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UNIVERSITI PUTRA MALAYSIA

CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF ARTOCARPUS ELASTICUS REINW EX BLUME

FAIQAH BINTI RAMLI

FS 2013 57

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CHEMICAL CONSTITUENTS AND

BIOLOGICAL ACTIVITIES OF ARTOCARPUS

ELASTICUS REINW EX BLUME

FAIQAH BINTI RAMLI

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2013

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CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF

ARTOCARPUS ELASTICUS REINW EX BLUME

By

FAIQAH BINTI RAMLI

Thesis submitted to the School of Graduate studies, Universiti Putra Malaysia in

fulfilment of requirements for the degree of Master in Science

May 2013

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All material contained within the thesis, including without limitation text, logos, icons,

photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless

otherwise stated. Use may be made of any material contained within the thesis for non-

commercial purposes from the copyright holder. Commercial use of the material may only

be made with the express, prior, written permission of Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfilment of

requirements for the degree of Master of Science

CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF

ARTOCARPUS ELASTICUS REINW. EX BLUME

By

FAIQAH BINTI RAMLI

May 2013

Chairman: Professor Mawardi Rahmani, PhD

Faculty : Science

Detailed phytochemical investigation has been carried out on leaves and bark of

Artocarpus elasticus (Moraceae) collected from two separate locations, Gunung

Nuang, Selangor and Sarawak. Various chromatography techniques such as thin

layer chromatography (TLC), gravity column chromatographic, vacuum column

chromatography, and chromatotron have been used to isolate several compounds of

different classes, such as triterpenes, chalcones, flavone, and

dihydrobenzoxanthones. Structural elucidations of the pure compounds were carried

out using spectroscopic techniques such as UV, IR, NMR, MS and also by

comparison with published data. The crude extracts and some isolated compounds

were screened for antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl assay,

cytotoxicity by using microculture tetrazolium salt assay and antimicrobial activity

using disc diffusion assay. The cell lines used in the cytotoxic assay were human

estrogen receptor (ER+) positive breast cancer (MCF-7), human estrogen receptor

(ER-) negative (MDA-MB 231), human hepatocarcinoma (HepG2), and normal

human cell (WLR-68). For the antimicrobial activity samples were tested against

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Bacillus ATCC, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus ATCC,

Staphylococcus aureus IMR, Staphylococcus pyogenes, Staphylococcus epidermidis,

S1211 IMR, Pseudomonas aeruginosa, Klebsiella sp., Pseudomonas multocida,

Enterobacter cloacae and Escherichia coli.

Isolation work on leaves of Artocapus elasticus collected from Gunung Nuang,

Selangor, Malaysia has led to the isolation and identification of three new

dihydrochalcones [elastichalcone A (105), elastichalcone B (109), elastichalcone C

(107)], cycloartocarpesin (111) along with stigmasterol (96) and p-hydroxybenzoic

acid (112). In contrast, detailed study on the bark of Artocarpus elasticus collected

from Sarawak afforded one new flavone, elastixanthone (113), together with the

known cycloartobiloxanthone (15), artobiloxanthone (79) and artonin E (80).

All compounds isolated showed interesting biological activity towards certain

bioassays. However, five of the prenylated flavonoids, cycloartobiloxanthone (15),

artonin E (80), elastichalcone B (109), cycloartocarpesin (111) dan elastixanthone

(113) exhibited good potential for further development as antioxidant agent with IC50

values of 40.02, 11.50, 11.30, 11.89 and 21.60 µg/ml, respectively. Cytotoxic assay

carried out on the isolated compound revealed that artonin E (80) possesses potent

cytotoxic activity against human estrogen receptor (ER+) positive breast cancer

(MCF-7) and human estrogen receptor (ER-) negative (MDA-MB 231) with IC50

value of 2.5 and 15 µg/ml, respectively. Similarly, artonin E (80) and elastichalcone

B (109) displayed broad spectrum of antimicrobial activities on the growth of

Bacillus ATCC, B. cereus, B. subtilis, S. aureus ATCC, S. aureus IMR, S. pyogenes,

E. coli and S. epidermidis.

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Abstrak tesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains.

