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Journal of Ethnopharmacology 135 (2011) 4854

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

j o u rn a l h o mep ag e : www.e l sev i e r. co m/ l o ca t e / j e t h p h arm

Antiplatelet effects of Cyperus rotundus and its component (+)-nootkatone

Eun Ji Seo a , c , Dong-Ung Lee e , Jong Hwan Kwak f , Sun-Mee Lee f , Yeong Shik Kim g , Yi-Sook Jung a , b , d ,a Department of Physiology, Ajou University School of Medicine, Suwon 443-749, Republic of Koreab Brain Korea 21 for Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Koreac Brain Korea 21 for Division of Cell Transformation and Restoration, Ajou University School of Medicine, Suwon 443-749, Republic of Koread Research Institute of Pharmaceutical Sciences and Technology, Ajou University, Suwon 443-749, Republic of Koreae Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Koreaf College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Koreag College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea

a r t i c l e i n f o

Article history:Received 6 November 2010Received in revised form 2 February 2011Accepted 17 February 2011Available online 24 February 2011

Keywords:Cyperus rotundus(+)-NootkatoneAntiplateletBleeding timeTerpenoidsSesquiterpenoids

a b s t r a c t

Ethnopharmacological relevance: Cyperus rotundus , a well-known oriental traditional medicine, has beenreported to exhibit wide spectrum activity in biological systems including the circulatory system, how-ever, little information is available on its antiplatelet activity. This study was undertaken to investigatethe antiplatelet effects of Cyperus rotundus EtOH extract (CRE) and its constituent compounds.Materialsand methods: The antiplatelet activitiesof CRE and its eightconstituent compoundswere evalu-ated by examining their effects on rat platelet aggregations in vitro and ex vivo , and on mice tail bleedingtimes.Results: During the in vitro platelet aggregation study, CRE showed signicant and concentration-dependent inhibitoryeffects on collagen-, thrombin-, and/or AA-induced platelet aggregation. Of its eightcomponents, (+)-nootkatonewas foundto havethe mostpotent inhibitoryeffect on collagen-, thrombin-,and AA-induced platelet aggregation. In addition, CRE- and (+)-nootkatone-treated mice exhibited sig-nicantly prolonged bleeding times. Furthermore, (+)-nootkatone had a signicant inhibitory effect onrat platelet aggregation ex vivo .Conclusions: This study demonstrates the antiplatelet effects of CRE and its active component (+)-nootkatone, and suggests that these agents might be of therapeutic benet for the prevention of platelet-associated cardiovascular diseases.

2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Arterial thrombosis is an acute complication that develops inassociation with the lesions of atherosclerosis and causes car-diovascular diseases, such as, ischemic heart disease and stroke(Ruggeri, 2002; Davi and Patrono, 2007 ). Platelets are key com-ponents of thrombosis, and may also participate in the progressionof atherosclerotic plaque ( Ruggeri, 2002; Jennings, 2009 ). Hence,the inhibition of platelet aggregation is important for preventingthe progression of atherosclerosis and arterial thrombosis, and asuitable antiplatelet therapy could be a cornerstone for the man-agement of atherosclerotic cardiovascular diseases ( Ruggeri, 2002;Gross and Weitz, 2009 ).

Antiplatelet agents, such as, aspirin and clopidogrel, are widelyused clinically and directly inhibit platelet aggregation ( Gaglia

Corresponding author at: Department of physiology, School of Medicine, AjouUniversity, Suwon 443-749, Republic of Korea. Tel.: +82 31 219 5044;fax: +82 31 219 5049.

E-mail address: [email protected] (Y.-S. Jung).

et al., 2010 ). However, the continuous use of these agents is lim-ited because they can induce resistance or adverse effects, suchas, headache, abdominal cramps, vomiting, and gastric ulceration(Angiolillo et al., 2007; Cannon et al., 2007 ). For these reasons, anumber of studies have been conducted to search for naturallyderived platelet aggregation inhibitors with fewer adverse effects.In particular, plant extracts offer a rich potential source of novelantiplatelet agents ( Tsai et al., 2000; Ballabeni et al., 2007 ), andsince many of these extracts are free from adverse effects and haveexcellentpharmacologicalactions,their use couldlead to thedevel-opment of new classes of safe antiplatelet agents ( Tognolini et al.,2006; Ryuet al., 2009 ). Furthermore, some avonoidsand polyphe-nols have been found to effectively inhibit platelet aggregation(Bucki et al., 2003 ), and therefore, much effort has focused on theidentication of plant-based materials with commercial potential.

