CHEMICAL CONSTITUENTS and BIOACTIVITY STUDIES of Artocarpus Fulvicortex F

CHEMICAL CONSTITUENTS AND BIOACTIVITY STUDIES OF Artocarpus fulvicortex F. M. JARRET, Artocarpus integer var. silvestris CORNER AND Artocarpus rigidus BLUME

MASURI KAMA BINTI KAMARUDDIN SHAH

UNIVERSITI TEKNOLOGI MALAYSIA CHEMICAL CONSTITUENTS AND BIOACTIVITY STUDIES OF Artocarpus fulvicortex F. M. JARRET, Artocarpus integer var. silvestris CORNER AND Artocarpus rigidus BLUME

MASURI KAMA BINTI KAMARUDDIN SHAH

A thesis submitted in fulfilment of the requirements for the award of the degree of Doctor of Philosophy (Chemistry)

Faculty of Science Universiti Teknologi Malaysia

MARCH 2019 iii

This is for you, my supermom Maziah, for your endless supports and prayers.

To my beloved dad Kamaruddin Shah and my brother Firuz Shah how I wish you could read this. I miss both o f you so much

To all my sisters that always there fo r me no matter how emotionally unstable I am at the time. Thanks fo r having me!

To all my friends that have been waiting for me to officially be “Dr. Surie” Even though it is sounds impossible initially but finally I am almost there!

To whom who is always be the driven force fo r me to finish this thesis This is fo r you iv

ACKNOWLEDGEMENT

Praise to our Almighty God for giving endless blessings throughout this academic programme. I would like to express my deepest appreciation to Prof. Dr. Hasnah Mohd. Sirat for her willingness to support of my research and writing, for her constant encouragement, for her valuable sharing of knowledge, positivity and guidance to help me fulfil the requirement in this PhD course. It was a great honour to finish this work under her supervision. A special thanks to my supervisor, Assoc. Prof. Dr. Shajarahtunnur Jamil for her care and supports.

Through sweat and tears, yet by my own desire to complete this research and it has been wonderful as I gain unconditional love and support from many people especially my family and friends. I want to take this opportunity to express deep sense of gratitude to my family and friends for the greatest moral supports along my PhD’s journey.

My appreciation also extends to all academic staffs of Department of Chemistry, Faculty of Science and postgraduate students for the helps and contribution to ease this study. Lastly, I would like to thanks to MyBrain15, Ministry of Higher Education for the funding throughout this doctor of philosophy research programme and make my dream come through. v

PREFACE

This thesis is the result of my work carried out in the Department of Chemistry, Universiti Teknologi Malaysia between September 2011 and September 2015 under supervision of Prof. Dr. Hasnah Mohd Sirat and Prof. Madya Dr. Shajarahtunnur Jamil. Part of my work described in this thesis have been reported in the following publications.

1 Shah, M. K. K., Sirat, H. M., and Jamil, S. (2016). Cholinesterase inhibitors from heartwood of Artocarpus fulvicortex F. M. Jarret (Moraceae). Jurnal Teknologi. 78(6), 185-189.

2 Shah, M. K. K., Sirat, H. M., Jamil, S., and Jalil, J. (2016). Flavonoids from the bark of Artocarpus integer var. silvestris and their anti-inflammatory properties. Natural Product Communications. 11(9), 1275-1278. vi

ABSTRACT

Investigations on the chemical constituents of A. fulvicortex, A. integer var. silvestris and A. rigidus have been carried out. All the extracts were obtained from maceration method by successive extractions using petroleum ether, dichloromethane, ethyl acetate and methanol as solvents. A total of sixteen compounds were successfully isolated; nine from A. fulvicortex, five from A. integer var. silvestris and two from A. rigidus. Purification of the extract from heartwood of A. fulvicortex led to the isolation of catechin, oxyresveratrol, lupeol-3-acetate and fridelin. 5-Hydroxy-(6:7,3':4')-di(2,2- dimethylpyrano)flavone, carpachromene, norartoarpetin, cycloartocarpesin and fridelin were isolated from the leaves ofA. fulvicortex. Purification on the barks extract of A. integer var. silvestris afforded one new pyranoflavone class named as methoxycyclocommunol along with four known flavonoids, heteroflavanone A, artonin F, cudraflavone C and cyclocommunol. Recrystallisation of a fraction from the ethyl acetate barks extract of A. rigidus gave artonin E while purification of the dichloromethane extract yielded cyclorigidol. All the pure compounds except cycloartocarpesin and cyclocommunol were tested for their anti-inflammatory properties using radioimmunoassay method on human whole blood. Compounds that inhibited prostaglandin E2 (PGE2) production greater than 55% were tested in serial concentration to determine the IC50 values. Cudraflavone C exhibited the strongest inhibition toward vasoactive PGE2 with IC50 value of 0.03 ^g/mL which is higher than positive control, indomethacin that gave IC50 value of 0.06 ^g/mL. Catechin and oxyresveratrol showed significant values of inhibition against butyrylcholinesterase enzyme in dose dependent manner. The IC50 values of catechin and oxyresveratrol are 25.0 mM and 3.13 mM, respectively. vii

ABSTRAK

Penyelidikan mengenai komposisi kimia ke atas A. fulvicortex, A. integer var. silvestris dan A. rigidus telah dijalankan. Semua ekstrak diperoleh dengan kaedah rendaman secara berturutan menggunakan petroleum eter, diklorometana, etil asetat dan metanol sebagai pelarut. Sejumlah enam belas sebatian kimia telah berjaya diasingkan iaitu sembilan daripada A. fulvicortex, lima daripada A. integer var. silvestris dan dua daripada A. rigidus. Penulenan ekstrak kulit batang A. fulvicortex telah memberikan katekin, oksiresveratrol, lupeol-3-asetat dan fridelin. 5-Hidroksi- (6:7,3':4')-di(2,2-dimetilpirano)flavon, karpakromena, norartokarpetin, sikloartokarpesin dan fridelin telah diasingkan daripada ekstrak daun A. fulvicortex. Penulenan ke atas ekstrak batang A. integer var. silvestris telah menghasilkan satu kelas piranoflavon baharu dinamakan sebagai metoksisiklokomunol dan empat flavonoid sedia ada iaitu heteroflavanon A, artonin F, kudraflavon C dan siklokomunol. Penghabluran semula fraksi daripada ekstrak etil asetat batang A. rigidus memberikan artonin E manakala penulenan ekstrak diklorometana memberikan siklorigidol. Semua sebatian tulen kecuali sikloartokarpesin dan siklokomunol telah dikaji sifat anti-inflamasi menggunakan kaedah radioimunocerakinan ke atas darah manusia. Sebatian yang merencatkan penghasilan prostaglandin E2 (PGE2) melebihi 55% telah diuji menggunakan kepekatan bersiri untuk menentukan nilai IC50. Kudraflavon C mempamerkan rencatan tertinggi ke atas vasoaktif PGE2 dengan nilai IC50 0.03 ^g/mL melebihi nilai IC50 0.06 ^g/mL kawalan positif indometasin. Katekin dan oksiresveratrol menunjukkan nilai perencatan yang signifikan terhadap enzim butirilkolinesterase dengan kebergantungan kepada dos. Nilai IC50 katekin dan oksiresveratrol masing-masing ialah 25.0 mM dan 3.13 mM. viii

TABLE OF CONTENT

CHAPTER TITLE PAGE

DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv PREFACE v ABSTRACT vi ABSTRAK vii TABLE OF CONTENTS viii LIST OF TABLES xii LIST OF SCHEMES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xvii LIST OF APPENDICES xx

1 INTRODUCTION 1.1 General Information 1 1.2 Problem Statements 3 1.3 Objectives 3 1.4 Scope of Study 4 1.5 Significance of Study 4

2 LITERATURE REVIEW 2.1 Botanical description of the plant 5 2.1.1 The Moraceae family 5 2.1.2 Artocarpus genus 6 2.2 Review on Recent Phytochemicals Study of 7 Artocarpus species 2.2.1 Flavonoids 8 ix

