广西甜茶调节脂代谢紊乱的物质基础及作用机制研究

Dyslipidemia is a major risk for hyperlipidemia, type 2 diabetes, metabolic syndrome and other major diseases of human factors, has become the biggest threat to human health. Leaves of Rubus suassimus, has multiple function, including “medicine, tea and sugar". However, the active substances as well as their mechanisms underlying are not clear. The project takes leaves of R. suavissmus as the research object, tries to establish of high fat diet-induced lipid metabolism disorders golden hamster model, the biological samples of animals will be analyzed before and after administration of R. suavissmus, respectively, through metabolomics research methods from endogenous metabolites level to screen the targets related to lipid metabolism pathway, and confirms by the results of biology research results. Combined with the determination of tissue structure, physiological and biochemical indexes, try to explore the action mechanism of R. suavissmus for lipid regulating effects from the whole animal, cell and molecules levels. Using the traditional phytochemistry research strategy to obtain different parts and compound monomer of R. suavissmus. Using the live cell model to guide the separation and purification of active compounds, and explore the action mechanism of active compounds by molecular biology method. Establish the HPLC fingerprint methods of R. suavissmus, and identify the quantify the major constituents of it; Combines the screening results of cell model and plasma pharmaceutical chemistry together to find out and identify the effective substances of R. suavissimus for regulation of dyslipidemia. The results will provide a basis for the further development and utilization of R. suavissmus.

脂代谢紊乱是高脂血脂、2型糖尿病、代谢综合症等人类重大疾病的主要危险因素，已成为人类健康的最大威胁。集“药、茶、糖”三重功效于一身的广西特色植物-甜茶具有调节脂代谢紊乱效果，但是药效物质基础和作用机理不明。本项目拟以甜茶为研究对象，采用高脂饮食建立脂代谢紊乱金黄地鼠模型，对给予甜茶前后动物模型生物样品进行分析，通过代谢组学从内源性代谢物筛选与脂代谢有相关的作用靶点并结合分子生物学的测定结果验证甜茶对脂代谢通路的变化规律,结合组织结构和生理生化指标的测定，从整体、分子和细胞水平探讨甜茶调节脂代谢紊乱的作用机制。采用传统提取分离方法，获得甜茶的不同部位和化合物单体,同时采用肝细胞模型指导化合物的分离纯化鉴定，并探讨其作用机制；建立指纹图谱分析方法，鉴定和测定其主要成分；结合活性筛选与血清药物化学研究结果，阐明甜茶血脂调节的物质基础。研究结果对进一步开发广西甜茶资源奠定了基础。

（1）采用高脂饮食金黄地鼠模型，对给予广西甜茶、甜茶素的生理生化指标进行了检测，明确了广西甜茶和甜茶素对脂代谢紊乱的调节作用。（2）采用1H NMR代谢组学方法，对不同组别金黄地鼠的血液样品进行分析，通过代谢组学从内源性代谢物筛选其质量标志物，并通过代谢通路分析明确其影响的代谢通路。（3）从分子生物学验证广西甜茶调节脂代谢的作用机制：通过影响脂质合成与代谢关键通路PPAR/SREBP上的相关基因转录和蛋白的表达，参与脂肪分化、脂肪酸生物合成和脂质代谢调节过程，改善脂代谢紊乱。（4）采用传统的化学分离方法获得广西甜茶的不同极性部位。同时采用3T3-L1模型，追踪其活性部位和成分，发现甜茶素单体、乙酸乙酯和正丁醇部位可促进3T3-L1细胞的分化，采用UPLC-QTOF-MS/MS技术对甜茶乙酸乙酯和正丁醇活性部位中的化学成分进行表征，步鉴定了50个化合物，其中乙酸乙酯部位47个，正丁醇部位23个，两个部位共有化合物20个。进一步采用系统化学分离方法，从乙酸乙酯和正丁醇部位共分离得到16个化合物，鉴定了其中的13个化合物。（5）采用UPLC-QTOF-MS/MS技术建立了广西甜茶指纹图谱分析方法，鉴定和推测了66个化合物。建立了甜茶UPLC含量测定的方法，测定了不同生长年限和采收时间甜茶中6个主要成分、总黄酮及总多酚的含量，评价了生长年限和采收时间对甜茶化学成分的影响。（6）开展体内代谢研究，大鼠灌胃分别给予甜茶和甜茶素后，收集血浆、尿、粪便及胆汁样品，以UPLC-QTOF-MS技术检测和分析了甜茶和甜茶素在大鼠体内的化学成分原形及代谢产物。同时开展了甜茶素的体内药代动力学研究。阐明了主要甜味和活性成分甜茶素在大鼠体内的主要代谢途径和方式。