蓝莓提取物抗氧化活性及对t-BHP诱导L-02细胞氧化损伤的保护作用

李舜尧; 郭小妮; 臧慧明; 李松; 辛秀兰; 张强

doi:10.13386/j.issn1002-0306.2025050250

蓝莓提取物抗氧化活性及对t-BHP诱导L-02细胞氧化损伤的保护作用

Antioxidant Activity of Blueberry Extract and Its Protection Effect on t-BHP-induced Oxidative Damage of L-02 Cells

摘要

摘要: 为明确蓝莓提取物中绿原酸同系物和花青素的组成及其对人L-02肝细胞的保护作用，本文对15个不同品种、产地和地理位置的蓝莓样品，采用有机溶剂提取和大孔吸附树脂分离的方法制得绿原酸提取物（chlorogenic acid extracts，CGAE）和花青素提取物（anthocyanin extracts，ANTE），在体外开展了自由基清除实验，L-02肝细胞抗氧化酶活实验。结果表明：15个蓝莓样品绿原酸和花青素两种提取物成分的平均含量为：绿原酸>新绿原酸>隐绿原酸>异绿原酸B>异绿原酸A>异绿原酸C，飞燕草素>牵牛花素>芍药色素>锦葵色素>矢车菊素。相关性分析表明：绿原酸、新绿原酸、隐绿原酸含量与DPPH、ABTS⁺自由基清除率呈显著负相关，与FRAP、RP呈显著正相关，飞燕草素-3-半乳糖苷和飞燕草素-3-阿拉伯糖苷含量与FRAP、RP呈显著正相关，与ABTS⁺自由基清除率呈负相关，但只有飞燕草素-3-阿拉伯糖苷显著，而飞燕草素-3-葡萄糖苷与ABTS⁺自由基清除率呈显著正相关，与FRAP、RP呈显著负相关。在矢车菊素、牵牛色素、芍药色素和锦葵色素中，仅矢车菊素-3-葡萄糖苷与FRAP、RP呈显著负相关，牵牛花素-3-半乳糖苷、牵牛花素-3-阿拉伯糖苷与FRAP、RP呈显著正相关，表明不同品种、不同产地和地理位置的蓝莓，其活性成分含量及抗氧化活性存在显著差异，并利用主成分分析（Principal Component Analysis，PCA）从统计学角度证实了不同品种的化学特征差异，构建细胞水平的功能性验证模型，优化并最终确定了造模条件为350 μmol/L t-BHP处理4 h，在细胞模型中验证了CGAE和ANTE提取物的保护作用，综上，20~60 μg/mL CGAE和15~25 μg/mL ANTE显著降低ROS水平的同时，显著提高GSH-Px和SOD的活性，且ANTE的抗氧化效果显著高于CGAE，本文筛选出高抗氧化活性蓝莓提取物，为蓝莓提取物的抗氧化应用研究提供参考。

Abstract: To elucidate the composition of chlorogenic acid homologs and anthocyanins in blueberry extracts and their protective effects on human L-02 hepatocytes, the present study utilized organic solvent extraction and macroporous adsorption resin separation to prepare chlorogenic acid extracts (CGAE) and anthocyanin extracts (ANTE) from 15 blueberry samples of distinct cultivars, origins, and geographical locations. In vitro experiments were conducted to assess free radical scavenging capacity and evaluate the activity of antioxidant enzymes in L-02 hepatocytes. The results showed that chlorogenic acid>neochlorogenic acid>cryptochlorogenic acid>isochlorogenic acid B>isochlorogenic acid A>isochlorogenic acid C and delphinidin>petunidin>peonidin>malvidin>cyanidin in the average content of chlorogenic acids and anthocyanins in 15 blueberry samples. Correlation analysis showed that the contents of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid were significantly negatively correlated with DPPH and ABTS⁺ radical scavenging rates, and significantly positively correlated with FRAP and RP. The contents of delphinidin-3-galactoside and delphinidin-3-arabinoside were significantly positively correlated with FRAP and RP, and negatively correlated with ABTS⁺ radical scavenging rate, but only the correlation for delphinidin-3-arabinoside was significant. In contrast, delphinidin-3-glucoside was significantly positively correlated with ABTS⁺ radical scavenging rate, and significantly negatively correlated with FRAP and RP. Among cyanidin, petunidin, peonidin, and malvidin, only cyanidin-3-glucoside was significantly negatively correlated with FRAP and RP, while petunidin-3-galactoside and petunidin-3-arabinoside were significantly positively correlated with FRAP and RP. The results indicated that the active component content and antioxidant activity of blueberries varied significantly depending on the different cultivars, growing regions, and geographical origins. Principal component analysis (PCA) was employed to statistically validate the distinct chemotypic profiles among different varieties. A functional validation model at the cellular level was established, and the optimal induction conditions were determined as treatment with 350 μmol/L t-BHP for 4 hours. Using this optimized model, the protective effects of both CGAE and ANTE extracts were rigorously evaluated. In summary, 20~60 μg/mL CGAE and 15~25 μg/mL ANTE significantly reduced ROS levels while significantly increasing the activities of GSH-Px and SOD, and the antioxidant effect of ANTE was significantly higher than that of CGAE. This study screened blueberry extracts with high antioxidant activity, providing a reference for research on the antioxidant applications of blueberry extracts.