KANDUANGAN KIMIA DAN AKTIVITI BIOLOGI DARIPADA

ARTOCARPUS ELASTICUS REINW. EX BLUME

Oleh

FAIQAH BINTI RAMLI

Mei 2013

Pengerusi: Profesor Mawardi Rahmani, PhD

Fakulti : Sains

Kajian fitokimia dan aktiviti biologi ke atas kulit batang dan daun Artocarpus

elasticus telah dijalankan. Pelbagai teknik kromatografi seperti turus graviti,

kromatografi turus vakum, kromatografi radial dan kromatografi lapisan nipis telah

digunakan untuk penulenan beberapa sebatian daripada kelas yang berbeza seperti

triterpena, calkon, flavon, dan xanton. NMR, MS, IR, UV dan juga pembandingan

dengan data literatur telah digunakan untuk pengenalpastian struktur sebatian yang

telah dipencilkan. Ekstrak mentah dan sebahagian sebatian yang telah dipencilkan

telah diuji aktiviti antioksidan, sitotoksik dan antimikrob dengan masing-masing

menggunakan kaedah 2,2-difenil-1-pikrilhidrazil (DPPH), mikrokultur tetrazolium

garam dan peresapan cakera. Sel yang digunakan untuk ujikaji sitotoksik adalah

kanser payudara (MCF-7), penerima estrogen negative manusia (MDA-MB 231),

hepatokarsinoma manusia (HepG2), dan sel manusia normal (WLR-68). Aktiviti

mikrob telah diuji ke atas Bacillus ATCC, Bacillus cereus, Bacillus subtilis,

Staphylococcus aureus ATCC, Staphylococcus aureus IMR, Staphylococcus

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pyogenes, Staphylococcus epidermidis, S1211 IMR, Pseudomonas aeruginosa,

Klebsiella sp., Pseudomonas multocida, Enterobacter cloacae dan Escherichia coli.

Kerja pemencilan ke atas daun Artocarpus elasticus yang diperoleh dari Gunung

Nuang, Selangor, Malaysia telah membawa kepada pemencilan tiga dihidrocalkon,

elasticalkon A (105), elasticalkon B (107), elasticalkon C (109) dan asid p-

hidroksibenzoik (112), sikloartokarpesin (111), dan stigmasterol (96). Kajian yang

sama ke atas kulit batang Artocarpus elasticus yang diperoleh dari Sarawak,

Malaysia telah membawa kepada pemencilan satu flavon baru elastixanton (113),

bersama dengan tiga flavon lain yang dikenali sebagai, cycloartobiloxanthone (15)

dan artobiloxanthone (79) dan artonin E (80).

Ujian aktiviti biologi telah dijalankan ke atas semua sebatian yang telah dipencilkan.

Walau bagaimanapun, lima flavon, sikloartobiloxanton (15), artonin E (80),

elasticalkon C (107), sikloartokarpesin (111), dan elastixanton (113) menunjukkan

potensi yang baik sebagai ajen antioksidan dengan IC50 40.02, 11.53, 11.30, 11.89

dan 21.60 µg/ml. Kaedah sitotoksik ke atas kesemua sebatian menunjukkan artonin

E (80) mempunyai aktiviti sitotoksik ke atas sel kanser payudara (MCF-7) dan

penerima estrogen negatif (MDA-MB 231) dengan nilai IC50 2.5 dan 15 µg/ml.

Namun begitu, hanya artonin E (80) dan elasticalkon B (109) sahaja menunjukkan

perencatan yang kuat ke atas pertumbuhan Bacillus ATCC, B. cereus, B. subtilis, S.

aureus ATCC, S. aureus IMR, S. pyogenes, E. coli dan S. epidermidis.

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ACKNOWLEDGEMENTS

I wish to extend my deepest gratitude and appreciation to my research supervisor,

Prof. Dr. Mawardi Rahmani for his constant guidance, support, encouragement, and

patience throughout the completion of this research project. My sincere thanks also

goes to my supervisory committee members Prof. Dr. Aspollah Mohd Sukari and Dr.

Abdah Md Akim for their thoughtful ideas and kind guidance through this project.

I would also like to thank my senior labmates, Mrs Kartinee, Nadiah, Kamilah,

Aizat, Winda, Maizatulakmal and Nazil for their priceless discussion and assistance.

