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Regular Article

Anti-platelet effects of Curcuma oil in experimental models of myocardialischemia-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 Indiab Department of Pharmacokinetics and Metabolism, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 Indiac Department of Pharmaceutics, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 Indiad Department of Medicinal and Process Chemistry, Central Drug Research Institute (CSIR), 1. M.G. Marg, Lucknow - (U.P) 226001 India

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

Article history:Received 28 May 2010Received in revised form 25 October 2010Accepted 8 November 2010Available online 8 December 2010

Keywords:C.oil/herbal medicamentMyocardial ischemia-reperfusionPlatelet activationPlatelet tyrosine phosphorylationThrombosis

Extensive research on the mechanism of action and medicinal importance of curcumin obtained fromturmeric (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 itsneuroprotective efcacy against rat cerebral ischemia-reperfusion injury; however its effect on myocardialreperfusion injury remains unexplored. In the present study, effect of C.oil (500 mg/kg, po) was evaluatedagainst myocardial ischemia-reperfusion induced injury in the rat model. C.oil failed to confer protectionagainst cardiac injury, however signicant reversal of ADP induced platelet aggregation (pb0.05) was evidentin the same animals. Moreover, collagen and thrombin induced platelet aggregation (pb0.001) as well astyrosine phosphorylation of various proteins in activated platelets was also suppressed. C.oil also offeredsignicant protection against collagen-epinephrine induced thromboembolism in mice as well as augmentedtotal time to occlusion against FeCl3 induced arterial thrombosis in rats. C.oil however had no effect oncoagulation parameters (TT, PT and aPTT) and exerted a mild effect on the bleeding time. Bioavailability ofC.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 hcorrelated with single 500 mg/kg dose regimen of C.oil. In the present study, C.oil thus seems to be anefcacious 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 fertileground for the source of a number of modern medicines. Besidesexpanding the herbal therapeutic and preventive armamentarium,traditional medicines also offer new avenues to identify newpharmacophores and novel drug targets [1]. Turmeric, derived fromthe rhizomes of Curcuma longa, is one of the oldest remedy used for

centuries in Southeast Asia. Besides its use in Indian cooking foravour and food preservation, turmeric has also been used exten-sively in Ayurvedic medicines to treat common ailments such asstomach upset, atulence, dysentery, ulcers, arthritis, sprains,wounds, acnes, and skin and eye infections [2]. It is attributed withnumerous pharmacological activities including antioxidant, antimi-crobial, anti-inammatory and anti-proliferative properties [3,4]. Thevolatile curcuma oil (C.oil) is however under scrutiny for variousbiological activities. Till date, Curcuma oil has been reported forantimicrobial, antifungal, antiviral [5], anti-inammatory, woundhealing activity and, of late, for its potent effect against human oralsubmucosal brosis [6]. Thus, further evaluations against variousbiological activities, fractionation and identication of the mechanismof 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 Bconsists 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

Thrombosis Research 127 (2011) 111118

Abbreviations: p.o., per oral; iNOS, inducible nitric oxide synthase; eNOS,endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitricoxide; ADP, adenosine diphosphate; PMA, 12-phorbol 13-myristate acetate; EGTA,ethylene glycol tetraacetic acid; HRP, horseradish peroxidase; CMC, carboxymethylcellulose; TTC, 2,3,5-triphenyl tetrazolium chloride; CK-MB, creatine kinase-MB; MPO,myeloperoxidase; HTA-Br, hexadecyl trimethyl ammonium bromide; PRP, plateletrich plasma; HEPES, (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); SDS,sodium dodecyl sulphate; PMSF, phenyl methyl sulphonyl uoride; BSA, bovine serumalbumin; MCAo, middle cerebral artery occlusion. Corresponding author. Department of Pharmacology, Central Drug Research

Institute (CSIR), Lucknow-226001, India. Tel.: +91 522 2612411 18x4254; fax: +91522 2623405.

E-mail address: [email protected] (M. Dikshit).

