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Int. J. Curr. Res. Biosci. Plant Biol. 2016, 3(2): 106-113
S. Arunima and T. Rajamohan (2016) / Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive Oil and
Sunflower Oil on Thrombotic Factors in Rats and In Vitro Platelet Aggregation
106
International Journal of Current Research in
Biosciences and Plant Biology
ISSN: 2349-8080 (Online) ● Volume 3 ● Number 2 (February-2016)
Journal homepage: www.ijcrbp.com
Original Research Article doi: http://dx.doi.org/10.20546/ijcrbp.2016.302.013
Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive
Oil and Sunflower Oil on Thrombotic Factors in Rats and In Vitro
Platelet Aggregation
Sakunthala Arunima and Thankappan Rajamohan*
Department of Biochemistry, University of Kerala, Thiruvananthapuram-695581, Kerala, India
*Corresponding author.
A b s t r a c t
Ar ti cle I nf o
Virgin coconut oil (VCO) extracted from fresh coconut kernel is becoming very
valuable because of its numerous beneficial properties. In the present study,
comparative effect of VCO with copra oil (CO), olive oil (OO) and sunflower oil (SFO)
on thrombotic factors and platelet aggregation were investigated. Male Sprague-Dawley
rats were fed test oils at 8% level for 45 days along with the synthetic diet. Results
demonstrated that compared to CO, a prolonged prothrombin time (PT) and activated
partial thromboplastin time (aPTT) were observed in VCO fed rats and was comparable
with OO and SFO. Supplementation of VCO reduced the coagulation factors namely
factor V, fibrin, fibrinogen and thromboxane B2 levels in plasma compared to those fed
CO, OO and SFO. Compared to other test oils, platelet aggregating tendency was also
reduced in VCO fed rats. The polyphenolic fraction (PF) isolated from VCO inhibited in
vitro platelet aggregation induced by ADP compared to PF from other oils. These results
indicated that supplementation of VCO has significant antithrombotic effect by
inhibiting the activation of platelets and coagulation factors compared to rats fed other
test oils.
Accepted: 26 January 2016
Available Online: 06 February 2016
K e y w o r d s
Coronary thrombosis
Olive oil
Platelet aggregation
Polyphenols
Thromboxane B2
Thrombotic factor
Introduction
Coronary thrombosis is a major cause of sudden cardiac
death, acute myocardial infarction, unstable angina
pectoris and silent myocardial ischaemia (Davies and
Thomas, 1984; De Wood et al., 1980; Fuster and
Chesebro, 1986; Gurfinkel et al., 1994). The thrombogenic
state arises when an imbalance exists between
procoagulant and profibrinolytic activity (Astrup, 1958;
Nossel, 1998). A significant component of
thrombogenesis is contributed by platelet and its reactivity
(Huo and Ley, 2004). Dietary fat appears to influence both
the atherosclerotic and thrombogenic components of
coronary heart disease (CHD) (Miller, 1997; Hornstra,
1980). Previous studies have shown that fatty acids affect
coagulation of blood (Leray, 2001; Tholstrup, 2003;
Nelson et al., 1997), but the effects of individual fatty
acids on haemostasis are still controversial (Hoak, 1997;
Knapp, 1997), which depends on both the fatty acid chain
length and the degree of saturation (McGregor et al., 1980;
Hornstra and Starrenburg, 1973). There is evidence that
dietary long-chain saturated fatty acids appear to increase
platelet aggregation whereas intake of short and medium-
chain fatty acids has been negatively correlated with
platelet aggregation (Takachi et al., 2004). Although
polyunsaturated fatty acids have been reported to reduce
platelet aggregation (Phang et al., 2013; Gao et al., 2013;
Moertl et al., 2011), but available evidence on this is
equivocal (Mutanen and Freese, 1996; Rand et al., 1988).
There are reports which suggest a possible relationship
between dietary fat, lipid peroxidation and thrombus
formation (Barrowcliffe et al., 1984).
