Research Article

Effects of 2-(1-Hydroxypentyl)-Benzoate on PlateletAggregation and Thrombus Formation in Rats

Yi Zhang, Ling Wang, Liying Zhang, and Xiaoliang Wangn

Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciencesand Peking Union Medical College, Beijing, China

Strategy, Management and Health Policy

Enabling

Technology,

Genomics,

Proteomics

Preclinical

Research

Preclinical Development

Toxicology, Formulation

Drug Delivery,

Pharmacokinetics

Clinical Development

Phases I-III

Regulatory, Quality,

Manufacturing

Postmarketing

Phase IV

ABSTRACT 2-(1-hydroxypentyl)-benzoate (dl-PHPB), a derivate of 3-n-butylphthalide (NBP), is a noveltherapeutic agent for treatment of cerebral ischemia. In the present study, the antiplatelet andantithrombotic activities of dl-PHPB were evaluated in ex vivo platelet aggregation and in vivoarteriovenous (A-V) shunt models. dl-PHPB inhibited platelet aggregation induced by adenosinediphosphate (ADP), arachidonic acid (AA), and collagen (COL) in a dose-dependent manner when givenorally (12.9–129.5 mg/kg). The inhibitory potency was similar to 3-n-butylphthalide (NBP) and aspirin(ASP). Inhibition on platelet aggregation was also observed after iv administration of dl-PHPB (1.29–12.9mg/kg). The time-course of these effects showed the maximal inhibition on platelet aggregation at 1 h afteroral administration and 30 min after iv injection. dl-PHPB (12.9–129.5 mg/kg, p.o.) caused dose-dependent inhibition of thrombus formation in the rat A-V shunt thrombosis model. These results show thatdl-PHPB is an orally and iv antiplatelet and antithrombotic agent and may be useful for treatment ofischemia stroke. Drug Dev Res 63:174–180, (2004). �c 2004 Wiley-Liss, Inc.

Key words: 2-(1-hydroxypentyl)-benzoate (dl-PHPB); platelet aggregation; thrombus formation; cerebral ischemia

INTRODUCTION

Ischemic stroke is initiated by a transient orpermanent reduction in cerebral blood flow (CBF) thatis restricted to the territory of the major brain artery.The decrease of CBF in ischemic regions may result inan energy failure and further leads to an activation ofthe toxic intracellular pathway [Jorgensen and Diemer,1982; Dirnagl et al., 1999; Hou and MacManus, 2002].In most cases, reduced CBF was caused by theocclusion of a cerebral artery either by embolus or bylocal thrombosis where platelets are considered to playa crucial role. Platelets contribute to normal hemostasisby adhering to subendothelial tissues following vasculardamage with the recruitment of additional platelets viathe process of aggregation. In pathological conditions,thrombogenic surfaces act as a nidus for plateletadherence and thrombus formation, resulting invascular occlusion and stroke [Becker, 1991; Hollopeteret al., 2001].Potassium 2-(1-hydroxypentyl)-benzoate

(dl-PHPB), derivated from 3-n-butylphthalide (NBP),is a newly synthesized compound that is underdevelopment as a therapeutic drug for cerebralischemia [Yang et al., 2002]. As reported, NBP is aprimary naphtha component from seeds of Apiumgraveolens Linn. The phase 3 clinic trail of NBP wascompleted in 2002 and is used for treatment ofischemic stroke in clinic. Many basic and clinic studies

DDR

Grant sponsor: National Natural Science Foundation ofChina; Grant number: 30271490.

nCorrespondence to: Xiaoliang Wang, Department ofPharmacology, Institute of Materia Medica, Chinese Academy ofMedical Sciences and Peking Union Medical College, 1 XianNong Tan Street, Beijing 100050, China. E-mail: wangxl@imm.ac.cn

Received 21 August 2004; Accepted 17 November 2004

Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ddr.10401

DRUG DEVELOPMENT RESEARCH 63:174–180 (2004)