2.2.1.1 Prenylated Flavone s 11 2.2.1.2 Pyranoflavones 19 2.2.1.3 Flavanones 20 2.2.1.4 Dihydrobenzoxanthones 22 2.2.2 Terpenes 23 2.2.3 Stilbenes 27 2.3 Bioactivities of Artocarpus species 30 2.3.2 Anti-inflammatory 31 2.3.3 Anti-cholinesterase 37

3 PHYTOCHEMICALS OF ARTOCARPUS FULVICORTEXF.M. JARRET, ARTOCARPUS INTEGER VAR SILVESTRIS CORNER AND ARTOCARPUS RIGIDUS BLUME 3.1 Phytochemicals of the Leaves of Artocarpus 39 fulvicortex F. M. Jarrett 3.1.1 Friedelin (86) 40 3.1.2 5-Hydroxy-(6:7,3':4')-di(2,2- 42 dimethylpyrano)flavone (42) 3.1.3 Carpachromene (43) 45 3.1.4 Cycloartocarpesin (44) 48 3.1.5 Norartocarpetin (119) 51 3.2 Phytochemicals of the Heartwoods of Artocarpus 53 fulvicortex F. M. Jarrett 3.2.1 Lupeol-3-acetate (88) 54 3.2.2 Fridelin (86) 55 3.2.3 P-Sitosterol (87) 56 3.2.4 Oxyresveratrol (99) 58 3.2.5 Catechin (70) 60 3.3 Phytochemicals of the Barks of Artocarpus 63 integer var. silvestris Corner 3.3.1 Heteroflavanone A (120) 63 3.3.2 Artonin F (29) 66 3.3.3 Methoxycyclocommunol (121) 69 3.3.4 Cudraflavone C (14) 82 3.3.5 Cyclocommunol (54) 84 x

3.4 Phytochemicals of the Barks of Artocarpus 87 rigidus BLUME 3.4.1 Cyclorigidol (60) 87 3.4.2 Artonin E (113) 90

4 BIOACTIVITIES OF THE PHYTOCHEMICAL COMPOUNDS FROM ARTOCARPUS FULVICORTEX F.M JARRET, ARTOCARPUS INTEGER VAR SILVESTRIS CORNER AND ARTOCARPUS RIGIDUS BLUME 4.1 Introduction 93 4.2 Prostaglandin Inhibition using 93 Radioimmunoassay Techniques 4.3 15-Lipoxygenase (15-LOX) Inhibition using 98 Cayman Screening Kit 4.4 Anti-cholinesterase activity 100

5 RESEARCH METHODOLOGY 5.1 General Experimental Procedures 103 Plant materials5.2 104 5.3 Isolation of Chemical Constituents from the 104 Leaves of A. fulvicortex JARRET 5.3.1 Friedelin (86) 104 5.3.2 5-Hydroxy-(6:7,3':4')-di(2,2- 105 dimethylpyrano)flavone (42) 5.3.3 Carpachromene (43) 106 5.3.4 Cycloartocarpesin (44) 106 5.3.5 Norartocarpetin (119) 107 5.4 Isolation of Chemical Constituents from the 107 Heartwood of A. fulvicortex JARRET 5.4.1 Lupeol-3-acetate (88) 107 5.4.2 Fridelin (86) 108 5.4.3 P-Sitosterol (87) 108 5.4.4 Oxyresveratrol (99) 109 5.4.5 Catechin (70) 109 5.5 Isolation of Chemical Constituents from the bark 110 of A. integer var. silvestris Corner xi

5.5.1 Heteroflavanone A (120) 110 5.5.2 Artonin F (29) 111 5.5.3 Methoxycyclocommunol (121) 111 5.5.4 Cudraflavone C (14) 112 5.5.5 Cyclocommunol (54) 112 5.6 Isolation of Chemical Constituents from the bark 113 of A. rigidus BLUME 5.6.1 Cyclorigidol (60) 113 5.6.2 Artonin E (113) 114 5.7 Anti-cholinesterase assays 115 5.7.1 Chemicals 115 5.7.2 TLC bioautographic AChE assays 115 5.7.3 Microplate test 116 5.8 Lipoxygenases Inhibition Assay 116 5.8.1 Lipoxygenase Inhibitory Activity using 116 Cayman Screening Kit 5.9 Pro staglandim Inhibition 117 5.9.1 Chemicals and reagents 117 5.9.2 Preparation of chemicals and reagents 117 5.9.3 Plasma blood preparation 118

5.9.4 Radioimmunoassay for Prostaglandin E2 118 (PGE2)

6 CONCLUSIONS AND RECOMMENDATIONS 6.1 Phytochemical Studies 120 6.2 Bioactivity Studies 121 6.3 Recommendations 121

REFERENCES 122 APPENDICES 133 xii

LIST OF TABLES

TABLE NO. TITLE PAGE 3.1 The NMR Spectral Data of (86) (400 MHz, CDCb) 42 and Fridelin Isolated from Eugenia chlorantha (600 MHz, CDCl3) [73] 3.2 The NMR Spectral Data of (42) (400 MHz, CDCb) 45 and 5-hydroxy-(6:7, 3':4')-di(2,2- dimethylpyrano)flavone Isolated from A. fulvicortex

(400 MHz, CDCl3) [7] 3.3 The NMR Spectral Data of (43) (400 MHz, CDCb) 48

3.4 The NMR Spectral Data of (44) (400 MHz, Acetone- 50 dtf) and Cycloartocarpesin Isolated from A. heterophyllus (300 MHz, DMSO-d6) [79] 3.5 The NMR Spectral Data of (119) (400 MHz, CDCb) 52

3.6 The NMR Spectral Data of (88) and Lupeol-3-acetate 55 Isolated from Phyllanthus reticulatus (400 MHz, CDCb) [81] 3.7 The NMR Spectral Data of (87) and P-sitosterol 57 Isolated from Rubus suavissimus (600 MHz, CDCb) [82] 3.8 The NMR Spectral Data of (99) (400 MHz, CDCb) 59 and Oxyresveratrol Isolated from Artocarpus lakoocha [84] 3.9 The NMR Spectral Data of (70) (400 MHz, Acetone- 62 d6) and Catechin (200 MHz, MeOH-d^) [85] xiii

3.10 The NMR Spectral Data of (120) (400 MHz, CDCb) 65 and Heteroflavanone A Isolated from Artocarpus heterophylus [87] 3.11 The NMR Spectral Data of (29) (400 MHz, Acetone- 69 d6) and Artonin F Isolated from Artocarpus communis [90] 3.12 1H NMR (400 MHz) and 13C NMR (100 MHz) of 72 Compound (121) 3.13 1H NMR (400 MHz) and 13C NMR (100 MHz) of 84 Compound (14) 3.14 The NMR Spectral Data of (60) (400 MHz, Acetone- 89 d6) and Cyclorigidol Isolated from Artocarpus rigida (300 MHz, CDCb) [33] 3.15 NMR Spectral Data for Artonin E (113) (400 MHz, 91 CDCl3)

4.1 IC50 values of active compounds from A. integer var. 96

silvestris on PGE2 inhibition in human blood. 4.2 Detection limit of crude extracts and isolated 101 compounds measured by TLC assay xiv

LIST OF SCHEMES

SCHEME NO. TITLE PAGE 3.1 The EIMS Fragmentation Pattern of Compound (42) 44

3.2 The EIMS Fragmentation Pattern of Compound (43) 47

3.3 Fragmentation pattern of Compound (44) 50

3.4 The EIMS Fragmentation Pattern of Compound (70) 61

3.5 The EIMS Fragmentation Pattern of Compound (120) 66

3.6 Fragmentation pattern of Compound (29) 67

3.7 Fragmentation Pattern of Compound (121) 71

3.8 Fragmentation pattern of Compound (14) 82

3.9 Fragmentation pattern of Compound (54) 86

3.10 Fragmentation pattern of Compound (113) 92

4.1 Ellman’s Method 100 xv

LIST OF FIGURES

FIGURE NO. TITLE PAGE 2.1 The Moraceae Family 6

2.2 Biosynthetic pathways of secondary metabolites [14] 7

2.3 C6-C3-C6 phenyl-benzopyran backbone 8

2.4 Biosynthetic Pathways of Flavonoid and Stilbenoids 9 [16]. 2.5 Biogenetic relationship of various classes of 10 flavonoids [15]. 2.6 Biosynthetic Pathways of Terpenoids [14] 24

