Abdelrahman Elnager, Abuzar Mahmoud
(2015)
Fibrinolytic and haemostatic activities of caffeic acid phenethyl ester and propolis from Malaysian stingless bee and romanian poplar in vitro.
Masters thesis, Universiti Sains Malaysia.
Abstract
Caffeic acid phenethyl ester (CAPE) is a phenolic derivative from propolis and
plants. The aims of this study include: (1) Development and validation of an in vitro
whole blood (WB) clot lysis procedure for fibrinolytic activity study, (2)
Investigation of fibrinolytic, antiplatelet and anticoagulant properties of CAPE,
Malaysian Tetratrigona itama (T.itama) and poplar propolis and (3) Determination
and quantification of CAPE compound in Malaysian T.itama propolis.
The WB clot lysis procedure was developed using a standardized unresected
retracted WB clot incubated in pooled platelet poor plasma (PPP) for varying
incubation times and successfully validated using streptokinase (SK). The fibrinolyic
activity was assessed by D-Dimer (DD), fibrin morphology by confocal microscopy
and WB clot weight. DD was measured photometrically by immuno-turbidometric
method. Fibrinolytic activity of CAPE, Malaysian T.itama and poplar propolis was
assessed by the new WB clot lysis procedure at different concentrations and different
times. Platelet activity study of CAPE was performed using different in vitro assays
including: 1) Platelet aggregation measurement by platelet aggregometry with
different types of agonists, adenosine diphosphate (ADP), arachidonic acid (AA) and
ristocetin. 2) Platelet activation markers (PAC-1 and P-selectin) expression by flow
cytometry. 3) P2Y12 receptor determination by Western blot (W.blot) technique. The
platelet study of Malaysian T.itama and poplar propolis was done by platelet
aggregometry. Thromboelastgraphy (TEG) parameters were recorded following WB
incubation with CAPE, Malaysian T.itama and poplar propolis. Quantitation of
CAPE in propolis was performed by Gas Chromatograph-Mass Spectrometer (GCMS)
analysis using constructed calibration curves on peak area versus various
concentrations of CAPE.
The mean differences of DD (μg/ml) levels were significantly different (p<0.05)
across samples incubated with different CAPE concentrations and both types of
propolis compared with normal control (PPP). The median pre and post-incubation
WB clot weights (gm) were significantly decreased for CAPE, Malaysian T.itama
and poplar propolis. Fibrin removal was observed microscopically and indicated
dose-dependent effects of CAPE compared with that of normal control at different
time. The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/ml.
The anti-platelet effect was observed by the techniques used in this study for CAPE
and both propolis. The ED50 of CAPE, Malaysian T.itama and poplar propolis
(based on platelet aggregation) was 7.31 μg/ml, 0.79 mg/ml and 0.86 mg/ml
respectively. TEG results showed fibrinolytic parameter (LY30), was significantly
different (p<0.17) from normal control samples incubated in different concentrations
of CAPE, Malaysian T.itama and poplar propolis. The antiplatelet effect of CAPE
may have contributed to the reduced maximum amplitude (MA) value of TEG assay.
The quantity of CAPE measured in Malaysian T.itama and poplar propolis were:
0.6 (0.1) and 29.7 (1.2) mg/g respectively.
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