Int. J. Curr. Res. Biosci. Plant Biol. 2016, 3(2): 106-113
S. Arunima and T. Rajamohan (2016) / Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive Oil and
Sunflower Oil on Thrombotic Factors in Rats and In Vitro Platelet Aggregation
107
Apart from fatty acids, antioxidant components present
in dietary oil may indirectly inhibit platelets through
scavenging of reactive oxygen species (ROS) (Petroni et
al., 1994). Previous studies from our laboratory have
reported that consumption of virgin coconut oil (VCO)
exerts significant antithrombotic effect compared to
copra oil (CO) in cholesterol fed rats. In the present
study, we investigated the comparative effect of
consumption of VCO with CO, monounsaturated fatty
acid (MUFA) rich olive oil (OO) and polyunsaturated
fatty acid (PUFA) rich sunflower oil (SFO) on
thrombotic factors and platelet aggregation in rats fed
normal diet.
Materials and methods
Chemicals
Adenosine 5′-diphosphate, factor II, fibrinogen,
hemoglobin, and other biochemicals were purchased
from Sigma Aldrich Co. (St Louis, MO, USA). All the
other chemicals used were of analytical grade.11-
dehydro TX B2 immunoassay kit was obtained from
Cayman Chemical Co. (Ann Arbor, MI, USA).
Extraction of virgin coconut oil and copra oil
The solid endosperm of mature coconut (West coast tall
variety) was crushed, made in to viscous slurry and
squeezed through cheese cloth to obtain coconut milk,
which was refrigerated for 48 hours, then subjected to
mild heating (50° C) in a thermostat oven. The obtained
VCO filtered through cheese cloth was used for the
present study (Nevin and Rajamohan, 2004). CO was
extracted from coconut meat, which was dried in sunlight
continuously for 4 days to remove moisture and the
resulting copra was pressed in a mill to obtain CO (Nevin
and Rajamohan, 2008).
Olive oil and sunflower oil
Olive oil and sunflower oil were purchased from the
local market.
Animals and diet
Male Sprague-Dawley rats (100 –120 g body weight)
bred in our department animal house were used for the
study. The animals were individually housed under
hygienic conditions in polypropylene cages in a room
maintained at an ambient temperature of 25°± 10°C with
a 12 h light and 12 h dark cycle. The rats were randomly
divided into four groups of six rats each. Each rat was
given a synthetic diet containing 8% dietary oils daily for
45 days (Table 1). Experimental groups were as follows:
Group I rats given CO, Group II rats given VCO, Group
III rats given OO and Group IV rats given SFO. All the
animal cares and procedures were according to the
guidelines of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA),
India. The entire experimental protocol was approved by
Institutional Animal Ethics Committee (IAEC),
University of Kerala. Food intake was monitored
routinely and body weight was determined weekly. After
45 days, animals were fasted overnight and sacrificed by
thiopentone sodium injection (>40 mg per kg body
weight) and the blood was collected for various
estimations.
Table 1. Formulation of synthetic diet.
Ingredients a
Group I
Group II
Group III
Group IV
Corn starch
71
71
71
71
Casein
16
16
16
16
Copra oil
8
--
--
--
Virgin coconut oil
--
8
--
--
Olive oil
--
--
8
--
Sunflower oil
--
--
--
8
Salt mixture
4
4
4
4
Vitamin mixture
1
1
1
1
a g per 100 g wet weight.
