目的 确定蚂蟥的最佳酶解工艺。方法 选取活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)3种抗凝活性指标，以外翻肠囊实验筛选蚂蟥酶解物在肠吸收前后最佳活性测定指标。采用胃蛋白酶、胰蛋白酶、复合酶(先胃蛋白酶后胰蛋白酶)对蚂蟥进行酶解，以筛选得到的最佳活性指标考察酶的种类、酶底比、酶解时间以及酶解温度对蚂蟥酶解工艺的影响。结果 TT是测定蚂蟥酶解物抗凝活性的最佳指标，采用胰蛋白酶对蚂蟥酶解可获得高活性的酶解产物，最佳的酶解条件为温度73℃，酶底比10%，酶解时间8 h。结论 TT作为抗凝指标进行酶解工艺考察，可反映整个凝血过程，结果更可靠，且该法操作简便，灵敏度高。所优选的酶解工艺简单可行，可为蚂蟥合理提取提供依据。

Objective To optimize the enzymolysis process for Whitmania pigra. Methods In order to select an appropriate index for study on the enzymolysis process, the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of enzymatic products were determined before and after intestinal absorption, respectively, using an isolated everted intestine model. The emzymolysis of W. pigra was carried out by pepsin, trypsin, and the complex enzyme of pepsin and trypsin. According to the selected indexes, the enzymolysis process was optimized under different conditions, such as the kind of enzyme, ratio of enzymes to substrates, time of enzymolysis, and temperature of enzymolysis. Results TT was an appropriate index. Enzymatic products with high activity could be obtained only using trypsin. After optimization, the best enzymolysis conditions were as follows: 10% trypsin was added in the solution of W. pigra, and the reaction was proceeded under 73℃ for 8 h. Conclusion TT is a reliable and sensitive anticoagulation index for enzymolysis. The enzymolysis technology is simple and stable, which could supply the supports for reasonable extraction of W. pigra.

北京市自然科学基金资助项目（7133247）；北京中医药大学药物分析教学团队；北京中医药大学自主课题（2014-JYBZZ-XS-117）