The present study shows that the antimicrobial compound carvacrol accumulates in hydrophobic phases. Comparison of the Po/w
of carvacrol with those of other compounds (Table ) shows that the determined value agrees with values found for similar compounds. The addition of alkyl groups to a molecule increases the P
, while the addition of a hydroxyl group or a system of delocalized electrons to a molecule decreases the P
. The Pmembrane/buffer
of carvacrol is lower than its Po/w
(3.26 versus 3.64). This result may be explained by the fact that PE liposomes are more hydrophilic than octanol due to the presence of hydrophilic head groups attached to the fatty acids. As was described by De Young and Dill (7
), there is a difference between the partitioning of a compound into membranes, which are interfacial phases, and an octanol phase, which is uniform throughout. Sikkema et al. (21
) and Machleidt et al. (16
) observed Pmembrane/buffer
s for cyclic hydrocarbon compounds that were lower than their Po/w
Comparison of the observed P (expressed as log P) of carvacrol with those of different compounds in relation to their structures
Weber et al. (30
) reported that compounds with a log Po/w
value higher than 3 partition deeply in the membrane. As a result of this partitioning of carvacrol, interactions of components (lipids and proteins) in the membrane are affected. Accumulated carvacrol occupies more than the normal amount of space between fatty acid chains, resulting in conformational changes of the phospholipid bilayer. A disturbance of the van der Waals interactions between the lipid acyl chains in the membrane causes fluidization of membrane lipids. This fluidization by carvacrol was described earlier (25
). In addition, an expansion of membranes was observed. The expansion did not increase further when we added carvacrol at concentrations above 0.50 μmol/mg of PE, which corresponded with 3 × 104
μg of carvacrol/g of PE (calculated on the basis of the P
of carvacrol in the liposomes). The maximum concentration of carvacrol in the liposomal membrane (6 × 104
μg/g of PE) was higher, which indicates that the maximum expansion is not solely determined by the maximum possible concentration of carvacrol in the membrane.
Due to the higher P
of cymene, more cymene is expected to accumulate in the membranes, which may explain the higher expansion of the membrane in the presence of cymene. This possibility agrees with the observations of Sikkema et al. (21
), who found that the expansion of the membrane increased when the log Po/w
of a compound increased. The marked preference of cymene (compared to that of carvacrol) for the hydrophobic phase also explains the observation that a maximum expansion is reached at a higher concentration of cymene than of carvacrol (2 and 0.5 μmol/mg of PE, respectively).
An expansion of the membrane may result in a destabilization of the membrane and, consequently, the leakage of ions. Both carvacrol and cymene cause a decrease in the membrane potential, suggesting leakage of ions. However, the addition of 0.125 mM cymene resulted in much higher concentrations of cymene in the membrane than the addition of 0.125 mM carvacrol as a result of the higher P
of cymene. In the presence of cymene, the membrane potential is probably decreased mainly by leakage of ions other than H+
, e.g., K+
, because only a very small effect on the ΔpH was observed. This absence of an effect on the ΔpH may explain why ATP pools were not affected by cymene. Cells can probably cope with the effect on membrane potential, as was also observed by the absence of an effect of cymene on the growth of B. cereus
. As described by Ultee et al. (26
), decreasing the membrane potential alone did not decrease the viability of B. cereus
), even though leakage of ions was observed. Vermuë et al. (29
) showed that a higher P
did not always result in the greater toxicity of a compound. Compounds with a log P
value above a certain maximum (approximately 4, dependent on the kind of compound and the organism tested) are not toxic to organisms. These results clearly show that both carvacrol and cymene have an effect on membrane integrity. However, destabilization of the membrane does not explain the higher antimicrobial activity of carvacrol than that of cymene. This indicates that another factor must be involved.
This factor is the presence of the hydroxyl group at the carvacrol molecule. The above-described observations (summarized in Table ) support the following model for the antimicrobial action of carvacrol, which is shown schematically in Fig. . A characteristic feature of a phenolic hydroxyl group is its significantly greater acidity than that of an aliphatic hydroxyl group (4
). We propose that carvacrol acts as a transmembrane carrier of monovalent cations by exchanging its hydroxyl proton for another ion such as a potassium ion. Undissociated carvacrol diffuses through the cytoplasmic membrane towards the cytoplasm and dissociates by releasing its proton to the cytoplasm. It may then return undissociated by carrying a potassium ion (or other cation) from the cytoplasm which is transported through the cytoplasmic membrane to the external environment. A proton is taken up, and carvacrol in the protonated form diffuses again through the cytoplasmic membrane and dissociates by releasing a proton to the cytoplasm. This hypothesis is supported by the observed efflux of K+
and influx of H+
in B. cereus
during exposure to carvacrol (26
). Given a pKa
of phenolic compounds of 10 (16
), 0.1% carvacrol will be dissociated at the hydroxyl group under the experimental conditions (pH 7). Cymene does not have this property of carvacrol because of the lack of a hydroxyl group. The large accumulation of cymene in the membrane probably causes sufficient expansion of the membrane and, consequently, spacing of phospholipids in the destabilized membrane through which ions can leak. However, the presence of the hydroxyl group seems to be more important for the antimicrobial activities of these compounds than the ability to expand and consequently to destabilize the membrane.
Summary of effects of carvacrol and cymene on different parameters in this studya
FIG. 8. Schematic overview of the hypothesized activity of carvacrol. Undissociated carvacrol diffuses through the cytoplasmic membrane towards the cytoplasm and dissociates, thereby releasing its proton to the cytoplasm. It then returns undissociated by carrying (more ...)
The above-described hypothesis is supported by the results with menthol, carvacrol methyl ester, and thymol. Although menthol has a hydroxyl group, it does not possess high antimicrobial activity. This lack is caused by the absence of a system of delocalized electrons (double bonds) and, consequently, the inability of the hydroxyl group to release its proton. Although the log P
value of menthol is not known, it is expected to be higher than the log P
of carvacrol because introduction of a system of delocalized electrons (as in carvacrol) lowers the P
. This finding indicates that the reduced antimicrobial activity of menthol is not caused by a reduced partition of menthol in the membrane compared to that of carvacrol. Like menthol, carvacrol methyl ester does not have the ability to release a proton and is therefore not antimicrobial. Similar to carvacrol, thymol contains both a hydroxyl group and a system of delocalized electrons and was found to possess strong antimicrobial activity. It was described earlier that the hydroxyl group (bound to a benzene ring) is important for the activities of some antimicrobial compounds and that these activities are enhanced by the presence of α-β double bonds (3
). Salih et al. (19
) showed that hydroxycinnamic acids showed antimicrobial activities even though their esters were not active.
The synergistic activity between carvacrol and cymene has been described previously (27
). Based on the knowledge obtained in the present study, we conclude that cymene probably acts synergistically with carvacrol by expanding the membrane, which results in the destabilization of the membrane. This supports the possible role of carvacrol as an exchanger of cations.
In conclusion, although both carvacrol and cymene cause destabilization of the membrane and a decrease in the membrane potential, the antimicrobial activity of carvacrol is most probably caused by an additional decrease in the ΔpH as a result of the presence of a hydroxyl group and a system of delocalized electrons. If we suppose that metabolism is aerobic, these events result in the absence of a proton motive force and, consequently, no more ATP can be synthesized. Depletion of ATP pools leads to impairment of essential processes in the cell and finally to cell death.