| The current quality control modes of Hirudo include traditional morphological identifica-tion,Chemical qualitative and quantitative identification.Thrombin titration methodfor dete-rmination of the content of Hirudo is the quality control standards in China Pharmacopeia2010, defining the Hirudo titer based on antithrombin titer. But this method has a lot of problems, such as need to human judgment, artificial operation fault create and so on. In addition, thrombin needs to dropexcess thrombin in the coagulation of fibrinogen. But for Hirudo, after dropping the last drop of thrombin, some will soon be solidified, some require more a long time to solidification, which shows the amount of remaining thrombin reaction system is different. There fore, the current quality control methods lackof accuracy and objecti-vity.Our group focuses on the main pharmacological effects of experimental researches before establish of quality control reaction system in vitro.Providing basic research information,to ensure the safe and effective use of Hirudo in clinical by design dosage and experiment indexesscientifically. Pharmacological study in vivo and vitro foundthat Hirudocan significa-ntly prolong activated partial thro mbop last in time (APTT) and thrombin time (TT), and there is a certain dose-effect relationship. This experiment based on Hirudo biological effects, bio-statistics as a tool, using specific experimental design to quantify the biological activity, establishing biological activity test methodology of Hirudo. The experiments included evaluation of Hirudo of pharmacological experiment in vivo and vitro, construction the bioa-ssay method of Hirudo in vitro, discussed separately below.First Part:Evaluation of Hirudo of Pharmacological experiment in vivoFirst, the effect of Hirudo and Maixuekang on hemorheology and blod coagulation system in acute blood stasis model of rats. The rat models were established by injection of Adr together with being put into ice-water for several minutes, investigated the change of the prothrombin time(PT), activated partial thro mbop lastin time (APTT), thrombin time(TT), plasma fibrinogen (FIB), and platelet aggregation rate in five minutes. The results showed that Hirudo and Maixuekang can prolong APTT and TT, and reduce blood viscosity and platelet aggregation rate in five minutes.So the mechanism of Hirudo and Maixuekang against acute blood stasis may be prolong APTT andTT,and reduce platelet aggregation rate.Secondly, the effect of Hirudo on blood coagulation system and platelet aggregation in the animal models with blood hypercoagulable state. The model of blood hypercoagulable state was established by intravenous injection of ellagic acid in mice and rats. Using docking method and the capillary tube method to measure bleeding time(BT) and coagulation time (CT) in mice and measuring the viscosity of whole blood and plasma, prothrombin time (PT), activated partial thro mbop last in time (APTT),thrombin time(TT)and plasma fibrinogen (FIB) in rats. Hirudo not only can prolong BT and CT of hypercoagulable mice, but also can reduce the viscosity of whole blood and plasma and prolong PT,APTT and TT of hypercoagulable rats. So the anticoagulation mechanism of Hirudo may be related to reduce platelet aggregation, lower whole blood and plasma viscosity, pro long clotting time and reduce blood.Thirdly, the research of anticoagulation effect of Hirudo in normal mice. After continuo us administration of mice, using docking method and the capillary tube method to measure bl-eeding time(BT) and coagulation time(CT), drawing blood from extracted eyeball to measure the prothrombin time (PT),activated partial thromboplastin time (APTT),thro-mbin time(TT)and plasma fibrinogen (FIB). Through the results we can see that Hirudo can prolong BT, CT, PT and APTT of normal mice, then it indicated that Hirudo has the significant effect on hemorheoogy and blod coagulation systemin normal mice.Second Part:Evaluation of Hirudo of Pharmacological experiment in vitroTo screen reaction system of the anticoagulation effect and establish reaction system of bioactivity assay of Hirudo in vitro, we choose six experiments in vitro:thrombin titration experiment, anti-platelet aggregation in vitro, prothrombin time (PT), activated partial thromboplastin time (APTT), thro mbin time (TT),fibrinogen-thrombin time(Fibg-TT).Based on these experiments in vitro, screening the better linear relationship between dose and effect experimental system.The problems of thrombin titration experiment are described previously.Platelet aggregation experiment in vitro, The results showed that different concentrations of Hirudo extracts can promote platelet aggregation, but it should be the opposite.PT experiment results showed that Hirudo were no significant effect in vitro. APTT, TT and Fibg-TT results all indicated that Hirudo extract presented a good linear relationship between dose and effect in a range of concentrations.Third Part:Construction the bioassay method of Hirudo in vitroScreening the optimal reaction system based on the results of pharmacological expe-riment in vivo and vitro, establish a new method for Hirudo bio-assay. The reaction sys-tems which results consistency in vivo and vitro:activated partial thromboplastin time (APTT),thr-ombin time (TT),fibrinogen-thrombin time(Fibg-TT).Choosing Fibg-TT reaction system to confirm Hirudo biological activity based on its good dose-response relationship, reproducibility and stability. Two types of Hirudo we were established bioassay methods because a big difference in anticoagulant effect between Whitmania pigra Whitman and Hirudo nipponica Whitman in vitro. Because anticoagulant activity of Whitmania pigra Whitman is not representative of Hirudo nipponica Whitman, so we choose traditional Chinese medicine as a standard. Then establishing the reaction system, studying dose-response relationship, creating and defining the potency of standard, finally this method is methodological study in laboratories.Meanwhile, The characteristics of the APTT reaction system is the good dose-response relationship in a certain range of concentrations. So we can establish a APTT method for Hirudo bio-assay as Fibg-TT. According to Fibg-TT Hirudo bioassay method, we can define the potency of Hirudo, FL%of experiment results was low(fess than10%) and the reliability test is also well, the method of Fibg-TT was accurate and reliable with good accuracy (RSD%=4.5,n=6;RSD%=3.6,n=6) and reproducibility (RSD%=3.8,n=6;RSD%=2.4,n=6) and intermediate precision (RSD%=1.7,n=6;RSD%=2.4,n=6). The method of Fibg-TT that can define the potency of Hirudo, therefore to establish standard substance of Hirudo based on prolonging coagulation time is scientific. A summary is given that the Fibg-TT method is simple, objective, accurate, reproducible, precise and the human factors affect little. So the method of Fibg-TT can distinguish the potency of different batches of Hirudo and can be used to control the quality and bioactivity of Hirudo. |
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