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ANTIOXIDANT, ANTI-ANGIOGENIC AND ANTI-
INFLAMMATORY ACTIVITIES OF
STANDARDIZED GYNURA SEGETUM LEAF
EXTRACTS
by
SEOW LAY JING
Thesis submitted in fulfillment of the requirements
for the degree of
Doctor of Philosophy
September 2014
ii
ACKNOWLEDGEMENT
This thesis is the end of my journey in obtaining my Ph.D. I am grateful for everyone
who has been there to support my journey towards the finished thesis. I’m indebted
to and grateful for the following persons.
First of all, I would like to express my sincere gratitude to my main supervisor Prof.
Dr. Mohd Zaini Asmawi for his invaluable guidance, patience and understanding,
comments and suggestions throughout the study. He has been a supportive adviser to
me throughout my graduate school career and always given me great freedom to
pursue independent work. I hope that one day I would become as good supervisor to
my students as he has been to me.
My co-supervisor, Prof. Dr. Amirin Sadikun has been always there to listen and give
advice. I am most appreciative of his guidance for invaluable lessons and the
freedom he granted to me during my work. I am most grateful for the time he spent
providing insightful critique and guidance when it was most necessary.
I would like to acknowledge the financial support given by MyBrain 15 (MyPhD)
scholarship sponsored by the Ministry of Higher Education (MOHE) Malaysia. The
given financial support takes away that sense of worry and concern away about
financial woes, without this concern, I am able to pay more attention to my studies
and do the best that I can.
iii
My deepest gratitude goes to the Dean of School of Pharmaceutical Sciences, USM
for allowing me the full use of facilities and equipment during these years. In
addition, many thanks go to the academic staffs, laboratory assistants and technicians
of the school for their kindness help, cooperation and contribution to the success of
this study.
My sincere thanks also goes to all of my friends and lab mates to support me for
giving hand in solving problem and inspiring working atmosphere during these years.
I would never forget all the chats and beautiful moments I shared with them. They
were fundamental in supporting me during these stressful and difficult moments.
Most importantly, none of this would have been possible without the love and
patience of my family. I am forever indebted to my parents for all they sacrificed for
me, and thanks also to my lovely brothers, who are so supportive in whatever I do in
my life. I know I always have my family to count on when times are rough.
Lastly, my deepest gratitude goes to all the people I have met along the way and have
contributed to the development of my research.
iv
TABLE OF CONTENTS
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iv
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xvi
LIST OF APPENDICES xx
ABSTRAK xxii
ABSTRACT xxiii
CHAPTER 1: INTRODUCTION
1.1 Background 1
1.2 Problem of Statement 5
1.3 Objectives 6
CHAPTER 2: LITERATURE REVIEWS
2.1 Description of Gynura segetum (Lour.) Merr. 7
2.2 Previous studies on Gynura segetum 8
2.3 Standardization 11
2.3.1 High performance thin layer chromatography (HPTLC) 14
2.3.2 High performance liquid chromatography (HPLC) 15
2.4 Antioxidant 17
2.4.1 The mechanism of actions of antioxidant 18
2.4.2 Methods of antioxidant activity assessment 19
v
2.4.3 Herbal medicines and antioxidant activities 22
2.5 Angiogenesis 23
2.5.1 Angiogenic factors and inhibitors 24
2.5.2 Methods of anti-angiogenic activity assessment 26
2.5.2(a) In vivo assay 26
2.5.2(b) In vitro assay 28
2.5.2(c) Ex vivo assay (organ culture) 30
2.5.3 Herbal medicines in anti-angiogenic activity 31
2.6 Inflammation 32
2.6.1 Mediators of inflammation 33
2.6.2 Mechanisms of anti-inflammatory action 34
2.6.3 Methods of anti-inflammatory activity assessment 35
2.6.3(a) In vitro assay 35
2.6.3(b) In vivo assay 36
2.6.4 Herbal medicines as a source of anti-inflammatory agent 38
CHAPTER 3: MATERIALS AND METHODS
3.1 Chemicals and solvents 39
3.2 Instruments 39
3.3 Plant materials 40
3.4 Plant extraction 40
3.5 HPTLC profiling of Gynura segetum’s leaves 41
3.5.1 Preparation of sample and standard solutions 41
3.5.2 High-performance thin layer chromatography (HPTLC) 41
analysis
3.6 Development and validation of HPLC method 42
vi
3.6.1 Preparation of sample and standard solutions 42
3.6.2 Chromatographic conditions 42
3.6.3 Method of validation 43
3.6.3(a) Linearity, limit of detection and quantification 43
3.6.3(b) Accuracy and precision 44
3.6.3(c) Recovery 44
3.6.4 Quantification of seneciphylline and senecionine in 44
Gynura segetum’s extract
3.7 Antioxidant studies 45
3.7.1 Chemicals/solvents and apparatus 45
3.7.2 Sample preparation 45
3.7.2(a) Liquid-liquid partition 45
3.7.2(b) Resin column chromatography 46
3.7.3 Determination of total phenolic content 46
3.7.4 Determination of total flavonoid content 47
3.7.5 Evaluation of antioxidant activity of Gynura segetum’s leaves 48
3.7.5(a) DPPH (1,1-diphenyl-2-picrylhydrazyl) radical- 48
scavenging assay
3.7.5(b) Beta carotene-linoleic acid assay 49
3.7.6 Statistical analysis 50
3.8 Anti-angiogenic studies 51
3.8.1 Chemicals/solvents and apparatus 51
3.8.2 In vivo CAM assay 51
3.8.2(a) Fertilized chicken eggs 51
3.8.2(b) Sample preparation 51
3.8.2(c) Experimental design 52
vii
3.8.2(d) Image analysis 54
3.8.3 Ex vivo rat aorta ring assay 56
3.8.3(a) Experimental design 56
3.8.3(b) Statistical analysis 57
3.8.4 Cytotoxicity (MTT Cell proliferation assay) 58
3.8.4(a) Chemicals, reagent and cell cultures 58
3.8.4(b) Test samples preparation 58
3.8.4(c) MTT assay 59
3.8.5 Enzyme-linked immunosorbant assay (ELISA) assay 60
for VEGF determination
3.8.5(a) Determination of VEGF concentration 60
3.8.5(b) Statistical analysis 60
3.9 Anti-inflammatory activities 61
3.9.1 Chemicals and solvents/apparatus 61
3.9.2 In vivo HET-CAM assay 61
3.9.2(a) Fertilized chicken eggs 61
3.9.2(b) Preparation of test samples 61
3.9.2(c) Experimental design 62
3.9.2(d) Statistical analysis 62
3.9.3 Cotton pellet-induced granuloma assay 64
3.9.3(a) Animals 64
3.9.3(b) Experimental design 64
3.9.3(c) Statistical analysis 65
3.9.4 In vitro anti-inflammatory screening 67
3.9.4(a) Enzyme-linked immunosorbant assay (ELISA) for 67
pro-inflammatory cytokines
viii
3.9.4(b) Cyclooxygenase assay (COX assay) 69
3.9.4(c) Statistical analysis 70
CHAPTER 4: RESULTS AND DISCUSSIONS
4.1 Chromatographic analysis of plant extract 71
4.1.1 HPTLC analysis of Gynura segetum’s leaves 71