0049-3848/$ see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.thromres.2010.11.007

Contents lists available at ScienceDirect

Thrombosis Research

j ourna l homepage: www.e lsev ie r.com/ locate / th romres


to the brain is facilitated and has been found to be protective againststroke [9]. Neuroprotective action of C.oil is due to its action at multipletargets that are activated in the ischemic and neurodegenerativedisorders [10]. C.oil signicantly reduced expression of iNOS, nNOS andeNOS, NO content as well as oxidative stress. Moreover, signicantinhibition of NO-induced peroxynitrite formation led to signicantreduction in theneuronal apoptosis [11]. C.oil thus seems tobepromisingagainst neuro-cerebrovascular disorders, however, its role againstmyocardial ischemia-reperfusion injury and intravascular thrombosisremains unexplored. Since cardiovascular diseases are the mostprevalent cause of morbidity and mortality worldwide [12,13], thepresent studywas thereforeundertaken to investigate the efcacyof C.oilagainst myocardial ischemia-reperfusion injury (MI/RP) and thrombosisin rats. Thepresent studywasalsoextended toassess its pharmacokineticproperties by measuring the circulating levels of marker compounds.

1. Materials and methods

Adenosine 5-diphosphate (ADP), thrombin, collagen, 12-phorbol13-myristate acetate (PMA), arachidonic acid, calcium ionophore(A23187), epinephrine, ferric chloride, heparin, apyrase, EGTA, aprotinin,ticlopidine hydrochloride, protease inhibitors and anti-mouse HRPconjugated secondary antibodywere procured from Sigma (USA). Equinetendon brillar Collagen type I was procured from Chrono-Log Corp(USA). STA thrombin reagents, Neoplastin CI plus, Fibri-Prest, CK Prestwere purchased from Stago (France). Aspirin was obtained as a gift fromAlta Laboratories (India). Warfarin was received as a gift from Themis(India). Anti phosphotyrosine clone PY20 and 4 G10 were obtained fromSanta Cruz biotechnology (USA) and Millipore (USA) respectively. Allother reagents used in the experiments were from Sigma-Aldrich (USA).Reference standards of ar-turmerone, ,-turmerone, Curlone and DHP(Internal Standard) were provided by the Medicinal and ProcessChemistry Division of CDRI, Lucknow, India. High-performance liquidchromatography (HPLC) grademethanol andacetonitrilewerepurchasedfrom Sisco Research Laboratories Pvt. Limited (Mumbai, India). AQ11Glacial acetic acid AR was purchased from E Merck Limited (Mumbai,India). Heparin sodium injection I.P. (1000 IU/mL) was purchased fromGland Pharma (Hyderabad, India).

1.1. Instrumentation

The samples were analyzed on an API-4000 mass spectrometerwith electrospray ionization and a triple quadrupole analyzer(Applied Biosystems, Toronto, Canada) coupled with HPLC systemconsisting of Series 200 pumps and auto sampler with temperaturecontrolled Peltier-tray and degasser (Perkin- Elmer instruments,Norwalk, USA) was used to inject 40 L aliquots of the processedsamples. The chromatographic separation was performed on aBrownlee C18 column (504.5 mm, 5.0 m) with guard column ofthe same make at the ambient temperature. Mobile phase consists ofacetonitrile andMilli-Q water (90:10 v/v) at a ow rate of 0.4 ml/min.Mobile phase was duly ltered through 0.22 m Millipore lter(Billerica, USA) and degassed ultrasonically for 15 min prior to use.Chromatographic runs were performed at room temperature byinjecting 20 L of the test samples. Auto-sampler carry-over effect wasdetermined by injecting the highest calibration standard followed byan injection of a blank sample.

1.2. Animals

Male Sprague Dawley/Wistar rats (220-300 g) and Swiss albinomice (20-25 g) were kept in polypropylene cages and maintained at240.5 C, 12 h day/night cycle and were provided with chow pelletsand water ad libitum. Prior approval from the Institutional AnimalEthics Committee (IAEC) was sought for maintenance, experimental

studies, euthanasia and disposal of carcass of animals. All theprocedures 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 atleast six rats. All the drugs, compounds or vehicle were administeredorally 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 thetemperature-controlled pad and the animals were ventilated with arodent ventilator (Harvard Apparatus, England) at tidal volume of1 ml /100 g body weight and rate of 80 breaths/min. Myocardialischemia was produced by one stage occlusion of the left anteriordescending coronary artery (LAD), 34 mm from its origin. Theanimals then underwent 30 min of ischemia and themyocardiumwasreperfused by releasing the suture for a period of 180 min. Successfulreperfusion was conrmed by the visualization of arterial blood owthrough the artery [14], while Sham operated animals were used ascontrol. At the end of reperfusion period (180 min), animals weresacriced for biochemical and histological studies.

