## 4 Discussion
Several factors may account for these differences, including diffusion limitations, medium composition and the physicochemical nature of essential oils. In solid media, the dilution of active compounds occurs in 10 mL of moltenagar, compared to only 1 mL of broth in the microdilution method. This ten-fold increase in volume can significantly lower the final concentration of active components, especially for volatile and hydrophobic substances. Additionally, the solid matrix impedes uniform diffusion of essential oils, which maylead to the underestimation of their actual antimicrobial potential. The selimitations are particularly relevant for lipophilic compounds with low solubility, suchas thymol and carvacrol [37; 38; 39]. In silico analysis provided additional insight into possible molecular mechanisms underlying the observed effects. Using Swiss Target Prediction, STRING and Reactome, we identified putative human protein targets of thymol, carvacrol and vitamin C. Thymol was associated with neuroreceptors and COX-2, indicating potential anti-inflammatory and sedative properties [48; 49]. Carvacrol interacted with TRP receptors and metabolic enzymes, suggesting immunomodulatory and stress response effects [50; 51]. Vitamin C primarily targeted oxidoreductaseases and enzymes involved in cell cycle regulation and antioxidant defense [52; 53]. The STRING analysis indicated that these compounds influence pathways related to inflammation, oxidative stress and immune modulation. Specifically, suppression of NF-\(\kappa\)B and activation of Nrf may contribute to reduced inflammation and enhanced cellular protection [54; 55; 56]. Vitamin C has also been associated with increased phagocytic activity and ROS production, mechanisms that play a critical role in innate immunity and are modulated under oxidative stress conditions [37; 38; 50; 56]. Previous studies have associated thymol and carvacrol with strengthening of the epithelial barrier and inhibition of biofilms [57, 58], although such effects were not directly investigated in this study. While in silico analyses do not confirm antimicrobial mechanisms directly, they support the hypothesis that combined agents act through complementary pathways. Previous reports have also highlighted the potential of natural antioxidants such as vitamin C to support epithelial integrity and immune resilience under stress conditions, adding an additional layer to the interpretation of these results [36, 50, 56]. The data align with previous findings indicating that multi-component natural mixtures exert broader antimicrobial activity and are less prone to resistance development compared to single-target agents [59]. The observed effects may also involve the enhanced penetration of essential oil components facilitated by vitamin C, leading to oxidative and metabolic stress within microbial cells. This mechanism is supported by previous reports on the ability of antioxidants to modulate microbial redox balance, indirectly contributing to membrane destabilization and intracellular damage [37, 38]. Thymol and carvacrol have been shown to disrupt bacterial membrane integrity and interfere with quorum-sensing pathways, both of which are critical for biofilm formation and antimicrobial resistance [60, 61]. For instance, thymol has been reported to inhibit the MexAB-OprM efflux pump and act as a quorum-sensing inhibitor in _Pseudomonas aeruginosa_, enhancing susceptibility to antibiotics [39, 61]. Finally, although the test strains used in this study (ATCC reference strains) do not carry known resistance genes, future work should involve multidrug-resistant clinical isolates to better evaluate therapeutic potential. In conclusion, our findings confirm that vitamin C can modulate the antimicrobial activity of essential oils, enhancing their effect in a strain-dependent manner.