2013 33 anti-angiogenic potential of ardisia crispa

UNIVERSITI PUTRA MALAYSIA

DAYANG ERNA ZULAIKHA AWANG HAMSIN

FPSK(m) 2013 33

ANTI-ANGIOGENIC POTENTIAL OF ARDISIA CRISPA ROOTS ETHANOLIC EXTRACT AND ITS QUINONE-RICH FRACTION IN MICE

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ANTI-ANGIOGENIC POTENTIAL OF Ardisia

crispa ROOTS ETHANOLIC EXTRACT AND ITS

QUINONE-RICH FRACTION IN MICE

DAYANG ERNA ZULAIKHA BT AWANG

HAMSIN

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2013

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ANTI-ANGIOGENIC POTENTIAL OF Ardisia crispa ROOTS ETHANOLIC

EXTRACT AND ITS QUINONE-RICH FRACTION IN MICE

By

DAYANG ERNA ZULAIKHA BT AWANG HAMSIN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

July 2013

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COPYRIGHT

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 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 fulfillment

of the requirement for the Degree of Master of Science

ANTI-ANGIOGENIC POTENTIAL OF Ardisia crispa ROOTS ETHANOLIC

EXTRACT AND ITS QUINONE-RICH FRACTION IN MICE

By


July 2013

Chair: Roslida Abd Hamid @ Abd Razak, PhD

Faculty: Medicine and Health Sciences

Angiogenesis is the process of blood vessel formation which plays a crucial role in

normal physiology, and also in the progression of various chronic diseases such as

cancer, arthritis and such. As targeting angiogenesis has become an important

strategy in the search of treatments of various debilitating ailments, it is a need-based

study to identify a natural source of anti-angiogenic agent that may halt the

progression of angiogenesis event. Ardisia crispa, locally known as “mata itik”

(Family: Myrsinaceae) has been used in traditional Malay medicine to treat various

ailments related to inflammation. Ardisia crispa roots have been shown to treat

various inflammation-related diseases in several previous studies. As angiogenesis is

strongly correlated with inflammation, the aim of the present study was to evaluate

anti-angiogenic potential of the hexane partition of Ardisia crispa roots ethanolic

extract (ACRH) and its quinone- rich fraction (QRF) on several experimental

models, namely Miles vascular permeability test, murine air pouch granuloma and

mouse sponge implantation test. Preliminary cyclooxygenase and soy lipoxygenase

inhibitory study were also conducted to elucidate the possible pathways involved.

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Preliminary phytochemical screening of ACRH indicated the abundant presence of

flavonoid, triterpene and tannin. Quinone- rich fraction (QRF) was separated from

ACRH (38.33% w/w) and further isolated to yield a compound, namely fAC-2,

indicated by a single TLC spot at Rf: 0.76. The compound was later found to be

impure, when later analysed with GC-MS. Nevertheless, fAC-2 was elucidated to

possess a major constituent of a benzoquinonoid compound (2-methoxy-6-undecyl-1,

4-benzoquinone), when compared with the standard data. Both ACRH and QRF were

also quantified using high performance liquid chromatography (HPLC). For toxicity

study, the LD50 value of ACRH was found to be 617.02 mg/kg. In Miles vascular

permeability assay, the lowest dose of both ACRH and QRF (10 mg/kg) produced

significant reduction in VEGF-induced hyperpermeability compared to vehicle

control. In murine air pouch granuloma, ACRH and QRF displayed significant and

dose-dependent angiogenic and inflammatory inhibition, in which significant

reduction of vascular index and granuloma tissue weight was observed at high dose

(100 mg/kg). ACRH and QRF were also shown to possess selective COX-2

inhibitory properties which were dose-dependent, though COX-1 inhibition was also

observed in a lower percentage. On the other hand, ACRH and QRF did not exhibit

LOX inhibitory activity. Interestingly, fAC-2 showed its selectivity towards the

inhibition of COX-2, instead of COX-1, and showed to be a moderate LOX inhibitor.

