凝血酶时间法的改进及对四物汤类方筛选研究

The Development of a Bioassay Called Thrombin Time and Application of the Bioassay in Study on Siwu Decoction and its Serial Decoctions

目的:改进凝血酶时间(TT)法,使其更适合体外凝血酶抑制剂筛选。并用此方法测定比较四物汤类方及方中代表性成分的抗凝血酶活性。方法:建立凝血酶浓度对lgTT延长率(%)的标准曲线,对影响凝血酶活性的相关因素进行考察,如血浆的储存条件;药物溶媒的选择;药物与凝血酶预温时间。并且测定比较四物汤(SW)、少腹逐瘀汤(SF)、香附四物汤(XF)、桃红四物汤(TH)、芩连四物汤(QL)的抗凝血酶活性。对方中代表性成分的抗凝血酶活性进行测定。结果:获得改进凝血酶时间法的适宜条件;筛选结果显示四物汤类方均有明显的抗凝血酶活性,抑制性的强弱顺序为QL(IC50=1.30 mg.mL-1)>SF(IC50=1.39 mg.mL-1)>XF(IC50=3.55 mg.mL-1)>SW(IC50=8.14 mg.mL-1)>TH(IC50=10.94 mg.mL-1);活性成分槲皮素也具有显著抗凝血酶活性。结论该研究建立了一种凝血酶抑制剂筛选的便捷方法,证实了四物汤类方及成分具有直接抗凝血酶的活性,为深入解析活血化瘀方中靶向凝血酶的活性物质奠定了基础。

Objective： A bioassay called thrombin time （TF） was developed for screening potential thrombin inhibitor and determining anti-thrombin activities of Siwu Decoction and its serial decoctions and some representative compositions. Methods： The lgTT prolongation rate was found to be linearly related to thrombin concentration. The effects of thrombin activities were studied, for example plasma storage methods and time, the chosen of solvent, drug and thrombin pre-heating time. Inhibitory activities of Siwu Decoction and its serial decoctions were compared including Shaofu Zhuyu Decoction （SF）, Xiangfu Siwu Decoction （XF）, Taohong Siwu Decoction （TH）, Qinlian Siwu Decoction （QL）. Inhibitory activities of some compositions were determined. Results The suitable condition of optimized bioassay Tr is got. Siwu Decoction and its serial decoctions have obvious anti-thrombin activities. Anti-thrombin activities of those decoctions are ranked in the order of QL（IC50 = 1.30 mg·mL^-1 ） 〉 SF（IC50 = 1.39 mg·mL^-1 ） 〉 XF（IC50 = 3.55 mg·mL^-1） 〉 SW（IC50 = 8.14 mg·mL^-1） 〉 TH（IC50 = 10.94 mg·mL^-1）. Active composition of quercetin also has antithrombin activities. Conclusion The optimized method is simple to screen thrombin inhibitor and lays the foundation for compositions of Siwu Decoction and its serial decoctions.