炒制麦芽中酚类物质释放特征及其调节肠道上皮细胞氧化应激作用机理的研究

Overnutrition leads to the disorder of intestinal metabolism, which causes excessive accumulation of free radicals in intestinal. Then, excessive free radicals induce enteritis, intestinal cancer, and so on. Barley malt products have antioxidant activity and could regulate the disorder of intestinal metabolism. However, it is not scientific to demonstrate that fried and dark barley malt have stronger functions of regulate the disorder of intestinal metabolism than that of raw barley malt from amylase. We found that the content of phenolics from barley malt was increased and the cellular antioxidant activity and the inhibition of colon cancer cells proliferation were enhanced. Phenolics play important role in regulating intestinal epithelial cells oxidative stress. Due to the ingredients of barley malt and the degradation products of phenolics under thermal processing are complex, the feature of phenolics degradation from barley malt during stir-frying, and the mechanism of phenolics regulates intestinal epithelial cells oxidative stress are blurry. In the study, we would employ High Performancee Liquid Chromatography-Mass Spectrometry to analyze free and bound phenolics compounds kinds, respectively. Then, comparing the mass spectrograph and statistically analyzing the data, the results would qualitatively and quantitatively explain the changes of ingredient and contents of phenolics degradation of barley malt during stir-frying. Then, we would analyze the kinetic changes of symbolictic phenolics and interaction with others components during stir-frying in macromolecular model. The results could explain the feature of phenolics release from barley malt and interaction between components during stir-frying. And the effects of barley malt phenolics on the antioxidant protease activities and expression of antioxidant proteins would be performed in vitro intestinal epithelial cell oxidation stress model. The results would explain the mechanism of phenolics regulate intestinal epithelial cells oxidative stress. Furthermore, this project would enrich the theory of precise nutrients from fried barley malt products.

营养过剩导致肠道代谢紊乱，诱使肠道自由基积累过多，诱发肠炎、肠癌等健康问题。麦芽具有调节肠道代谢紊乱的营养作用，仅以淀粉酶不能科学的解释炒制麦芽比生麦芽调节肠道代谢紊乱功效强的作用机理。申请人发现炒制提升麦芽酚类物质含量、细胞抗氧化活性和抑制结肠癌细胞增殖能力，酚类物质对调节肠道氧化应激具有重要影响，限于麦芽成分的复杂性和热加工下酚类物质的不确定性，炒制麦芽中酚类物质释放特征及其调节肠道上皮细胞氧化应激机理尚未清晰。本申请拟采用液质联用高通量技术分析炒制麦芽中游离态、结合态酚，通过谱图对比、统计分析和大分子模型，定性、定量的分析酚类物质种类和含量变化特征、热动力学变化、组分间相互作用，揭示炒制麦芽中酚类物质降解释放特征。采用肠道上皮细胞氧化应激模型，研究麦芽酚类物质对氧化还原酶活性及相关蛋白表达的影响，阐释调节肠道上皮细胞氧化应激的物质基础和作用机理，为探索炒制麦芽精准营养成分提供依据。

营养过剩导致肠道代谢紊乱，导致肠道自由基过多，诱发肠炎、肠癌等问题。麦芽具有调节胃肠道代谢紊乱的作用，本项目以种不同温度处理的生麦芽、炒麦芽、焦麦芽三种为研究对象，针对不同温度处理后麦芽对人体胃肠道有不同功效的现象，探索麦芽中酚类物质在炒制作用下释放特征及其调节肠道氧化应激的物质基础和作用机制，从化学成分特征、活性成分筛选、体外功效作用学等方面开展了系统研究探索。采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Orbitrap HRMS)对结合多酚成分进行鉴定及分析，鉴定出46种物质；以筛选出的12个差异性成分构建18×12数据矩阵，利用该差异性成分的数据矩阵建立麦芽及其炒制品的PCA鉴别模型，并选择标志性成分阿魏酸、异阿魏酸、香豆酸和ECG验证其对Caco-2细胞氧化应激的调控作用，发现其对AAPH诱导的氧化应激模型具有调控作用。并选择麦芽中含有的三种绿原酸衍生物，采用体外酶法、细胞模型及分子模型计算手段对其调控肠道代谢酶的作用，结果显示，对α-葡萄糖苷酶抑制活性依次为异绿原酸A >绿原酸> 洋蓟酸；荧光分析表明，它们以1:1的摩尔比与α-葡萄糖苷酶形成络合物，发挥抑制作用，异绿原酸A的结合能力最强，其次为绿原酸和洋蓟酸；分子对接结果明确了咖啡基分布对α-葡萄糖苷酶抑制活性的影响；在HepG2细胞中，异绿原酸A也表现出最佳的葡萄糖消耗，细胞毒性可忽略不计，这可能与其在细胞内清除活性氧的能力有关。采用Osborne法分别从三种麦芽中直接提取多酚-蛋白复合物及除去酚之后的蛋白质，通过对比三种麦芽中不同提取物中总酚含量、可溶性蛋白质含量、蛋白质相对分子量，初步探讨炒制对麦芽中多酚-蛋白复合物的互作的影响，研究结果表明，麦芽中酚类物质对麦芽的生物活性具有重要的贡献。