安石榴苷的抗菌活性筛选及对金黄色葡萄球菌的作用机制研究

Screening of Antibacterial Activity of Punicalagin and Its Mechanism of Action against Staphylococcus Aureus

目的:研究植物鞣质主要单体成分安石榴苷对5种不同菌种的微生物抑制活性,并初步探讨其对金黄色葡萄球菌Staphylococcus aureus的抑菌机制。方法:采用微量稀释法,测定安石榴苷对金黄色葡萄球菌等5种细菌的最小抑菌浓度(MIC),从细菌生长曲线、碱性磷酸酶(AKP)活性、260 nm物质、钾离子(K+)泄漏及胞内外ATP酶含量,结合流式细胞术和SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)谱带,考察安石榴苷对菌体生长特性、膜损伤及可溶性蛋白的影响,并通过透射电镜直接观察菌体形态变化。结果:安石榴苷总体对革兰阳性菌抑制作用较革兰阴性菌强;药物作用金黄色葡萄球菌后,细菌的生长及活力相比对照组受到明显抑制,胞内AKP、核酸、K+及ATP酶均有不同程度释放,SDS-PAGE结果显示经安石榴苷处理的菌体蛋白条带模糊或消失,且细菌超微结构受到破坏。结论:安石榴苷对5种菌株具有不同程度的抗菌活性,能够显著影响金黄色葡萄球菌的细胞壁完整性及细胞膜通透性,且对菌体蛋白具有一定抑制作用。

Objective: To study the microbial inhibitory activity of the main component of plant tannin,punicalagin,against five different strains,and to explore its antibacterial mechanism against Staphylococcus aureus. Methods: The minimum inhibitory concentration( MIC) of punicalagin against five strains of Staphylococcus aureus was determined by micro-dilution method. From bacterial growth curve,alkaline phosphatase( AKP) activity,260 nm substance,potassium ion leakage and intracellular and extracellular ATPase contents,and combined with flow cytometry and SDS polyacrylamide gel electrophoresis( SDS-PAGE) bands,the effects of punicalagin on cell the growth characteristics,membrane damage and soluble protein were investigated,and the bacteria morphology changes were directly observed under a transmission electron microscope. Results: Punicalagin inhibited above five strains to different extents. The inhibitory effect on Gram-positive bacteria was stronger than that on Gram-negative bacteria. After the agent was applied to Staphylococcus aureus,the growth and vitality of the bacteria were significantly higher than that of the control group. Intracellular AKP,nucleic acid,potassium ions and ATPase had different degrees of excretion. SDS-PAGE results showed that the phenolic acid-treated cell protein bands were blurred or disappearred.Conclusion: The five strains of punicalagin possess different degrees of antibacterial activity,which can significantly affect the cell wall integrity and cell membrane permeability of Staphylococcus aureus,and exhibite a certain inhibitory effect on bacterial proteins.