:

1. To standardize the methanolic extract of P. longifolia leaf

2. To evaluate the in vitro antioxidant activity, in vivo hepatoprotective activity

and to determine total phenolic and flavonoid contents of methanolic extract

of P. longifolia leaf

3. To evaluate the potential genoprotective effect of the methanolic extract of P.

longifolia leaf

4. To evaluate in vivo radioprotective activities of the methanolic extract of P.

longifolia leaf using an animal model.

5. To isolate and identify of active compound/ fraction with antioxidant activity

from the methanolic extract of P. longifolia leaf

7

CHAPTER 2.0: LITERATURE REVIEW

2.1 Radiation

Human is constantly exposed to lethal radiation either from planned radiation, such

as during radiotherapy or unplanned radiation, such as the nuclear industry, sun’s

radiation and natural background radiation emanating from the earth or other

radioactive sources. Once exposed to this lethal radiation, it will cause various

adverse implications in our bodily system by the deposition of energy directly into

the bio macromolecules, which leads to the production of free radicals, as shown in

Figure 2.1. The free radicals are fundamental in modulating various biochemical

processes and represent an essential part of aerobic life and metabolism (Tiwari,

2001). The most common Reactive Oxygen Species (ROS) include superoxide

anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-), which result

from the cellular redox processes. At low or moderate concentrations, ROS exert

beneficial effects on cellular response and the immune function, however, at high

levels, these radicals become toxic and disrupt the antioxidant defence system of the

body, which may lead to “oxidative stress” (Pham-Huy et al., 2008). These reactive

oxygen species, in turn, react with different bio-molecules viz., lipid, DNA, proteins

and inflict oxidative damage in them (Figure 2.1).

The mediated reactions of major reactive oxygen species (ROS) include lipid

peroxidation, removal of thiol group from cellular and membrane proteins, strand

breaks and base alterations leading to DNA damage (Shukla and Gupta, 2010). After

the widespread realization concerning the adverse effects from lethal irradiation

various safety measurements were introduced to overcome this problem. However,

the radioprotective system developed against the lethal irradiation, most of the time,

8

Figure 2.1: Production of free radical and related damages by lethal irradiation.

A. In response to irradiation, lysosomal proteases such as cathepsins are shuttled

from the lysosomal lumen to the cytosol, resulting in autophagic cell death

9

B. The cell damage arises from damage to DNA due to base damages, double

strand breaks (DSB) or single strand breaks (SSB).

C. In the presence of DNA damage, p53-dependent gene transcription is

increased and ubiquitin-dependent degradation of the protein is blocked

D. Leading to induction of apoptosis and/or cell cycle arrest

E. Failure to activate the DNA repair mechanism in the cell leads to DNA

mutation and tumourigenesis as a consequence.

F. Proapoptotic BCL-2 family members Bak and Bax sensitize the mitochondria

to calcium-mediated fluxes and cytochrome c release. Cytochrome c together

with Apaf-1 activates a cascade of caspases, resulting in D) apoptosis.

10

is burdensome to use or less practical in various situations, such as during space

travel. Therefore, medicinal plants rich with various phytochemicals with

antioxidant properties could serve as an alternative radioprotective agent and could

be the most practical strategy to protect from lethal irradiation, which leads to

various diseases including cancer. The development of safe, non-toxic, cheap,

reliable and accessible radioprotective agents is crucial to overcome radiation related

problems, especially for patients undergoing radiotherapy. Plants will be the ideal

source to achieve this noble intention. In 1948, for the first time, Patt et al. (1949)

discovered that cysteine (Figure 2.2) was a radioprotector agent and proved that it

protected mice against the harmful effects of X irradiation. Ever since then, a

number of compounds have been evaluated by various scientists from various

sources including plants for the development of a radioprotective agent. Medicinal

plants remain the exclusive choice for the development of a safe and effective non-

toxic radioprotector since most of the medicinal plants are rich with antioxidant

phytochemicals.

2.2 Ionization and formation of free radicals

When cells are exposed to radiation they interact with target atoms and deposit the

energy resulting in ionization or excitation. Subsequently, the absorbed energy starts

to damage the molecules directly or indirectly. The damage occurs directly through

the ionization of atoms in the key molecules in the biological system, which leads to

functional alteration of the molecule. Absorption of energy is enough to get rid of an

electron, which results in bond breaks in the molecules. Conversely, the indirect

mechanism involves ionization in the cytoplasm, which produces reactive free

radicals of which the toxicity to the essential molecules results in an adverse effect

and biological effects, as shown in Figure 2.1.