�c 2004 Wiley-Liss, Inc.

have proved that NBP is a potentially beneficial andpromising drug treatment of stroke with multipleactions that affect different pathophysiological pro-cesses, such as improving microcirculation, decreasingbrain infarct volume, regulating energy metabolism,and especially inhibiting platelet aggregation andreducing thrombus formation [Chong and Feng,1997; Yan et al.,1998; Xu and Feng, 2000, 2001; Dongand Feng, 2002]. In our previous research, it was foundthat dl-PHPB could transform quickly to NBP whengiven to rat orally or intravenously and it could alsodecrease the infarct volume and improve neurologicaldeficits in the middle cerebral artery occlusion(MCAO) model of the rat. In the present study, weused the ex vivo platelet aggregation method and thearteriovenous shunt model to evaluate the antiplateletand antithrombotic effects of dl-PHPB.

MATERIALS AND METHODS

Animals and Compounds

Male Wistar rats (280–320 g) were individuallyhoused with food and water available ad libitum in atemperature-controlled environment at 201C. Experi-ments were performed in accordance with the guide-lines established by the National Institutes of Healthfor the care and use of laboratory animals and wereapproved by the Animal Care Committee of ChineseAcademy of Medical Sciences.

dl-PHPB and NBP (Fig. 1) were provided by TheDepartment of Synthetic Pharmaceutical Chemistry,Institute of Materia Medica, Chinese Academy ofMedical Sciences and Peking Union Medical College,with a purity of 99.9%. Doses of dl-PHPB (12.9, 38.8,129.5 mg/kg) used to treat orally were equimolar to thedoses of NBP (10, 30, 100 mg/kg), and the doses of dl-PHPB (1.3, 3.9, 12.9 mg/kg) used i.v. were equimolar tothe doses of NBP (1, 3, 10 mg/kg), respectively. Aspirin(ASP), adenosine diphosphate (ADP), arachidonic acid(AA), and collagen (COL) were purchased from SigmaChemical Co. (St. Louis, MO). dl-PHPB was dissolvedin 0.9% saline. NBP was dissolved in pure soybean oil

(p.o.) or in component solvent (PEG400:H2O¼ 1:3)(i.v.). ASP was dissolved in 0.9% saline after dispersedby tween-80. All drugs were administered to rats in avolume of 5 ml/kg body weight (p.o.) or 1 ml/kg bodyweight (i.v.).

Preparation of Platelet-Rich Plasma and PlateletAggregation

To obtain platelet-rich plasma (PRP), animalswere anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and blood samples were collected into 3.8%sodium citrate (9:1 v/v) from the common carotidartery. Samples were centrifuged at 1,000 rpm for 5min at room temperature. PRP was isolated and theremainder centrifuged at 3,000 rpm for 10 min toobtain platelet-poor plasma (PPP). The PRP wasadjusted with PPP so as to obtain platelet counts of 3� 1011/l. Platelet aggregation was determined byBorn’0s [1962] method using a four-channel aggreg-ometer (model PAT-4A, Meguro-Ku, Tokyo, Japan).The 200-ml aliquots of PRP contained in microcuvetteswere preincubated at 371C for 5 min, and 10 ml inducer(ADP, AA, or COL, final concentration 3.75 mmol/l, 0.5mmol/l, or 7.5 mg/ml, respectively) added to each cell.Samples were agitated at a rate of 1,000 rpm with astirrer bar in each microcuvette at 371C. Plateletaggregation was determined by calibrating the equip-ment at 0% light transmission for PRP and at 100% forPPP. The aggregation curve was recorded for 5 min andthe peak value was received and served as the maximalaggregation [Hoffmann et al., 1998].

