Antimicrobial activity of dragonhead, sage, oregano, hyssop and mint essential oils

Agar diffusion has been the standard method for determination of antimicrobial activity, but Eloff showed that it is very insensitive and introduced a microdilution technique for 96-well plates using p-iodonitrotetrazolium violet (INT) as a growth indicator and this method was suitable for testing plant extracts [1]. Another well known growth indicator is resazurin [2]. The great challenge when testing essential oils with a 96-well plate method was to find a suitable solvent, which is miscible with water and not toxic to the bacteria. The solvent has to be miscible with water because the bacterial growth medium is water based and the indicator is water soluble. The effect of acetone, ethyl acetate, methanol and different concentrations of ethanol and DMSO as such and supplemented with 0.5% Tween 80, were tested on Escherichia coli (FOMK). Acetone, ethanol and DMSO were non toxic in concentrations less than 6%.

Essential oils were isolated by hydrodistillation from dragonhead (Dracocephalum moldavica L.), sage (Salvia officinalis), oregano (Origanum vulgare L.), hyssop (Hyssopus officinalis L.) and two mint species (Mentha longifolia (L.) Hudson and M. x piperita L.). The oil composition was studied by GC-MS. For the antimicrobial assay a 20% stock solution of each essential oil was prepared in ethanol, diluted 1:15 in Mueller-Hinton broth by vortexing and diluted further 1:1 four or five times. Ampicillin (1mg/ml and 0.1mg/ml) was used as positive control and resazurin 0.04mg/ml as growth indicator. The plates were incubated over night (ca 18h) at 37^oC in a shaker. The minimum inhibitory concentration (MIC) value was defined as the lowest concentration that inhibited visible growth and they were in the range of 0.15–1% v/v. The most effective oil, as expected, was oregano because of its content of carvacrol.

References: 1. Eloff, J.N. (1998) Planta Medica 64: 711–713.

2. Sarker, S.D. et al. (2007) Methods 42: 321–324.