1.4. Assessment of Infarct Size

The hearts were cut transversely across the left ventricle to obtainslices ~0.1 cm in thickness. Slices were placed in 1% pre-warmed TTC at37 C for 45 min followed by rinsing with distilled water to remove anytraces of TTC. The Viable tissue stains pale red and the dead tissueremains uncolored. Infarct sizewas observed under surgicalmicroscope(Leica) and quantied (Leica Qwin software). Percentage infarct size(%IS) is the percentage area of whole section of myocardium thatstained 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 (A) per min wasmeasured spectrophotometrically using SHIMADZU UV-Visible Spec-trophotometer (UV-1201) at 340 nm.

Cardiac tissue was weighed, snap frozen and homogenized inpotassium phosphate buffer, followed by mixing with 1% HTA-Br andwas left overnight at 4 C, subsequently centrifuged at 12000 rpm for10 min at the room temperature. MPO activity wasmeasured by adding50 l of supernatant to a reaction mixture containing O-dianisidinedihydrochloride (7.09 mM) and H2O2 (44 mM) and potassium phos-phate buffer (50 mM, pH 6). The change in absorbance (E) wasmeasured at1 min interval over 5 minat460 nmat37 C byusingmolarcoefcient [=10,062(Mcm) -1 ] for oxidized O-dianisidinedihydrochloride [16].

1.6. Platelet aggregation and Coagulation parameters

Rats were anaesthetized under anesthetic ether and blood (9 ml)was drawnby cardiac puncture andmixedwith 1 ml of 1.9% tri- Sodiumcitrate. Blood was centrifuged at 300 g for 20 min and the platelet richplasma (PRP) was collected for aggregation and immunoblottingstudies. Platelet aggregation in rat PRP was monitored according tothe protocol described earlier [17]. Aggregation was induced byadenosine-5-diphosphate (ADP (10 M)), thrombin (0.64U/ml),collagen (10 g/ml), calcium ionophore A23187 (2.5 g/ml) or PMA(1.5 M) 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 partialthromboplastin time (aPTT) and thrombin time (TT) were measured

112 P. Prakash et al. / Thrombosis Research 127 (2011) 111118


in the plasma within 2 h of sample collection from all the groups. Allthe assays were performed by using commercially available kits as permanufacturer's instructions (Stago, France) and were measured byusing a Semi automated Coagulometer (Start4, Young Instruments,Stago, France) [18].

1.7. Immunoblotting

Acid-citrate-dextrose was added to platelet rich plasma and wasspun at 800 g for 10 min. Platelets were then washed twice withbuffer (20 mM HEPES, 138 mM NaCl, 2.9mMKCl, 1mMMgCl2,0.36 mM NaH2PO4, 1 mM EGTA, 4.77 mM trisodium citrate, and2.35 mM citric acid, 5mMglucose and Apyrase 1U/ml, pH 6.5) andthe cells were nally suspended in the HEPES-buffered Tyrodesolution (pH 7.4) at 2108 platelets/mL [19].

Platelet activation was triggered in washed platelets by theaddition of ADP, collagen or thrombin. The reaction was stopped bythe sample buffer (2% SDS, 0.062 M Tris-HCl, 0.01% Bromophenol blue,10% glycerol, 20% -mercaptoethanol, pH 6.8) [20] containing 2 mMPMSF, 10 mM sodium uoride and 1 mM sodium orthovanadate. Thesamples 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 withthe 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 (dilut-ed 1:10000 in TBST) for 2 h and immunoreactive bands were detectedby enhanced chemiluminescence.

1.8. Antithrombotic efcacy of C.oil

1.8.1. Collagen-epinephrine induced thrombosis in miceTo assess the antithrombotic efcacy of C.oil, mice were divided

into vehicle, aspirin and C.oil treated groups, and each group includedten animals. Pulmonary thromboembolismwas induced by injecting amixture of collagen (150 g/ml) and adrenaline (50 g/ml) into thetail 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 beenreported as percentage protection, which represents protectionagainst collagen and epinephrine induced thrombosis and expressedas [18]:

Percent Protection = 1Ptest = Pcontrol 100

Ptest - number of animals paralyzed/dead in test compound-treatedgroup; Pcontrol - number of animals paralyzed/dead in vehicle treatedgroup.

