Eupolyphaga Bionic Stomach Enzyme Was Purified And Separated From The Basic Research Material

Objective:To study the process conditions of extracting Eupolyphaga seu Steleophaga by bionic enzymatic hydrolysis. Separate anticoagulant component from Eupolyphaga seu Steleophaga Walker and investigate the physicochemical properties and stability of it. Study the effect of polypeptide F2-2in Eupolyphaga seu Steleophaga on activating blood circulation、dilating vascular and modulating blood contents of NO/ET-1、t-PA/PAI.Methods:Basing on the confirmed hydrolysis temperature and taking the prothrombin time (PT), activated partial thromboplastin time (APTT) as indexes, the ratio of enzymes to substrates and the time of enzymatic hydrolysis were selected, and the extractive technique was verified by taking the platelet aggregation rate, and blood clots-fibrinolytic dynamic figure as the indexes; Sephadex gel chromatography was applied to separate The pepsin hydrolysate with the index of anticoagulant activity; The effect of polypeptide F2-2in Eupolyphaga seu Steleophaga on activating blood circulation was evaluated in vitro and in vivo by taking the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time(TT), plasma fibrinogen(FIB),platelet aggregation rate, blood clots-fibrinolytic dynamic figure as indexes. The functions of anticoagulant component F2-2on dilating blood vessels and protecting endothelial cells was investigated based on indexes of vascular dilatation ratio NO/ET-1、t-PA/PAI content in rat plasma.Result:(1) The best enzymatic hydrolysis condition was as follow:Eupolyphaga seu Steleophaga was warm-up in artificial gastric juice (37℃) for30min and then hydrolyzed with addition of pepsin (ratio of pepsin to substratewas2.0%) for3hours, and then the residue was hydrolyzed in artificial intestinal juice (45℃) and trypsin (ratio of trypsin to substrate was4%) for2hours.(2) Separated with Sephadex G-50and G-25, polypeptide F2-2was proved to be the most effective anticoagulant component in Eupolyphaga seu Steleophaga.(3) Polypeptide F2-2showed favorable activities in prolonging PT、APTT、TT, decrease FIB、declining the platelet aggregation rate and the largest of blood coagulation in vitro. As to the result in vivo, PT and APTT were prolonged, FIB was increased, and platelet aggregation rate and the largest of blood coagulation were declined, no significant difference was found in TT.(4) polypeptide F2-2was able to dilate the rat artery in vitro and increase the content of PAI in vivo, no significant difference was found in NO. ET-1and t-PA. Conclusion:The best enzymatic hydrolysis condition of Eupolyphaga seu Steleophaga was determined and an effective anticoagulant component F2-2was separated from Eupolyphaga seu Steleophaga.F2-2was proved to be able of anticoagulating both in vitro and in vivo and dilating the rat artery in vitro. our efforts could be a reference for further study on material base of Eupolyphaga seu Steleophaga.