Biochemical investigation
Citrated plasma was used for the determination of
coagulation parameters viz., prothrombin time, activated
partial thromboplastin time, fibrinogen, fibrin, factor V and
11-dehydro thromboxane B2. Prothrombin time (PT) was
estimated using liquiplastin kit from Tulip Diagnostics (P)
Ltd, Goa, India (Hull et al., 1982). Activated partial
thromboplastin time (aPTT) was estimated using Liquicelin
kit from Tulip Diagnostics (P) Ltd, Goa, India (Hoffmann
Int. J. Curr. Res. Biosci. Plant Biol. 2016, 3(2): 106-113
S. Arunima and T. Rajamohan (2016) / Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive Oil and
Sunflower Oil on Thrombotic Factors in Rats and In Vitro Platelet Aggregation
108
and Meulendijk, 1978). Citrated plasma was diluted with 2
mL of isotonic saline and from this fibrin was estimated
according to the method described by King and Wootton
(1959). Plasma fibrinogen levels were estimated as
described by Clauss (1957) using fibrinogen kit from Tulip
Diagnostics (P) Ltd, Goa, India. Factor V was assayed by
the method of Daniel (1955). The concentration of 11-
dehydro thromboxane B2 in plasma was determined by EIA
kit purchased from Cayman Chemical Co.USA (Takasaki,
1991).The absorbance was read at 412 nm and
concentration of each sample was obtained from the
standard curve.
Platelet preparation and platelet aggregation test
Blood was collected in anticoagulant solution (2.4%
sodium citrate, 1.5% citric acid and 1.8% dextrose). The
ratio of the blood to anticoagulant solution was
approximately 5:1 and the platelet rich plasma (PRP) was
separated by centrifugation at 1850 rpm for 7 minutes.
PRP was centrifuged at 4500 rpm for 18 minute to
sediment the platelets (Chopra, 1999). The platelet
sediment was dispersed in washing buffer composed of
113 mM NaCl, 4.3 mM KH2PO4, 4.3 mM Na2HPO4,
24.44 mM NaH2PO4 and 5.5 mM dextrose (pH 6.5) and
the platelets were collected after centrifugation at 900 g
for 10 minutes. The platelet aggregating activity was
measured by spectrophotometric method as described by
Joseph et al. (2005).
Platelet aggregation in vitro
In vitro platelet aggregation was performed by the
method of Duttaroy and Jorgensen (2004). Polyphenols
from test oils were extracted according to the method
described previously (Arunima and Rajamohan, 2013).
250 μL of platelet suspension was incubated with 50μg
of polyphenol fraction isolated from test oils and allowed
to stand for 3 minutes. Absorbance of the sample and
control were measured at 600 nm immediately after the
addition of agonist (10 µM ADP) and at 120 minutes in a
spectrophotometer. The values were expressed as
percentage aggregation.
Statistical analysis
Statistical differences were determined using one way
ANOVA followed by Duncan's, post-hoc test to identify
the differences using SPSS 11.5 (SPSS Inc., Chicago IL,
USA). Differences of p < 0.05 were considered to be
significant. Data are reported as mean ± SEM unless
otherwise stated.
Results
Effect of VCO on blood coagulation parameters
Fig. 1 summarizes the levels of prothrombin time (PT)
and activated partial thromboplastin time (aPTT) in rats
fed test oils. A prolonged PT and aPTT were observed in
VCO fed rats when compared to those fed CO. But no
significant difference in PT and aPTT were observed
among rats fed VCO, OO and SFO. VCO supplementation
significantly (p <0.05) decreased the factor V levels when
compared to rats fed other test oils. Factor V levels were
also significantly (p <0.05) decreased in CO fed rats in
comparison to those fed OO and SFO. But
supplementation of SFO increased the factor V levels
than OO fed rats (Fig. 2). The concentration of
fibrinogen was significantly (p <0.05) decreased in VCO
fed rats when compared to other oil fed rats. Fibrinogen
levels were also found to be decreased in CO fed rats
compared to those fed OO and SFO. Rats fed OO
showed increased levels of fibrinogen compared to SFO
fed rats. The fibrin levels were also significantly
decreased in VCO fed rats compared to other oil fed rats.
But there was no significant difference in fibrin levels
among rats fed CO, OO and SFO (Table 2).
Table 2. Concentration of fibrinogen (mg/dL) and fibrin (mg/dL).