4.1.2 HPLC analysis of of Gynura segetum’s leaves 75
4.1.3 Validation of HPLC method 78
4.1.3(a) Linearity, limit of detection (LOD) and quantification 78
(LOQ)
4.1.3(b) Accuracy and precision 79
4.1.3(c) Recovery 79
4.1.4 Quantification of seneciphylline and senecionine in Gynura 81
segetum’s extract
4.2 Antioxidant activity 82
4.2.1 Total phenolic and flavonoid content 82
4.2.2 Evaluation of antioxidant activity of Gynura segetum’s leaves 85
4.2.2(a) DPPH (1,1-diphenyl-2-picrylhydrazyl) radical- 85
scavenging assay
4.2.2(b) Beta carotene-linoleic acid assay 88
4.2.3 Correlation between total phenolic, total flavonoid contents 90
and antioxidant assay
4.2.4 Discussions 92
4.3 Evaluation of anti-angiogenic activitiy of Gynura segetum’s leaves 97
4.3.1 In vivo CAM screening 97
4.3.2 Ex vivo rat aorta ring assay 104
4.3.3 Cytotoxicity 107
ix
4.3.4 Determination of VEGF concentration 108
4.3.5 Discussions 111
4.4 Evaluation of anti-inflammatory activity of Gynura segetum’s leaves 115
4.4.1 HET-CAM assay 115
4.4.2 Cotton pellet-induced granuloma assay 121
4.4.3 In vitro assays 123
4.4.3(a) ELISA assay for pro-inflammatory cytokines 123
4.4.3(b) Cyclooxygenase assay (COX assay) 124
4.4.4 Discussions 126
CHAPTER 5 – GENERAL DISCUSSIONS 132
CHAPTER 6 – CONCLUSIONS 135
REFERENCES 138
APPENDICES 155
LIST OF PUBLICATIONS 187
x
LIST OF TABLES
Page
Table 2.1 Examples of (A) activators and (B) inhibitors of 25
angiogenesis and their known functions
Table 2.2 Summary of important in vivo models of inflammation 37
Table 3.1 Score system for the evaluation of the anti-inflammatory 63
effect of sodium dodecyl sulfate (SDS) irritated
chorioallantoic membranes
Table 4.1 Intra-day and Inter-day precision and accuracy value of 80
seneciphylline and senecionine
Table 4.2 Recovery value for seneciphylline and senecionile 80
Table 4.3 Total phenolic and flavonoid content of various extract 83
and fractions of Gynura segetum
Table 4.4 EC50 values of DPPH scavenging and beta-carotene 87
antioxidant activity of various extracts and fractions
from Gynura segetum’s leaf
.
Table 4.5 Pearson’s correlation coefficients (r) for relationship 91
between total phenolic content, total flavonoid content
and antioxidant activities based on DPPH and beta-
carotene assays
Table 4.6 CAM assay: The percent inhibition of blood vessel on 98
CAM treated with various extracts and fractions of
Gynura segetum leaf
Table 4.7 IC50 values of various extracts and fractions of Gynura 100
segetum leaf in CAM assay
Table 4.8 Percentage inhibition of suramin and methanol extracts 105
of Gynura segetum leaf on microvessel outgrowth in rat
aortic rings
Table 4.9 Percentage inhibition of different concentration of 107
methanol extract of Gynura segetum leaf on HCT 116
colon cancer cells
Table 4.10 The inhibitory effect of methanol extracts of Gynura 109
segetum’s leaf on VEGF activity in HCT 116 colon
cancer cells
xi
Table 4.11 Average score and percent inhibition of CAM treated with 116
various extracts and fractions of Gynura segetum by
HET-CAM assay
Table 4.12 Cotton Pellet assay: Inhibition of granuloma tissue 121
formation of Indomethacin and methanol extracts of
Gynura segetum’s leaf
xii
LIST OF FIGURES
Page
Figure 1.1 Flow chart of antioxidant, anti-angiogenic and anti- 4
inflammatory activities of standardized Gynura segetum
leaf extracts
Figure 2.1 Gynura segetum (Lour.) Merr 7
Figure 2.2 Chemical structures of some isolated compounds of 9
Gynura segetum
Figure 3.1 Overview step by step of the image processing of the 55
anti-angiogenic effects of Gynura segetum’s leaf on
CAM. The original images of before (A) and after
treatments (B) were first processed. C and D represent
the cropped image in focus of the treatment area before
and after. E and F are skeletonized image of the
vasculature after color threshold adjustment before and
after treatment. G and H show the area point focusing
on the area of before and after treatment
Figure 3.2 Cotton pellet-induced granuloma assay: 66
A) The fur on abdominal area was shaved; B) Two
pouches were made; C) Cotton pellet implanted and the
pouches were stitched close
Figure 4.1 Developed TLC plates of the leaves of Gynura segetum 73
and reference standards under 254 nm (A) and UV 366
nm (B). Tracks 1 and 2 show the standards seneciphylline
(SP) and senecionine (SO); tracks 3-5 methanol extract of
the leaves of Gynura segetum
Figure 4.2 HPTLC scan track of methanol extract of the leaves of 74
Gynura segetum and reference standards seneciphylline
(SP) and senecionine (SO) at 254 nm
Figure 4.3 HPLC chromatogram of reference standards (A) and 76
methanol extract of Gynura segetum’s leaves (B)
Figure 4.4 HPLC chromatogram of methanol extract of Gynura 77
segetum’s leaves (A) and spiked methanol extract with
reference standards (B)
Figure 4.5 DPPH free radical scavenging activity of crude extracts 86
(A) and fractions of Gynura segetum (B). All values are
represented as the mean ± SD (n=3). (PE: petroleum ether
extract; CHCl3 ext: chloroform extract; MeOH: methanol
extract; CHCl3 fraction: chloroform fraction; EA fraction:
xiii
ethyl acetate fraction; BuOH fraction: n-butanol fraction;
R1:20% methanol eluate; R2:40% methanol eluate; R3:
60% methanol eluate; R4:80% methanol eluate; R5:100%
methanol eluate)
Figure 4.6 Beta-carotene antioxidant activity of crude extracts (A) 89
and fractions of Gynura segetum (B). All values are
represented as the mean ± SD (n=3). (PE: petroleum
ether extract; CHCl3 ext: chloroform extract; MeOH:
methanol extract; CHCl3 fraction: chloroform fraction;
EA fraction: ethyl acetate fraction; BuOH fraction:
n-butanol fraction; R1:20% methanol eluate; R2:40%
methanol eluate; R3:60% methanol eluate; R4:80%
methanol eluate; R5:100% methanol eluate)
Figure 4.7 Inhibition of blood vessel on CAM treated with 12.5 to 99
100 µg of crude extracts (A), fractions from liquid-liquid
partition (B) and methanolic fractions (C) of Gynura
segetum leaf compared with positive control, SU5416.
All values are represented as the mean ± SD (n=6). (PE:
petroleum ether extract; CHCl3: chloroform extract;
MeOH: Methanol extract; CHCl3 fraction: chloroform
fraction; EA fraction: ethyl acetate fraction; BuOH fraction:
n-butanol fraction; R1:20% methanol eluate; R2:40%
methanol eluate; R3:60% methanol eluate; R4:80%
methanol eluate; R5:100% methanol eluate)
Figure 4.8 Images of CAM treated with 12.5-100 µg positive control, 101
SU5416. A-D show images of CAM before treatment and
E-H represent the CAM after treatment, respectively.