11

Figure 2.2: Chemical structure of Cysteine

12

2.2.1 Free radicals and cell death

DNA damage within the cell may occur as a result of a direct radiation hit or

indirectly from free radicals (ROS). Eukaryotic cells typically respond to radiation

by activating the DNA repair pathways and cell cycle checkpoints, followed by

either full biological recovery or cell death (Ozben, 2007) (Figure 2.1). Radiation-

induced ROS production can lead to cell death through several mechanisms

including apoptosis, necrosis and autophagy (Ozben, 2007; Azad et al., 2009;

Wochna et al., 2007).

Apoptosis is a type I programmed cell death that occurs through two main

pathways, triggered either by the release of apoptotic proteins from the mitochondria

(intrinsic pathway) or by death-receptor ligation (extrinsic pathway) (Edinger and

Thompson, 2004). Apoptosis is depicted by membrane blebbing, early collapse of

the cytoskeleton, externalisation of phosphatidylserine (PS) on the cell surface,

cytoplasmic shrinkage, chromatin condensation, and, subsequently, the formation of

apoptotic bodies. In contrast to apoptosis, necrosis is regarded as a passive form of

cell death. Necrotic cells swell and lose their membrane integrity, then lyse and

release their contents into the extracellular space, causing inflammation and damage

to the surrounding tissue (Edinger and Thompson, 2004). In many cases, apoptosis

and necrosis may occur sequentially or simultaneously within the same tissue due to

irradiation. Through a series of well-designed studies, Wochna et al. (2007)

hypothesised that the switch from apoptotic to necrotic cell death involves not only a

diminution in cellular adenosine triphosphate (ATP) during cellular dysfunction, but

also an explosion of intracellular ROS.

Mitochondria organelles are the energy powerhouse of the cell. Irradiation

causes lipid peroxidation of membrane phospholipids and compromised membrane

13

integrity resulting in the release of small molecules including cytochrome c (Liu et

al., 1996) from the intermembrane space and apoptosis-inducing factor AIF (Susin et

al., 1999), resulting in cell death. The pro-apoptotic BCL-2 family members are

mediators of cell death that reside upstream of the mitochondria (Tsujimoto, 2003).

In response to irradiation, the p53 tumour suppressor induces the expression of a

number of damage induced genes regulating apoptosis, including death receptors and

proapoptotic members of the Bcl-2 family, Bax and Bak (Chipuk et al., 2004). The

p53-induced apoptosis proceeds through a series of events from the liberation of

cytochrome c from the mitochondria to the activation of caspase cascades (Villunger

et al., 2003).

Autophagy or type II programmed cell death is caspase independent and does

not involve DNA fragmentation. In autophagic cell death, organelles in the

cytoplasm, including mitochondria, are sequestered in an autophagosome, which

then fuses with the lysosomes (Azad et al., 2009). Lysosomal proteases, cathepsins,

will be shuttled from the lysosomal lumen to the cytoplasm in response to ROS. The

hydroxide produced, as in mitochondria by ROS, diffuses into lipofuscin-loaded

lysosomes, and the hydroxide causes damage to the lysosomal membranes, which

causes the leak of lysosomal enzymes. The lysosomal enzymes permeabilise the

mitochondrial membranes, resulting in the release of cytochrome c, the apoptosis-

inducing factor (AIF), and the second mitochondria-derived activator of caspase

(Smac)/direct inhibitor of apoptosis protein binding protein with low pI (DIABLO),

hence triggering cell death (Ghavami et al., 2010; Szumiel, 2011).

2.2.2 Free radicals and cancer

However, the irradiated cells that escape cell death may undergo mutation, which

creates an error in the DNA blueprint leading to altered gene expression and protein

14

modification; peptide bond cleavage and cross linking, for example, may affect

protein localization, interactions and enzyme activity. Although ROS-mediated DNA

damage may enable cells to function partially and proliferate, they eventually

develop into cancer, especially if the regulation of the tumour suppressor genes is

impaired (Wu, 2006). The high levels of ROS in cancer cells can further contribute

to oxidative stress, which may further stimulate tumour growth, invasion,

angiogenesis and metastasis (Wu, 2006; Girdhani et al., 2005).

The level of ROS production and antioxidant signalling appear to be altered

in malignant cells, contributing to cancer progression. However, the results from

different studies have been paradoxical, for instance, superoxide dismutase (SOD)

expression has been shown to decrease cancer cell proliferation and tumorigenicity

in vitro (Oberley, 2005), albeit its expression was found to be associated with bad

prognosis in patients with gastric cancer (Kim, 2002).