Groups
Fibrinogen
Fibrin
I
273.12 ± 27.76 a
17.61 ± 1.6 a
II
237.67± 21.69 b
15.38 ±1.55 b
III
319.68± 29.09 c
19.64 ± 1.66 a,c
IV
275.26± 28.38 d
18.32 ± 1.79 a,c
Values are mean of six rats ± SEM, values not sharing a common superscript differs significantly at p <0.05. Group I – 8% CO
fed rats; Group II – 8% VCO fed rats; Group III – 8% OO fed rats; Group IV – 8% SFO fed rats.
Int. J. Curr. Res. Biosci. Plant Biol. 2016, 3(2): 106-113
S. Arunima and T. Rajamohan (2016) / Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive Oil and
Sunflower Oil on Thrombotic Factors in Rats and In Vitro Platelet Aggregation
109
Fig. 1: Prothrombin time and activated partial thromboplastin
time. Values are mean of six rats ± SEM, values not sharing a
common superscript differs significantly at p <0.05.Group I –
8% CO fed rats; Group II – 8% VCO fed rats; Group III – 8%
OO fed rats; Group IV – 8% SFO fed rats.
Fig. 2: Concentration of factor V. Values are mean of six rats
± SEM, values not sharing a common superscript differs
significantly at p <0.05.Group I – 8% CO fed rats; Group II –
8% VCO fed rats; Group III – 8% OO fed rats; Group IV – 8%
SFO fed rats.
Fig. 3: Concentration of 11-dehydro TX B2 in plasma. Values
are mean of six rats ± SEM, values not sharing a common
superscript differs significantly at p <0.05.Group I – 8% CO
fed rats; Group II – 8% VCO fed rats; Group III – 8% OO fed
rats; Group IV – 8% SFO fed rats.
Effect of VCO on 11-dehydro thromboxane B2 in
plasma
Concentration of 11-dehydro thromboxane B2 (11-
dehydro TX B2) were significantly (p <0.05) decreased
in VCO fed rats compared to those fed other test oils.
Supplementation of CO decreased the 11-dehydro TX B2
levels when compared to rats fed OO and SFO. But there
was no significant difference in 11-dehydro TX B2 levels
among OO and SFO fed rats (Fig. 3).
Effect of VCO on platelet aggregation in vivo and
in vitro
The platelet aggregating tendency was significantly
lowered in VCO supplementation compared to other oil
fed rats (Fig. 4). There was no significant difference in
the aggregation tendency among rats fed CO and SFO.
Results from in vitro analysis indicated that polyphenol
fraction (PF) extracted from VCO significantly (p <0.05)
decreased ADP induced platelet aggregation than other
test oils. But there was no significant difference in
platelet aggregating tendency among PF isolated from
CO, OO and SFO (Fig. 5).
Fig. 4: Platelet aggregation induced by ADP in vivo. Values
are mean of six rats ± SEM, values not sharing a common
superscript differs significantly at p <0.05.Group I – 8% CO
fed rats; Group II – 8% VCO fed rats; Group III – 8% OO fed
rats; Group IV – 8% SFO fed rats.
Fig. 5: Platelet aggregation induced by ADP in vitro. Values
are mean of six experiments. Values not sharing a common
superscript differs significantly at p <0.05.Group I-Platelet rich
plasma (PRP) + ADP (10μM) + 50 μg CO PF; Group II- PRP
+ ADP (10μM) + 50 μg VCO PF; Group III- PRP + ADP
(10μM) + 50 μg OO PF; Group IV- PRP + ADP (10μM) + 50
μg SFO PF.
