Cell culture and Caco-2 Transwell model

The Caco-2 cells were purchased from ATCC (Manassas, VA) and cultured in DMEM high glucose medium with 15% FBS, 1% NEAA and 100 U/mL penicillin and streptomycin. Cell culture was conducted at 37 °C with 5% CO₂.

1 × 10⁵ cells/cm² were seeded into Transwell polycarbonate insert filters with the pore size of 0.4 μm (Corning Costar Corporation, Cambridge, MA) and cultured for 21 days. In the first seven days, the medium was replaced freshly every two days, and then daily. The transepithelial electrical resistance (TEER) of the monolayer cells was measured using Millicell ERS-2 (Millipore Corporation, Billerica, MA), the cells can be used for the transport experiment when TEER exceeding 400 Ω·cm².

Before the transport experiment, the Caco-2 cell monolayers were washed with warm (37 °C) Hanks’ balanced salt solution (HBSS) twice and incubated at 37 °C for 20 min. Then, the cell monolayers were incubated with paeoniflorin in fresh medium at 37 °C. The incubation volume of apical (AP) and basolateral (BL) was 0.5 and 1.5 mL, respectively. After incubating for the indicated time, 100 μL aliquot of the incubation solution was withdrawn and replaced with fresh HBSS (37 °C). Verapamil (50 μM) was added to both sides of cell monolayers and incubated for 30 min to inhibit the activity of P-gp. The permeability of paeoniflorin (10 μM) in all of the above conditions for both directions, i.e., from the AP side to the BL side and from the BL side to the AP side, was measured after incubation for 30, 60, 90 and 120 min at 37 °C. The efflux activity of P-gp was validated using a typical P-gp substrate rhodamine (10 μM).

The values of P_app and efflux ratio were calculated with the following equation:

where P_{app BA} is the apparent permeability coefficient from the BL side to the AP side (cm/s) and P_{app AB} is the apparent permeability coefficient from the AP side to the BL side (cm/s). ΔQ/Δt (μmol/s) is the rate at which the compound appears in the receiver chamber, C₀ (μM) is the initial concentration of the compound in the donor chamber and A (cm²) represents the surface area of the cell monolayer.