胡桃醌对微生物的氧化损伤机理及1,4-萘醌类化合物抑菌构效关系研究

Juglone, namely 5-hydroxy-1,4-naphthoquinone, extracted from the juglans mandshurica maxim, root, leaves, and green hull of walnut, has been proved to be of a wonderful inhibiting microbes capacity to G+ bacterials, so as to be a promising phytochemical in developing natural bactericides, food preservatives, and antifungal medicaments for healing skin infections. However, its inhibiting mechanism remains unclear. This project will explore the inhibiting effects of juglone on bacterial, especially on cell wall and membrance, and its antimicrobes mechanism using Staphylococcus aureus as a pattern bacterial. Differential proteomics analysis will be used to confirm the differential protein in cell wall, membrance, and protein in cytoplasm. Meanwhile, the structural-efficiency relationship will be studied by comparing the inhibiting effect among juglone and eight 1,4-naphthoquinone compounds by using semi-empirical method, and the key factors that influence inhibiting effect will be confirmed. The results could provide theoretical foundation for high-throughput screening and synthesizing high-efficiency bactericides in the future.

核桃楸、根、叶片及青果皮中含有的胡桃醌（5-羟基-1,4-萘醌）被证实对革兰氏阳性菌具有优良的抑菌能力，在天然杀菌剂、防腐剂、治疗真菌性皮肤病等方面均有很大的发展潜力，但其抑菌机理尚不明确。本项目以对胡桃醌敏感的金黄色葡萄球为目标菌，以胡桃醌对细胞壁、细胞膜的损伤机理研究为切入点，采用蛋白质组学的方法研究胡桃醌对微生物细胞壁蛋白、细胞膜蛋白和菌体内蛋白表达的差异，解析胡桃醌对金黄色葡萄球菌的抑菌机理；同时采用半经验法对比分析胡桃醌及另外8种1,4-萘醌化合物对金黄色葡萄球菌抑菌效果的构效关系，明确影响胡桃醌类化合物抑菌效果的关键因素，为未来高通量筛选和合成胡桃醌类高效杀菌剂提供可供借鉴的理论依据。

青龙衣是胡桃外部的一层青皮，胡桃醌（5-羟基-1,4-萘醌）是其中主要成分。研究表明其具有良好的抗肿瘤、抗疟疾、抗病毒、杀虫、化感活性。然而其对典型食源性病原微生物的作用机制与构效关系的研究却鲜有报道。本课题选择金黄色葡萄球菌与大肠杆菌这两种常见的食源性致病菌，观察其在胡桃醌干预后的表观和生物学变化，同时利用蛋白质组学技术分析其蛋白层面的变化，从表观到分子多维度的揭示胡桃醌对两种微生物的损伤机制；此外，采用3D-QSAR构效关系模型进行分析，为提高1,4萘醌类物质的抗菌活性提供理论基础。实验结果表明，胡桃醌可以诱导金黄色葡萄球菌胞内氧化还原酶上调表达，制造胞内过氧化环境，进而阻碍细胞壁合成，蛋白质合成，三羧酸循环，DNA的复制与转录，同时增加细胞膜渗透性。对于大肠杆菌，胡桃醌可以抑制其脱氢酶和细胞色素氧化酶的表达，进而抑制能量代谢；诱导RNA合成和核糖体装配，进而抑制菌体转录。选取19种1,4萘醌类物质，测定其对金黄色葡萄球菌的IC50与MIC活性，选用CoMFA（Q2=0.681，主成分数4，R2=0.961）与CoMSIA（Q2=0.731，主成分数2，R2=0.895）模式进行3D-QSAR模型构建，结果表明在1,4萘醌的2位增加疏水性残基的体积对增加其活性有利。本研究在分子水平上揭示了胡桃醌的抗菌机制，提出了提高1,4萘醌类物质抗菌活性的可行途径，为其在食品防腐保鲜及临床应用上奠定了理论基础。