Thus, it can be concluded that Ardisia crispa roots showed potential anti-angiogenic

properties by partly mediating COX-2 activity, as shown in the in vitro screening,

and it is postulated that fAC-2 (2-methoxy-6-undercyl-1, 4-benzoquinone) displays

dual COX-2 and LOX once it is purely isolated in a large scale and tested in vivo.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

POTENSI AKAR Ardisia crispa EKSTRAK ETANOL DAN FRAKSI KAYA

KUINON SEBAGAI PERENCAT PEMBENTUKAN SALUR DARAH KE

ATAS MENCIT

Oleh


Julai 2013

Pengerusi: Roslida Abd Hamid @ Abd Razak, PhD

Fakulti: Perubatan dan Sains Kesihatan

Pembentukan salur darah merupakan satu proses yang penting dalam fisiologi

normal, dan juga penting dalam perkembangan pelbagai jenis penyakit kronik seperti

kanser, radang otot, dan sebagainya. Oleh sebab pensasaran pembentukan salur darah

semakin menjadi strategi yang penting dalam pencarian rawatan bagi pelbagai

penyakit, adalah penting untuk kajian dijalankan bagi mengenal pasti sumber

semulajadi bagi agen perencat pembentukan salur darah, yang mampu merencat

perkembangan proses yang berlaku di dalam penghasilan salur darah. Ardisia crispa

(Famili: Myrsinaceae) yang juga dikenali sebagai pokok mata itik, telah lama

digunakan dalam perubatan tradisional Melayu untuk mengubati penyakit yang

berkaitan dengan keradangan. Akar Ardisia crispa telah terbukti mampu merawat

pelbagai penyakit yang berkaitan dengan keradangan melalui beberapa kajian yang

dijalankan sebelum ini. Oleh sebab terdapat korelasi yang kuat antara keradangan

dan pembentukan salur darah, objektif penyelidikan ini dilakukan adalah untuk

mengetahui sama ada partisi heksana (ACRH) daripada ekstrak mentah akar pokok

Ardisia crispa, beserta fraksi kaya kuinon (QRF) mempunyai aktiviti merencat

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pembentukan salur darah dalam beberapa model eksperimen, termasuk ujian

ketelapan vaskular Miles, granuloma kantung udara murin dan ujian implan span

dalam mencit. Kajian awal terhadap penglibatan enzim siklooksigenase (COX) dan

lipoksigenase (LOX) juga dijalankan untuk mengenalpasti mekanisma yang mungkin

terlibat dalam perencatan formasi salur darah. Kajian awal terhadap komposisi

fitokimia ACRH menunjukkan bahawa ACRH mengandungi sebatian-sebatian

flavonoid, triterpena dan tannin yang banyak. Fraksi kaya kuinon (QRF) telah

dipisahkan daripada ACRH (38.33% w/w) dan seterusnya diasingkan untuk

menghasilkan satu sebatian utama (fAC-2) berdasarkan bintik tunggal pada plat

Kromatografi Lapisan Nipis (KLN) pada Rf:0.76. Sebatian tersebut kemudiannya

didapati tidak tulen melalui analisis Kromatografi Gas-Spektroskopi Jisim (KG-SJ).

Walaubagaimanapun sebatian benzokuinon iaitu 2-metoksi-6-undesil-1,4-

benzokuinon dibuktikan merupakan komponen utama dalam sebatian tersebut,(fAC-

2) apabila dibandingkan dengan data piawai. ACRH dan QRF juga diukur dengan

menggunakan kromatografi cecair prestasi tinggi (KCPT). Untuk kajian ketoksikan,

nilai LD50 bagi ACRH telah dikenalpasti sebagai 617.02 mg/kg. Dalam ujian

ketelapan vaskular Miles, dos terendah bagi ACRH and QRF (10 mg/kg)

menghasilkan pengurangan yang signifikan dalam ketelapan hiper yang dirangsang

oleh VEGF. Dalam granuloma kantung udara murin, ACRH and QRF menunjukkan

kebergantungan dos yang signifikan dalam perencatan formasi salur darah dan

keradangan, di mana pengurangan yang signifikan dalam indeks vaskular, dan juga

berat kasar tisu granuloma juga dapat diamati. Aktiviti perencatan formasi saluran

darah oleh ACRH and QRF telah terbukti disebabkan oleh enzim siklooksigenase-2

(COX-2) secara selektif, walaupun aktiviti enzim siklooksigenase-1 (COX-1) juga

direncat, dalam peratusan yang lebih rendah. Walaubagaimanapun, sebatian fAC-2

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telah terbukti menunjukkan sifatnya yang selektif terhadap perencatan aktiviti COX-

