Journal of Ethnopharmacology 135 (2011) 4854

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

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 Korea Brain Korea 21 for Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea c Brain Korea 21 for Division of Cell Transformation and Restoration, Ajou University School of Medicine, Suwon 443-749, Republic of Korea d Research Institute of Pharmaceutical Sciences and Technology, Ajou University, Suwon 443-749, Republic of Korea e Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea f College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea g College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
b

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Cyperus rotundus, a well-known oriental traditional medicine, has been reported to exhibit wide spectrum activity in biological systems including the circulatory system, however, little information is available on its antiplatelet activity. This study was undertaken to investigate the antiplatelet effects of Cyperus rotundus EtOH extract (CRE) and its constituent compounds. Materials and methods: The antiplatelet activities of CRE and its eight constituent compounds were evaluated by examining their effects on rat platelet aggregations in vitro and ex vivo, and on mice tail bleeding times. Results: During the in vitro platelet aggregation study, CRE showed signicant and concentrationdependent inhibitory effects on collagen-, thrombin-, and/or AA-induced platelet aggregation. Of its eight components, (+)-nootkatone was found to have the most potent inhibitory effect on collagen-, thrombin-, and AA-induced platelet aggregation. In addition, CRE- and (+)-nootkatone-treated mice exhibited signicantly prolonged bleeding times. Furthermore, (+)-nootkatone had a signicant inhibitory effect on rat 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.

Article history: Received 6 November 2010 Received in revised form 2 February 2011 Accepted 17 February 2011 Available online 24 February 2011 Keywords: Cyperus rotundus (+)-Nootkatone Antiplatelet Bleeding time Terpenoids Sesquiterpenoids

1. Introduction Arterial thrombosis is an acute complication that develops in association with the lesions of atherosclerosis and causes cardiovascular diseases, such as, ischemic heart disease and stroke (Ruggeri, 2002; Davi and Patrono, 2007). Platelets are key components of thrombosis, and may also participate in the progression of atherosclerotic plaque (Ruggeri, 2002; Jennings, 2009). Hence, the inhibition of platelet aggregation is important for preventing the progression of atherosclerosis and arterial thrombosis, and a suitable antiplatelet therapy could be a cornerstone for the management of atherosclerotic cardiovascular diseases (Ruggeri, 2002; Gross and Weitz, 2009). Antiplatelet agents, such as, aspirin and clopidogrel, are widely used clinically and directly inhibit platelet aggregation (Gaglia

Corresponding author at: Department of physiology, School of Medicine, Ajou University, Suwon 443-749, Republic of Korea. Tel.: +82 31 219 5044; fax: +82 31 219 5049. E-mail address: yisjung@ajou.ac.kr (Y.-S. Jung). 0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.02.025

et al., 2010). However, the continuous use of these agents is limited because they can induce resistance or adverse effects, such as, headache, abdominal cramps, vomiting, and gastric ulceration (Angiolillo et al., 2007; Cannon et al., 2007). For these reasons, a number of studies have been conducted to search for naturally derived platelet aggregation inhibitors with fewer adverse effects. In particular, plant extracts offer a rich potential source of novel antiplatelet agents (Tsai et al., 2000; Ballabeni et al., 2007), and since many of these extracts are free from adverse effects and have excellent pharmacological actions, their use could lead to the development of new classes of safe antiplatelet agents (Tognolini et al., 2006; Ryu et al., 2009). Furthermore, some avonoids and polyphenols have been found to effectively inhibit platelet aggregation (Bucki et al., 2003), and therefore, much effort has focused on the identication of plant-based materials with commercial potential. Cyperus rotundus is a sedge of the Cyperaceae family, and grows naturally in tropical, subtropical, and temperate regions. It is one of the earliest known and most important edible herbs, and has been reported to exhibit wide spectrum activity in biological systems, including antioxidant and anti-inammatory effects

