连翘抑制酪氨酸酶活性体外实验反应体系研究

The Reaction System for the Study of Forsythia suspensa on Tyrosinase Inhibitory Effect

目的研究连翘抑制酪氨酸酶活性实验在微孔板检测系统中适宜的反应体系。方法以连翘两个极性部位的萃取物为受试对象,将反应总体积定为200μl,保持底物L-酪氨酸的体积40μl不变,依次变化酪氨酸酶和溶剂二甲亚砜的体积,在37℃环境下每2 min检测一次吸光度A475,持续40 min,以不含药时吸光度的变化值或含药时的酪氨酸酶活性抑制率作为参考指标,从酶体积、二甲亚砜体积及孵育时间等变化因素分析此实验适宜的反应体系。结果①吸光度值随着酶体积的增加而增高。酶体积为70μl时,吸光度值最高。不同酶体积的反应体系分别在20～26 min之间出现最大吸光度变化速率。②吸光度值随着二甲亚砜体积增加而增高。二甲亚砜终体积分数为5%时,样品溶解程度和线性关系均较2%时好。结论此实验适宜的反应体系为:底物L-酪氨酸40μl,酶体积70μl或以下,加入适量pH 6.8缓冲液和受试药物,使二甲亚砜的终体积分数为5%,体系总体积为200μl,恒温37℃下孵育20～26 min之后检测。此反应体系为下一步研究连翘抑制酪氨酸酶活性实验提供基础,也为其他药物的同类实验提供方法学参考。

Objective To study the proper reaction system for Forsythia suspensa on tyrosinase inhibitory effect. Methods Subjects were different polar extracts from Forsythia suspensa.The total volume of reaction system was 200 μl and L-tyrosine was 40μl,the volume of tyrosinase and DMSO changed,and the absorbance of samples was measured every 2 minutes at 475nm under the condition of 37 °C for 40 minutes.Took the change of absorbance without drug or the inhibition effect of tyrosinase with drugs as reference indexes,the proper reaction conditions of system were analyzed according to the changing factors from tyrosinase,DMSO and reaction time. Results The absorbance of samples were increased with the increasing volume of tyrosinase and DMSO.The highest absorbance appeared when the volume of tyrosinase was 70 μl.The solubility of samples and the linear relation were better when the final volume fraction of DMSO was 5% than that was 2%.Maximum rate of absorbency change appeared in catalytic reaction during 20 to 26 minutes. Conclusion The proper reaction conditions of system including L-tyrosine 40 μl,tyrosinase 30～70 μl,or less proper buffer and drug.The total volume is 200μl and DMSO is 5%,measuring the absorbance at 475nm under the condition of 37℃ for more than 20～26 minutes.The reaction system will lay the foundation for further research on the tyrosinase inhibitory effect of Forsythia suspensa and provide methodology reference for other durgs on congener research.