Dotted circles identify the anti-angiogenic effect of SU5416
Figure 4.9 Images of CAM treated with 12.5-100 µg chloroform 101
extract of Gynura segetum leaf. A-D show images of
CAM before treatment and E-H represent the CAM
after treatment, respectively. Dotted circles identify the
anti-angiogenic effect of chloroform extract
Figure 4.10 Images of CAM treated with 12.5-100 µg methanol extract 103
of Gynura segetum leaf. A-D show images of CAM before
treatment and E-H represents the CAM after treatment,
respectively. Dotted circles identify the best anti-
angiogenic effect of methanol extract in dose-dependent
manner
Figure 4.11 Images of CAM treated with 12.5-100 µg CAM treated 103
with chloroform fraction of Gynura segetum leaf. A-D
show images of CAM before treatment and E-H represent
the CAM after treatment, respectively. Dotted circles
identify the anti-angiogenic effect of chloroform fraction
xiv
Figure 4.12 Rat aortic ring assay: 106
The effect of different concentrations 50-200 μg/mL of
methanol extract of Gynura segetum leaf and suramin
on the microvessels outgrowth in rat aorta rings. Control
rings are shown; B) suramin (100 μg/mL) as positive
control; C, D and E) Rings treated with 200, 100 and 50
μg/mL of methanol extract
Figure 4.13 Dose response curve of methanol extract of Gynura 108
segetum leaf in MTT assay on HCT 116 colon cancer cells
Figure 4.14 Inhibitions on VEGF activity by different concentrations 110
of methanol extract of Gynura segetum’s leaf and
rosmarinic acid (100 µg/mL). ME 400, ME 200, ME 100
and ME 50 indicate methanol extract in concentration of
400, 200, 100 and 50 ug/mL. Values are the mean ± SD
(n=3); *** p<0.001 and * p<0.05 are significant differences
as compared with the control group
Figure 4.15 The vasculature of CAM before (A, C) and after (B, D) 117
SDS-induced irritation. Dotted circles identify the SDS-
induced irritation. The granuloma is strongly vascularized,
as the arrow indicates
Figure 4.16 The vasculatures of CAM before (A) and after being 117
exposure to SDS together with indomethacin (B).
Dotted circles identify the anti-inflammatory effect of
indomethacin. The network of blood vessels is normal
and no granuloma can be observed
Figure 4.17 The vasculatures of CAM before (A) and after being 118
exposure to SDS together with methanol extract of
Gynura segetum leaf (B). Dotted circles identify the anti-
inflammatory effect of methanol extract. The methanol
extract neutralized the formation of blood vessels around
the granuloma and the capillaries network appeared normal
Figure 4.18 The vasculature of CAM before (A, C, E) and after being 118
exposure to SDS together with fractions of Gynura segetum
leaf (B chloroform fraction; D ethyl acetate fraction; F
n-butanol fraction). Dotted circles identify the anti-
inflammatory effect of fractions. A thin network of
capillaries is formed starlike, as the arrow indicates
Figure 4.19 The vasculature of CAM before (A-E) and after being 120
exposure to SDS together with methanolic fractions of
Gynura segetum leaf (F-J). Dotted circles identify the
anti-inflammatory effect of methanolic fractions (R1=20%
methanol eluate; R2=40% methanol eluate; R3=60%
methanol eluate; R4=80% methanol eluate; R5=100%
xv
methanol eluate). The granuloma is poorly vascularized
and the network of capillaries is formed starlike, as the
arrow indicates
Figure 4.20 Inhibitory effect of methanol extract of Gynura segetum’s 122
leaf on granuloma tissue formation in rats. A shows images
of cotton pellets dissected out from the rats on the 8th
day of
treatment with graded doses of methanol extract. B represents
weights of dried cotton pellets that were taken out from the
rats treated with extract (ME 125, 250 and 500 mg/kg),
standard drug indomethacin (5 mg/kg) and control. The
results are represented as mean ± SD (n=6). *** shows
statistical significance of the treatment groups with p<0.001
when compared with control
Figure 4.21 Inhibitory effect of methanol extract of Gynura segetum 123
on the levels of tumor necrosis factor alpha (TNF-ɑ) and
interleukin-1 (IL-1) in the plasma samples of rats treated
with 125, 250 and 500 mg/kg of extract (treatment groups)
and 5 mg/kg of indomethacin (reference group). The results
are presented as mean ± SD (n=3). *** shows statistical
difference between the treatment groups and control at a
significance of p<0.001
Figure 4.22 The inhibitory effect of standard drugs aspirin (for COX-1), 125
celecoxib (for COX-2) and methanol extract of Gynura
segetum on the activities of cyclooxygenase (COX-1 and
COX-2) enzymes in an in vitro colorimetric assay. The
standard drugs were taken in the concentration of 200
µg/mL whereas the extract (ME) was tested in the
concentrations of 50 and 200 µg/mL. The results are
presented as mean ±SD (n=3).*** presents a statistical
significance of treatment groups with p<0.001 when
compared with control
xvi
LIST OF ABBREVIATIONS
AA Arachidonic acid
ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid))
AChE Anti-cholinesterase
anti-CD31 Anti-cluster of differentiation 31
AP-1 Activating protein-1
bFGF Basic Fibroblast Growth Factor
BHA Butylated hydroxyanisole
BHT Butylated hydroxytoluene
BrdU Bromodeoxyuridine
CAM Chick embryo chorioallantoic membrane
CCD-18Co Human colon fibroblast cell line
COX Cyclooxygenase
CUPRAC Cupric reducing antioxidant power
CV Coefficient of variation
DNA Deoxyribonucleic acid
DMSO Dimethyl sulfoxide
DPPH 1,1-diphenyl-2-picrylhydrazyl
ELISA Enzyme-linked immunosorbant assay
FBS Fetal bovine serum
FGF Fibroblast growth factors
FRAP Ferric reducing antioxidant power
EC50 Effective concentration 50
HET-CAM Hen’s egg chorioallantoic membrane test
xvii
HCT116 Human colon cancer
HGF Hepatocyte growth factor
HIF Hypoxia-inducible factors
HPLC High performance liquid chromatography
HPTLC High performance thin layer chromatography
IC50 Inhibitory concentration 50
IFN Interferon
iNOS Inducible nitric oxide synthase
IL-1 Interleukin-1
IL-6 Interleukin-6
IL-8 Interleukin-8
LOD Limit of detection
LOQ Limit of quantification
LOX Lipoxygenase
LTB4 Leukotriene B4
M Molar
MCP1 Monocyte chemotactic protein-1
MMPs Matrix metalloproteinases
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
n Number of samples
NaCl Sodium chloride
NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells
NIH National Institutes of Health
NO Nitric oxide
NOS Nitric oxide synthase
xviii
NSAIDS Non-steroidal anti-inflammatory drugs
OD Optical density
ORAC Oxygen radical absorption capacity
PBS Phosphate buffer saline
PDGF Platelet-derived growth factor
PG Propyl gallate
PGD2 Prostaglandin D2
PGE2 Prostaglandin E2
PGF2α Prostaglandin F2α
PGH2 Prostaglandin H2
pH Power of the concentration of the Hydrogen ion
PlGF Placental Growth Factor
PLA2 phospholipase A2
r2 correlation coefficients
Rf Retardation factor
ROS Reactive oxygen species
S.D. Standard deviation
SDS Sodium dodecyl sulfate
TGF Transforming growth factor
TIMP Tissue inhibitor of metalloproteinase
TLC Thin layer chromatography
TNF-α Tumor necrosis factor alpha
TRAP Total Radical-Trapping Antioxidant Parameter
TWEEN Polysorbate
U/mL Units per milliliter
xix
UV Ultraviolet
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptors
v/v Volume to volume
°C Degree Celsius
xx
LIST OF APPENDICES
Page
Appendix A Animal Ethical approval letter 156
Appendix B Calibration curves of the reference standards senecionine 158
(A)and seneciphylline (B).