Cyperus rotundus is a sedge of the Cyperaceae family, andgrows naturally in tropical, subtropical, and temperate regions. Itis one of the earliest known and most important edible herbs, andhas been reported to exhibit wide spectrum activity in biologi-cal systems, including antioxidant and anti-inammatory effects

0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.

doi: 10.1016/j.jep.2011.02.025
http://dx.doi.org/10.1016/j.jep.2011.02.025http://dx.doi.org/10.1016/j.jep.2011.02.025http://www.sciencedirect.com/science/journal/03788741http://www.elsevier.com/locate/jethpharmmailto:[email protected]://dx.doi.org/10.1016/j.jep.2011.02.025http://dx.doi.org/10.1016/j.jep.2011.02.025mailto:[email protected]://www.elsevier.com/locate/jethpharmhttp://www.sciencedirect.com/science/journal/03788741http://dx.doi.org/10.1016/j.jep.2011.02.025


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E.J. Seo et al. / Journal of Ethnopharmacology 135 (2011) 4854 49

(Ardestani and Yazdanparast, 2007; Kilani et al., 2008; Kilani-Jaziriet al., 2009 ). Cyperus rotundus continues to be used as a traditionalmedicine to improve blood circulation, particularly, in gynecolog-ical diseases caused by blood stagnation ( Yang, 1997; Elizabeth,2002 ). The constituents of Cyperus rotundus have been previ-ouslyexamined for monoterpenes and sesquiterpenes ( Kilaniet al.,2008 ). It has been reported that terpenoids from Annona squamosahave inhibitory effects on platelet aggregation ( Yang et al., 2002 ).In order to evaluate the effects of Cyperus rotundus on platelet-related cardiovascular diseases, we investigated whether Cyperusrotundus EtOH extract (CRE) has antiplatelet effects in vitro andin vivo . Furthermore, we examined the effects of compounds of Cyperus rotundus to identify the constituent which is associatedwith its bioactivities.

2. Materials and methods

2.1. Materials and animals

Collagen, ADP, thrombin, and arachidonic acid (AA) werepurchased from Chrono-Log Co. (Harvertown, PA, USA). Dimethyl-sulfoxide (DMSO), polyethylene glycol (PEG), Tween-80, -nicotinamide adenine dinucleotide (reduceddisodiumsalt hydrate,

-NADH), and pyruvic acid were purchased from Sigma (St. Louis,MO, USA).

SpragueDawley (SD) rats and ICR mice were purchased fromthe Samtako Laboratory Animal Center (Republic of Korea), andhoused in a conventional animal facility with free access to foodand water in a temperature and relative humidity monitored andcontrolled environment under articial lighting (12 h of light perday). Animals were allowed to acclimatize for at least 7 days beforeexperiments. All animals related study protocols were conductedin accordance with the guidelines for the Care and Use of Labo-ratory Animals published by the US National Institute of Health(NIH Publication No. 85-23, revised 1996), and were approved bythe Committee on Animal Research at Ajou Medical Center, AjouUniversity.

2.2. Extraction and isolation

2.2.1. Plant collectionThe rhizomes of Cyperus rotundus , which are cultivated in

Koryung (Republic of Korea), were provided by Je-Hyun Lee (Col-lege of Oriental Medicine, Dongguk University, Gyeongju, Republicof Korea). A voucher specimen has been deposited at the Herbar-iumof the Traditional Herb Research Center, Korean Food andDrugAdministration.

2.2.2. ExtractionAir-dried and chopped rhizomes of Cyperus rotundus (3.0 kg)

were extracted twice with hot 70% EtOH (10L) for 3 h in a waterbath. The EtOH extract obtained was evaporated to dryness ina rotary evaporator (Eyela N-1, Japan) under reduced pressure(400 mmHg) using a water aspirator, affordingcrudeextract (342 g,11.4% yield) of Cyperus rotundus.

