基于Caco-2细胞的远东拟沙丁鱼蛋白多肽抗氧化活性研究

Studies on Antioxidant Activity for Polypeptide from Sardinops sagax Based on a Caco-2 Cell

为探究远东拟沙丁鱼蛋白多肽在细胞中的抗氧化活性(CAA),以荧光探针DCFH-DA被氧化成的二氯荧光素(DCF)的荧光值为指标,检测细胞中的活性氧含量,通过检测添加物蛋白多肽对Caco-2细胞内活性氧的清除能力,来评估其抗氧化活性。首先对Caco-2细胞的接种量、培养时间和DCFH-DA的培养浓度及时间进行优化,研究不同质量浓度的远东拟沙丁鱼蛋白多肽对Caco-2细胞抗氧化活性的影响。结果显示,Caco-2细胞的最佳接种量为5×105个/m L;DCFH-DA的最佳浓度为100μmol/L,最佳培养时间60 min,整个试验在细胞接种3 d内完成最佳;远东拟沙丁鱼蛋白多肽的质量浓度为200μg/m L,其细胞抗氧化活性的最强。试验结果表明:远东拟沙丁鱼蛋白多肽具有一定的抗氧化活性,可作为功能性食品配料应用于抗氧化保健品中,为其开发与高值利用提供了一条新途径。

Studies on antioxidant activity for polypeptide from Sardinops sagax based on a Caco-2 Cell. A dichlorofluorescein diacetate（DCFH-DA） is probed in the cell and oxidized to fluorescent compound 2′, 7′-dichlorofluorescin（DCF） by reactive oxygen induced by 2,2-azobios-（2-amidinopropane） dihydrochloride（AAPH）. With fluorescence value as index, reactive oxygen content in Caco-2 cell was measured. In the present of the sample, its antioxidant activity was evaluated by determination of the intracellular reactive oxygen scavenging ability. In the study, the best conditions,such as Caco-2 cells seeding density, cell culture time, concentration and time of DCFH-DA were optimized. Results showed that the optimal experimental conditions were seeding density of 5 ×10^5 well/mL, cell culture time of 3 d, 100μmol/L DCFH-DA in Caco-2 cells for 60 min. Based on the above optimal conditions, scavenging reactive oxygen ability of the different mass concentrations of the polypeptide from Sardinops sagax was investigated. Its antioxidant activity was the best at concentration of polypeptide of 200 μg/mL. Therefore, polypeptide from Sardinops sagax has certain antioxidant activity, can be used as functional food ingredients in antioxidant supplements, which is provides a new way for its high-used and development.