Appendix C LOD and LOQ studies 159
Appendix D Calibration curve of gallic acid for total phenolic study 160
(A) and quercetin for total flavonoids study (B).
Appendix E Raw data for total phenolic study 161
Appendix F Raw data for total flavonoid study 162
Appendix G Raw data for DPPH free radical scavenging activity 163
Appendix H Raw data for Beta carotene-linoleic acid antioxidant study 169
Appendix I CAM assay
Figure 1: Blank control 174
Figure 2: Effect of CAM treated with petroleum ether extract 174
Figure 3: Effect of CAM treated with ethyl acetate fraction 175
Figure 4: Effect of CAM treated with butanol fraction 175
Figure 5: Effect of CAM treated with 20% methanol eluate 176
Figure 6: Effect of CAM treated with 40% methanol eluate 176
Figure 7: Effect of CAM treated with 60% methanol eluate 177
Figure 8: Effect of CAM treated with 80% methanol eluate 177
Figure 9: Effect of CAM treated with 100% methanol eluate 178
Appendix J Raw data for rat aorta ring assay 179
Appendix K Raw data for MTT assay of methanol extract in HCT 116 180
colon cancer cells (A) and normal cell lines (B)
Appendix L Raw data for VEGF concentration 181
xxi
Appendix M HET CAM assay: CAM treated with crude extracts (A: 182
petroleum ether extract; B: chloroform extract)
Appendix N Raw data of the weight of cotton pellet dissected out from 183
rats
Appendix O Raw data of TNF-α and Interleukin-1 levels in the blood 184
samples of the rats implanted with cotton pellet
Appendix P Raw data of cyclooxygenase assay 186
xxii
AKTIVITI ANTIOKSIDAN, ANTI-ANGIOGENIK DAN ANTI-INFLAMASI
EKSTRAK TERPIAWAI DAUN GYNURA SEGETUM
ABSTRAK
Gynura segetum (daun dewa dalam bahasa Malaysia) ialah herba, dipercayai berguna
untuk rawatan kanser dan inflamasi. Dalam kajian ini, aktiviti anti-inflamasi atas
ekstrak terpiawai daun Gynura segetum dan juga kemungkian hubungan dengan
aktiviti antioksidan, anti-angiogenik dan sitotoksik telah disiasat. Fraksi etil asetat
menunjukkan aktiviti antioksidan yang terkuat berdasarkan asai memerangkap DPPH
dan asai kapasiti pelunturan beta karotena dan berkaitan terus dengan jumlah
kandungan fenolik dan flavonoid. Ekstrak metanol adalah yang paling aktif dalam
menghalang pertumbuhan saluran darah dalam asai anak ayam embrio membran
korioalantoik (CAM) dan asai aorta tikus cincin. Ini menunjukkan ia mempunyai
aktiviti anti-angiogenik. Ekstrak metanol tidak sitotoksik dan penyekatan aktiviti
faktor pertumbuhan endotel vaskular (VEGF) adalah salah satu mekanisme tindakan
untuk anti-angiogenik. Ekstrak metanol mempunyai aktiviti anti-inflamasi kerana ia
menghalang kerengsaan oleh SDS-teraruh dalam ujian telur ayam membran
korioalantoik (HET-CAM) dan pembentukan granuloma dalam asai pelet kapas-
teraruh granuloma pada tikus. Aktiviti anti-inflamasi ekstrak metanol ditunjukkan
melalui perencatan pada tahap pro-inflammasi sitokin tumor nekrosis faktor-α (TNF-
α), interleukin-1 (IL-1) dan enzim siklooksigenase-2 (COX-2). Oleh itu, penemuan
daripada kajian ini mencadangkan bahawa daun Gynura segetum adalah sumber
antioksidan semula jadi dan mempunyai aktiviti anti-angiogenik yang mungkin
menjelaskan aktiviti anti-inflamasi kronik dan potensi dalam anti-kanser.
xxiii
ANTIOXIDANT, ANTI-ANGIOGENIC AND ANTI-INFLAMMATORY
ACTIVITIES OF STANDARDIZED GYNURA SEGETUM LEAF EXTRACTS
ABSTRACT
Gynura segetum (daun dewa in Malay) is a herb, reputed of value among others for
treatment of inflammation and cancer. In the present study, the anti-inflammatory
activity of the standardized leaf extracts of Gynura segetum were investigated as well
as its possible relation to antioxidant, anti-angiogenic and cytotoxic activities. The
ethyl acetate fraction showed the strongest antioxidant activity based on DPPH
scavenging activity and beta carotene bleaching capacity assays and directly related
to its total phenolic and flavonoid contents. The methanol extract was the most
active in inhibiting the blood vessels growth in chick embryo chorioallantoic
membrane (CAM) assay and rat aortic ring assay. It shows it has anti-angiogenic
activity. It is non-cytotoxic and suppression of vascular endothelial growth factor
(VEGF) activity is one of the anti-angiogenic mechanisms of action. The methanol
extract possesses anti-inflammatory activity as it inhibited SDS-induced irritation in
hen’s egg chorioallantoic membrane (HET-CAM) test and cotton pellet-induced
granuloma formation in rat. Its anti-inflammatory activity is mediated through the
inhibition of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and
interleukin-1 (IL-1) and cycloxygenase-2 (COX-2) enzyme activities. Taken
together, the present study suggests that Gynura segetum’s leaf is a natural source of
antioxidants and possesses anti-angiogenic activity which may explain its chronic
anti-inflammatory activity and anti-cancer potential.
1
CHAPTER 1
INTRODUCTION
1.1 Background
Herbal medicines are the therapeutic experiences of generations of practicing
physicians of traditional medicine over hundreds of years and they are known to be
oldest health care products that have been used by mankind all over the world to treat
various types of ailments (Torey et al., 2010).
The demand for medicine from plants has increased and the global awakening of
herbal medicines is also reflected in Malaysia. Medicinal plants grown in Malaysia
have also received attention as new resources for alternative medicines over the past
years (Sone et al., 2011). Currently, attention is being focused on the investigation
of the efficacy of traditionally used plants as they are affordable and have fewer
adverse effects (Masresha et al, 2012).