2.3 Radioprotection mechanisms by plant extract or compounds

Numerous investigations on radioprotection mechanisms have been carried out in

several biological systems and the following radioprotection mechanisms have been

proposed from these studies: free radical scavenger, repair by hydrogen donation to

target molecules, formation of mixed sulphides, delaying of cellular division and

induction of hypoxia in the tissue (Varanda and Tavares, 1998). The mechanism of

free radical scavenger suggests that medicinal plants will donate electrons to the free

radicals and form a stable compound incapable of reacting with other cellular

components. This mechanism prevents the free radicals from reacting with the vital

cell components. Another mechanism that has been proposed is the repair by

hydrogen donation. If a R-H molecule is converted into a radical R by exposure to

radiation, the antioxidant plant extract or compound can donate a hydrogen atom to

15

this radical, restoring it to its original state (Biaglow, 1987), which is not vulnerable

to the vital components of our bodily system. In addition, the mechanism of the

formation of mixed sulphides suggests aminothiols, which involves radioprotector

binding to cellular components. According to this proposed mechanism, the

sulphydryl compound of medicinal plants form mixed disulphides with sulphydryl

compounds of cellular proteins. Once the free radicals generated by irradiation attack

the disulphides, the sulphur atoms will be reduced and the other sulphur atom will be

oxidized (Varanda and Tavares, 1998). This mechanism prevents the free radicals

from reacting with the vital cell components because if the sulphur atom of the

protein is reduced by the free radicals and the sulphur atom of the protective agent is

oxidized, the protein is not damaged.

Delaying of cellular division and granting additional time for repairing DNA

damage caused by irradiation has been considered a potentially important

mechanism in radioprotection activity. For this type of mechanism, Brown (1967)

proposed that the sulphydryl compounds of the radioprotective agents will bind to

the cellular DNA and inhibit its replication and provide additional time for repair of

the damaged DNA. Protection by the induction of hypoxia in the tissue has also been

considered a potentially important mechanism in radioprotection activity. Oxidation

of the radioprotective agents uses enough oxygen to reduce its tension, and it has

already been revealed that hypoxia is radioprotective. Moreover, the induction of

hypoxia in tissue in certain conditions may contribute to radioprotection.

Nevertheless, other mechanisms might be involved, since some compounds exhibit

radioprotective activity without altering the oxygen tension on the tissue (Varanda

and Tavares, 1998). There is evidence of the existence of more than one

radioprotective mechanism of a certain compound, and that one of the compounds

16

might be more or less important, depending on the irradiated system and on the

specific radiation conditions (Prasad, 1982).

2.4 Plant as anti-radiation sources

2.4.1 Traditional usage of medicinal plant as radioprotective agent

For eons, plants and plant products have been infused in human life, as palatable and

remedial sources. Traditional healers exploited plants to treat various maladies long

before the discovery of drugs (Cragg et al., 1997). Moreover, the conventional plant

preparations are also demonstrated to be non-toxic or less-toxic, considering their

derivation from natural resources.

Gingko biloba is one of the world’s ancient trees and is believed to have

survived an atomic bomb explosion dropped on Hiroshima on 6 August 1945 by the

Americans (Anonymous, 2013a). The surviving trees were found near the blast

centre and appeared to sprout without major deformations. The observation

substantiates the plant’s amazing resistance to mutagen agents like radiation

(Pickstone, 2010). On a different occasion, the Buddhist monks took delight in

tending to these trees by preserving them near the pagodas in China’s Imperial

Gardens and on sacred grounds to ward off fire. G. biloba is also denoted as a

symbol of longevity.

Although folklore does not directly imply that plants impart a radioprotective

effect, much evidence has been found of their incorporation in ceremonies and

rituals in which specific plants are utilized. The Tulsi or Ocimum sanctum, for

example, is worshipped along with milk, yogurt, honey and Ganga (river) water,

which are consumed by the devotees at the end of the ritual (McGuire, 2012). The

ancient Indian legend states that this Queen of Herbs came as an incarnation of the

Hindu goddess Tulsi and is favoured by the Lord Vishnu, Krishna and Ram (Miller

17

and Miller, 2003). A plant with radioprotective effect can also be identified with the

presence of other properties, such as anti-inflammatory, antioxidant, antimicrobial

and immune modulatory (Jagetia, 2007). Likewise, Tulsi, within the confinement of

Ayurveda, was used to regulate fever, relieve coughs and flu, and mobilize mucus in

bronchitis and asthma. The leaves especially were used to treat tuberculosis and

ringworm of the skin. The tulsi oil is rich in vitamin C, carotene, calcium and

phosphorus and is also believed to possess other properties including antibacterial,

antifungal and antiviral (Anonymous, 2013b).

