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Berberine,berberrubine,thalifendine,demethyleneberberine,jatrorrhizine,and columbamine are six natural protoberberine alkaloid (PA) compounds that display extensive pharmacological properties and share the same protoberberine molecular skeleton with only slight substitution differences.The oral delivery of most PAs is hindered by their poor bioavailability,which is largely caused by P-glycoprotein (P-gp)-mediated drug efflux.Meanwhile,P-gp undergoes large-scale conformational changes (from an inward-facing to an outward-facing state) when transporting substrates,and these changes might strongly affect the P-gp-binding specificity.To confirm whether these six compounds are substrates of P-gp,to investigate the differences in efflux capacity caused by their trivial structural differences and to reveal the key to increasing their binding affinity to P-gp,we conducted a series of in vivo,in vitro,and in silico assays.Here,we first confirmed that all six compounds were substrates of P-gp by comparing the drug concentrations in wild-type and P-gp-knockout mice in vivo.The efflux capacity (net efflux) ranked as berberrubine ＞ berberine ＞columbamine ～ jatrorrhizine ＞ thalifendine ＞ demethyleneberberine based on in vitro transport studies in Caco-2 monolayers.Using molecular dynamics simulation and molecular docking techniques,we determined the transport pathways of the six compounds and their binding affinities to P-gp.The results suggested that at the early binding stage,different hydrophobic and electrostatic interactions collectively differentiate the binding affinities of the compounds to P-gp,whereas electrostatic interactions are the main determinant at the late release stage.In addition to hydrophobic interactions,hydrogen bonds play an important role in discriminating the binding affinities.