茶皂素对临床常见致病菌的抗菌活性及作用机制研究  

Study on the antibacterial activity and mechanism of tea saponin against common clinical pathogens

作　　者：刘静 谢朋飞[1] 蔡延渠[2,3,4] LIU Jing;XIE Pengfei;CAI Yanqu(Department of Pharmacy,First People's Hospital of Chenzhou,Chenzhou423000,China;Center for Drug Research and Development,Guangdong Pharmacological University,Guangzhou 510006,China;Class III Laboratory of Modern Chinese Medicine Preparation,State Administration of Traditional Chinese Medicine of the P.R.C,Guangzhou 510006,China;Key Laboratory of Modern Chinese Medicine of Education Department of Guangdong Province,Guangzhou 510006,China)

机构地区：[1]郴州市第一人民医院药学部,湖南郴州423000 [2]广东药科大学新药研发中心,广东广州510006 [3]国家中医药管理局中药制剂实验室(三级),广东广州510006 [4]广东省教育厅现代中药重点实验室,广东广州510006

出　　处：《广东药科大学学报》2021年第5期35-41,共7页Journal of Guangdong Pharmaceutical University

基　　金：湘南学院科学研究项目(2019XJ58);广东省教育厅“2017年度广东省省级大学生创新创业训练计划”项目(201710573009)。

摘　　要：目的评价茶皂素对临床常见致病菌的抗菌活性,并分析其对金黄色葡萄球菌、福氏痢疾杆菌的作用机制。方法(1)以表皮葡萄球菌、金黄色葡萄球菌、肺炎克雷伯菌、变形杆菌、沙门氏菌、铜绿假单胞菌、大肠杆菌、福氏痢疾杆菌为受试菌株,采用K-B纸片法测定茶皂素对不同致病菌株的抑菌环直径,同时通过微量稀释法测定最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC);(2)以金黄色葡萄球菌、福氏痢疾杆菌为受试菌株,采用酶标仪测定不同培养时间点的A600值,评价茶皂素对致病菌的生长特性及细胞壁通透性的影响;采用荧光分光光度计测定不同时间的荧光强度,评价茶皂素对细胞膜电位的影响;采用酶标仪测定培养6 h后的细胞外核酸及蛋白质含量,评价茶皂素对细胞内容物的影响。结果(1)体外抗菌活性:茶皂素对表皮葡萄球菌、金黄色葡萄球菌的敏感性最强,其次为变形杆菌、沙门氏菌、铜绿假单胞菌、福氏痢疾杆菌,最弱为肺炎克雷伯菌、大肠杆菌,茶皂素对受试菌株的抑菌环直径范围为10.86~17.31 mm,MIC值范围为0.20~1.56 mg/mL,MBC值范围为0.39~3.13 mg/mL。(2)抗菌作用机制:与空白组相比,茶皂素组(MIC、2MIC)能够显著抑制金黄色葡萄球菌、福氏痢疾杆菌的生长(P<0.01),同时破坏细胞壁和细胞膜引起胞外AKP、核酸、蛋白质含量显著提高(P<0.01),胞内荧光强度显著下降(P<0.01)。结论茶皂素对临床常见致病菌株(尤其是革兰阳性菌)均表现出较好的抗菌活性,其抗菌作用机制与破坏菌体细胞壁和细胞膜结构与功能的完整性有关,表现为细胞外AKP、核酸、蛋白质含量上升,细胞膜电位下降。Objective To evaluate the antibacterial activity of tea saponin against common clinical pathogens,and analyze its mechanism against Staphylococcus(S.)aureus and Shigella(S.)flexneri.Methods(1)S.epidermidis,S.aureus,Klebsiella(K.)pneumoniae,Proteus,Salmonella,Pseudomonas(P.)aeruginosa,Escherichia(E.)coli and S.flexneriwere taken as the tested strains.The inhibitory ring diameter of tea saponin against different pathogenic strains was determined by K-B paper method.The microdilution method was used to determine minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC).(2)Using S.aureus and S.flexneri as test strains,the A600 values at different culture time points were measured by microplate reader to evaluate the effects of tea saponin on the growth characteristics and cell wall permeability of pathogenic bacteria.The fluorescence intensity at different time was measured by fluorescence spectrophotometer to evaluate the effect of tea saponin on cell membrane potential.The contents of extracellular nucleic acid and protein after 6 h culture were measured by microplate reader to evaluate the effect of tea saponin on cell contents.Results(1)Antibacterial activity in vitro:Tea saponin had the strongest sensitivity to S.epidermidis and S.aureus,followed by Proteus,Salmonella,P.aeruginosa and S.flexneri,and the weakest was K.pneumoniae and E.coli.The antibacterial ring diameter of tea saponin to the tested strains was 10.86~17.31 mm,the MIC value was 0.20~1.56 mg/mL,and the MBC value was 0.39~3.13 mg/mL.(2)Antibacterial mechanism:Compared with the blank group,tea saponin group(MIC,2MIC)significantly inhibited the growth of S.aureus and S.flexneri(P<0.01),destroyed the cell wall and cell membrane,which led to increase in extracellular AKP,nucleic acid and protein content(P<0.01),and decrease in intracellular fluorescence intensity(P<0.01).Conclusion Tea saponin shows good antibacterial activity against common clinical pathogens,especially Gram-positive bacteria,which is related to destruction of th

关 键 词：茶皂素 临床致病菌 抗菌活性 作用机制 

分 类 号：R285.5[医药卫生—中药学]