Radiation interacts and distresses the atoms that compose the cells. The

affected atoms will subsequently form free radicals that disrupt molecules, cells,

tissues and organs that eventuate to the detriment of the organism (USNRC

Technical Training Center, 2013). Since free radicals are responsible for inducing

radiation-damage, the radioprotective property of Panax ginseng is associated either

directly or indirectly with its free radical scavenging capability (Lee et al., 2005).

Ginseng refers to the root and the rhizome of Panax ginseng C.A. Meyer

(Araliaceae), which have been conventionally utilised by the Chinese for more than

200 years. The Chinese believe that ginseng is a reservoir with a range of

pharmacological roles, such as restorative, tonic, nootropic and anti-aging (Lee et

al., 2005).

2.4.2 Medicinal plant with radioprotective effects

Naturally occurring herbs constitute a wide variety of antioxidants, such as alpha

carotene, ascorbic acids, flavones, flavanones, flavanols, stilbenoids, anthocyanins,

phenolic acids, etc., which are reported to have a broad spectrum of radiation

absorption properties (Bajpai et al., 2005; Ashawat et al., 2006; Nichols and Katiyar,

2010; Vaid and Katiyar, 2010). In addition, it has been shown that these

18

phytoconstituents have a synergistic photo-protective effect and can be used as

sunscreen to protect cellular damage of the skin from radiation light exposure

(Campos et al., 2006; Afaq et al., 2003). The radioprotective effect of

phytochemicals has gained popularity in skin care and attention has been focused in

developing topical formulations, which can be used as complementary as well as

alternate medicine to heal and rejuvenate skin from various disorders (Griffiths et

al., 2005; Kapoor et al., 2009; Saraf and Kaur, 2010; Svobova et al., 2003). Some of

the medicinal plants with radioprotective properties – antioxidant, anti-inflammatory

and immunomodulatory – are listed in Table 2.1.

2.5 Antiradiation compounds

Antiradiation compounds are studied by in vitro and in vivo tests that assess some of

these aspects. Assay of free radicals and antioxidant assay of pharmacological agents

are suggested as a good means for evaluating the radioprotective potential (Jagetia,

2007). The polyphenolic compounds, especially flavonoids, ubiquitously present in

plants, have been reported to possess various beneficial biological properties, most

of which are attributed to antioxidant activity. It is not surprising that radioprotective

potential has been reported for extracts of herbs containing flavonoids, as well as for

individually isolated flavonoids. The radioprotective effect of two extracts of

Caesalpinia digyna and the isolated compound bergenin were compared using in

vitro methods by Singh et al. (2009). The in vitro approach compared the protective

action against the damaging effect of protein carbonylation in bovine serum albumin,

lipid peroxidation in liposomes, and DNA breakage in pBR322 plasmid. The study

showed that the flavonoid, bergenin, from the plant is equally potent in inhibiting

DNA damage as the extracts, albeit the extracts were more potent in protecting the

proteins and lipids. The pBR322 model was also used in

19

Table 2.1: Plant with radioprotective activity or antioxidant activity

Plant species Scientific names component activity Reference

Tomato Solanum Lycopersicum Carotenoids –

lycopenes

antioxidant (Griffiths et al., 2005; Saraf and Kaur, 2010; Ravichandran et al., 2005)

Carrot Daucus carota β-carotene antioxidant (Griffiths et al., 2005; Svobova et al., 2003)

Papaya Carica papaya L-ascorbic acid Antioxidant and photoprotective

(Vile , 1997)

orange Citrus sinensis L-ascorbic acid antioxidant

(Cimino et al., 2007)

Lemon Citrus limon L-ascorbic acid antioxidant (Apak et al., 2007)

Mango Mangifera indica L-ascorbic acid antioxidant

with anti-inflammatory and immunomodulatory activities.

(Song et al., 2013)

Pomegranate Punica granatum ascorbic acid antioxidant (Kumar et al., 2009)

Celery

Apium graveolens Flavones –

5,7,4’-

antioxidant and ROS scavenger

(Griffiths et al., 2005; Svobova et al., 2003)

20

trihydroxystilibine

Red clover Trifolium pratense Isoflavone –

Genistein

Inhibit UV induced peroxidase production

(Widyarini et al., 2001)

Soybean Glycine max Anthocyanin Photo protective of UV radiation

(Tsoyi et al., 2008)

Green tea Camellia sinensis Flavanol –

Epigallocatechin gallate


(Katiyar et al., 2000; Katiyar et al., 2001; Katiyar and Elmets, 2001; Higdon, 2007; Li et al., 2009; Sharangi, 2009; Kaur and Saraf, 2011b)