2.2.3. IsolationDried extract of Cyperus rotundus (342 g) was dissolved in

distilled water (1L), and 800mL of the resulting solution was con-secutively partitioned using hexane, CH 2 Cl2 , EtOAc, and n -BuOH(each 800mL), and obtained hexane (34.61g), CH 2 Cl2 (10.71 g),EtOAc (3.70 g), n -BuOH (18.31g), and H 2 O (204.51 g) fractions.Because hexane fraction was the major fraction (over 50%) amongfour fractions except H 2 O fraction, we primarily focused on hex-

ane fraction for further isolation. The hexane fraction (34.61g)

was subfractionated on a silica gel column using a stepwise elu-tion procedure using hexane:CH 2 Cl2 (3:1 and 1:1, v/v), CH 2 Cl2 ,and hexane:CH 2 Cl2 :MeOH (10:10:1, v/v), and subfractions of hex-ane fraction were then combined according to their TLC (Thinlayer chromatography) patterns. Each of these subfractions wasfurther chromatographed on a silica gel column using a gradienthexaneEtOAc mixture to give valencene (1.03 g, 0.3%), -pinene(0.14 g, 0.04%), limonene (0.20 g, 0.06%), p-cymene (0.07 g, 0.02%)and 1,8-cineole (0.07g, 0.02%). These ve components were iden-tied by comparing their spectral data with published values(Minyard et al., 1965; Majetich et al., 1985; Miyazawa et al.,1989; Guerrini et al., 2006 ). (+)-Nootkatone (1.6 g, 0.47%) wasobtained as yellowish needles by silica gel column chromatogra-phy using stepwise elution (hexane:EtOAc = 15:1, 7:1, 3:1, and 1:1,v/v) from another subfraction, and was also identied by compar-ing its spectral data with previously reported values ( Miyazawaet al., 2000 ). Caryophyllene oxide (1.7g, 0.5%) was also obtainedby silica gel column chromatography from another subfractionusing a hexane/EtOAc gradient and 8090% acetonitrile (RP-18)(Thebtaranonth et al., 1995 ). Coumarin (0.03g, 0.008%) ( Chan et al.,1977 ) was obtained by Sephadex LH-20 (CH 2 Cl2 :MeOH = 1:1) col-umn chromatography from the other subfraction. The purities of above isolated compounds were determined by GCMS (HP 6890series) to be valencene 90.6%, -pinene 98.1%, limonene 97.2%, p-cymene 99.0%, and 1,8-cineole 98.0%, (+)-nootkatone 98.6%,caryophyllene oxide 89.5%, and coumarin 99.0%.

(+)-Nootkatone : C15 H22 O; Colorless oil; [ ]20 D + 195.5 (CHCl 3 ,c = 0.01); 1 H NMR (500 MHz, CDCl 3 ) : 0.97 (3H, d, J = 6.4Hz, H-14),1.12 (3H, s, H-15), 1.202.60 (methylene + methine), 1.74 (3H, s,H-13), 4.73 (2H, br s, H-12), 5.77 (1H, s, H-1); 13 C NMR (125MHz,CDCl3 ) : 14.9 (C-14), 16.8 (C-15), 20.8 (C-13), 31.6 (C-8),33.0(C-6),39.3 (C-5),40.3(C-4),40.4(C-7), 42.1 (C-3),43.9(C-9),109.2 (C-12),124.7 (C-1), 149.1 (C-11), 170.5 (C-10), 199.6 (C-2); EI-GC/MS m/z :218 [M] + (18), 147 (base peak).

2.3. Preparations of samples

For in vitro experiments, CRE was dissolved in 10% Tween 80-saline. The eight components isolated from Cyperus rotundus weredissolvedin DMSO. Thenal concentration of DMSO in platelet sus-pension never exceeded 1%. For ex vivo and in vivo experiments,(+)-nootkatone was suspended in 70% PEG. All agents were pre-pared immediately prior to use.

2.4. Preparation of platelets

Platelet-rich plasma was prepared as described previously( Junget al., 2002 ). Briey, SD rats, weighing 200250g, were lightlyanesthetized with diethyl ether. Blood was collected in a syringecontaining 3.8% sodium citrate (1:9, v/v) from the abdominal aorta,andthen centrifugedat 150 g for10minatroomtemperature.The

supernatant (platelet-rich plasma, PRP) obtained was used in theaggregation study. The platelet count in PRPwas nallyadjustedtoabout 2 10 8 cells/mL with Tyrode solution (pH 7.4, NaCl 11.9 mM,KCl 2.7mM, MgCl 2 2.1mM, NaH 2 PO4 0.4mM, NaHCO 3 11.9mM,glucose 11.1mM) containing bovine serum albumin (3.5mg/mL).

