Study On Oligopeptide With Anticoagulant Activity From Whitmania Pigra Whitman

Objective:Traditional Chinese medicine leeches refer to dried bodies of three species of animals usually, including Hirudo nipponica Whitman, Whitmania pigra Whitman and Whitmania acranulata Whitman. Leechs are widely used in treatment of cardiovascular and cerebrovascular diseases by activating blood circulation to dissipate blood stasis, and the curative effect isobviously. Whitmania pigra Whitman is the main breed among them applied in clinical. To probe the anticoagulative material foundation of Whitmania pigra Whitmanarger in clinical, we studied the anticoagulant active components from Whitmania pigra Whitman.Methods:1We compared the anticoagulant activity of three different methods of extracting anticoagulant active components from Whitmanis pigra Whitmanwith physiological saline extraction method from fresh Whitma pigra. The extraction methods include unheated, heated extraction from dried Whitman pigra and heated extraction from skimmed dried Whitman pigra. We also evaluate three methods including the thrombin titration method, the APTT kit method and the PT kit method,which are applied to compare the anticoagulant activity and found out the most accuracy and speedy one.2We tested the levels of PT, APTT, FIB and plasma viscosity by in vitro anticoagulant experiment in rats to evaluate the anticoagulant activity of the extracts using the best method.3We separated the hirudo extraction by using Gel Chromatography and ion exchange chromatography which include GE Sephadex G50Medium water as eluting agent, DEAE-cellulose52Buffer salt as gradient elution, Sephadex G50desalination, GE SephadexLH-20water as eluting agent in order to study on anticoagulative material foundation, anticoagulant activity as a guidance.4We measured the molecular weights of the active monomer by LC-MS and tested the anticoagulant activity of the active monomer.Result:1The extracts of three extraction methods all have strong anticoagulant activity, while the heated extraction from skimmed dried Whitman pigra has the strongest anticoagulant activity, and has Statistically significant compared with unheated and heated extraction. the test conclusions by thrombin titration method, APTT kit method and PT kit method have the consistency, The improvement of thrombin titration method which is used to test leeches anticoagulant activity in Chinese Pharmacopoeia made the experimental phenomenology easily visible and the experimental result easy to evaluate.2Experiment of leeches extraction in vitro in rats showed that it can extend PT and APTT, it prompte that leeches extraction can disturb internal blood coagulation factors and exogenous clotting factors, restrain prothrombin to thrombin, suppressed the transformation process of FIB to fibrin, significantly decreased FIB content in plasma, but the effect on plasma viscosity is not obvious.3The experiments acquire three active sites which had strong anticoagulant activity, the activated monomers are gotten by using HPLC which is polypeptide on the basis of physical and chemical identification. The molecular weight are722.6and1880.6.Conclusion:1The anticoagulant activity of extraction from Whitmania pigra Whitman is not destroyed by heating. The extraction ratio of anticoagulant activity increases by degreased with ligarine and can reduce the distraction of color.2The hrombin titration method may be used as a initial method to assess the anticoagulant activity, which is stable, accurate, convenient and easy to operate, while the APTT kit method and the PT kit methods may be applied as a further detect methods.3The hrombin titration method in Chinese Pharmacopoeia, using the white porcelain plate as experimental carrier instead of test tube, monitoring by dissecting needle to make the experiment end point clea, which can be as conventional method in pharmacopoeia.4The Whitmania pigra Whitman contains anticoagulant oligopeptides, whose molecular weights are722.954and1880.6. We suppose preliminarily that the heavier (MW=1880.6) oligopeptide is one of the anticoagulant activity material. The oligopeptide will be sequencing, synthesize, in vitro and in vivo activity study, lay the foundation for drug development in Whitmania pigra Whitman.