ANTI-PLATELET AGGREGATION ACTIVITY OF MELALEUCA BRACTEATA
VAR.REVOLUTION GOLD DERIVED BETULINIC ACID AND ITS DERIVATIVES
BY
OSUNSANMI FOLUSO OLUWAGBEMIGA (Msc UNILAG)
201200147
A DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENT FOR THE
DEGREE OF DOCTORATE OF SCIENCE IN THE DEPARTMENT OF
BIOCHEMISTRY AND MICROBIOLOGY, FACULTY OF SCIENCE AND
AGRICULTURE, UNIVERSITY OF ZULULAND, KWADLANGEZWA, SOUTH AFRICA
SUPERVISOR: PROF A.R. OPOKU CO-SUPERVISOR: PROF F.O SHODE
NOVEMBER 2015
1-1
DECLARATION
The experimental work described in this dissertation was conducted in the Department
of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of
Zululand, from January 2013 to November 2015, under the supervision of Prof. A.R.
Opoku and co-supervision of Prof. F.O. Shode
This study represents the original work by the author which has not been submitted in
any form to another University. Any use of the work of others has been properly cited
and acknowledged in the text.
I, Osunsanmi Foluso Oluwagbemiga firmly declare the above statement to be true.
--------------------------------------------------------
Foluso Oluwagbemiga Osunsanmi
--------------------------------------------------------
Prof. A.R. Opoku
--------------------------------------------------------
Prof. F.O. Shode
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DEDICATION
This research work is dedicated to the Almighty God, my beloved late father Deacon
David Olanrewaju Osunsanmi, my mother Mrs Benedicta Omolara Osunsanmi, my
dearest wife Mrs Adesola Sekinat Osunsanmi and my children Obanioluwa and
Olaloluwa.
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ACKNOWLEDGEMENTS
I am deeply grateful to the Almighty God who has kept me alive to the completion of this
study.
I would like to express my gratitude to my supervisor, Prof A. R. Opoku for his guidance,
love, encouragement and patience throughout this work.
I am indebted to my co-supervisor Professor F. O. Shode for his scientific guidance,
support and encouragement during this research work.
I wish to express my thanks to Prof Basson, Dr A. Kappo, Dr A.R Mosa and other staff
members of the Department of Biochemistry and Microbiology, University of Zululand
for their support, and to the Research Office of the University of Zululand for funding this
research work.
I am forever grateful to my parents (Late Deacon David Olanrewaju Osunsanmi and Mrs
Bendeicta Omolara Osunsanmi) for their moral and financial support throughout this
work; my dearest wife (Mrs Adeshola Sekinat Osunsanmi) for her love and
encouragement; my children (Obanioluwa and Olaoluwa) for their patience and
understanding; and my siblings (Dr (Mrs) Oluwaseyi Modupe Ajayi, Engineer Akinyemi
Olabode Osunsanmi, Dr (Mrs) Monisola Itohan Ikhile, Engineer Oluwafemi Sunday
Osunsanmi and Mr Temidayo Oluwasola Osunsanmi) for their prayers and financial
assistance.
I would like to thank Dr Ogunyinka
’s
family for their encouragement and support. I would
also like to thank my friends: Rexwhite Enakrire, Rekha Dunpall, Wole Durodolu, Pst
Babatunji Oyinloye, Wole Aremu, Zanele Dube, Kayode Oki, Pst Charles Akappo, Pst
and Mrs Foli, Dr and Pst (Mrs) Adetiba, Dr and Mrs Muyiwa Olowe, John Luke, Mr
Albert Ikhile, Pst and Pst (Mrs) Johnny, Pst and Pst (Mrs) D.O Adelaja, Pst and Pst
(Mrs) Tom Mensah, Prof and Dr (Mrs) Dapo Longe and host of others for their prayers
and support throughout this research work.
