Comprehensive Strategy Integrating Fingerprint Analysis and Multicomponent Quantification for Quality Control and Anti‐Osteoporotic Activity Evaluation of Cnidii Fructus Before and After Processing by Wine Steaming

Wine-steaming of Cnidii Fructus is a traditional processing method believed to enhance efficacy, but its mechanistic basis remains unverified. This study integrated HPLC-based chemical profiling with in vivo anti-osteoporotic evaluation in zebrafish to elucidate the effects of processing. Comparative fingerprinting and multicomponent quantification delineated distinct chemical profiles between raw Cnidii Fructus (RCF) and wine-steamed Cnidii Fructus (WCF). Multivariate analysis (PCA, OPLS-DA) pinpointed six differential coumarins. Quantitative analysis confirmed a marked decrease in imperatorin but increases in osthole, alloimperatorin, and xanthotoxol after processing. Critically, the WCF extract demonstrated superior efficacy over RCF in promoting bone mineralization and enhancing alkaline phosphatase (ALP) activity in an osteoporotic zebrafish model. The differential compounds counteracted dexamethasone-induced impairment, with xanthotoxol showing the most potent osteogenic activity, followed by alloimperatorin, diosmin, osthole, and imperatorin. These findings establish that wine-steaming potentiates the anti-osteoporotic activity of WCF by modulating its coumarin composition, providing a chemical and pharmacological rationale for its traditional use and quality control.