Validation of the AlamarBlue® Assay as a Fast Screening Method to Determine the Antimicrobial Activity of Botanical Extracts

Plant compounds are a potential source of new antimicrobial molecules against a variety of infections. Plant extracts suppose complex phytochemical libraries that may be used for the first stages of the screening process for antimicrobials. However, their large variability and complexity require fast and inexpensive methods that allow a rapid and adequate screening for antimicrobial activity against a variety of bacteria and fungi. In this study, a multi-well plate assay using the AlamarBlue® fluorescent dye was applied to screen for antimicrobial activity of several botanical extracts and the data were correlated with microbial colony forming units (CFU). This correlation was performed for three pathogenic model microorganisms: Escherichia coli (Gram negative bacteria), Staphylococcus aureus (Gram positive bacteria) and for the yeast-like fungi Candida albicans. A total of ten plant extracts from different Mediterranean plants, including several Cistus and Hibiscus species, were successfully tested. HPLC-DAD-ESI-MS/MS analysis was utilized for the characterization of the extracts in order to establish structure-activity correlations. The results show that extracts enriched in ellagitannins and flavonols are promising antibacterial agents against both Gram positive and Gram negative bacteria. In contrast, phenolic acids, anthocyanidins and flavonols may be related to the observed antifungal activity.

植物化合物是对抗多种感染性疾病的新型抗菌分子的潜在来源。植物提取物构成复杂的植物化学物质库，可用于抗菌剂筛选流程的初始阶段。然而，植物提取物的高度多样性与复杂性，亟需快速且低成本的方法，以实现对多种细菌和真菌的抗菌活性进行高效且充分的筛选。本研究采用基于阿蓝玛蓝（AlamarBlue®）荧光染料的微孔板分析法，对多种植物提取物的抗菌活性进行筛选，并将检测数据与微生物菌落形成单位（CFU）进行关联分析。本次关联分析针对三种致病模式微生物展开：革兰氏阴性菌大肠埃希氏菌（Escherichia coli）、革兰氏阳性菌金黄色葡萄球菌（Staphylococcus aureus），以及类酵母真菌白色念珠菌（Candida albicans）。研究共对10种来自不同地中海植物的提取物顺利完成检测，其中包括多种岩蔷薇属（Cistus）和木槿属（Hibiscus）物种。本研究采用高效液相色谱-二极管阵列检测器-电喷雾电离串联质谱（HPLC-DAD-ESI-MS/MS）分析法对提取物进行表征，以建立构效关联。结果表明，富含鞣花单宁与黄酮醇的提取物，对革兰氏阳性菌及革兰氏阴性菌均展现出良好的抗菌应用前景。相较而言，酚酸类、花青素类与黄酮醇类成分或与本次检测到的抗真菌活性相关。