2.16 Problem statement
The diseases associated with platelet aggregation are among the leading causes of
death in the world today; these include heart attacks, stroke, pulmonary hypertension
and angina pectoris. Antiplatelet aggregation therapy treatment and prevention has
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undergone dramatic changes and improvement over the years. Various synthesized
drugs have been formulated for the treatment of diseases associated with platelet
aggregation which have been shown to be effective, but are unfortunately not without
side effects. These problems have created the need to formulate new drugs with
improved clinical safety and efficacy at a reduced cost. This study aims to investigate
the antiplatelet aggregation activities of betulinic acid and derivatives from Melaleuca
bracetata var. revolution gold.
2.17 Aims and objectives
2.17.1 Aims
This project aims to extract and isolate betulinic acid from Melaleuca bracteata var.
revolution gold , and synthesize some of its derivatives, which will be evaluated for their
antiplatelet aggregation, anti-inflammatory activities and possibly determine the
apparent mechanism of action.
2.17.2 Objectives
The objectives of this study are to:
Collect and identify Melaleuca bracteta var. revolution gold
Extract, isolate and characterize betulinic acid
Synthesize betulinic derivatives
Investigate the antithrombin properties of the compounds
Investigate the antiplatelet aggregation activities of the compounds using blood
platelets from Wistar rats
Investigate the anti-inflammatory activities of the compounds
Investigate the mechanisms of action of the compounds
Evaluate the cytotoxicity potential of the compounds using human hepatocellular
carcinoma (HepG2) and human embryonic kidney (HEK293)
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2.18 Research hypothesis
Pentacyclic triterpenes have platelet aggregation inhibitory potential, therefore Betulinic
acid and its derivatives exhibit anti platelet aggregation activities.
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Chapter three
3.
Materials and methods
The materials used for this research work are listed below and a brief methodology is
also given. The full details of reagent preparations and the methodology used are
presented in Appendix A and B respectively.
3.1
Materials
3.1.1
List of equipment
Incubator (Labcom)
Rotor evaporator (Heidolph
—
Laborota 4000)
pH meter (Hanna Instruments)
Centrifuge- 5404R Eppendorf (Merck )
Platform shaker - Labcon (Polychem supplies)
BiotekElx 808 UI plate reader (Biotek Instrument Suppliers)
U-bottom 96-well plate (Sigma)
Spectrophotometer
—
Spekol 1300 (Polychem supplies)
Barnstead or Electothermal digital melting point apparatus (Thermo Scientific)
Dissecting set (Laboratory and Scientific Equipment Company (PTY) (Lasec)
Columns of different sizes (Merck)
Nuclear magnetic resonance (Bruker)
Infrared spectroscopy (Perkin Elmer)
Ulta-violet visible spectroscopy ( Perkin Elmer)
Grinder
—
IKA (Werek)
Microwave oven
–
Defy model DMO 353
Light microscope
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3.1.2
Chemicals and reagents (see Appendix A for reagent details)
The chemical solvents and regents for this project were all of analytical grade.
3.1.2.1
Chemicals supplied by sigma-Aldrich, St Louis, MO, USA
Deuterated dimethyl sulfoxide d
6
, deuterated Chloroform, Thrombin, Adenosine
diphosphate, Citric acid, Methanol, Ethyl acetate, Glucose, Epinephrine, Dextrose,
Trizma HCl, 2-Thiobarbituric acid, Ferric chloride, Ferrozine (Benzenesulfonic acid, 4,4'-
(3-(2-pyridinyl)-1,2,4-triazine-5,6-diyl) bis- disodium salt), n
–
hexane, Ferrous chloride,
Acetic anhydride, Mercury (II) chloride, Potassium iodide, Potassium persulfate, and
Potassium ferrocyanide.
