Thrombosis Research 127 (2011) 111–118

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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

Anti-platelet effects of Curcuma oil in experimental models of myocardial

ischemia-reperfusion and thrombosis
Prem Prakash a, Ankita Misra a, William R. Surin a, Manish Jain a, Rabi S. Bhatta b, Raghvendra Pal c,
Kanwal Raj d, Manoj K. Barthwal a, Madhu Dikshit a,⁎
a
Department of Pharmacology, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) – 226001 India
b
Department of Pharmacokinetics and Metabolism, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) – 226001 India
c
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

a r t i c l e i n f o a b s t r a c t

Article history: Extensive research on the mechanism of action and medicinal importance of curcumin obtained from
Received 28 May 2010 turmeric (Curcuma longa) has unfolded its potential therapeutic value against many chronic ailments.
Received in revised form 25 October 2010 Curcuma oil (C.oil), the highly lipophilic component from Curcuma longa has been documented for its
Accepted 8 November 2010
neuroprotective efficacy against rat cerebral ischemia-reperfusion injury; however its effect on myocardial
Available online 8 December 2010
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
Keywords:
C.oil/herbal medicament
against cardiac injury, however significant reversal of ADP induced platelet aggregation (p b 0.05) was evident
Myocardial ischemia-reperfusion in the same animals. Moreover, collagen and thrombin induced platelet aggregation (p b 0.001) as well as
Platelet activation tyrosine phosphorylation of various proteins in activated platelets was also suppressed. C.oil also offered
Platelet tyrosine phosphorylation significant protection against collagen-epinephrine induced thromboembolism in mice as well as augmented
Thrombosis 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, α,β-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),
α,β-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
efficacious and safe anti-platelet agent which was protective against intravascular thrombosis.
© 2010 Elsevier Ltd. All rights reserved.

The diversity of traditional medicinal plants has been a fertile
ground for the source of a number of modern medicines. Besides
expanding the herbal therapeutic and preventive armamentarium,
traditional medicines also offer new avenues to identify new
pharmacophores and novel drug targets [1]. Turmeric, derived from
the rhizomes of Curcuma longa, is one of the oldest remedy used for
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 fluoride; 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).
centuries in Southeast Asia. Besides its use in Indian cooking for
flavour and food preservation, turmeric has also been used exten-
sively in Ayurvedic medicines to treat common ailments such as
stomach upset, flatulence, dysentery, ulcers, arthritis, sprains,
wounds, acnes, and skin and eye infections [2]. It is attributed with
numerous pharmacological activities including antioxidant, antimi-
crobial, anti-inflammatory and anti-proliferative properties [3,4]. The
volatile curcuma oil (C.oil) is however under scrutiny for various
biological activities. Till date, Curcuma oil has been reported for
antimicrobial, antifungal, antiviral [5], anti-inflammatory, wound
healing activity and, of late, for its potent effect against human oral
submucosal fibrosis [6]. Thus, further evaluations against various
biological activities, fractionation and identification of the mechanism
of action prior its therapeutic use are needed.
Three fractions have been isolated so far from Curcuma oil.
Fraction A is enriched with ar-turmerone and turmerone, fraction B
consists of curcumene and zingiberine, while fraction C has germacrone,
curcumerone, zedoarone, sedoarondiol, isozedoaronidiol, curcumenone,
and curlone [7,8]. Since C oil is highly lipophilic in nature its accessibility