2.5. In vitro platelet aggregation study

Platelet aggregation studies were performed using the tur-bidimetric method described by Mustard et al. (1972) . PRP wasstimulated with different aggregating agents at the followingnal concentrations; collagen 2 g/mL, thrombin 0.4 U/mL, or AA100 M. Platelet aggregation was recorded for 5 min after plateletstimulation. Aggregation was measured and expressed as a percent

change in light transmission, with the value for the blank sample


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50 E.J. Seo et al. / Journal of Ethnopharmacology 135 (2011) 4854

Fig. 1. Dose-dependent inhibitoryeffectsof Cyperus rotundus EtOHextract(CRE) on plateletaggregation in vitro . Platelet-richplasma (PRP) werepreincubated for 5 min withvarious concentrations of CRE at 37 C before being induced to aggregate by collagen 2 g/mL (A), thrombin 0.4U/mL (B), or arachidonic acid 100 M (C). Data are expressedas means SEM. *P < 0.05 versus vehicle.

(buffer without platelets) set at 100%. For in vitro inhibition stud-ies, PRP was preincubated with different concentrations of CRE orthe eight components for 5 min in the cuvette of an aggregome-ter before being stimulated with the aggregating agents describedabove.

2.6. Determination of cytotoxicity

The cytotoxic effects of samples on platelets were determined

by measuring lactate dehydrogenase (LDH) leakage from platelets,as described previously ( Lee et al., 2009; Ryu et al., 2009 ). Afterincubating rat PRP at 37 C for 5 min with vehicle or samples, itwas centrifuged at room temperature for 1 min at 10,000 g . Tomeasure LDH release, 25 L aliquots of supernatant were collectedfrom each group to 96-well, and mixed with 100 L of NADH solu-tion (0.03% -NADH in phosphate buffer) and 25 L of pyruvatesolution (22.7 mM pyruvic acid in phosphate buffer) at room tem-perature.Reductions in absorbance at 340nm dueto theconversionof NADH to NAD+ were measured to determine LDH activity insupernatant. LDH leakages were expressed as percentages of totalenzyme activity measured in platelets completely lysed with 0.2%Triton X-100.

2.7. Ex vivo platelet aggregation study

(+)-Nootkatone (3, 10, or 30 mg/kg) or aspirin (30 mg/kg) wasadministered once by oral gavage in 0.5mL of 70% PEG (vehicle) toeach rat 2 h before experiments. Control animals received vehicleonly. Blood samples were collected and platelet aggregation wasperformed as described above in 2.4. and 2.5 .

2.8. In vivo mice tail bleeding times

Bleeding times were determined as previously described ( Choet al., 2008 ). Male ICR mice, weighing 3540g, were used in thisexperiment. Mice were fasted overnight before experiments. Twohours after administering CRE (3, 10, or 30 mg/kg), (+)-nootkatone

(3, 10, or 30 mg/kg), or aspirin (50 mg/kg) orally, mice were then

anesthetized with sodium pentobarbital (75 mg/kg), and individu-ally placed on a hotplate to control body temperature at 37 C. Ineach case, the tail was transected 3 mm from its tip with a razorblade, and then immersed in a 15-mL clear conical tube containingnormal saline prewarmed to 37 C. Times to blood ow cessation(dened as no bleeding for 15 s) were measured.

2.9. Statistical analysis

Allresultsareexpressedas means

SEMof atleastfourdifferentexperiments. One way ANOVA followed by Dunnetts test and/orthe paired t -test were used for the analysis. P -values of


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Fig. 3. Effects of EtOH extract (CRE) and the eight compounds of Cyperus rotunduson LDH release from platelets. LDH release was measured after incubating rat PRP(2 10 8 cells/mL) for 5 min with vehicle or samples. Data represent means SEM.*P < 0.05 versus vehicle.

Table 1IC50 values of Cyperus rotundus EtOH extract (CRE) on in vitro platelet aggregationinduced by collagen, thrombin, and arachidonic acid (AA).