3.1.2.2
Chemicals supplied by Merck, Darmstadt, Germany
Glacial acetic acid, calcium chloride, hydrochloric acid, tri-Sodium citrate, sodium
hydroxide, sodium chloride, silica gel 60 0.040-0.063 mm (230-400 mesh ASTM), silica
gel 60 0.063-0.200 mm (70-230 mesh ASTM), sulphuric acid silica gel 60 0.2-0.5 mm
(30-70 mesh ASTM), acid purified sand, chloroform, hexane, ethylacetate, TLC
aluminium sheets 20x20 cm, and Silica gel 60 F254.
3.1.2.3
Chemicals supplied by other sources
disodium hydrogen phosphate (Lab. Consumables and Chemical Supplies)
dipotassium hydrogen phosphate (Associated Chemical Enterprises)
ammonium solution (NT Supplies)
ethylenediaminetetra-acetic acid (Associated Chemical Enterprises)
S-2238 (Chromogenix) (Instrumentation Laboratory Company)
sodium dihydrogen phosphate (Lab. Consumables and Chemical Supplies)
3.2
Methods (see Appendix B for details)
3.2.1
Collection and identification of plants
The leaves of Melaleuca bracteata var. revolution gold were harvested from trees
growing around the University of Zululand, KwaDlangezwa campus, South Africa.
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These leaves were taken to the Department of Botany, University of Zululand for
identification and voucher specimen (VN 0256) was deposited at the University
herbarium.
3.2.2
Extraction and isolation of betulinic acid
The method described by Habila and colleagues (2011) was used to extract betulinic
acid from Melaleuca bracteata var. revolution gold. The leaves were allowed to air dry at
room temperature and then extracted by cold maceration in dichloromethane (1.5 w/v)
at room temperature (5L X 3) for 24 hours. The combined filtrate was concentrated
under a reduced pressure by using the rotator evaporator at 40
o
C and allowed to air
dry at room temperature, yielding 0.6 % of the crude extract. The mass obtained from
the crude extract was washed with n-hexane (80 %) twice to remove oily materials. A
portion (5 g) of the residue was subjected to chromatographic separation on a silica gel
(60-120 mesh) column (20 x 5.5 cm) and eluted with a gradient of hexane/ethyl acetate
(8:2 to 7:3) for the isolation of betulinic acid. Eighty fractions of eluates (20 ml) were
collected and monitored with thin-layer chromatography. Similar fractions containing the
desired compound were combined. These were further concentrated by a rotator
evaporator at 40
o
C and recrystallized in methanol. The isolated compound was
characterized by spectral analysis (NMR, IR and MS).
3.2.3
Preparation of betulinic derivatives
The method described by Adrine et al (2012) was adopted, with slight modification, to
synthesize acetyl derivatives of BA (Figure 3.1). A portion of betulinic acid (2 g) was
mixed with pyridine (10 ml) and acetic anhydride (12 ml) in a round bottom flask. This
mixture was then refluxed in a fume cupboard for 8 hours at 40
0
C. Distilled water (25
ml) was used to terminate the reaction. The mixture was stirred for 45 minutes and
filtered. The filtrate was rinsed with HCl (12 %) to remove pyridine and then air-dried at
room temperature. The compound was purified by subjecting it to chromatographic
separation on silica gel (60 x 120 mesh) columns (20 x 5.5 mm) and by using the n-
hexane and acetyl acetate solvent system (8:2 to 7:3) to elute. A total of 50 fractions of
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eluates (20 ml) were collected and similar fractions (based on thin-layer
chromatography) were combined and concentrated in vacuo at 40
o
C. The compound
was recrystallized in methanol to form a yellowish powder. The spectral analysis (NMR,
IR, and MS) was used to confirm the compound.
