基于代谢组学与血清药物化学整体辨识波棱瓜子抗胆汁淤积药效物质基础及其作用机制研究

Seeds of Herpetospermum caudigerum Wall are widely used as an effective Tibetan folk medicine “Bo-ling-gua-zi” for treatment of liver and gallbladder diseases, which is similar to “Chi-ba” disease in traditional Tibetan medicine. In our previous study, it was found that ethyl acetate fraction and petroleum ether fractions from the seeds of H. caudigerum exhibited a dose-dependently protective effect on ANIT-induced liver injury in cholestatic rats. However, the marital basis is still unclear. On the basis of our substantial work previously, under the guidance of the theory of Tibetan Medicine and the metabolic process of drugs in vivo, an integrative study on identifying the material basis of H. caudigerum by combined method of metabolomics and serum pharmacochemistry is proposed. After systemic isolation and identification of the chemical constituents of H. caudigerum seeds, modern analytical method, such as UPLC-QTOF-MS is applied in the metabolomics study to detect the potential biomarkers from rat blood, urine and liver tissue. And their metabolic pathways are suggested. Drug metabolites in the levels of blood absorb will also be analyzed by means of liquid chromatography combined Mass method, which will be correlated with biomarkers to obtain the potential active constituents in vivo by means of informatics analysis. After the marital basis in vitro is obtained and its anti-cholestasis in ANIT-induced cholestatic rats is confirmed, the primary mechanisms against anti-cholestasis will be studied via FXR and Nrf2/ARE signaling pathways. According to understand the intrinsic link of “disease-biomarkers- in vivo active constituents- in vitro material basis”, the therapeutic compounds with clear mechanisms in H. caudigerum for treatment of cholestasis can be clarified overall.

波棱瓜子藏医临床用于治疗“赤巴病”（部分相当于胆汁淤积）疗效确切。课题组前期也证实其能显著改善ANIT诱导的大鼠急性肝内胆汁淤积，但药效物质基础不明。本项目结合藏医药理论，提出基于代谢组学与血清药物化学方法整体辨识波棱瓜子药效物质基础的研究思路。拟在对化学成分深入研究基础上，运用UPLC-QTOF-MS等手段，通过代谢组学分析大鼠血液、尿液、肝组织生物标志物及其代谢通路；通过血清药物化学分析鉴定药物体内入血成分；将入血成分与生物标志物相关联获得体内效应物质；追踪制备其对应于药材的潜在药效物质基础并进行药效学验证；在此基础上探讨药效物质基础通过调控核受体FXR和Nrf2/ARE通路共同作用的机制。本研究实施可揭示波棱瓜子抗胆汁淤积“疾病-生物标志物-体内效应物质-药效物质基础”内在联系，阐明体现藏医药特点、化学成分明确、疗效确切、机制清楚的波棱瓜子抗胆汁淤积药效物质基础。

胆汁淤积是由于胆汁生成障碍或胆汁流动及代谢障碍所导致的肝脏病理症状。目前临床上抗胆汁淤积的药物仍然十分缺乏。波棱瓜子（Herpetospermum caudigerum Wall 或Herpetospermum pedunculosum (Ser.) C.B. Clarke）藏医临床用于治疗“赤巴病”（部分相当于胆汁淤积）疗效确切，课题组发现其能显著改善ANIT诱导的大鼠急性肝内胆汁淤积，但药效物质基础不明。本项目整合了基于体内代谢过程的药物血清化学方法、中药化学、中药药理学、药代动力学等技术方法，研究波棱瓜子的抗胆汁淤积的药效物质基础。主要研究内容及结果如下：（1）分别考察了波棱瓜石油醚部位（PEE）和乙酸乙酯部位（EAE）抗胆汁淤积活性，其中乙酸乙酯部位具有更好的抗胆汁淤积作用；（2）利用GC-MS分析了PEE成分，鉴定出49个化合物，主要为长链不饱和脂肪酸类物质；利用LC-MS分析了EAE成分，鉴定了14个化合物，主要是木脂素类；（3）应用LC-MS从EAE含药血浆中检测到4个原型成分，胆汁中检测到5个原型成分，尿液中检测到9个原型成分，粪便中检测到5个原型成分；对EAE不同入血时间的体内成分也进行了分析；（4）从波棱瓜中共分离鉴定了132个化合物，包括波棱瓜子中分离鉴定的88个化合物（60个木脂素，24个新化合物）和茎藤中44个化合物（22个木脂素，5个新化合物）；（5）利用高内涵成像对分离到的木脂素类化合物进行FXR激动活性筛选，14个单体化合物表现出显著FXR激动活性；（6）以活性最好的2个木脂素波棱甲素（HPE）与去氢双氢伯醇（DA）开展抗胆汁淤积研究，DA和HPE均能显著逆转ANIT引起的大鼠肝内胆汁淤积，是波棱瓜子发挥疏肝利胆和保护胆汁淤积引起的肝损伤的活性成分，作用机制是通过激动FXR受体，进而调控其下游靶蛋白的表达来抑制胆汁酸的合成，促进胆汁酸的转运减轻胆汁淤积；（7）应用LC-MS等对波棱内酯I与hepetotriol进行药代动力学研究，拟合药时曲线、计算药动学参数、分析组织分布情况与推导体内代谢途径。本项目揭示了波棱瓜子木脂素类成分，尤其是HPE与DA是其主要药效物质基础，可通过激活FXR调节下游基因发挥抗胆汁淤作用。本项目的完成基本阐明了波棱瓜子抗胆汁淤积药效物质基础，对于将波棱瓜子开发为治疗胆汁淤积的创新中药奠定了重要基础。