Collagen Thrombin AA

CRE IC50 ( g/mL) 86.7 0.7 208.4 0.9 70.8 0.3

Values are presented as mean

SEM.

inhibitory effect on collagen (2 g/mL)-, thrombin (0.4U/mL)-, andAA (100 M)-inducedplatelet aggregation (100 0.0%, 89.0 6.7%,and 84.9 15.1%, % of inhibition, respectively; Fig. 1). IC50 (half inhibitory concentration) values of CRE for collagen-, thrombin-,and AA-induced platelet aggregation are shown in Table 1 .

3.2. Effect of CRE on bleeding times

Bleeding times provide a useful means of estimating plateletfunction and studying the in vivo effects of compounds that inter-fere with platelet aggregation ( Praga et al., 1972 ). Therefore, wedetermined bleeding times in ICR mice administered with CRE (3,

10,or 30mg/kg) toevaluate its in vivo antiplatelet effect. CREsignif-icantly prolonged bleeding times as compared with vehicle alone.

Table 2IC50 values of the eight compounds of Cyperus rotundus on in vitro platelet aggrega-tion induced by collagen or thrombin.

Compounds Collagen ( M) Thrombin ( M)

4-Cymene >100 >100-Pinene >100 >100

1,8-Cineole >100 >100Limonene >100 >100Valencene >100 >100

(+)-Nootkatone 54.3

0.2 34.3

0.3Caryophyllene oxide 91.2 0.3 73.6 0.4Coumarin >100 >100Aspirin 130.5 16.1 1810.5 7.0

Values are presented as mean SEM.

Furthermore, this effect of CRE was greater than that of aspirin, awell-known antiplatelet drug used as a positive control ( Fig. 2).

3.3. In vitro antiplatelet effects of the eight compounds of Cyperusrotundus

The present study primarily focused on hexane fraction forfurther isolation because hexane fraction was the major frac-tion (over 50%) among four fractions from CRE, and obtainedeight compounds, which included three monoterpenes and threesesquiterpenes. The antiplatelet effects of eight compounds wereexamined using rat PRP. IC 50 values of compounds with respectto collagen (2 g/mL)- and thrombin (0.4U/mL)-induced aggrega-tion areshownin Table 2 . Of the compoundstested, (+)-nootkatoneinhibited platelet aggregation with greater potency than that of aspirin. Furthermore, (+)-nootkatone dose-dependently inhibitedAA (100 M)-induced platelet aggregation ( Fig. 4).

To determine whether the eight compounds have cytotoxic-ity that may affect to the antiplatelet effect, we examined theireffects on LDH release (an index of cellular injury) from platelets.However, the amount of LDH released was not signicantly alteredby CRE or the eight compounds for 5 min at the highest concen-

trations examined ( Fig. 3), whereas digitonin (40 M; a positivecontrol) signicantly increased LDH release. These ndings show

Fig. 4. Dose-dependent inhibitory effects of (+)-nootkatone on in vitro platelet aggregation. (A) The chemical structure of (+)-nootkatone. PRP was preincubated for 5 minwith various concentrations of (+)-nootkatone at 37 C before aggregation induction with collagen 2 g/mL (B), thrombin 0.4U/mL (C), or arachidonic acid 100 M (D). Data

represent means SEM. *P < 0.05 versus vehicle.


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Fig. 5. Effect of (+)-nootkatone on ex vivo platelet aggregation. (+)-Nootkatone (3,10,or 30mg/kg)or aspirin(30 mg/kg)were orallyadministeredto SDrats2 h beforethe experiments. PRP was stimulated with collagen (2 g/mL). Data are expressedas means SEM (n = 68). * P < 0.05 versus vehicle.

that neither CRE nor the eight compounds affected cell membraneintegrity.

3.4. Ex vivo platelet response to (+)-nootkatone administration

The inhibitory effect of (+)-nootkatone on ex vivo platelet aggre-gation is shown in Fig. 5 . Isolated platelets from rats administratedwith (+)-nootkatone (3, 10, or 30mg/kg; p.o.) showed a great ten-dency to inhibit collagen (2 g/mL)-induced platelet aggregation,which concurred with the results obtained when PRP was directlyincubated with (+)-nootkatone. Aspirin (30 mg/kg) also signi-cantly inhibited ex vivo platelet aggregation.