Gynura segetum, from family Compositae is a cultivated species and can be found
growing in the tropical regions in Indonesia and Malaysia. G. segetum has drawn a
lot of attention due to its uses in traditional medicine. The plant is known for its
traditional use for the treatment of cancer, inflammation, diabetes, hypertension and
skin afflictions. A variety of chemical compounds have been identified from this
genus such as flavonoids, pyrolizidine alkaloids, triterpenes, steroids and
sesquiterpenoids (Zhu et al., 2013). In spite of numerous phytochemical studies,
there has been no systematic approach to understanding the ethno pharmacological
activity of this plant.
2
In previous study, crude leaf extracts of G. segetum showed potential anti-angiogenic
effect in chick embryo chorioallantoic membrane (CAM) model (Seow, 2010). Anti-
angiogenic strategies are emerging as an important tool for the treatment of cancer
and inflammatory diseases. Angiogenesis and inflammation are codependent
processes, which involves both augmentation of cellular infiltration and proliferation
and overlapping roles of regulatory growth factors and cytokines (Jackson et al.,
1997). Many compounds that inhibit angiogenesis have also shown to block
inflammatory responses due to this interconnection.
Angiogenesis in inflammatory diseases contribute to tissue growth, disordered tissue
perfusion, abnormal ossification (formation of bony substance) and enhanced
responses to normal or pathological stimuli (Walsh and Pearson, 2001).
Angiogenesis inhibitor may reduce inflammation and also help to restore appropriate
tissue structure and function. Most of the research on anti-angiogenic agents has
been directed towards inflammation treatment (Kim et al., 2004; Ahn et al., 2007;
Jung et al., 2009; Krenn and Paper, 2009). Collectively, this suggested that many
herbs and their phytochemicals may exert both anti-angiogenic effects and anti-
inflammatory activity. Thus, there is an interest to discover local medicinal plant for
anti-angiogenic and anti-inflammatory effects.
Hence, the present study was carried out to expand the study on the anti-angiogenic
and anti-inflammatory activities of the various extracts and fractions of G. segetum’s
leaf, and investigate the probable mechanism of action of the most active
extract/fraction. Standardization of the G. segetum’s leaf extract by chemical
profiling (High performance thin layer chromatography), quantification of two
3
marker compounds using a validated HPLC (high performance liquid
chromatography) method and biological profiling (determination of total phenolic
and flavonoids content, and antioxidant activity) were also carried out. The
experiments of the present study were summarized in Figure 1.1.
4
Figure 1.1: Flow chart of antioxidant, anti-angiogenic and anti-inflammatory activities of standardized Gynura segetum leaf extracts
' I Antioxidant activity I
Total Phenolic content
I Total Flavonoid content I I DPPH scavenging assay I I
Beta carotene-linoleic acid assay
I Extraction I
I Standardization I
Anti-angiogenic activity
I 1 l
CAM assay I Rat aortic ring assay
I I \II
I Cytotoxicity I \ I/
Mechanism (VEGF concentration)
' Anti-inflammatory activity I
It
I I I I HET-CAM COX assay assay
It
I Cotton pellet granuloma assay I
l Cotton pellet Mechanism
weight (Pro-inflammatory cytokines )
5
1.2 Problem of Statements
Gynura segetum from family Compositae was claimed to possess various medicinal
values including possible treatment of cancer, diabetes, inflammation and
hypertension (Suharmiati and Maryani, 2003). Although this plant is traditionally
used in treatment of cancer and inflammatory disorders, but to the best of our
knowledge, its anti-cancer potential and anti-inflammatory activity has not yet been
confirmed scientifically. The present studies focus on both in vivo and in vitro anti-
angiogenic and anti-inflammatory activities in order to further investigate the
potential of this plant. The standardization and antioxidant activities of the G.
segetum’s leaf extract were also performed to evaluate its possible relation to the
anti-angiogenic and anti-inflammatory activities.
6
1.3 Objectives
The objectives of the present study are:
1. To standardize the G. segetum leaf extracts by using phytochemical and
chromatographic methods (HPTLC and HPLC).
2. To evaluate antioxidant potential of standardized leaf extracts of G. segetum
by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity
and beta-carotene-linoleate model assays and their correlation with total
phenolic and flavonoid content.
3. To determine the anti-angiogenic activity of standardized leaf extracts of G.
segetum by different experimental models (in vivo chicken chorioallantoic
membrane assay and ex vivo rat aortic ring assay) and their possible
mechanism of action.
4. To study the in vivo anti-inflammatory effects of standardized leaf extracts of
G. segetum by different experimental models (hen’s egg chorioallantoic
membrane test and cotton pellet granuloma model in rats) and their possible
mechanism of action.
7
CHAPTER 2
LITERATURE REVIEWS
2.1 Description of Gynura segetum (Lour.) Merr.
G. segetum from family Compositae is a cultivated species and can be found growing
in the tropical regions in Indonesia and Malaysia. The leaves are shortly petiole,
fleshy with serrated margin and both surfaces are pubescent (Figure 2.1). The upper
surface is green and lower surface is purplish green. The stem branches profusely,
purplish green in color and slightly hairy. The yellowish orange flowers have a
strong and foetid odour. The plants produce numerous roots which are attached to a
tuber. The plants collected from Jabatan Pertanian Relau, Penang were identified by
the botanist (Mr. V. Shanmugan) from School of Biological Sciences, Universiti
Sains Malaysia and a voucher specimen has been deposited at the herbarium of
School of Biological Sciences, University Sains Malaysia with reference number
11013.
Figure 2.1: Gynura segetum (Lour.) Merr
8
2.2 Previous studies on Gynura segetum
A literature survey indicated that lack of research on G. segetum plant. To date, only
few investigations on phytochemical studies of G. segetum have been reported. The
structures of some isolated compounds of G. segetum are shown in Figure 2.2.
Alkaloid compound senecionine was elucidated and reported by Liang et al. (1984).
Succinic acid, D-mannitol, thymine, adenine and pyrrolizidine alkaloids were
isolated and identified by Liu et al. (1988) from aerial portion of G. segetum plant.
Six alkaloids were isolated by Yuan et al. (1990) and four of them were identified as
follows: senecionine, seneciphylline and seneciphyllinine. In Yang et al. (2009), a
rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method
was established for simultaneous determination of senecionice, seneciplylline and
senecionine N-oxide in G. segetum.
Zhu et al. (2013) reported the isolation of nine new compounds from G. segetum for
the first time. Their structures were identified to be stigmasterol, isoarborinol,
arborinol, zhebeiresinol, lumichrome, (2S,3S,4R,8E)-2-[(2R)-2-
hydroxypalmitoylamino]-8-octadecene-1.3.4-triol, syringic acid, vanillic acid, trans-
p-hydroxycinnamic acid on the basis of mass and NMR spectra.
