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目的对中药水蛭品种之一的宽体金钱蛭鲜品药材中抗凝血的活性成分进行提取、分离及纯化,并进行抗凝活性检测。方法采用凝胶色谱等方法对活性成分进行提取、分离及纯化;采用HPLC对单体蛋白进行纯度检查;采用SDS-聚丙烯酰胺凝胶电泳法和质谱法测定蛋白单体的分子量;通过单体蛋白对家兔全血凝固时间的影响和对家兔血浆复钙时间的影响进行抗凝血活性检测。结果从宽体金钱蛭新鲜药材中分离出3个蛋白质单体,分子量分别为12042.57 Da、15580 Da、24416 Da,经检测有两个蛋白单体具有抗凝血活性。结论从非吸血类水蛭药材的宽体金钱蛭体内也可分离出具有抗凝血作用的活性物质。
OBJECTIVE To extract, separate and purify the anticoagulant active ingredients from the medicinal materials of Hirudo kaempferi, one of the Chinese traditional medicinal materials, and measure anticoagulant activity. Methods The active components were extracted, separated and purified by gel chromatography and other methods. The purity of the protein was determined by HPLC. The molecular weight of protein was determined by SDS-polyacrylamide gel electrophoresis and mass spectrometry. The influence of protein on the whole blood coagulation time of rabbits and the influence on the time of calcium recalcification in rabbits were tested for anticoagulant activity. Results Three protein monomers were isolated from the fresh Radix Hirudoides. Their molecular weights were 12042.57 Da, 15580 Da and 24416 Da, respectively. Two protein monomers were tested for anticoagulant activity. Conclusion It is also possible to separate active substances with anti-clotting activity from the body of Hirudinia longus which is not a blood-sucking leech.