Int. J. Curr. Res. Biosci. Plant Biol. 2016, 3(2): 106-113
S. Arunima and T. Rajamohan (2016) / Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive Oil and
Sunflower Oil on Thrombotic Factors in Rats and In Vitro Platelet Aggregation
110
Discussion
Results obtained indicate that supplementation of VCO
had a significant beneficial effect on blood coagulation
factors in rats fed normal diet. PT and the aPTT are
global coagulation tests used to assess the coagulation
system. PT, which measures the clotting time of plasma
in the presence of thromboplastin, was determined to
assess the efficiency of extrinsic system, while, aPTT
depends on substances normally present in blood for its
activity and to assess the intrinsic pathway. Studies
suggested that short aPTT probably represent an increase
in the procoagulant potential (McKenna et al., 1977) and
were associated with an increased risk of thrombosis
(Landi et al., 1992; Gallus et al., 1973). In the present
study, a prolonged PT and aPTT were observed in VCO
fed rats when compared to CO fed rats. Studies revealed
that apart from fatty acids, the unsaponifiable
components present in dietary oils have a role in
regulating the coagulation system. There are reports that
polyphenols possess antithrombotic effects, it was
evidenced by a prolonged PT and aPTT (Kim et al.,
2012). Chemical analysis of the test oils has revealed that
VCO by wet processing retains higher amounts of
polyphenols and tocopherols than other test oils
(Arunima and Rajamohan, 2013). These higher amounts
of polyphenols present in VCO may prolong the PT and
aPTT and which may be one of the reasons for its
antithrombotic potential.
The factor V levels were found to be decreased in VCO
fed rats compared to other groups. Factor V is a 330-kDa
glycoprotein synthesized in the liver and is released in
the bloodstream as a single-chain inactive pro-cofactor.
After limited proteolysis by thrombin or factor Xa, factor
V is converted to its activated form, factor Va (Monkovic
and Tracy, 1990). The factor Va acts as a non enzymatic
cofactor of factor Xa in the conversion of prothrombin to
thrombin (Nesheim et al., 1979; Rosing et al., 1980).
The lesser levels of factor V observed in VCO fed rats
reflects reduced thrombotic risk. Moreover, low levels of
both fibrin and fibrinogen observed in VCO fed rats are
associated with decreased clotting of the blood. A raised
plasma fibrinogen concentration is a powerful predictor
of risk of fatal CHD (Miller et al., 1996). Clotting
response is enhanced by activated blood platelets and the
fibrin formed reinforces the fragile platelet mass to a
stabilized thrombus of great pathological significance
(Mosesson, 2005). Elevated fibrin concentration, lowered
prothrombin time and activated partial thromboplastin
time are an indication of hypercoagulability (Curnow
et al., 2007; McKenna et al., 1977).
Blood clotting is affected by many substances within our
body, which depends on a balance between substances
that promote coagulation and those that inhibit it (Lipe
and Ornstein, 2011). Thromboxane A2 (TX A2)
produced by activated platelets, has prothrombotic
properties, stimulating activation of new platelets as well
as increasing the platelet aggregation. There are reports
that thromboxane-dependent platelet activation enhances
cerebral thrombosis (Patrono et al., 1991). Moreover, TX
A2 acts as both a vasoconstrictor and platelet activator
(Ellis et al., 1976). TX A2 has a short half-life in the
body and is rapidly hydrolyzed to thromboxane B2 (TX
B2).11-dehydro TX B2 is a metabolite of TX B2 with a
circulating half-life (t½) of 45 minutes; its measurement
in plasma or urine will give a time-integrated indication
of TX A2 production (Catella et al., 1986). An increased
production of TX B2 might be contributory to
thrombosis (Saldeen et al., 1983). Our results revealed
significant decrease in 11-dehydro TX B2 levels in VCO
fed rats as compared to those fed other oils. There are
reports that saturated fats have an inhibitory effect on TX
B2 production (Steel et al., 1990), while, unsaturated
fatty acids viz., oleic, linoleic and arachidonic acid
enhances the production of TX B2 (Ishitsuka et al., 2004;
Muakkassa et al., 1991; Whelan et al., 1993). But
supplementation of n-3 PUFA reduces TX B2 production
(Vilaseca et al., 1990).