Previous study demonstrated that the leaf extracts of G. segetum inhibited the
sprouting of blood vessels growth in chick embryo chorioallantoic membrane (CAM)
model. The anti-angiogenic activity of G. segetum provided a pharmacological basis
on its folkloric
9
Figure 2.2: Chemical structures of some isolated compounds of Gynura segetum
Senecionine
HO
Stigmasterol
o---CH3
HO
,~
CH3 .. ·~,,,H
0 0
Zhebeiresinol
Seneciphyllinine
OH 0 ~
~CH3 NH VH 7
HO ~ CH3
HO. (2S,3S, 4R,8E)-2 -[ (2R)-2 -hydroxypalmitoy !amino ]-8-octadecene-1.3.4-triol
,o~o H,c ~X ' cH,
0 OH
Lumichrome
Syringic acid
Isoarborinol
Arborinol
~OH HO~
OCH3
Vanillic acid
~OH HOV
Trans-p-hydroxycinnamic acjd
10
use for the treatment of inflammatory diseases and cancer (Seow, 2010). Angiogenic
factors have a central role in promoting cancer and a strategic position in the
regulation of angiogenesis, eventually stimulating endothelial cells that are
genetically stable in comparison with the genetically labile tumor cells (Schenone et
al., 2007). New blood vessels can maintain the chronic inflammatory state by
transporting inflammatory cells to the site of inflammation and supplying nutrients
and oxygen to the inflamed tissue. The inflamed tissues are induced to release large
quantities of angiogenic factors (VEGF) and cytokines (Walsh and Pearson, 2001).
Therefore, anti-angiogenic therapy is a promising approach for the treatment of
cancer and other angiogenesis-dependent diseases i.e. chronic inflammation.
Preliminary phytochemical screening revealed the presence of alkaloids, terpenes,
flavonoids, tannins and saponins in leaves of G. segetum (Seow, 2010). On the other
hand, the presence of phenolic compounds in the plants indicates that this plant may
be anti-microbial agent and this agreed with the findings of Seow (2010). Moreno et
al. (2006) suggested that the antimicrobial action of phenolic compounds was related
to inactivation of cellular enzymes, which depended on the rate of penetration of the
substance into the cell or caused by membrane permeability changes. Increased
membrane permeability is a major factor in the mechanism of antimicrobial action,
where phenolic compounds disrupt the membranes and causing loses of membrane
permeability and eventually the death of cell (Cetin-Karaca, 2011).
11
2.3 Standardization
Standardization of herbal medicines means confirmation of its identity and
determination of its quality and purity (Pandey and Tripathi, 2014). Plants contain
phytochemical that can be used for therapeutic purpose or as precursors for
pharmaceutical synthesis. The world market demand for herbs and their products has
increased tremendously in recent years. Therefore, methods for standardization of
herbal raw materials and herbal formulations are developed.
Pharmacopoeias carry monographs for herbs and herbal products to maintain their
quality. Several pharmacopoeias including United States Pharmacopoeia, Indian
Pharmacopoeia, European Pharmacopoeia, and British Pharmacopoeia do cover
monographs and quality control tests for a few medicinal plants. There many changes
are being incorporated into the different pharmacopoeias, as reflected by the
availability, development, and subsequent refinement of analytical techniques
(Rajani and Kanaki, 2008). In the present study, the standardization and validation
of analytical procedures were carried out according to the European Pharmacopoeia.
Standardization of herbal medicines involves the collection of information and
application of strict quality control measurement from the growing of a medicinal
plant to the finished therapeutic substance (Garg et al., 2012). The standardization
of plant includes a complete description of the starting plant materials, control and
monitoring of numerous factors namely growing conditions, harvesting time,
harvested plant part, microscopy and macroscopic analysis, ash values and extractive
values, moisture content determination, drying methods, storage, extraction process,
chromatographic and analytical evaluation which are required for providing
12
consistency of the quality of an plant extract (Bele and Khale, 2011; Garg et al.,
2012).
Most of the medicinal plants have definite specific chemical constituents to which
their biological or pharmacological activity is attributed (Pandey and Tripathi, 2014).
Pharmacological properties of a plant extract depend on phytochemical constituents
present therein. Without a consistent quality of a phytochemical mixture, a
consistent pharmacological effect is not achieved (Mosihuzzaman and Choudhary,
2008; Maiti et al., 2011). Phytochemical standardization comprises all the possible
information with regard to the chemical constituents present in herbal medicines. The
phytochemical evaluations include preliminary screening for the presence of
different chemical groups (e.g. alkaloids, flavonoids, saponins, triterpenic acids,
tannins and etc.), quantification of chemical of interest and establishment of
fingerprint profiles based upon single or multiple markers (Kulkarni et al., 2014).
Chromatographic fingerprinting plays an important role in the quality control of
complex herbal medicines (Kamboj and Saluja, 2013). A fingerprint refers to the
common peaks of chromatograms which can be used to characterize and determines
the concentration of a set of characteristic chemical substances in an herb (Kulkarni
et al., 2014). Using this technique, the identification of various chemical markers of
the herbal medicines can be easily done and it also serves as a tool for authentication
and quality control of herbal drugs (Kasar et al., 2013).
Chromatographic fingerprint of herbal medicine is a chromatographic pattern
produced from extract of some specific chemical constituents which may be
13
pharmacologically active or have some chemical characteristics (Kulkarni et al.,
2014). Standardized extracts are high-quality extracts containing consistent levels of
specified marker/bioactive compounds (Bandaranayake, 2006). There are two types
of standardized extract, which are active constituent extract and marker compound
extract. Active constituent extract contains the constituents with known therapeutic
effect which can be used to standardize a biological activity. However, marker
compound is not necessarily active compounds, as chemically defined constituents,
which are used for control purpose (Kamboj and Saluja, 2013).
Sensitivity is the major concern for the detection of various chemical markers in
extract. Thus, chromatographic techniques in combination with different detections
are the preferred techniques. High performance thin layer chromatography (HPTLC)
and High performance liquid chromatography (HPLC) are suitable analytical method
for the separation and quantitative determination of various chemical markers of
herbal medicines.
14
2.3.1 High performance thin layer chromatography (HPTLC)
HPTLC has recently emerged as an useful tool for fingerprinting and quantification
of marker compounds in herbal medicines (Bala and Saini, 2013). In its traditional
form, thin layer chromatography has use in identification of botanical raw materials
(Kasar et al., 2013). The modern HPTLC offers better resolution which allows a
quantitative analysis at micro and even in nano gram levels (Kamboj and Saluja,
2013). A wide spectrum of constituents can be detected at the same time in a single
run in an experiment by HPTLC and the flexibility of detection are particularly
useful for fingerprinting analysis.
The modern HPTLC can generate a chromatographic fingerprint in the form of an
unique sequence of peaks corresponding to the analyzed sample in its fullness
(Nicoletti, 2011). The resulted chromatogram can be evaluated based on number,
sequence, color and relative position of zones (Rf) with respect to zone obtained on
the same plate with chemical reference materials (Kasar et al., 2013). The results are
not only reported as peak data, but can also conveniently be presented as colorful
images, their absorption spectra, λ max and shoulder inflections of all the resolved
bands (Kamboj and Saluja, 2013).
Collectively, fingerprinting of HPTLC is proved to be a linear and precise method
for herbal identification and can be further used in authentication and
characterization of the medicinally important plant (Sushma et al., 2013). However,
HPTLC method has few limitations, such as the limited developing distance and it is
often not possible to obtained baseline separation of all components of such complex
samples as medicinal plant materials (Kasar et al., 2013). Also, the sensibility of
15
HPTLC is still not comparable with HPLC. In HPTLC, two closely related
compounds may not be appropriately resolved and hence migrate as a single peak.