1.8.2. Ferric chloride induced arterial thrombosis in ratsSD 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 bloodow. A square (11mm) piece of Whatmann Chromatography paperwas immersed in 20% FeCl3 solution for 5 min and placed on thecarotid artery and blood ow was monitored. Thrombosis wasmonitored as the reduction in carotid artery blood ow and thetime at which the blood-ow velocity was reduced to zero wasrecorded as time to occlusion (TTO) [21].

1.8.3. Bleeding time in miceThe tail of mice (approximately 2 mm from tip) was incised with a

sharp razor blade. The time elapsed from the tip incision to thestoppage of bleeding was determined as the bleeding time. Thechange in bleeding time was compared from that of vehicle treated

mice and results have been depicted as percent increase from control[18].

1.8.4. Plasma pharmacokineticsThe in vivo oral pharmacokinetic study was performed in male SD

rats. C.oil was administered orally at a dose of 500 mg/kg. Bloodsamples were collected into microfuge tubes containing heparin as ananti-coagulant at pre-dened time intervals. Plasmawas harvested bycentrifuging the blood at 13000 rpm for 10 min and stored frozen at 7010 C until analysis. Plasma (100L) samples analysis was doneusing validated LC-MS/MS method. Along with the plasma samples,QC samples were distributed among calibrators and unknownsamples.

1.8.5. Statistical analysisValues have been reported as the MeanSEM in control and drug

treated groups. Comparisons between the different groups wereperformed by one way ANOVA and differences were consideredsignicant at pb0.05.

2. Results

2.1. Effect of C.oil on myocardial ischemia reperfusion injury in rat

TTC staining of coronary artery ligated and reperfused hearts wasused to assess the infarct area (pale white), non-infarct area (redcolored, Fig. 1A) and percent infarct size (%IS, Fig. 1B). Infarct sizefollowingMI/RP was 21%2% in control group, which was reduced to81% with ramipril pre-treatment (pb0.01, Fig. 1B). However C.oil(500 mg/kg (p.o. for 3 days) or aspirin (30 mg/kg) treated ratsexhibited no signicant reduction in infarct area in MI/RP group(Fig. 1B).

Ischemia-reperfusion mediated cell death was assessed bymeasuring creatine kinase-MB (CK-MB) activity. A signicant increasein serum CK-MB activity was observed after myocardial ischaemia-reperfusion (45040 Vs 876 U/L, Fig. 1C). Administration oframipril prevented rise of CK-MB activity (25411 U/L), while C.oilas well as aspirin failed to prevent the elevation in CK-MB followingMI/RP (Fig. 1C).

The accumulation of polymorphonuclear leucocytes (PMNs) wasmonitored by measuring MPO activity in infarct and non-infarctzones. MPO activity in the vehicle treated ischemic zone was 1032 M/min/100 mg tissue, which was signicantly more (pb0.001) tothe sham-operated group 182 M/min/100 mg tissue. Ramipriltreatment blunted the increase in MPO activity (533 M/min/100 mg tissue), while C.oil or aspirin (1043 M/min/100 mg tissue)did not prevent rise in the MPO activity Fig. 1D, suggesting that C.oilfailed to offer cardioprotection against MI/RP injury in the rats.

2.2. Effect of C.oil on platelet activation and coagulation cascadefollowing MI/RP

ADP induced aggregagtionwas signicantlymore (pb0.05) inMI/RPrats (553%) as compared to vehicle treated sham operated rats(42.02%, Fig. 2A). Moreover, the observed platelet hyperactivation inMI/RP ratswaspersistent even inwashedplatelet suspension in absenceof any plasma components. ADP induced aggregation in washedplatelets of MI/RP and sham operated rats was 692% and 554%(Pb0.05) respectively (data not shown). Both the standard drugs,aspirin and ramipril were also effective in restoring the platelet functionto those of sham operated groups (Fig. 2A). C.oil (500 mg/kg)pretreatment signicantly reduced ADP (5 M) induced plateletaggregagtion in PRP in comparison to thevehicle treatedMI/RP controls,indicating that C.oil might exhibit a specic anti-platelet mechanism ofaction.

