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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
DAYANG ERNA ZULAIKHA BT AWANG HAMSIN
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
DAYANG ERNA ZULAIKHA BT AWANG HAMSIN
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
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
Huzwah Khaza’i, PhD
Senior Lecturer
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
Khalit Mohamad, PhD
Associate Professor
Universiti Malaya
Malaysia
(External Examiner)
NORITAH OMAR, PhD
Assoc. Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
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
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Latifah Saiful Yazan, PhD Associate Professor,
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Che Norma Mat Taib
Senior Lecturer
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Saidi Moin, PhD
Associate Professor,
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies,
Universiti Putra Malaysia
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
4.8 High performance liquid chromatography
(HPLC) fingerprint of QRF.
103
4.9 High performance liquid chromatography
(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
4.15 Microphotograph of a section showing
occasional blood vessels around gel
impregnated with VEGF and ACRH. Red
arrows indicted the blood vessels (x200).
116
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4.16 Microphotograph of a section showing
occasional blood vessels around gel
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
various concentrations.
122
4.19 COX-1 and COX-2 inhibition of ACRH in
various concentrations.
123
4.20 COX-1 and COX-2 inhibition of QRF in
various concentrations.
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
COX-2 Cyclooxygenase-2
COX-3 Cyclooxygenase-3
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-12 Interleukin-12
IL-13 Interleukin-13
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