Study On Antithrombotic And Antiplatelet Aggregation Activities Of Curdione

With the development of economy and society, cardiovascular diseases becomethe most serious disease to human body. According to the report of The World HealthOrganization, more than1/3of the deaths were caused by cardiovascular disease in theworld every year. It is well known that most of the cardiovascular diseases wereassociated with thrombogenesis and thromboembolism. Therefore, a great deal ofefforts are focused on antithrombotics which are natural, high-performance and littleside-effect, for treatment of cardiovascular diseases.Rhizoma Curcumae （Ezhu in Chinese） is the dry rhizomes derived from Curcumawenyujin Y.H. Chen et C. Ling, Curcuma phaeocaulis Val. and Curcuma kwangsiensisS.G. Lee et C.F. Liang, which has been used in removing blood stasis and alleviatingpain for over thousands of years. As a common traditional Chinese medicine （TCM） toremove blood stasis and alleviate pain, however, to date, the pharmacologicantithrombotic and anti-platelet aggregation activities of its ingredient have not beeninvestigated. The main experimental information was as follows:1. Isolation of curdioneCurdione was isolated from the essential oil in Curcuma wenyujin using silica-gelcolumn chromatography method to give four fractions, which were identified as curdione, curcumol, isocurcumol and germacrone. Their purities are above98%. Thecontent of curdione in the essential oil of Curcuma wenyujin was about11%. The resultshowed that silica-gel column chromatography offered a simple, rapid and highlysensitive method to isolate various components from Zedoary oil.2. Experimental study on anticoagulation and antithrombosis activities of curdionein rhizoma curcumaeIt was aimed to investigate the antithrombosis activity and its possible mechanismof curdione. The antithrombosis activity of curdione was studied using tail thrombosiscaused by carrageenan. The coagulation time was determined using capillary methodin mice. The prothrombin time （PT）, activated partial thromboplastin time （APTT） andthrombin time （TT） were measured by reagent kits in rats. The contents of NO andP-selectin were measured by ELISA. The concentrations of TXB₂and6-keto-PGF₁αinplasma were determined by radioimmunoassay （RIA）. As a result, at three doses ofcurdione, it could significantly inhibit thrombosis of mouse in vivo. At the dose of100mg/kg、200mg/kg, curdione could prolong the coagulation time in mice. Curdionecould significantly affect APTT and TT in a dose dependent manner in rats, but had noeffect on PT. Curdione at30mg/kg、60mg/kg decreased the level of P-selectin and theratio of TXB₂/6-keto-PGF₁α. At the dose of60mg/kg、120mg/kg, curdione couldincrease the content of NO in rats. These findings suggested that curdione couldpossess the anticoagulation and antithrombotic activity, which its anticoagulationactivity might be related to the intrinsic coagulation path. Moreover, its mechanismsmay also be related to inhibit platelet aggregation and increase the release of NO.3. Inhibitory effect of Curdione on platelet aggregationIt was aimed to investigate the effect of curdione on anti-platelet aggregation. Tostudy the inhibitory effect of curdione on platelet aggregation, we monitored humanplatelet aggregation induced by thrombin （0.3U/ml）, PAF （0.375μg/ml）, ADP （10μM）and AA （0.1mg/ml） using Born method. The P-selectin express was measured by flow cytometry analysis. The platelet [Ca²⁺]i was determined by double beam fluorescencespectrophotometer method. The cAMP level was measured by ELISA. This studyutilized the various of selective inhibitors to investigate the role of p38MAPK, ERK andJNK by regulating PAF-induced platelet aggregation using Born method. As a result,curdione preferentially inhibited PAF-and thrombin-induced platelet aggregation in aconcentration dependent manner （IC50:60-80μM）, whereas much higher concentrationsof curdione were required to inhibit aggregation induced by ADP and AA. Curdioneinhibited P-selectin expression in activated platelets. Furthermore, Curdione increasedcAMP level and attenuated intracellular Ca²⁺mobilization in platelets that was inducedby PAF. Moreover, MAPK signal path could regulate PAF-induced platelet aggregation.These findings suggested curdione could posses antiplatelet aggregation activity. Theinhibitory mechanism of curdione on platelet aggregation may increase cAMP levelsand subsequently inhibit intracellular Ca²⁺mobilization. But the in-depth mechanism ofanti-platelet aggregation effects whether curdione could regulate MAPK signal path isneeded to be studied.