My sincere appreciation is extending to the lab assistants in the Department of

Chemistry, Faculty of Science especially En. Zainal for GC-MS, En Johadi, En Fadli

and Miss Shareena for their guidance and advice on handling the NMR machine,

Mrs Ros for IR and Mr. Shaharudin for UV machine. Special thanks to Dr. Najihah

and Hazirah for helping me in the cytotoxic and antimicrobial activity.

Last but not least, my deepest gratitude go to my parents, Mrs Zubaidah Ali and En

Ramli as well as my siblings, Filzani, Firhan, and Haiqal for the encouragement and

support toward my successful research.

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I certify that an Examination Committee has met on date of viva to conduct the final

examination of name of student on his degree thesis entitled "Chemical Constituents

And Biological Activities Of Artocarpus elasticus Reinw ex Blume" inaccordance with

Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Putra

Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the

candidate be awarded the relevant degree.

Members of the Examination Committee are as follows:

Mohamed Ibrahim bin Mohamed Tahir, PhD

Senior Lecturer

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Intan Safinar binti Ismail, PhD

Senior Lecturer

Faculty of Science

Universiti Putra Malaysia

(Internal Examiner)

Gwendoline Ee Cheng Lian, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Internal Examiner)

Farediah binti Ahmad, PhD

Associate Professor

Faculty of Science

Universiti Teknologi Malaysia

(External Examiner)

___________________________

BUJANG BIN KIM HUAT, PhD

Professor/Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted

as fulfilment of the requirement for the degree of Master of Science. The members of the

Supervisory Committee were as follows:

Mawardi Rahmani, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Aspollah Mohd Sukari

Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

Abdah Md Akim

Senior Lecturer

Faculty of Medicine and Health Science

Universiti Putra Malaysia

(Member)

___________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that this thesis is my original work except for quotation and citations, which

have been duly acknowledged. I also declared that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other Institutions.

__________________________

FAIQAH BINTI RAMLI

Date: 22 May 2013

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK v

ACKNOWLEDGEMENT vii

APPROVAL vii

DECLARATION ix

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xviii

CHAPTER

1 INTRODUCTION

1.1 General Introduction 1

1.2 Problem statement 4

1.3 Objective and Scope of Study 5

1.4 Significance of Study 5

2 LITERATURE REVIEW

2.1 Botanical aspects of plants

2.1.1 Moraceae Family 6

2.1.2 Artocarpus genus 7

2.2 Chemical constituents

2.2.1 Diversity Of Secondary Metabolites 8

2.2.2 Chemical Constituents from Artocarpus Genus. 13

3 METHODOLOGY

3.1 Plant material 33

3.2 Instrument 33

3.2.1 Infrared Spectroscopy (IR) 33

3.2.2 Ultraviolet (UV) 33

3.2.3 Mass spectra (MS) 34

3.2.4 Nuclear Magnetic Resonance (NMR) 34

3.2.5 Melting point 34

3.3 Chromatography Method 34

3.3.1 Column Chromatography 35

3.3.2 Chromatotron 36

3.3.4 Thin layer chromatography (TLC) 36

3.4 Extraction and Isolation of leaves of Artocarpus

elasticus.

36

3.4.1 Extraction of the leaves 36

3.4.2 Fractionation and isolation of hexane extract 38

3.4.3 Fractionation and isolation of chloroform extract 41

3.4.4 Fractionation and isolation of methanol extract 45

3.5 Extraction and Isolation of bark of Artocarpus elasticus. 47

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3.5.1 Fractionation and isolation of ethyl acetate

