## 3 Discussion  
In the present study, eight different EOs distilled from plants obtained of two genotypes belonging to the subspecies _Origanum heracleoticum_ L. and to the hybrid _Origanum vulgare ssp. viridium \(\times\) Origanum vulgare ssp. hirthum_ were characterized according to their chemical composition and several biological activities. First, chemical analyses allowed for highlighting the differences in the profiles of the two genotypes and evaluating the variability within each genotype depending on the cultivation location. EOs from the two genotypes showed differences not only in their predominant compound, carvacrol or thymol, but also in their overall profile complexity, which was more pronounced in the thymol chemotype. The profile of the EOs from the _Origanum heracleoticum_ L. subspecies, described as belonging to the thymol chemotype, was in accordance with the data reported in the literature on the same subspecies of wild and cultivated oregano plants grown in the same geographic area of Sicily [20; 21; 28]. No previous information was available in the literature, to the best of our knowledge, on the profile of the hybrid _Origanum vulgare ssp. viridium \(\times\) Origanum vulgare ssp. hirthum_, described as belonging to the carvacrol chemotype. In both groups, the occurrence of relatively high levels of \(\gamma\)-terpinene and p-cymene was consistent with the biosynthetic pathway proposed for the main compounds carvacrol/thymol, by which \(\gamma\)-terpinene is first converted to p-cymene and, in turn, further converted to carvacrol or thymol by two distinct enzymes [29]. Interestingly, while most monoterpene and sesquiterpene hydrocarbons appeared to be positively correlated among themselves and with thymol, \(\beta\)-caryophyllene was positively correlated with carvacrol and negatively correlated with the other sesquiterpenes. From the point of view of relationships with biological activities, it is interesting to note that the level of the most active EO compound was significantly higher in the hybrid _Origanum vulgare ssp. viridium \(\times\) Origanum vulgare ssp. hirthum_. than in the subspecies _Origanum heracleoticum_ L., which might suggest a higher potential in the former. On the contrary, the profile of EOs from the subspecies _Origanum heracleoticum_ L. was more complex than the other in terms of the number of minor constituents detected. Moreover, chemical characterization provided detailed information on the enantiomeric distribution of chiral compounds in the aerial parts of the considered oregano genotypes and in the corresponding EOs. This piece of information may be important not only because optical isomers can have different odors and, in general, different biological properties, but also because it provides useful information for uncovering the biosynthetic and geographical origins of the considered EOs as well as their authenticity  
Figure 6: Study of NF-kB pathway gene expression in Caco-2 cells treated with (**A**) CAR1 and/or TNF-\(\alpha\) and (**B**) THY5 and/or TNF-\(\alpha\). Caco-2 cells were pre-treated for 1 h with 0.02% CAR1 or THY5 and then treated for 1 h with 15 ng/mL TNF-\(\alpha\). IkB\(\alpha\), cIAP2, IL-18, IL-6 and IL-8 gene expression was analyzed by real-time PCR. Data are expressed as the fold change (log2) \(\pm\) SD of two independent experiments, carried out in triplicate. Means without a common letter significantly differ, \(p\) < 0.05.  
and also for investigating technological treatments to which EOs could be subjected [30]. In the oregano EOs analyzed in this study, chiral compounds were all present at a relatively low level (<1%), whereas all major compounds were achiral. This would suggest a minor relevance of the enantiomeric characterization for these oregano genotypes.