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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。
The establishment of a HPLC direct determination of three kinds of water-soluble components of Salvia thrombin inhibition. Based on the reaction of chromogenic substrate and thrombin, the product p-nitroaniline was separated from the sample solution by ethyl acetate-semitransparent extraction, and then the product p-nitroaniline was determined by HPLC. After investigation, the concentration of thrombin was determined to be 5 U · m L -1, the pH value of the buffer solution was 8.3, and incubated at 37 ℃ for 5 min after the start of the reaction, which was determined to be the best within 3 h. The newly established HPLC was used to determine the inhibitory rates of Salvia miltiorrhiza in Salvia miltiorrhiza Bunge, salvianolic acid A and salvianolic acid B to 3.06%, 77.77% and 2.35%, respectively. The results showed that salvianolic acid A salvianolic acid A has a direct inhibitory effect on thrombin, Danshensu and salvianolic acid B direct inhibition of thrombin was not significant. The method can eliminate interference of the sample itself in determining the absorption wavelength, sensitive and low consumption, and can be used for screening single or multiple direct antithrombin active ingredients in the Chinese traditional medicine extract.