Potential multiple bioactive components from Sinopodophyllum hexandrum explored by affinity ultrafiltration with four drug targets

: Sinopodophyllum hexandrum (S. hexandrum) is a typical Chinese herbal medicine with multiple components and pharmacological activities. However, the specific phytochemicals responsible for its anti-proliferative, anti-inflammatory and anti-viral effects remain unexplored. : This study aimed to explore the specific bioactive phytochemicals from S. hexandrum responsible for its multiple remarkable pharmacological in an effort to construct and decipher its empirical therapeutic roles. : Bio-affinity ultrafiltration (AUF) with multiple drug targets corresponding to its empirical anti-proliferative, anti-inflammatory and anti-viral activities was developed to screen for potential bioactive ligands from S. hexandrum, and then in vitro bioactivity assays or molecular docking methods were used to verify those bioactive ligands. : The integrative analytical strategy was developed to rapidly screen and identify bioactive ligands from S. hexandrum. The in vitro anti-proliferative, COX-2 and ACE2 inhibitory assays of ligands were further verified by SRB cell proliferation and cytotoxicity detection, COX-2 and ACE2 inhibitor screening kits, respectively. Molecular docking analysis was also implemented by the AutoDockTools 1.5.6 software. : Ten, seven, nine and nine phytochemicals were screened out and identified as the potential Topo I, Topo II, COX-2 and ACE2 ligands, respectively. Hereinto, podophyllotoxin and quercetin with higher EF values displayed strong inhibitory effects on A549 and HT-29 cells comparable with etoposide and 5-FU as the positive controls. Furthermore, compared with indomethacin at 0.73 ± 0.07 μM, kaempferol and podophyllotoxin with higher EF values exerted stronger inhibitory effects with IC₅₀ values at 0.36 ± 0.02 μM and 10.49 ± 0.61 μM, respectively. Isorhamnetin with a higher EF value exerted stronger inhibitory effect against ACE2 with IC₅₀ value at 118.46 ± 14.78 μM in contrast to podophyllotoxin with relatively lower EF value at > 970.00 μM. In addition, the optimal binding sites and mode of action between multiple drug targets and bioactive ligands were determined by molecular docking. : This study showcased a quick and reliable experimental strategy for unveiling the underlying mechanism of S. hexandrum, which could also provide valuable information for better understanding the therapeutic targets and ligands of other herbal medicines.