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、200mg·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℃、120r·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600nm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260nm处测定A₂₆₀值，以A₂₆₀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595nm处测定A₅₉₅值，检测胞外可溶性蛋白含量；培养8h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、200mg·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100mg·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 to 0.5 MIC, 1.0 MIC and 2.0 MIC, and 1.0×10⁸ CFU·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 2h, the supernatant was centrifuged, and the activity of alkaline phosphatase (AKP) was determined by kit method. The supernatant was sampled every 1h and centrifuged. The A₂₆₀ value was determined by UV-VIS spectrophotometer at 260 nm. The A₂₆₀ 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. A₅₉₅ value was determined at 595 nm to detect extracellular soluble protein content. After 8h 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 amount of extracellular DNA/RNA macromolecules was significantly increased (P<0.01), and the extracellular soluble protein content was significantly increased (P<0.01). The total protein expression of thallus decreased significantly. Conclusion The antibacterial effects of the EGP on S.aureus may be related to the damage of cell wall and cell membrane structure and the inhibition of bacterial protein.

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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）