2.7 Structure of Costunolide 33

2.8 Arachidonic pathways in nociception [64] 36

2.9 Common substitution patterns for prostaglandins (PG) 36

2.10 Structure of PGE2 37

2.11 Role of oxidative stress (cyclooxygenase and lip- 38 oxygenase) and acetylcholineesterase in neuro generation [72] 3.1 Isolated Compounds from the Crude Extracts of the 40 Leaves of A. fulvicortex F. M. Jarrett. 3.2 Isolated Compounds from the Crude Extracts of the 53 Heartwoods of A. fulvicortex F.M. Jarrett. 3.3 Isolated Compounds from the Crude Extracts of the 63 Barks of A. integer var. silvestris 3.4 UV Spectra for Methoxycyclocommunol (121), a) 73

NaOH analysis, b) NaOAc/ H3BO3 analysis and c) AlCb/HCl analysis xvi

3.5 IR Spectrum of Methoxycyclocommunol (121) 74

3.6 MS Spectrum of Methoxycyclocommunol (121) 75

3.7 1H NMR Spectrum of Methoxycyclocommunol (121) 76

3.8 HMBC Spectrum of Methoxycyclocommunol (121) 77

3.9 COSY Spectrum of Methoxycyclocommunol (121) 78

3.10 13C NMR Spectrum of Methoxycyclocommunol (121) 79

3.11 DEPT Spectra of Methoxycyclocommunol (121) 80

3.12 HMQC Spectrum of Methoxycyclocommunol (121) 81

3.13 Isolated Compounds from the Crude Extracts of the 87 Barks of A. rigidus 4.1 Percentage inhibition of isolated compounds towards 95

prostaglandin 2 (PGE2) 4.2 Comparison Cholineasterase Inhibition of Catechin (85) 102 and Oxyresveratrol (133) with Galanthamine (180) xvii

LIST OF ABBREVIATIONS

a Alpha 1D 1 dimensional 2D 2 dimensional Abs Absorbance AChe Acetylcholinesterase p Beta br Broad BuChe Butyrylcholinesterase 13C Carbon-13 cm centimeter 5 Chemical shift CHCl3 Choroform CC Column Chromatography COSY Correlation Spectroscopy J Coupling constant COX Cyclooxygenase DEPT Distortionless Enhancement by Polarization Transfer Distortionless Enhancement by Polarization Transfer Including DEPTQ the Detection of Quaternary Nuclei d doublet dd doublet of doublets ddd doublet of doublets of doublets EI Electron Impact ELISA Enzyme Linked Immunosorbent Assay EtOAc Ethyl acetate EDTA Ethylene Diamine Triacetic Acid FTIR Fourier Transform Infrared xviii

Y Gamma

IC50 Half maximal inhibitory concentration Hz Hertz HMBC Heteronuclear Multiple Bond Correlation HMQC Heteronuclear Multiple Quantum Coherence HSQC Heteronuclear Single Quantum Coherence IR Infrared X lamda LPS Lipopolysaccharide LOX Lipoxygenase LSC Liquid Scintillation Counter L Liter MS Mass Spectrometry m/z Mass to change ratio MHz megahertz m.p Melting point MeOH Methanol ^ micro mm milimeter mg milligram mL Millilitre M molar M+ Molecular ion m multiplet nm Nanometer NMR Nuclear Magnetic Resonance ppm Part per million PE Petroleum ether PG Prostaglandin 1H Proton QSAR Quantitative Structure Activity Relationship q quartet RIA Radioimmunoassay xix

ROS Reactive Oxygen Species R/ Retention factor RDA Retro-Diels-Alder s singlet SD Standard deviation TLC Thin Layer Chromatography t triplet UV Ultraviolet VLC Vacuum Liquid Chromatography xx

LIST OF APPENDICES

APPENDIX TITLE PAGE 1 MS Spectrum of Fridelin (86) 133 2 IR Spectrum of Fridelin (86) 134 3 13C NMR Spectrum of Fridelin (86) 135 4 DEPT Spectra of Fridelin (86) 136 5 1H NMR Spectrum of Fridelin (86) 137

6 COSY Spectrum of Fridelin (86) 138 7 HMQC Spectrum of Fridelin (86) 139 8 HMBC Spectrum of Fridelin (86) 140

9 IR Spectrum of 5-Hydroxy-(6:7,3':4')-di(2,2- 141 dimethylpyrano)flavone (42) 10 1H NMR Spectrum of 5-Hydroxy-(6:7,3':4')- 142 di(2,2-dimethylpyrano)flavone (42) 11 1H NMR spectrum of 5-Hydroxy-(6:7, 3':4')- 143 di(2,2-dimethylpyrano)flavone (42) - expansion 12 COSY Spectrum of 5-Hydroxy-(6:7,3':4')-di(2,2- 144 dimethylpyrano)flavone (42) 13 13C NMR spectrum of 5-Hydroxy-(6:7,3':4')- 145 di(2,2-dimethylpyrano)flavone (42) 14 DEPT Spectra of 5-Hydroxy-(6:7,3':4')-di(2,2- 146 dimethylpyrano)flavone (42) 15 HMQC Spectrum of 5-Hydroxy-(6:7, 3':4')- 147 di(2,2-dimethylpyrano)flavone (42) 16 MS Spectrum of 5-Hydroxy-(6:7,3':4')-di(2,2- 148 dimethylpyrano)flavone (42) 17 HMBC Spectrum of 5-Hydroxy-(6:7,3':4')- 149 di(2,2-dimethylpyrano)flavone (42) 18 UV of spectrum of Carpachromene (43) 150 19 IR Spectrum of Carpachromene (43) 151 20 1H NMR Spectrum of Carpachromene (43) 152 21 COSY Spectrum of Carpachromene (43) 153 22 13C NMR Spectrum of Carpachromene (43) 154 23 DEPT Spectra of Carpachromene (43) 155 24 HMQC Spectrum of Carpachromene (43) 156 25 HMBC spectra of Carpachromene (43) 157 26 MS Spectrum of Carpachromene (43) 158 27 IR Spectrum of Cycloartocarpesin (44) 159 28 UV Spectrum of Cycloartocarpesin (44) 160 29 1H NMR Spectrum of Cycloartocarpesin (44) 161 30 COSY Spectrum of Cycloartocarpesin (44) 162 31 13C NMR spectrum of Cycloartocarpesin (44) 163 32 DEPT Spectra of Cycloartocarpesin (44) 164 33 HMQC Spectrum of Cycloartocarpesin (44) 165 34 HMBC Spectrum of Cycloartocarpesin (44) 166 35 MS Spectrum of Cycloartocarpesin (44) 167 36 MS Spectrum of Norartocarpetin (119) 168 37 IR Spectrum of Norartocarpetin (119) 169 38 1H NMR of Norartocarpetin (119) 170 39 COSY of Norartocarpetin (119) 171 40 13C NMR of Norartocarpetin (119) 172 41 HMQC of Norartocarpetin (119) 173 42 HMBC of Norartocarpetin (119) 174 43 MS Spectrum of Lupeol-3-acetate (88) 175 44 1H NMR Spectrum of Lupeol-3-acetate (88) 176 45 13C NMR Lupeol-3-acetate (88) 177 46 DEPT Spectra of Lupeol-3-acetate (88) 178 47 COSY of Lupeol-3- acetate (88) 179 48 HMQC of Lupeol-3-acetate (88) 180 49 HMBC of Lupeol-3-acetate (88) 181 50 MS Spectrum of P-Sitosterol (87) 182 51 IR Spectrum of P-Sitosterol (87) 183 52 1H NMR Spectrum of P-Sitosterol (87) 184 53 1H NMR Spectrum of P-Sitosterol (87) - 185 expansion 54 COSY Spectrum of P-Sitosterol (87) 186 55 13C NMR Spectrum of P-Sitosterol (87) 187 56 13C NMR Spectrum of P-Sitosterol (87)- 188 expansion 57 DEPT Spectra of P-Sitosterol (87) 189 58 HMQC of P-Sitosterol (87) 190 59 HMBC of P-Sitosterol (87) 191 60 IR Spectrum of Oxyresveratrol (99) 192 61 MS Spectrum of Oxyresveratrol (99) 193 62 1H NMR Spectrum of Oxyresveratrol (99) 194 63 13C NMR Spectrum of Oxyresveratrol (99) 195 64 HMBC Spectrum of Oxyresveratrol (99) 196 65 DEPT Spectra of Oxyresveratrol (99) 197 66 COSY Spectrum of Oxyresveratrol (99) 198 67 HMQC Spectrum of Oxyresveratrol (99) 199 68 MS Spectrum of Catechin (70) 200 69 IR Spectrum of Catechin (70) 201 70 1H NMR Spectrum of Catechin (70) 202 71 1H NMR Spectrum of Catechin (70) - expansion 203 72 13C NMR Spectrum of Catechin (70) 204 73 DEPT Spectra of Catechin (70) 205 74 COSY Spectrum of Catechin (70) 206 75 HMQC Spectrum of Catechin (70) 207 76 HMBC Spectrum of Catechin (70) 208 77 UV Spectrum of Catechin (70) 209 78 IR Spectrum of Heteroflavanone A (120) 210 79 1H NMR Spectrum of Heteroflavanone A (120) 211 80 13C NMR Spectrum of Heteroflavanone A (120) 212 81 DEPT Spectra of Heteroflavanone A (120) 213 82 HMQC Spectrum of Heteroflavanone A (120) 214 83 COSY Spectrum of Heteroflavanone A (120) 215 84 HMBC Spectrum of Heteroflavanone A (120) 216 85 MS Spectrum of Heteroflavanone A (120) 217 86 MS Spectrum of Artonin F (29) 218 87 IR Spectrum of Artonin F (29) 219 88 1H NMR Spectrum of Artonin F (29) 220 89 13C NMR Spectrum of Artonin F (29) 221 90 DEPT Spectra of Artonin F (29) 222 91 HMBC Spectrum of Artonin F (29) 223 92 COSY Spectrum of Artonin F (29) 224 93 HMQC Spectrum of Artonin F (29) 225 94 UV Spectrum of Artonin F (29) 226 95 UV Spectrum of Artonin F (29) a) NaOH 227

