Purification of Total Flavonoids from Gnaphalium affine and Analysis on Its Antioxidant Stress Activity

Gnaphalium affine is rich in flavonoids with diverse biological activities, but research on its protective effects against cellular oxidative damage remains limited. To further explore its utilization value, this study aimed to optimize the purification process of total flavonoids from Gnaphalium affine, investigate its chemical composition, and deeply explore its protective mechanism against oxidative stress damage. In this study, Gnaphalium affine was used as the raw material. After deep eutectic solvent heating extraction, the optimal macroporous adsorption resin was screened and the purification process was determined. LC-MS/MS was employed for qualitative and quantitative analysis of the purified total flavonoids to clarify their chemical composition. An oxidative stress model induced by H₂O₂ in HepG2 cells was used to investigate the protective effects of the purified Gnaphalium affine total flavonoids against cellular oxidative damage. The results show that, through screening, D101 is the most suitable resin for purifying Gnaphalium affine flavonoids, with 60% ethanol solution as the desorption solvent. Dynamic adsorption-desorption experiments determined the optimal sample 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, hyperin, quercetin, apigenin, and scutellarin, etc. Among these, hyperoside is the most abundant, with a content of (391.91 ± 40.69) μg/g. Purified total flavonoids from Gnaphalium affine can significantly increase cell viability after H₂O₂-induced oxidative damage, effectively scavenge excess reactive oxygen species (ROS), and reduce lactate dehydrogenase (LDH) release from damaged cells. Concurrently, they enhanced the activities of superoxide dismutase (SOD) and catalase (CAT), as well as glutathione (GSH) levels. Real-time quantitative polymerase chain reaction (qPCR) results indicate that Gnaphalium affine total flavonoids can mitigate oxidative stress by regulating the Keap1/Nrf2 signaling pathway. These findings demonstrate that Gnaphalium affine total flavonoids possess strong antioxidant activity, providing a reference for the development of related products.