Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity.

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol,
p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the
Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action,
the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring
the release, following exposure to the compounds under study, of the water-soluble fluorescent marker
carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition
(phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/
stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine
multilamellar vesicles as model membranes was monitored by means of differential scanning
calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and
water solubility of the compounds examined. We observed that thymol is considerably more toxic
against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory
against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentrationdependent
CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on
PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from
PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles.
Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol
and p-cymene) pH values, very likely acting as substitutional impurities. Taken together, our findings
lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene
may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane
of the microorganism. In addition to being related to the physicochemical characteristics of the
compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic
composition and net surface charge of the microbic membranes. Furthermore, the compounds might
cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular
sites critical for antibacterial activity.