High‐Efficiency Screening of Compounds Targeting Postmenopausal Osteoporosis in Cuscutae Semen Based on Affinity Ultrafiltration‐UPLC‐QE‐Orbitrap‐MS Platform

ABSTRACTIntroductionPostmenopausal osteoporosis, a skeletal disorder induced by estrogen deficiency, is commonly addressed in clinical practice through the use of Cuscutae semen and its compound formulations, owing to their significant therapeutic efficacy.ObjectiveThis study sought to systematically identify bioactive compounds present in Cuscutae semen that interact with estrogen receptor β (ESR2) using Affinity Ultrafiltration combined with Ultra‐Performance Liquid Chromatography‐Quadrupole‐Orbitrap Mass Spectrometry (UPLC‐QE‐Orbitrap‐MS) to find possible therapy options for PMOP.MethodsThe Cuscutae semen extract was fractionated using a C18 column to obtain fractions based on distinct polarity. Preliminary evaluation of anti‐osteoporotic activity was conducted in MC3T3‐E1 cells by assessing osteoprotegerin (OPG) expression. Affinity Ultrafiltration integrated with UPLC‐QE‐Orbitrap‐MS was utilized to screen for ligands binding to ESR2, followed by molecular docking to validate the interaction mechanisms. The osteogenic effects of the identified compounds were further confirmed through CCK‐8 proliferation assays, OPG quantification, and alkaline phosphatase (ALP) activity analysis.ResultsThe fraction of Cuscutae semen extract eluted with 100% methanol exhibited significant anti‐osteoporotic activity. Three flavonoids—Astragalin, Isorhamnetin, and Quercitrin—that interact with ESR2 were successfully identified. In vitro validation demonstrated the efficacy of these compounds.ConclusionThis study presents a comprehensive strategy that integrates Affinity Ultrafiltration, UPLC‐QE‐Orbitrap‐MS, and bioactivity validation to efficiently identify ESR2‐targeted postmenopausal osteoporosis compounds in Cuscutae Semen. The findings offer both theoretical and empirical foundations for the development of innovative therapeutics for postmenopausal osteoporosis.