extract

49

3.5.2 Fractionation and isolation of methanol

extract

52

3.6 Bioactivity Test Procedures 55

3.6.1 DPPH free radical scavenging activity 55

3.6.2 Cytotoxicity 56

3.6.3 Antimicrobials 58

4 RESULTS AND DISCUSSION

4.1 Isolation of chemical constituents from A. elasticus 60

4.2 Chemical constituents from leaves of A. elasticus 61

4.2.1 Characterization of Elastichalcone A (105) 62

4.2.2 Characterization of Elastichalcone C (107) 77

4.2.3 Characterization of Elastichalcone B (109) 92

4.2.4 Characterization of Cycloartocarpesin (111) 104

4.2.5 Characterization of Stigmasterol (96) 115

4.2.6 Characterization of p-hydroxybenzoic acid (112) 120

4.3 Chemical constituents of bark of A.elasticus 124

4.3.1 Characterization of Elastixanthone (113) 124

4.3.2 Characterization of Artonin E (80) 136

4.3.3 Characterization of Cycloartobiloxanthone (15) 146

4.3.4 Characterization of Artobiloxanthone (79) 157

4.4 Biosynthetic pathway for selected isolated flavonoids 168

4.5 Biological Activity Results 169

4.5.1 Antioxidant Activity (DPPH Assay) 169

4.5.2 Cytotoxic Activity (MTT Assay) 171

4.5.3 Antimicrobial Activity (Disc Diffusion Assay) 175

5 CONCLUSIONS 179

BIBLIOGRAPHY 181

APPENDICES 186

BIODATA OF STUDENT 192

LIST OF PUBLICATIONS 193

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LIST OF TABLES

Table Page

4.1 Compounds isolated from A. elasticus 61

4.2 NMR spectral data of elastichalcone A (105) 65

4.3 NMR spectral data of elastichalcone C (107) 80

4.4 NMR spectral data of elastichalcone B (109) 94

4.5 NMR spectral data of cycloartocarpesin (111) 106

4.6 NMR spectral data of stigmasterol (96) 116

4.7 NMR spectral data of elastixanthone (113) 127

4.8 NMR spectral data of artonin E (80) 138

4.9 NMR spectral data of cycloartobiloxanthone (15) 149

4.10 NMR spectral data of artobiloxanthone (79) 160

4.11 The IC50 values of crude extracts and isolated compound from A.

elasticus against DPPH radical.

170

4.12 Cytotoxic activity of crude extracts and pure compound on MCF-

7, MDA-MB-231, HepG2 and WRL68 for 24 hours.

173

4.13 The diameter inhibition zone (in mm) of isolated compound

against selected microbes.

176

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LIST OF FIGURES

Figure Page

2.1 The derivation of the flavonoid and isoflavonoid ring system 8

2.2 The biosynthesis of chalcones 9

2.3 The biogenetic relationship of the flavonoids 9

2.4 The reaction catalysed by chalcone-flavonone isomerase 11

2.5 The biosynthesis of flavones 12

2.6 The biosynthesis of mulberrin (10) and artocarpin (12) 13

3.1 Isolation work of Artocarpus elasticus leaves 37

3.2 Isolation work of Artocarpus elasticus bark

48

4.1 IR spectrum of elastichalcone A (105) 66

4.2 UV spectrum of elastichalcone A (105) 66

4.3 EIMS spectrum of elastichalcone A (105) 66

4.4 HREIMS spectrum of elastichalcone A (105) 67

4.5 Selected HMBC ( ) correlations of elastichalcone A (105) 67

4.6 COSY ( ) correlations of elastichalcone A (105) 67

4.7 1H-NMR spectrum of elastichalcone A (105) 68

4.8 13

C-NMR spectrum of elastichalcone A (105) 69

4.9 DEPT-NMR spectrum of elastichalcone A (105) 70

4.10 COSY-NMR spectrum of elastichalcone A (105) 71

4.11 HMQC-NMR spectrum of elastichalcone A (105) 72

4.11.1 Expanded HMQC-NMR spectrum of elastichalcone A (105) 73

4.12 HMBC-NMR spectrum of elastichalcone A (105) 74

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4.12.1 Expanded HMBC-NMR spectrum of elastichalcone A (105) 75

