超滤亲和液质联用技术筛选显齿蛇葡萄叶中酪氨酸酶抑制剂  

Screening and identification of natural inhibitors of tyrosinase from Ampelopsis grossedentata using ultra-filtration affinity-liquid chromatography-mass spectrometry

作　　者：周慧吉 张璐 李廷钊 李波 ZHOU Hui-ji;ZHANG Lu;LI Ting-zhao;LI Bo(Amway(China)R&D Center,Shanghai 201203,China;Amway(China)Botanical R&D Center,Wuxi 214145,China)

机构地区：[1]安利(中国)研发中心,上海201203 [2]安利(中国)植物研发中心,江苏无锡214145

出　　处：《中草药》2021年第22期6796-6805,共10页Chinese Traditional and Herbal Drugs

基　　金：国家重点研发计划中医药现代化研究重点专项(2018YFC1706800)。

摘　　要：目的采用超滤亲和-液质联用技术(ultra-filtration affinity-liquid chromatography-mass spectrometry,UF-LC-MS)结合体外酶活性实验,筛选药茶两用植物显齿蛇葡萄Ampelopsis grossedentata叶(藤茶)中的酪氨酸酶抑制剂。方法通过体外酶活实验发现藤茶具有很好的酪氨酸酶抑制活性,采用UF-LC-MS筛选并鉴定藤茶中1个主要组分对酪氨酸酶的活性具有抑制作用,并鉴定为二氢杨梅素。进一步通过体外荧光光谱、圆二色谱和分子对接技术研究了二氢杨梅素对酪氨酸酶的分子作用机制。结果发现二氢杨梅素对酪氨酸酶具有较高的抑制作用,对单酚酶及双酚酶的半数抑制浓度(median inhibition concentration,IC_(50))分别为37.73、18.87μg/mL。分子模拟结果显示,二氢杨梅素可以进入酪氨酸酶的活性中心,与氨基酸残基形成4个氢键,改变酶活性中心的构象,从而导致酪氨酸酶催化活性下降。此外,二氢杨梅素能明显抑制B16黑色瘤细胞的增殖,并且显著降低黑色素合成。结论为利用UF-LC-MS和分子对接技术筛选药用植物中酪氨酸酶抑制剂提供了参考,也为后续研究和开发藤茶及二氢杨梅素用于治疗皮肤色素沉着性疾病提供理论基础。Objective To screen tyrosinase inhibitors from the leaf extract of Ampelopsis grossedentata(vine tea)by ultra-filtration affinity-liquid chromatography-mass spectrometry(UF-LC-MS)technique combined with enzyme activity in vitro.Methods The inhibitory activity of vine tea extract was evaluated by enzyme activity test in vitro.Meanwhile one major component from vine tea was screened by UF-LC-MS and identified as potential tyrosinase inhibitor,which was identified as dihydromyricetin.The molecular mechanism of dihydromyricetin on tyrosinase was further studied by in vitro fluorescence spectroscopy,circular dichroism and molecular docking techniques.Results The tyrosinase inhibitor was identified as dihydromyricetin,and IC_(50) of monophenolase and diophenolase were 37.73μg/mL and 18.87μg/mL,respectively.The docking results demonstrated that dihydromyricetin entered the active center of tyrosinase,forming four hydrogen bonds with the amino acid residues,and changed the conformation of the active center,which lead to inactivation of tyrosinase.In addition,dihydromyricetin could significantly inhibit the proliferation of B16 melanoma cells,then dramatically reduce the synthesis of melanin.Conclusion Our research provided theoretical reference for screening natural and high affinity tyrosinase inhibitors from herbal medicines by using UF-LC-MS and molecular docking,which could provide a basis for the subsequent research and development of vine tea and dihydromyricetin for the treatment of skin pigmentation diseases.

关 键 词：超滤亲和-液质联用 显齿蛇葡萄 二氢杨梅素 酪氨酸酶 分子对接 B16黑色瘤细胞 

分 类 号：R284.1[医药卫生—中药学]