LIST OF FIGURES
Figure 2.1 Platelet structure ....................................................................................... 2-24
Figure 2.2: Mechanism of platelet activation .............................................................. 2-25
Figure 2.3: Representative scanning electron microscopic (ScEM) showing activated
platelets (5HT and ET1) .......................................................................... 2-26
Figure 2.4: Platelet receptors-ligand interactions. Overview of well-known receptors on
platelets and mode of activation .............................................................. 2-27
Figure 2.5: Processes of platelets aggregation .......................................................... 2-36
Figure 2.6: Biology of homeostasis system ................................................................ 2-38
Figure 2.7: Mechanism of acetylchilinesterase ........................................................... 2-41
Figure 2.8: The Cyclic AMP Pathway ......................................................................... 2-44
Figure 2.9: The four major pathways for Arachidonic acid metabolism ...................... 2-46
Figure 2.10: The difference between COX-1 and COX-2 ........................................... 2-47
Figure 2.11: Oxidative stress ...................................................................................... 2-49
Figure 2.12: Diagram that shows how SOD and Catalase carry out their functions ... 2-52
Figure 2.13: Structures of Aspirin ............................................................................... 2-54
Figure 2.14: Structure of Clopidogrel.......................................................................... 2-55
Figure 2.15: Structure of Dipyridamole ....................................................................... 2-56
Figure 2.16: Structure of Ticlopidine........................................................................... 2-57
Figure 2.17: Structure of Cilostazol ............................................................................ 2-58
Figure 2.18: Structure of sarpogrelate ........................................................................ 2-59
Figure 2.19: Structure of picotamide .......................................................................... 2-60
Figure 2.20: Structure of Beraprost ............................................................................ 2-60
Figure 2.21: Structure of Trapidil ................................................................................ 2-61
Figure 2.22: Structure of squalene ............................................................................. 2-64
1-17
Figure 2.23: Structure of Cholesterol.......................................................................... 2-64
Figure 2.24: Structure of Lanostrane .......................................................................... 2-64
Figure 2.25: Structure of Cycloartane......................................................................... 2-64
Figure 2.26: Structure of Betulinic acid ....................................................................... 2-65
Figure 2.26: Melaleuca bracteata var. Revolution Gold .............................................. 2-66
Figure 3.1: Schematic diagram for synthesis of BAA ................................................. 3-74
Figure 3.2: Schematic diagram for synthesis of BAA/OAA from BA/OA ..................... 3-75
Figure 3.3: Animal model chart for inflammation evalution ......................................... 3-81
Figure 4.1: Chemical structure of BA .......................................................................... 4-84
Figure 4.2: Chemical structure of BAA ....................................................................... 4-85
Figure 4.3: Chemical structure of OA ......................................................................... 4-86
Figure 4.4: Chemical structure of OAA ....................................................................... 4-86
Figure 4.5: Antithrombin activity of the isolated compounds. ..................................... 4-91
Figure 4.6a: Platelet aggregation induced by collagen. .............................................. 4-93
Figure 4.6b: Platelet aggregation induced by ADP. .................................................... 4-94
Figure 4.6c: Platelet aggregation induced by thrombin. ............................................. 4-95
Figure 4.6d: Platelet aggregation induced by epinephrine. ........................................ 4-96
Figure 4.7: ATP activity of the isolated compounds. .................................................. 4-97
Figure 4.8: Acetylcholinesterase inhibition activity of the compounds. ....................... 4-98
Figure 4.9: Percentage phosphodiesterase inhibition activity of the compounds. ...... 4-99
Figure 4.10: The compound inhibition of Calcium levels in cytosol. ......................... 4-100
Figure 4.11: Bleeding time for the isolated compounds. ........................................... 4-101
Figure 4.12: Inflammation evaluation of the isolated compounds. ............................ 4-102
Figure 4.13: Percentage COX inhibitory activity of the isolated compounds. ........... 4-103
1-18
Figure 4.14: Percentage SOD stimulatory activity of the isolated compounds. ........ 4-104
Figure 4.15: Catalase stimulatory activity of the isolated compounds. ..................... 4-105
