陈皮黄酮抑制肠道菌群TMAO生成及预防动脉粥样硬化机理研究

Recent research has proved that human microbiota can metabolize L-carnitine, existing in red meat, to trimethylamine (TMA), which in turn is oxidized to trimethylamine oxide (TMAO) by liver enzyme flavin-containing monooxygenases (FMOs). TMAO has been shown to promote atherosclerosis and subsequent development of heart disease. Antibiotics has shown effective inhibition of TMA and TMAO production in vivo, however, they have much detrimental property for long term usage. Therefore, it is important and pivotal to explore the mechanism of inhibiting TMAO production in human microbiota with food components, which will serve as a foundation for further research and exploration in functional foods. The aim of this study is to investigate the inhibition mechanism of TMA and TMAO in mice microbiota by nobiletin and aged-tangerine peels, which contain multiple polymethoxyflavones and nobiletin is a major representative. We will use a C57BL/6 male mouse model feeding with L-carnitine to mimic the diet of ominivores to induce the elevated TMA and TMAO levels in vivo. Then we will attempt to inhibit the production of TMA and TMAO in mouse microbiota with nobiletin and citrus peel flavonoids to establish the mechanism based relationship between microbiota adjustment and functional food ingredients. We will also study the details of microbiota environment changes caused by nobiletin and citrus peel flavonoids and to elucidate the mechanism of L-carnitine (red meat) induced TMA production in mouse microbiota including the study of the elevated oxidative potential in microbiota in L-carnitine challenged mice. The ultimate goal of this study is to prove the theory that food ingredients such as nobiletin and mixed flavonoids from aged tangerine peel can have adjustable effect on microbiota and also can further inhibit the production of TMA and TMAO from meat consumption to reduce and prevent the occurrence of heart disease such as atheroscerosis.

随着人们生活节奏的加快和生活水平的提高，以肉类为主导的饮食方式日益普遍，由此引发的动脉粥样硬化和肥胖等代谢综合症已成为必须重视的健康主题。最近研究证明，红肉饮食后，肠道菌群代谢其中的左旋肉碱（L-Carnitine)，生成三甲胺（TMA）,三甲胺经肝脏氧化后生成氧化三甲基胺（TMAO）， TMAO 是诱发动脉粥样硬化的独立因素，针对性地开发有效控制TMAO产生的保健手段是当务之急。我们预实验发现药食两用食材-陈皮及其主要活性成分陈皮黄酮可以改变肠道菌群的构成、降低血液中的TMAO水平和减轻体重。因此本项目拟利用已建立的陈皮黄酮分析分离体系及生物活性发现平台，用左旋肉碱喂食小鼠模拟食肉饮食，探讨陈皮及其黄酮组分如何与肠道菌群相互作用，以及这种相互作用模式对TMAO产生的影响，从而在理论上揭示陈皮及其功能组分预防动脉粥样硬化的机理，为基于陈皮及其活性成分为基础的补充膳食开发提供依据。

课题以药食两用食材陈皮及其主要黄酮成分川陈皮素为基础旨在明确其能否抑制肠道菌群TMAO生成及预防动脉粥样硬化及其机理。实验主体结果总结如下：（1）利用不同分离方法，高效分离、鉴定了陈皮黄酮及其组成，成功大量分离了川陈皮素等单体。确定了准确快速分析陈皮黄酮的高效液相色谱、高效逆流逆流色谱分析方法，并对14种国产陈皮及橘柑皮中9中陈皮黄酮的单体含量进行了准确的检测和定量分析，为陈皮和橘柑皮的黄酮与活性关系的测定与阐明奠定了基础。（2）在上述分离获得陈皮黄酮单体的基础上，我们利用酵母模型比较了陈皮多甲氧黄酮nobiletin和tangeritin和它们的5-去甲基衍生物的抗氧化能力及其机制。我们发现，这些陈皮黄酮组分都能够有效拮抗膜脂质过氧化和清除细胞内过多的氧自由基，从而发挥细胞保护作用。此外，5-去甲基衍生物在抗氧化能力方面要优于对应的nobiletin和tangeritin。机理上，还原酶Ctt1 和GSH是这些陈皮黄酮抗氧化作用的主要靶点。（3）在明确陈皮黄酮抗氧化机制的基础上，我们利用左旋肉碱摄入啮齿类动物动脉粥样硬化模型探讨陈皮黄酮（以川陈皮素为代表）的心血管防护作用。我们以肠道菌群的调节和TMA/TMAO为主要关注指标，证实陈皮及其黄酮组分能够有效地预防心血管炎症和动脉粥样硬化，陈皮及其黄酮能有效的改善左旋肉碱摄入所致的啮齿类动物肠道菌群构成改变，降低体内TMA和TMAO含量，抑制血管内皮细胞的过度氧化应激，从而有效地预防心血管炎症（动脉粥样硬化）。.综上所述，本项目证实了陈皮黄酮组分调节肠道菌群、抑制肠道菌群代谢产生TMA和TMAO的机理，提供陈皮中黄酮组分的保健功能理论依据，在机理研究的基础上，系统深入研究功能食品的组分与健康保健关系，例如陈皮黄酮与其预防动脉粥样硬化和预防肥胖功能。本项目的相关成果将为有效开发功能食品提供理论指导，为寻找调节肠道菌群、有效抑制肠道菌群产生TMA和TMAO的食物组分奠定理论基础，同时为未来陈皮功能食品产品开发提供科学依据。本项目相关成果除可指导人们合理饮食，达到通过膳食来增强体质和预防疾病的目的，还将对提升我国优势植物资源的转化和深加工利用水平也将产生深远意义。