4.12.2 Expanded HMBC-NMR spectrum of elastichalcone A (105) 76

4.13 IR spectrum of elastichalcone C (107) 81

4.14 UV spectrum of elastichalcone C (107) 81

4.15 EIMS spectrum of elastichalcone C (107) 81

4.16 HREIMS spectrum of elastichalcone C (107) 82

4.17 Selected ( ) HMBC correlations of elastichalcone C (107)

82

4.18 COSY ( ) correlations of elastichalcone C (107) 82

4.19 1H-NMR spectrum of elastichalcone C (107) 83

4.20 13

C-NMR spectrum of elastichalcone C (107) 84

4.21 DEPT-NMR spectrum of elastichalcone C (107) 85

4.22 COSY-NMR spectrum of elastichalcone C (107) 86

4.23.1 Expanded HMQC-NMR spectrum of elastichalcone C (107) 87

4.23.2 Expanded HMQC-NMR spectrum of elastichalcone C (107) 88

4.24.1 HMBC-NMR spectrum of elastichalcone C (107) 89

4.24.2 Expanded HMBC-NMR spectrum of elastichalcone C (107) 90

4.24.3 Expanded HMBC-NMR spectrum of elastichalcone C (107) 91

4.25 IR spectrum of elastichalcone B (109) 95

4.26 UV spectrum of elastichalcone B (109) 95

4.27 EIMS spectrum of elastichalcone B (109) 96

4.28 HREIMS spectrum of elastichalcone B (109) 96

4.29 Selected HMBC ( ) correlations of elastichalcone B (109) 97

4.30 COSY ( ) correlations of elastichalcone B (109) 97

4.31 1H-NMR spectrum of elastichalcone B (109) 98

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4.32 13

C-NMR spectrum of elastichalcone B (109) 99

4.33 DEPT-NMR spectrum of elastichalcone B (109) 100

4.34 COSY-NMR spectrum of elastichalcone B (109) 101

4.35 HMQC-NMR spectrum of elastichalcone B (109) 102

4.36 HMBC-NMR spectrum of elastichalcone B (109) 103

4.37 IR spectrum of cycloartocarpesin (111) 107

4.38 UV spectrum of cycloartocarpesin (111) 107

4.39 EIMS spectrum of cycloartocarpesin (111) 108

4.40 1H-NMR spectrum of cycloartocarpesin (111) 109

4.41 13

C-NMR spectrum of cycloartocarpesin (111) 110

4.42 DEPT-NMR spectrum of cycloartocarpesin (111) 111

4.43 COSY-NMR spectrum of cycloartocarpesin (111) 112

4.44 HMQC-NMR spectrum of cycloartocarpesin (111) 113

4.45 HMBC-NMR spectrum of cycloartocarpesin (111) 114

4.46 IR spectrum of stigmasterol (96) 117

4.47 EIMS spectrum of stigmasterol (96) 117

4.48 1H-NMR spectrum of stigmasterol (96) 118

4.49 13

C-NMR spectrum of stigmasterol (96) 119

4.50 IR spectrum of p-hydroxybenzoic acid (112) 121

4.51 EIMS spectrum of p-hydroxybenzoic acid (112) 121

4.52 1H-NMR spectrum of p-hydroxybenzoic acid (112) 122

4.53 13

C-NMR spectrum of p-hydroxybenzoic acid (112) 123

4.54 IR spectrum of elastixanthone (113) 128

4.55 UV spectrum of elastixanthone (113) 128

4.56 EIMS spectrum of elastixanthone (113) 129

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4.57 HREIMS spectrum of elastixanthone (113) 129

4.58 Selected HMBC ( ) correlations of elastixanthone (113) 130

4.59 COSY ( ) correlations of elastixanthone (113) 130

4.60 1H-NMR spectrum of elastixanthone (113) 131

4.61 APT-NMR spectrum of elastixanthone (113) 132

4.62 COSY-NMR spectrum of elastixanthone (113) 133

4.63 HMQC-NMR spectrum of elastixanthone (113)

134

4.64 HMBC-NMR spectrum of elastixanthone (113)

135

4.65 IR spectrum of artonin E (80) 139

4.66 UV spectrum of artonin E (80) 139

4.67 EIMS spectrum of artonin E (80) 140

4.68 1H-NMR spectrum of artonin E (80) 141

4.69 APT-NMR spectrum of artonin E (80) 142

4.70 COSY-NMR spectrum of artonin E (80) 143

4.71 HMQC-NMR spectrum of artonin E (80) 144

4.72 HMBC-NMR spectrum of artonin E (80) 145

4.73 IR spectrum of cycloartobiloxanthone (15)

150

4.74 UV spectrum of cycloartobiloxanthone (15)

150

4.75 EIMS spectrum of cycloartobiloxanthone (15)

151

4.76 1H-NMR spectrum of cycloartobiloxanthone (15)

152

4.77 APT-NMR spectrum of cycloartobiloxanthone (15)

153

4.78 COSY-NMR spectrum of cycloartobiloxanthone (15)

154

4.79 HMQC-NMR spectrum of cycloartobiloxanthone (15) 155

4.80 HMBC-NMR spectrum of cycloartobiloxanthone (15) 156

4.81 IR spectrum of artobiloxanthone (79) 161

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4.82 UV spectrum of artobiloxanthone (79) 161

