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Thrombosis Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
Department of Pharmacology, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 India
Department of Pharmacokinetics and Metabolism, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 India
Department of Pharmaceutics, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 India
d
Department of Medicinal and Process Chemistry, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 India
b
c
a r t i c l e
i n f o
Article history:
Received 28 May 2010
Received in revised form 25 October 2010
Accepted 8 November 2010
Available online 8 December 2010
Keywords:
C.oil/herbal medicament
Myocardial ischemia-reperfusion
Platelet activation
Platelet tyrosine phosphorylation
Thrombosis
a b s t r a c t
Extensive research on the mechanism of action and medicinal importance of curcumin obtained from
turmeric (Curcuma longa) has unfolded its potential therapeutic value against many chronic ailments.
Curcuma oil (C.oil), the highly lipophilic component from Curcuma longa has been documented for its
neuroprotective efcacy against rat cerebral ischemia-reperfusion injury; however its effect on myocardial
reperfusion injury remains unexplored. In the present study, effect of C.oil (500 mg/kg, po) was evaluated
against myocardial ischemia-reperfusion induced injury in the rat model. C.oil failed to confer protection
against cardiac injury, however signicant reversal of ADP induced platelet aggregation (p b 0.05) was evident
in the same animals. Moreover, collagen and thrombin induced platelet aggregation (p b 0.001) as well as
tyrosine phosphorylation of various proteins in activated platelets was also suppressed. C.oil also offered
signicant protection against collagen-epinephrine induced thromboembolism in mice as well as augmented
total time to occlusion against FeCl3 induced arterial thrombosis in rats. C.oil however had no effect on
coagulation parameters (TT, PT and aPTT) and exerted a mild effect on the bleeding time. Bioavailability of
C.oil, as assessed by monitoring ar-turmerone, ,-turmerone and curlone, was 13%, 11% and 7% respectively,
indicating high systemic exposure. Moreover, longer mean residence time (MRT) of ar-turmerone (13.2 h),
,-turmerone (11.6 h) and Curlone (14.0 h) and plasma elimination half lives in the range of 5.5 to 7.2 h
correlated with single 500 mg/kg dose regimen of C.oil. In the present study, C.oil thus seems to be an
efcacious and safe anti-platelet agent which was protective against intravascular thrombosis.
2010 Elsevier Ltd. All rights reserved.
Abbreviations: p.o., per oral; iNOS, inducible nitric oxide synthase; eNOS,
endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitric
oxide; ADP, adenosine diphosphate; PMA, 12-phorbol 13-myristate acetate; EGTA,
ethylene glycol tetraacetic acid; HRP, horseradish peroxidase; CMC, carboxymethyl
cellulose; TTC, 2,3,5-triphenyl tetrazolium chloride; CK-MB, creatine kinase-MB; MPO,
myeloperoxidase; HTA-Br, hexadecyl trimethyl ammonium bromide; PRP, platelet
rich plasma; HEPES, (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); SDS,
sodium dodecyl sulphate; PMSF, phenyl methyl sulphonyl uoride; BSA, bovine serum
albumin; MCAo, middle cerebral artery occlusion.
Corresponding author. Department of Pharmacology, Central Drug Research
Institute (CSIR), Lucknow-226001, India. Tel.: +91 522 2612411 18x4254; fax: +91
522 2623405.
E-mail address: madhu_dikshit@cdri.res.in (M. Dikshit).
0049-3848/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2010.11.007
in the plasma within 2 h of sample collection from all the groups. All
the assays were performed by using commercially available kits as per
manufacturer's instructions (Stago, France) and were measured by
using a Semi automated Coagulometer (Start4, Young Instruments,
Stago, France) [18].
1.7. Immunoblotting
Acid-citrate-dextrose was added to platelet rich plasma and was
spun at 800 g for 10 min. Platelets were then washed twice with
buffer (20 mM HEPES, 138 mM NaCl, 2.9mMKCl, 1mMMgCl2 ,
0.36 mM NaH2PO4, 1 mM EGTA, 4.77 mM trisodium citrate, and
2.35 mM citric acid, 5mMglucose and Apyrase 1U/ml, pH 6.5) and
the cells were nally suspended in the HEPES-buffered Tyrode
solution (pH 7.4) at 2 108 platelets/mL [19].
Platelet activation was triggered in washed platelets by the
addition of ADP, collagen or thrombin. The reaction was stopped by
the sample buffer (2% SDS, 0.062 M Tris-HCl, 0.01% Bromophenol blue,
10% glycerol, 20% -mercaptoethanol, pH 6.8) [20] containing 2 mM
PMSF, 10 mM sodium uoride and 1 mM sodium orthovanadate. The
samples were immediately boiled for 3 min and were run on SDSPAGE (8%), transferred on a nitrocellulose membrane (Bio-Rad,
Hercules, CA), blocked with TBST (10 mM Tris-base, 100 mM NaCl,
and 0.01% Tween 20) containing 5% BSA for 1 h and then probed with
the primary antibodies for 2 h: anti-p-Tyr (PY20:4 G10 1:1) and anti-actin (diluted 1:10000 in TBST). Membranes were washed,
incubated with horseradish peroxidase-linked anti-mouse IgG (diluted 1:10000 in TBST) for 2 h and immunoreactive bands were detected
by enhanced chemiluminescence.
