回毒银花散中各药及药物不同配比对耐甲氧西林金黄色葡萄球菌的作用研究  

Effects of the various herbs and different proportions of the herbs in Huidu Yinhua powder on methicillin⁃resistant Staphylococcus aureus

作　　者：李雨芬 姜爽[1] 宋伍 姜涛[1] 刘畅[1] 周豪芳 唐亚婷 魏琳 苏鑫[1] LI Yufen;JIANG Shuang;SONG Wu;JIANG Tao;LIU Chang;ZHOU Haofang;TANG Yating;WEI Lin;SU Xin(Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区：[1]长春中医药大学,长春130117

出　　处：《中国比较医学杂志》2024年第2期63-71,共9页Chinese Journal of Comparative Medicine

基　　金：吉林省教育厅科学技术研究项目(JJKH20230985KJ)。

摘　　要：目的研究《外科正宗》中回毒银花散对耐甲氧西林金黄色葡萄球菌(methicillin⁃resistant Staphylococcus aureus,MRSA)的抑菌能力以及毒力因子α-溶血素(α⁃hemolysin,Hla)活性和生物被膜形成的抑制作用,同时探究回毒银花散最佳配比为古方新用提供实验支撑。方法通过最低抑菌浓度(minimum inhibitory concentration,MIC)、最低杀菌浓度(minimum bactericidal concentration,MBC)、纸片扩散法(K⁃B法)分析回毒银花散及组方中各味药对MRSA菌株USA300的抑制效果,溶血实验、Hla中和实验、Hla寡聚实验、免疫印记(Western blot)实验验证药物以何种形式抑制毒力因子Hla的活性,生物被膜形成实验评价回毒银花散对生物被膜的抑制效果,最后正交实验探究回毒银花散的最佳配比。结果回毒银花散抑制MRSA菌株,MIC_(90)为64 mg/mL,MBC为256 mg/mL,抑菌圈直径为(7.50±0.50)mm。回毒银花散还通过抑制Hla的释放抑制Hla的活性,最小有效浓度(minimum effective concentration,MEC)为16 mg/mL,抑制生物被膜形成的MEC为8 mg/mL。回毒银花散中金银花、黄芪只影响MRSA溶血活性以及生物被膜形成但不抑制细菌的生长,两药溶血活性MEC以及生物被膜形成MEC均为32 mg/mL;甘草抑菌能力较强,MIC_(90)为8 mg/mL,生物被膜MEC为1 mg/mL,在亚抑菌浓度下未表现出抑制溶血活性。最后正交实验显示,当回毒银花散中金银花、黄芪、甘草比例为1∶2∶4时,MIC_(90)为16 mg/mL,溶血活性MEC为8 mg/mL,生物膜形成MEC为4 mg/mL,均为9组中最低。结论回毒银花散在亚抑菌浓度下可影响MRSA的溶血活性以及生物被膜形成,其中金银花、黄芪、甘草的最佳比例为1∶2∶4。Objective To study the inhibitory effect of Huidu Yinhua powder from the Orthodox Manual of External Medicine on methicillin⁃resistant Staphylococcus aureus(MRSA),virulence factorα⁃hemolysin(Hla)activity,and biofilm formation,and to explore the optimal ratios of Huidu Yinhua powder and provide experimental support for its use.Methods The inhibitory effects of Huidu Yinhua powder and the herbs in the formula on USA300 were analyzed by the minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),and disk diffusion assay(K⁃B method).Hemolysis,neutralization,oligomerization,and Western blot assays were used to verify in which form the drug inhibits the activity of virulence factorα⁃hemolysin(Hla).A biofilm assay was performed to evaluate the inhibitory effect of Huidu Yinhua powder on biofilm.Orthogonal experiments were performed to explore the optimal ratio of Huidu Yinhua powder.Results Huidu Yinhua powder inhibited the MRSA strain with a MIC_(90) of 64 mg/mL and an MBC of 256 mg/mL with antibacterial circle diameter of(7.50±0.50)mm.Huidu Yinhua powder inhibited Hla activity by inhibiting Hla secretion.The minimum effective concentration(MEC)was 16 mg/mL,and the MEC of biofilm was 8 mg/mL.In Huidu Yinhua powder,honeysuckle and astragalus only affected the hemolytic activity of MRSA and biofilm formation without inhibiting bacterial growth.The hemolytic activity and biofilm of MEC were both 32 mg/mL.Glycyrrhiza had a strong bacterial inhibitory capacity with a MIC_(90) of 8 mg/mL and biofilm MEC of 1 mg/mL without showing inhibitory hemolytic activity at subinhibitory concentrations.The orthogonal experiment showed that,at a ratio of honeysuckle,astragalus,and glycyrrhiza in Huidu Yinhua powder of 1∶2∶4,the MIC_(90) was 16 mg/mL,MEC of hemolytic activity was 8 mg/mL and that of biofilm was 4 mg/mL,both of which were the lowest among the nine groups.Conclusions Huidu Yinhua powder affects the hemolytic activity and biofilm formation of MRSA at subinhibitory concentrations with the

关 键 词：金黄色葡萄球菌 耐甲氧西林金黄色葡萄球菌 回毒银花散 α-溶血素 生物被膜 

分 类 号：R-33[医药卫生]