基于刃天青微平板法检测金黄色葡萄球菌对天然产物的敏感性

Susceptibility tests of Staphylococcus aureus to natural products using the resazurin method

关键词:  刃天青  最低抑菌浓度  金黄色葡萄球菌  天然产物  抑菌活性

Key words:  resazurin  minimum inhibitory concentration  Staphylococcus aureus  natural products  antibacterial activity

摘要:

[目的] 以刃天青为检测试剂，评价大黄酸、棉子酚、盐酸小檗碱、兰雪醌、槲皮素5种天然产物对金黄色葡萄球菌的抑制作用。[方法] 比较刃天青浓度、菌悬液浓度、培养基种类和反应时间对吸光值的影响，优化刃天青的工作条件。刃天青法进行药敏性实验得到五种天然产物的最低抑菌浓度（MIC），求得毒力回归方程、相关系数（R²）和半抑菌浓度（IC₅₀）。通过Z值判断刃天青显色法适用于高通量筛选药物的可行性。[结果] 当刃天青孔内终浓度为20μg/mL，培养基为LB，菌悬液浓度为5×10⁵CFU/mL，反应时间9h时适用于金黄色葡萄球菌的药敏性实验。刃天青显色法与微量肉汤法检测得到的五种天然产物的抑菌结果具有一致性。[结论] 五种天然产物均具有一定的抑菌活性。刃天青显色法具有准确性好、灵敏度高及高通量的特点，适用于天然产物抗菌活性的筛选。

Abstract:

[Objective] A resazurin-based microtiter method was developed for the quantitative monitoring the inhibitory effects of rhein,gossypol,berberine hydrochloride,plumbagin and quercetin. [Methods] The conditions of resazurin assay were optimized by evaluating influences of the concentration of resazurin,the concertration of bacteria,the species of mediums and rection time on the optical density. The resazurin-based microtiter method was used to drug susceptibility tests to obtain the minimum inhibitory concentration(MIC),toxic regression equation, coefficient of association (R²) and semi-inhibitory concentration(IC₅₀). The feasibility of high throughput drug screening was evaluated by Z value. [Results] The result showed the concentration of resazurin was 20 μg/mL,the medium was LB,the concertration of bacteria was 5×10⁵CFU/mL,rection time was 9 h. The results obtained by the two methods were consistent. [Conclusion] Five kinds of natural products have a certain inhibitory effect. And the resazurin- based assay can be used successfully to assess antibacterial properties of natural products,along with the characteristics of good accuracy,high sensitivity and high throughput.