一种测定抑制凝血酶活性成分的HPLC方法

A new method on investigate chemical constituents which have anti-thrombin effect by HPLC

通过建立一种HPLC直接测定丹参3种水溶性成分对凝血酶的抑制作用。以生色底物与凝血酶反应为基础,通过乙酸乙酯-半微量萃取法实现产物对硝基苯胺与样品溶液分离,然后采用HPLC测定产物对硝基苯胺。经考察确定体系凝血酶浓度5 U·m L^(-1),缓冲溶液p H为8.3,反应开始后37℃下孵育5 min,3 h以内测定为最佳。用新建立的HPLC测定中药丹参中丹参素钠、丹酚酸A与丹酚酸B对凝血酶的抑制率,分别为3.06%,77.77%,2.35%。结果表明丹参水溶性成分中丹酚酸A对凝血酶有直接抑制作用,丹参素与丹酚酸B对凝血酶直接抑制作用不显著。该方法可排除样品本身在测定波长吸收的干扰,灵敏、低耗,可用于筛选中药提取物中单个或多个直接抗凝血酶有效成分。

An in vitro anti-thrombin bioassay was developed to investigate the chemical constituents which have anti-thrombin effect from the water soluble components of Salvia miltiorrhiza. Using Chromozym TH as a probe combined with ethyl acetate Semi-micro ex- traction was applied to measure p-nitroaniline by HPLC. According to the results, the inactivationrate of thrombin by sodium danshen- su, salvianolic acid A and salvianolic acid B under a given set of conditions were 3.06%, 77. 77% and 2. 35%, respectively. In the water-soluble components, salvianolic acid A has a direct inhibition of thrombin, while sodium danshensu and salvianolic acid B have no significant effect on thrombin. The method is sensitive and low consumption. It can eliminate the interference absorbed for the sam- ple itself which can be used for screening single or multiple direct antithrombin active ingredient of herbal extract.