苦丁冬青苦丁茶咖啡酰奎尼酸类物质与α-淀粉酶的相互作用特性

Interaction Properties of Caffeoylquinic Acid Derivatives from Ilex kudingcha C. J. Tseng with α-Amylase

探讨苦丁冬青苦丁茶中咖啡酰奎尼酸(caffeoylquinic acids,CQA):3-CQA、4-CQA、5-CQA、3,4-di CQA、3,5-di CQA和4,5-di CQA对α-淀粉酶的体外抑制活性,并采用荧光光谱法和圆二色谱法分析CQA与α-淀粉酶的相互作用特性,使用修正后的Stern-Volmer方程与van’t Hoff方程探讨CQA-酶之间的结合常数、结合位点数及热力学参数。结果表明:3-CQA、4-CQA、5-CQA、3,4-di CQA、3,5-di CQA和4,5-di CQA对α-淀粉酶均有较强的抑制作用,半抑制浓度(IC_(50))分别为1.54、1.05、1.28、0.96、0.33、0.64 mg/m L;CQA与α-淀粉酶发生结合反应,生成稳定的复合物,从而造成酶分子内部荧光发生猝灭;热力学参数分析表明CQA与α-淀粉酶主要靠疏水作用力结合,反应自发进行。此外,圆二色谱结果显示CQA的结合引起α-淀粉酶二级结构的变化,破坏了酶蛋白的天然构象,从而降低了酶的催化活性。

The inhibitory effects of six caffeoylquinic acid（CQA） derivatives（3-CQA, 4-CQA, 5-CQA, 3,4-di CQA, 3,5-di CQA and 4,5-di CQA） against α-amylase were studied comparatively by inhibitory activity assay. Furthermore, the potential interaction mechanisms between CQA derivative and α-amylase were investigated by fluorescence quenching and circular dichroism（CD） spectroscopy. The binding parameters were calculated according to modified Stern-Volmer equation, and the thermodynamic parameters were determined by the van＇t Hoff equation. The results showed that all CQA derivatives exhibited inhibitory effects on α-amylase and the half inhibitory concentrations（IC_（50）） were 1.54, 1.05, 1.28, 0.96, 0.33 and 0.64 mg/m L, respectively. CQA derivatives interacted with α-amylase, forming stable complexes and leading to fluorescence quenching. Thermodynamic analysis indicated that the interaction process was spontaneous, and hydrophobic force might be primarily responsible for the interaction. In addition, the CD spectra suggested that the binding of CQA derivatives to the enzyme induced the change of protein structure, thus destabilizing the enzyme and reducing its activity.