赪桐提取物抗菌活性及其对金黄色葡萄球菌的抗菌机理

Antimicrobial activity of extracts from Clerodendrum japonicum and its antibacterial mechanism on Staphylococcus aureus

【目的】揭示赪桐提取物(Extracts from Clerodendrum japonicum,EFC)对金黄色葡萄球菌(Staphylococcus aureus)的抗菌机理,为临床抗病原菌感染药及植物杀菌剂的研发提供理论依据。【方法】采用纸片扩散法和微量肉汤稀释法测定金黄色葡萄球菌、伤寒沙门菌和枯草芽孢杆菌对EFC的敏感性,明确EFC的抗菌活性;通过试剂盒、流式细胞仪及扫描电镜等测定EFC作用后金黄色葡萄球菌细胞膜、细胞壁、三羧酸(TCA)循环、可溶性蛋白、胞内活性氧(ROS)水平、细胞凋亡及形态结构的变化,研究EFC的抗菌机理。【结果】EFC对金黄色葡萄球菌、伤寒沙门菌和枯草芽孢杆菌3种供试细菌均具有抗菌活性,其中对金黄色葡萄球菌的抗菌活性最强,其抑菌圈(IZ)、最小抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为19.04 mm、0.50 mg/mL和1.00 mg/mL。经EFC作用10 h后,金黄色葡萄球菌的乳酸脱氢酶(LDH)、钾离子(K+)及碱性磷酸酶(AKP)外泄分别显著增加72.0%、43.0%和78.0%(P<0.05,下同),琥珀酸脱氢酶(SDH)和苹果酸脱氢酶(MDH)活性分别显著下降80.0%和36.4%。金黄色葡萄球菌经EFC作用24 h后,与对照组相比,5MIC(2.50 mg/mL)、10MIC(5.00 mg/mL)和20MIC(10.00 mg/mL)组的胞外蛋白含量分别上升28.6%、41.8%和61.5%,胞内蛋白含量分别下降40.9%、61.3%和82.5%,胞内ROS水平分别增加9.1%、33.5%和51.0%,细胞凋亡率分别增加24.0%、50.6%和72.8%。经EFC作用24 h后,扫描电镜下的菌体形态结构不规则、萎缩、畸形。【结论】EFC通过破坏金黄色葡萄球菌细胞壁和细胞膜的完整性及通透性,影响蛋白合成,导致TCA循环减慢而发挥抑菌作用。

【Objective】To reveal the antibacterial mechanism of extracts from Clerodendrum japonicum(EFC)against Staphylococcus aureus,and to provide a theoretical foundation for the research and development of clinical anti-pathogenic bacteria infection drugs and plant fungicides.【Method】The sensitivity of S. aureus,Salmonella typhimurium and Bacillus subtilis to EFC was determined by disk diffusion method and microbroth dilution method,and the antibacterial activity of EFC was determined. The changes of cytomembrane,cytoderm,tricarboxylic acid(TCA)cycle,soluble protein,intracellular reactive oxygen species(ROS)level,apoptosis and morphological structure of S. aureus treated with EFC were determined by kit,flow cytometry and scanning electron microscope to study the antibacterial mechanism of EFC.【Result】EFC had antibacterial activity against S. aureus,S. typhimurium and B. subtilis,among which the antibacterial activity against S. aureus was the strongest,and its bacteriostatic zone(IZ),minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)were 19.04 mm,0.50 mg/mL and 1.00 mg/mL,respectively. After 10 h of treatment with EFC,the leakage of lactate dehydrogenase(LDH),potassium ion(K+)and alkaline phosphatase(AKP)of S. aureus increased significantly by 72.0%,43.0% and 78.0%,respectively(P<0.05,the same below). The activities of succinate dehydrogenase(SDH)and malate dehydrogenase(MDH)decreased significantly by 80.0% and 36.4%,respectively. After S. aureus was treated with EFC for 24 h,compared with the control group,the extracellular protein content of 5 MIC,10 MIC and 20 MIC groups increased by 28.6%,41.8% and 61.5%,respectively,while the intracellular protein content decreased by 40.9%,61.3% and 82.5%,respectively. The intracellular ROS level increased by 9.1%,33.5%and 51.0%,and the apoptosis rate increased by 24.0%,50.6% and 72.8%,respectively. After 24 h of treatment with EFC,the morphology and structure of the bacteria were irregular,atrophied and deformed under scanning electron microscope.【Conclusion】By destroying the completeness and permeability of the cytoderm and cytomembrane of S. aureus,EFC affecting protein synthesis which leads to the slowdown of TCA circulation and hence plays a role in antibacterial action. Therefore,strengthening the research on the antibacterial components of EFC is an important way to develop new,efficient and low-toxic anti-pathogenic drugs and plant fungicides.