analysis, b) NaOAc/ H3BO3 analysis and c) AlCb/HCl analysis 96 UV Spectrum of Cudraflavone C (14) 228 97 IR Spectrum of Cudraflavone C (14) 229 98 MS Spectrum of Cudraflavone C (14) 230 99 1H NMR Spectrum of Cudraflavone C (14) 231 100 1H NMR Spectrum of Cudraflavone C (14) - 232 expansion 101 13C NMR Spectrum of Cudraflavone C (14) 233 102 13C NMR Spectrum of Cudraflavone C (14) - 234 expansion 103 DEPTQ Spectra of Cudraflavone C (14) 235 104 COSY Spectrum of Cudraflavone C (14) 236 105 HSQC Spectrum of Cudraflavone C (14) - 237 expansion 106 HMBC Spectrum of Cudraflavone C (14) 238 107 HMBC Spectrum of Cudraflavone C (14) - 239 expansion 108 IR Spectrum of Cyclocommunol (54) 240 109 1H NMR Spectrum of Cyclocommunol (54) 241 110 COSY Spectrum of Cyclocommunol (14) 242 111 UV Spectrum of Cyclocommunol (54) 243 112 13C NMR Spectrum for Cyclocommunol (14) 244 113 DEPTQ Spectra for Cyclocommunol (54) 245 114 HSQC Spectrum of Cyclocommunol (54) 246 115 HMBC Spectrum of Cyclocommunol (54) 247 116 MS Spectrum of Cyclocommunol (54) 248 117 IR Spectrum of Cyclorigidol (60) 249 118 MS Spectrum of Cyclorigidol (60) 250 119 13C NMR Spectrum of Cyclorigidol (60) 251 120 DEPT Spectra of Cyclorigidol (60) 252 121 1H NMR Spectrum of Cyclorigidol 253 122 1H NMR Spectrum of Cyclorigidol (60) - 254 expansion 123 COSY Spectrum of Cyclorigidol (60) 255 124 HMBC Spectrum of Cyclorigidol (60) 256 125 HMQC Spectrum of Cyclorigidol (60) 257 126 UV Spectrum of Cyclorigidol (60) 258 127 IR Spectrum of Artonin E (113) 259 128 1H NMR Spectrum of Artonin E (113) 260 129 13C NMR Spectrum of Artonin E (113) 261 130 DEPT Spectra of Artonin E (113) 262 131 COSY Spectrum of Artonin E (113) 263 132 HMQC Spectrum of Artonin E (113) 264 133 HMBC Spectrum of Artonin E (113) 265 134 UV Spectrum of Artonin E (113) 266 135 MS Spectrum of Artonin E (113) 267 CHAPTER 1

INTRODUCTION

1.1 General Information

Medicinal plants are generally known as “Chemical Goldmines” as they contain natural bioactive chemicals that could give benefits to their users by acting as tools for maintaining overall health and vitality [1]. The bioactive chemicals which are acceptable to human and animal systems also work as the secondary metabolites within the plants. Like the human beings, plants are complete organisms that adapted and survived by developing their own natural defences against their enemies [2].

The natural production of the precious bioactive chemicals by medicinal plants creates the intention of many scientists to study it thoroughly. The study of plant is called ethnopharmacology or frequently known as ethno-medicine [3]. Over 250,000 plants species on earth, more than 80,000 are potential to become the herbal medicine. Approximately, 5000 species are extensively used as phytomedicine or the practice of using any parts of the plant and were studied in details [1, 2].

Generally, natural occurring compounds can be divided into three broad categories. First category is related to primary metabolites in which the compounds play important roles in metabolism and cells production. Another two categories are high molecular weight polymeric materials and secondary metabolites. The most attractive part to be discovered is the secondary metabolites because they can be only characterised in limited range of species [4]. Our nature which is extremely complex 2 and more challenging enhanced the scientists to reveal one by one of the special properties of the secondary metabolites.

Initially, the scientists and doctors began to make a connection between chemistry and diseases by focusing on phytochemical studies of secondary metabolites that may lead to the discovery of new drug candidates. The phytochemicals can be used unmodified as drugs, as precursor for the partial synthesis of drugs or as total synthesis of the new drugs [5]. As the medicinal field developed, many sophisticated and powerful weapons were invented to help people against unpredictable diseases, example the radiotherapy, chemotherapy, steroidal painkillers and antibiotics [2]. More or less the inventions could give bad effects towards the consumers.

As the result, many people prefer traditional alternative and complementary medicines especially herbal medicine, as it is safe for human consumption to boost the immune systems. Nowadays, the research approaches are focusing on the development of herbal medicines as the new pharmaceutical drugs. This creates high demand for novel bioactive compounds from medicinal plants for various purposes [6].

However, even though the utilisation of natural ingredients in product formulations is a popular trend amongst the manufacturers, the terms conventional drugs and herbal medicine usually are misunderstood. Although many of the prescriptions and over-the-counter drugs consumed today are originally come from the plant, conventional drugs are synthetic chemicals which must be approved by the Food and Drug Administration (FDA) for their specific uses and to provide a desired response when it is consumed.

