多孔板-MTT比色法评价植物和微生物代谢产物的抗真菌活性

Evaluation of Antifungal Activity of Plant and Microbial Metabolites by Microplate-MTT Colorimetric Assay

多孔板-MTT比色法测定植物和微生物代谢产物对真菌抑制活性的步骤为:在多孔板的每孔中依次加入浓度为105孢子/mL的供试真菌孢子悬液90μL,不同浓度的药液10μL。25℃暗培养48 h,然后每孔中加入8mg/mL的MTT溶液10μL,继续培养10 h后,离心去上清,加入DMSO 150μL,振荡30 min,离心后上清液在510nm测定吸光值。采用上述条件测定了白屈菜红碱对稻瘟病菌和西瓜枯萎病菌的MIC值分别为80和1.5μg/mLI,C50值分别为21.99和0.78μg/mL;Diepoxinζ对稻瘟病菌的MIC和IC50值分别为200和96.21μg/mL。多孔板-MTT比色法为快速有效地筛选和评价植物和微生物抗真菌活性成分创造了条件。

An optimal experimental procedure with the microplate-MTT colorimetric assay was developed,that was as follows：90 μL of fungal spore suspension at concentration of 105 spores/mL was added in each well of the 96-well microplate first;10 μL of the sample solution with its different concentration was then added,and cultivated for 48 h at 25 ℃ in darkness;10 μL of MTT solution at 8 mg/mL was added into each well,and incubated for another 10 h at 25 ℃ and centrifuged;The supernatant was removed,and 150 μL of DMSO was added;After shaking for another 30 min at 15 rpm,light absorption values at 510 nm were obtained for the supernatant transferred in a new plate with a microplate spectrophotometer.With the optimal MTT assay mentioned above,the minimum inhibitory concentration（MIC） values of chelerythrine from Macleaya cordata on Fusarium oxysporum f.sp.niveum and Magnaporthe oryzae were 80 and 1.5 μg/mL respectively,and the median inhibitory concentration（IC50） values were 21.99 and 0.78 μg/mL,correspondingly.The MIC and IC50 values of diepoxin ζ from the endophytic fungus Dzf12 on Magnaporthe oryzae were 200 and 96.21 μg/mL.It indicated that the antifungal metabolites from plants and microbes could be evaluated fast and micro-quantitatively by using microplate-MTT colorimetric assay that is favorable for screening antifungal components with a high throughout.