1-4
LIST OF ABBREVIATIONS
12-KETE 12-ketoeicosatetraenoic acid
AD Alzheimer’s disease
ADP Adenosine diphosphate
ATP Adenosine triphosphate
BA Betulinic acid
BAA 3-
β acetylbetulinic acid
CaCl
2
Calcium chloride
COX-1 Cyclooxgenase 1
COX-2 Cyclooxgenase 2
DMSO Dimethyl sulfoxide
EDTA Ethylenediaminetetra-acetic acid
EETs Epoxyeicosatrienoic acids
Factor II Prothrombin
Factor VII Proconvertin factor
Factor XI Plasma thromboplastin antecedent
Factor XIII Fibrin stabilizing factor
Factor XIIIa Fibrin stabilizing factor A
Fctor X Stuart- Power factor
FLAP 5-lipoxygenase-activating protein
GPIb/IX Platelet glycoprotein GPIb/IX complex
H
2
SO
4
Sulfuric acid
1-5
HEK293 Human embryonic kidney cells
HEPG2 Human hepatocellular carcinoma cells
HETEs Hydroxyeicosatetraenoic acids
HPETE Hydroxyperoxyeicosatetraenoic acid
HSC Human stem cell
IC50 Inhibitory concentration with 50%
IR Infra-red Spectroscopy
LD Lethal dose
LOX Lipoxygenase
LT Leukotriene
MK Megarkaryocytes
MS Mass Spectroscopy
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
NaCl Sodium chloride
NMR Nuclear magnetic resonance spectroscopy
NMR Nuclear Magnetic Resonance
OA Oleanolic acid
OAA 3-
β acetyloleanolic acid
PDE Phosphodiesterase
PG Prostaglandin
RANTES Regulated on Activation Normal T Expressed and Secreted
ROS Reactive oxygen species
SOD Superoxide dismutase
TPO Thrombopoetin
1-6
TXA
2
Thromboxane A
2
VWf Von Willebrand factor
WHO World Health Organisation
1-7
ABSTRACT
Abnormal Platelet aggregations are implicated in the onset of cardiovascular diseases
which are the leading cause of death and disability globally. Management of
pathological platelet aggregation with medicinal plants is a promising approach in
treatment of cardiovascular diseases.
In this study, betulic acid (BA) and a mixture of betulinic acid and oleanolic acid (BA/OA)
isolated from Melaleuca bracteata leaf extract and their acetyl derivatives (3-
β
acetylbetulinic acid) (BAA), (3-
β acetylbetulinic acid and 3
-
β acetyloleanolic acid
mixture) (BAA/OAA) were investigated for their antiplatelet aggregation, anti-
inflammatory, anticoagulant, anti-oxidant and cytotoxicity activity. The compound
structures were confirmed through spectral nuclear magnetic resonance (NMR), mass
(MS) and infrared (IR) spectroscopy data analysis. The antiplatelet aggregation
activities of the compounds were evaluated against four agonists (thrombin, collagen,
adenosine diphosphate and epinephrine) used separately to induce platelet
aggregation. The ability of the compounds to separately inhibit the hydrolysis of
chromogenic substrate was used for antithrombin activity of the triterpenoids. The
release of ATP and calcium mobilization from the cytosol, as platelets aggregate, was
investigated using a commercial kit and Fura 2/AM respectively. The anti-
acetylcholinesterase activity of the triterpenes was also investigated using a commercial
kit. The compounds were fed to rats and the tail bleeding time was used to determine
the ex vivo anticoagulation activity of the triterpenoids. The anti-inflammatory activity of
the triterpenes was investigated using the cotton pellet-induced granuloma model in
rats. The homogenates from the granuloma tissues were used to determine the effect of
the test compounds on catalase (CAT) and superoxide dismutase (SOD) activities. The
in vitro effect of the triterpenes on cyclooxygenase COX-1 and COX-2 activity was
investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
cytotoxicity assay was used to investigate the cytotoxic effect of the triterpenoids
against carcinoma (HEPG2) and human embryonic (HEK293) cell lines.
All the test compounds exhibited significant anti-platelet aggregation activity, albeit to
1-8
different degrees of efficacy. BAA showed the highest antiplatelet aggregation activity
regardless of the agonists. Coupled with its anti-platelet aggregation activity, BAA also
exhibited significant anti-inflammatory, antithrombin, acetylcholinesterase inhibition,
phosphodiesterase inhibition, calcium mobilization inhibition, inhibition of the release of
ATP from dense granules, anticoagulant, cyclooxgensase (COX-2) activity inhibition,
and iron chelating activities. BAA also significiantly stimulates SOD and CAT activity. In
addition to the efficacy, the weak cytotoxicity of triterpenoids indicated their safety as an
antiplatelet agent.
It was concluded that BAA could be served
as a template for the synthesis of safer anti-
platelet agent.
1-9
CONTRIBUTION TO KNOWLEDGE
Publications
Osunsanmi, F.O., Soyinbe, O.S. Ogunyinka, I.B.,Mosa, R.A.,Ikhile, M.I., Ngila,
J.C., Shode, F.O., Opoku, A.R. (2015). Antiplatelet aggregation and cytotoxic
activity of betulinic acid and its acetyl derivative from Melaleuca bracteata.
Journal of Medicinal Plant Research. 9(22): 647-854.
Penduka, D., Gasa, N.P., Hlongwane, M.S., Mosa, R.A., Osunsanmi, F.O., and
Opoku, A.R. (2015). The antibacterial activities of some plant-derived triterpenes.
