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.

This study use Guangdong Zhaoqing kapok as raw material. After ultrasonic-assisted alcohol extraction, the most suitable macroporous resin type was investigated, and the purification process of the crude kapok flavonoids was determined. The inhibitory effect of purified kapok flavonoids on Staphylococcus and Propionibacterium acnes was measured by tests of antibacterial activity in vitro. The inhibition rate of kapok flavonoids on tyrosinase was determined by in vitro whitening activity method, and the whitening activity of kapok flavonoids was further verified by zebrafish embryo melanin inhibition test method. Finally, the scavenging ability of kapok flavonoids to free radi-cals such as ABTS·⁺ and DPPH was investigated by in vitro antioxidant activity assay. By comparing the adsorption and desorption rates of 8 macroporous resins to kapok flavonoids, D101 was screened out as the best resin type and volumetric concentration of ethanol for the best analytical solution is 60%. The optimal adsorption-analytical conditions are: the sample volume is 2 BV, and the elution volume of the analytical solution is 0.625 BV. After purification, the purity of kapok flavone increased from 32.5% to 98.2%. The IC₅₀ values of the purified kapok flavone for the free radical scavenging rate of ABTS·⁺ and DPPH are (616.90±11.15) mg/L and (11.80±0.62) mg/L, respectively. The IC₅₀ of kapok flavone on tyrosinase inhibition is (1.69±0.01) g/L. When the concentration of kapok flavone is 1.0 g/L, the inhibition rate of zebrafish embryo melanin is 12%, with significant whitening activity. The inhibition zone experiment shows that when the mass concentration of kapok flavonoids is 12.5 g/L, it is moderately sensitive to Propionibacterium acnes and highly sensitive to Staphylococcus epidermidis, and the antibacterial effect shows a dose-dependent effect. The inhibitory effect of kapok flavonoids on the inflammatory factors TNF-α and IL-6 secreted by RAW264.7 cells is detected by ELISA, which shows when the mass concentration of kapok flavonoids is more than 0.5 g/L, it can significantly inhibitthe secretion of inflammatory cytokines TNF-α; when the mass concentration of kapok flavonoids is 0.25 g/L and above, the secretion of inflammatory factor IL-6 can be significantly inhibited.

Ferulic acid (FA) is a common dietary polyphenol which is widely found in plant tissues. It has various biological activities such as antioxidant, anti-inflammatory, antithrombotic, and hypoglycemic. Due to the biological activities of dietary polyphenols largely depend on their digestion and absorption in vivo, studies on the intestinal metabolites of dietary polyphenols have gradually attracted researchers’interest in recent years. Ferulic acid cannot be absorbed in the stomach and small intestine, but it can be converted into a series of hydroxyphenylpropionic acid compounds under the action of colonic esterases. This study selected three main colonic metabolites of FA, namely, 3-(3,4-dihydroxyphenyl) propionic acid (3,4diOHPPA), 3-(3-hydroxyphenyl) propionic acid (3OHPPA) and 3-phenylpropionic acid (PPA), to evaluate their antioxidant and antitumor activities. The results show that the antioxidant activity of metabolite 3, 4diOHPPA is superior to that of FA and metabolites 3OHPPA and 3PPA. FA and its three metabolites can significantly inhibit the proliferation of HepG2 with EC50 values of 1.82 mmol/L (FA), 0.74 mmol/L (3, 4diOHPPA), 7.77 mmol/L (3PPA) and 4.52 mmol/L (3PPA), respectively. Cell-cycle experiments show that FA and its three metabolites can regulate HepG2 cell cycle progression in an orderly manner, blocking the cell cycle in G2 or S phase. FA and its three metabolites can also induce apoptosis of HepG2 in a dose-dependent manner. Among them, FA and 3,4diOHPPA can increase the total apoptosis rate of HepG2 cells to 15.47% and 71.84% (4.23% for the control). After a pretreatment for 24 h, FA exerts its antiproliferative effects by upregulating Bax, p53 genes and downregulating CDK-2, CDK-4 genes; 3,4diOHPPA inhibits the proliferation of HepG2 by upregulating Bax, caspase-3 genes and downregulating CDK-2, CDK-4 genes. All these findings show that the anti-oxidative and anti-HepG2 cell proliferation ability of the metabolite 3,4diOHPPA is superior to that of FA. This research provides theoretical support for the antitumor activity of FA and its colonic metabolites, revealing the health benefits of FA intestinal metabolites.