槲皮素-3-O-葡萄糖苷抑制α-乳白蛋白非酶糖基化机制分析

Mechanism of Action of Quercetin-3-O-glucoside against the Non-enzymatic Glycosylation ofα-Lactalbumin

以牛α-乳白蛋白(bovineα-lactalbumin,α-La)和果糖为非酶糖基化模型,分析槲皮素-3-O-葡萄糖苷(quercetin-3-O-β-D-glucuronide,Q3G)对α-La非酶糖基化过程中果糖胺和5-羟甲基糠醛(5-hydroxymethylfurfural,5-HMF)形成及游离氨基含量和褐变度的影响,然后结合分子对接和质谱鉴定技术进一步揭示其对非酶糖基化的抑制机理。结果表明:Q3G能显著减轻糖基化过程中的褐变程度,增加游离氨基含量,抑制果糖胺和5-HMF的形成,延缓糖基化反应的进程,且Q3G添加量为2 mmol/L时效果最好。分子对接结果表明Q3G可通过氢键、范德华力和疏水相互作用与α-La的活性氨基酸残基进行结合,影响α-La和果糖的糖基化反应,缓解糖基化反应的进程。同时,超高效液相色谱-四极杆-静电场轨道离子阱串联质谱分析也发现Q3G屏蔽了糖基化的潜在活性位点Lys93和Lys108,且新增糖基化位点Lys98的糖基化活性较弱,可达到抑制非酶糖基化的作用。本研究可为Q3G作为潜在抗糖基化剂的开发提供理论基础。

In this study,the effect of quercetin-3-O-β-D-glucuronide(Q3G)on the formation of fructosamine and 5-hydroxymethylfurfural(5-HMF),the amount of free amino groups and browning degree in a non-enzymatic glycosylation model consisting of bovineα-lactalbumin(α-La)and fructose were evaluated.In addition,the inhibitory mechanism of Q3G against non-enzymatic glycosylation was explored by molecular docking and mass spectrometry(MS).The results indicated that Q3G could significantly reduce the browning degree,increase the free amino groups,inhibit the formation of fructosamine and 5-HMF,and delay the process of glycosylation reaction.The effect was most pronounced at 2 mmol/L Q3G.Molecular docking analysis indicated that Q3G could bind with the active amino acid residues ofα-La through hydrogen bonds,van der Waals force and hydrophobic interactions,delaying the process of glycosylation reaction.Meanwhile,by ultra-high performance liquid chromatography-quadrupole-orbitrap tandem mass spectrometry(UPLC-Q-Orbitrap MS/MS)analysis,it was found that Q3G shielded the potential glycosylation sites Lys93 and Lys108,and the activity of the glycosylation site Lys98 was weak,indicating inhibited non-enzymatic glycosylation reaction.This study can provide a theoretical basis for the development of Q3G as a potential anti-glycosylation agent.