In the other words, these pharmaceutical products are based on the single isolated compound of the plants. Meanwhile, herbal medications are taken from the natural chemicals within a plant either the extract is taken in its original form, combined with other herbal extracts, or in its purified form. This is due to plants comprise of myriad active components which work together to develop the medicinal actions [2]. 3

The phytochemical study of plant generally is the preliminary study to identify the active compounds that may contribute to certain specific properties such as anti microbial, anti-oxidant, anti-inflammatory, anti-cancer and many more. In this research, Artocarpus fulvicortex F. M. Jarret, A.integer var. silvestris Corner, and A. rigidus Blume belong to Moraceae family were chosen as the samples.

1.2 Problem Statements

Besides the appreciable importance as a source of edible fruit, Artocarpus species have been reported to have abundance of fascinating and variety of chemical constituents. Some of the metabolites possess remarkable bioactivities that might be considered as the potential medicinal drugs in the future. There is a great opportunity and need to study further on this genus and identify the bioactive compounds from selected three species which are A. fulvicortex, A.integer var. silvestris and A. rigidus.

Phytochemical study on first species, A. fulvicortex collected from different locality with the previous study was performed on two parts, leaves and heartwood [7]. The second species, A. integer var. silvestris which was believed to possess interesting secondary metabolites as this species considered as underutilised species and the study on its main variety revealed the isolation of abundance phenolic isolates [8]. Since there are no publication regarding the phytochemicals and bioactivities on A. rigidus collected from Malaysia it was chosen to be investigated in this research, since different locality could effects the production of the secondary metabolites.

1.3 Objectives

The objectives of the research are: 1. To extract, isolate and identify the phytochemicals of A. fulvicortex, A.integer var. silvestris and A. rigidus. 4

2. To elucidate the structure of isolated compounds using spectroscopic technique including infrared, nuclear magnetic resonance, ultraviolet spectroscopies and mass spectrometry. 3. To screen and evaluate the ability of the phytochemicals as well as the crude extracts as anti-inflammatory agents and enzyme inhibitors.

1.4 Scope of Study

Studies were carried out to identify the phytochemicals from three Artocarpus species and their biological activities. The crude extracts were obtained by cold extraction method. Then, the purification was carried out by successive column chromatography after being fractionated by vacuum liquid chromatographic technique. The bioactivity studies of pure compounds focused on anti-inflammatory activity using several assays in expression of enzyme inhibitory such as cyclooxygenase, lipoxygenase and cholinesterase.

1.5 Significance of Study

The importance of this research is to isolate the bioactive compounds that may lead to the production of new nature-based products. Begins with the isolation and identification of the chemical constituents in the Artocarpus species, then chemistry of the compounds can be studied for the evaluation of their bioactivities. This research may reveal the secondary metabolites that might be useful for human being. Those secondary metabolites can also be synthesised chemically or biologically. The bioactive constituents obtained will be used as the basis for product formulation in the agro-based industries. This research can also contribute to the database of secondary metabolites of the plants. REFERENCES

1. Joy, P. P., Thomas, J., Mathew, S., and Skaria, B. P. (1998). Zingiberaceous Medicinal and Aromatic Plants. India: Aromatic and Medicinal Plants Research Station. 2. Connors, M. S., and Altshuler, L. (2009). Guide to Herbal Remedies. USA: Advantage Quest Publications. 1-17. 3. Ahmed, Z., and Dar, A. (2008). Advances in Natural Products Importance in Health and Economy. Delhi: Daya Publishing House. 213-218. 4. Hanson, J. R. (2003). Natural Products the Secondary Metabolites. Cambridge, UK: The Royal Society of Chemistry. 1-30. 5. Jantan, I. (2004). Medicinal Plant Research in Malaysia: Scientific Interests and Advances. Jurnal Sains Kesihatan Malaysia. 2 (2), 27-46. 6. Mailina, J., Nor Azah, M. A., Mastura, M., Mazura, P., Fadzureena, J., and Siti Humeirah, A. G. Utilisation of Phytoextracts and Essential Oils from Malaysian Zingiberaceae and Annonaceae for The Development of Cosmeceutical Products. Forest Research Institute Malaysia (FRIM). 7. Jamil, S., Taher, M., Sirat, H. M. and Othman, N. A. 2012. Flavonoids and Triterpenes from the leaves of Artocarpus fulvicortex. Nat Prod Commun. 7(12). 1587-1588. 8. Wang, M., and Zerega, N. (2014). Origin and diversity of an underutilized tropical fruit tree, Cempedak (Artocarpus integer, Moraceae), and its putative wild relative (Artocarpus integer var. silvestris). Oral Session: Botany 2014. Boise, ID, USA. 9. Jones, A. M. P., Ragone, D., Tavana, N. G., Bernotas, D. W., Murch, S. J. (2011). Beyond the Bounty: Breadfruit (Artocarpus altilis) for food security and novel foods in the 21st Century. Ethnobot Res Appl. 9, 129-150. 10. Jagtap, U. B., Bapat, V. A. (2010). Artocarpus: A review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol. 129. 142-166. 123

11. Whitmore, T. C. (1978) Tree Flora o f Malaya (Volume 3). Longman Malaysia Sdn. Bhd. Kuala Lumpur. 12. Soepadmo, E., and Saw, L. G. (2000) Tree Flora o f Sabah and Sarawak (Volume 3). Ampang Press Sdn. Bhd. Kuala Lumpur. 13. Wyatt-Smith, J. (1999) Pocket Check List o f Timber Trees (3rd revision). Forest Research Institute Malaysia. Malaysia. 14. Ribera, A. E., and Zuniga, G. (2012). Induced plant secondary metabolites for phytopatogenic fungi control: a review. J. Soil Sci. Plant Nutr. 2 (4), 893-911. 15. Rao, V. (2012). Phytochemicals - A Global Perspective o f Their Role in Nutrition and Health. InTech. Croatia. 16. Kodan, A., Kuroda, H., and Sakai, F. (2002). A stilbene synthase from Japanese red pine (Pinus densiflora): Implications for phytoalexin accumulation and down- regulation of flavonoid biosynthesis. Proc Natl Acad Sci. 99(5). 3335-3339. 17. Hakim, A. (2010). Diversity of secondary metabolites from Genus Artocarpus (Moraceae). Nusantara Bioscie. 2 (3), 146-156. 18. Shen, G., Huhman, D., Lei, X., Snyder, J., Sumner, L. W., and Dixon, R. A. (2012). Characterization of an Isoflavonoid-Specific Prenyltransferase from Lupinus albus. Plant Physiol. 159, 70-80. 19. Venkataraman, K. (1972). Wood Phenolics in the Chemotaxonomy of the Moraceae. Phytochemistry. 11, 1571-1586. 20. Botta, B., Vitalib, A., Menendezc, P., Misitia, D., and Monachea, G. D. (2005). Prenylated Flavonoids: Pharmacology and Biotechnology. Curr Med Chem. 12, 713-739. 21. Lathiff, S. M. A., Jemaon, N., Abdullah, S. A. and Jamil, S. (2015). Flavonoids from Artocarpus anisophyllus and their Bioactivities. Nat Pro Commun. 10(3), 393-396. 22. Shamaun, S. S., Rahmani, M., Hashim, N. H., Ismail, H. B. M., Sukari, M. A., Lian. G. E. C., and Go, R. (2010). Prenylated flavones from Artocarpus altilis. J Nat Med. 64, 478-481. 23. Zong-Ping, Z., Yang, X., Chuan, Q., Shuang, Z., Xiaohong, G., Yingying, L., Guobin, X., Mingfu, W., and Jie, C. (2014). Characterization of Antiproliferative Activity Constituents from Artocarpus heterophyllus. J Agric Food Chem. 62, 5519-5527. 124

