Establishment of a method for determination of biological activity of leech active peptide
Objective The anticoagulant activity of three kinds of leech peptides(Whitmania pigra Whitman,Whitmania acran-ulata Whitman and Hirudo nipponica)was systematically evaluated in vitro.Based on this,a new method for determining the bio-logical activity of leech active peptides was established.Methods In vitro bioactivity assays APTT,PT,TT and fibrinogen-thrombin time(Fibg-TT)were used to evaluate the active poly-peptides of W.pigra Whitman(APP),active poly-peptides of W.acranulata Whitman(APA)and active poly-peptides of H.nipponica Whitman(APH).A new system for the determina-tion of biological activity using leech active peptides as the object of study was developed.By establishing leech standards,defi-ning and measuring potency and examining methodology,a suitable method for the determination of biological activity of leech ac-tive peptides was established.Results APP,APA and APH were all able to significantly prolong plasma-TT and Fibg-TT times.As the Fibg-TT method uses fibrinogen instead of animal plasma,experimental errors caused by individual animal varia-bility are avoided.Therefore,in this study,Fibg-TT was used as the assay method,and through optimization experiments,a modified Fibg-TT method with a fibrinogen content of 0.2％ was finally selected to establish the biological activity evaluation method for APP,APA and APH.The potency determination results as well as the reliability test were good.The results of the methodological investigation indicate that the method is feasible,reproducible and accurate,and that a complete operational proto-col has been established,which can be used as a biological activity evaluation method for APP,APA and APH.Conclusion A new method for the determination of leech active peptide bioactivity,the modified Fibg-TT method,its accuracy,repeatability and feasibility are good,which can complement the study of the Chinese Pharmacopoeia for the determination of leech bioactivity and respond to the true potent substances of leech in activating blood circulation and resolving blood stasis.
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