Fluorescein Transport Assay in Hesperidin-Treated Caco-2 Cells

Fluorescein transport experiments were performed in the hesperidin‐pretreated Caco‐2 cell monolayers using the Ussing Chamber system (Model U‐2500, Warner Instrument Co., Holliston, MA, USA). Caco‐2 cell monolayers in the transwell inserts were gently rinsed with Hanks's balanced salt solution (HBSS) buffer [pH 7.4, adjusted with 10 mm 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid, HEPES] to remove any free test compound before carefully mounting the inserts in the Ussing Chamber. HBSS buffer aliquots (6.0 mL) were added to the apical and basolateral sides to equilibrate the monolayers for 10 min. The transport assay was started by replacing the apical buffer with fresh HBSS buffer containing 100 μm fluorescein. During the transport experiment, both sides were continuously bubbled with a mixture of O₂:CO₂ (95:5). Sample aliquots of 100 μL were collected from the basolateral side every 5 min for 30 min, and the same sample volume of fresh HBSS was simultaneously added to the basolateral side at each time point. The fluorescence of the collected samples was measured with a fluorescence spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The apparent permeability coefficient (P_app) was calculated using the following equation: $${\mathrm{P}}_{\mathrm{app}}\left(\mathrm{cm}/\mathrm{s}\right)=\frac{V}{A{C}_0}\frac{\mathrm{d}C}{\mathrm{d}t}$$ where V is the assay solution volume in the basolateral compartment (6 mL); A is the surface area of the membrane (0.2826 cm²); C₀ is the initial concentration in the apical compartment (mmol); dC/dt is the change in concentration in the basolateral compartment over time (mmol·s⁻¹). The relative P_app of fluorescein in Caco‐2 cell monolayers pretreated with the test compound (hesperidin) was expressed as percentage (%) of the control P_app.