Study on the active ingredients and mechanism of Huyang Yangkun Formula for treating Premature Ovarian Insufficiency via Chemical profiling, network pharmacology, and experimental validation

Huyang Yangkun Formula (HYF) is a Chinese herbal remedy used for premature ovarian insufficiency (POI), though its active ingredients and mechanisms are not well understood.This study aims to identify HYF's chemical components and investigate its mechanism in treating POI. Using UHPLC-QE Focus HRMS, chemical profiling and quantification were conducted. The therapeutic effects and target validation of HYF were examined using a POI rat model, human ovarian granulosa cells (COV434), and network pharmacology. Molecular docking was used to predict the affinity of active compounds for the key target TP53. 125 compounds were identified in HYF, including flavonoids, organic acids, saponins and phenylethanoid glycosides. By using network pharmacological analysis, a total of 123 potential targets for the HYF treatment of POI were identified. PIK3R1, AKT1, EGFR, MMP2 and TP53 were deduced to be core targets of HYF for treating POI, and the main pathway included HIF-1, PI3K-Akt and P53 signaling pathway. HYF enhances follicle development and ovarian function by reducing apoptosis in ovarian granulosa cells in VCD-POI rats. In vitro, HYF decreased VCD-induced COV434 cell death. qRT-PCR and WB experiments identified the P53-mitochondrial apoptosis signaling pathway as the main target, with molecular docking showing Hyperoside, Isochlorogenic acid B, and Baohuoside I having the highest binding affinity to TP53.The potential active components and mechanisms of HYF in relation to POI were investigated through chemical profiling, network pharmacology, and both in vitro and in vivo experimental validation.