鼠曲草总黄酮的纯化及其抗氧化应激活性分析

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

摘要/Abstract

摘要：

鼠曲草富含的黄酮类化合物具有多种生物活性，但对其在细胞氧化损伤保护作用方面的研究仍较为有限。为进一步开发其利用价值，该研究通过优化鼠曲草总黄酮纯化工艺，探究其化学成分，并深入分析其对氧化应激损伤的保护作用机制。该研究以鼠曲草为原料，通过低共熔溶剂加热提取后，筛选出最适大孔吸附树脂，并确定纯化工艺；利用液相色谱-串联质谱联用技术（LC-MS/MS）对纯化后的总黄酮进行定性和定量分析，明确其化学组成；以H₂O₂诱导HepG2细胞氧化应激模型，来探究纯化后鼠曲草总黄酮对细胞氧化损伤的保护作用。结果表明，通过筛选确定D101为最理想的鼠曲草黄酮纯化树脂，体积分数为60%的乙醇溶液作为解吸溶剂，通过动态吸附-解吸确定上样量和洗脱液用量分别为124 mL和200 mL。从鼠曲草总黄酮中分离鉴定出11种黄酮化合物，主要有木犀草素、金丝桃苷、槲皮素、芹菜素、野黄芩苷等，其中金丝桃苷含量最高，达到（391.91±40.69） μg/g。纯化后的鼠曲草总黄酮能显著提高H₂O₂诱导的细胞氧化损伤后的存活率，并且能够有效清除过量的活性氧（ROS），减少细胞损伤导致的乳酸脱氢酶（LDH）释放，提高超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性以及谷胱甘肽（GSH）含量。且实时荧光定量聚合酶链式反应（qPCR）结果表明，鼠曲草总黄酮可以通过调节Keap1/Nrf2信号通路来抵抗氧化应激反应。该研究结果表明鼠曲草总黄酮具有良好的抗氧化活性，为鼠曲草相关产品的开发提供了参考依据。

关键词: 鼠曲草, 黄酮, 纯化, 氧化应激, 抗氧化

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

中图分类号:

TS201.2

郑必胜, 徐燕婷, 徐秋雄, 范鑫吕慧. 鼠曲草总黄酮的纯化及其抗氧化应激活性分析[J]. 华南理工大学学报(自然科学版), 2025, 53(10): 174-182.

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.

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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