0049-3848/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2010.11.007
112 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118
to the brain is facilitated and has been found to be protective against
stroke [9]. Neuroprotective action of C.oil is due to its action at multiple
targets that are activated in the ischemic and neurodegenerative
disorders [10]. C.oil significantly reduced expression of iNOS, nNOS and
eNOS, NO content as well as oxidative stress. Moreover, significant
inhibition of NO-induced peroxynitrite formation led to significant
reduction in the neuronal apoptosis [11]. C.oil thus seems to be promising
against neuro-cerebrovascular disorders, however, its role against
myocardial ischemia-reperfusion injury and intravascular thrombosis
remains unexplored. Since cardiovascular diseases are the most
prevalent cause of morbidity and mortality worldwide [12,13], the
present study was therefore undertaken to investigate the efficacy of C.oil
against myocardial ischemia-reperfusion injury (MI/RP) and thrombosis
in rats. The present study was also extended to assess its pharmacokinetic
properties by measuring the circulating levels of marker compounds.
1. Materials and methods
Adenosine 5’-diphosphate (ADP), thrombin, collagen, 12-phorbol
13-myristate acetate (PMA), arachidonic acid, calcium ionophore
(A23187), epinephrine, ferric chloride, heparin, apyrase, EGTA, aprotinin,
ticlopidine hydrochloride, protease inhibitors and anti-mouse HRP
conjugated secondary antibody were procured from Sigma (USA). Equine
tendon fibrillar Collagen type I was procured from Chrono-Log Corp
(USA). STA thrombin reagents, Neoplastin CI plus, Fibri-Prest, CK Prest
were purchased from Stago (France). Aspirin was obtained as a gift from
Alta Laboratories (India). Warfarin was received as a gift from Themis
(India). Anti phosphotyrosine clone PY20 and 4 G10 were obtained from
Santa Cruz biotechnology (USA) and Millipore (USA) respectively. All
other reagents used in the experiments were from Sigma-Aldrich (USA).
Reference standards of ar-turmerone, α,β-turmerone, Curlone and DHP
(Internal Standard) were provided by the Medicinal and Process
Chemistry Division of CDRI, Lucknow, India. High-performance liquid
chromatography (HPLC) grade methanol and acetonitrile were purchased
from Sisco Research Laboratories Pvt. Limited (Mumbai, India). AQ11
Glacial acetic acid AR was purchased from E Merck Limited (Mumbai,
India). Heparin sodium injection I.P. (1000 IU/mL) was purchased from
Gland Pharma (Hyderabad, India).
1.1. Instrumentation
The samples were analyzed on an API-4000 mass spectrometer
with electrospray ionization and a triple quadrupole analyzer
(Applied Biosystems, Toronto, Canada) coupled with HPLC system
consisting of Series 200 pumps and auto sampler with temperature
controlled Peltier-tray and degasser (Perkin- Elmer instruments,
Norwalk, USA) was used to inject 40 μL aliquots of the processed
samples. The chromatographic separation was performed on a
Brownlee C18 column (50 × 4.5 mm, 5.0 μm) with guard column of
the same make at the ambient temperature. Mobile phase consists of
acetonitrile and Milli-Q water (90:10 v/v) at a flow rate of 0.4 ml/min.
Mobile phase was duly filtered through 0.22 μm Millipore filter
(Billerica, USA) and degassed ultrasonically for 15 min prior to use.
Chromatographic runs were performed at room temperature by
injecting 20 μL of the test samples. Auto-sampler carry-over effect was
determined by injecting the highest calibration standard followed by
an injection of a blank sample.
1.2. Animals
Male Sprague Dawley/Wistar rats (220-300 g) and Swiss albino
mice (20-25 g) were kept in polypropylene cages and maintained at
24 ± 0.5 °C, 12 h day/night cycle and were provided with chow pellets
and water ad libitum. Prior approval from the Institutional Animal
Ethics Committee (IAEC) was sought for maintenance, experimental
studies, euthanasia and disposal of carcass of animals. All the
procedures involved were subject to IAEC guidelines.
1.3. Myocardial Ischemia Reperfusion injury in rat
Wistar rats were divided into vehicle (0.25% CMC) treated sham,
vehicle treated MI/RP, ramipril (3 mg/kg), aspirin (30 mg/kg) or C.oil
(500 mg/kg) treated MI/RP groups, and each group consisted of at
least six rats. All the drugs, compounds or vehicle were administered
orally for 3 days and the animals were subjected to MI/RP procedure.
Rats were anesthetized with ketamine (80 mg/kg) and xylazine
(10 mg/kg). The animals were tied in the supine position on the
temperature-controlled pad and the animals were ventilated with a
rodent ventilator (Harvard Apparatus, England) at tidal volume of
1 ml /100 g body weight and rate of 80 breaths/min. Myocardial
ischemia was produced by one stage occlusion of the left anterior
descending coronary artery (LAD), 3–4 mm from its origin. The
animals then underwent 30 min of ischemia and the myocardium was