Milk thistle Silybum marianum Stilbenoid-

Silybin,silibinin,

silidianin,

Silychristin

anti-inflammatory and immunomodulatory

(Katiyar, 2002; Fguyer et al., 2003; Vaid and Katiyar, 2010)

Grape Vitis vinifera Stilbenoid-

Resveratrol, Flavanol -proanthocyanidin


(Afaq et al., 2003; Saraf and Kaur, 2010; Aziz et al., 2005; Mantena and Katiyar, 2006)

Apple Malus domestica Flavanoid-Quercetin antioxidant (Erden Inal et al., 2001; Korac and Khambholja, 2001)

Boldo Peumus boldus Quercetin, Flavanol- antioxidant and anti- (Peter et al., 2006; Russo et al.,

21

catechin; aporphine inflammatory 2011)

Turmeric Curcuma longa Phenolic -curcumin anti-inflammatory, antiproliferative, Photoprotective effect

(Saraf and Kaur, 2010; Garcia Bores and Avila, 2008)

Aloe vera Aloe barbadensis antraquinones Cellular repair (West and Zhu, 2003)

Rhubarb Rheum rhaponticum stilbene antioxidant and ROS scavenger

(Silveira et al., 2013)

22

assessing the protective effect of pure compounds isolated from Phyllanthus amarus

(Londhe et al., 2009). The flavonoids, quercetin 3-O-glucoside followed by rutin,

offered the greatest protection on DNA as seen by the decrease in the nicked circular

form of plasmid. However, the ellagitannins, namely amariin, 1-galloyl-2,3-

dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin,

corilagin, phyllanthusiin D were also effective. The protective effects of these

compounds on protein and lipids damage by radiation were assessed by using rat liver

mitochondria. The compounds, rutin and repandusinic acid offered maximum

protection against lipid damage whereas protection against carbonyl formation in

proteins was highest in rutin, phyllanthusiin D, geraniin and quercetin 3-O-glucoside.

The effects of flavonoids have also been studied by using in vivo techniques.

For example, various doses of preparation containing 12 flavonoids (FAC) from seeds

of Astragalus complanatus protected mice from radiation damage (Qi et al., 2011).

Basically, FAC increased the survival rate of irradiated mice and had a protective

effect on haematopoietic tissue and the immune system. The alkaline comet assay,

which involves single cell electrophoresis was able to show the protective effect

against DNA damage in mouse liver cells by the FAC. Studies on radioprotection

have also taken advantage of the availability of synthetic drugs that have been used

clinically in humans. The protective effect of troxerution, a flavonoid derivative used

for treating venous disorders, was also ascertained by using the comet assay. In this

study, the method assessed the protection against DNA damage in mice blood, bone

marrow and tumour cells (Maurya et al., 2004).

23

2.5.1 Modern technique for evaluation of radioprotective activity of medicinal

plants

In this section various reliable methods available for the study of radioprotective

activity, such as plant sample extraction techniques, in vitro and in vivo

radioprotective activity were analysed and compared. Figure 2.3 shows the various

steps involved in the evaluation of the medicinal plants for radioprotective activity

properties.

2.5.2 Extraction

The first step in the process of screening medicinal plants for radioprotective activity

is extraction. Extraction is the separation of medically active portions of plant tissue

using selective solvents through various standard procedures. The extraction

technique using an appropriate solvent system separates the soluble plant metabolites

and leaves behind the insoluble cellular marc. The products obtained from plants are

relatively complex mixtures of metabolites, in liquid or semisolid state or in dry

powder form and are intended for oral or external use (Handa, 2008). The general

techniques of plant extraction include maceration, percolation, digestion, hot

continuous extraction (Soxhlet) and ultrasound extraction (sonication). In addition,

modern extraction methods have been developed, which include microwave-assisted

extraction and superficial fluid extraction. The fundamental operations of extraction

include pre-washing, drying of plant materials or freeze drying, grinding to obtain a

homogenous sample and often improving the analytical extraction, and also

increasing the contact of the sample surface with the solvent system (Sasidharan et

al., 2012). The selection of a proper extraction method is the most important part of

any experiment in order to obtain therapeutically potential active constituents from

the plant sample. The standardization of active compounds

24

Plant • Extraction • Standardization

in vitro test

• Antioxidant assay• Comet assy• Plasmid relaxation assay• Allium cepa assay

in vivo test

• Endogenous spleen colony forming unit (CFU) assay

• Whole- body survival and body weight

• Gastrointestinal damage assay

Figure 2.3: Various steps involved in the development and evaluation of radioprotective property of medicinal plants

assay

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