Transport of amentoflavone across the blood-brain barrier in vitro

The biflavone amentoflavone is an ingredient of Hypericum perforatum L. (Clusiaceae), a plant which is widely used for the treatment of mild to moderate depression. Amentoflavone inhibits the binding of flumazenil to the benzodiazepine binding site of the GABA(A)-receptor (IC50 = 14.9 nM). Since it has to pass the blood-brain barrier (BBB) before reaching this receptor, the penetration of [H-3]-amentoflavone through BBB was studied in an in vitro model consisting of primary cell cultures of porcine brain capillary endothelial cells (BCEC). Concentration-dependent uptake (37-2000 nM) was neither saturable nor temperature-sensitive indicating passive diffusion as the major uptake mechanism. This finding was confirmed by transport experiments through BCEC monolayers (> 2 % of applied dose was transported after 30 min). Co-administration of Hypericum extract increased amentoflavone transport significantly (amentoflavone alone: permeability coefficient P-app = 4.59(.)10(-6) cm/s; co-administrated sucrose: P-app = 3.22(.)10(-6) cm/s; amentoflavone together with hypericum: P-app = 6.74(.)10(-6) cm/s, co-administrated sucrose P-app = 5.49(.)10(-6) cm/s) indicating that Hypericum constituents enhance amentoflavone transport possibly by modulating paracellular permeability. Experiments with the P-glycoprotein (P-gp) overexpressing cell line P388-MDR showed that amentoflavone uptake was significantly enhanced by addition of the P-gp inhibitor verapamil, suggesting a P-gp mediated back-transport out of the cells. In conclusion, our findings show, that amentoflavone is able to pass the blood-brain barrier in vitro by passive diffusion.