2.12. Permeability through the Blood–Brain-Barrier (PAMPA-BBB)

To evaluate the effective permeability (Pe) of the H. physodes extract and physodic acid, the Parallel Artificial Membrane Permeability Assay (PAMPA) for the Blood–Brain Barrier (BBB) was used (Pion Inc., Billerica, MA, USA). The stock solutions of acetone extract from H. physodes and from physodic acid were prepared with DMSO (3 mg/0.1 mL or 1.5 mg/0.1 mL, respectively) and diluted with Prisma buffer (pH = 7.4; from Prisma HT, Pion Inc.) to obtain the donor solutions for physodic acid: 0.45 mg/mL (30 µL of stock/1000 µL of buffer for physodic acid) or 0.9 mg/mL (60 µL of stock/1000 µL of buffer for physodic acid), and 0.9 mg/mL (30 µL of stock/1000 µL H. physodes acetone extract). Then, 180 µL of the donor solution were added to the donor wells. Subsequently, each filter membrane of the top plate was coated with 5 μL BBB-1 lipid solution (Pion Inc.) and the acceptor well was filled with 200 µL BSB (Brain Skin Buffer, Pion Inc.). The acceptor plate and the donor plate were sandwiched together and incubated for 4 h at 37 °C. After incubation, the sandwiched plates were separated, and concentrations were determined using the HPLC method (HPLC Prominence-i LC-2030C, Shimadzu) [36]. Effective permeability (Pe) of the compounds was calculated by using the following equation:

where Pe is the effective permeability coefficient (cm/s); V_D—donor volume; V_A—acceptor volume; C_eq—equilibrium concentration, Ceq=CD×VD+CA×VAVD+VA,; S—membrane area; t—incubation time (in seconds).

Compounds with Pe (10⁻⁶ cm/s) > 1.5 are classified as high permeation predicted, with Pe (*10⁻⁶ cm/s) < 1.5 classified as low permeation predicted [36,37]. Samples were analyzed in triplicate and the average was reported.