113P. Prakash et al. / Thrombosis Research 127 (2011) 111118


Furthermore, signicant decrease in prothrombin time wasobserved inMI/RP rats (180.3 sec) in comparison to Sham operatedanimals (210.5 sec) following cardiac injury. However there was no

signicant difference in prothrombin time among ramipril (190.4 sec), aspirin (190.2 sec), or C.oil treated (220.6 sec) groups,in comparison to vehicle treated MI/RP rats (Fig. 2B).

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 MeanSEM. *pb0.05,***pb0.001 vs Sham, ###pb0.001 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 ratspretreated 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 byvarious 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 MeanSEM. (*pb0.05, **pb0.01 & ***pb0.001 vscontrol, #pb0.05 vs MI/RP). (n=6 animals/group).



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 collected1 h and 24 h post oral dosing and the platelet rich plasma wasseparated from the blood. C.oil signicantly reduced ADP (313%),collagen (287%) and thrombin (345%) induced aggregation incomparison to control (Fig. 2C). The inhibitory effect of C.oil on plateletaggregation was sustained even after 24 h of C.oil administration. C oilexerted signicant inhibitory effect against ADP, collagen and thrombininduced aggregation (pb0.001, Fig. 2C), while PMA, calcium ionophore(A23187) and arachidonic acid induced aggregation were not affected(Fig. 2C).

Moreover, thrombin time (TT), prothrombin time (PT) andactivated partial thromboplastin time (aPTT) in control group andC.oil treated groups at both 500 mg/kg and 1 g/kg dose remainedunaltered and there was no evident modication of the coagulationcascade 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 waseventually evaluated following ADP, thrombin and collagen mediatedplatelet activation. Platelets exhibit high level of non-receptor proteintyrosine kinase (PTK) activity [22]. The activation of platelets withagonists such as thrombin, collagen and ADP leads to several events(including shape change, granule secretion, binding of solublebrinogen to its receptor and aggregation) that are primarilyregulated through a number of intracellular signalling proteins,including tyrosine kinases. In the present study, collagen, ADP andthrombin induced phosphorylation of multiple proteins in plateletsranging from ~120-90, 80-85, 70-75 and 60-55 kDa after 1 min ofstimulation was monitored (Fig. 3A, B). Aspirin and C.oil (500 mg/kgand 1 g/kg) reduced tyrosine phosphorylation of various proteins inADP, thrombin and collagen stimulated platelets (Fig. 3A, B). The

ability of C.oil to prevent protein tyrosine phosphorylation correlatedwith its potency to inhibit platelet aggregation.

2.5. Antithrombotic efcacy of C.oil

2.5.1. Collagen-epinephrine induced thrombosis in miceEffect of C.oil was evaluated against collagen-epinephrine induced

thrombosis inmiceat three timepoints (1, 2 and24 h,p.o.) at 500 mg/kgand at 1 g/kg dose. The protection obtained at 500 mg/kg, 1 h, p.o. was437%, which was comparable to the effect of aspirin (383%).However, at 1 g/kg the protectionwas signicantlymore in comparisonto aspirin treated group (635% vs 383%, Fig. 4A). Efcacy of C.oil(1 g/kg) remained consistent even after 2 (603%) and 24 h (505%)of its administration, while the plasma levels of its marker componentsreachedmaximumat2 h, suggesting that theanti-thrombotic efcacyofC.oil was either due to some other components or the level achieved at1 h was sufcient to protect animals against thrombosis and furthermodulation in the level did not affect anti-thrombotic efcacy.

2.5.2. Ferric chloride induced thrombosis in ratsApplication of ferric chloride to the adventitial surface of the

carotid artery led to the formation of a stable thrombus. Completecessation of blood ow in the vehicle treated control group was at141 min. C.oil signicantly augmented the time to occlusion(TTO) at 500 mg/kg and 1 g/kg dose (p.o., 1 h). Anti-platelet drugslike aspirin (161 min) and ticlopidine (132 min) did notenhance TTO at the doses used. Prolongation in TTO observedafter pretreatment with C.oil was 223 min and 202 min at500 mg/kg and 1 g/kg (1 h) respectively (Fig. 4B). The delay inocclusion time was signicantly more than aspirin and ticlopidinetreated groups and remained consistent even after 24 h.

