## 4 Discussion
CVL, known for its modulatory effects on ion channels, may initially enhance the movement of ions across the cell membrane, resulting in an increased Isc. The subsequent reduction in Isc might be associated with cellular adaptation or regulatory mechanisms. It is possible that prolonged exposure to CVL triggers feedback mechanisms that downregulate ion transport, leading to a decrease in Isc. The observed biphasic response evoked by CVL shares similarities with the response induced by 100 \(\mathrm{\SIUnitSymbolMicro mL}\) of _T. spicata_ ethanolic extract. This is not surprising, since CVL is the most abundant component of the ethanolic extract, and therefore it is conceivable that it could play a key role in determining the electrical changes recorded in our studies. Both TE and CVL initially increased Isc, suggesting a commonality in their early effects on ion transport. The subsequent reduction in Isc in the second phase might involve shared or convergent signaling pathways. The ability of the _T. spicata_ to increase the ionic permeability and the transepithelial resistance was maintained after simulated gastrointestinal digestion (FMDT sample), although with lower efficacy due to the partial loss of phytochemical compounds during digestion, as reported by our previous study [28], and CVL, which was lower in the digested sample than the ethanolic extract. However, when pre-digested _T. spicata_ were subjected to human microbial fermentation, mimicking the physiological interaction between digested food and healthy human gut microbiota, an effect on Caco-2 monolayers was not observed. This could be also explained by the dramatic reduction in CVL in the fermented sample. The results of this series of experiments demonstrate that although the fecal slurry (used as negative control) does not have any significant influence on intestinal function, it has a strong impact on the ability of _T. spicata_ to modulate the permeability of the intestinal barrier, and its function, since the biological effects observed in non-fermented _T. spicata_ (TE and FMDT) on the Isc and Rt were completely abolished. Taken together, we can observe a correlation between CVL and the effects on Caco-2 monolayers, and therefore, we could speculate that CVL plays a key role in determining the biological effects of _T. spicata_. Although no studies have elucidated the effects of _T. spicata_ on Caco-2 cells, a few studies have reported the beneficial effects of CVL. For example, CVL at lower concentrations exerted a cytoprotective and DNA-protection effect on free radical-injured Caco-2 cells [49; 50]. The dual-phase response in Caco-2 monolayers exposed to FMTD at 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL likely involves intricate interactions between _T. spicata_ components and the gut, in terms of polyphenic metabolites and digestion, leading to distinct cellular responses. This response differs from the effects seen with _T. spicata_ ethanolic extract or CVL, indicating unique microbial-plant-metabolite interactions, supported by the different levels of polyphenols and polyphenol metabolites. The gut microbiome is known for its dynamic metabolic activities. Metabolites produced during fecal medium preparation may contain active metabolites (i.e., enzymes) that could interact with _T. spicata_, and which may play a role in modulating cellular responses. Furthermore, microbial signals could potentially interact with the cellular pathways involved in ion transport. To exclude such effects, we also tested the effects of fecal slurry (FS) on Caco-2 monolayers, and found no significant effect. Although no significant changes in the amount of polyphenols (TPC) or the radical scavenging activity (DPPH) were observed between FMTD and FSTD, the CVL content in FMTD was ten times higher than in FSTD. This crosstalk and the content of CVL may contribute to the changes observed in Isc. Unravelling these mechanisms will enhance our understanding of how _T. spicata_'s different interactions shape cellular responses in the gut epithelium. _T.