多光谱法研究二氢杨梅素对胰脂肪酶的抑制作用  

Prohibition Mechanism Between Dihydromyricetin and Pancreatic Lipase by Multiple-Spectroscopy and Molecular Docking

作　　者：孟晓慧 黄旭波[1] 夏张晨 徐娟[2] 王衍彬[1] 程俊文[1] 杨柳[1] 贺亮[1] MENG Xiao-hui;HUANG Xu-bo;XIA Zhang-chen;XU Juan;WANG Yan-bin;CHENG Jun-wen;YANG Liu;HE Liang(The Key Laboratory of Biochemical Utilization of Zhejiang Province,Zhejiang Academy of Forestry,Hangzhou 310023,China;Zhejiang Provincial Key Laboratory of Resources Protection and Innovation of Traditional Chinese Medicine,Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]浙江省森林资源生物与化学利用重点实验室,浙江省林业科学研究院,浙江杭州310023 [2]浙江省特色中药资源保护与创新利用重点实验室,浙江农林大学食品与健康学院,浙江杭州311300

出　　处：《光谱学与光谱分析》2025年第1期107-116,共10页Spectroscopy and Spectral Analysis

基　　金：浙江省省属科研院所扶持专项(2024F1065-3);国家自然科学基金项目(31571795)资助。

摘　　要：藤茶中主要黄酮类物质——二氢杨梅素(DMY)具有多种生物活性,而其降血脂机理鲜有报道。采用紫外光谱研究了DMY对胰脂肪酶(PL)的抑制作用,结合荧光光谱、同步荧光光谱、三维荧光光谱及分子对接模拟法研究了二者之间的相互作用及机理。紫外光谱测得DMY对PL的半抑制浓度为2.6×10^(-4)mol·L^(-1),表明其对PL具有较好的抑制作用,采用Lineweaver-Burk方程得出该抑制剂为竞争性作用,其抑制常数为6×10^(-4)mol·L^(-1);通过Stern-Volmer方程和静态猝灭双对数公式对293和310 K两者互作的荧光光谱计算,结果表明DMY能猝灭PL的自发荧光,荧光猝灭方式为静态猝灭,猝灭常数K_(SV)与温度负相关;结合常数K a与温度正相关,结合位点数n约为1,说明DMY与PL结合并形成稳定的配位复合物;采用Van't Hoff方程求出结合过程中的热力学参数ΔS=0.2014 J·mol^(-1)·K^(-1),ΔH=32.311 kJ·mol^(-1),ΔG小于0,表明两者通过疏水作用力自发结合且放热;由F rster非辐射能量转移理论计算出结合距离r=1.475 nm,说明DMY与PL结合生成复合物时发生了非辐射能量转移;同步荧光光谱结果表明,DMY与PL的相互作用对PL的二级构象发生了明显改变,主要作用于色氨酸(Tyr)附近。紫外光谱进一步揭示DMY可使PL发生π→π跃迁,导致其氨基酸残基的微环境变化;三维荧光光谱显示DMY可使PL两个特征峰(peak1,peak2)分别红移10和5 nm,荧光强度分别下降51.38%和41.93%,证实两者发生疏水作用后PL的微环境极性增大,构象改变;分子对接模拟结果表明,DMY结合位点位于PL中由PHE77、PHE215、TYR114、ILE209、PRO180氨基酸形成的疏水口袋内,其A环C5、B环C4′、C环C3、C环C O的-OH分别与氨基酸HIS263、TYR114、SER152、PHE215形成氢键,与氨基酸LRU213、GLU179、ALA178、THR78产生范德华力。实验数据有助于深入解DMY的降血脂分子作用机制,其独特的结构为天然抑制剂的药物合成和筛选提A natural plant named vine(Ampelopsis grossedentata)has been proven to exsert various bioactivities due to its major component of dihydromyricetin(DMY),but there is little information on its hypolipidemic function.In this study,the inhibition behavior of DMY based on pancreatic lipase(PL)assay was investigated by ultraviolet spectroscopy followed by a series of multiple-spectroscopy measurements including fluorescence spectroscopy,synchronous fluorescence spectroscopy and 3D fluorescence spectroscopy as well as the DMY-PL interaction mechanism by molecular docking.The half inhibitory concentration(IC_(50))of PL detected by UV spectroscopy was 2.6×10^(-4)mol·L^(-1),showing its satisfactory lipid-lowering capacity on PL.The calculation of the Lineweaver-Burk equation indicated their interaction type was competitive inhibition with the inhibition constant of 6×10^(-4)mol·L^(-1).The Stern-Volmer equation and static quenching double logarithmic formula analyzed the fluorescence spectra.The results suggested that DMY could significantly quench PL's self-luorescence and its fluorescence quenching constant K_(SV)was negatively sensitive to temperature,revealing that the fluorescence quenching process belonged to static quenching.The value of 1 for the binding site and positive relation of K a to temperature demonstrating PL might combine one DMY to produce a stable complex,which was further evidenced by K q values exceeding 2.0×10^(10)L·mol^(-1)·s^(-1).According to the Van't Hoff equation,the results of thermodynamic parametersΔS=0.2014 J·mol^(-1)·K^(-1),ΔH=32.311 kJ·mol^(-1)andΔG<0 under 293 and 310 K elaborated that the binding force was mainly hydrophobic force and a spontaneous and exothermic process.The binding distance r=1.475 nm reflected the possible non-radiative energy transfer from PL to DMY based on the theory of F rster's non-radiative energy transfer.Both synchronous fluorescence spectroscopy and UV spectroscopy results ascertained that the amino acid residue microenvironment and secondary stru

关 键 词：二氢杨梅素 胰脂肪酶 多光谱法 分子对接模拟 

分 类 号：O657.3[理学—分析化学]