黑莓黄酮的自由基清除活性与防护肝细胞氧化损伤研究

Studies on Free Radical Scavenging and Oxidative Damage Protection Activities of Blackberry Flavonoid

基于超声辅助提取技术,采用单因素和正交试验优化黑莓黄酮的提取工艺。试验结果表明:黑莓中黄酮物质的最优提取条件是:温度70℃,提取时间150 min,乙醇体积分数80%,料液比1∶30 g/m L。在此条件下,黑莓黄酮的得率为2.23 mg/g,是优化前的2.01倍。在此基础上研究了黑莓黄酮的自由基清除活性,结果显示:黑莓黄酮的总抗氧化能力等价于14.89μg trolox,且黑莓黄酮清除DPPH自由基、ABTS自由基的IC50值分别为0.57mg/m L,0.49 mg/m L。进一步研究了黑莓黄酮对肝细胞氧化损伤的保护作用,结果表明过氧化氢(H2O2)能引起肝细胞LO2发生氧化损伤(细胞存活率为60.6%),80μg/m L的黑莓黄酮能显著抑制H2O2引发的细胞氧化损伤(细胞存活率为85.04%)。深入探究黑莓黄酮的作用机理发现,H2O2能引发肝细胞LO2中产生大量的活性氧自由基ROS(与正常对照组相比,荧光强度为276.62%);80μg/m L的黑莓黄酮能显著抑制H2O2引发的ROS(与正常对照组相比,荧光强度为139.19%),从而防护肝细胞的氧化损伤。

This study investigated the extraction process of flavonoid from blackberry. According to the single factor and the orthogonal experiment, the optimum conditions for extraction were as follows： water bath at 70 ℃, ultrasonic extracting for 150 min, the material-liquid ratio of 1∶30（g/m L） and the extraction solvent of ethanol of 80%; Under this condition, the flavonoid can be arrived to 2.23 mg/g, which is 2.01 times as optimization before. We then studied the free radical scavenging activities of blackberry flavonoid, and found that total antioxidant capacity of blackberry flavonoid is equivalent to 14.89 μg trolox. Moreover, flavonoid can significantly scavenge DPPH（IC500.57 mg/m L） and ABTS（IC500.49 mg/m L）. We further studied the protection activities of blackberry flavonoid against oxidative damage. We found that H2O2 can induce oxidative damage in LO2 cells（with 60.6% cell viability compared to control）, and blackberry flavonoid（80 μg/m L） significantly inhibited H2O2- induced oxidative damage（cell viability 85.04%）. Finally, we found that H2O2 can induce ROS increase in LO2 cell, with ROS fluorescence intensity 276.62% compared to control. Blackberry flavonoid（80 μg/m L） significantly inhibited H2O2-caused ROS increase in LO2 cells, with ROS fluorescence intensity 139.19% compared to control.