Investigation of antifungal activity mechanisms of alpha-pinene, eugenol, and limonene

Plant essential oils are preferred in cosmetics, medicine, food, and beverage industries for various purposes. α-Pinene is found mainly in eucalyptus oils, eugenol is the active ingredient in clove oil, and limonene is the major component in the oil of citrus fruit peels. In this study, we aimed to determine the antifungal activity of α-pinene, eugenol, and limonene against Saccharomyces cerevisiae yeast cells. Besides, we focused on revealing the target side of the compounds on the yeast cells. 
Firstly, the antifungal activity of compounds was tested via minimum inhibitory concentration (MIC) measurement. After that, we performed a sorbitol effect assay to understand whether it acts on the cell wall or not. With sorbitol, the MIC values were not changed. It means that they are not effective on the yeast cell wall. Then, we measured the extracellular conductivity increase upon treatment with the compounds to understand the effect on the cell membrane. Eugenol and limonene were not changed the extracellular conductivity, and there was no ion leakage from the cell membrane. On the other hand, α-pinene damaged the yeast cell membrane causing a sudden increase in conductivity due to ion leakage. An ergosterol effect assay with α-pinene was performed to detect cell membrane disruption via ergosterol or not. With ergosterol, the MIC value was not changed. α-Pinene must have another target than the ergosterol in the yeast cell membrane. Finally, revealing the mode of action of compounds against yeast cells will provide new insights into their usage in various fields.