Arteriovenous (A-V) Shunt Induced ThrombusFormation in Rat

One hour after administration (compounds orvehicle), rats were anaesthetized with sodium pento-barbital (50 mg/kg, i.p.) and surgery of A-V shunt wereperformed. An 8-cm tube was inserted between the leftjugular vein and right common carotid artery. Thesaline-filled shunt was assembled by connecting twocannula with a slightly curved 6-cm-long polyethylenetubing (internal diameter 2 mm) containing a 6-cm-long 0 braided cotton thread (diameter 0.25 mm). Theextracorporeal circulation was maintained for 15 min,and the thrombus formed on the cotton thread duringthis period. The shunt was then removed and thethread with its associated thrombus was withdrawn andimmediately weighed. The wet weight of thrombus wasdetermined by subtracting the weight of thread fromthe total wet weight [Damiano et al., 2001; Umar et al.,2003].Fig. 1. Structures of dl-PHPB and NBP.

EFFECTS OF dl-PHPB ON PLATELET AGGREGATION 175

Statistical Analysis

The results are expressed as mean 7 S.E.M.Data were statistically analyzed using one-way ANOVAfollowed by Dunnet’s test for comparing the treatmentgroups and control group. Results were considered toshow a significant difference when Po0.05.

RESULTS

Platelet Aggregation

Time-course of effect of dl-PHPB (p.o.) on ex vivoplatelet aggregation induced by ADP in rats

To investigate the time-course of the antiplateleteffects of dl-PHPB, ADP (final concentration 3.75mmol/l) -induced platelet aggregation in rats was used.Rats were randomly distributed into seven experimen-tal groups: six treated and one control groups. Treatedgroups orally received dl-PHPB (129.5 mg/kg) in avolume of 5 ml/kg body weight 0.5, 1, 1.5, 2, 3, and 4 hbefore the experiment, while the control groupreceived an equivalent volume of saline. The resultsare shown in Figure 2. dl-PHPB significantly inhibitedplatelet aggregation from 0.5 to 1.5 h after treatmentwith the maxim inhibitory effect being observed 1 hafter treatment (Po0.01 vs. control). No significanteffects were found 2 h after drug administration.

Effect of dl-PHPB, NBP and ASP (p.o.) on ex vivoplatelet aggregation induced by ADP, AA and COL inrats

The effects of dl-PHPB, NBP, and ASP on the exvivo platelet aggregation are shown in Figure 3. Ratswere randomly divided into 10 experimental groups:three dl-PHPB-treated groups (12.9, 38.8, 129.5 mg/kg), three NBP-treated groups (10, 30, 100 mg/kg),three ASP-treated groups (10, 30, 100 mg/kg), and onecontrol group. All groups were given compounds or

vehicle orally by gavage in a volume of 5 ml/kg. Onehour after oral administration, rats were anaesthetizedand blood samples were collected as describedpreviously. Platelet aggregation induced by ADP, AA,and COL was quantified by Born0s method asdescribed above.

dl-PHPB dose-dependently inhibited plateletaggregation. At the highest dose, dl-PHPB inhibitedADP, AA, or COL-induced aggregation by 26%(Po0.001), 20% (Po0.01) and 12% (Po 0.01),respectively. Platelet aggregation induced by the threeagents was dose-dependently inhibited by NBP andASP (10, 30, 100 mg/kg, p.o.). The inhibitory potencyof dl-PHPB was similar to NBP and ASP, except theeffect of ASP on platelet aggregation induced by AAwas especially potent.

Time-course of effect of dl-PHPB (i.v.) on ex vivoplatelet aggregation induced by ADP in rats

ADP (3.75 mmol/l)-induced platelet aggregationafter the administration of a single dose of dl-PHPB(12.9 mg/kg iv.) at 5, 15, 30, 45, 60, 120, and 180 min)was quantified (Fig. 4). Inhibitory effects occurred at15 and 30 min after administration with a maximaleffect at 30 min (Po 0.01 vs. control ). No significanteffects were observed at 45 min after administration.

