基于生物活性方法对地龙药材质量评价研究

Quality evaluation study of Pheretima based on the bioassay method

目的:建立地龙蚓激酶生物效价测定方法,通过测定地龙的蚓激酶生物活性,比较不同基原、不同炮制方法地龙的差异性,进而对地龙产地加工与应用提出一定的理论依据和建议。方法:用标准曲线法计算蚓激酶生物效价,从方法耐用性、专属性、线性范围、精密度、回收率等方面对的地龙中蚓激酶生物效价测定的琼脂糖纤维蛋白平板法进行了方法学考察.并分别测定不同来源不同炮制方法的地龙的蚓激酶生物活性。结果:不同琼脂糖、不同纤维蛋白原对本法测定影响较小,阴性对照无干扰;本法线性范围为12.5~400U,回收率为86.16%,精密度试验RSD为0.98%;地龙药材的蚓激酶生物活性在低于60度时基本稳定。考察不同来源、不同炮制方法地龙22批,表明地龙蚓激酶生物效价差异很大。结论:琼脂糖纤维蛋白平板法可以快速、简便、准确地测定蚓激酶的生物活性,用标准曲线法可以控制测定的误差,本法可评价不同来源和不同炮制方法地龙的蚓激酶生物活性,指导和规范研发、产地加工、生产和临床单位对地龙正确合理的使用。

Objective : To establish the titer test method of lumbrokinase in Pheretima, then to compare the differences of Pheretima with different origins and different processing methods by determining the bioactivity of lumbrokinase, and to provide the scientific basis and advices for processing and application of Pheretima. Methods : The lumbrokinase titer was calculated by the standard curve method, and the agarose-fibrin plate method for lumbrokinase titer test was validated on robustness, specificity, linear range, precision and recovery, etc. Then the bioactivity of lumbrokinase in Pheretima with different origins and processing methods was determined and compared. Results : The different agarose and fibrinogen had the less effect on the establish method, and the negative control test showed no interference. The linear range fell into 12.5-400 U, the recovery was 86.16%, and the RSD of precision was 0.98%. The bioactivity of lumbrokinase was stable when the temperature was below 60 ℃. The test results of 22 batches of Pheretima with different origins and processing methods showed that there existed big difference in the lumbrokinase titer. Conclusion : The agarose-fibrin plate method can be served as a stable and reliable quality evaluation method to determine the bioactivity of lumbrokinase, and the errors can be reduced by the standard curve method. The established method can be used to evaluate the bioactivity of lumbrokinase in Pheretima with different origins and processing methods, and to guide the R&D, collection and processing, production and rational clinical use of Pheretima.