目的 研究藏藥二十五味鬼臼丸對(duì)金黃色葡萄球菌的抑菌作用，初步探討相關(guān)抑菌機(jī)制。方法 采用瓊脂平板打孔法，測(cè)定二十五味鬼臼丸（50、100、200mg·mL^-1）對(duì)金黃色葡萄球菌的抑菌圈大小。通過(guò)微量肉湯稀釋法，測(cè)定最小抑菌濃度（MIC）。調(diào)整肉湯培養(yǎng)基中的二十五味鬼臼丸濃度為0.5MIC、1.0MIC和2.0MIC，分別加入1.0×10⁸CFU·mL^-1菌懸液，以不含藥液而含菌量相同的培養(yǎng)基作為對(duì)照組，在37℃、120r·min^-1恒溫培養(yǎng)箱中振蕩培養(yǎng)，每小時(shí)取樣，用紫外可見(jiàn)分光光度計(jì)在600nm處測(cè)定吸光度（A₆₀₀）值，繪制細(xì)菌的生長(zhǎng)曲線；每2小時(shí)取樣，離心取上清液，試劑盒法測(cè)定堿性磷酸酶（AKP）活性；每小時(shí)取樣，離心取上清液，用紫外可見(jiàn)分光光度計(jì)在260nm處測(cè)定A₂₆₀值，以A₂₆₀值表示DNA/RNA大分子相對(duì)量；每小時(shí)取樣，離心取上清液，加入考馬斯亮藍(lán)G250溶液，于595nm處測(cè)定A₅₉₅值，檢測(cè)胞外可溶性蛋白含量；培養(yǎng)8h后，離心留沉淀，進(jìn)行SDS-PAGE電泳，檢測(cè)菌體蛋白含量。結(jié)果 二十五味鬼臼丸50、100、200mg·mL^-1對(duì)金黃色葡萄球菌的抑菌圈直徑分別為（12.33±0.75）、（16.33±0.41）、（19.17±0.68）mm；對(duì)金黃色葡萄球菌的MIC為100mg·mL^-1；與對(duì)照組相比，二十五味鬼臼丸組的金黃色葡萄球菌生長(zhǎng)顯著減緩（P＜0.05），胞外的AKP活性顯著升高（P＜0.05），胞外DNA/RNA大分子相對(duì)量顯著升高（P＜0.01），胞外可溶性蛋白含量顯著升高（P＜0.01），菌體總蛋白表達(dá)量明顯減少。結(jié)論 二十五味鬼臼丸對(duì)金黃色葡萄球菌具有抑菌作用，其抗菌作用可能與致細(xì)菌細(xì)胞壁、細(xì)胞膜結(jié)構(gòu)破損，且對(duì)菌體蛋白具有一定抑制作用有關(guān)。

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.

R285.5

國(guó)家自然科學(xué)基金資助項(xiàng)目（81470247）；西藏自治區(qū)科技廳中央引導(dǎo)地方科技（XZ202202YD0020C）