Effect of dl-PHPB and NBP (i.v.) on ex vivo plateletaggregation induced by ADP, AA and COL in rats

The effects of dl-PHPB and NBP on the ex vivoplatelet aggregation are shown in Figure 5. Rats wererandomly divided into seven experimental groups:three dl-PHPB-treated groups (1.3, 3.9, 12.9 mg/kg),three NBP-treated groups (1, 3, 10 mg/kg), and onecontrol group. All groups were administered withcompound or vehicle intravenously in a volume of 1ml/kg. Blood samples were collected 30 min afteradministration, then PRP and PPP were prepared.ADP, AA, and COL were used to induce plateletaggregation that was quantified by Born0s method asdescribed above.

dl-PHPB and NBP both inhibited plateletaggregation induced by ADP, AA, and COL in adose-dependent manner. Only the middle and thehighest doses were significant (Po 0.05 vs. control ). Atthe highest dose, dl-PHPB inhibited ADP, AA andCOL induced platelet aggregation by 30% (Po 0.001),35% (Po0.01) and 19% (Po 0.01), respectively. Theinhibitory potency of the two compounds were similarat corresponding doses.

Fig. 2. Time-course of the effect of dl-PHPB (129.5 mg/kg, p.o.) on exvivo platelet aggregation induced by ADP (final concentration 3.75mmol/l) in rats. Data is presented as mean 7 S.E.M.(n¼8). nPo0.05and nnPo0.01 vs. control (vehicle-treated group).

176 ZHANG ET AL.

Effect of dl-PHPB, NBP and ASP (p.o.) on thrombusformation induced in rat arteriovenous (A-V) shunt

The antithrombotic activities of dl-PHPB, NBP,and ASP were observed in the rat A-V shunt model

(Fig. 6). Rats were randomly divided into ten experi-mental groups: three dl-PHPB-treated groups (12.9, 38.8,129.5 mg/kg), three NBP-treated groups (10, 30, 100 mg/kg), three ASP-treated groups (10, 30, 100 mg/kg), and

Fig. 3. Effect of dl-PHPB, NBP, and ASP on ex vivo platelet aggregation induced by ADP (A), AA (B), and COL (C) in rats. Single doses of dl-PHPB (doses displayed represent the actual doses of 12.9, 38.8, 129.5 mg/kg, respectively), NBP or ASP were orally administered 1 h beforeplatelet aggregation was measured. The final concentrations of ADP, AA, and COL were 3.75 mmol/l, 0.5 mmol/l, and 7.5 mg/ml separately. Datais presented as mean 7 S.E.M. (n¼10). nPo0.05, nnPo0.01, and nnnPo0.001 vs. control (vehicle-treated group).

Fig. 4. Time-course of the effect of dl-PHPB (12.9 mg/kg, i.v.) on ex vivo platelet aggregation induced by ADP (final concentration 3.75 mmol/l)in rats. Data are presented as mean 7 S.E.M. (n¼5). nnPo0.01 vs. control (vehicle-treated group).

EFFECTS OF dl-PHPB ON PLATELET AGGREGATION 177

one control group. All groups were given compound orvehicle orally by gavage in a volume of 5 ml/kg. Onehour after oral administration, rats were anaesthetized

and surgery of A-V shunt was performed. At doses of12.9, 38.8, 129.5 mg/kg (p.o.), dl-PHPB dose-depen-dently decreased thrombus weight significantly from

Fig. 5. Effect of dl-PHPB and NBP on ex vivo platelet aggregation induced by ADP (A), AA (B), and COL (C) in rats. Single doses of dl-PHPB(doses displayed represent the actual doses of 1.3, 3.9, and 12.9 mg/kg, respectively) and NBP were intravenously administered 30 min beforeplatelet aggregation was measured. The final concentrations of ADP, AA, and COL were 3.75 mmol/l, 0.5 mmol/l, and 7.5 mg/ml separately. Dataare presented as mean 7 S.E.M. (n¼10). nPo0.05, nnPo0.01, and nnnPo0.001 vs. control (vehicle-treated group).