2.5.3. Bleeding time in miceBleeding time in control vehicle group was 41 min, which was

signicantly prolonged in the aspirin treated group (84 min). C.oil,after 1 h, led to 18% and 25% increase in the bleeding time at 500 mg/kg

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 phosphorylationwas detected by immunoblotting with anti-phosphotyrosine 4 G10 and PY20.The blots shown are representative of three separate experiments.



and 1 g/kg respectively. Bleeding time remained mildly affected evenafter 24 h (Fig. 4C).

2.5.4. Quantication of bisabolane sesquiterpenoid markers in C.oilPrincipal component analysis of bisabolane sesquiterpenoid

markers ar-turmerone, , -turmerone and curlone in C.oil was doneby the HPLC-tandem mass spectrometry technique. Table 1 showsabundance of ar-turmerone, ,-turmerone and curlone in C.oil,amounting to nearly 45 to 55% of total oil content.

2.5.5. Plasma PharmacokineticsThe in vivo pharmacokinetic studies further explored the oral

bioavailability of ar-turmerone (12.66%), which was substantiallyhigher than , -turmerone (10.47%) and curlone (6.82%). Plasmaelimination half life of ar-turmerone (7.2 h) was also higher than, -turmerone (5.6 h) and Curlone (6.8 h). Peak plasma levels ofar-turmerone (386.030.04 ng/ml), , -turmerone (389.761.0 ng/ml) and Curlone (27.22.7) were observed at 2 h (Fig. 5 A,B, C). Overall systemic availability of ar-turmerone,, -turmerone andcurlone was found to be 3534.43, 2882.4 and 287.5 h*ng/mL respec-tively and the threemarkers were detectable in plasma up to 48, 36 and24 h respectively indicating their prolonged systemic availability.

3. Discussion

Curcumin and curcuma oil, the two major components isolatedfrom Curcuma longa L. (Zingiberaceae), exhibit important therapeuticpotential [23]. Extensive research within the past decade has

established curcumin as a pleotropic molecule, which is useful forneurodegenerative, cardiovascular, pulmonary, metabolic, arthriticand autoimmune diseases [24]. Studies using C.oil exhibited neuro-protective effects against cerebral stroke in rat MCAo model [911].However effect of C.oil was not investigated against myocardialischemia-reperfusion injury. In the present study, C.oil failed tosalvage the injured myocardium at the same dose which was mostprotective against cerebral ischemia [11]. Curcumin was foundprotective against myocardial ischemia in cats [25]. C.oil however,failed to exhibit cardio-protective activity, as evident from unalteredinfarct size, MPO activity in the infarct tissue [26] and CK-MB releasein the serum following MI/RP in rats. The widely used antithromboticdrug aspirin was also ineffective in ameliorating the reperfusionmediated injury. The neuroprotective effects of C.oil on the basis ofavailable data might be attributed to the reduction of NOS expression,NO mediated peroxynitrite formation, oxidative stress and neuronalapoptosis [11]. Reports from literature in rat MI/RP model paradox-ically indicate that unlike cerebral ischemia, induction of iNOSprevented cardiac injury in rats [27]. Reported attenuation of NOSexpression by Coil might be the reason for its ineffectiveness in theprevention of MI/RP injury in the rat model.

In the present study, MI/RP was accompanied with plateletactivation in response to ADP, both in the presence and absence ofplasma, indicating that the platelet activation subsequent to reperfu-sion injury was not due to plasma derived factors. This is in closeagreement with the clinical scenario in which enhanced plateletaggregation has been observed in patients with recent coronaryevents [28,29]. Anti-platelet effect of curcumin is already well-established in diverse experimental settings [3032]. It was furthermimicked by C.oil in reversing ADP mediated platelet aggregation inMI/RPmodel, whichwas comparable to the inhibitory effect conferredby aspirin. This paved the way for detailed investigation ofantithrombotic properties of C.oil in rats after oral administration.C.oil dose dependently reduced ADP, collagen and thrombininduced platelet aggregation, which indicated the presence of potentanti-platelet compounds as its constituents. C.oil contains ar-turmerone,,-turmerone and curlone as three major components amounting to

Fig. 4. Effect of C.oil on arterial thrombosis and hemostasis in animal models. Bar diagram representing (A) percent protection in collagen-epinephrine induced pulmonarythromboembolism 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 inmice 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 asMeanSEM (*pb0.05 from aspirintreated group).