Blood platelets are known to play a role in the regulation
of hemostasis and thrombosis (Véricel et al., 2004) and
consequently in the major cardiovascular complications
(Massberg et al., 2002). Platelet-dependent thrombus
formation plays an essential role in the manifestation of
ischemic heart syndrome (Meade et al., 1986; Heinrich
et al., 1994). Platelets are thought to initiate a series of
intricate reactions by adhering to the injured arterial
lining, aggregating irreversibly to form a platelet plug,
and releasing vasoactive metabolites and hydrolytic
enzymes that might in turn alter both the function and
structure of the vessel (Colman, 1975). Platelets mediate
both thrombotic occlusion of the entire epicardial
coronary artery and also accumulate in the
microcirculation resulting in impairment of
microcirculation and provoking myocardial ischemia
during reperfusion (Gawaz, 2004).
Studies suggest that individual saturated fatty acids
differently affect platelet aggregation capacity (Fuhrman
et al., 1986; Renaud et al., 1986). There are reports that
chain length of fatty acids plays an important role in
platelet aggregation and the inhibitory effect on platelet
aggregation was increased with increase in chain length
Int. J. Curr. Res. Biosci. Plant Biol. 2016, 3(2): 106-113
S. Arunima and T. Rajamohan (2016) / Supplementation of Virgin Coconut Oil Compared with Copra Oil, Olive Oil and
Sunflower Oil on Thrombotic Factors in Rats and In Vitro Platelet Aggregation
111
up to C 14 (Kitagawa et al., 1984). Fatty acid analysis of
test oils revealed that oil extracted from coconut mostly
consists of short and medium chain fatty acids (Arunima
and Rajamohan, 2013). In vivo experiments have shown
that feeding medium chain triglycerides (MCT)
decreases thrombosis formation in rats (Kaunitz, 1986).
Intervention studies have shown that increased platelet
aggregation was found in linoleic acid enriched diets
(Mutanen and Freese, 1996). Apart from fatty acids,
polyphenols and tocotrienols are known to inhibit
platelet aggregation (De Lange et al., 2007; Qureshi
et al., 2011). Chemical analysis of these test oils has
revealed that VCO by wet processing contain increased
polyphenolic contents (84 mg/100 g oil), which is
significantly (p <0.05) higher than other test oils viz., CO
(64·4 mg/100 g oil), OO (75·63 mg/100 g oil) and SFO
(55·26 mg/100 g oil).
HPLC analysis of the phenolic fraction of VCO has
revealed the presence of caffeic acid, p-coumaric acid,
ferulic acid, (+)-catechin hydrate and syringic acid
compared with CO, OO and SFO (Arunima and
Rajamohan, 2013), which may have a synergistic effect
on platelet aggregation. In addition, the nonsaponifiable
fraction of VCO contains appreciably higher amounts of
antioxidants, namely vitamin E (33·12 mg/100 g oil) and
b-carotene (196mg/100 g oil) (Arunima and
Rajamohan, 2012). Increased amounts of these non-
saponifible components present in VCO may partly be
responsible for the decreased platelet aggregation
compared with other oils. The inhibitory effect of VCO
on platelet aggregation was confirmed by the in vitro
platelet aggregation assay using polyphenol fraction
isolated from test oils. A decreased aggregation rate was
observed with VCO polyphenols on ADP induced in
vitro platelet aggregation.
These results demonstrated that supplementation of VCO
have a significant antithrombotic effect compared to
those fed CO,OO and SFO, which is characterized by an
increased fibrinolytic activity as well as decreased rate
of platelet aggregation.
Conflict of interest statement
Authors declare that they have no conflict of interest.
Acknowledgement
Financial assistance in the form of a research fellowship
from the University of Kerala (grant number 5825/2009)
to S. Arunima is gratefully acknowledged; the University
of Kerala had no role in the design, analysis or writing of
this article. All authors read and approved the final
manuscript. The authors declare that there are no real or
perceived conflicts of interest.
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How to cite this article:
Arunima, S., Rajamohan, T., 2016. Supplementation of virgin coconut oil compared with copra oil,
olive oil and sunflower oil on thrombotic factors in rats and
in vitro
platelet aggregation. Int. J.
Curr. Res. Biosci. Plant Biol. 3(2), 106-113. doi: http://dx.doi.org/10.20546/ijcrbp.2016.302.013