24. Wen-Chun, L., Cheng-Wei, T., Chun-Ching, L., Feng-Lin, Y., and Horng-Huey, K. (2013). Prenylated flavonoids from Artocarpus altilis: Antioxidant activities and inhibitory effects on melanin production. Phytochemistry. 8, 78-88. 25. Nguyen, N. T., Nguyen, M. H. K., Nguyen, H. X., Bui, N. K. N., and Nguyen, M. T. T. (2012). Tyrosinase Inhibitors from the Wood of Artocarpus heterophyllus. J Nat Prod. 75, 1951-1955. 26. Arung, E. T., Shimizu, K., Tanaka, H., Kondo, R. (2010). 3-Prenyl luteolin, a new prenylated flavone with melanin biosynthesis inhibitory activity from wood of Artocarpus heterophyllus. Fitoterapia. 81, 640-643. 27. Dej-adisai, S., Meechai, I., Puripattanavong, J., and Kummee, S. (2013). Antityrosinase and antimicrobial activities from Thai medicinal plants. Arch Pharm Res. 37(4), 473-483. 28. Minakawa, T., Toume, K., Arai, M. A., Koyano, T., Kowithayakorn, T., and Ishibashi, M. (2013). Prenylflavonoids isolated from Artocarpus champeden with TRAIL-resistance overcoming activity. Phytochemistry. 96. 299-304. 29. Panthong, K., Tohdee, K., Hutadilok-Towatana, N., Voravuthikunchaib, S. P., and Chusri, S. (2013) Prenylated Flavone from Roots of a Hybrid between Artocarpus heterophyllus and Artocarpus integer and its Biological Activities. J Braz Chem Soc. 24(10), 1656-1661. 30. Mei-Hua, Y., Ting, Z., Gui-Rui, Y., Hong-Xun, Y., He-Yao, W., and Ai-Jun, H. (2012). New Isoprenylated Flavones and Stilbene Derivative from Artocarpus hypargyreus. Chem Biodivers. 9(2), 394-402. 31. Pethakamsetty, L., Ganapaty, S. and Bharathi, K. M. (2013). Phytochemical and Biological Examination of the Root Extract of Artocarpus Hirsuta, Lam. Int J Bioassays. 2(4), 735-738. 32. Bourjot, M., Apel1, C., Martin, M., Grellier, P., Nguyen, V. H., Gueritte, F., and Litaudon, M. (2010). Antiplasmodial, Antitrypanosomal, and Cytotoxic Activities of Prenylated Flavonoids Isolated from the Stem Bark of Artocarpus styracifolius. Planta Med. 76, 1600-1604. 33. Ren, Y., Kardono, L. B. C., Riswan, S., Chai, H., Farnsworth, N. R., Soejarto, D. D., Blanco, E. J. C., and Kinghorn, A. D. (2010) Cytotoxic and NF-KB Inhibitory Constituents of Artocarpus rigida. J Nat Prod. 73, 949-955. 125

34. Maneechai, S., De-Eknamkul, W., Umehara, K., Noguchi, H., and Likhitwitayawuid, K. (2012). Flavonoid and stilbenoid production in callus cultures of Artocarpus lakoocha. Phytochemistry. 81, 42-49. 35. Chin-Lin, H., Ming-Huan, S., Jer-An, L., Gow-Chin, Y., and Song-Chwan, F. (2011). Cytotoxic effects of geranyl flavonoid derivatives from the fruit of Artocarpus communis in SK-Hep-1 human hepatocellular carcinoma cells. Food Chem. 127(1), 127-134 36. Mai, N. T. T., Hai, N. X., Phu, D. H., Trong, P. N. H., and Nhan, N. T. (2012). Three new geranyl aurones from the leaves of Artocarpus altilis. Phytochem Lett. 5(3), 647-650. 37. Gang, R., Heng-Yun, X., Zhi-Cheng, H., Rong-Hua, L., Zhi-Wang, Z., Hui-Lian, H., Feng, S., and Ming, Y. (2013). A new isoprenylated flavone from the root bark of Artocarpus styracifolius. Biochem Syst Ecol. 46, 97-100. 38. Huihui, T., Ping, W., Lidong, L., and Xiaoyi, W. (2011). Stilbenes and flavonoids from Artocarpus nitidus subsp. lingnanensis. Fitoterapia. 82, 662-665. 39. Jer-An, L., Song-Chwan, F., Chi-Hao, W., Shang-Ming, H., and Gow-Chin, Y. (2011). Anti-inflammatory Effect of the 5,7,40-Trihydroxy-6-geranylflavanone Isolated from the Fruit of Artocarpus communis in S100B-Induced Human Monocytes. JAgric Food Chem. 59, 105-111. 40. Hongjie, Z., Minghua, Q., Yegao, C., Jinxiong, C., Yun, S., Cuifang, W. and Harry, H. S. F. (2011). Plant Terpenes. Cock, E. I. (Ed.) The Encyclopedia o f Life Support Systems. India: EManuscript Publishing Services. 41. Ramli, F., Rahmani, M., Kassim, N. K., Hashim, N. H., Sukari, M. A., Akim, M. A., and Go, R. (2013). New diprenylated dihydrochalcones from leaves of Artocarpus elasticus. Phytochem Lett. 6, 582-585. 42. Gang, R., Zhi, C. H., Heng, Y. X., Jia-Bing, P., Rong-Hua, L., Hui-Lian, H., and Feng, S. (2013). Chemical constituents from the fruiting branches of Artocarpus nanchuanensis endemic to China. Biochem Syst Ecol. 51, 98-100. 43. Iverson, C. D., Zahid, S., Yimeng, L., Shoqafi, A. H., Ata, A., and Samarasekera, R. (2010). Glutathione S-transferase inhibitory, free radical scavenging, and anti- leishmanial activities of chemical constituents of Artocarpus nobilis and Matricaria chamomilla. Phytochem Lett. 3, 207-211. 44. Gorham, J. (1980). The stilbenoids. In progress in phytochemistry. 6, 203-252. 126

45. Namdaung, U., Athipornchai, A., Khammee, T., Kuno, M., and Suksamrarn, S. (2018). 2-Arylbenzofurans from Artocarpus lakoocha and methyl ether analogs with potent cholinesterase inhibitory activity. Eur. J. Med. Chem. 143.1301 1311. 46. Zhang, P. Z., Gua, J., and Zhang, G. L. (2015). Novel stilbenes from Artocarpus nanchuanensis. J Asian Nat Prod Res. 17(3), 217-223. 47. Roberts, E., Magis, A., Ortiz, J. O., Baumeister, W., and Luthey-Schulten, Z. (2011). Noise Contributions in an Inducible Genetic Switch: A Whole-Cell Simulation PLoS Comput Biol. 7(3). 48. Tunev, S. S., Hastey, C. J., Hodzic, E., Feng, S., Barthold, S. W., and Baumgarth, N. (2011). Lymphoadenopathy during Lyme Borreliosis is caused by Spirochete Migration- Induced Specific B Cell Activation. PLoSPathog. 7(5). 49. Kinnings, S. L., Liu, N., Buchmeier, N., Tonge, P. J., Xie, L., and Bourne, P. E. (2009). Drug Discovery Using Chemical Systems Biology: Repositioning the Safe Medicine Comtan to Treat Multi-Drug and Extensively Drug Resistant Tuberculosis. PLoS Comput Biol. 5(7). 50. Efferth, T., and Koch, E. (2011). Complex Interactions between Phytochemicals. The Multi-Target Therapeutic Concept of Phytotherapy Home. Curr Drug Targets. 12(1), 122-132. 51. Pereira, V. J., and Kaplan, M. A. C. (2013). The High Bioactivity of Artocarpus - An Exotic Genus. Floresta e Ambiente. 20(1), 1-15. 52. Rayburn, E. R., Ezell, S. J., and Zhang, R. (2009). Anti-Inflammatory Agents for Cancer Therapy. Mol Cell Pharmacol. 1(1), 29-43. 53. Chung, M. I., Ko, H. H., Yen, M. H., Lin, C. N., Yang, S. Z., Tsao, L. T., and Wang, J. P. (2000). Artocarpol A, a Novel Constituent with Potent Anti Inflammatory Effect, Isolated from Artocarpus rigida. Helv Chim Acta. 83, 1200 1204. 54. Kuan, Y. H., Lin, R. H., Tsao, L. T., Lin, C. N., and Wang, J. P. (2005). Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways. Br. J. Pharmacol. 145, 460-468. 55. Kuan, Y. H., Lin, R. H., Lin, H. Y., Huang, L. J., Tsai, C. R., Tsao, L. T., Lin, C. N., Chang, L. C., and Wang, J. P. (2006). Activation of phosphoinositide 3-kinase 127

