Development of a novel online coupling of HPLC-FLU, a fast bioactive targeting and guided purification of type II 5α reductase inhibitors from Picea wilsonii

Seeking type II 5α reductase (5-AR2) inhibitors from natural products (NPs) has emerged as a promising strategy to combat androgen metabolism related diseases. However, there is no universally applicable method for identifying and isolating 5-AR2 inhibitors to date. In this study, an online HPLC-5AR system was constructed for the first time by integrating HPLC-FLU and 5-AR2 solution, enabling the qualitative recognition of 5-AR2 inhibitors in NPs. Taking Picea wilsonii ( P. wilsonii ) as an example, three bioactive peaks were recognized via the online HPLC-5AR system, and then three high purity 5-AR2 inhibitors were obtained by medium and high-pressure liquid chromatography, named pungenin, astringe, and isorhapontin. Molecular docking analysis showed that pungenin (-7.72 kcal/mol), astringe (-9.36 kcal/mol), and isorhapontin (-9.11 kcal/mol) exhibited strong binding affinity with 5-AR2. Enzyme activity assay revealed their IC 50 values were 30.6 μM, 12.6 μM, and 89.9 μM, respectively. In addition, in vitro experiments revealed that these inhibitors also capable of interfering with 5-AR2 from protein and transcriptional levels, which in turn affects its biological function. Overall, this study will further lead to the high value utilization and economic value assessment of P. wilsonii . This novel online HPLC-5AR system has been demonstrated to be convenient and efficient for the rapid recognition of 5-AR2 inhibitors from NPs, which holds significant promise for the treatment of androgen metabolism related diseases, warranting further exploration and broader application in future research. • A novel HPLC-5AR system was first constructed and applied to the rapid recognition of 5-AR2 inhibitors in P. wilsonii . • This is the first report of three 5-AR2 inhibitors (pungenin, astringe, and isorhapontin) from P. wilsonii . • Astringe showed excellent inhibitory activity against 5-AR2 with an IC 50 value of 12.6 μM.