Fig. 6. Effect of dl-PHPB, NBP, and ASP on thrombus formation induced in rat arteriovenous shunt. Single doses of dl-PHPB, NBP, or ASP wereorally administered 1 h before extracorporeal blood circulation. Data are presented as mean 7 S.E.M. (n¼10). nPo0.05 and nnPo0.01 vs.control (vehicle-treated group).

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a control of 48.2 mg to 39.9, 36.8, and 31.8 mg,respectively (Po0.05 vs. control). At the correspondingdoses (10, 30, 100 mg/kg, p.o.), NBP and ASP also haddose-dependent antithrombitic effects except for thelower dose of ASP (10 mg/kg).

DISCUSSION

Arterial thrombus frequently induces irreversibledamage or infarction in target organs, e.g., heart andbrain, that can lead to death or permanent disability.Since arterial thrombi are composed predominantly ofplatelets under pathological conditions of disturbedblood flow and shear stress, platelet activation for thedevelopment plays an important role [Toshihiko et al.,1995; Molina et al., 2000]. Platelets are activated byvarious endogenous factors and a platelet-rich throm-bus is formed subsequently. Agents with antiplateletand antithrombotic properties could have a potentiallytherapeutic role for circulatory diseases [Hiroyukiet al., 1996].

Many different antiplatelet drugs are marketed.Aspirin is the prototype of antiplatelet drugs due of itsefficacy, safety, and low cost. Nevertheless, drug-related adverse effects have been reported for allavailable antiplatelet agents such as gastrointestinaldisturbances, bleeding disorders, and rash [Lanas et al.,1996; Masamitsu et al., 1997]. Thus, the search for neweffective and safer antiplatelet agents is still beingupdated.

dl-PHPB, a potassium salt modified from NBPmay convert into NBP in vivo when given orally orintravenously. The present results show that a singleoral dose of dl-PHPB (12.9–129.5 mg/kg) significantlyinhibited ex vivo platelet aggregation induced by ADP,AA, and COL in a dose-dependent manner in rats. Atthe highest dose, dl-PHPB inhibited ADP, AA, orCOL-induced aggregation by 26, 20, and 12%,respectively, being more effective than COL in thismodel. Similar results were obtained after iv adminis-tration. dl-PHPB at the highest dose inhibited ADP,AA, and COL induced platelet aggregation by 30, 35,and 19%, respectively.

Maximal inhibition on platelet aggregation ofdl-PHPB (129.5 mg/kg, p.o.) was observed at 1 hafter treatment with significant inhibition up to 1.5 hafter dosing. No significant effects were found 2 h aftertreatment, suggesting a reversible inhibition of plateletaggregation. Because of this property, dl-PHPB mightavoid some adverse effects of common antiplateletagents, e.g., bleeding disorders, as has already beenobserved for NBP [Peng et al., 2004]. The similarity inthe time-effect relationship of dl-PHPB on the ADP-

induced platelet aggregation was observed afterintravenous injection.

Our data showed that dl-PHPB was moreeffective in inhibiting ADP-induced platelet aggrega-tion indicating that an ADP and ADP-sensitive P2Yreceptors mediating inhibitory effects are associatedwith the antiplatelet effects of dl-PHPB. ADP, releasedfrom damaged vessels and red blood cells, inducesplatelet aggregation through activation of the integrinGPIIb-IIIa and subsequent binding of fibrinogen. ADPis also secreted from platelets on activation, providingpositive feedback that potentiates the actions of manyplatelet activators [Hollopeter et al, 2001].