Afri J tradit Complement Altern Med. 12(6):180-188.
Soyinbe, O.S. Myeni, C.B., Osunsanmi, F.O., Lawal, O.A., Opoku, A.R. (2015).
Antimicrobial and efflux pumps inhibitory activities of Eucalyptus grandis
essential oil against respiratory tract infectious bacteria. Journal of Medicinal
Plant Research, 9(10): 343-348.
Osunsanmi, F.O., Soyinbe, O.S. Ogunyinka, I.B., Mosa, R.A., Ikhile, M.I.,
Ngila, J.C., Shode, F.O., Opoku, A.R. (2015).
Antiplatelet Aggregation Activity of
Betulinic Acid, Oleanolic Acid, Maslinic Acid and derivatives from Medicinal
Plants. Afri J tradit Complement Altern Med. 12(6):180-188. (Editorial
consideration)
Osunsanmi F. Oluwagbemiga, Soyingbe, O.S, Shode E.A, Rebamang, A.M
and Opoku A.R. Antioxidant and cytotoxic activity of Betulinic acid and
derivatives from Melaleuca bracteata var revolution gold. Journal of Natural
Products (Manuscript in preparation).
Osunsanmi F. Oluwagbemiga, Opoku A.R, Soyingbe, O.S, Antiplatelet activity
of betulinic acid and betulinic acetate extracted from Melaleuca bracteata var.
revolution gold is involved in the suppression of intracellular calcium mobilization
and ATP release. African journal of Pharmacy and Pharmacology (Manuscript
in preparation).
1-10
Conferences
Osunsanmi, F.O., Soyinbe, O.S., Shode, F.O., Opoku, A.R. Antiplatelet
Aggregation Activity of Betulinic Acid, Oleanolic Acid, Maslinic Acid and
derivatives from Medicinal Plants. SAAB Symposium, 12-16 January 2015.
Venda, South .Africa.
Osunsanmi, F.O., Soyinbe, O.S. Ogunyinka, I.B., Mosa, R.A., Ikhile, M.I., Ngila,
J.C., Shode, F.O., Opoku, A.R. Antiplatelet aggregation and cytotoxic activity of
betulinic acid and its acetyl derivative from Melaleuca bracteata var. revelovtion
gold. Global summit on Herbal and Natural remedies. October 26-27, 2015,
Chicago, USA.
Osunsanmi, F.O., Shode, F.O., Opoku, A.R. Anti-inflammatory activity of
betulinic acid and its acetyl derivative from Melaleuca bracteata. SAAB
Symposium, 10-13 January 2016. Free State, South .Africa.
1-11
Table of Contents
ACKNOWLEDGEMENTS
1-3
LIST OF ABBREVIATIONS
1-4
ABSTRACT
1-7
CONTRIBUTION
TO
KNOWLEDGE
1-9
CHAPTER ONE
1-20
1.
I
NTRODUCTION
1-20
1.1S
TRUCTURE OF THE THESIS
1-21
CHAPTER TWO
1-22
2.
L
ITERATURE REVIEW
2-22
2.1
Platelet structure
2-23
2.2
Platelet activation
2-25
2.3
Platelet receptors
2-26
2.3.1
Collagen receptors
2-27
2.3.2
Platelet CD148 receptors
2-29
2.3.3
C-type lectin-like receptor 2 (CLEC-2)
2-29
2.3.4
Platelet integrin αII
b
β
3
receptor
2-29
2.3.5
Integrin α
v
β
3
2-30
2.3.6
P2Y1 receptors
2-30
2.3.7
P2Y12 receptors
2-30
2.3.8
P2X1 receptors
2-31
2.3.9
Thromboxane (TXA
2
) receptors
2-31
2.3.10
Prostaglandin E
2
(PGE
2
) receptors
2-31
2.3.11
Prostaglandin I
2
(PGI
2
)
2-32
2.3.12
Thrombin receptors
2-32
2.3.13
Eph kinases
2-32
2.3.14
Gas 6 (growth arrest-specific gene 6)
2-33
2.3.15
P-selectin receptors
2-33
2.4
Platelet aggregation
2-35
2.5
Hemostatic system
2-37
1-12
2.6
Acetylcholinesterase
2-39
2.7
Phosphodiesterase
2-42
2.8
Inflammation
2-45
2.8.1
Cyclooxygenase (COX-1 and COX-2)
2-46
2.9
Reactive oxygen species
2-48
2.9.1
Antioxidants
2-50
2.10
Treatment of platelet aggregation
2-52
2.10.1
Aspirin
2-52
2.10.2
Clopidogrel
2-54
2.10.3
Dipyridamole
2-55
2.10.4
Ticlopidine
2-56
2.10.5
Cilostazol
2-57
2.10.6
Sarpogrelate
2-58
2.10.7
GPIIb/IIIa Receptors Antagoinst
2-59
2.10.8
Picotamide
2-59
2.10.9
Beraprost
2-60
2.10.10
Trapidil
2-61
2.11
Recent developments in platelet aggregation inhibitors
2-61
2.12
Medicinal plants in traditional medicine
2-62
2.13
Triterpenoids
2-63
2.14
Betulinic acid
2-65
2.15
Melaleuca bracteata
2-66
2.15.1
Scientific classification of Melaleuca bracteata
2-67
2.15.2
Some other Melaleuca genuses
2-67
2.15.3
Economic importance of Melaleuca bracteata var. revolution gold
2-68
2.16
Problem statement
2-68
2.17
Aims and objectives
2-69
2.17.1
Aims
2-69
2.17.2
Objectives
2-69
2.18
Research hypothesis
2-70
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CHAPTER THREE
2-71
3.