3.5. Effect of (+)-nootkatone on bleeding times

We also determined bleeding times in ICR mice administeredwith (+)-nootkatone to evaluate its in vivo antiplatelet effect. (+)-Nootkatone signicantly prolonged bleeding times as compared

with vehicle alone, and mice administered with (+)-nootkatone at30mg/kg had bleeding times similar to those of mice administeredwith aspirin at 50 mg/kg ( Fig. 6).

4. Discussion

Platelets are responsible for the formation of pathogenicthrombi that cause the acute manifestations of atherothrom-botic cardiovascular diseases. Increased platelet aggregation andatherosclerosis are two essential contributors to the onset anddevelopment of the cardiovascular diseases, which remain lead-ing causes of death ( Davi and Patrono, 2007 ). Therefore, muchresearch has been carried out to develop antiplatelet agents withimproved efcacy to prevent and/or treat atherothrombotic car-

diovascular diseases ( Gross and Weitz, 2009 ). The present studydemonstrates the antiplatelet effects of CRE and its active com-ponent (+)-nootkatone, and suggests that CRE and (+)-nootkatonehave therapeutic potential for the prevention of platelet-relatedcardiovascular diseases.

Several studies reported that uncontrolled blood pressure isassociated with thrombotic and atherosclerotic complications ( Lipet al., 1997; Felmeden and Lip, 2005 ), and that the pathophys-iological changes in high blood pressure are contributed to anintravascular microenvironment which promotes platelet aggre-gation and thrombus formation ( Lip et al., 2001 ). Moreover, theincreased platelet activitywas foundin patients suffering fromvas-cular injury and hypertension ( Felmeden and Lip, 2005 ). Hence,antiplatelettherapy hasbeen supposed to be benecialfor patients

withhypertension. Indeed, it has beenreported thatseveral antihy-

Fig.6. Effect of (+)-nootkatone on micetail bleedingtimes.(+)-Nootkatone (3,10, or30mg/kg)or aspirin (50mg/kg)wereorally administeredto ICRmice2 h before theexperiments. Data are expressed as means SEM (n = 68). * P < 0.05 versus vehicle.

pertensive agents, including amlodipine, have antiplatelet activity(Wallen et al., 1995; Chou et al., 1999 ) Cyperus rotundus , a widelyused oriental medicine, has been known to elicit various benecialeffects including anti-inammatory effect, and it continues to beused to improve bloodcirculation.Furthermore, its blood pressure-lowering effect has been reported in animals and humans ( Singhet al., 1970; Elizabeth, 2002 ). In the present study, we found thatCyperus rotundus has an antiplateleteffectbased on theresults thatCREinhibitedthe plateletaggregations induced bycollagen, throm-bin and AA in vitro . The antiplatelet activity of CRE was furtherconrmed by its prolongation effect on bleeding time in vivo .

Of theeightcomponents of thehexane fraction of Cyperus rotun-dus examined, (+)-nootkatone was found to be the most potent,in terms of inhibiting platelet aggregation. (+)-Nootkatone is awell-known sesquiterpenoid, and the sesquiterpenoids are thelargest subclass of terpenoids ( Sowden et al., 2005 ). Terpenoidsare found in almost every plant and have diverse bioactivities

that range from anti-inammatory, antioxidant and antiplateleteffects ( Wagner and Elmadfa, 2003 ). Although previous studies onsesquiterpenoids, such as ergolide and parthenolide, have mainlyfocused on anti-inammatory activity ( Salminen et al., 2008 ), thepresent study demonstrates for the rst time that (+)-nootkatonehas considerable antiplatelet activity; inhibition of platelet aggre-gation and prolongation of bleeding time.

