| The scorpion Buthus maresii Karsch belonging to the Buthidae family is poisonous,passes through liver-channel and is pungent in flavor and in property.There are three effects in pharmacopeia,relieving spasm by calming endogenous wind,detoxicating and resolving swelling,activating the channels to alleviate pain.In Chinese traditional medicine,it has been used to be pill,pulvis and decoction in the clinical application.At present,the study of extracting technology to Scorpion includes decoction,ethanol extraction and enzymatic hydrolysis,which is the most common application.There has rarely reported about concentrating methods except heating.As is known to everyone,it is easy to lose its activity for protein by heating.Therefore,it is imperative to explore new methods of extraction and separation of the Scorpion.Modern research has shown that the main medicinal component of the Scorpion is protein,which has a variety of pharmacological effects such as anti-tumor,analgesic,antiepileptic.The composition of the medicinal components,especially the active ingredients of anticoagulant and antithrombotic,remains unclear.The Pharmacodynamic material foundation and mechanism are still not deep enough.Therefore,this project chooses Scorpion as a representation,aiming to its active ingredient using a coupling system,wet ultra-fine grinding technology and ultrafiltration.The study of Scorpion is raised to a new level along with the depth study of anticoagulant and antithrombotic effect and the mechanism of action.The content and results of paper are as follows:1 The feasibility of extracting and concentrating the Scorpion rapidly is investigated in the paper.The experiment is about the differences of wet ultra-fine grinding and decoction in chemical composition and pharmacodynamic properties.Vivo and in vitro anticoagulant,antithrombotic activity tests prove its feasibility.The results show that it is feasible to be used in Scorpion’s extracting and refining.2 Choosing protein extracting rate and membrane flux as indicators,process parameters of wet ultra-fine grinding and ultrafiltration are optimized respectively by different methods.Apart from this,the basic theory of datas for the coupling technologies are also be provided.3 Polypeptides hydrolyzed by Pepsin are assessed to be the active site by preliminary screening.Taking the tablet dissolve laps area of the fibrinogen as the indicator,the parameters,such as enzyme dosage,reaction temperature,the optimum pH value and time,are optimized in the whole process by orthogonal experimental method.Determine the optimal process parameters of the enzyme protein active components prepared.4 DEAE-Sepharose FF ion-exchange chromatography,Sephadex G-75,25gel chromatography technology,the RP-HPLC reverse phase chromatography and LC/MS are used to purify and identify the active component.At the same time,it is that by the SDS-PAGE gel electrophoresis we study the fibrinolytic mechanism of the purified components.The results show that 16 polypeptides are obtained by these methods,and the purification factor reach up to 1836 times.The active component is to play its role of antithrombotic activity by degrading fibrinogen and fibrin.The innovations of this article lie in the following ways:(1)studying the feasibility of two coupling technologies,Wet ultra-fine grinding technology and ultrafiltration,in Scorpion’s extracting and separating,exploring a new method for animal drugs’ further development.(2)Inspecting important process parameters of new Scorpion extraction method,which is favor extended to the industrial production.(3)identifying and studying in-depth of the active compositions and mechanisms in anti-coagulant and antithrombotic effect of Scorpion,isolating and purifying 16 kinds of high-temperature mixture of polypeptides,for the first time,founding a similar role of fibrinolysin----not only the direct degradation of fibrinogen,but also the active role of the degradation of fibrin,providing certain guiding significance for the clinical application of Scorpion. |
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