Podophyllum sinense (P. sinense) has been used as a traditional herbal medicine for ages due to its extensive pharmaceutical activities, including antiproliferative, anti-inflammatory, antiviral, insecticidal effects, etc. Nevertheless, the specific bioactive constituents responsible for its antiproliferative, anti-inflammatory, and antiviral activities remain elusive, owing to its complicated and diversified chemical components. In order to explore these specific bioactive components and their potential interaction targets, affinity ultrafiltration with multiple drug targets coupled with high performance liquid chromatography/mass spectrometry (UF–HPLC/MS) strategy was developed to rapidly screen out and identify bioactive compounds against four well-known drug targets that are correlated to the application of P. sinense as a traditional medicine, namely, Topo I, Topo II, COX-2, and ACE2. As a result, 7, 10, 6, and 7 phytochemicals were screened out as the potential Topo I, Topo II, COX-2, and ACE2 ligands, respectively. Further confirmation of these potential bioactive components with antiproliferative and COX-2 inhibitory assays in vitro was also implemented. Herein, diphyllin and podophyllotoxin with higher EF values demonstrated higher inhibitory rates against A549 and HT-29 cells as compared with those of 5-FU and etoposide. The IC(50) values of diphyllin were calculated at 6.46 ± 1.79 and 30.73 ± 0.56 μM on A549 and HT-29 cells, respectively. Moreover, diphyllin exhibited good COX-2 inhibitory activity with the IC(50) value at 1.29 ± 0.14 μM, whereas indomethacin was 1.22 ± 0.08 μM. In addition, those representative constituents with good affinity on Topo I, Topo II, COX-2, or ACE2, such as diphyllin, podophyllotoxin, and diphyllin O-glucoside, were further validated with molecular docking analysis. Above all, the integrated method of UF–HPLC/MS with multiple drug targets rapidly singled out multi-target bioactive components and partly elucidated their action mechanisms regarding its multiple pharmacological effects from P. sinense, which could provide valuable information about its further development for the new multi-target drug discovery from natural medicines.
Exploring Multifunctional Bioactive Components from Podophyllum sinense Using Multi-Target Ultrafiltration