基于计算机模拟和实验研究探索类胡萝卜素和酚类化合物的抗氧化协同作用机理

The mixtures of phytochemicals with suitable ratios in diets were effective to enhance the synergistic antioxidant effects and have protective effects on human health. However, the complex phytochemical combinations with different ratios showed significant differences of antioxidant activity. For now, less studies were focus on and could well explain the synergistic antioxidant mechanism of phytochemicals (especially with different ratios). It has been shown that the synergistic antioxidant activity may be related to the changes of reaction energy and phytochemical structures, which were caused by the molecular interactions. In this study, the synergistic antioxidant effects of several phytochemical standards (carotenoids and phenolics, with different ratios) were evaluated by the cell-based antioxidant activities (CAA). H2O2 was used in CAA model to induce the free radicals such as •OH, •O2 , HOO•, and the combinations with great synergistic antioxidant effects were therefore picked up by CAA and CI (combination index). Moreover, the structure and the ratio of phytochemicals in cells was also measured by LC-MS for the molecular simulation analysis. The changes of molecular energy (kinetic energy, potential energy, reactive energy, etc.) of the reaction were carried out by the molecular dynamics simulation. Then, the quantum chemistry was used to measure the changes of phytochemical chemical structures. Finally, the in vitro chemical based antioxidant activity were investigated to verify the mechanisms in terms of synergistic effects, energy and structural changes. The main objective of this study is therefore to reveal the in vitro chemical based synergistic antioxidant mechanisms via the molecular interactions in different carotenoids and phenolics. The results would be used to explain the antioxidant interactions of phytochemicals with different ratios as well as the synergistic effects of antioxidants in diets.

从果蔬中摄入的天然植物化学物常以多组分的形式存在，并多有抗氧化协同作用。但植物化学物复配比例的不同会直接影响其抗氧化效果，且至今尚无系统理论能解释这种增效现象。抗氧化化学协同作用的本质是分子间相互作用引起的能量和结构变化。本项目拟以类胡萝卜素和酚类化合物为研究对象，用H2O2诱导细胞产生自由基，结合细胞内抗氧化能力和CI联合指数评价方法，筛选出具有协同作用的植物化学物复配组合。并根据这些组合在细胞内的比例，采用分子动力学模拟和量子化学分析，研究它们与自由基反应过程中能量和结构的变化规律。最后，将植物化学物以细胞内的比例与自由基进行体外化学反应，以实际能量和结构变化验证分子模拟的理论结果。本项目拟从分子间相互作用的本质，解释植物化学物与自由基的体外化学反应过程，旨在揭示不同比例复配后植物化学物的抗氧化化学协同作用规律及机理，为诠释膳食中不同抗氧化组分之间的增效现象提供理论指导。

番茄红素、叶黄素、绿原酸和飞燕草素是食品中含量丰富且分布广泛的抗氧化物质，具有抗氧化、抗炎症、降低心血管疾病等多种疾病的发病率等功能。本研究以上述四种天然植物化学物为研究对象，采用ABTS和DPPH两种体外化学实验方法，分析四种植物化学物的体外抗氧化相互作用，采用H2O2诱导H9c2大鼠心肌细胞建立氧化损伤模型，研究复配前后对H9c2细胞氧化损伤的保护作用。在此基础上，研究番茄红素和叶黄素的细胞内吸收与降解,并基于Nrf2通路分析植物化学物的抗氧化相互作用机制。并采用量子化学密度泛函理论(DFT)，通过Chemdraw 2D和3D软件构建合适的分子模型，运用高斯软件得到键长、红外光谱、前线轨道能量等结构特征和分子能量参数；通过计算这四种化合物与羟基自由基、超氧阴离子自由基、氢过氧自由基反应的过渡态能垒，表征了四种化合物的不同位点的反应活性，为阐明植物化学物抗氧化相互作用机制和开发相应的功能食品提供理论依据。主要研究内容及结果如下：.1、四种天然植物化学物以不同比例复配后，采用CAA法和联合指数法共同测定其抗氧化相互作用。结果表明，四种天然植物化学物通过直接降低H9c2细胞ROS水平发挥抗氧化相互作用。在不同的酶体系中，显著协同、拮抗组的抗氧化酶活性与抗氧化理论值有所不同。.2、通过高液相色谱探究植物化学物组合中另一组分对番茄红素和叶黄素的影响。结果表明，植物化学物组合的抗氧化相互作用也会受细胞吸收率的影响。.3、根据密度泛函理论，计算了植物化学物与超氧阴离子自由基和氢过氧自由基在水、甲醇溶剂中的反应焓值。结果表明，飞燕草色素、天竺葵色素和超氧阴离子反应后，负电荷从自由基转移到了半醌结构上。β-胡萝卜素和番茄红素在苯溶剂中最易与超氧阴离子自由基发生反应，而在水溶剂中比较容易和氢过氧自由基发生反应。.4、通过计算可能的活性位点与羟自由基的过渡态反应，结果表明，过渡态反应所需的活化能与其键长的计算结果是一致。键长越长，该键越容易发生抽氢反应从而生成水。

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