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(Ardestani and Yazdanparast, 2007; Kilani et al., 2008; Kilani-Jaziri et al., 2009). Cyperus rotundus continues to be used as a traditional medicine to improve blood circulation, particularly, in gynecological diseases caused by blood stagnation (Yang, 1997; Elizabeth, 2002). The constituents of Cyperus rotundus have been previously examined for monoterpenes and sesquiterpenes (Kilani et al., 2008). It has been reported that terpenoids from Annona squamosa have 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 Cyperus rotundus EtOH extract (CRE) has antiplatelet effects in vitro and in vivo. Furthermore, we examined the effects of compounds of Cyperus rotundus to identify the constituent which is associated with its bioactivities. 2. Materials and methods 2.1. Materials and animals Collagen, ADP, thrombin, and arachidonic acid (AA) were purchased from Chrono-Log Co. (Harvertown, PA, USA). Dimethylsulfoxide (DMSO), polyethylene glycol (PEG), Tween-80, nicotinamide adenine dinucleotide (reduced disodium salt hydrate, -NADH), and pyruvic acid were purchased from Sigma (St. Louis, MO, USA). SpragueDawley (SD) rats and ICR mice were purchased from the Samtako Laboratory Animal Center (Republic of Korea), and housed in a conventional animal facility with free access to food and water in a temperature and relative humidity monitored and controlled environment under articial lighting (12 h of light per day). Animals were allowed to acclimatize for at least 7 days before experiments. All animals related study protocols were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996), and were approved by the Committee on Animal Research at Ajou Medical Center, Ajou University. 2.2. Extraction and isolation 2.2.1. Plant collection The rhizomes of Cyperus rotundus, which are cultivated in Koryung (Republic of Korea), were provided by Je-Hyun Lee (College of Oriental Medicine, Dongguk University, Gyeongju, Republic of Korea). A voucher specimen has been deposited at the Herbarium of the Traditional Herb Research Center, Korean Food and Drug Administration. 2.2.2. Extraction Air-dried and chopped rhizomes of Cyperus rotundus (3.0 kg) were extracted twice with hot 70% EtOH (10 L) for 3 h in a water bath. The EtOH extract obtained was evaporated to dryness in a rotary evaporator (Eyela N-1, Japan) under reduced pressure (400 mmHg) using a water aspirator, affording crude extract (342 g, 11.4% yield) of Cyperus rotundus. 2.2.3. Isolation Dried extract of Cyperus rotundus (342 g) was dissolved in distilled water (1 L), and 800 mL of the resulting solution was consecutively partitioned using hexane, CH2 Cl2 , EtOAc, and n-BuOH (each 800 mL), and obtained hexane (34.61 g), CH2 Cl2 (10.71 g), EtOAc (3.70 g), n-BuOH (18.31 g), and H2 O (204.51 g) fractions. Because hexane fraction was the major fraction (over 50%) among four fractions except H2 O fraction, we primarily focused on hexane fraction for further isolation. The hexane fraction (34.61 g)