How does dl-PHPB inhibit AA-induced plateletaggregation? Arachidonate metabolism plays an im-portant role in platelet function and thrombus forma-tion. Thromboxane A2 (TXA2) and prostaglandin I2(PGI2) are major metabolites of cyclooxygenase activa-tion in platelets and in endothelial cells, respectively.TXA2 is a potent platelet aggregating and vasoconstric-tor, while PGI2 is a powerful antiplatelet and vasodi-lator agent [Peng et al., 2004]. Previous studiesindicated that NBP decreased the production ofTXA2 in cerebral cortex cells, and increased theproduction of PGI2, therefore, decreasing the ratio ofTXA2 in platelets to PGI2 in cerebral cortex cells[Chong and Feng, 1997]. Such activities may be alsoinvolved in the antiplatelet effects of dl-PHPB.

Comparing the antiplatelet effects between oraland intravenous treatment, we also found that,although there was no significant difference betweendl-PHPB and NBP at the corresponding doses,inhibition of dl-PHPB was slightly lower than that ofNBP when given orally, a reflection of bioavailability.

The A-V shunt model is widely used andreproducible for evaluation antithrombotic agents.Both dl-PHPB and NBP inhibited thrombus formationin a dose-dependent manner with similar efficacies.The relative importance of a coagulation system in theA-V shunt model has been demonstrated in previousstudies [Peters et al., 1991; Kaul et al., 1996; Hoffmannet al., 1997].

In conclusion, dl-PHPB significantly inhibitsplatelet aggregation ex vivo either by oral or by ivadministration. It also potently inhibits thrombusformation in the A-V shunt model in vivo. Accordingto previous research, the mechanisms of NBP onantiplatelet and antithrombotic effects were related toits actions of increasing the ratio of PGI2/TXA2,enhancing the release of cyclic AMP, and inhibitingthe release of 5-HT, among others [Chong and Feng,1997; Xu and Feng, 2001]. As dl-PHPB may be a pro-drug of NBP, it should have similar effects ex vivo or invivo. Nevertheless, additional studies are needed to

EFFECTS OF dl-PHPB ON PLATELET AGGREGATION 179

completely elucidate the mechanism accounting for theantiplatelet and antithrombotic effects of dl-PHPB.

ACKNOWLEDGMENTS

We thank professor Jinghua Yang, SyntheticPharmaceutical Chemistry Department, Institute ofMateria Medica, for providing dl-PHPB and NBP.

REFERENCES

Becker RC. 1991. Seminars in thrombosis, thrombolysis andvascular biology. Cardiology 79:49–63.

Born GV. 1962. Aggregation of blood platelets by adenosinediphosphate and its reversal. Nature 194:927–929.

Chong ZZ, Feng YP. 1997. Effects of dl-3-n-butylphthalide onproduction of TXB2 and 6-keto-PGF1 alpha in rat brain duringfocal cerebral ischemia and reperfusion. Acta Pharmacol Sin18:505–508.

Damiano BP, Mitchell JA, Giardino E, Corcoran T, Haertlein BJ, deGaravilla L, Kauffman JA, Hoekstra WJ, Maryanoff BE, Andrade-Gordon P. 2001. Antiplatelet and antithrombotic activity of RWJ-53308, a novel orally active glycoprotein IIb/IIIa antagonist.Thromb Res 104:113–26.

Dirnagl U, Iadecola C, Moskowitz MA. 1999. Pathobiology ofischaemic stroke: an integrated view. Trends Neurosci 22:391–397.

Dong GX, Feng YP. 2002. Effects of NBP on ATPase and anti-oxidant enzymes activities and lipid peroxidation in transient focalcerebral ischemic rats. Acta Acad Med Sinicae 24:93–97.

Hiroyuki N, Hiroaki N, Yasumichi I, Hisao T, Minoru S. 1996. Invivo evaluation of antiplatelet agents in gerbil model of carotidartery thrombosis. Stroke 27:1099–1104.

