Evaluation of antibacterial potential and toxicity of plant volatile compounds using new broth microdilution volatilization method and modified MTT assay

With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128 μg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95 μg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71 μg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024 μg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.

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