多酚氧化酶抑制剂高良姜素抗黑色素瘤机制研究

Inhibitory effect of galangin on polyphenol oxidase and study of mechanism

目的:研究高良姜素对多酚氧化酶(PPO)的抑制作用机制,并探讨了高良姜素与多酚氧化酶的结合机制,以期获得高良姜素抗人恶性黑色素瘤细胞株A375机理。方法:分光光度法检测高良姜素对PPO的抑制作用和抑制作用类型;荧光光谱法和分子对接法检测高良姜素与PPO酶的结合方式;MTT法测定高良姜素人恶性黑色素瘤细胞株A375增殖的抑制作用,Transwell法检测高良姜素对A375细胞侵袭的抑制作用。结果:高良姜素是一种竞争型的PPO抑制剂,对PPO氧化酶的半抑制率IC_(50)为(47.86±3.33)μmol/L,抑制常数Ki为(24.83±1.45)μmol/L;高良姜素能够有效猝灭PPO的荧光,高良姜素与PPO的结合常数Ka为(4.67±0.43)×10~4L/mol;高良姜素能够与PPO的活性中心铜离子发生结合作用,并与催化基团His259形成氢键;高良姜素能够明显的抑制A375细胞的增殖和转移,且细胞中的PPO活性和黑色素的合成量降低。结论:高良姜素是一个竞争性的PPO酶抑制剂,对调节黑色素水平起到重要作用,为临床抗皮肤癌提供了理论基础。

Objective： To investigated the activity inhibition and inhibitory type of polyphenol oxidase （PPO） induced by galangin and the interaction mechanism of galangin with polyphenol oxidase was preliminarily indicated, and prove the related mechanism of galangin on proliferation of on human melanoma A375 cells. Methods： The activity inhibition and inhibitory type of PPO induced by galangin were investigated by spectrophotometric method, and interation mechanism of galangin with PPO was preliminarily indicated by fluorescence quenching and molecular docking, and chelating copper ions with the inhibitory mechanism of galangin on polyphenol oxidase was measured. Results： Galangin was a competitive inhibitor,the IC50 and Ki on PPO were obtained to be （47. 86± 3.33 ） and （24. 83 ± 1.45 ） ±mol/L, respectively. Fluorescence spectrum indicated the fluorescence of PPO was quenched effectively by galangin and the binding constant Ka was obtained to be （4. 67±0. 43 ） × 10^4 L/mol. Chelating copper ions and molecular simulation further showed that galangin was combined with active center of copper ions, and formed hydrogen bonds with catalytic site His259. Luteolin could induce the apoptosis of A375 cells significantly, and the tyrosinase activity and melanin synthesis were decreased. Conclusion： Galangin as a competitive polyphenol oxidase inhibitor and reduced the activity of polyphenol oxidase, which provides the theoretical basis for the clinical anti skin cancer.