reperfused by releasing the suture for a period of 180 min. Successful
reperfusion was confirmed by the visualization of arterial blood flow
through the artery [14], while Sham operated animals were used as
control. At the end of reperfusion period (180 min), animals were
sacrificed for biochemical and histological studies.
1.4. Assessment of Infarct Size
The hearts were cut transversely across the left ventricle to obtain
slices ~0.1 cm in thickness. Slices were placed in 1% pre-warmed TTC at
37 °C for 45 min followed by rinsing with distilled water to remove any
traces of TTC. The Viable tissue stains pale red and the dead tissue
remains uncolored. Infarct size was observed under surgical microscope
(Leica) and quantified (Leica Qwin software). Percentage infarct size
(%IS) is the percentage area of whole section of myocardium that
stained with TTC [15].
1.5. Biochemical Estimation
CK-MB concentration in serum was analyzed as per manufac-
turer's protocol (Merck). The change in absorbance (ΔA) per min was
measured spectrophotometrically using SHIMADZU UV-Visible Spec-
trophotometer (UV-1201) at 340 nm.
Cardiac tissue was weighed, snap frozen and homogenized in
potassium phosphate buffer, followed by mixing with 1% HTA-Br and
was left overnight at 4 °C, subsequently centrifuged at 12000 rpm for
10 min at the room temperature. MPO activity was measured by adding
50 μl of supernatant to a reaction mixture containing O-dianisidine
dihydrochloride (7.09 mM) and H2O2 (44 mM) and potassium phos-
phate buffer (50 mM, pH 6). The change in absorbance (ΔE) was
measured at 1 min interval over 5 min at 460 nm at 37 °C by using molar
coefficient [ε = 10,062 × (M × cm) -1 ] for oxidized O-dianisidine
dihydrochloride [16].
1.6. Platelet aggregation and Coagulation parameters
Rats were anaesthetized under anesthetic ether and blood (9 ml)
was drawn by cardiac puncture and mixed with 1 ml of 1.9% tri- Sodium
citrate. Blood was centrifuged at 300 g for 20 min and the platelet rich
plasma (PRP) was collected for aggregation and immunoblotting
studies. Platelet aggregation in rat PRP was monitored according to
the protocol described earlier [17]. Aggregation was induced by
adenosine-5’-diphosphate (ADP (10 μM)), thrombin (0.64U/ml),
collagen (10 μg/ml), calcium ionophore A23187 (2.5 μg/ml) or PMA
(1.5 μM) and was monitored on a dual channel aggregometer
(Chrono log Corp.,US). At least 6 animals were used for each group.
Coagulation parameters, prothrombin time (PT), activated partial
thromboplastin time (aPTT) and thrombin time (TT) were measured
 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118
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 finally 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% β-mercaptoethanol, pH 6.8) [20] containing 2 mM
PMSF, 10 mM sodium fluoride and 1 mM sodium orthovanadate. The
samples were immediately boiled for 3 min and were run on SDS-
PAGE (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-
β-actin (diluted 1:10000 in TBST). Membranes were washed,
incubated with horseradish peroxidase-linked anti-mouse IgG (dilut-
ed 1:10000 in TBST) for 2 h and immunoreactive bands were detected
by enhanced chemiluminescence.
1.8. Antithrombotic efficacy of C.oil
1.8.1. Collagen-epinephrine induced thrombosis in mice
To assess the antithrombotic efficacy 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 μg/ml) and adrenaline (50 μg/ml) into the
tail vein to achieve final 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 = ð1−Ptest = Pcontrol Þ × 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-120A-
CPx, CBI-8000, Crystal Biotech, USA) was placed to record the blood
flow. 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 flow was monitored. Thrombosis was
monitored as the reduction in carotid artery blood flow and the
time at which the blood-flow 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-defined 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 (100μL) 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
significant 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 significant 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 significant increase
in serum CK-MB activity was observed after myocardial ischaemia-
reperfusion (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 μM/min/100 mg tissue, which was significantly more (p b 0.001) to
the sham-operated group 18 ± 2 μM/min/100 mg tissue. Ramipril
treatment blunted the increase in MPO activity (53 ± 3 μM/min/
100 mg tissue), while C.oil or aspirin (104 ± 3 μM/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 significantly 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 significantly reduced ADP (5 μM) induced platelet
aggregagtion in PRP in comparison to the vehicle treated MI/RP controls,
indicating that C.oil might exhibit a specific anti-platelet mechanism of
action.
114 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118