was subfractionated on a silica gel column using a stepwise elution procedure using hexane:CH2 Cl2 (3:1 and 1:1, v/v), CH2 Cl2 , and hexane:CH2 Cl2 :MeOH (10:10:1, v/v), and subfractions of hexane fraction were then combined according to their TLC (Thin layer chromatography) patterns. Each of these subfractions was further chromatographed on a silica gel column using a gradient hexaneEtOAc 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 (0.07 g, 0.02%). These ve components were identied 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%) was obtained as yellowish needles by silica gel column chromatography using stepwise elution (hexane:EtOAc = 15:1, 7:1, 3:1, and 1:1, v/v) from another subfraction, and was also identied by comparing its spectral data with previously reported values (Miyazawa et al., 2000). Caryophyllene oxide (1.7 g, 0.5%) was also obtained by silica gel column chromatography from another subfraction using a hexane/EtOAc gradient and 8090% acetonitrile (RP-18) (Thebtaranonth et al., 1995). Coumarin (0.03 g, 0.008%) (Chan et al., 1977) was obtained by Sephadex LH-20 (CH2 Cl2 :MeOH = 1:1) column chromatography from the other subfraction. The purities of above isolated compounds were determined by GCMS (HP 6890 series) 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 (CHCl3 , c = 0.01); 1 H NMR (500 MHz, CDCl3 ) : 0.97 (3H, d, J = 6.4 Hz, 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 (125 MHz, 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 80saline. The eight components isolated from Cyperus rotundus were dissolved in DMSO. The nal concentration of DMSO in platelet suspension never exceeded 1%. For ex vivo and in vivo experiments, (+)-nootkatone was suspended in 70% PEG. All agents were prepared immediately prior to use. 2.4. Preparation of platelets Platelet-rich plasma was prepared as described previously (Jung et al., 2002). Briey, SD rats, weighing 200250 g, were lightly anesthetized with diethyl ether. Blood was collected in a syringe containing 3.8% sodium citrate (1:9, v/v) from the abdominal aorta, and then centrifuged at 150 g for 10 min at room temperature. The supernatant (platelet-rich plasma, PRP) obtained was used in the aggregation study. The platelet count in PRP was nally adjusted to about 2 108 cells/mL with Tyrode solution (pH 7.4, NaCl 11.9 mM, KCl 2.7 mM, MgCl2 2.1 mM, NaH2 PO4 0.4 mM, NaHCO3 11.9 mM, glucose 11.1 mM) containing bovine serum albumin (3.5 mg/mL). 2.5. In vitro platelet aggregation study Platelet aggregation studies were performed using the turbidimetric method described by Mustard et al. (1972). PRP was stimulated with different aggregating agents at the following nal concentrations; collagen 2 g/mL, thrombin 0.4 U/mL, or AA 100 M. Platelet aggregation was recorded for 5 min after platelet stimulation. Aggregation was measured and expressed as a percent change in light transmission, with the value for the blank sample

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Fig. 1. Dose-dependent inhibitory effects of Cyperus rotundus EtOH extract (CRE) on platelet aggregation in vitro. Platelet-rich plasma (PRP) were preincubated for 5 min with various concentrations of CRE at 37 C before being induced to aggregate by collagen 2 g/mL (A), thrombin 0.4 U/mL (B), or arachidonic acid 100 M (C). Data are expressed as means SEM. *P < 0.05 versus vehicle.

(buffer without platelets) set at 100%. For in vitro inhibition studies, PRP was preincubated with different concentrations of CRE or the eight components for 5 min in the cuvette of an aggregometer before being stimulated with the aggregating agents described above. 2.6. Determination of cytotoxicity

anesthetized with sodium pentobarbital (75 mg/kg), and individually placed on a hotplate to control body temperature at 37 C. In each case, the tail was transected 3 mm from its tip with a razor blade, and then immersed in a 15-mL clear conical tube containing normal saline prewarmed to 37 C. Times to blood ow cessation (dened as no bleeding for 15 s) were measured. 2.9. Statistical analysis

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). After incubating rat PRP at 37 C for 5 min with vehicle or samples, it was centrifuged at room temperature for 1 min at 10,000 g. To measure LDH release, 25 L aliquots of supernatant were collected from each group to 96-well, and mixed with 100 L of NADH solution (0.03% -NADH in phosphate buffer) and 25 L of pyruvate solution (22.7 mM pyruvic acid in phosphate buffer) at room temperature. Reductions in absorbance at 340 nm due to the conversion of NADH to NAD+ were measured to determine LDH activity in supernatant. LDH leakages were expressed as percentages of total enzyme 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) was administered once by oral gavage in 0.5 mL of 70% PEG (vehicle) to each rat 2 h before experiments. Control animals received vehicle only. Blood samples were collected and platelet aggregation was performed as described above in 2.4. and 2.5. 2.8. In vivo mice tail bleeding times Bleeding times were determined as previously described (Cho et al., 2008). Male ICR mice, weighing 3540 g, were used in this experiment. Mice were fasted overnight before experiments. Two hours 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

All results are expressed as means SEM of at least four different experiments. One way ANOVA followed by Dunnetts test and/or the paired t-test were used for the analysis. P-values of <0.05 were considered statistically signicant. 3. Results 3.1. In vitro antiplatelet effect of CRE We evaluated the antiplatelet effect of CRE using rat PRP. Our results showed that CRE at 300 g/mL had a maximum

Fig. 2. Effect of Cyperus rotundus EtOH extract (CRE) on mice tail bleeding times. CRE (3, 10, or 30 mg/kg) or aspirin (50 mg/kg) were orally administered to ICR mice 2 h before the experiments. Data are expressed as means SEM (n = 68). *P < 0.05 versus vehicle.

