BackgroundDysosma versipellis (D. versipellis) and Phyllanthus urinaria (P. urinaria) are two typical Chinese herbs with diverse chemical composition and affluent pharmacological activities. Yet, the specific bioactive components with anti-inflammatory and anti-viral effects and corresponding mechanisms remain unclear. PurposeThe aim of this study was to screen for the specific constituents with anti-inflammatory and anti-viral activities from the anti-HBV formulation. Study designThe anti-viral and anti-inflammatory activities of D. versipellis, P. urinaria and the anti-HBV formulation extracts were evaluated by in vitro ACE2 and COX-2 inhibitory assays. Affinity ultrafiltration combining liquid chromatography / mass spectrometry (UF-LC/MS) with COX-2, ACE2, and NTCP was conducted according to its empirical traditional use, and then molecular docking analysis was applied to validate the activity of the ligands. MethodsAn efficient strategy was employed to screen bioactive compounds from the anti-HBV formulation. Corresponding activities of the extracts or phytochemicals were confirmed by inhibitor screening kits or docking simulation. ResultsA total of 8, 3 and 8 phytochemicals exerting distinct affinities with COX-2, ACE2 or NTCP were rapidly screened out and characterized from D. versipellis, P. urinaria and the anti-HBV formulation, respectively. Thereafter, the interaction between the specific binding ligands, namely dehydropodophyllotoxin, β-apopicropodophyllin, quercetin, and COX-2, ACE2 or NTCP was further simulated by docking analysis, and the results were well correspondent with the UF results. Noticeably, the interaction network among multiple component-target-activity correlations was for the first time constructed in an effort to elucidate the mechanisms of action exerting its therapeutic effects for the anti-HBV formulation, which was also better in accordance with the traditional Chinese medicine (TCM) theory. ConclusionThe present study showcased an integrative strategy that could offer not only a puissant tool to screen for active constituents from this anti-HBV formulation, and further help to unveil its underlying mechanisms, but also provide an alternative platform for discovering new candidate drugs for anti-inflammation and anti-virus from other medicinal plants in the near future.