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).

Furthermore, significant decrease in prothrombin time was significant difference in prothrombin time among ramipril (19 ±
observed in MI/RP rats (18 ± 0.3 sec) in comparison to Sham operated 0.4 sec), aspirin (19 ± 0.2 sec), or C.oil treated (22 ± 0.6 sec) groups,
animals (21 ± 0.5 sec) following cardiac injury. However there was no in comparison to vehicle treated MI/RP rats (Fig. 2B).

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).
 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118
2.3. Effect of C.oil on platelet aggregation (ex vivo) in rats
To systematically analyse the effect of C.oil on platelet aggregation,
rats were thus treated with C.oil (500 mg/kg) and blood was collected
1 h and 24 h post oral dosing and the platelet rich plasma was
separated from the blood. C.oil significantly reduced ADP (31 ± 3%),
collagen (28 ± 7%) and thrombin (34 ± 5%) induced aggregation in
comparison to control (Fig. 2C). The inhibitory effect of C.oil on platelet
aggregation was sustained even after 24 h of C.oil administration. C oil
exerted significant inhibitory effect against ADP, collagen and thrombin
induced aggregation (pb 0.001, Fig. 2C), while PMA, calcium ionophore
(A23187) and arachidonic acid induced aggregation were not affected
(Fig. 2C).
Moreover, thrombin time (TT), prothrombin time (PT) and
activated partial thromboplastin time (aPTT) in control group and
C.oil treated groups at both 500 mg/kg and 1 g/kg dose remained
unaltered and there was no evident modification of the coagulation
cascade even after 24 h of its administration (data not shown).
2.4. Platelet protein tyrosine phosphorylation after C.oil treatment
Effect of C.oil on platelet signal transduction pathways was
eventually evaluated following ADP, thrombin and collagen mediated
platelet activation. Platelets exhibit high level of non-receptor protein
tyrosine kinase (PTK) activity [22]. The activation of platelets with
agonists such as thrombin, collagen and ADP leads to several events
(including shape change, granule secretion, binding of soluble
fibrinogen to its receptor and aggregation) that are primarily
regulated through a number of intracellular signalling proteins,
including tyrosine kinases. In the present study, collagen, ADP and
thrombin induced phosphorylation of multiple proteins in platelets
ranging from ~ 120-90, 80-85, 70-75 and 60-55 kDa after 1 min of
stimulation was monitored (Fig. 3A, B). Aspirin and C.oil (500 mg/kg
and 1 g/kg) reduced tyrosine phosphorylation of various proteins in
ADP, thrombin and collagen stimulated platelets (Fig. 3A, B). The
Fig. 3. Effect of C.oil on protein tyrosine phosphorylation.Washed platelets from control and treated rats were stimulated with (A) ADP (10 μM), thrombin (1U/ml) and (B) collagen
(10 μg/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.
115
ability of C.oil to prevent protein tyrosine phosphorylation correlated
with its potency to inhibit platelet aggregation.
2.5. Antithrombotic efficacy of C.oil
2.5.1. Collagen-epinephrine induced thrombosis in mice
Effect of C.oil was evaluated against collagen-epinephrine induced
thrombosis in mice at three time points (1, 2 and 24 h, p.o.) at 500 mg/kg
and at 1 g/kg dose. The protection obtained at 500 mg/kg, 1 h, p.o. was
43± 7%, which was comparable to the effect of aspirin (38 ± 3%).
However, at 1 g/kg the protection was significantly more in comparison
to aspirin treated group (63 ± 5% vs 38± 3%, Fig. 4A). Efficacy of C.oil
(1 g/kg) remained consistent even after 2 (60 ± 3%) and 24 h (50 ± 5%)
of its administration, while the plasma levels of its marker components
reached maximum at 2 h, suggesting that the anti-thrombotic efficacy of
C.oil was either due to some other components or the level achieved at
1 h was sufficient to protect animals against thrombosis and further
modulation in the level did not affect anti-thrombotic efficacy.
2.5.2. Ferric chloride induced thrombosis in rats
Application of ferric chloride to the adventitial surface of the
carotid artery led to the formation of a stable thrombus. Complete
cessation of blood flow in the vehicle treated control group was at
14 ± 1 min. C.oil significantly augmented the time to occlusion
(TTO) at 500 mg/kg and 1 g/kg dose (p.o., 1 h). Anti-platelet drugs
like aspirin (16 ± 1 min) and ticlopidine (13 ± 2 min) did not
enhance TTO at the doses used. Prolongation in TTO observed
after pretreatment with C.oil was 22 ± 3 min and 20 ± 2 min at
500 mg/kg and 1 g/kg (1 h) respectively (Fig. 4B). The delay in
occlusion time was significantly more than aspirin and ticlopidine
treated groups and remained consistent even after 24 h.
2.5.3. Bleeding time in mice
Bleeding time in control vehicle group was 4 ± 1 min, which was
significantly prolonged in the aspirin treated group (8± 4 min). C.oil,
after 1 h, led to 18% and 25% increase in the bleeding time at 500 mg/kg
116 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118