2, berbanding COX-1. Sebatian fAC-2 juga menunjukkan perencatan yang baik

terhadap enzim LOX soya. Kesimpulannya, akar Ardisia crispa menunjukkan

potensi sebagai perencat formasi salur darah, dengan sebahagiannya melalui

perencatan aktiviti enzim COX secara selektif, terbukti melalui ujian penyaringan in

vitro. Walaubagaimanapun, sebatian fAC-2 berpotensi untuk menjadi agen perencat

dwienzim COX-2 dan LOX setelah diasingkan dan ditulenkan melalui skala besar

dan diuji secara in vivo pada masa yang akan datang.

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ACKNOWLEDGEMENTS

Bismillahirrahmanirrahim.

I would like to sincerely thank my supervisor, Dr Roslida Abd Hamid and co-

supervisors, Dr Latifah Saiful Yazan and Dr Che Norma Mat Taib for the kind

assistance and guidance throughout my journey of completing this Master’s degree. I

am also grateful to be having such a helpful group of peoples in Pharmacology

Laboratory; many thanks to Joyce Yeong, Hamizah Sulaiman, Wan Nurul Husna

Nordin, fellow labmates, and laboratory staffs for their kind support and generous

help.

I would like to dedicate my special thanks to my family and husband, Awang Arshad

Awang Kamis for giving me endless support from the start until the completion of

my Master’s degree. Without the kind encouragement and prayers from them, I

would not be able to carry on and complete my Master’s degree successfully. I

appreciate the loving and kind support given very much.

Last but not least, I would also like to express my gratitude to Ministry of Higher

Education and UNIMAS for giving me the SLAB scholarship to further my study.

Without the opportunity given, I will not be able to taste the intense satisfaction of

completing a research and I will never experience the challenges that have made me

grow so much to become a better person I am today.

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"A path without a heart is never enjoyable. On the other hand, a path with heart is

easy— it does not make a warrior work at liking it; it makes for a joyful journey; as

long as a man follows it, he is one with it."

Carlos Castaneda

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I certify that a Thesis Examination Committee has met on 20 June 2013 to conduct

the final examination of Siti Zaleha binti Raduan on her thesis entitled “Anti-Acute

Inflammatory Effect of Hibiscus rosa-sinensis L. and Hibiscus rosa-sinensis var.

alba Flower and Leaf Ethanol Extracts and Its Mechanism of Action in Rats” in

accordance with the Universities and University Colleges Act 1971 and the

Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The

Committee recommends that the student be awarded the Master of Science

Members of the Thesis Examination Committee were as follows:

Cheah Yoke Kqueen, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Asmah Rahmat, PhD

Professor



(Internal Examiner)

Huzwah Khaza’i, PhD

Senior Lecturer



(Internal Examiner)

Khalit Mohamad, PhD

Associate Professor

Universiti Malaya

Malaysia

(External Examiner)

NORITAH OMAR, PhD

Assoc. Professor and Deputy Dean

School of Graduate Studies


Date: 16 August 2013

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

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

the Supervisory Committee were as follows:

Roslida Abd Hamid @ Abd Razak, PhD

Senior Lecturer



(Chairman)

Latifah Saiful Yazan, PhD Associate Professor,



(Member)

Che Norma Mat Taib

Senior Lecturer



(Member)

Saidi Moin, PhD

Associate Professor,



(Member)

____________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies,


Date: 16 August 2013

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

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

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

any other institution.

_____________________________

DAYANG ERNA ZULAIKHA

AWANG HAMSIN

Date: 12 July 2013

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LIST OF TABLES

Table Page

2.1 Events of angiogenesis. 10

2.2 Angiogenic activators and its functions. 14

2.3 Angiogenic inhibitors and its functions. 17

2.4 Ardisia species and its medicinal properties. 48

3.1 Colour indications and the interpretation for

different qualitative phytochemical tests.

