3.2.8.2. Evaluation of SRO (Fatty Acids), PCA, and LYC Enteric Absorption Rate

Based on dose-response curve information and their posology, digested formulations were added to the apical side of the in vitro intestinal epithelium, while HBSS buffer supplemented with 1% BSA was placed in the basolateral compartment. Due to the lipophilicity of formulation active components, 1% BSA was added to the basolateral compartment for improving their absorption rate. According to the literature (Fossati et al., 2008) [37] the addition of BSA improves the correlation between absorption occurring in Caco-2 cell monolayer and humans. After 3 h incubation, apical and basolateral solutions were collected and fatty acids, proanthocyanidin and lycopene content was determined by GC/MS and HPLC respectively. Absorption rate of SRO fatty acids, PCA and LYC is expressed as percentage of absorption, derived from three independent experiments.

## 3.2.8.3. Barrier Integrity and Cell Viability

After exposure to digested formulations, cell viability and barrier integrity of the intestinal epithelium model were evaluated. Briefly, at the end of the incubation with the digested formulations, the in vitro intestinal epithelia were washed twice with pre-warmed HBSS and equilibrated in the same buffer for 30 min. Once equilibrated, epithelia barrier integrity was evaluated by measuring the trans-epithelial electrical resistance (TEER) of the cell monolayer with an ERS2 Voltohmmeter (Millipore), equipped with a chopstick electrode. Intestinal epithelium model paracellular permeability was determined with Lucifer Yellow (LY), a fluorescent polar tracer unable to pass through intact tight junctions. Paracellular permeability was measured by adding 0.5 mL of 100 μg/mL LY in HBSS in the apical compartment and 1.5 mL of HBSS in the basolateral compartment. After 1-h incubation, the basolateral fractions were collected and their fluorescence measured with a spectrofluorometer (Synergy 4, Biotek). Apparent permeability coefficient (Papp, cm/s) was calculated with the following formula:

$$P\_{\rm app} = (\Delta \mathbf{C} \cdot \mathbf{V}) / (\Delta \mathbf{t} \cdot \mathbf{A} \cdot \mathbf{C}\_0) \tag{1}$$

where ΔC/Δt is the flow of the molecule being transported across the monolayer during the incubation time (mM/s), V is the volume of the basolateral compartment (cm3), A is the area of the membrane (cm2), C0 is the initial concentration of the molecule in the apical compartment. Finally, cell viability was evaluated by using MTS assay as described above.
