Liu Manjuan, Seidel Veronique, Katerere David R, Gray Alexander I
Natural Products Research Group, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR, UK.
Methods. 2007 Aug;42(4):325-9. doi: 10.1016/j.ymeth.2007.02.013.
Screening plant extracts for antifungal activity is increasing due to demand for new antifungal agents, but the testing methods present many challenges. Standard broth microdilution methods for antifungal susceptibility testing of available antifungal agents are available now, but these methods are optimised for single compounds instead of crude plant extracts. In this study we evaluated the standard NCCLS method as well as a modification which uses spectrophotometric determination of the end-points with a plate reader. We also evaluated another standard method, the EUCAST method, which is a similar microdilution assay to the NCCLS method, but uses a larger inoculum size and a higher glucose concentration in the medium as well as spectrophotometric end-point determination. The results showed that all three methods had some drawbacks for testing plant extracts and thus we modified the NCCLS broth microdilution method by including a colorimetric indicator-resazurin for end-point determination. This modified method showed good reproducibility and clear-cut end-point, plus the end-point determination needed no instruments. It enabled us to evaluate the activity of a selection of extracts from six Combretaceous plants against three Candida spp. and thus provided pharmacological evidence for some traditional uses of these plants while assisting the identification of the active ingredients.
由于对新型抗真菌剂的需求不断增加，筛选植物提取物的抗真菌活性的工作日益增多，但测试方法面临诸多挑战。目前已有用于现有抗真菌剂抗真菌药敏试验的标准肉汤微量稀释法，但这些方法是针对单一化合物而非粗植物提取物进行优化的。在本研究中，我们评估了标准的美国国家临床实验室标准化委员会（NCCLS）方法以及一种改进方法，该改进方法使用酶标仪通过分光光度法测定终点。我们还评估了另一种标准方法——欧洲抗菌药物敏感性试验委员会（EUCAST）方法，它是一种与NCCLS方法类似的微量稀释试验，但使用更大的接种量、培养基中更高的葡萄糖浓度以及分光光度法测定终点。结果表明，这三种方法在测试植物提取物时都存在一些缺点，因此我们通过加入比色指示剂刃天青用于终点测定来改进NCCLS肉汤微量稀释法。这种改进后的方法显示出良好的重现性和明确的终点，而且终点测定无需仪器。它使我们能够评估六种使君子科植物的部分提取物对三种念珠菌属菌株的活性，从而为这些植物的一些传统用途提供了药理学证据，同时有助于鉴定其活性成分。