67

3.2 Experimental design for Miles vascular

permeability test.

75

3.3 Experimental design for mouse implantation

method.

79

3.4 Experimental design for murine air pouch

granuloma.

82

3.5 Protocols involved in each wells. 86

4.1 Extracts and properties of ACRE and ACRH. 90

4.2 Qualitative phytochemical analysis of ACRH 90

4.3 Chemical characteristics of compound

resembling AC-2 (standard compound) in

rich fraction

95

4.4 Mass breakdown of a major compounds

consisted in AC-2.

97

4.5 Quantification of modified Miles assay

performed in mice after induction of vessel

permeability using VEGF in ACRH and

QRF; extracted dye contents were quantified

by measuring at 620 nm.

107

4.6 Effects of hexane fraction of Ardisia crispa

roots (ACRH) and its quinone-rich fraction

(QRF) on vascular index (VI) in murine air

pouch granuloma

110

4.7 Effects of hexane fraction of Ardisia crispa

roots (ACRH) and its quinone-rich fraction

(QRF) on granuloma tissue dry weight (g) in

murine air pouch granuloma

112

4.8 Mean vascular density (MVD) following

various treatments

114

4.9 IC50 of different treatments in cyclooxygenase

inhibitory assay

119

4.10 Percentage of inhibitions of COX-1 and

COX-2 following treatments.

120

4.11 IC50 of different treatments in soy LOX

inhibitory assay.

126

4.12 The percentage of inhibition of soy

lipoxygenase following different treatment

127

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LIST OF FIGURES

Figure Page

2.1 Sequential steps of angiogenesis. 9

2.2 Conversion of arachidonic acids into it

different metabolites by COXs and LOXs and

clinically used classes of inhibitors.

23

2.3 Products of arachidonic metabolism via COX-

1 or COX-2 activity.

25

2.4 The examples of classical NSAIDs. (a)

Aspirin; (b) Ibuprofen; and (c) Indomethacin.

36

2.5 The examples of COX-2 inhibitors. (a)

Celecoxib; (b) Rofecoxib

39

2.6 Selective 5-lipoxygenase (5-LOX) inhibitor

(Zileuton).

41

2.7 Chemical structure of licofelone (ML-3000). 43

2.8 Chemical structures of Flavocoxid; (a)

baicalin; (b) catechin.

44

2.9 Ardisia crispa plant. 51

2.10 Illustration of Ardisia crispa (Synonym:

Ardisia crenata Sims 2). (1) roots systems;

(2) flowering branchlet; (3) fruiting branch.

52

2.11 Chemical structures of AC7-1 (2-methoxy-6-

tridecyl-1,4-benzoquinone).

55

2.12 Chemical structures of; (a) Ardisiacrispin A;

(b) Ardisiacrispin B.

56

2.13 Chemical structure of FR900359. 57

3.1 A summarised figure of the assays conducted

to evaluate anti-angiogenesis activity of

Ardisia crispa roots extract and rich fraction.

60

3.2 The flow chart of Ardisia crispa roots

extraction, fractionation and isolation.

64

3.3 Depiction of TLC plate. (a), (b) and (c) were

the initial spots while (i) and (ii) were spots

developed

70

3.4 Flow chart of LD50 determination of ACRH

crude extract.

74

3.5 Flow chart of vascular permeability test of

ACRH and its rich fraction.

78

3.6 Treatments in murine air pouch granuloma. 84

4.1 TLC-guided chromatographic isolation (AC-2

Rf: 0.76).

92

4.2 Comparison of Rf of benzoquinonoid

compound in AC-2 with similar compound in

94

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fraction 3 (QRF), and fAC-2(Benzoquinonoid

rich fraction). Spots were resolved using

chloroform as developing solvent.

4.3 fAC-2 obtained following refractionation of

QRF, in dark yellow colour.