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 FeCl3-
induced 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).

and 1 g/kg respectively. Bleeding time remained mildly affected even
after 24 h (Fig. 4C).
2.5.4. Quantification of bisabolane sesquiterpenoid markers in C.oil
Principal component analysis of bisabolane sesquiterpenoid
markers ar-turmerone, α, β-turmerone and curlone in C.oil was done
by the HPLC-tandem mass spectrometry technique. Table 1 shows
abundance of ar-turmerone, α,β-turmerone and curlone in C.oil,
amounting to nearly 45 to 55% of total oil content.
2.5.5. Plasma Pharmacokinetics
The in vivo pharmacokinetic studies further explored the oral
bioavailability of ar-turmerone (12.66%), which was substantially
higher than α, β-turmerone (10.47%) and curlone (6.82%). Plasma
elimination half life of ar-turmerone (7.2 h) was also higher than
α, β-turmerone (5.6 h) and Curlone (6.8 h). Peak plasma levels of
ar-turmerone (386.0 ± 30.04 ng/ml), α, β-turmerone (389.7 ±
61.0 ng/ml) and Curlone (27.2 ± 2.7) were observed at 2 h (Fig. 5 A,
B, C). Overall systemic availability of ar-turmerone, α, β-turmerone and
curlone was found to be 3534.43, 2882.4 and 287.5 h*ng/mL respec-
tively and the three markers were detectable in plasma up to 48, 36 and
24 h respectively indicating their prolonged systemic availability.
3. Discussion
Curcumin and curcuma oil, the two major components isolated
from Curcuma longa L. (Zingiberaceae), exhibit important therapeutic
potential [23]. Extensive research within the past decade has
Table 1
Quantification of bisabolane sesquiterpenoid markers in C.oil by HPLC- tandem mass
spectometry.
Marker-1 Marker-2
Ar-turmerone (%) α,β-turmerone (%)
21 to 25 20-28
established curcumin as a pleotropic molecule, which is useful for
neurodegenerative, cardiovascular, pulmonary, metabolic, arthritic
and autoimmune diseases [24]. Studies using C.oil exhibited neuro-
protective effects against cerebral stroke in rat MCAo model [9–11].
However effect of C.oil was not investigated against myocardial
ischemia-reperfusion injury. In the present study, C.oil failed to
salvage the injured myocardium at the same dose which was most
protective against cerebral ischemia [11]. Curcumin was found
protective against myocardial ischemia in cats [25]. C.oil however,
failed to exhibit cardio-protective activity, as evident from unaltered
infarct size, MPO activity in the infarct tissue [26] and CK-MB release
in the serum following MI/RP in rats. The widely used antithrombotic
drug aspirin was also ineffective in ameliorating the reperfusion
mediated injury. The neuroprotective effects of C.oil on the basis of
available data might be attributed to the reduction of NOS expression,
NO mediated peroxynitrite formation, oxidative stress and neuronal
apoptosis [11]. Reports from literature in rat MI/RP model paradox-
ically indicate that unlike cerebral ischemia, induction of iNOS
prevented cardiac injury in rats [27]. Reported attenuation of NOS
expression by Coil might be the reason for its ineffectiveness in the
prevention of MI/RP injury in the rat model.
In the present study, MI/RP was accompanied with platelet
activation in response to ADP, both in the presence and absence of
plasma, indicating that the platelet activation subsequent to reperfu-
sion injury was not due to plasma derived factors. This is in close
agreement with the clinical scenario in which enhanced platelet
aggregation has been observed in patients with recent coronary
events [28,29]. Anti-platelet effect of curcumin is already well-
established in diverse experimental settings [30–32]. It was further
mimicked by C.oil in reversing ADP mediated platelet aggregation in
MI/RP model, which was comparable to the inhibitory effect conferred
by aspirin. This paved the way for detailed investigation of
antithrombotic properties of C.oil in rats after oral administration.
Marker-3 C.oil dose dependently reduced ADP, collagen and thrombin
Curlone (%) induced platelet aggregation, which indicated the presence of potent
anti-platelet compounds as its constituents. C.oil contains ar-turmerone,
2.3-3.0
α,β-turmerone and curlone as three major components amounting to
 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118 117

