Abstract:

Using Gnaphalium affine as the raw material, total flavonoids were extracted via a deep eutectic solvent-assisted heating method. The optimal macroporous adsorption resin was systematically screened, and the purification parameters were subsequently optimized. LC-MS/MS analysis was employed to characterize the chemical composition of purified flavonoids through qualitative and quantitative profiling. An oxidative stress model induced by H₂O₂ in HepG2 cells was employed to investigate the protective effects of the purified Gnaphalium affine total flavonoids against cellular oxidative damage. The results show that, through screening, D101 was identified as the most suitable resin for purifying Gnaphalium affine flavonoids, with a 60% ethanol solution as the elution solvent. The dynamic adsorption-desorption process determined the optimal loading volume and eluent volume to be 124 mL and 200 mL, respectively. Eleven flavonoid compounds were isolated and identified from the total flavonoids of Gnaphalium affine, primarily including luteolin, hyperoside, quercetin, apigenin, and scutellarin, among others. Among these, hyperoside was found to be the most abundant, with a content of 391.91 ± 40.69 μg/g. The purified Gnaphalium affine total flavonoids significantly enhanced cell viability under H₂O₂-induced oxidative stress.  These flavonoids effectively scavenged excess ROS, reduced LDH release, and upregulated SOD and CAT. Additionally, they increased GSH content, demonstrating potent antioxidative and cytoprotective effects. Furthermore, quantitative PCR results indicated that Gnaphalium affine total flavonoids could mitigate oxidative stress by modulating the Keap1/Nrf2 signaling pathway. These findings provide a reference for the development of antioxidant functional products derived from Gnaphalium affine.

Key words: Gnaphalium affine, flavonoid, purification, oxidative damage, antioxidant