## Results
### Effects of T. spicata Ethanolic Extract and Carvacrol on the Intestinal Barrier Function  
To evaluate the effect of _T. spicata_ ethanolic extract (TE) on the intestinal barrier function, the electrophysiological properties of Caco-2 monolayers were investigated using the Using chamber technique. This technique monitors concurrent changes in short-circuit current (Isc), a measure of net electrogenic transepithelial ion flux, as well as transepithelial resistance (Rt), as a measure of barrier integrity. Following an equilibration period of 15 min, the baseline values of short-circuit current (Isc) and transepithelial resistance (Rt) were 2.94 \(\pm\) 0.23 mA/cm\({}^{2}\) and 517 \(\pm\) 31.8 \(\Omega\)/cm\({}^{2}\), respectively (n = 37). The addition of increasing concentrations of TE at the apical side of Caco-2 monolayers increased the Isc when applied at 25 and 50 \(\mu\)g/mL (+24.8% and +82.7% compared to baseline, respectively), without affecting the Rt (Figure 7B-D). Figure 6: Concentration (mg/kg) of carvacrol determined by GC-MS in fecal medium and _T. spicata_ digest (FMTD), fecal slurry (FS) and fecal slurry and _T. spicata_ digest (FSTD) samples. Statistical differences were evaluated by One-way ANOVA followed by [PERSON]’s test. Asterisk (*) indicates statistically significant differences (_p_-value < 0.05). Of note, higher concentrations of TE (100 and 200 ug/mL) evoked biphasic responses, with a significant and transient increase in the Isc (+175.6% and 161.1%) not associated with changes in the Rt (\(-\)2.21 and \(-\)0.72%), followed by a slight increase in the Isc at 100 ug/mL (+22.1%) and a decrease at 200 ug/mL (\(-\)38.7%) accompanied by a significant increase in the Rt (+14.5% and +25.1%) (Figure 7A,C,D). The dose-response curve of TE was characterized by measuring the percentage change in short-circuit current with increasing concentrations of TE. The EC\({}_{50}\) value, representing the concentration at which Isc exhibits a 50% change from baseline, was determined to be 51.02 ug/mL (Figure 7B). Given that CVL is the major component of TE (constituting about 25% of TE [15]), our specific aim was to determine whether the response observed after the addition of TE could be attributed to the presence of CVL. To address this, we investigated the effect of CVL by using 6.25, 12.5, and 25 and 50 ug/mL, which correspond to the amounts of CVL contained in the different doses of the ethanolic extract tested. As illustrated in Figure 8B,C, the application of increasing concentrations of CVL at the apical side resulted in an increase in Isc (+39% and +52,4% for 6.25 and 12.5 ug/mL, respectively) without any change in  
Figure 7: Effect of apical addition of _T. spicata_ ethanolic extract on Caco-2 monolayers. (**A**) Representative traces of short circuit current (Isc) and transepithelial resistance (Rt) after _T. spicata_ ethanolic extract (TE) addition (100 ug/mL). (**B**) Dose–response curve of _T. spicata_ ethanolic extract-evoked Isc changes. The plots show the means (represented by black dots) \(\pm\) the SEM of Isc measured at each concentration. The half-maximal inhibition constant (EC\({}_{50}\)) for TE was 51.02 ug/mL. (**C,D**) Changes in the electrophysiological parameters induced by apical TE addition. Results are expressed as percentages of the Isc and Rt basal values. Sample size is represented by the number at the bottom of each bar. Values are reported as mean values \(\pm\) SEM, with the control represented by a white bar and TE addition at concentrations of 25, 50, 100, and 200 ug/ml depicted by light gray and dark gray bars for the first and second phases, respectively. ## \(p<0.01\), ## \(p<0.001\) vs. basal condition calculated by one-way analysis of variance (ANOVA) with [PERSON]’s multiple comparison test; * \(p<0.05\), *** \(p<0.001\) values calculated by one-way analysis of variance (ANOVA) with [PERSON]’s multiple comparison test, indicating how much the results differ from those of other treatments. the Rt.