Fig. 5. Plasma concentration time profile of (A) Ar-turmerone, (B) α,β-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 benefits [7,8]. Ar-turmerone has been shown to
inhibit collagen (IC50, 14.4 μM) and arachidonic acid (IC50, 43.6 μM)
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 anti-
platelet 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
tyrosine residues which regulate various platelet functional responses
[34]. The tyrosine phosphorylation was significantly attenuated after
the C.oil treatment, thus demonstrating its involvement in the
modulation of specific signaling mechanisms during platelet activation.
Anti-thrombotic activity of C.oil appears to be platelet mediated as
under similar experimental conditions coagulation parameters (TT, PT
and aPTT) in the rat plasma were not altered.
The ex vivo anti-platelet property of C.oil observed in rats was
further translated and verified in animal models of thrombosis. The
time course of the antithrombotic effect of C.oil after oral adminis-
tration was studied in the mice model of collagen-epinephrine
induced pulmonary thromboembolism. The model is characterized
118 P. Prakash et al. / Thrombosis Research 127 (2011) 111–118
by the massive activation of circulating platelets and the widespread
formation of platelet thrombi in the microcirculation of the lungs
leading to the disseminated pulmonary microembolism and hind
limb paralysis [35]. Protective effect of C.oil observed after 1 h of oral
administration was similar after 2 h, when the peak plasma levels
of its individual components were achieved. Moreover significant
antithrombotic effect was observed even up to 24 h after its
administration. Therefore, further investigations pertaining to the
evaluation of antithrombotic efficacy of C.oil were conducted after 1 h
of oral administration in other experimental models. C.oil appeared to
be more potent than aspirin and ticlopidine in the ferric chloride
induced arterial thrombosis model, where the thrombus has been
characterized to be platelet rich. The arterial thrombosis model is
characterized by arterial damage and platelet deposition in the
context of elevated shear stress. Moreover, C.oil marginally prolonged
bleeding time as in stark distinction to aspirin which augmented it
almost two fold. Other prevalent anti-platelet drugs are also known to
adversely affect the bleeding time. These evidences provide intriguing
possibility that C.oil might be more relevant for pathologic thrombus
formation with less impact on normal hemostasis, an exciting
prospect for the future potential antithrombotic therapies.
The pharmacokinetic profiling in rats further revealed the extent
of absorption, higher bioavailability and prolonged systemic avail-
ability of all the three markers indicating that these might be playing
an important role in exerting broad ranging therapeutic benefits of C.
oil. Interestingly, after achieving peak plasma level at 2 h, the levels of
ar-turmerone and α, β-turmerone were maintained in the range of
100 to 135 ng/ml from 8 h to 18 h, while that of Curlone plasma levels
in this time zone ranged from 8 to16 ng/ml.
The present study thus demonstrates that C.oil mediated
antithrombotic action was primarily due to the inhibition of
platelet activation, while it had no effect on MI/RP induced injury at
the dosage used. C.oil being a rich source of numerous bioactive phyto-
chemicals needs to be characterized further by evaluating all the
components for their anti-platelet, anti-coagulant or fibrinolytic
activity, in order to establish its potential therapeutic use for
cardiovascular disease and thrombotic disorders.
Conflict of interest
The authors have no conflict of interest.
Acknowledgment
The authors are thankful to Council of Scientific and Industrial
Research (CSIR), India and Central Drug Research Institute (CDRI),
India for financial assistance. Authors wish to thank Dr. GK Jain, Head
Pharmaceutics Division CDRI, Lucknow for the help and support.
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