红茶和绿茶多酚对猪胰腺α-淀粉酶抑制效果的动力学研究

Dynamic Investigation of Inhibitory Effects of Black Tea and Green Tea Polyphenols on Porcine Pancreatic α-amylase

目的:研究红茶和绿茶抑制剂对猪胰腺α-淀粉酶催化水解淀粉的抑制效果和特异性。方法:实验在25℃和pH6.9条件下进行。混合0.7 mL不同浓度的淀粉和0.2 mL红绿茶(7.5 g/L),添加0.1 mLPPA(0.04 g/L),进行5 min孵化反应。通过SmartSpec Plus Spectrophotometer不间断540 nm波长下,测量PPA催化生成的麦芽糖的吸光度增加值。通过动力曲线的直线段斜率获得反应速度,动力学参数(Vmax和Ks)由Lineweaver-Burk图求出,抑制类型通过统计学分析确定。结果:无抑制剂和红茶抑制剂的最大反应速度(Vmax)之间的差异具有统计学意义(P<0.05),无抑制剂和绿茶抑制剂的酶-底物解离常数(Ks)之间的差异具有统计学意义(P<0.05)。红茶抑制剂解离常数(Ki)值高于绿茶(P<0.05)。结论:红茶抑制为非竞争抑制型,绿茶抑制为竞争抑制型。红茶抑制剂既能与自由酶活化中心位点结合,也能与酶-底物上的位点结合,而绿茶抑制剂仅能与活化中心位点结合。

Objective： To study the effectiveness and specificity of black tea and green tea inhibition of starch hydrolysis that was catalyzed by porcine pancreatic α-amylase（PPA）.Methods： Kinetic experiments were carried out at 25℃and pH 6.9.Starch（0.7 mL） with different concentration and black tea and green tea（0.2 mL,7.5 mg/mL） were mixed together and the reaction was initiated by adding PPA（0.1 mL,0.04 mg/mL） to the incubation medium for 5 min.The increase of absorbance resulting from the PPA liberated maltose was measured continuously at 540nm using SmartSpec Plus Spectrophotometer. The initial velocity was determined from the slope of linear part of the kinetic curves. Kinetic parameters （ Vmax, Ks ） were calculated using Lineweaver - Burk plots. The type of inhibition was determined by statistical analysis. Results： There were significant differences between maximum velocity （Vmax） in the absence and presence of black tea inhibitor （ P 〈 0.05 ） and between the dissociation constants of enzyme - substrate （ Ks ） in the absence and presence of green tea inhibitor （ P 〈 0.05 ）. The dissociation constant of black tea inhibitor （ Ki ） was much higher than that of green tea inhibitor. Conclusion： Acording to the statistical analysis, the black tea inhibition was belong to non - competitive inhibition type, and the green tea inhibition was belong to competitive inhibition type. The black tea inhibitor could bind either to the active site or to the other site of enzyme - substrate to give rise to enzyme - inhibitor and enzyme - substrate - inhibitor complexes, and the green tea inhibitor could only bind to the active site.