This situation would result into misleading results in quantitative analysis, especially
if one of the non-resolved compounds is an unknown impurity or a degradation
product (Hewala et al., 2012). Therefore, most fingerprinting analysis is typically
based on HPLC.
2.3.2 High performance liquid chromatography (HPLC)
HPLC is one of the most important tools of analytical chemistry and widely used for
fingerprinting study of herbal plants. HPLC mainly utilizes a column that holds
packing material (stationary phase), a pump that moves the mobile phase through the
column, and a detector that shows the retention times of the molecules (Malviya et
al., 2010). The separation of sample is based on the differences in the rates of
migration through the column arising from different partition of the sample between
the stationary and mobile phase. Depending upon the partition behavior of different
components, elution at different time takes place (Gupta et al., 2012).
Analytical method development and validation are key elements of any
pharmaceutical development program (Singh, 2013). HPLC analysis method is
developed to ensure the identity, purity and quantification of the chemical
constituents in herbal plant. All analytical methods that are intended to be used for
analyzing any chemical constituents in plant material will need to be validated
(Gupta et al., 2012). The validation practice demonstrated that an analytic method
measures the correct constituent in correct quantity, and in the proper range for the
intended samples (Julia et al., 2011; Khan et al., 2012).
16
The validation of analytical method is performed according to the ICH (International
Conference on the Harmonization of Technical Requirements for the Registration of
Pharmaceuticals for Human Use) guidelines (ICH, 2002). The various validation
parameters include linearity, accuracy, limits of detection and quantification (LOD
and LOQ), precision, robustness and selectivity or specificity (Singh, 2013).
Reproducible quality of HPLC results can only be obtained if proper attention has
been paid to the method development, validation and system’s suitability to carry out
the analysis (Singh, 2013). The validated methods provide emerging knowledge
supporting the standardization of herbal plant and the method could be utilized for
quality check of various pharmaceutical and nutraceutical preparations developed
from the herbal plants (Masresha et al., 2012). The herbal plants will be earlier
tested in animal experiments and further tested in a clinical study in order to prove its
safety and efficacy.
17
2.4 Antioxidant
Antioxidants are defined as molecules which can be safely interact with free radicals
and terminate the chain reaction before vital molecules are damaged (Nidhi and
Gauray, 2012). Antioxidants can also be molecules which prevent or inhibit free
radical formation, involved in the defense mechanism of the organism against the
pathologies associated to the attack of free radicals (Pisoschi and Negulescu, 2011).
Antioxidants can be derived from natural sources (vitamins, flavonoids, anthocyanins,
some mineral compounds), but can also be synthetic compounds, like butylated
hydroxyl anisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), etc
(Pisoschi and Negulescu, 2011). BHA and BHT are widely used in food industry as
antioxidants in order to reduce the damage caused by free radicals. However, the
toxicological research demonstrated that these synthetic antioxidants possess toxic,
pathogenic and carcinogenic effects (Ganie et al., 2011; Jiang et al., 2013).
Therefore, interest in finding natural antioxidants from plant materials has drawn
more and more attention (Safaei-Ghomi et al., 2009).
The use of traditional medicine is widespread and plants are potential source of
natural antioxidants that might serve as leads for the development of novel drugs
(Conforti et al., 2008). Plant-derived antioxidants have been shown to function as
singlet and triplet oxygen quenchers, free radical scavengers, peroxide decomposers,
enzyme inhibitors and synergists (Lai and Palanisamy, 2012). Antioxidants such as
the vitamins A, C and E, beta carotene, flavonoids, mineral selenium have potential
to prevent oxidative damage and demonstrated their potent antioxidant activities both
18
in vitro and in vivo (Mokhtar et al., 2008; Pandey and Rizvi, 2009; Hamid et al.,
2010).
2.4.1 The mechanism of actions of antioxidant
Free radicals are considered as unstable molecule, which react quickly with other
compounds, trying to capture the needed electron to gain stability (Nidhi and Gauray,
2012). Free radicals of highly reactive oxygen species (superoxide, O2˙ ; hydroxyl,
OH˙; peroxyl, ROO˙; peroxinitrite, ˙ONOO and nitric oxide, NO˙) produced
through oxidative process (Abdel-Hameed, 2009). Oxidative stress occurs when
there is an imbalance between free radical reactions and the scavenging capacity of
antioxidative defense mechanism of the organism (Seifu et al., 2012). A number of
biochemical processes can initiate oxidation, which proceeds continuously in the
presence of (a) suitable substrate(s), until a blocking defense mechanism occurs
(Pisoschi and Negulescu, 2011).
Antioxidants are compounds or systems that delay autoxidation by inhibiting the
formation of free radicals by several mechanisms: initiation, propagation, and
termination (Hamid et al., 2010) as shown below. The oxidation may be initiated by
the action of external agents such as heat, light or ionizing radiation or by chemical
initiation involving metal ions or metalloproteinase (Pisoschi and Negulescu, 2011).
a) Initiation: free radicals are formed from molecules that readily give up electrons.
(1) RH → R˙+ H˙
(2) R˙ + O2 → ROO˙
(3) 2ROOH → ROO˙ + RO˙ + H2O
19
b) Propagation: the free radicals are alternately expended and formed.
(1) R˙ + O2 → ROO˙
(2) ROO˙ + RH → ROOH + R˙
(3) RO˙ + RH → ROH + R˙
c) Termination: involve the combination of radicals to form non-radical products.
(1) R˙ + R˙ → R – R
(2) R˙ + ROO˙ → ROOR
(3) ROO˙ + ROO˙ → ROOR + O2
2.4.2 Methods of antioxidant activity assessment
Antioxidants affect the process of lipid oxidation at different stages due to the
differences in their mode of action (Ghasemzadeh et al., 2010). There is no single
method that can provide unequivocal measurement of antioxidant activity and the
best solution is to use various methods instead of a one-dimension approach
(Carocho and Ferreira, 2013).
a) The 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method
The ABTS assay is a colorimetric assay in which the ABTS radical decolorizes in the
presence of antioxidants (carotenoids, phenolic compounds and others) (Moon and
Shibamoto, 2009). The ABTS cation radical (ABTS•+) which absorbs at 743 nm
(giving a bluish-green colour) is formed by the loss of an electron by the nitrogen
atom of ABTS. In the presence of hydrogen donating antioxidant, the nitrogen atom
quenches the hydrogen atom, yielding the solution decolorization (Pisoschi and
Negulescu, 2011).
