鹿药抗真菌有效部位筛选及其作用机制研究  

Active fraction selection of Smilacina japonica A.Gray and its antifungal mechanisms

作　　者：刘伟 赵然然 张梦莎 罗凯英 庞廷松 赵鹏帅 周翼鹏 LIU Wei;ZHAO Ran-ran;ZHANG Meng-sha;LUO Kai-ying;PANG Ting-song;ZHAO Peng-shuai;ZHOU Yi-peng(Faculty of Pharmacy,School of Food and Biological Engineering,Shaanxi University of Science&Technology,Xi′an 710021,China;Department of Medicinal Chemistry and Pharmaceutical Analysis,School of Pharmacy,Fourth Military Medical University,Xi′an 710032,China)

机构地区：[1]陕西科技大学食品与生物工程学院药学系,陕西西安710021 [2]空军军医大学药学院药化和药分教研室,陕西西安710032

出　　处：《陕西科技大学学报》2021年第6期58-64,共7页Journal of Shaanxi University of Science & Technology

基　　金：国家自然科学基金项目(81701981);国家级大学生创新创业训练计划项目(S202010708026);陕西科技大学博士科研启动基金项目(2016BJ-52)。

摘　　要：旨在筛选鹿药抗真菌的有效部位,初步探讨鹿药抗真菌有效部位的作用机制.首先采用75%乙醇加热回流提取与石油醚、乙酸乙酯、正丁醇、水萃取相结合的方法对鹿药进行提取分离制备鹿药正丁醇萃取物、石油醚萃取部位、乙酸乙酯萃取部位、水萃取部位,通过微量液基稀释法、生长实验、时间-杀菌曲线实验、生物被膜实验、棋盘式微量液基稀释法等考察鹿药不同萃取部位的抗真菌和抗生物被膜活性,CCK8法考察鹿药部位的细胞毒性,并采用点板实验和激光共聚焦显微镜初步研究鹿药部位对真菌细胞壁的作用.结果发现鹿药的正丁醇部位具有良好的抗真菌和抗生物被膜活性,MIC值范围为104~832 μg/mL,其抗真菌活性浓度低于细胞毒性浓度.此外,鹿药的正丁醇部位与两性霉素B或阿尼芬净联用具有协同抗真菌活性.初步机制研究显示鹿药正丁醇作用后的真菌对细胞壁抑制剂和两性霉素B敏感性增加,且会引起真菌几丁质含量增加.因此,鹿药的正丁醇部位可能是其抗真菌的有效部位,其抗真菌活性可能与影响细胞壁尤其是增加几丁质含量有关.This study aimed to explore active fraction of Smilacina japonica A.Gray with antifungal activity and its antifungal mechanisms.The rhizomes of Smilacina japonica A.Gray was extracted with 75%ethanol under reflux.Then the extract was partitioned with petroleum ether,ethyl acetate,n-butanol and water successively.Microdilution method,growth test,time-killing curve study,anti-biofilm formation assay and checkerboard microdilution assay were used to investigate the antifungal and antibiofilm activities of extracts of Smilacina japonica A.Gray.Spot assay and confocal laser scanning microscope were used to explore its antifungal mechanisms.n-butanol extract of Smilacina japonica A.Gray showed potent antifungal and antibiofilm activities with MICs ranged from 104μg/mL to 832μg/mL,and its MICs were lower than the concentration of its cytotoxicity.In addition,n-butanol extract of Smilacina japonica A.Gray dispalyed antifungal synergism with amphotericin B and anidulafungin.After treated with n-butanol extract of Smilacina japonica A.Gray,the sensitivities of fungi to cell wall inhibitors and amphotericin B were increased,and the chitin content was also augmented.Therefore,The n-butanol extract of Smilacina japonica A.Gray was its active fraction with antifungal activities,and its antifungal mechanism might be related to the effect on the cell wall and the increase of chitin content.

关 键 词：鹿药 抗真菌 抗生物被膜 有效部位 作用机制 

分 类 号：R978.5[医药卫生—药品]