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Table 2 IC50 values of the eight compounds of Cyperus rotundus on in vitro platelet aggregation induced by collagen or thrombin. Compounds 4-Cymene -Pinene 1,8-Cineole Limonene Valencene (+)-Nootkatone Caryophyllene oxide Coumarin Aspirin Fig. 3. Effects of EtOH extract (CRE) and the eight compounds of Cyperus rotundus on LDH release from platelets. LDH release was measured after incubating rat PRP (2 108 cells/mL) for 5 min with vehicle or samples. Data represent means SEM. *P < 0.05 versus vehicle. Table 1 IC50 values of Cyperus rotundus EtOH extract (CRE) on in vitro platelet aggregation induced by collagen, thrombin, and arachidonic acid (AA). Collagen CRE IC50 ( g/mL) 86.7 0.7 Thrombin 208.4 0.9 AA 70.8 0.3 Collagen ( M) >100 >100 >100 >100 >100 54.3 0.2 91.2 0.3 >100 130.5 16.1 Thrombin ( M) >100 >100 >100 >100 >100 34.3 0.3 73.6 0.4 >100 1810.5 7.0

Values are presented as mean SEM.

Furthermore, this effect of CRE was greater than that of aspirin, a well-known antiplatelet drug used as a positive control (Fig. 2). 3.3. In vitro antiplatelet effects of the eight compounds of Cyperus rotundus The present study primarily focused on hexane fraction for further isolation because hexane fraction was the major fraction (over 50%) among four fractions from CRE, and obtained eight compounds, which included three monoterpenes and three sesquiterpenes. The antiplatelet effects of eight compounds were examined using rat PRP. IC50 values of compounds with respect to collagen (2 g/mL)- and thrombin (0.4 U/mL)-induced aggregation are shown in Table 2. Of the compounds tested, (+)-nootkatone inhibited platelet aggregation with greater potency than that of aspirin. Furthermore, (+)-nootkatone dose-dependently inhibited AA (100 M)-induced platelet aggregation (Fig. 4). To determine whether the eight compounds have cytotoxicity that may affect to the antiplatelet effect, we examined their effects on LDH release (an index of cellular injury) from platelets. However, the amount of LDH released was not signicantly altered by CRE or the eight compounds for 5 min at the highest concentrations examined (Fig. 3), whereas digitonin (40 M; a positive control) signicantly increased LDH release. These ndings show

Values are presented as mean SEM.

inhibitory effect on collagen (2 g/mL)-, thrombin (0.4 U/mL)-, and AA (100 M)-induced platelet 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 platelet function and studying the in vivo effects of compounds that interfere with platelet aggregation (Praga et al., 1972). Therefore, we determined bleeding times in ICR mice administered with CRE (3, 10, or 30 mg/kg) to evaluate its in vivo antiplatelet effect. CRE significantly prolonged bleeding times as compared with vehicle alone.

Fig. 4. Dose-dependent inhibitory effects of (+)-nootkatone on in vitro platelet aggregation. (A) The chemical structure of (+)-nootkatone. PRP was preincubated for 5 min with various concentrations of (+)-nootkatone at 37 C before aggregation induction with collagen 2 g/mL (B), thrombin 0.4 U/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 30 mg/kg) or aspirin (30 mg/kg) were orally administered to SD rats 2 h before the experiments. PRP was stimulated with collagen (2 g/mL). Data are expressed as means SEM (n = 68). *P < 0.05 versus vehicle.

