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

doi:10.12141/j.issn.1000-565X.250082

Abstract

Abstract:

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.

Key words: Gnaphalium affine, flavonoids, purification, oxidative stress, antioxidation

CLC Number:

TS201.2

ZHENG Bisheng, XU Yanting, XU Qiuxiong, FAN Xinlühui. Purification of Total Flavonoids from Gnaphalium affine and Analysis on Its Antioxidant Stress Activity[J]. Journal of South China University of Technology(Natural Science Edition), 2025, 53(10): 174-182.

Figures/Tables 9

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树脂类型	极性	吸附率/%	解吸率/%
D101	非极性	83.22±0.51^a	90.55±0.90^a
HPD-100	非极性	74.91±0.39^c	85.34±0.73^b
AB-8	弱极性	74.39±1.17^c	84.19±1.10^b
ADS-17	中极性	78.13±0.89^b	86.16±1.27^b
NKA-9	极性	75.33±0.82^c	91.42±1.02^a
S-8	极性	75.22±0.64^c	89.13±1.18^a

物质名称	标准曲线方程	分子式	m/z（母离子）	m/z（特征碎片离子）	含量/（μg·g^-1）
木犀草素	y = 12 020.625 30x+4 729.323 55，R²= 0.999 59	C₁₅H₁₀O₆	285.0	133.0	289.57±7.50
金丝桃苷	y = 43.555 58x+221.583 28，R²= 0.997 56	C₂₁H₂₀O₁₂	465.1	314.9	391.91±40.69
槲皮素	y = 12 322.286 34x+8 480.388 26，R²= 0.999 7	C₁₅H₁₀O₇	301.1	151.0	228.44±4.12
圣草酚	y = 4 904.533 10x+17 18.43 311，R²= 0.998 34	C₁₅H₁₂O₆	287.1	135.0	184.53±8.95
芹菜素	y = 10 546.478 98x+3 461.369 17，R²= 0.999 78	C₁₅H₁₀O₅	269.1	117.0	165.30±7.52
野黄芩苷	y = 10 471.028 93x+2 570.485 38，R²= 0.999 58	C₂₁H₁₈O₁₂	461.1	285.1	238.24±20.13
槲皮素-3-O-葡萄糖醛酸苷	y = 17 298.137 41x+1 083.097 28，R²= 0.99 951	C₂₁H₁₈O₁₃	477.1	301.0	235.30±20.54
异鼠李素	y = 21 684.797 56 x+3 282.213 40，R²= 0.999 84	C₁₆H₁₂O₇	315.1	300.0	53.97±0.78
绣线菊苷	y = 6.077 28e4 x+20 680.97 361，R²= 0.998 88	C₂₁H₂₀O₁₂	463.1	301.0	40.17±1.20
二氢槲皮素	y = 4 097.122 65 x-6 642.076 74，R²= 0.999 8	C₁₅H₁₂O₇	303.1	125.0	13.55±0.53
芹菜素-7-O-葡萄糖醛酸苷	y = 14 375.38 407 x+4 848.90 482，R²= 0.999 54	C₂₁H₁₈O₁₁	445.1	269.1	17.25±0.88