Hoffmann P, Bernat A, Savi P, Herbert JM. 1997. Antiplatelet andantithromboticefficacy of SR 121787, a nonpeptide orally activeglycoprotein IIb/IIIa anagonist, in Rabbits: comparison withclopidogrel and aspirin. J Cardiovasc Pharmacol 30:360–366.

Hoffmann P, Bernat A, Savi P, Herbert JM. 1998. Prevention ofthrombosis and enhancement of thrombolysis in the rabbits by SR121787, a glycoprotein IIb/IIIa anagonist. J Pharmacol Exp Ther286:670–675.

Hollopeter G, Jantzen HM, Vincent D, Li G, England L,Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D,Conley PB. 2001. Identification of the platelet ADP receptortargeted by antithrombotic drugs. Nature 409:202–207.

Hou ST, MacManus JP. 2002. Molecular mechanisms of cerebralischemia-induced neuronal death. Int Rev Cytol 221:93–147.

Jorgensen MB, Diemer NH. 1982. Selective neuron loss aftercerebral ischemia in the rat: possible role of transmitterglutamate. Acta Neurol Scand 66:536–546.

Kaul S, Makkar RR, Nakamura M, Litvack FI, Shah PK, ForresterJS, Hutsell TC, Eigler NL. 1996. Inhibition of acute stentthrombosis under high-shear flow conditions by a nitric oxidedonor, DMHD/NO. An ex vivo porcine arteriovenous shunt study.Circulation 94:2228–2234.

Lanas AI, Arroyo MT, Esteva F, Cornudella R, Hirschowitz BT,Saina R. 1996. Aspirin related gastrointestinal bleeders have anexaggerated bleeding time response due to aspirin use. Gut39:654–660.

Masamitsu S, Yoshiharu T, Kazuo U, Mitsuyoshi N. 1997.Antithrombotic effects of KBT-3022, a novel antiplatelet agent,in an arterial thrombosis model in the guinea-pig. Drug DevelopRes 40:217–222.

Molina V, Arruzazabala ML, Carbajal D, Mas R, Valdes S. 2000.Antiplatelet and antithrombotic effect of D-003. Pharmacol Res42:137–143.

Peng Y, Zeng XG, Feng YP, Wang XL. 2004. Antiplatelet andantithrombotic activity of L-3-butylphthalide in rats. J CardiovascPharmacol 43:876–881.

Peters RF, Lees CM, Mitchell KA, Tweed MF, Talbot MD, WallisRB. 1991. The characterisation of thrombus development in animproved model of arterio-venous shunt thrombosis in the rat andthe effects of recombinant desulphatohirudin, heparin, andiloprost. Thromb Haemost 65:268–274.

Toshihiko I, Hideki K, Masaru T. 1995. Platelet activation in thecerebral circulation in different subtypes of ischemic stroke andBinswanger’s Disease. Stroke 26:52–56.

Umar A, Guerin V, Renard M, Boisseau M, Garreau C, Begaud B,Molimard M, Moore N. 2003. Effects of armagnac extracts onhuman platelet function in vitro and on rat arteriovenous shuntthrombosis in vivo. Thromb Res 110:135–140.

Xu HL, Feng YP. 2000. Inhibitory effects of chiral 3-n-butylphtha-lide on inflammation following focal ischemic brain injury in rats.Acta Pharmacol Sin 21:433–438.

Xu HL, Feng YP. 2001. Effects of 3-n-butylphthalide on thrombosisformation and platelet function in rats. Acta Pharm Sin 36:329–333.

Yan CT, Feng YP, Zhang JT. 1998. Effects of dl-3-n-butylphthalideon regional cerebral blood flow in right middle cerebral arteryocclusion rats. Acta Pharmacol Sin Mar 19:117–120.

Yang JH, Wang XL, Xu ZB, Peng Y. 2002. Novel salts of 2-(a-hydroxypentyl) benzoic acid, the methods for preparation and theapplication of these salts (patent). PCT/CN02/00320.

180 ZHANG ET AL.