Fig. 6. Effect of (+)-nootkatone on mice tail bleeding times. (+)-Nootkatone (3, 10, or 30 mg/kg) or aspirin (50 mg/kg) were orally administered to ICR mice 2 h before the experiments. Data are expressed as means SEM (n = 68). *P < 0.05 versus vehicle.

that neither CRE nor the eight compounds affected cell membrane integrity. 3.4. Ex vivo platelet response to (+)-nootkatone administration The inhibitory effect of (+)-nootkatone on ex vivo platelet aggregation is shown in Fig. 5. Isolated platelets from rats administrated with (+)-nootkatone (3, 10, or 30 mg/kg; p.o.) showed a great tendency to inhibit collagen (2 g/mL)-induced platelet aggregation, which concurred with the results obtained when PRP was directly incubated with (+)-nootkatone. Aspirin (30 mg/kg) also signicantly inhibited ex vivo platelet aggregation. 3.5. Effect of (+)-nootkatone on bleeding times We also determined bleeding times in ICR mice administered with (+)-nootkatone to evaluate its in vivo antiplatelet effect. (+)Nootkatone signicantly prolonged bleeding times as compared with vehicle alone, and mice administered with (+)-nootkatone at 30 mg/kg had bleeding times similar to those of mice administered with aspirin at 50 mg/kg (Fig. 6). 4. Discussion Platelets are responsible for the formation of pathogenic thrombi that cause the acute manifestations of atherothrombotic cardiovascular diseases. Increased platelet aggregation and atherosclerosis are two essential contributors to the onset and development of the cardiovascular diseases, which remain leading causes of death (Davi and Patrono, 2007). Therefore, much research has been carried out to develop antiplatelet agents with improved efcacy to prevent and/or treat atherothrombotic cardiovascular diseases (Gross and Weitz, 2009). The present study demonstrates the antiplatelet effects of CRE and its active component (+)-nootkatone, and suggests that CRE and (+)-nootkatone have therapeutic potential for the prevention of platelet-related cardiovascular diseases. Several studies reported that uncontrolled blood pressure is associated with thrombotic and atherosclerotic complications (Lip et al., 1997; Felmeden and Lip, 2005), and that the pathophysiological changes in high blood pressure are contributed to an intravascular microenvironment which promotes platelet aggregation and thrombus formation (Lip et al., 2001). Moreover, the increased platelet activity was found in patients suffering from vascular injury and hypertension (Felmeden and Lip, 2005). Hence, antiplatelet therapy has been supposed to be benecial for patients with hypertension. Indeed, it has been reported that several antihy-

pertensive agents, including amlodipine, have antiplatelet activity (Wallen et al., 1995; Chou et al., 1999) Cyperus rotundus, a widely used oriental medicine, has been known to elicit various benecial effects including anti-inammatory effect, and it continues to be used to improve blood circulation. Furthermore, its blood pressurelowering effect has been reported in animals and humans (Singh et al., 1970; Elizabeth, 2002). In the present study, we found that Cyperus rotundus has an antiplatelet effect based on the results that CRE inhibited the platelet aggregations induced by collagen, thrombin and AA in vitro. The antiplatelet activity of CRE was further conrmed by its prolongation effect on bleeding time in vivo. Of the eight components of the hexane fraction of Cyperus rotundus examined, (+)-nootkatone was found to be the most potent, in terms of inhibiting platelet aggregation. (+)-Nootkatone is a well-known sesquiterpenoid, and the sesquiterpenoids are the largest subclass of terpenoids (Sowden et al., 2005). Terpenoids are found in almost every plant and have diverse bioactivities that range from anti-inammatory, antioxidant and antiplatelet effects (Wagner and Elmadfa, 2003). Although previous studies on sesquiterpenoids, such as ergolide and parthenolide, have mainly focused on anti-inammatory activity (Salminen et al., 2008), the present study demonstrates for the rst time that (+)-nootkatone has considerable antiplatelet activity; inhibition of platelet aggregation and prolongation of bleeding time. To compare the potency between CRE and (+)-nootkatone, we recalculated the M concentration of (+)-nootkatone into g/mL, and got IC50 values of (+)-nootkatone for collagen- and thrombininduced 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 shown in Figs. 2 and 6, CRE showed greater potency than (+)-nootkatone in prolongation of bleeding time. This discrepancy of the results might be due to the other constituents rather than (+)-nootkatone present in the CRE, such as valencene and caryophyllene oxide, which might contribute to the antiplatelet activity of CRE. In particular, (+)-valencene, the one of eight compounds from Cyperus rotundus, is known as the biosynthetic precursor of (+)-nootkatone, and the biosynthesis and biotransformation of the (+)-valencene to (+)-nootkatone has also been demonstrated (Fraatz et al., 2009). These reports suggest that (+)-valencene may contribute to the antiplatelet activity of CRE through metabolism in vivo. Taken together, CRE may be more effective than (+)-nootkatone in terms of antiplatelet activity in vivo, while (+)-nootkatone alone appears to be more potent than CRE in vitro.