1.8. Antithrombotic efcacy of C.oil
1.8.1. Collagen-epinephrine induced thrombosis in mice
To assess the antithrombotic efcacy of C.oil, mice were divided
into vehicle, aspirin and C.oil treated groups, and each group included
ten animals. Pulmonary thromboembolism was induced by injecting a
mixture of collagen (150 g/ml) and adrenaline (50 g/ml) into the
tail vein to achieve nal doses of collagen (1.5 mg/kg) and adrenaline
(0.5 mg/kg) to induce hind limb paralysis or death. Results have been
reported as percentage protection, which represents protection
against collagen and epinephrine induced thrombosis and expressed
as [18]:
Percent Protection = 1Ptest = Pcontrol 100
Ptest - number of animals paralyzed/dead in test compound-treated
group; Pcontrol - number of animals paralyzed/dead in vehicle treated
group.
1.8.2. Ferric chloride induced arterial thrombosis in rats
SD rats were anesthetized by urethane (1.25 g/kg, ip), carotid
artery was carefully dissected and a pulsed Doppler Probe (DBF-120ACPx, CBI-8000, Crystal Biotech, USA) was placed to record the blood
ow. A square (1 1mm) piece of Whatmann Chromatography paper
was immersed in 20% FeCl3 solution for 5 min and placed on the
carotid artery and blood ow was monitored. Thrombosis was
monitored as the reduction in carotid artery blood ow and the
time at which the blood-ow velocity was reduced to zero was
recorded as time to occlusion (TTO) [21].
1.8.3. Bleeding time in mice
The tail of mice (approximately 2 mm from tip) was incised with a
sharp razor blade. The time elapsed from the tip incision to the
stoppage of bleeding was determined as the bleeding time. The
change in bleeding time was compared from that of vehicle treated
113
mice and results have been depicted as percent increase from control
[18].
1.8.4. Plasma pharmacokinetics
The in vivo oral pharmacokinetic study was performed in male SD
rats. C.oil was administered orally at a dose of 500 mg/kg. Blood
samples were collected into microfuge tubes containing heparin as an
anti-coagulant at pre-dened time intervals. Plasma was harvested by
centrifuging the blood at 13000 rpm for 10 min and stored frozen at
70 10 C until analysis. Plasma (100L) samples analysis was done
using validated LC-MS/MS method. Along with the plasma samples,
QC samples were distributed among calibrators and unknown
samples.
1.8.5. Statistical analysis
Values have been reported as the Mean SEM in control and drug
treated groups. Comparisons between the different groups were
performed by one way ANOVA and differences were considered
signicant at p b 0.05.
2. Results
2.1. Effect of C.oil on myocardial ischemia reperfusion injury in rat
TTC staining of coronary artery ligated and reperfused hearts was
used to assess the infarct area (pale white), non-infarct area (red
colored, Fig. 1A) and percent infarct size (%IS, Fig. 1B). Infarct size
following MI/RP was 21% 2% in control group, which was reduced to
8 1% with ramipril pre-treatment (p b 0.01, Fig. 1B). However C.oil
(500 mg/kg (p.o. for 3 days) or aspirin (30 mg/kg) treated rats
exhibited no signicant reduction in infarct area in MI/RP group
(Fig. 1B).
Ischemia-reperfusion mediated cell death was assessed by
measuring creatine kinase-MB (CK-MB) activity. A signicant increase
in serum CK-MB activity was observed after myocardial ischaemiareperfusion (450 40 Vs 87 6 U/L, Fig. 1C). Administration of
ramipril prevented rise of CK-MB activity (254 11 U/L), while C.oil
as well as aspirin failed to prevent the elevation in CK-MB following
MI/RP (Fig. 1C).
The accumulation of polymorphonuclear leucocytes (PMNs) was
monitored by measuring MPO activity in infarct and non-infarct
zones. MPO activity in the vehicle treated ischemic zone was 103
2 M/min/100 mg tissue, which was signicantly more (p b 0.001) to
the sham-operated group 18 2 M/min/100 mg tissue. Ramipril
treatment blunted the increase in MPO activity (53 3 M/min/
100 mg tissue), while C.oil or aspirin (104 3 M/min/100 mg tissue)
did not prevent rise in the MPO activity Fig. 1D, suggesting that C.oil
failed to offer cardioprotection against MI/RP injury in the rats.