O
H
3
C
O
CH
3
O
HO
CH
3
CH
3
CH
3
H
CO
2
H
H
CH
3
H
3
C
H
3
C
CH
2
H
O
CH
3
CH
3
CH
3
H
CO
2
H
H
CH
3
H
3
C
H
3
C
CH
2
H
OH
O
Acetic anhydride
BA
BAA
Pyridine
Figure 3.1: Schematic diagram for synthesis of BAA
3.2.4
Isolation of betulinic and oleanolic acid
The method described by Habila et al., 2011 was followed, with slight modification, to
extract a mixture of betulinic and oleanolic acid from Melaleuca bracetata var. revolution
gold. The leaves (600 g) were allowed to air dry and were extracted by cold maceration
in ethyl acetate at room temperature (5L X 3) for 72 hours. The filtrate was concentrated
on a rotator evaporator at 40
o
C and dried at room temperature (25
o
C) to yield 8 % of
the crude extract. The crude extract was defatted using n-hexane. The crude extract
(6g) was then subjected to chromatographic separation on silica gel (60x120 mesh)
columns (20 x 5.5 cm) and solvent system hexane/ethylacetate (8:2 to 7:3). A total of 46
fractions of eluates (2 ml) were collected. Similar fractions were combined by monitoring
with thin-layer chromatography. This fraction was concentrated and recrystallized with
methanol. Spectral analysis (NMR, IR, and MS) was carried out on the compounds for
confirmation.
3.2.5
Preparation of betulinic and oleanolic acids derivatives
The method described by Andrine et al., 2012, as modified and described in section
3.2.3 above, was employed to prepare the acetyl derivatives of the two acids mixtures
(Figure 3.2). The spectral analysis (NMR, IR, MS) was carried out on the compound for
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confirmation.
Figure 3.2: Schematic diagram for synthesis of BAA/OAA from BA/OA
3.2.6
Structural elucidation
All NMR experiments were conducted on a 400 MHz Bruker Ultrashield spectrometer.
BA and the BA/OA mixture were dissolved separately in a mixture (1:2) of deuterated
chloroform and methanol-d4, whereas BAA and BAA/OAA were separately dissolved
separately in deuterated chloroform. Infrared spectra were recorded with a PerkinElmer
Spectrum FTIR spectrophotometer. Mass data was run on the Agilent 1100 series
LC/MSD trap system Electrospray ionization. All solvents and reagents were purchased
from Sigma-Aldrich and were used as received. Melting points were recorded on an
Electrothermal (Thermoscientific) digital melting point apparatus and were uncorrected
3.2.7
Experimental animals
Ethical clearance (UZREC 171110-030 PGD 2014/53; see Appendix D) for
experimental animals was obtained from the Research Animal Ethic Committee (RAEC)
of the University of Zululand. The guidelines for the proper care of animals and
conducting of animal experiments were followed. Sprague
–
Dawley rats (9 weeks, 230-
260 kg) of both sexes were collected from the animal house in the Department of
Biochemistry and Microbiology, University of Zululand. The animals were housed in
C
H
3
CH
3
CH
3
CH
3
CH
3
CO
2
H
CH
3
C
H
3
H
H
H
HO
O
O
CH
3
O
C
H
3
N
C
H
3
CH
3
CH
3
CH
3
CH
3
CO
2
H
CH
3
C
H
3
H
H
H
O
O
O
H
Oleanolic acid (OA)
+
C
H
3
CH
3
CH
3
CH
3
CH
3
CO
2
H
H
H
H
H
HO
CH
2
C
H
3
C
H
3
CH
3
CH
3
CH
3
CH
3
CO
2
H
H
H
H
H
O
CH
2
C
H
3
O
O
H
3-
-acetonyloleanolic acid (OAA)
+
Betulinic acid (BA)
3-
-acetonylbeutilinic acid (BAA)
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standard cages and were maintained under standard environmental conditions (room
temperature, with 12: 12 light: dark cycle), having access to normal pellet feeds and
drinking water.
3.2.8
In vitro antiplatelet aggregation study
3.2.8.1
Preparation of compounds and derivatives for anti-platelet aggregation
study
Various concentrations of the compounds (1 mg/ml, 3 mg/ml, 5 mg/ml and 10 mg/ml)
were prepared by dissolving the compounds and their derivatives in a 1 % DMSO that
contained a few drops of tween 20.