Figure 4.16: Percentage iron chelation of the isolated compounds. ......................... 4-106
Figure 4.17: The microscopic pictures of platelet aggregation treated with isolated
compounds (10mg/ml) at a magnification of x1500. .............................. 4-108
Figure B1: Calibration curve of SOD concentration (mg/ml) against absorbance (nm). . 6-
157
Figure C1.1a:
1
H-NMR spectrum of BA .................................................................... 6-158
Figure C1.1b:
1
H-NMR spectrum of BA .................................................................... 6-159
Figure C1.2a:
13
C-NMR spectrum of BA .................................................................. 6-160
Figure C1.2b:
13
C-NMR spectrum of BA.................................................................... 6-161
Figure C1.3a: IR spectrum of BA ............................................................................. 6-162
Figure C1.3b: IR spectrum of BA ............................................................................. 6-163
Figure C1.4a: Mass spectroscopy of BA .................................................................. 6-164
Figure C1.4b: Mass spectroscopy of BA .................................................................. 6-165
Figure C2.1a:
1
H-NMR spectrum of BAA ................................................................. 6-166
Figure C2.1b:
1
H-NMR spectrum of BAA ................................................................ 6-167
Figure C2.2:
13
C-NMR spectrum of BAA .................................................................. 6-168
Figure C2.3a: IR spectrum of BAA ........................................................................... 6-169
Figure C2.3b: IR spectrum of BAA ........................................................................... 6-170
Figure C2.4a: Mass spectroscopy of BAA ................................................................ 6-171
Figure C2.4b: Mass spectroscopy of BAA ................................................................ 6-172
Figure C3.1a:
1
H-NMR spectrum of BA/OA .............................................................. 6-173
Figure C3.1b:
1
H-NMR spectrum of BA/OA .............................................................. 6-174
Figure C3.2a:
13
C-NMR spectrum of BA/OA ............................................................ 6-175
1-19
Figure C3.2b:
13
C-NMR spectrum of BA/OA ............................................................ 6-176
Figure C3.3a: IR spectrum of BA/OA ....................................................................... 6-177
Figure C3.3b: IR spectrum of BA/OA ....................................................................... 6-178
Figure C3.4a: Mass spectroscopy of BA/OA ............................................................ 6-179
Figure C3.4b: Mass spectroscopy of BA/OA ............................................................ 6-180
Figure C3.4c: Mass spectroscopy of BA/OA ............................................................ 6-181
Figure C4.1:
1
H-NMR spectrum of BAA/OAA ........................................................... 6-182
Figure C4.2a:
13
C-NMR spectrum of BAA/OAA ........................................................ 6-183
Figure C4.2b:
13
C-NMR spectrum of BAA/OAA ........................................................ 6-184
Figure C4.3a: IR spectrum of BAA/OAA ................................................................... 6-185
Figure C4.3b: IR spectrum of BAA/OAA ................................................................... 6-186
Figure C4.3b: IR spectrum of BAA/OAA ................................................................... 6-187
Figure C4.4a: Mass spectroscopy of BAA/OAA ....................................................... 6-188
Figure C4.4b: Mass spectroscopy of BAA/OAA ....................................................... 6-189
Figure C4.4a: Mass spectroscopy of BAA/OAA ....................................................... 6-190
Figure C4.4b: Mass spectroscopy of BAA/OAA ....................................................... 6-191
Figure C4.4c: Mass spectroscopy of BAA/OAA ....................................................... 6-192
Figure C4.4d: Mass spectroscopy of BAA/OAA ....................................................... 6-193
Figure C4.4d: Mass spectroscopy of BAA/OAA ....................................................... 6-194
1-20
Chapter one
1.
Introduction
More than 17 million people die of cardiovascular diseases (such as pulmonary
hypertension, stroke, heart attacks, and angina pectoris) annually and this number is
expected to grow to more than 23.6 million by 2030 (Mozaffarian et al., 2014). A
substantial number of the deaths can be attributed to pathological platelet aggregation
which forms clots (thrombosis) within the blood vessel and disrupts the ease of blood
circulation. A clot within the vessel can break and begin to travel around the body
leading to an embolus formation (Furies and Furies, 2008). Unfortunately, most of the
currently used antiplatelet agents have been reported with undesirable side effects and
drugs resistance (Armani et al., 2009). Therefore provision of optimal protection from
thrombosis or embolism with no risk of side effects on the body system is the new
frontier of research in antiplatelet therapy. This requires the identification of agents that
can block undesired pathological thrombosis without altering the physiological
protection of homeostasis.