94

4.4 Gas chromatogram of AC-2 (reference)

separated using gas chromatography

technique. Peak 2 was found to be compatible

with 2-methoxy-6-undecyl-1, 4-benzoquinone

based on its similar molecular ion peak and

consistent mass fragmentation.

98

4.5 Spectral mass breakdown of a major

compound of AC-2 at min 39.536.

99

4.6 Spectral mass breakdown of a major

compound in AC-2 at min 42.992.

100

4.7 High performance liquid chromatography

(HPLC) fingerprint of ACRH.

102


(HPLC) fingerprint of QRF.

103


(HPLC) fingerprint for AC-2.

104

4.10 Graphical method for determination of LD50. 105

4.11 Percentage of VEGF-induced vascular

permeability suppression (%) following pre-

treatment of ACRH and QRF at three

different doses (10, 30, and 100 mg/kg),

compared to positive control (indomethacin

10 mg/kg). The treatment groups were

compared with indomethacin as vehicle

control was used in the calculation of

vascular permeability suppression (%). The

formula was [(ODvehicle control-

ODtreatment)/ODvehicle control] x 100%.

108

4.12 Microphotograph of a section showing blood

vessels around gel impregnated with VEGF

alone. Red arrow indicated the blood vessels

(x200).

115

4.13 Microphotograph of a section showing blood

vessels around gel impregnated with VEGF

and DMSO (vehicle). Red arrows indicated

the blood vessels (x200).

115

4.14 Microphotograph of a section showing

occasional blood vessels around gel

impregnated with VEGF and licofelone. Red

arrows indicated the blood vessels (x200).

116



impregnated with VEGF and ACRH. Red

arrows indicted the blood vessels (x200).

116

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impregnated with VEGF and QRF. Red

arrows indicated the blood vessels (x200).

117

4.17 COX-1 and COX-2 inhibition of Aspirin in

various concentrations.

121

4.18 COX-1 and COX-2 inhibition of Celecoxib in


122

4.19 COX-1 and COX-2 inhibition of ACRH in


123

4.20 COX-1 and COX-2 inhibition of QRF in


124

4.21 COX-1 and COX-2 inhibition of fraction AC-

2 (fAC-2) in various concentrations.

125

.

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LIST OF ABBREVIATIONS

5-HETE 5-Hydroxyeicosatetraenoic acid

5-HPETE 5-hydroperoxyeicosatetraenoic acid

5-LOX 5-lipoxygenase

ACR Ardisia crispa roots

ACRH Ardisia crispa roots hexane extract

ACUC Animal Care and Use Committee

Ang1 Angiopoietin 1

ANOVA Analysis of Variance

bFGF Basic Fibroblast Growth Factor

COX Cyclooxygenase

COX-1 Cyclooxygenase-1



COXIB Cyclooxygenase inhibitor

CFR Code of Federal Regulation

DAD Diode array detector

DMSO Dimethyl sulfoxide

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

FAP Familial Adenomatous Polyposis

FCA Freund’s Complete Adjuvant

FDA Food and Drug Administration

FGF Fibroblast Growth Factor

FLAP 5-Lipoxygenase Activating Protein

HGF Hepatocyte growth factor

HPLC High Performance Liquid Chromatography

IFN-α Interferon- α

IFN-β Interferon- β

IFN-γ Interferon- γ

IL-1 Interleukin-1

IL-10 Interleukin-10



IL-1β Interleukin-1β

IL-4 Interleukin-4

IL-6 Interleukin-6

IL-8 Interleukin-8

LAK Lymphocyte-activated Killer

LIF Leukemia inhibitory factor

LOX Lipoxygenase

LSD Least Significant Difference

LT Leukotriene

LTA4 Leukotriene A4

LTB4 Leukotriene B4

LTC4 Leukotriene C4

LTD4 Leukotriene D4

MCP-1 Monocyte Chemotactic Protein-1

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MMP Matrix Metalloproteinase

MMP-2 Matrix Metalloproteinase-2

MMP-8 Matrix Metalloproteinase-8

MVD Mean Vascular Density

NOS Nitric Oxide Species

NRP-1 Neuropilin-1

NSAID Non-Steroidal Anti-Inflammatory Drug

oxLDL Oxidized Low Density Lipoprotein

p.o. per os (orally)