20
b) The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method
DPPH (2,2-diphenyl-1-picrylhydrazyl) is characterized as stable free radical and able
to accept an electron or hydrogen radical (Kchaou et al., 2013). This colorimetric
assay uses the DPPH radical, which changes from purple to yellow in the presence of
antioxidants (Moon and Shibamoto, 2009). When DPPH reacts with a hydrogen
donor, the reduced (molecular) form (DPPH) is generated, accompanied by the
disappearance of the violet color. Therefore, the absorbance diminution depends
linearly on the antioxidant concentration (Pisoschi and Negulescu, 2011).
c) The ferric reducing antioxidant power (FRAP) method
The FRAP method is characterized by the reduction of iron, Fe3+
to Fe2+
depending
on the available reducing species followed by the alteration of color from yellow to
blue and analyzed through a spectrophotometer (Antolovich et al., 2002). Reducing
power appears to be related to the extent of conjugation in phenols as well as the
number of hydroxyl constituents (Schaich, 2006). Further, the assay reaction must
be carried out at acidic pH in order to maintain iron solubility (Craft et al., 2012).
d) The oxygen radical absorption capacity (ORAC) assay
This method measures the antioxidant scavenging activity against the peroxyl radical
(ROO˙), induced by 2,2’-azobis-(2-amidino-propane) dihydrochloride (AAPH), at
37ºC. Fluorescein is used as the fluorescent probe. The loss of fluorescence is an
indicator of the extent of the decomposition, from its reaction with the peroxyl
radical (Thaipong et al., 2006; Denev et al., 2010). Any antioxidant species present
in the reaction mixture will undergo hydrogen atom transfer mechanism with the
peroxyl radicals and delay the reduction of the fluorescent signal (Craft et al., 2012).
21
e) The total radical-trapping antioxidant parameter (TRAP) assay
The TRAP assay is used to determine the total antioxidant activity, based on
measuring oxygen consumption during a controlled lipid oxidation reaction induced
by thermal decomposition of AAPH (2,2’-Azobis(2amidinopropane)hydrochloride)
(Antolovich et al., 2002). The basic reactions of the assay are similar to those of
ORAC. The TRAP assay involves the initiation of lipid peroxidation by generating
water-soluble peroxyl radicals and is sensitive to all known chain breaking
antioxidants, but it is relatively complex and time-consuming to perform, requiring a
high degree of expertise and experience (Prior et al., 2005).
f) The cupric reducing antioxidant power (CUPRAC) assay
The method relies on the reduction of copper, Cu (II) to Cu (I) by the antioxidants
present in the sample. Then, bathocuproine (2,9-dimethyl-4,7-diphenyl-1,10-
phenanthroline) forms a complex with Cu (I) and has a maximum absorbance at 490
nm (Huang et al., 2005). Copper has a lower redox potential than iron, so its
reactions are more selective e.g. sugars and citric acid which commonly interferences
with iron in FRAP, are not oxidized in CUPRAC. The low redox potential enhances
redox cycling, so copper reduction may be an even more sensitive indicator of
potential pro-oxidant activity of antioxidants (Prior et al., 2005).
g) Beta-carotene linoleic acid method/conjugated diene assay
The basis of the beta-carotene bleaching assay is the discoloration of beta-carotene in
a reaction with a linoleic acid free radical. A radical is formed upon the removal of a
hydrogen atom located between two double bonds in linoleic acid. Beta-carotene has
22
a maximum absorbance at 470 nm. Antioxidants present in the reaction solution can
prevent the degradation of beta-carotene (Fu et al., 2013).
2.4.3 Herbal medicines and antioxidant activities
To date, numerous of plants in Malaysia have been studied for their antioxidant
activity. Garcinia mangostana, Mangifera indica, Nephelium lappaceum,
Anacardium occidentale, Piper betle and Syzygium aquem exhibit high antioxidant
activity and proposed to be an alternative source of polyphenols (Mokhtar et al.,
2008; Lai and Palanisamy, 2012). Compounds that have several or many phenolic
hydroxyl substituents are often referred to as polyphenols, which have strong
hydrogen atom donating activity to terminate the chain reactions of free radicals and
inhibit oxidation (Maestri et al., 2006).
An antioxidant activity has been reported in a number of Gynura species. Caffeic
acid and quercetin 3-O-rutinoside isolated from Gynura formosana Kiamnra were
found to show good antioxidant activity against DPPH and hydroxyl radicals (Hou et
al., 2005). The antioxidant potency of Gynura procumbens leaves were investigated
by Rosidah et al. (2008); Akowuah et al. (2009) and Shwter et al. (2014), suggest
that phenolic compounds in these plants provide substantial antioxidant activity. The
Gynura medica leaf extract showed a moderate radical scavenging activity, which
agreed with the moderate level of flavonoid compounds existing in the plant;
kaempferol-3-O-glucoside was the major compound of the plant (Liu et al., 2010).
Meanwhile, the ethyl acetate extract of Gynura bicolor with the highest total
phenolic content among these species extracts generally showed the strongest ability
in scavenging DPPH radicals and metal chelating activity (Teoh et al., 2013).
23
2.5 Angiogenesis
Angiogenesis is a progressive, multistep physiological process by which new blood
vessels are generated from pre-existing vasculature (Deryugina and Quigley, 2008).
Angiogenesis plays a central role in various physiological processes within human
body, such as, embryonic development, menstrual cycle, and wound repair.
Unregulated angiogenesis is intensively involved in many pathological conditions
including psoriasis, retinopathy and cancer and various ischemic and inflammatory
diseases (Choe and Lee, 2007; Yoo and Kwon, 2013).
The process of angiogenesis can be simply described into three major steps including:
i) Initiation of the angiogenic response: Angiogenesis is initiated in response to
hypoxia, by the release of hypoxia inducible factors (HIF), which facilitate the
release of angiogenic stimulators, which in turn lead to endothelial cell
activation. Activated endothelial cell secrete matrix metalloproteinases (MMPs)
and plasminogen activators (PAs), which degrade the extracellular tissue to
facilitate endothelial penetration. Both PAs and MMPs are secreted together
with their inhibitors, ensuring a stringent control of local proteolytic activity
(Bisht et al., 2010).
ii) Endothelial cell migration, proliferation and tube formation: Extracellular matrix
degradation results in an increased concentration of various growth factors,
which stimulate endothelial cell migration and proliferation. After the initial
period of migration, rapid endothelial cell proliferation begins, thus increasing
the rate of sprout elongation. These processes are also mediated by cell adhesion
24
molecules (Integrin αvβ3and αvβ5, vascular endothelial cadherin, vascular cell
adhesion molecule-1, P-selectin, and E-selectin) (Bisht et al., 2010).
iii) Maturation of the neovasculature: The final phase of the angiogenic process
involves maturation of the neovasculature. After endothelial cell proliferation
and maturation, and the formation of endothelial tube structures, surrounding
vessel layers composed of mural cells (pericytes in small vessels and smooth
muscle cells in large vessels), need to be recruited. Endothelial cells may
accomplish this via the synthesis and secretion of platelet-derived growth factor
(PDGF), a mitogen and chemo attractant for a variety of mesenchyme cells.
Subsequent differentiation of the mural precursor cells into pericytes and smooth
muscle cells is believed to be a cell-cell contact dependent process (Griffioen
and Molema, 2000). Finally, when sufficient neovascularization has occurred,
the angiogenic factors are down regulated or the local concentration of the
inhibitors increases. As a result, the endothelial cells become quiescent (Bisht et
al., 2010).
2.5.1 Angiogenic factors and inhibitors
Angiogenesis is a tightly controlled by a complex balance between the pro-
angiogenic and anti-angiogenic factors (Tahergorabi and Khazaei, 2012). A number
of endogenous angiogenic factors and inhibitors have been discovered and identified,
that regulate the balance between the pro- and anti-angiogenic states (Carmeliet et al.,
2000). Some of the most important factors and inhibitors are shown in Table 2.1.
