| Leeches are commonly used in traditional Chinese medicine, its clinical efficacy, characteristics and mechanism of action is more clear, widespread clinical application, according to the2010edition of "Chinese Pharmacopoeia" provides a method of quality control of its targets in anti-thrombin and traditional physicochemical identification of these leeches are not effectively control the clinical efficacy and safety applications. This paper aims to examine the basis of the consolidated activity of leech blood circulation, including leeches on rheology, antithrombotic and anticoagulant activity experiment, a comprehensive evaluation of leeches and leech vivo efficacy experiments through consistency analysis after the establishment of the biological activity of leeches quality control methods and methodological visits in order to promote scientific leech, a comprehensive evaluation of the clinical application of leeches to effectively control the effectiveness and safety.This thesis,doublely guided by theories of Traditional Chinese Medicine and bioassay statistics, was a exploratory research on the establishment and methodological study of the activated partial thromboplastin time(APTT)-based Leech biological activity. The main contents and results were shown as follow:The fist part General topicSince the issue is completed by two graduate students together, so the first task of the overall overview of experimental drugs overview of what they have learned, and describes the contents of the paper related experimentsThe second part Leech in vivo pharmacodynamics researchFirst, experimental studies on the rats models of blood stasis syndrome:To establish six groups rats blood stasis syndrome models(Ice group、Adr group. Dextran group. Hyperlipemia group. Hyperlipemia-Ice group and Blood Deficiency-blood Stasis group) to compare the changes of blood viscosity、plasma viscosity、red blood cell deformability and aggregation index. hematocrit(HCT). platelet aggregation. prothrombin time(PT). activated partial thromboplastin time (APTT) and fibrinogen (Fibg) contents.As the results,Adr group. Hyperlipemia group. Hyperlipemia-Ice group and Blood Deficiency-blood Stasis group Compared have improved hemorheology and anticoagulant activity,especially the Adr group. Dextran group significantly inproved the indexes of red blood cells. Ice group significantly inproved anticoagulant activity.The model of Adr group is good.Second, the effect of Leech and maixuekang on hemorheology in acute blood stasis model of rats After two weeks of continuous administration,the models of rats were prepared by injection of Adr together with being put into ice-water for several minuters.The whole blood viscosity,plasma viscosity,haematocrit(HCT), red cell deformation index and red cell assembling index,were tested to observe the effects of Leech on the hemorheology of blood stasis rats. As the results, compared with the model group, Leech and Maixuekang can reduce blood viscosity,platelet aggregation rate.Leech and Maixuekang can improve hemorheology and the function of blood coagulation.Third, effect of Leech on experimental thrombosisThe way of by-passed artery and vein、electrical stimulation of common carotid artery and induced at mouse tail by subcutaneous injection of carrageenan was used to form in vivo thrombosis, to study the effect of Leech and maixuekang on experimental thrombosis.As the results,the wet and dry weight of thrombosis and TT of all dosages of Leech and the mid dosages of maixuekang respectively in the model of by-passed artery and vein, had significant difference in comparison with the controlled group. The high and mid dosages of hirudo and the high dosages of maixuekang could significantly prolongate the period of in vivo thrombosis(25%、50%、95%) but the mid and low dosages of maixuekang could significantly prolongate the period of in vivo thrombosis(95%)in the model of electrical stimulation of common carotid artery, and all dosages of Leech and maixuekang had significant difference in FIB. Leech and maixuekang in each dosages could obviously decrease the black tail length and clotting time(24h.48h) in the model of induced at mouse tail by subcutaneous injection of carrageenan. Leech and maixuekang has inhibiting action on thrombosis.The third part Leech in vitro pharmacodynamics researchFirst, the fibrinogen plate methodThe immune pervasion reaction has been carried out on the f ibrinogen plate with the fibrinogen as the substrate.The hirudin activityw as evaluated by using the residual of act ivity of thrombin. After reacting with the leech,consultation was done on the standard curve about the thrombin activity obtained from the concent ration dependence of the diameter of the deposit circles.Second,different batches leech thrombin titration and APTT assay results compareComparison of measurement results can be seen from three groups leech source APTT method wherein Hirudo Japan was " exponential " leech leech and the lancet is "S"-shaped curve, the method of the APTT measurement results, it was found to dose ratio of the three leech:willow leech:leech=1:2:2. And willow leech leech leech may have thought the same, or nearly the same efficacy, and willow leech leech can be applied clinically.Thrombin titration experiments, the measurement results for the anticoagulant effect of the strength of Hirudo> Hubei leech> Anhui, Fujian willow=leech leech, Hirudo anticoagulant effect and far greater than the leech leech and willow. By titration with thrombin results contrast shows that the determination of pharmaceutical ingredients both methods differ only focuses on the leech thrombin antithrombin ingredients, is single, and APTT method is to measure the anticoagulant effect on the overall level of leeches, which The method has certain advantages. Third, different batches of leech thrombin titration assay results compare with APTTThrough literature, the stability of hirudin at70-80℃,theoretically inactivated hirudin,the anticoagulant effect was measured with APTT leech thrombin titration method after passing through the high-temperature treatment,and compare the two.Will be transferred to the oven to100℃and150℃,respectivery,are drying time1h,2h,4h, and8h, thrombin titration assays and APTT assay performed after the removal, and set the blank control group.Thrombin titration of the measurement results, along with changes in temperature and time, and willow leech leech titer basically did not change, and the role of anticoagulation leech Hirudo has decreased at100℃,150℃while basically no anti-coagulation. This paper argues that antithrombin and willow leech leech ingredients are basically the same, but with antithrombin Hirudo and different ingredients.APTT assay results, along with changes in temperature and time, the leech, consistent changes in drug and willow leech Hirudo three basic sources,100℃when the ups and downs of drug APTT values have changed, and to150℃, the APTT values reduced basic blank value, basically no anticoagulant effect, this paper considers three anti APTT consistent composition, three equally effectiveThe fourth part The establishment and methodological study of the APTT-based Leech biological activityAPTT dose-effect relationship study law, because its different sources leech different base curve, Japan Hirudo establish an experimental system, leech leech with willow create another experimental system. Select the appropriate test for APTT method linear range investigated, the standard definition of potency, potency test, thereby establishing leech bioassay method APTT methodology and conducted laboratory study methodology, including examine the accuracy and reproducibility study, intermediate precision study, stability study and investigation methods applicability five aspects, which initially established a leech in vitro bioassay method APTT method. |
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