A spectrum-effect based method for screening active components and elucidating processing-enhanced pro-osteoblast proliferation activity of Epimedii folium using UPLC-DAD fingerprint and GC-IMS.

Epimedii Folium (EF) is a widely used traditional herbal medicine. Compared with the raw material, its processed products exhibit enhanced clinical efficacy, yet the mechanism underlying this processing-enhanced bioactivity remains poorly understood. In this study, a spectrum-effect relationship strategy was adopted to analyze compositional variations in processed EF and identify the key active constituents that enhance osteoblast proliferation. Raw EF, mutton fat-processed EF (MEF), and butter-processed EF (BEF) were analyzed in this study. Chemical fingerprints for 30 samples (10 per group) using UPLC-DAD (for flavonoids) and GC-IMS (for VOCs). Pro-osteoblast proliferation was tested on rat osteoblasts (ROBs). Orthogonal partial least squares (OPLS) analysis was performed to screen for potential active components, which were subsequently putatively annotated via UPLC-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Differences in color, VOCs, and flavonoid fingerprints were observed between raw and processed EF. Notably, processed EF exhibited significantly higher pro-osteoblast proliferative activity. Among the 29 common peaks, 16 were positively correlated with the proliferation of ROBs, and 10 of these had a VIP value >1. A total of seven flavonoid mono- and diglycosides, including icariin and baohuoside I, were successfully annotated, and their contents increased remarkably after processing. Both compounds were confirmed to promote ROBs proliferation in vitro. Simulated processing experiments supported that the conversion of flavonoid polyglycosides into lower-glycosylated derivatives with higher pro-proliferative activity is a major contributor to the observed activity enhancement, although contributions from other processing-induced changes cannot be excluded. The spectrum-effect method effectively screened active components responsible for the enhanced efficacy of processed EF. Thus, processing-mediated transformation of flavonoid glycosides appears to be a central factor underlying the improved bioactivity, providing a scientific basis for the rational application of processed EF.