Natural products, which are chemical compounds or substances produced by living
organisms offer new opportunities for the treatment of antiplatelet aggregation. The
medicinal properties of various plants traditionally used to cure different ailments have
been well documented (George et al., 2001). In South Africa, which is a developing
nation, indigenous African medicinal plants are used alongside western allopathic
medicine to treat ailments (Van Wyk et al., 2004).
This present study investigated the antiplatelet aggregation activity of betulinic acid and
its acetyl derivatives from Melaleuca bracteata Revolution Gold.
1-21
1.1Structure of the thesis
This thesis consist six chapters and appendices:
Chapter One gives a brief background and motivation of the study
Chapter Two gives the literatures review and. also described the aim and objectives of
the study.
Chapter Three gives the materials and methods used to conduct all the experiments in
the study.
Chapter Four gives the results obtained from the study.
Chapter Five gives the overall discussion of the results
Chapter Six gives the conclusion obtained from the results and suggestion for further
studies.
2-22
Chapter two
2.
Literature review
Blood platelet hyperactivity is implicated in atherosclerosis plaques which are the major
cause of cardiovascular diseases such as stroke, heart attack and pulmonary
hypertension. Cardiovascular diseases are one of the leading causes of mortality
(Patrono, 2001).
Platelets, initially
called ―
dust of the blood
‖
, were discovered by James Homer Wright to
be produced by megakaryocytes from bone marrow. He used W
rights’ stain
to
distinguish the similarities in the morphology between megakaryocytes and platelets
(Kuter, 1996). In 1837, Osler described the structure of platelets, whereas Bizzozero
described their anatomy and was the first to identify megarkaryocytes in bone marrow,
but never identified them as the precursor of platelets (Kuter, 1996).. Bizzozero
recognized that platelets were responsible for hemostatic and thrombosis formation and
demonstrated that platelets adhere to ruptured endothelial blood vessels to form
aggregates (Kuter, 1996)..
Platelets are anucleated cells that have a discoid shape, with a diameter of 1- 3 µm.
They are formed from the cytoplasm of megakaryocytes (Kellie et al., 2013). The
megakaryocytes are the largest cell (50- 100 µm) in which 0.01 % of nucleated cells are
accounted for in the bone marrow (Pease, 1956). During platelet formation,
megakaryocytes undergo two major stages. In the first stage, megakaryocytes
’
DNA
replicates without cellular in a process known as endomitosis. This stage requires
megakaryocytes growth factors but takes longer days to complete. The MKs cytoplasma
proliferate and enlarge as it is filled with platelet specific granules, protein cytoskeletals
and membranes for the platelet assembly process. The second stage takes an hour for
completion, the MKs firstly remodel their cytoplasm to form proplatelets, which later
transform into preplatelets. The matured preplatelets then elongate and divide
repeatedly to form discoid platelets from their tips (Richardson et al., 2005). During the
development of Platelets, their granular contents are received from the MK cell body.
Platelets are then released into the blood circulation along with the red blood cells
2-23
(RBC) and white blood cells (WBC) (May et al., 1998) . Human platelets have a 7-9 day
life span within the blood stream after formation, whereas rodents
’ platelets
only have 4-
5 days to survive (Aster, 1967; Jackson and Edward, 1977).
2.1
Platelet structure
Platelets are anucleated cells that comprise of organelles. These organelles are divided
into three zones, each with specific functions (Figure 2.1).
The first is a peripheral zone containing glycocalyx, which is a thick coat around the
platelet membranes (Moake et al., 1988). The platelet membranes consist of a lipid
bilayer that comprises of lipoproteins, glycolipids and glycoproteins. These
glycoproteins are reported to be responsible for platelet antigenicity, cellular tissue
compatibility and blood group (Matzdorff, 2005). The platelet glycoproteins function as
receptors that aid in the transmission of external impulses through the cell membrane.
The glycoprotein Ib (GPIb) enhances platelet adherence to the sub-endothelium through
von Willebrand factor (vWF) binding. The platelet membranes house glycoprotein GP
IIb/IIIa which serves as a receptor for fibrinogen, fibronectin and vWF during high shear
to initiate platelet aggregation. They also house the receptors serotonin, ADP, thrombin,
collagen and epinephrine that further strengthen platelet aggregation.
