麦胚清蛋白抗氧化肽的筛选及对细胞氧化损伤的保护作用

Screening of Wheat Germ Albumin-Derived Antioxidant Peptides and Its Protective Activity against Cellular Oxidative Damage

本研究运用超滤与凝胶层析等分离纯化技术逐级分离麦胚清蛋白中性蛋白酶酶解产物,以氧化自由基吸收能力(oxygen radical absorbance capacity,ORAC)与2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid),ABTS)阳离子自由基清除率为评价指标,筛选具有较高抗氧化活性的产物组分,并以该组分为研究对象,利用液相色谱-串联质谱结合Peptide Ranker数据库活性预测技术筛选出具有高抗氧化活性的小肽,并通过高糖诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)及H2O2诱导HepG2细胞与VSMCs建立细胞氧化应激模型,评价小肽对细胞氧化损伤的保护作用。结果表明,经超滤与层析筛选出分子质量较小的V组分抗氧化活性最高,液相色谱-串联质谱鉴定出该组分主要由20个小肽组成,PeptideRanker预测与抗氧化实验验证发现肽序列LNYPPY(765.3 Da)抗氧化活性最高(ORAC 3.01μmol TE/μmol)、ABTS阳离子自由基清除率(95.58%)。肽序列LNYPPY对H2O2诱导的HepG2细胞和VSMCs氧化损伤具有很好的保护作用,但对高糖诱导的VSMCs异常增殖以及活性氧水平上升无明显影响。综上,肽LNYPPY具有良好的体外抗氧化活性,且对H2O2诱导的细胞氧化损伤具有保护作用,研究可为麦胚清蛋白的开发利用提供重要的理论参考。

In this study,neutral protease enzymatic hydrolysates of wheat germ albumin were separated step by step using ultrafiltration and chromatography techniques.Oxygen radical absorbance capacity(ORAC)and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid(ABTS)cation radical scavenging capacity were used as antioxidant activity evaluation indicators for selecting hydrolysate components with high antioxidant activity.The selected component was served as the initial material to further screen small peptides with high antioxidant activity by liquid chromatography-tandem mass spectrometry(LC-MS/MS)combined with PeptideRanker database activity prediction technology.High glucose-induced vascular smooth muscle cells(VSMCs)and H2O2-induced HepG2 and VSMCs were served as oxidative stress injury models to explore the protective effect of obtained small peptides.Results:Component V with small molecular weight obtained by ultrafiltration and chromatography had the highest antioxidant activity.Twenty small peptides were identified with LC/MS-MS analysis from this fraction.PeptideRanker prediction and antioxidant experiment validation showed that the peptide sequence LNYPPY(765.3 Da)had the highest antioxidant activity(ORAC was 3.01μmol TE/μmol),and ABTS cation radical clearance rate was 95.58%).This peptide had a good protective effect on the H2O2-induced oxidative damage in HepG2 cells and vascular smooth muscle cells(VSMCs),but had no significant effect on the abnormal proliferation and the increase of reactive oxygen species(ROS)levels in VSMCs induced by high glucose.In conclusion,the peptide LNYPPY has good antioxidant properties in vitro and has protective effect against cellular oxidative damage induced by H2O2,which provides an important theoretical reference for the development and utilization of wheat germ albumin.