地龙抗凝血活性体外评价方法的建立

Research on the Determination of in Vitro Anticoagulant Activity of Earthworms

关键词:  威廉环毛蚓  可溶性蛋白  纤维蛋白原-凝血酶时间  比活

Key words:  Pheretima guillelmi  soluble protein  fibrinogen-thrombin time  specific activity

摘要:

目的 建立一种基于全自动血凝分析仪测定纤维蛋白原-凝血酶时间的地龙及其相关制剂的抗凝血活性的体外评价方法。方法 以威廉环毛蚓提取物为研究对象，采用BCA蛋白测定法测定可溶性蛋白含量，以抗凝血活性为指标，建立纤维蛋白原-凝血酶时间法测定活性，以活性浓度与可溶性蛋白浓度的比值（比活）作为活性评价指标。结果 10批地龙提取物可溶性蛋白含量为（26.56±3.57）%，比活为（34.64±3.98）U·μg^-1。其比活大小与蚓激酶标准品（43.60 U·μg^-1）相当，且是市售蚯蚓酶（22.17 U·μg^-1）比活的1.5倍。结论 采用BCA蛋白测定法测定地龙提取物可溶性蛋白，方法简便、可靠；所建立的纤维蛋白原-凝血酶时间法，操作简便，人为影响因素小，结果客观准确，可用于实时测定，重复性和仪器精密度良好，线性范围较广，具有可行性，可用于地龙及其制剂活性的评价。

Abstract:

OBJECTIVE To establish a method to evaluate the anticoagulant activity of earthworms and corresponding preparations based on the measurement of fibrinogen-thrombin time with automatic hemagglutination analyzer. METHODS Pheretima guillelmi extracts was used as test sample, and BCA method was applied to measure the content of soluble protein, and fibrinogen-thrombin time method was established to evaluate the antithrombin activity of earthworm, using the ratio of active concentration to soluble protein concentration as the index. RESULTS The protein content of ten bathes earthworms was (26.56±3.57)%. The specific activity of these earthworms was (34.64±3.98)U·μg^-1, which was similar to the activity of lumbrokinase (43.60 U·μg^-1), and 1.5-fold of purchased earthworm enzyme (22.17 U·μg^-1). CONCLUSION BCA method is applied to measure the soluble protein content of earthworms, and it is verified to be reliable and convenient. The established fibrinogen-thrombin time method express to be convenient operation, objective result and real-time measurement properties, with satisfactory methodological evaluation results, and can be used to evaluate the bioactivity of earthworms and related preparations.