PAI-1 Plasminogen Activator Inhibitor-1

PBS Phosphate Buffer Saline

PDGF Platelet-derived Growth Factor

PECAM Platelet Endothelial Cell Adhesion Molecule

PGD2 Prostaglandin D2

PGE2 Prostaglandin E2

PGF2 Prostaglandin F2

PGF2α Prostaglandin F2α

PGG2 Prostaglandin G2

PGI2 Prostaglandin I2

pO2 partial Oxygen

QRF Quinone Rich Fraction

Rf Retention factor

Rt Retention time

SEM Standard Error of Mean

SPARC Secreted Protein Acidic and Rich in Cysteine

TAE Tris-acetate-EDTA

TGF-h Transforming Growth Factor-h

TIMP Tissue Inhibitor of Metalloproteinases

TLC Thin Layer Chromatography

TMPD Tetramethylphenylenediamine

TNF-α Tumor Necrosis Factor- α

TXA2 Thromboxane-A2

USC United States Code

VE-cadherin Vascular Endothelial-cadherin

VEGF Vascular Endothelial Growth Factor

VEGFR Vascular Endothelial Growth Factor Receptor

VEGI Vascular Endothelial Growth Inhibitor

VI Vascular Index

WHO World Health Organization

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TABLE OF CONTENTS

Page

CHAPTER

1 INTRODUCTION

1.1 Background

1.2 Objectives

1.3 Hypothesis

1

1

4

5

2 LITERATURE REVIEW

2.1 Angiogenesis

2.1.1 Introduction

2.1.2 Angiogenic cascades

2.1.3 Angiogenic mediators

2.1.4 Therapeutic intervention

2.2 Inflammation

2.2.1 Inflammation and angiogenesis

2.2.2 Mechanism of inflammation

2.2.3 Arachidonic acid metabolizing enzymes

2.2.4 Inflammatory mediators

2.2.5 Therapeutic intervention

2.3. Medicinal food

2.3.1. Introduction

2.3.2 Genus Ardisia

2.3.3 Ardisia crispa

6

6

6

7

11

18

20

20

22

22

30

34

45

46

50

3 METHODOLOGY

3.1 Materials, apparatus and equipments

3.1.1 Materials

3.1.2 Apparatus and equipments

3.2 Phytochemistry of Ardisia crispa roots

3.2.1 Flow chart

3.2.2 Collection, extraction and fractionation of

Ardisia crispa roots

3.2.3 Phytochemical analysis of ACRH

3.2.4 Isolation of rich fraction and bioactive

compound using column chromatography

60

61

61

62

64

65

66

68

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vii

APPROVAL ix

DECLARATION x

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF ABBREVIATIONS xv

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3.2.5 Thin layer chromatography (TLC)

3.2.6 High performance liquid chromatography

(HPLC)

3.2.7 Gas chromatography-mass spectrometry (GC-

MS)

3.3 In vivo studies

3.3.1 Subject

3.3.2 Location of experiment

3.3.3 LD50 determination

3.3.4 Miles vascular permeability test

3.3.5 Mouse sponge implantation method

3.3.6 Murine air pouch granuloma

3.4 In vitro studies

3.4.1 Cyclooxygenase inhibitory assay

3.4.2 Soy lipoxygenase inhibitory assay

3.5 Statistical analysis

69

71

72

73

73

73

73

75

79

82

85

85

87

88

4 RESULTS

4.1 Extraction, fractionation and isolation of rich fraction of

rich fractions and bioactive compounds in Ardisia

crispa roots

4.2 Lethal dose 50 (LD50) determination

4.3 Miles vascular permeability test

4.4 Murine air pouch granuloma

4.5 Mouse sponge implantation method

4.6 Cyclooxygenase inhibitory assay

4.7 Soy lipoxygenase inhibitory assay

89

89

105

106

109

113

118

126

5 DISCUSSION 128

6 CONCLUSION 146

REFERENCES 149

APPENDICES 183

BIODATA OF STUDENT 188

PUBLICATIONS

189

2013 33 anti-angiogenic potential of ardisia crispa

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