## 3 Discussion
Another Interesting aspect to consider in terms of antimicrobial activity is the effect of the minor components present in EOs [3]. [PERSON] et al. [36] investigated the antimicrobial effect of EOs extracted from _Origanum vulgare ssp. hirthum_, _Origanum dictamus_and a commercial oregano EO, concluding that these oils, although different in chemical composition, exhibited similar antibacterial activities. The authors attributed this behaviour to the action of minor compounds that could have their own activity or act in synergy or antagonism with the main ones. Several studies have reported synergistic activity among EO components: for example, the presence of carvacrol and \(p\)-cymene causes a major destabilization of the bacterial membrane and a reduction in membrane potential [37; 38]. The results of the present study confirmed that the diversity in the chemical profile between the two genotypes did not produce marked differences in antibacterial activity. The potential applications of EOs as food preservatives as well as food ingredients cannot be separated from the evaluation of their impact on intestinal health following their intake. Thus, in this study, we investigated possible adverse and beneficial actions of EOs on the intestinal mucosa since it represents the first site of impact between ingested food and the organism. To such purposes, human intestinal epithelial Caco-2 cells were used from a well characterized enterocyte-like cell line capable of expressing the morphological and functional differentiation features typical of mature enterocytes, including cell polarity and a functional brush border [39]. The Caco-2 cell line has been extensively used as a reliable in vitro system to study cell toxicity, inflammation and intestinal injury induced by pathogens [40]. The effect of oregano EOs on intestinal cells is quite unexplored, except for a couple of papers evaluating antioxidant activities [41; 42]; yet, the few available studies are focused on isolated components, such as thymol and/or carvacrol, with a particular emphasis on cell toxicity [43]. Another study conducted on undifferentiated Caco-2 cells treated up to 48 h with relatively high concentrations (2.5 mM and below) of carvacrol and thymol demonstrated heavy perturbation of the cellular ultrastructure and apoptosis [44]. However, it should be emphasized that the activity of pure compounds (singly or in mixtures) can be very different from that of complete EOs, which also contain a variety of secondary compounds. The incubation of Caco-2 cells in the presence of both CAR1 and THY5 caused a significative rapid and strong reduction in TEER values accompanied by a high passage of the paracellular marker phenol red at the higher concentrations tested (0.1%), approximately corresponding to 5.2 mM of carvacrol and 3.6 mM of thymol for CAR1 and THY5, respectively. Although this trend was relatively less sharp for the THY treatment at a 0.025% concentration, this sudden and strong toxic response likely indicated cell death occurrence, suggesting unspecific cytotoxicity effects, probably due to EO's ability to permeabilize mitochondrial membranes leading to cell death [45]. This was also confirmed by [PERSON] and co-workers [46], who observed a high necrosis rate in Caco-2 cells treated for 1 h with oregano EO at concentrations comparable to ours. However, at concentrations lower than 0.02%, both CAR1 and THY5 did not affect Caco-2 monolayer integrity for up to 24 h of incubation, indicating a complete absence of adverse effects under these experimental conditions and likely suggesting a level of safe concentrations at which EOs do not alter intestinal integrity. To the best of our knowledge, this is the first study evaluating the intestinal permeability effects of oregano EOs in human intestinal cells.