M
ATERIALS AND METHODS
3-71
3.1
Materials
3-71
3.1.1
List of equipment
3-71
3.1.2
Chemicals and reagents (see Appendix A for reagent details)
3-72
3.2
Methods (see Appendix B for details)
3-72
3.2.1
Collection and identification of plants
3-72
3.2.2
Extraction and isolation of betulinic acid
3-73
3.2.3
Preparation of betulinic derivatives
3-73
3.2.4
Isolation of betulinic and oleanolic acid
3-74
3.2.5
Preparation of betulinic and oleanolic acids derivatives
3-74
3.2.6
Structural elucidation
3-75
3.2.7
Experimental animals
3-75
3.2.8
In vitro antiplatelet aggregation study
3-76
3.2.9
Antithrombin activity (chromogenic: S2238)
3-77
3.2.10
Determination of calcium levels in cytosol
3-77
3.2.11
ATP release assay
3-78
3.2.12
Determination of phosphodiesterase activity
3-78
3.2.13
Tail bleeding time assay
3-79
3.2.14
Cytotoxity test
3-79
3.2.15
Anti- acetylcholine esterase activity of betulinic acid and its derivate
3-79
3.2.16
Iron (Fe
2+
) chelation
3-80
3.2.17
Anti-inflammatory evaluation
3-80
3.2.18
In vitro cyclooxygenase (COX-1 and COX-2) inhibition assay
3-82
3.2.19
Superoxide dismutase (SOD) activity
3-82
3.2.20
Catalase activity
3-83
3.2.21
Statistical analysis
3-83
CHAPTER FOUR
3-84
4.
R
ESULTS
4-84
4.1
Compound Identification
4-84
4.2
Cytotoxicity assay
4-90
1-14
4.3
Anti-thrombin activity
4-91
4.4
The Anti-Platelet aggregation studies
4-92
4.5
ATP Assay
4-97
4.6
The Anti-acetylcholinesterase activity
4-97
4.7
Phosphodiesterase inhibitory activity
4-99
4.8
Calcium levels in cytosol
4-100
4.9
Tail Bleeding time (ex vivo)
4-101
4.10
Anti-inflammation activity
4-102
4.11
Cyclooxygenase evaluation
4-103
4.12
Superoxide Dismutase activity
4-104
4.13
Catalase activity
4-105
4.14
Iron chelation
4-106
4.15
The microscopic pictures of platelet aggregation treated with isolated compounds
4-107
CHAPTER FIVE
4-109
5.
D
ISCUSSION
5-109
CHAPTER SIX
5-116
6.
C
ONCLUSION
6-116
6.1
Recommendation for further studies
6-116
R
EFERENCES
6-117
APPENDICES
A
PPENDIX
A
6-152
A
PPENDIX
B
6-153
A
PPENDIX
C
6-158
A
PPENDIX
D
6-195
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LIST OF TABLES
Table 4.1:
13
C-NMR (100 MHz) spectral data for 1 and 2 .......................................... 4-87
Table 4.2: 13C-NMR (100 MHz) spectral data for samples 3 and 4 .......................... 4-89
Table 4.3: The IC
50
(µg/ml) of betulinic acid and 3-
β acetylbetu
linic acid on HEK293 and
HEPG2 cells ............................................................................................ 4-90
Table 4.4: The IC
50
values of antithrombin activity in the isolated compounds........... 4-92
Table 4.5: The IC50 values of betulinic acid and 3-
β acetylbetulinic on platelet
aggregation inhibition .............................................................................. 4-92
Table 4.6: The IC
50
values of percentage phosphodiesterase inhibition of the
compounds .............................................................................................. 4-99
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