and Src family kinase is required for respiratory burst in rat neutrophils stimulated with artocarpol A. Biochem Pharmacol. 71, 1735-1746. 56. Su, B. N., Cuendet, M., Hawthorne, M. E., Kardono, L. B. S., Riswan, S., Fong, H. H. S., Mehta, R. G., Pezzuto, J. M., and Kinghorn, A. D. (2002). Constituents of the Bark and Twigs of Artocarpus dadah with Cyclooxygenase Inhibitory Activity. J Nat Prod. 65, 163-169. 57. Jantan, I., Yasin, Y. H. M., Jamil, S., Sirat, H., Basar, N. (2010). Effect of prenylated flavonoids and chalcones isolated from Artocarpus species on platelet aggregation in human whole blood. J Nat Med. 64, 365-369. 58. Sritularak, B., Tantituvanont, A., Chanvorachote, P., Meksawan, K., Miyamoto, T., Kohno, Y., and Likhitwitayawuid, K. (2010). Flavonoids with free radical scavenging activity and nitric oxide inhibitory effect from the stem bark of Artocarpus gomezianus. J Med Plants Res. 4(5), 387-392. 59. Han, A. R., Kang, Y. J., Windono, T., Lee, S. K., and Seo, S. K. (2006). Prenylated Flavonoids from the Heartwood of Artocarpus communis with Inhibitory Activity on Lipopolysaccharide-Induced Nitric Oxide Production. J Nat Prod. 69, 719-721. 60. Wei, B. L., Weng, J. R., Chiu, P.H., Hung, C. F., Wang, J. P., and Lin, C. N. (2005). Anti-inflammatory flavonoids from Artocarpus heterophyllus and Artocarpus communis. JAgric Food Chem. 53(10), 3867-3871. 61. Perez, G. (2001). Anti-Inflammatory Activity of Compounds Isolated from Plants. Sci World J. 1, 713-784. 62. Smith, H. S. (2006). Arachidonic Acid Pathways in Nociception. J Support Oncol. 4(6), 277-287. 63. Klein, D. R. (2012). Organic Chemistry. United States of America: John Wiley & Sons, Inc. 64. Rainsford, K. D. (2007). Anti-Inflammatory Drugs in the 21st Century. R. E. Harris (Ed.). Inflammation in the Pathogenesis o f Chronic Diseases (pp 3-27). United Kingdom: Springer. 65. Ricciotti E., and FitzGerald G. A. (2011). Prostaglandins and Inflammation. Arterioscler Thromb Vasc Biol. 31(5), 986-1000. 66. Jalil, J., Sabandar, C. W., Ahmat, N., Jamal, J. A., Jantan, I., Aladdin, N., Muhammad, K., Buang, F., Mohamad, H. F., and Sahidin, I. (2015). Inhibitory

Effects on Triterpenoids from Dillenia serrata (Dilleniaceae) on prostaglandin E2 128

production and quantitative HPLC analysis of its koetjapic acid and betulinic acid contents. Molecules. 20, 3206-3220. 67. Mashima, R., and Okuyama, T. (2015). The role of lipoxygenases in pathophysiology; new insights and future perspectives. Redox Biol. 6. 297-310. 68. Nassar, G.M., Morrow, J.D., Roberts, L.J., II, Lakkis, F. G., and Badr, K. F. Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes. (1994). J Biol Chem. 269, 27631-27634. 69. Suardiaz, R., Jambrina, P. G., Masgrau, L., Gonzalez-Lafont, A.,Rosta, E., and Lluch, J.M. (2016). Understanding the Mechanism of the Hydrogen Abstraction from Arachidonic Acid Catalyzed by the Human Enzyme 15-Lipoxygenase-2. A Quantum Mechanics/Molecular Mechanics Free Energy Simulation. J. Chem. Theory Comput. 12 (4). 2079-2090. 70. Bishnoi, M., Patil, C. S., Kumar, A., and Kulkarni, S. K. (2009). Relative Role of Cyclooxygenase-2 (COX-2) Inhibitors and Lipoxygenase (LOX) Inhibitors in Aging Induced Dementia and Oxidative Damage. Indo J. Chem. 9, 328-331. 71. Hussain, T., Tan, B., Yin, Y., Blachier, F., Tossou, M. C. B., and Rahu, N. (2016). Oxidative Stress and Inflammation: What Polyphenols Can Do for Us?. Oxidative Medicine and Cellular Longevity. 72. Popa-Wagner, A., Mitran, S., Sivanesan, S., Chang, E., and Buga, A. M. (2013). ROS and Brain Diseases: The Good, the Bad, and the Ugly. Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity. 73. Susidarti, R. A., Rahmani, M., Ali, A. M., Sukari, M. A., and Ismail, H. B. M. (2007). Friedelin from Kelat Merah (Eugenia chlorantha Duthie). Majalah Obat Traditional. (Volume 11). 74. Sousa, G. F., Duarte, L. P., Alcantara, A. F. C., Silva, G. D. F., Vieira-Filho, S. A., Silva, R. R., Oliveira, D. M., and Takahashi, J. A. (2012). New Triterpenes from Maytenus robusta: Structural Elucidation Based on NMR Experimental Data and Theoretical Calculations. Molecules. 17, 13439-13456. 75. Queiroga, C. L., Silva, G. F., Dias, P. C., Possenti, A., and de Carvalho, J. E. (2000). Evaluation of the antiulcerogenic activity of friedelan-3P-ol and friedelin isolated from Maytenus ilicifolia (Celastraceae). JEthnopharmacol. 72, 465-468. 76. Picker, K., Ritchie, E., and Taylor, W. C. (1976). The Chemical Constituents of Australian Flindersia Species. XXI* an Examination of the Bark and the Leaves of F. laevicarpa. Aust. J. Chem. 29. 2023-2036. 129

77. Ali, M. I., Ahmed, Z., Waffo, A. F., and Ali, M. S. (2010). Flavonoids from Erythrina vogelii (Fabaceae) of Cameroon. Nat Prod Commun. 5(6), 889-892. 78. Ersam, T., Achmad, S. A., Ghisalberti, E. L., Hakim, E. H., Makmur, L., and Syah, Y. M. (2002). A New Isoprenylated Chalcone, Artoindonesianin J, from root and tree bark of Artocarpus bracteata. J Chem Res. 4, 186-187. 79. Zheng, Z. P., Cheng, K. W., To, J. T. K., Li, H., and Wang, M. (2008). Isolation of tyrosinase inhibitors from Artocarpus heterophyllus and use of its extract as antibrowning agent. Mol. Nutr. Food Res. 52, 1530-1538. 80. Soekamto, N. H., Nafie, N. L., Mandey, F. W., and Garson, M. (2009). Norartocarpetin, Flavone Derivative from Leaves of Artocarpus fretessi. Indo. J. Chem. 9 (2), 328-331. 81. Jamal, A. K., Yaacob, W. A., and Din, L. (2008). A Chemical Study on Phyllanthus reticulatus. JPhys Sci. 19(2), 45-50. 82. Chaturvedula, V. S. P., and Prakash, I. (2012). Isolation of Stigmasterol and P- Sitosterol from the dichloromethane extract of Rubus suavissimus. Int Curr Pharm J. 1(9), 239-242. 83. Pierre, L. L., and Moses, M. N. (2015). Isolation and Characterisation of Stigmasterol and P-Sitosterol from Odontonema Strictum (Acanthaceae). JInnov Pharm Biol Sci. 2(1), 88-95. 84. Povichit, N., Phrutivorapongkul, A., Suttajit, M., and Leelapornpisid, P. (2010). Antiglycation and antioxidant activities of oxyresveratrol extracted from the heartwood of Artocarpus lakoocha Roxb. Maejo Int. J. Sci. Technol. 4(03), 454 461. 85. He, S. H., Seung, H. J., and Yong, S. K. (2007). Cytotoxic constituents from the stem of Rhododendron mucronulatum. Kor. J. Pharmacogn. 38(3), 227-233. 86. Atomssa, T., and Gholap, A. V. (2015). Characterization and determination of catechins in green tea leaves using UV-visible spectrometer. J. Eng. Technol. Res. 7(1), 22-31. 87. Chai, M. L., and Chun, N. L. (1993). Two 2’,4’,6’-Trioxygenated Flavanones From Artocarpus heterophyllus. Phytochemistry. 33(4), 909-911. 88. Hakim, E. H., Aripin, A., Achmad, S. A., Aimi, N., Kitajima, M., Makmur, L., Mujahidin, D., Syah, Y. M., and Takayama, H. (2001). Artoindonesianin-E suatu senyawa baru turunan flavanon dari tumbuhan Artocarpus champeden. Proc. ITB. 33(3), 69-73. 130