The second zone, called the Sol-Gel zone or cytoskeleton, contains microtubules and
microfilaments. The microtubules encase each platelet, thereby giving them a discoid
shape. The microfilaments are found in the cytoplasm, and they contain contractile
protein (actin and myosin). The actin accounts for 20 - 30 % of platelet protein, whereas
myosin composes 2 - 5%.
The third zone, called organelle, is the site of most platelet metabolic activity. Platelets
have three types of storage granule; these include Alpha granules, dense granules and
lysosomes. The Alpha granules are most abundant (20 - 200 per platelet) whereas the
dense granules are only at about 2 - 10 per platelet. The storage granules have
substance with mitogenic, angiogenesis, platelet proaggregatory and vasoconstriction
stimulating effects (Hartwig, 1991). Alpha granules are composed of different
membrane proteins. Proteomic studies indicated that alpha granules contain numerous
2-24
proteins (Coppinger et al., 2004). These proteins include angiogenic, and
antiangiogenic, pro-inflammatory and anti-inflammatory, coagulant and anticoagulant,
proteases and proteases inhibitor. The contradictory action of the alpha granules
component has raised a lot of questions on how they effectively manage their biological
functions (Italiano et al., 2008; Blair and Flaumenhaft, 2009). It was reported that
different alpha granules have distinct components. The Immunofluorescence
microscopy was used to demonstrate that fibrinogen and Von Willebrand factor (vWf)
are located in different granules (Sehgal and Storrie, 2007). The dense granules contain
polyphosphates, adenine nucleotide, cations, and amines such as histamine and
serotonin. These are released during platelet activation to recruits more platelets to the
site of damaged subendothelium (Sigel and Corfu, 1996). Platelets cargo two types of
lysosome (primary and secondary lysosomes). They contain cathepsin, acid hydrolases,
CD63 and LAMP-2. They play major roles in endosomal digestion by breaking down of
substances ingested by pinocytosis and phagocytosis (Flaumenhaft, 2013).
Figure 2.1 Platelet structure (adapted from www.blogspot.co.za)
2-25
2.2
Platelet activation
In a healthy endothelium vessel, microtubules help to keep platelets inactive by
maintaining their discoid shape. The healthy endothelium releases prostacyclin
(prostaglandin I2) and inhibits the release of activating factors from platelet granules.
But, once the endothelials are insulted through rupture, platelets lose shape, adhere
and spread over the injury site to stop bleeding or initiate the healing process. Platelet
activation entails (a) platelet aggregation, (b) secretion, (c) stimulation of biochemical
pathways to produce thromboxane and other agonists that amplify more platelet
aggregations, and (d)
activation of major integrin (αIIbβ3)
and Von Willebrand factor
(vWF) receptors (Harrison et al., 1997).
Figure 2.2: Mechanism of platelet activation (adapted from Jagroop et al., 2000)
During platelet activation (Figure 2.2) biochemical events are stimulated, due to an
increase in Ca
2+
mobilization into the cytoplasm. The platelets lose their disc shape to
form finger
–
like projections (pseudopodia) from the cell peripheral and extended broad
lamellae (Jagroop et al., 2000). The flattened platelets cause the organelle and granules
to concentrate to the centre giving a
―
fried egg
‖
appearance (Figure 2.3). The
compacted platelets, along with their extended dendrites, enhance adhesion to the
damaged endothelium (Grundmann et al., 2003). The influx of Ca
2+
into the cytoplasm
2-26
are instigated by the activation of the phospholipase C pathway. The phospholipase
hydrolyzes polyphosphoinostide (P
1
P
2
) into inositotriphosphate (IP
3
) and diacylglycerol
(Knofler et al., 1998). The soluble IP3 diffuses into the cytoplasm to bind to dense
granules and release stored calcium (Escolar et al., 1999). The G-prot
eins (βγ), coupled
with serpentine receptors, also activate the phospholipase C pathway. The serpentine
receptors include ADP receptors (P
2
Y
1
and P
2
Y
12
), 5HT receptors and protease
activated receptors (PAR
1
and PAR
3
).
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