4.83 EIMS spectrum of artobiloxanthone (79) 162

4.84 1H-NMR spectrum of artobiloxanthone (79) 163

4.85 APT-NMR spectrum of artobiloxanthone (79) 164

4.86 COSY-NMR spectrum of artobiloxanthone (79) 165

4.87 HMQC-NMR spectrum of artobiloxanthone (79) 166

4.88 HMBC-NMR spectrum of artobiloxanthone (79) 167

4.89 Biosynthetic pathway of dihydrobenzoxanthones 168

4.90 Reaction between DPPH radical and antioxidant to form DPPH 169

4.91 The chemical structure and cytotoxic activities against MCF-7 of

artonin E (80), cycloartobiloxanthone (15) and artobiloxanthone

(79)

175

4.92 Structure of artonin E (80) with attachment of chromene ring at C-8

contribute to stronger antimicrobial activity compared to

elastichalcone B (109)

178

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LIST OF ABBREVIATIONS

δ Chemical shift in ppm

λmax Maximum wavelength in mm

13C Carbon-13

COSY Correlated spectroscopy

DEPT Distortionless Enhancement By Polarization Transfer

EIMS Electrom Impact Mass Spectroscopy

GC-MS Gas Chromatography-Mass Spectroscopy

1H proton

HMBC Heteronucluear Multiple Bond Connectivity

HMQC Heteronucluear Multiple Quantum Coherence

IC50 Inhibition concentration at 50 percent

M.S Mass spectrometry

m/z Mass per charge

IR Infrared

UV ultraviolet

APT Attached proton test

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CHAPTER 1

INTRODUCTION

1.1 Introduction

It is a well known fact that plants play an important role in daily human life by

providing oxygen, food, wood, furniture, rubber and are even used in traditional

medicines which contribute to the economic development in certain countries

(Raskin et al., 2002). Studies have exhibited that chronic diseases such as stroke,

myocardial infarction, certain type of cancers, diabetes mellitus, Alzheimer’s

disease, allergy, septic shock, osteoporosis, neurodegeneration and some other

diseases associated with aging are easier to prevent than to cure, and that

consumption of phytochemical-rich fruits and vegetables, along with culinary herbs

and spices, can reduce the risk of developing such condition (Vickery et al., 1981).

Over the centuries, drugs were entirely of natural origin and composed of herbs,

animal products, and inorganic materials. Early remedies may have combined these

ingredients, but it is certain that those treatments were effective, leading to early

herbals. As chemical techniques improve, the active constituents were isolated from

plants, structurally characterized, and further synthesized in the laboratory.

Sometimes, more effectively, better drugs were produced by structural

modifications, or by total synthesis of analogues of the active principles. Even so,

herbal remedies are also enjoying revival as modern drugs (Dewick, 2001).

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In Malaysia, 55.4% are still covered by tropical forest all over the country. Studies

on local Malaysian plants such as turmeric (Curcuma domestica), betel leaf (Piper

betel), pandan leaf (Panadanus odorus), asam gelugur (Garnicia atroviridis),

mengkudu (Morinda citrifolia), pegaga (Centella asiatica), ginger (Zingiber

officinale), and cassava shoot (Manihot asculenta) have been shown to exhibit

various good biological activity (Sies, 1996).

The world has witnessed intense research on Artocarpus genus which is distributed

in South East Asia including Malaysia. Artocarpus species are large evergreen trees

that have edible fruits. They are use for furniture and building materials due to their

strong and durable dark-colored wood (Jayasinghe et al., 2006; Hakim et al., 2005). The

fruits, roots, buds and leaves are also widely used as traditional medicine against

various diseases such as malaria fever, liver cirrhosis, hypertension, and diabetes.

Regarding the chemical constituent, many chalcones, flavanoids, xanthones and

triterpenes have been reported from various Artocarpus species. Recent

investigations show that the flavanoids that present in both lower and higher plants

posses antioxidant, anticancer, antiplatelet, anti-inflammatory effects and protects

the body from mutagens such as smoke and other pollutants (Radwan et al., 2009).

