2.6.4. Parallel Artificial Membrane Permeability Assay for the Blood–Brain Barrier (PAMPA-BBB)

Parallel artificial membrane permeation assay (PAMPA-BBB) was performed as a prediction in vitro mechanism of BBB permeability according to the method proposed by Di et al. (2003) [55]. Previously, the BBB solution was prepared by dissolving 8 mg of PBL and 4 mg of cholesterol in 600 μL n-dodecane. Ethanolic olive leaves extract was dissolved in buffer (5 mM pH 7.4) as an initial solution at 10 mg/mL (EtOH/buffer, 1:1, v/v). Then, the filter membrane of the donor microplate was impregnated with 5 μL of BBB solution and the acceptor microplate was filled with 350 μL of buffer. Then, 200 μL of initial solution was added to the donor microplate which was assembled like a sandwich over the acceptor microplate. The microplate was incubated in the dark for 4 h at 37 °C and continuous agitation. After the incubation process, 200 μL were taken from each microplate, placed into a vial, dried, and reconstituted in 50 μL of EtOH to obtain donor and acceptor solutions. Both solutions were injected in the GC-qTOF-MS system to identify and compare the compounds present in both microplates. Permeability across the artificial BBB was calculated according to Chen et al. (2008) [56] in Equation (6):

where P_e is permeability (cm/s), V_D and V_A are donor and acceptor well volume, and correspond to 0.35 and 0.2 mL, respectively. C_D(t) is compound concentration in the donor well at time t and C_A(t) is compound concentration in the acceptor well at time t. A is effective filter area = ƒ ∗ 0.3 cm², t = incubation time = 14,400 s, and C_e is the concentration in equilibrium calculated by C_e = [C_D(t) ∗ V_D + C_A(t) ∗ V_A]/(V_D + V_A).