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Under pathologic conditions, platelet aggregation involves multiple agonists, and in particular, thrombin has great activity and binds to protease-activated receptor-1 (PAR-1) on the surfaces of platelets, which causes platelet aggregation (Jennings, 2009). Collagen, which also induces platelet activation, is a strong thrombogenic agent, and in damaged endothelium promotes platelet adhesion to subendothelium by binding to several receptors, such as, integrin 2 1 and glycoprotein VI (GPVI), on platelet surfaces, thus, inducing platelet aggregation (Varga-Szabo et al., 2008). AA also induces platelet aggregation. In fact, upon exposure to activating agonists, platelets liberate AA stored as phospholipid in the platelet plasma membrane, and this AA is then converted by cyclooxygenase and TXA2 synthase into thromboxane A2 (TXA2 ), which acts as a positive feedback mediator for the activation and recruitment of more platelets (Jennings, 2009). Accordingly, platelets are activated by multiple physiological agonists that interact with specic platelet receptors, and thus, antiplatelet agents that act only at one site are limited in terms of preventing the formation of pathogenic thrombi. Indeed, it has been reported that antiplatelet therapy with aspirin, which inhibits only the AA/cyclooxygenase pathway, is not sufcient to prevent ischemic events in patients with atherothrombotic cardiovascular disease (Yusuf et al., 2001). Furthermore, dual antiplatelet therapy with aspirin plus clopidogrel, a P2Y12 -receptor antagonist, has been shown to be more effective at reducing ischemic events in patients with atherothrombotic cardiovascular diseases than aspirin alone (Chen et al., 2005). Thus, available evidence suggests that antiplatelet agents acting at multiple sites are likely to provide more effective arterial thrombosis treatments. Hence, it appears that CRE and (+)-nootkatone, which can inhibit multiple sites of platelet aggregation, may be useful for the prevention of atherothrombotic diseases. Although we did not include a mechanistic study to identify the signaling pathways responsible for the inhibition of platelet aggregation by (+)-nootkatone, recent studies have reported that (+)-nootkatone is an activator of adenosine monophosphateactivated protein kinase (AMPK), which was suggested to be linked to the inhibition of platelet aggregation (Fleming et al., 2003; Murase et al., 2010). AMPK is also known to promote cardiovascular homeostasis by ensuring an optimum redox balance in the cardiovascular system, and AMPK dysfunction is believed to underlie several cardiovascular pathologies (Shirwany and Zou, 2010). It is evident that further study is needed to determine whether AMPK-activating activity of (+)-nootkatone is associated with its antiplatelet activity. In conclusion, the present study suggests that the antiplatelet activities of CRE and its constituent (+)-nootkatone may be therapeutically useful for the treatment of atherothrombotic diseases. Acknowledgements This work was supported by a grant from the Korea Food and Drug Administration (2009) for studies on the identication of the efcacy of biologically active components from oriental herbal medicines, and supported by Technology Development Program (308013-3) for Food, Ministry for Food, Agriculture, Forestry and Fisheries, and the Center for Biological Modulators of the 21st Century Frontier R&D Program established by the Ministry of Science and Technology (CBM34-B3003-01-02-00), Republic of Korea. References
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