长春中医药大学
吉林省教育厅科学技术研究项目
Changchun University of Chinese Medicine
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(MRSA)的抑菌能力以及毒力因子α-溶血素(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(MIC)、最低杀菌浓度(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、免疫印记分析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,MIC90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(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,MIC90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、甘草的比例为1：2：4时,MIC90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 ability of Huidu Yinhua Powder, from “Orthodox Manual of External Medicine”, on methicillin?resistant staphylococcus aureus (MRSA) as well as the inhibitory effect of virulence factor α-hemolysin (Hla) activity and biofilm formation, and at the same time, to explore the optimal ratios of Huidu Yinhua Powder, to provide experimental support for the new use of the ancient formula. Methods: The inhibitory effects of Huidu Yinhua Powder and the herbs in the formula on USA300 were analyzed by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and disk diffusion assay (K-B method). Hemolysis assay, neutralization assay, Oligomerization assay, Western Blot (WB) to verify in which form the drug inhibits the activity of the virulence factor alpha-hemolysin (Hla). The biofilm assay was performed to evaluate the inhibitory effect of Huidu Yinhua Powder on biofilm, while finally orthogonal experiments were performed to explore the optimal ratios of Huidu Yinhua Powder. Results: Huidu Yinhua Powder inhibited the MRSA strain with a MIC90 of 64 mg/mL and an MBC of 256 mg/mL, with antibacterial circle diameter of 7.50±0.50 mm. Huidu Yinhua Powder inhibits Hla activity by inhibiting Hla secretion and the minimum effective concentration (MEC) was 16 mg/mL, while the MEC of biofilm was 8 mg/mL. In Huidu Yinhua Powder, Honeysuckle and Astragalus only affected the hemolytic activity of MRSA and the formation of biofilm without inhibiting the growth of bacteria, the hemolytic activity MEC and the biofilm MEC of both of them were 32 mg/mL. Glycyrrhiza had a strong bacterial inhibitory capacity, with a MIC90 of 8 mg/mL and a biofilm MEC of 1 mg/mL, without showing inhibitory hemolytic activity at subinhibitory concentrations. The final orthogonal experiment showed that when the ratio of honeysuckle: astragalus: glycyrrhiza in Huidu yinhua Powder was 1:2:4, the MIC90 was 16 mg/mL, the hemolytic activity MEC was 8 mg/mL, and the biofilm MEC was 4 mg/mL, all of which were the lowest among the nine groups. Conclusion: Huidu Yinhua Powder affects the hemolytic activity and biofilm formation of MRSA at sub inhibitory concentrations, with the optimal ratio of honeysuckle, astragalus, and glycyrrhiza being 1:2:4.
