基于代谢组学技术的连钱草治疗胆固醇结石药效物质基础及作用机制研究

As the improvement of living standards, the incidence of cholesterol gallstone (CG) showed a rising trend year by year. High recurrence rate, residual stone rate and complications were the clinical dilemma in hepatobiliary surgery. Therefore, an urgent need for new drug for treatment of CG. Glechoma longituba has significant therapeutic efficacy of CG. But the material basis of its efficacy and action mechanism are not clear. The purpose of the present project was to use the in vivo CG model, metabolomics approach and knockout-fishing/spectral efficacy integration and characterization method for establishment of the integrated identification strategy of active chemical group based on the efficacy weighting, which contributes to pick out the active constituents in Glechoma longituba. In the meantime, the project discloses the chemical profile and in vivo metabolism profile of Glechoma longituba, and ultimately the quality control method based on the efficacy weighting was constructed for Glechoma longituba by summing the values of the product for the efficacy weighting and the corresponding concentrations of all active compounds. The study focus on cholesterol transportation and metabolism and gallbladder dynamics, the key point of gallstone formation. the mechanism research of Glechoma longituba on cholesterol gallstones is comprehensively studied. Ultimately providing strong scientific evidence for quality control and clinical application of Glechoma longituba.

随着我国人民生活水平的提高，胆固醇结石发病率呈不断上升趋势，目前手术治疗的高复发率、残石率、并发症一直是肝胆外科临床难题，因此寻找新的治疗药物备受关注。连钱草具有显著的治疗胆固醇结石功效，但其药效物质基础和作用机制尚不清楚，本项目拟基于已有工作基础，利用胆固醇结石模型、代谢组学技术和成分缺失-捕获/谱效集成表征技术，建立基于活性贡献指数的连钱草有效成分群的识别体系，全成分表征连钱草体外化学成分谱和体内代谢谱，开展连钱草多成分整合药代动力学研究，通过桥接有效成分的活性强弱和浓度而构建基于药效权重的连钱草质量控制方法，在胆固醇结石形成中的关键环节--胆固醇转运代谢和胆囊动力学变化上对连钱草治疗胆固醇结石作用机制系统阐述，为连钱草的质量控制和临床应用提供科学依据。

本研究采用HPLC-MS技术对连钱草70%乙醇提取物的化学成分进行定性定量分析，共鉴定了120个化学成分，包括31个酚酸类化合物、77个黄酮类成分和10个没食子酸类化合物，其中65个化合物首次在连钱草中发现，揭示了连钱草的化学物质基础。应用UHPLC-MS/MS技术对连钱草70%乙醇提取物中15个主要成分进行了多成分定量研究，迷迭香酸的含量最高，为连钱草的质量控制提供了快速、有效的分析方法。在明确化学成分的基础上，基于UHPLC-QTOF-MS/MS技术结合诊断离子和中性丢失策略研究连钱草70%乙醇提取物中酚酸类和黄酮类成分体内代谢，在血浆、尿液、胆汁和粪便中鉴定了13个原型化合物和78个代谢产物，其中82个代谢产物经尿液排出体外，19个代谢产物经胆汁排出体外，仅有4个代谢产物经粪便排出，代谢产物主要来自迷迭香酸、木犀草素和芹菜素的葡萄糖醛酸化、甲基化和硫酸酯化。连钱草70%乙醇提取物和迷迭香酸可降低C57BL/6J小鼠胆固醇结石形成率，降低血清TG、TC、LDLC 浓度，升高HDLC溶度，调节脂质代谢，同时降低HMGCoA还原酶和NPC1L1的表达。本项目初步揭示了连钱草抗胆固醇结石作用的科学内涵，为连钱草的资源开发和利用提供了科学依据。