89. Kweon, M. H., Kwak, J. H., Ra, K. S., Sung, H. C., and Yang, H. C. (1996). Structural Characterization of a Flavanoid Compound Scavenging Superoxide Anion Radical Isolated from Capsella bursa-pastoris. J. Biochem. Mol. Biol. 29(5), 423-428. 90. Hano, Y., Yamagami, Y., Kobayashi, M., Isohata, R., and Nomura, T. (1990). Artonins E and F, Two New Prenylflavones from the Bark of Artocarpus communis Forst. Heterocycles. 31(5). 879- 882 91. Chun, N. L., and Wen, L. S. (1992). Pyranoflavonoids from Artocarpus communis. Phytochemistry. 31(8). 2922-2924. 92. Hano, Y., Matsumoto, Y., Shinohara, K., Sun, J. Y., and Nomura, T. (1990). Cudraflavones C and D, Two New Prenylflavones from the Root Bark of Cudrania Tricuspidata (Carr.) Bur. Heterocycles. 31(7), 1339-1344. 93. Wang, L., Wang, H. Q., and Chen, R. Y. (2007). Studies on chemical constituents from bark of Morus nigra. Zhongguo Zhong Yao Za Zhi. 32(23), 2497-2499 94. Jayasinghe, U. L. B., Samarakoon, T. B., Kumarihamy, B. M. M., Hara, N., and Fujimoto, Y. (2008). Four New Prenylated Flavonoids and Xanthones from the Root Bark of Artocarpus nobilis. Fitoterapia. 79, 37-41. 95. Suhartati, T., Achmad, S. A., Aimi, N., Hakim, E. H., Kitajima, M., Takayama, H., and Takeya, K. (2001). Artoindonesianin L, A New Prenylated Flavone with Cytotoxic Activity from Artocarpus rotunda. Fitoterapia. 72. 912-918. 96. Darwish, I. A. (2006). Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances. Int J Biomed Sci. 2(3), 217-235. 97. Fowler, E., and Cheng, N. (1983). Comparison of Radioimmunoassay and Elisa Methods for Detection of Antibodies to Chromatin Components. Journal o f Immunological Methods. 62, 297-303. 98. Grange, R.D., Thompson J. P., and Lambert, D. G. (2014) Radioimmunoassay, enzyme and non-enzyme-based Immunoassays. Br J Anaesth. 112(2), 213-216. 99. Middleton, E., J., Kandaswami, C., and Theoharides, T. C. (2000). The Effects of Plant Flavonoids on Mammalian Cells: Implications for Inflammation, Heart Disease and Cancer. Pharmacol. Rev. 52(4), 673-751. 100. Lago, J. H. G., Toledo-Arruda, A. C., Mernak, M., Barrosa, K. H., Martins, M. A., Tiberio, I. F. L. C., and Prado, C. M. (2014). Structure-Activity Association of Flavonoids in Lung Diseases. Molecules. 19, 3570-3595. 131

101. Rosenkranz, H. S., and Thampatty, B. P. (2012). SAR: Flavonoids and COX-2 Inhibition. Oncol Res. 13(12), 529-535. 102. Saadawi. S., Jalil, J., Jasamai, M., and Jantan, I. (2012). Inhibitory Effects on Acetylmelodorinol, Chrysin and Polycarpol from Mitrella kentia on

Prostaglandin E2 and Thromboxane B2 Production and Platelet Activating Factor Receptor Binding. Molecules. 17. 4824-4835. 103. Blanco, F. J., Guitian, R., Moreno, J., de Toro, F. J., and Galdo, F. (1999). Effect of anti-inflammatory drugs on COX-1 and COX-2 activity in human articular chondrocytes. J Rheumatol. 26. 1366-1373. 104. Sautebin, L., Ialenti, A., Ianaro, A., and Di Rosa, M. (1998). Relationship between Nitric Oxide and Prostaglandins in Carrageenan Pleurisy. Biochem Pharmacol. 55(7), 1113-1117. 105. Dobrian, A. D., Lieb, D. C., Coleb, B. K., Taylor-Fishwick, D. A., Chakrabarti, S. K., and Nadler, J. L. (2011). Functional and pathological roles of the 12- and 15-Lipoxygenases. Prog Lipid Res. 50(1), 115-131. 106. Xu, Z. G., Li, S. L., Lanting, L., Kim, Y. S., Shanmugam, N., Reddy, M. A., and Natarajan, R. (2006). Relationship between 12/15-lipoxygenase and COX-2 in mesangial cells: potential role in diabetic nephropathy. Kidney Int. 69, 512-519. 107. Sadik, C. D., Sies, H., and Schewe, T. (2003). Inhibition of 15-lipoxygenases by flavonoids: structure-activity relations and mode of action. Biochem Pharmacol. 65, 773-781. 108. Noguchi, N., Yamashita, H., Hamahara, J., Nakamura, A., Kuhn, H., and Niki, E. (2002).The Specificity of Lipoxygenase-Catalyzed Lipid Peroxidation and the Effects of Radical-Scavenging Antioxidants. Biol Chem. 383, 619-626. 109. Ellman, G. L., Courtney, K. D., Andres, V. Jr., and Featherstone, R. M. A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity. Biochem Pharmacol. 7, 88-95. 110. Das, S., Laskar, M. A., Sarker, S. D., Choudhury, M. D., Choudhury, P. R., Mitra, A., Jamil, S., Lathiff, S. M. A., Abdullah, S. A., Basar, N., Nahar, L., and Talukdar, A. D. (2017) Prediction of anti-Alzheimer’s activity of flavonoids targeting acetylcholinesterase in silico. Phytochem Anal. 28 (4). 324-331. 111. Rahman, A., Parveen, S., Khalid, A., Farooq, A., and Choudhary, M. I. (2001). Acetyl and butyrylcholinesterase-inhibiting triterpenoid alkaloids from Buxus papillosa. Phytochemistry. 58(6), 963-968. 132

112. Orhan, I., Kartal, M., Tosun, F., and Sener, B. (2007). Screening of Various Phenolic Acids and Flavonoid Derivatives for their Anticholinesterase Potential. Z. Naturforsch. 62, 829-832. 113. Rhee, I. K., Rijn, R. M., and Verpoorte, R. (2003). Qualitative Determination of False-positive Effects in the Acetylcholinesterase Assay using Thin Layer Chromatography. Phytochem. Anal. 14, 127-131. 114. Chagas-Paula, D. A. 1, Zhang, T., Costa, F. B. D., and Edrada-Ebel, R. (2015). A Metabolomic Approach to Target Compounds from the Asteraceae Family for Dual COX and LOX Inhibition. Metabolites. 5, 404-430.