3.2.8.2
Preparation of blood platelets
The blood platelets were obtained according to the method described by Tomita et al.
(1983), with slight modification. Eight Sprague-Dawley rats were sacrificed by cervical
dislocation, and blood (5 ml) was collected surgically from the abdominal aorta and
mixed (5:1 v/v) with an anticoagulant (acid-dextrose anticoagulant, 0.085 mM citric acid,
2 % dextrose). The blood was centrifuged at 1200 rpm for 15 minutes and at 2200 rpm
for 3 minutes consecutively. The supernatant was collected and centrifuged again at
3200 rpm for 15 minutes. The supernatant was discarded and the sediment (platelets)
re-suspended in 5 ml washing buffer (PH 6.5). The washed platelets were centrifuged
again at 3000 rpm for 15 minutes and the supernatant discarded. The platelets were
suspended in a buffer (at pH 7.4, containing 0.14 M NaCl, 15 mM Tris- HCl, 5 mM
glucose). The working solution was prepared by further diluting (1:10) the platelets with
resuspending buffer and supplementary calcium chloride (0.4 ml, 10 µl CaCl
2
).
3.2.8.3
Anti-platelet aggregation evaluation
The method of Mekhfi and co-workers (2004) was used, with slight modification. The
antiplatelet aggregation activity of the compounds and their derivatives was separately
tested on collagen (10 µg/ml), thrombin (5 µg/ml), ADP (10 µg/ml), and epinephrine (10
µg/ml) induced platelet aggregation. The platelets (200 µl) and 20 µl of the different
concentrations of the compounds and derivatives (1, 3, 5 and 10 mg/ml) were
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separately pipetted into 96-well micro plates and pre-incubated at 37
o
C for 5 minutes.
The agonist (20 µl) was then added to the mixture to induce platelet aggregation.
Platelet aggregation inhibitory activity of the compounds was determined by the Biotek
plate reader by following the changes in absorption at 415 nm for 20 minutes at 30
second intervals. DMSO (1 %) served as a negative control, whereas aspirin served as
the positive control.
3.2.9
Antithrombin activity (chromogenic: S2238)
The method described by Rob et al., (1997) was adopted, with slight modification. The
various test compounds (1, 3, 5, 10 mg/ml) were prepared for antithrombin activity by
separately solubilizing them in 5 % DMSO. These were then diluted to a final
concentration of 1 % DMSO with a Tris-HCl buffer (175 mM NaCl, 50 µl Tris-HCl, 7.5
mM EDTA, pH 7.4). A portion (50 µl) from the prepared solution was then added to
thrombin (10 µl) in 96-well plates. The mixture was left for 10 minutes at room
temperature before adding chromogenic substrate (190 µl; 0.76 M). The reaction was
read at 412 nm for 8 minutes at 1 minute intervals using the Biotek ELx 808 UI plate
reader. DMSO (2 % v/v) in saline solution was used as a negative control.
3.2.10 Determination of calcium levels in cytosol
The level of calcium in cytosol was determined with Fura-2/AM following the method of
Kim et al., (2006). The platelets were incubated with 5 µM of Fura-2/AM for 30 minutes
at 37
o
C and then washed. The Fura-2-loaded, washed platelets were pre-incubated
with the compounds (1, 3, 5 and 10 mg/ml) for 3 minutes at 37
o
C in the presence of 1
mM CaCl
2.
The Platelets were stimulated to aggregate with thrombin for 5 minutes. The
Fluorescence Spectrofluorometer was used to measure the fluorescence signals from
the platelet suspension. The fluorescence emission was determined at 510 nM, with
continuous excitation at 340nM and 380nM and changing every 0.5s.
