An LC‐MS Based Metabolomic Approach Coupled With Bioassay‐Guided Fractionation to Identifying Bioactive Biflavonoids From <i>Selaginella Willdenowii</i>

The wide-ranging applications of biflavonoids (BFVs) enriched in Selaginella plants, particularly in cuisines and medicines have attracted interest in the isolation of bioactive BFVs. However, identification of bioactive compounds in a complex mixture is a crucial challenge in medicinal plant research. The data set of Selaginella willdenowii fractions having anti-inflammatory activity combined with their LC-MS-based metabolomic profiles was analyzed by the multivariate analysis. The findings suggested that tetrahydrobilobetin and 2'',3''-dihydrobilobetin were the potential anti-inflammatory agents. Furthermore, robustaflavone-7''-methyl ether (9c) demonstrated the strongest cytotoxicity against three cancer cell lines (A549, HepG2, and T47D) with high selectivity for A549 cells. Further mechanistic investigation indicated that 9c induced apoptotic cell death through elevation of reactive oxygen species (ROS) level in A549 cells. Notably, this work highlights the potential of applying a metabolomic strategy coupled with bioassay-guided isolation, thereby accelerating research in drug discovery and providing a valuable source of ingredients for the development of value-added food products.