2.2. Effect of C.oil on platelet activation and coagulation cascade
following MI/RP
ADP induced aggregagtion was signicantly more (pb 0.05) in MI/RP
rats (55 3%) as compared to vehicle treated sham operated rats
(42.0 2%, Fig. 2A). Moreover, the observed platelet hyperactivation in
MI/RP rats was persistent even in washed platelet suspension in absence
of any plasma components. ADP induced aggregation in washed
platelets of MI/RP and sham operated rats was 69 2% and 55 4%
(Pb 0.05) respectively (data not shown). Both the standard drugs,
aspirin and ramipril were also effective in restoring the platelet function
to those of sham operated groups (Fig. 2A). C.oil (500 mg/kg)
pretreatment signicantly reduced ADP (5 M) induced platelet
aggregagtion in PRP in comparison to the vehicle treated MI/RP controls,
indicating that C.oil might exhibit a specic anti-platelet mechanism of
action.
Fig. 1. Effect of C.oil in myocardial ischemia-reperfusion mediated cardiac injury (A) TTC stained sections of the heart obtained from vehicle, MI/RP, ramipril (3 mg/kg), aspirin
(30 mg/kg), and C.oil (500 mg/kg) treated rats followed by coronary artery ligation and reperfusion. Bar diagrams representing (B) percent infarct size. (C) Serum CK-MB level.
(D) Myeloperoxidase activity in myocardial ischemic zone (left ventricle) following coronary artery ligation and reperfusion. Results are expressed as Mean SEM. *p b 0.05,
***p b 0.001 vs Sham, ###p b 0.001 vs MI/RP. (n = 8-10 animals/group).
Fig. 2. Effect of C.oil on platelet aggregation and coagulation cascade in rats, ex vivo. Bar diagram representing (A) ADP induced platelet aggregation, (B) Prothrombin time, in rats
pretreated with vehicle, ramipril (3 mg/kg), aspirin (30 mg/kg), and C.oil (500 mg/kg) following coronary artery ligation and reperfusion and, (C) platelet aggregation induced by
various agonists in rats pretreated with vehicle, C.oil (500 mg/kg) and C.oil (1 g/kg), p.o., 1 h, (ex vivo). Results are expressed as Mean SEM. (*p b 0.05, **p b 0.01 & ***p b 0.001 vs
control, #p b 0.05 vs MI/RP). (n = 6 animals/group).
115
Fig. 3. Effect of C.oil on protein tyrosine phosphorylation.Washed platelets from control and treated rats were stimulated with (A) ADP (10 M), thrombin (1U/ml) and (B) collagen
(10 g/ml) for 1 min and samples were lysed at different time points. Protein tyrosine phosphorylation was detected by immunoblotting with anti-phosphotyrosine 4 G10 and PY20.
The blots shown are representative of three separate experiments.
Fig. 4. Effect of C.oil on arterial thrombosis and hemostasis in animal models. Bar diagram representing (A) percent protection in collagen-epinephrine induced pulmonary
thromboembolism in mice pretreated with Aspirin (30 mg/kg), C.oil (500 mg/kg) and C.oil (1 g/kg) p.o.1, 2 and 24 h. (n = 40 animals/group) (B) Total time to occlusion in FeCl3induced arterial thrombosis in rats pretreated with Aspirin (30 mg/kg), Ticlopidine (200 mg/kg), C.oil (500 mg/kg) and C.oil (1 g/kg) (n = 8-10 animals/group) (C) Bleeding time in
mice treated with Aspirin (30 mg/kg), C.oil (500 mg/kg, p.o.,1 h), C.oil (500 mg/kg, p.o., 24 h) and C.oil (1 g/kg, p.o., 1 h). Results are expressed as Mean SEM (*p b 0.05 from aspirin
treated group).
Table 1
Quantication of bisabolane sesquiterpenoid markers in C.oil by HPLC- tandem mass
spectometry.
Marker-1
Marker-2
Marker-3
Ar-turmerone (%)
,-turmerone (%)
Curlone (%)
21 to 25
20-28
2.3-3.0
117
Fig. 5. Plasma concentration time prole of (A) Ar-turmerone, (B) ,-Turmerone (C) Curlone, after administration of single dose of C.oil (500 mg/kg) in rats by oral route. Results at
each time point are expressed as Mean SEM.
nearly 45 to 55% of total oil, and are supposedly responsible for its broad
range of therapeutic benets [7,8]. Ar-turmerone has been shown to
inhibit collagen (IC50, 14.4 M) and arachidonic acid (IC50, 43.6 M)
induced platelet aggregation [33]. However, C.oil collectively reduced
thrombin, collagen and ADP induced aggregation, while it was not
effective against AA, PMA or A23187, suggesting that the overall antiplatelet effect of C.oil was possibly at the membrane/receptor level,
unlike curcumin, which modulates cycloxygenase (COX) activity. It also
seems that apart from ar-turmerone other constituents might also
possess platelet inhibitory activity, necessitating detailed exploration of
C.oil components for their anti-platelet effects. Platelet stimulation with
ADP, collagen and thrombin, phosphorylated multiple proteins at