藏药二十五味鬼臼丸对金黄色葡萄球菌的抑菌作用及机制研究  

Study on antibacterial effects and mechanism of Tibetan medicine Ershiwuwei Guijiu Pills on Staphylococcus aureus

作　　者：赵解南 朱彦蓉 袁志兵 张继稳[1,2] 谢和兵 ZHAO Jienan;ZHU Yanrong;YUAN Zhibing;ZHANG Jiwen;XIE Hebing(School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Yangtze Delta Drug Advanced Research Institute,Nantong 226133,China;Jiangsu Shenhou Pharmaceutical Research Co.,Ltd.,Nantong 226133,China;Rexplo Medical Laboratory of Guangxi Medical University,Nanning 530000,China)

机构地区：[1]江西中医药大学药学院,江西南昌330004 [2]南通市海门长三角药物高等研究院,江苏南通226133 [3]江苏神猴医药研究有限公司,江苏南通226133 [4]广西医科大学睿谷医学检验实验室,广西南宁530000

出　　处：《药物评价研究》2023年第1期78-84,共7页Drug Evaluation Research

基　　金：国家自然科学基金资助项目(81470247);西藏自治区科技厅中央引导地方科技(XZ202202YD0020C)。

摘　　要：目的研究藏药二十五味鬼臼丸对金黄色葡萄球菌的抑菌作用,初步探讨相关抑菌机制。方法采用琼脂平板打孔法,测定二十五味鬼臼丸(50、100、200 mg·mL^(-1))对金黄色葡萄球菌的抑菌圈大小。通过微量肉汤稀释法,测定最小抑菌浓度(MIC)。调整肉汤培养基中的二十五味鬼臼丸浓度为0.5MIC、1.0MIC和2.0MIC,分别加入1.0×108CFU·mL^(-1)菌悬液,以不含药液而含菌量相同的培养基作为对照组,在37℃、120 r·min^(-1)恒温培养箱中振荡培养,每小时取样,用紫外可见分光光度计在600 nm处测定吸光度(A600)值,绘制细菌的生长曲线;每2小时取样,离心取上清液,试剂盒法测定碱性磷酸酶(AKP)活性;每小时取样,离心取上清液,用紫外可见分光光度计在_(260) nm处测定A_(260)值,以A_(260)值表示DNA/RNA大分子相对量;每小时取样,离心取上清液,加入考马斯亮蓝G250溶液,于595 nm处测定A595值,检测胞外可溶性蛋白含量;培养8 h后,离心留沉淀,进行SDS-PAGE电泳,检测菌体蛋白含量。结果二十五味鬼臼丸50、100、200 mg·mL^(-1)对金黄色葡萄球菌的抑菌圈直径分别为(12.33±0.75)、(16.33±0.41)、(19.17±0.68)mm;对金黄色葡萄球菌的MIC为100 mg·mL^(-1);与对照组相比,二十五味鬼臼丸组的金黄色葡萄球菌生长显著减缓(P<0.05),胞外的AKP活性显著升高(P<0.05),胞外DNA/RNA大分子相对量显著升高(P<0.01),胞外可溶性蛋白含量显著升高(P<0.01),菌体总蛋白表达量明显减少。结论二十五味鬼臼丸对金黄色葡萄球菌具有抑菌作用,其抗菌作用可能与致细菌细胞壁、细胞膜结构破损,且对菌体蛋白具有一定抑制作用有关。Objective To study the antibacterial effects of the Tibetan medicine Ershiwuwei Guijiu Pills (EGP) on Staphylococcus aureus and preliminarily explore its related bacteriostatic mechanism.Methods The size of the antibacterial zone of EGP (50,100,and 200 mg·mL^(-1)) against Staphylococcus aureus was determined by agar plate drilling method.The minimum inhibitory concentration (MIC) was determined by micro broth dilution method.The concentration of EGP in broth medium was adjusted to0.5 MIC,1.0 MIC and 2.0 MIC,and 1.0×10~8CFU·mL^(-1)bacterial suspension was added,respectively.The medium containing no pharmaceutical solution but the same amount of bacteria was used as the control group,which was oscillated and cultured in a constant temperature incubator at 37℃and 120 r·min^(-1),with samples taken every 1 h.Uv-vis spectrophotometer was used to measure the absorbance (A600) value at 600 nm to draw the growth curve of bacteria.Samples were taken every 2 h,the supernatant was centrifuged,and the activity of alkaline phosphatase (AKP) was determined by kit method.The supernatant was sampled every1 h and centrifuged.The A_(260)value was determined by UV-VIS spectrophotometer at _(260) nm.The A_(260)value was used to represent the relative quantity of DNA/RNA macromolecules.Samples were taken every 1 h,supernatant was centrifuged and added into Coomassie bright blue G250 solution.A595value was determined at 595 nm to detect extracellular soluble protein content.After 8 h culture,centrifuge precipitation and SDS-PAGE electrophoresis were performed to detect the protein content of thalli.Results The diameters of the inhibition zone of EGP at concentrations of 50,100 and 200 mg·mL^(-1)against Staphylococcus aureus were (12.33±0.75) mm,(16.33±0.41) mm and (19.17±0.68) mm,respectively.The MIC against Staphylococcus aureus was 100 mg·mL^(-1).Compared with the control group,the growth of S.aureus was significantly slowed down (P<0.05),the extracellular AKP activity was significantly increased (P<0.05),the relative amou

关 键 词：二十五味鬼臼丸 金黄色葡萄球菌 最小抑菌浓度 碱性磷酸酶 可溶性蛋白 

分 类 号：R285.5[医药卫生—中药学]