## 4 Discussion
### Mode of Antifungal Action of LIN
Their results were consistent with ours and showed that MIC value of terpene also increased in the medium with exogenous ergosterol. This was an increase in the MIC from 64 \(\mathrm{\SIUnitSymbolMicro g}\)/mL to \(>\)1024 \(\mathrm{\SIUnitSymbolMicro g}\)/mL. Thus, it was confirmed that LIN affects membrane ergosterol. However, the mechanisms of these interactions are not yet fully understood. According to our results, LIN has a fungicidal effect and probably acts on cell membrane ergosterol, but the exact mechanisms of its action are not known by us. However, there are some reports about the effect of this terpene on the fungus cell. In order to investigate its mechanisms of action, some authors [16] analyzed the inhibition of ergosterol synthesis. The treatment of _C. albicans_ and _C. neoformans_ strains with LIN at a concentration of395 ug/mL resulted in a 38% and 57% inhibition of ergosterol synthesis, respectively. In the case of _C. neoformans_, even in subinhibitory concentrations, LIN reduced sterol content. This indicates that it may act in the ergosterol biosynthesis pathway. However, in another study [33], this terpen was not effective in reducing the ergosterol content in _C. albicans_, even at a higher concentration (8 mg/mL). Moreover, molecular docking of LIN with proteins necessary for the biosynthesis and maintenance of the cell wall and the integrity of the fungal plasma membrane showed the possibility of the interaction of this terpen with three important enzymes: 1,3-\(\beta\)-glucan synthase, lanosterol 14-\(\alpha\)-demethylase, and D-14-sterol reductase [26]. Recently, LIN was found to exhibit antifungal activity by arresting the _C. albicans_ cell cycle. In addition, it induces a reduction in cell size and abnormal germination and inhibition of _C. albicans_ germ tube formation (which plays an important role in the virulence) [1; 55; 56]. LIN also displays anticandidal activity toward the cells in biofilms [1; 51]. Some studies confirmed that LIN interfered with the initial stages of _Candida_ biofilm formation and was also effective against the artificial biofilms of _C. albicans_[16]. Its antibiofilm potential should be further explored as a therapeutic strategy against biofilm-associated _C. albicans_ infections [57]. It should be added that due to the multi-drug resistance exhibited by _Candida_ biofilms, biofilm cells are able to survive up to 1000-fold higher concentrations of antifungal agents than those inhibiting non-biofilm planktonic cells [58; 59]. Subsequent results exhibited that LIN at a concentration of 0.5% killed 100% of the _C. albicans_ ATCC 3153 cells within 30 s. At lower concentrations (even at 0.016%), it inhibits germ tube formation and hyphal elongation of _C. albicans_, showing that it is active toward fungal dimorphism. As a result, it can reduce the progression of fungi and the spread of infection in host tissues [51]. In addition, the exposure of cells to LIN induced decrements in the colony-forming ability, a decrease in the levels of superoxide anion radical and total reactive oxygen species (ROS), and increases in the concentration of peroxides and lipid peroxides, showing oxidative stress induction. LIN treatments resulted also in different adaptive modifications of the antioxidant system [56]. Moreover, LIN at a concentration of 125 ug/mL significantly increased the chromosomal damage of _Candida_ spp. [60].