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The following formula was used to determine the Ca
2+
:
Ca
2+
in cytosol= 224nM x (F-Fmin) / (Fmax-F)
224nM was the dissociation constant of the Fura-2-Ca
2+
complex, while F minimum and
F maximum represent the fluorescence level of intensity at very low and high intensity
concentrations and Ca
2+
concentrations respectively. Fmax was given a fluorescence
intensity at 510 nm of the Fura-2- Ca
2+
complex after the platelet suspension containing
1 mM of CaCl
2
had been solubilized by Triton X-100 (0.1 %). Fmin was the fluorescence
intensity at 510nm of the Fura-2-Ca
2+
complex after the platelet suspension containing
20 mM Tris/3 mM of EDTA had been solubilized by Triton X-100 (0.1%). F was the
fluorescence intensity of the Fura-2-complex at 510 nm after the stimulation of platelet
suspension containing 1 mM CaCl
2
with thrombin, with and without the compounds.
3.2.11 ATP release assay
Platelets were pre-incubated for 3 minutes with various concentrations of the
compounds (1, 3, 5 and 10 mg/ml) and were then stimulated to aggregate by collagen
following the method of PaI et al., (2011). The reaction was terminated and the samples
were centrifuged. The supernatants were then used for the assay. The ATP release was
measured in an Luminometer (Biotek plate reader ELx808) using the ATP assay kit.
3.2.12 Determination of phosphodiesterase activity
The phosphodiesterase inhibitory activity of the compounds was determined by
following the method of Razzell (1963), with slight modification. The reaction mixtures
consist of
50 μl of
0.5 mM p-Nph-
5′
-TMP,
100 μl
of 0.1 M TrisHCl (pH 8.9),
25 μl of
various concentrations (0.1 mg/ml, 0.5 mg/ml, 1 mg/ml) of the compound and
25 μl
of
the enzyme. The mixture was then incubated at 37°C for 15 minutes. The reaction was
stopped with 50 μl of
0.2 N NaOH. Absorbance was read at 400 nm and percentage
inhibition of the enzyme calculated. Caffeine served as a positive control.
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3.2.13 Tail bleeding time assay
Rat tail bleeding time was measured by following the method of Wang et al., (2004),
with slight modification. Twenty Sprague-Dawley rats were divided into four groups of
five each. Groups one to four were administered with the compounds (10 mg/kg, 50
mg/kg, and 250 mg/kg) and aspirin (40 mg/kg) respectively, and after 2 hours the
animals were anaesthetized with sodium pentobarbital (50 mg/kg). Anesthetized rats
were placed on a hotplate and the tails thermostated at 37
o
C. The bleeding time was
assessed by amputating 5mm of the tail tip with a scalpel and was blotted onto filter
paper every 30 seconds until the paper no longer stained with blood. The period
between amputation and when bleeding stopped was taken as bleeding time.
3.2.14 Cytotoxity test
The MTT [3-(4, 5-dimethylthiazol-2yl)-2-5-diphenyltetrazoliumbromide] cytotoxicity
proliferation assay was used to measure the toxicity of betulinic acid and its derivatives
by determining the absorbance of the cells in a culture (Mosman, 1983). The cells which
were used for this assay are the human embryonic kidney cells (HEK293) and human
hepatocellular carcinoma cells (HepG2). The cells were cultured in a 25 cm
2
flask to
confluence, which was then trypsinised and plated into 48-well plates, a 2.5 x 10
4
seeding density per well and incubated overnight at 37
o
C. Two exposure periods of 24
hours and 48 hours were chosen to determine the toxicity of betulinic acid and its
derivatives along with the positive control containing the cultured cells and the medium.
The negative control contained the medium and samples. The percentage cell growths
were calculated against the medium as a triplicate reading mean ± SD. The results were
stated as concentrations to reduce the absorbance of treated cells by 50 % with
reference to the control (untreated cells) or cancer cell growth inhibitor (D
50
) and the
lethal dose IC
50
values (µg/ml) of the compound derived from the growth curve.
3.2.15 Anti- acetylcholine esterase activity of betulinic acid and its derivate
The effects of the betulinic acid and its derivatives on acetylcholinesterase activity were
determined using acetylthiocholine kits based on manufacturer (sigma-Aldrich, St Louis,
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MO, USA) procedure.
3.2.16 Iron (Fe
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