## 3 Results and Discussion
### Absorption Evaluation Using the In Vitro Dynamic Intestinal Model
Its absorption mechanism is not yet well known, and only recently some authors have suggested that QA controversial data on permeability and absorption could be explained by an active efflux mechanism, acting on QA with a potential saturation. This mechanism seems to be concentration dependent, as demonstrated by experiments on Caco-2 cells testing low concentrations (\(\geq\)10 uM) [46]. Our results could be due to QA potential interaction with the RPMI-1640 medium, giving the formation of stable complexes, often polymeric structures, which could involve a QA \(\alpha\)-hydroxycarboxylic group in which its electron-attractive nature increases the strength of the carboxylic acid [47; 48]. This does not affect QA solubility, but compromises its detection by RP-HPLC-DAD using our method. Caffeic acid (CA) (Figure 1b) is well known to have a very low permeability across Caco-2 cells and simulated intestinal digestion studies present in the literature have shown that CA is one of the least bioaccessible polyphenols. Two different absorption mechanisms have been proposed: one is an active transport by the monocarboxylic acid transporter (MCT), and the other via paracellular diffusion (as chlorogenic acid) [39; 49; 50; 51]. In particular, in vitro experiments on Caco-2 cells performed in static conditions and in vivo experiments on animal models have shown that CA can be classified as a poorly absorbed compound (intestinal absorption: 0-20%) in the first hour of the intestinal digestion phase [49], as our results confirmed. In fact, 91.48% \(\pm\) 4.92 and 86.14% \(\pm\) 7.15 of CA was present in the medium after 4 and 6 h of contact with Caco-2 cells, respectively. Therefore, the absorbed CA fraction by Caco-2 cells was approximately 10-15% (Table 1). Rosmarinic acid (RA) (Figure 1c) has been classified as having a low intestinal permeability as well, but it can be considered one of the most bioaccessible polyphenols (50% absorbed compound). The paracellular diffusion seemed to be the main absorption process, but an active transport has also been supposed [39; 52; 53; 54]. Efflux transporters and TJ proteins seemed to have an important role in the RA absorption [53]. In vivo studies \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & & \multicolumn{3}{c}{**Time (h)**} \\ \hline
**Compound** & **1** & **2** & **4** & **6** & **24** \\
**(0.1 mg/mL)** & & & & & \\ \hline
**Quinic acid** & 0 & 0 & 0 & 0 & 0 \\ \hline
**Caffeic acid** & 98.48 \(\pm\) 1.62 & 96.67 \(\pm\) 2.14 & 91.48 \(\pm\) 4.92 & 86.14 \(\pm\) 7.15 & 52.52 \(\pm\) 2.31 \\ \hline
**Rosmarinic acid** & 93.15 \(\pm\) 3.58 & 86.23 \(\pm\) 4.05 & 68.20 \(\pm\) 3.38 & 59.53 \(\pm\) 0.98 & 0 \\ \hline
**Quercetin** & 99.55 \(\pm\) 4.15 & 94.10 \(\pm\) 5.36 & 49.16 \(\pm\) 3.73 & 37.15 \(\pm\) 2.68 & 0 \\ \hline
**Rutin** & 96.67 \(\pm\) 1.66 & 96.58 \(\pm\) 1.00 & 90.98 \(\pm\) 7.08 & 90.74 \(\pm\) 4.70 & 86.85 \(\pm\) 8.63 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Percentage of the non-absorbed fraction of each compound at different monitoring times. have confirmed that RA is quite rapidly metabolized during digestion [54]. In particular, literature data have shown that RA has a very high resistance to digestion at the intestinal level, thanks to its stability at neutral/alkaline pH. According to [PERSON] et al., RA stability rate, corresponding to its concentration before and after in vitro intestinal digestion ratio, was approximately 78% after 2 h, as also confirmed by our data indicating 86.23% \(\pm\) 4.05 non-absorbed RA at 2 h (Table 1) [55]. Our results indicated that the absorption increases over time reaching approximately 40% after 6 h and it exhibited a complete metabolization after 24 h in accordance with data obtained on animal models [56]. In particular, quercetin (QRC) and rutin (RU) have a great and important impact on the gastrointestinal tract, and QRC was especially reported to protect and stabilize TJ proteins of the Caco-2 cell monolayer [36].