高效液相色谱法快速测定降血压肽的血管紧张素转换酶抑制活性

Fast Determination of Inhibitory Activity of Antihypertensive Peptides by High Performance Liquid Chromatography

建立了体外快速测定降血压肽的血管紧张素转换酶抑制活性的高效液相色谱分析方法。以马尿酰组氨酰亮氨酸(HHL)为反应底物,血管紧张素转换酶(ACE)为催化剂,反应所生成的马尿酸为测定指标,未加酶抑制剂的反应为空白对照。使用Symmetry C18分析型色谱柱(5μm 3.9×150 mm,),柱温25℃,流动相A为乙腈(含0.05%三氟乙酸),流动相B为超纯水(含0.05%三氟乙酸),采用梯度洗脱方式,流速0.8 mL/min,检测波长228 nm。在马尿酸浓度为12.5～200μg/mL时,马尿酸浓度与其峰面积呈良好的线性关系(R^2=0.9999);该方法具有良好的准确性和重现性,对马尿酸的回收率为93.23%～100.25%。高效液相色谱法和紫外比色法对血管紧张素转换酶抑制活性无显善差异(P>0.05)。结果显示,高效液相色谱法具有检测快速、操作简便、灵敏度高等优点,为降血压产品的研制提供了简便可靠的检测手段。

Objective： High performance liquid chromatography method was established for the anlysis of angiotensin converting enzyme （ACE） inhibitory activity of antihypertensive peptides fast. Method： N-hippuryl-His-Leu tetrahydrate as the reaction substrate and hippuric acid as the reaction product. The chromatographic conditions were as follows： column, Symmetry C18 （3.9 × 150mm,5μm） column temperature, 25℃; 0. 05% acetonitrile in mobile phase A and 0.05% mobile phase B; flow rate 0. 8mL/min detection wavelength,228nm. Results： An excellent linearity over the range of 12.5-200 μg/mL（R2=0.9999） was observed. This assay method was with high accuracy and repeatability, recovery efficiency 93.23% 100.25%. There is no significant difference for angiotensin converting enzyme inhibitory activity by high performance liquid chromatography and ultraviolet colorimetry method. Conclusion： The high performance liquid chromatography avoided manipulative complexity of ultraviolet colorimetry method with quick analysis, simple manipulation and high sensitiveness. This reliable method was used for analysis of ACE activity of all kinds of antihypertensive peptide.