| 邢梦雨,夏道宗,田崇梅,等.枫杨叶提取物抑制金黄色葡萄球菌的机制研究[J].浙江中医药大学学报,2019,43(1):19-25, 33. |
| 枫杨叶提取物抑制金黄色葡萄球菌的机制研究 |
| Inhibitory Mechanism of Pterocarya Stenoptera Leaves Extract Against Staphylococcus Aureus |
| DOI：10.16466/j.issn1005-5509.2019.01.004 |
| 中文关键词: 枫杨 金黄色葡萄球菌 抑菌机制 细胞膜完整性 DNA片段化 |
| 英文关键词: Pterocarya stenoptera Staphylococcus aureus antibacterial mechanism cell membrane integrity DNA fragmentation |
| 基金项目:国家自然科学基金面上项目(81673656、81374048) |
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| 中文摘要: |
| [目的] 研究枫杨叶提取物对金黄色葡萄球菌的抑菌作用及相关机制。[方法] 通过纸片扩散法比较不同溶剂枫杨叶提取物对金黄色葡萄球菌的抑制效果；以微量稀释法测定枫杨叶醇提取物的最小抑菌浓度(minimum inhibitory concentration，MIC)与最小杀菌浓度(minimum bactericidal concentration，MBC)；以生长曲线法比较不同浓度枫杨叶醇提取物对细菌生长的影响；以扫描电镜观察该提取物对细菌菌体形态的影响；以碘化丙啶(propidium iodide，PI)和SYTO9荧光染料染色观察确定该提取物对菌体细胞膜的影响；以琼脂糖凝胶电泳检测该提取物对细菌基因组DNA的作用。[结果]纸片抑菌与微量稀释实验证实枫杨叶水提取物无抑菌效果，而醇提取物具有明显的抑菌效果，其MIC和MBC分别为125mg·L-1和1 600mg·L-1。枫杨叶醇提取物作用后，细菌进入对数生长期的时间延迟，培养至2h时各组OD600值均低于空白对照组，差异均有统计学意义(P＜0.01)。培养至4h开始，各时点2×MIC枫杨叶醇提取物处理组OD600值均低于1×MIC处理组，4×MIC处理组均低于2×MIC处理组，差异均有统计学意义(P＜0.01)。培养12h后，不同浓度枫杨叶醇提取物处理组菌量较空白对照组明显减少(P＜0.01)，且均进入稳定期，而空白对照组菌量在24h时仍呈增长趋势。说明枫杨叶醇提取物可以明显抑制金黄色葡萄球菌的增殖，而且抑制作用存在量效关系。扫描电镜观察发现，枫杨叶醇提取物作用后，金黄色葡萄球菌出现菌体变形、塌陷甚至表面溶解等现象；荧光探针实验进一步说明枫杨叶醇提取物可破坏金黄色葡萄球菌细胞膜与菌体结构的完整性，且效果具有剂量和时间依赖性。琼脂糖凝胶电泳表明枫杨叶醇提取物可使细菌基因组DNA发生损伤。[结论] 枫杨叶醇提取物对金黄色葡萄球菌生长具有显著的抑制作用，其机制可能与破坏细胞膜完整性和菌体结构，损伤基因组DNA有关。 |
| 英文摘要: |
| [Objective] To study the antibacterial activity and related mechanism of Pterocarya stenoptera(P. stenoptera) leaves extract against Staphylococcus aureus(S. aureus). [Method] The inhibitory effect of different solvent extracts on S. aureus was compared by disc diffusion method; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of P. stenoptera leaves alcohol extract were determined by micro-dilution method; growth curve method was used to determine the effect of bacterial growth with different concentrations; the effect to bacterial morphology was observed by scanning electron microscopy; the effect of extract on the cell membrane was determined by staining with propidium iodide(PI) and SYTO9 fluorescent dyes and the effect of extract on bacterial genomic DNA by agarose gel electrophoresis. [Results] It showed that P. stenoptera leaves water extract had no antibacterial effect, but alcohol extract had obvious antibacterial effect, and the MIC and MBC were 125mg·L-1 and 1 600mg·L-1, respectively. Treated with P. stenoptera leaves alcohol extract, there was delay in logarithmic growth phase of S. aureus. At 2h, the OD600 values of each group were lower than blank control group, and the difference was statistically significant(P＜0.01). At 4h, the OD600 values of the 2×MIC P. stenoptera leaves alcohol extract treatment group was lower than 1×MIC group, 4×MIC group was lower than 2×MIC group. The difference was statistically significant (P＜0.01). At 12h, the amount of the different concentrations of P. stenoptera leaves alcohol extract treatment group was significantly lower than blank control group (P＜0.01), and both entered a stable period, while the amount of blank control group still showed an increasing trend at 24 hours. It is indicated that the P. stenoptera leaves alcohol extract can significantly inhibit the proliferation of S. aureus, and showed dose-effect relationship. The morphology was greatly damaged of S. aureus like deformation, collapse, even surface dissolution could be found under scanning electron microscope. Fluorescence probe experiments further indicated that P. stenoptera leaves alcohol extract could destroy the integrity of cell membrane and structure, and has dose-time dependence. Agarose gel electrophoresis experiments showed that P. stenoptera leaves alcohol extract could damage bacterial genomic DNA. [Conclusion] The P. stenoptera leaves alcohol extract has a significant inhibitory effect on the growth of S. aureus, and its mechanism may related to the damage of cell membrane integrity, structure and genomic DNA. |
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