柑橘多甲氧基黄酮硫酸酯代谢产物肠道水解机制研究

Polymethoxyflavones (PMFs) are unique and important flavonoids in citrus, which have various good health-promoting effects. Our previous studies have shown that the non-bioactive conjugated metabolites would be converted to free form with potent health-promoting effects by gut microbiota, and reabsorbed by gut. Therefore, hydrolysis of conjugated metabolites in gut plays a critical important role in the in vivo health-promoting functions of PMFs. However, hydrolysis progress in gut for sulfated PMFs is lack of scientific study, especially the hydrolysis mechanism of sulfated metabolites is not clear. Hence, in this project, typical PMFs compound, nobiletin, will be chosen as a model, and the hydrolysis of sulfated nobiletin metabolites will be deeply explored from three aspects including sulfate hydrolysis gut microbiota, gene and sulfatase. Metabonomics, transcriptomics and flow cytometry are used to elucidate the sulfate hydrolysis gut microbiota; sulfatase coding gene cluster is obtained through whole genome sequencing and chromosome walking technique of PCR, and the gene structure will be analyzed; combined with CRISPR/Cas9, heterologous expression and analysis of sulfatase properties, the hydrolysis activity of sulfatase will be investigated. Hydrolysis mechanism of sulfated PMFs metabolites would be systematically elicited, which will facilitate realizing targeted regulation of the in vivo health-promoting functions of PMFs and other food components with similar metabolic progress. At the same time, the results will also provide important scientific basis and theoretical guidance for the development of nutritional and health-promoting foods.

多甲氧基黄酮（PMFs）是柑橘独有的一类主要黄酮物质，具有多种良好的健康功效。我们前期研究发现，失活的PMFs结合态代谢产物在肠道菌群作用下转化为功效显著的游离态并能被重新吸收。因此，肠道内结合态代谢产物的水解对其健康功效的发挥至关重要，但水解过程缺乏科学研究，特别是硫酸酯代谢产物的水解机制尚不明晰。基于此，本项目以典型PMFs川陈皮素为例，从“水解菌—基因—酶”三个层面深入探索：采用代谢组学和宏转录组学，结合流式细胞分选技术，分离并验证肠道内硫酸酯水解菌；采用全基因组测序和染色体步移PCR技术，获得硫酸酯酶基因簇并对其进行深入解析；采用CRISPR/Cas9技术、异源表达，并通过硫酸酯酶性质分析进一步明确其水解活性。项目将系统阐明肠道内PMFs硫酸酯代谢产物的水解机制，有助于实现PMFs（及具有类似生理过程的食品组分）体内健康功效的靶向调控，为营养健康食品开发提供重要科学依据和理论指导。

柑橘黄酮是重要的膳食黄酮来源，具有多种良好的营养健康功能。我们前期研究发现，柑橘黄酮的体内代谢及其与肠道菌群的相互作用在其体内营养健康功能发挥中具有重要意义。本项目利用小鼠和体外厌氧发酵模型，解析了柑橘黄酮的体内代谢，发现黄酮糖苷经口腔摄入后，难以被吸收，主要存在肠道内；进入大肠后，在肠道菌群的作用下，被水解为苷元、乙酰化、葡萄糖醛酸化等产物，再被进一步水解。以高脂饮食ApoE-/-小鼠为模型，发现了4种主要柑橘黄酮均可以有效缓解小鼠动脉粥样硬化症状，并探讨其构效关系，明确了4种主要柑橘黄酮的动脉粥样硬化缓解功能强弱依次为柚皮苷>橙皮苷>柚皮素>橙皮素，提示糖苷的存在有利于功效的发挥，且4'-位取代基也会显著影响其功效。联合微生物组学、代谢组学、western blot和RT-PCR等技术，首次揭示了柑橘黄酮糖苷主要在肠道内通过调节BSH产生菌的丰度，下调FXR/FGF15基因的表达，进而上调肝脏内CYP7A1蛋白的表达，从而促进胆固醇合成胆汁酸，降低体内胆固醇，发挥动脉粥样硬化缓解功能。利用体外厌氧培养技术，从健康成人粪便中分离多株肠道菌，并对其形态特征进行表征，以期得到参与黄酮代谢的肠道菌，并解析其结构功能。以上研究结果将为柑橘黄酮体内营养健康机制以及靶向调控策略开发提供科学指导，并为其在功能食品乃至个性化营养食品中的开发利用奠定基础。