## 4 Discussion
### Mode of Antifungal Action of LIN  
The next stage of our studies was the evaluation the mechanism of action of LIN on the cell of _Candida_ spp. We tried to assess whether its anticandidal activity is related to interaction with the structure of the cell wall and/or cell membrane of yeasts [26; 38; 39; 40; 41; 52]. First, the effect of the LIN on their cell wall was studied. This structure is very specific; it protects the fungal cell from environmental stresses and allows it to interact with its environment. The cell wall and its synthesis is characteristic of fungi, which makes them a very important target for antifungal drugs. Sorbitol, on the other hand, is a factor that induces a certain degree of cellular stress that can cause inhibition of cell growth in the presence of different cell wall inhibitors [5]. It stabilizes fungal protoplasts, which protects their cell wall against external factors. If a compound damages the cell wall, its MIC value in the presence of an osmotic medium increases [38; 39; 41]. This assay showed that the MICs of LIN did not vary in the presence of sorbitol and were independent of its presence in the medium for any of the five tested _Candida_ spp. strains. It suggests that terpene does not act by inhibiting the mechanisms that control cell wall synthesis, but rather by affecting other targets. Nystatin had a similar effect. In the case of both LIN and nystatin, the presence of sorbitol did not interfere with their anticandidal activity. Their MIC values were unchanged, indicating no effect on the structure of the cell wall. The results are consistent with data obtained for citral and geraniol (also terpenes), which indicated antifungal potential but demonstrated no effect on cell walls [38]. Activity of LIN on the cell wall of _C. albicans_ ATCC 76485 and clinical _C. albicans_ isolate was also studied by [PERSON] et al. [26]. Their results were different compared to ours. The MIC value of LIN for both fungi increased from 64 \(\mathrm{\SIUnitSymbolMicro g}\)/mL to \(>\)1024 \(\mathrm{\SIUnitSymbolMicro g}\)/mL in medium with sorbitol, indicating that terpene interferes with cell viability through certain molecular mechanisms that may involve the _C. albicans_ cell wall and compromise its integrity. Subsequent studies involved evaluating whether LIN affects ergosterol in the cell membrane. Cell membrane integrity is responsible for cell function [53]. Ergosterol and enzymes involved in its biosynthesis are important targets for some antifungals, such as polyenes or azoles [38; 41]. This sterol maintains cell function, and its integrity is involved in cell division, cell signaling, the regulation of membrane proteins, membrane fluidity, and endocytosis. The basis of this study was that the addition of exogenous ergosterol to the medium can increase the MIC value for compounds targeting this sterol in the cell membrane [5; 54]. Our results indicated that the cell membrane of studied yeasts may be more sensitive to the action of LIN, as its MIC value was 2-4 times higher in the medium with ergosterol compared to the medium without sterol. Slightly higher MIC increases were found for _C. albicans_ compared to non-_albicans Candida_ spp. The increase in the MIC of LIN in the presence of ergosterol indicated that this terpen may affect the plasma membrane integrity of _Candida_. In the case of nystatin (binding ergosterol found in lipid bilayer membranes), an 8-16-fold increase in MIC values was indicated. Similar results for nystatin were observed by [PERSON] and [PERSON] [41], in whom the MIC value of the antibiotic against _C. albicans_ increased 16-fold in the presence of the sterol. The obtained data suggest that LIN may affect the fungal cell membrane and bind to this sterol in the membrane, leading to cell membrane disintegration, increasing the permeability of \(\mathrm{Ca^{2+}}\) and \(\mathrm{K^{+}}\) ions, proteins or radicals, and potentially leading to cell death [5; 6; 38; 39; 52]. [PERSON] et al. [26] evaluated the effect of LIN on the cell membrane of _C. albicans_.