广豆根内生真菌中抗菌活性代谢产物的发现及相关作用机制研究

Bacteria infection has been posing a threat to human health. In recent years, with extensive use of antimicrobial agents, the bacterial resistance has become an increasing trend, and creates great difficulties for the treatment of infectious diseases. Therefore, the growing problems of bacterial resistance and side effects of traditional antibiotics need us to find more antimicrobial agents with better antibacterial activities and fewer side effects. Isolation and screening of compounds with effective antibacterial activities from metabolites of endophytic fungi has become an important method for antimicrobial agents’ researches. Sophora tonkinensis is a traditional medicine plant used for treating bacteria infection. Series endophytic fungi with antibacterial activities had been isolated from Sophora tonkinensis during the previous research work. And a series of cyclic peptides, sesquiterpenes and phthalides had been isolated from the metabolites of the fungus GDG102. This project is to find antimicrobial metabolites by chromatographic technology from the fermentation of four endophytic fungi, which were isolated from Sophora tonkinensis. The method of double dilution was adopted to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of active compounds to selected bacteria. And the effects of selected bacteria morphology were observed by scanning electron microscope. Meanwhile, β-galactosidase, alkaline phosphatase and conductivity were employed for the determination of active compounds on selected bacteria’s cell membrane and cell wall. Finally, the effects of protein synthesis and nucleic acid structure of selected bacteria were detected by gel electrophoresis. This research will help us to find lead compounds with antimicrobial activities, and will lay the foundation of the reasonable development of Sophora tonkinensis.

细菌感染是常见的病症。而细菌耐药性问题的突出和现有抗菌药物的毒副作用迫切需要我们寻找更多抗菌效果好、毒副作用少的抗菌药物。从植物内生真菌中发现有效的抗菌活性化合物已成为当前抗菌药物研究的重要途径之一。广豆根是民间常用抗菌中药。项目组前期研究工作表明，广豆根中共生有大量抗菌活性的内生真菌，通过发酵培养，已从真菌GDG102代谢产物中分离得到结构新颖、具有较好抗菌活性的环肽、倍半萜、苯肽等化合物。本项目以广豆根中4株抗菌活性菌株为研究对象，采用植物化学和药理学结合的方法，从真菌代谢产物中寻找结构新颖、具有良好抗菌活性的化合物；通过电导率法、酶活测定法、扫描电镜法、SDS-PAGE电泳法从细菌的形态结构、细胞膜和细胞壁、蛋白质合成和核酸结构的变化等方面探究具有较好应用前景的抗菌化合物的作用机制。本研究为从广豆根内生真菌中筛选抗菌药物先导化合物，为药用植物广豆根的合理开发利用提供科学参考。

本项目以广豆根中12株内生真菌为研究对象，采用植物化学和药理学结合的方法，通过大规模发酵培养和条件优化，运用多种色谱技术，从真菌的代谢产物中分离鉴定化合物154个，其中新化合物29个。采用二倍稀释法，对获得的化合物进行抗菌活性筛选，筛选出具有良好抗菌活性的化合物。对于具有较好应用前景的代表性化合物，从细菌的细胞壁和细胞膜变化等方面，对抗菌机制进行初步探究。本研究为从广豆根内生真菌中筛选抗菌药物先导化合物，为药用植物广豆根的合理开发利用提供科学参考。项目已基本完成了原申请书中预定的研究内容；相关研究工作已发表论文12篇，包括SCI 1区论文1篇，SCI 3区论文4篇，SCI 4区论文5篇，中文核心论文2篇；申请发明专利2项；参加国内学术交流3次。依托本项目，已培养硕士研究生5人，顺利毕业5人。通过本项目的实施，项目负责人和项目组成员在科研能力等方面得到了较大程度的提高。