Leech Anticoagulant Active Ingredient Research

The Whitmania pigra Whitman is dry body of Hirudinidea animals.It is Chinese traditional medicine for removing blood stasis in Chinese pharmacopoeia. Pharmacology experimentation indicate: the Whitmania pigra Whitman has much pharmacological activity, such as antithrombotic, anticoagulant, antiplatelet and so on. This experimentation investigate mainly the chemical compsition of anticoagulant in whitmania pigra Whitman for knowing the substance foundation of pharmacological effect.This experimentation use flash animal of whitmania pigra Whitman to study the component for avoiding the effect of preparation.Firstly , regarding clotting time as index to select the extracting solution, viz reviewing respectively the effect of rabbit clotting time of distilled water extract, N. S extract, ethanol extract (30%, 50%, 75%, 95%) , n-butanol extract, n-hexane extract, PBS extract, then select the N. S extract, secondly, regarding content of protein and anticoagulant value as index to select the extracting technics by orthgonal experimental design, which is extract two times by N. S, first is 5 times , second is 3 times, and soaking 2h each times (4°C) . Adopting salting out to purify simplely the extraction. Thirdly, adopting respectively CM-Sepharose , DEAE-Sephadex A-50, Sephacryl S-200HR, Sepharose F. F and preparative electrophoresis to purify further it, lastly obtaining 10 compound, certaining 9 is protein, furthermore 3 from 9 (MH-3, MH-6, MH-7) can elongate the clotting time and resume calcium time of blood plasma of rabbit, the effect of MH-6 and MH-3 are stronger than MH-7. The pharmacology experimentation indicate: MH-6 and MH-3 can elongate evidencely the bleeding time of sniping tail of mice and the clotting time by glass capillany method, which can make sure that MH-6 and MH-3 have antithrombin activity. Determining the molecular weight of 8 protein by SDS-PAGE, which are 15580, 16960, 20230,22020,24420,31880,33770,46440, and determining the molecular weight of MH-2 by Mass-spectrometry, the molecular weight is 12043. Analyzing the constitute of amino acid in MH-3, MH-6 and MH-7 by amino acidic analysis antomatist.Comparing the whitmania pigra Whitman with Hirudo nipponica inantithrombin activity, in the constitute of amino acid and in fingerprintings of electrophoresis. Investigating the constitute of fatty acid in whitmania pigra Whitman and the fingerprintings of electrophoresi. Studying the quality standard of the whitmania pigra Whitman extraction.