柚皮挥发性有机化合物与牛乳铁蛋白相互作用分子机制及指纹图谱  

Molecular mechanism and fingerprint of interactions of whole matter group of volatile organic compounds in pomelo peels binding with bovine lactoferrin

作　　者：耿照明 周清滕 郭明[1] 胡智燕[1] 朱杰丽 GENG Zhao-ming;ZHOU Qing-teng;GUO Ming;HU Zhi-yan;ZHU Jie-li(College of Chemistry and Materials Engineering,Zhejiang Agriculture and Forestry University,Hangzhou 311300,China;Zhejiang Academy of Forestry,Hangzhou 310023,China)

机构地区：[1]浙江农林大学化学与材料工程学院,浙江杭州311300 [2]浙江省林业科学研究院,浙江杭州310023

出　　处：《中国药理学与毒理学杂志》2022年第10期754-767,共14页Chinese Journal of Pharmacology and Toxicology

基　　金：浙江省基础公益研究计划项目(LGN20B070001)。

摘　　要：目的探究柚皮挥发性有机化合物(PVOC)与牛乳铁蛋白(BLF)相互作用分子机制及指纹图谱。方法利用顶空固相微萃取气相色谱-质谱(HS-SPME/GC-MS)技术分别获得琯溪蜜柚、南非葡萄柚和以色列青柚(S1~S9)PVOC物质组与BLF间相互作用指纹图谱并筛选活性PVOC物质组。通过分子对接技术建立PVOC物质组与BLF间结合的分子模型,结合荧光发射、同步荧光、圆二色性光谱及三维荧光光谱技术和动力学模拟交叉验证PVOC物质组与BLF相互作用的分子机制。结果通过HS-SPME/GC-MS技术共鉴定出S1~S9中21种共有成分,并鉴定出D-柠檬烯(Dt)、乙酸芳樟酯(La)和圆柚酮(Nt)为活性特征成分。PVOC物质组与BLF的分子对接模型表明,Dt上的双键及环己烯与BLF上的Trp560和Ala558联合产生交叉疏水作用,同时周围还存在Thr636,Thr527和Arg531等氨基酸残基通过范德华力与之结合。La分子被包裹在BLF由Trp560,Thr636,Ser519,Thr527,Leu639,Gly525,Tyr524,Gly528,Glu521,Lys522,Arg531及Ala558形成的疏水口袋中;Nt分子被包裹在由Asp509,Glu521,Asp508,Trp560,Ser519,Lys520,Gly528,Thr527,Lys637,Glu635,Thr636,Ala558,Arg531,Glu353及Lys522形成的疏水口袋中。光谱结果表明,PVOC物质组能够引起BLF内源荧光猝灭,Dt和La在BLF中存在独立结合位点与BLF形成复合物,是静态猝灭机制,Nt在BLF中则有多个结合位点且存在协同关系;Dt,La,Nt和柚皮PVOC物质组与BLF间的作用为非辐射能量转移,BLF二级结构改变,α-螺旋松动和β-折叠结构增多。计算结果表明,BLF与PVOC物质组中含量最少的Nt亲和性最强,但结合力不强,对La的亲和性最弱,对含量最多的Dt亲和性较弱,但结合力最强,结合率54.66%。结论BLF与柚皮PVOC物质组的选择结合强度取决于PVOC分子的结构与含量,BLF更倾向选择结合含有羰基氧结构的醛酮类PVOC分子。PVOC物质组导入后引起BLF二级结构的松动、伸展,构象改变是范德华力、疏水力和氢键共�OBJECTIVE To explore the molecular mechanism and interaction fingerprint between volatile organic compounds in pomelo peels(PVOCs)and bovine lactoferrin(BLF).METHODS Headspace solid phase microextraction gas chromatography-mass spectrometry(HS-SPME/GC-MS)was used to obtain the interaction fingerprint of PVOCs from Guanxi honey pomelo,South African grapefruit and Israel green pomelo(3 samples for each kind,S1-S9)with BLF,and the active PVOCs substances were screened.A molecular model of PVOCs binding with BLF was established by molecular docking technology,and the molecular mechanism of the interactions between PVOCs and BLF was cross-verified by combining fluorescence emission,synchronous fluorescence,three-dimensional fluorescence,circular dichroism spectroscopy and kinetic simulation.RESULTS Twenty-one common components of PVOCs in S1-S9 were identified by HS-SPME/GC-MS,and D-limonene(Dt),linalool acetate(La)and naringone(Nt)were confirmed as the characteristic components.The molecular docking model of PVOCs interacting with BLF showed that the double bond and cyclohexene on Dt combined with Trp560 and Ala558 on BLF to produce cross-hydrophobic interaction.At the same time,amino acids Thr636,Thr527,Arg531 and other amino acids were bound to them by van der Waals forces.La molecule was encapsulated in hydrophobic pockets formed by Trp560,Thr636,Ser519,Thr527,Leu639,Gly525,Tyr524,Gly528,Glu521,Lys522,Arg531 and Ala558.The Nt molecule was wrapped in the hydrophobic pockets composed of Asp509,Glu521,Asp508,Trp560,Ser519,Lys520,Gly528,Thr527,Lys637,Glu635,Thr636,Ala558,Arg531,Glu353 and Lys522.The spectral results showed that PVOCs could cause endogenous fluorescence quenching of BLF.Dt and La had independent binding sites of BLF to form a complex,which was a static quenching mechanism.Nt had multiple binding sites in BLF and there was a synergistic relationship.The interactions between Dt,La,Nt,PVOCs and BLF were non-radiation energy transfer,and the secondary structure change of BLF.CONCLUSION The binding strength

关 键 词：柚皮挥发性有机化合物 牛乳铁蛋白 顶空固相微萃取气相色谱-质谱联用 多光谱 分子建模 

分 类 号：R285[医药卫生—中药学]