Table 1Quantication of bisabolane sesquiterpenoid markers in C.oil by HPLC- tandem massspectometry.

Marker-1 Marker-2 Marker-3

Ar-turmerone (%) ,-turmerone (%) Curlone (%)

21 to 25 20-28 2.3-3.0



nearly 45 to 55% of total oil, and are supposedly responsible for its broadrange of therapeutic benets [7,8]. Ar-turmerone has been shown toinhibit collagen (IC50, 14.4 M) and arachidonic acid (IC50, 43.6 M)induced platelet aggregation [33]. However, C.oil collectively reducedthrombin, collagen and ADP induced aggregation, while it was noteffective against AA, PMA or A23187, suggesting that the overall anti-platelet effect of C.oil was possibly at the membrane/receptor level,unlike curcumin, whichmodulates cycloxygenase (COX) activity. It alsoseems that apart from ar-turmerone other constituents might alsopossess platelet inhibitory activity, necessitating detailed exploration ofC.oil components for their anti-platelet effects. Platelet stimulationwithADP, collagen and thrombin, phosphorylated multiple proteins at

tyrosine residues which regulate various platelet functional responses[34]. The tyrosine phosphorylation was signicantly attenuated afterthe C.oil treatment, thus demonstrating its involvement in themodulation of specic signaling mechanisms during platelet activation.Anti-thrombotic activity of C.oil appears to be platelet mediated asunder similar experimental conditions coagulation parameters (TT, PTand aPTT) in the rat plasma were not altered.

The ex vivo anti-platelet property of C.oil observed in rats wasfurther translated and veried in animal models of thrombosis. Thetime course of the antithrombotic effect of C.oil after oral adminis-tration was studied in the mice model of collagen-epinephrineinduced pulmonary thromboembolism. The model is characterized

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 ateach time point are expressed as MeanSEM.



by the massive activation of circulating platelets and the widespreadformation of platelet thrombi in the microcirculation of the lungsleading to the disseminated pulmonary microembolism and hindlimb paralysis [35]. Protective effect of C.oil observed after 1 h of oraladministration was similar after 2 h, when the peak plasma levelsof its individual components were achieved. Moreover signicantantithrombotic effect was observed even up to 24 h after itsadministration. Therefore, further investigations pertaining to theevaluation of antithrombotic efcacy of C.oil were conducted after 1 hof oral administration in other experimental models. C.oil appeared tobe more potent than aspirin and ticlopidine in the ferric chlorideinduced arterial thrombosis model, where the thrombus has beencharacterized to be platelet rich. The arterial thrombosis model ischaracterized by arterial damage and platelet deposition in thecontext of elevated shear stress. Moreover, C.oil marginally prolongedbleeding time as in stark distinction to aspirin which augmented italmost two fold. Other prevalent anti-platelet drugs are also known toadversely affect the bleeding time. These evidences provide intriguingpossibility that C.oil might be more relevant for pathologic thrombusformation with less impact on normal hemostasis, an excitingprospect for the future potential antithrombotic therapies.

The pharmacokinetic proling in rats further revealed the extentof absorption, higher bioavailability and prolonged systemic avail-ability of all the three markers indicating that these might be playingan important role in exerting broad ranging therapeutic benets of C.oil. Interestingly, after achieving peak plasma level at 2 h, the levels ofar-turmerone and , -turmerone were maintained in the range of100 to 135 ng/ml from 8 h to 18 h, while that of Curlone plasma levelsin this time zone ranged from 8 to16 ng/ml.

The present study thus demonstrates that C.oil mediatedantithrombotic action was primarily due to the inhibition ofplatelet activation, while it had no effect on MI/RP induced injury atthe dosage used. C.oil being a rich source of numerous bioactive phyto-chemicals needs to be characterized further by evaluating all thecomponents for their anti-platelet, anti-coagulant or brinolyticactivity, in order to establish its potential therapeutic use forcardiovascular disease and thrombotic disorders.

Conict of interest

The authors have no conict of interest.

Acknowledgment

The authors are thankful to Council of Scientic and IndustrialResearch (CSIR), India and Central Drug Research Institute (CDRI),India for nancial assistance. Authors wish to thank Dr. GK Jain, HeadPharmaceutics Division CDRI, Lucknow for the help and support.

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