To compare the potency between CRE and (+)-nootkatone, werecalculated the M concentration of (+)-nootkatone into g/mL,and got IC 50 values of (+)-nootkatone for collagen- and thrombin-induced platelet aggregation to be 11 g/mL and 34.3 g/mL,respectively, and those of CRE were 86.7 g/mL and 208.4 g/mL,respectively, as shown in Tables 1 and 2 . As per this comparison,(+)-nootkatone was likely to be more potent than CRE in in vitro

platelet aggregation. However, in in vivo experiments, as shownin Figs. 2 and 6 , CRE showed greater potency than (+)-nootkatonein prolongation of bleeding time. This discrepancy of the resultsmight be due to the other constituents rather than (+)-nootkatonepresent in the CRE, such as valencene and caryophyllene oxide,which might contribute to the antiplatelet activity of CRE. In par-ticular, (+)-valencene, the one of eight compounds from Cyperusrotundus , is known as thebiosynthetic precursorof (+)-nootkatone,and the biosynthesis andbiotransformation of the (+)-valencene to(+)-nootkatone has also been demonstrated ( Fraatz et al., 2009 ).These reports suggest that (+)-valencene may contribute to theantiplatelet activity of CRE through metabolism in vivo . Takentogether, CRE may be more effective than (+)-nootkatone in termsof antiplatelet activity in vivo , while (+)-nootkatone alone appears

to be more potent than CRE in vitro .


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Underpathologic conditions, platelet aggregation involves mul-tiple agonists, and in particular, thrombin has great activity andbinds to protease-activated receptor-1 (PAR-1) on the surfacesof platelets, which causes platelet aggregation ( Jennings, 2009 ).Collagen, which also induces platelet activation, is a strong throm-bogenic agent, and in damaged endothelium promotes plateletadhesion to subendothelium by binding to several receptors, suchas, integrin 2 1 and glycoprotein VI (GPVI), on platelet surfaces,thus, inducing platelet aggregation ( Varga-Szabo et al., 2008 ). AAalso induces platelet aggregation. In fact, upon exposure to acti-vating agonists, platelets liberate AA stored as phospholipid inthe platelet plasma membrane, and this AA is then converted bycyclooxygenase and TXA 2 synthase into thromboxane A 2 (TXA2 ),which acts as a positive feedback mediator for the activationand recruitment of more platelets ( Jennings, 2009 ). Accordingly,platelets are activated by multiple physiological agonists thatinteract with specic platelet receptors, and thus, antiplateletagents that act only at one site are limited in terms of prevent-ing the formation of pathogenic thrombi. Indeed, it has beenreported that antiplatelet therapy with aspirin, which inhibitsonly the AA/cyclooxygenase pathway, is not sufcient to preventischemic events in patients with atherothrombotic cardiovasculardisease ( Yusuf et al., 2001 ). Furthermore, dual antiplatelet ther-apy with aspirin plus clopidogrel, a P2Y 12 -receptor antagonist,has been shown to be more effective at reducing ischemic eventsin patients with atherothrombotic cardiovascular diseases thanaspirin alone ( Chen et al., 2005 ). Thus, available evidence sug-gests that antiplatelet agents acting at multiple sites are likely toprovide more effective arterial thrombosis treatments. Hence, itappears that CRE and (+)-nootkatone, which can inhibit multiplesites of platelet aggregation, may be useful for the prevention of atherothrombotic diseases.

Although we did not include a mechanistic study to identifythe signaling pathways responsible for the inhibition of plateletaggregation by (+)-nootkatone, recent studies have reported that(+)-nootkatone is an activator of adenosine monophosphate-activatedprotein kinase (AMPK), which was suggestedto be linked

to the inhibition of platelet aggregation ( Fleming et al., 2003;Murase et al., 2010 ). AMPK is also known to promote cardiovas-cular homeostasis by ensuring an optimum redox balance in thecardiovascular system, and AMPKdysfunction is believed to under-lie several cardiovascular pathologies ( Shirwany and Zou, 2010 ).It is evident that further study is needed to determine whetherAMPK-activating activity of (+)-nootkatone is associated with itsantiplatelet activity.

In conclusion, the present study suggests that the antiplateletactivities of CRE and its constituent (+)-nootkatone may be thera-peutically useful for the treatment of atherothrombotic diseases.

Acknowledgements

This work was supported by a grant from the Korea Food andDrug Administration (2009) for studies on the identication of the efcacy of biologically active components from oriental herbalmedicines, and supported by Technology Development Program(308013-3) for Food, Ministry for Food, Agriculture, Forestry andFisheries, and the Center for Biological Modulators of the 21st Cen-tury Frontier R&D Program established by the Ministry of Scienceand Technology (CBM34-B3003-01-02-00), Republic of Korea.

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