Plants are potential sources of natural antioxidant. They produce various

antioxidative compounds to counteract reactive oxygen species (ROS) in order to

survive. ROS include free radicals such as hydroxyl radicals (OH·) and superoxide

anion radicals (O2-

), and non free-radicals such as H2O2 and single hydrogen are

various forms of activated oxygen. Studies have shown that ROS can cause aging,

cancer and many other diseases as they can induce some oxidative damage to

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biomolecules such as lipids, nucleic acids, proteins and carbohydrates (Choi et al.,

2006; Fareidoon et al., 2007). Several studies had been conducted to estimate the

relationship between phenolic compounds and antioxidant activity. The phenolic

compound effect as antioxidant has been demonstrated in many systems through in

vitro studies as in human low density lipoprotein and liposomes (Katalinic et al.,

2004).

Antimicrobial resistance is a global concern since it is now recognized that resistant

microorganisms are prevalent in both the inpatient and outpatient population. The

world currently face antibiotic-resistant microbes such as Mycibacterium

tuberculosis, Staphyloccus aureus, and Streptococcus pneumonia that quickly evolve

pathways around singular antimicrobial agents. Plants already demonstrated the

sensible strategy of using multiple biologically active chemicals to outsmart the

adaptable microbial pests (Blondeau et al., 1999). It is shown that green tea

(Camellia sinesis) extract is effective in the prevention of dental cavity because of

the antibacterial activity of the flavour compounds and the antiplaque activity of the

polyphenols extracted from the plant (Cseke et al., 2006).

Although several new antimicrobial drugs have recently been introduced and

additional ones are forthcoming, experience suggests that, as new drugs become

widely deployed, resistance to these agents will emerge and spread as well.

Successful control of antibiotic resistance will require both the continued

development of new drugs and the judicious use of our current arsenal of antibiotics

(Blondeau et al., 2000).

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Taxol is a unique taxane diterpene amide that possesses antitumor and antiluekemic

properties. Until recently, taxol could not be synthesized, and the most economical

taxol was from Pacific yew (Taxus brevifolia). Thus, to obtain this anticancer drug,

large areas of Pacific yew forests in United States were destroyed.

Flavonoids extracted from several plants display antimutagenic and anticancer

activity through modulation of cell-signalling pathways related to cell growth and

proliferation. Thus, recent years the world has witness rapid bioactivity research on

flavonoid for new and alternative compound that can be used as cure (Cseke et al.,

2006).

1.2 Problem Statement

Several constituents including chalcones, flavanoids, xanthones and triterpenes

isolated from Moraceae plants were reported to have biological activities such as

antifungal, antibacterial, antimalarial and cytotoxic activities. Some of the

compounds isolated are particularly important and relevant for many applications

such as prevention of cancer or stroke (Hakim, 2010). Demand for natural

ingredients which are reported to be safe and health-promoting property is increasing

due to the awareness of consumers on health diet (Raskin et al., 2002). Hence, the

past few decades have witnessed intense research devoted to the antioxidant,

cytotoxic, antimicrobial and anti-inflammatory properties of phenolic compounds

found in Artocarpus species (Jagtap et al., 2010). Thus, bioassay activities of

Artocarpus elasticus are of interest.

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1.3 Objectives and Scope of Study

The objective of this study is to extract and isolate the compounds from two different

parts of Artocarpus elasticus, which were collected from Gunung Nuang, Selangor

and Sarawak, respectively. Further, biological activity of crude extracts and the

compounds will be tested using DPPH, MTT and disc diffusion assays. The scope of

the study is the extraction of the plants was done by cold percolation method using

solvent of increasing polarity (hexane, chloroform/ethyl acetate, and methanol) for

three days and three times each. The pure compounds were isolated using various

chromatography techniques such as thin layer chromatography (TLC), gravity

column chromatographic, vacuum liquid chromatography (VLC) and chromatotron.

The structures of the isolated compounds will be determined spectroscopically by

interpreting various spectral data including NMR, MS, IR, and UV and through

comparison with data of related compounds in the literature. The biological activity

of the crude extracts and pure compounds were tested using the DPPH, MTT and

disc diffusion method.

1.4 Significance of Study

To best of our knowledge, phytochemical studies on leaves of the plant have not

been done before. However, previous studies reported the isolation of flavonoid

derivatives and their interesting biological activities from bark of A. elasticus. The

chemical deviation and biological activities of the isolated compounds were

determined.

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