龙须菜中蛋白酪氨酸磷酸酶1B抑制剂的靶向追踪研究

Protein tyrosine phosphatase 1B (PTP1B) is an accepted drug target for diabetes and obesity. Discovery for highly effective inhibitors of PTP1B has a promising application in diabetes and obesity therapy. Our prophase research showed that the extracts of Gracilaria lemaneiformis have the PTP1B inhibitory activities. Based on the results of previous research, this study is to discover the PTP1B inhibitors from Gracilaria lemaneiformis by bioassay-guided separation method in vitro and in vivo. This method will compare the difference between incubation solution and blank solution of extracts or the incubation solution and blank solution of metabolites by LC-MS. To establish the rational separation method, the properties of target compounds, such as molecular weight, chromatography retention and partition coefficient, can be determined using small amount of mixed sample, and the active compounds will be separated by combination chromatography efficiently. The structures can be identified by MS and NMR, and the activity of the compound will be tested. This research will discover the highly active PTP1B inhibitors and lay a foundation for further study of their function and molecular mechanism, it also can establish a method for rapid acquisition of the active compounds from natural products.

蛋白酪氨酸磷酸酶1B（PTP1B）是公认的治疗糖尿病和肥胖症的药物作用靶点，寻找其高活性抑制剂对于糖尿病和肥胖症等疾病的治疗有着广阔的应用前景。在前期工作中发现龙须菜提取物具有抑制PTP1B的活性，在此基础上，本项目拟通过体内外靶向追踪的方法，利用LC-MS检测提取物与靶蛋白的孵育液和空白样品（即原提取物）中化学成分的差异，及体内代谢产物与靶蛋白孵育液和空白样品（即原代谢产物）中化学成分的差异，发现具有PTP1B抑制活性的化合物。通过少量混合样品对目标化合物的性质（分子量、正反相液相色谱保留、分配系数等）进行测定，合理制定分离方法，通过组合色谱的方法对活性物质进行高效分离，并通过质谱、核磁共振等方法鉴定其结构，并进行活性验证。通过本项目的研究不仅可以从龙须菜中发现高活性的PTP1B抑制剂，为其进一步的功能、分子机制研究奠定物质基础，而且可以建立快速获取天然产物中的活性化合物的方法。

龙须菜Gracilaria lemaneiformis具有悠久的食用和药用历史，具有降糖潜质，但是其降糖物质基础研究相当匮乏，有效成分不明确，针对此现状，本项目采用蛋白酪氨酸磷酸酶1B（PTP1B）为靶点，对龙须菜中的降糖物质基础展开了靶向追踪研究，主要从三个方面展开：a. 龙须菜中化学成分提取和活性筛选；b. 龙须菜活性部位的体内外靶向追踪研究；c. 多种色谱法相结合的龙须菜活性部位的分离研究。并取得了以下结果：a. 筛选得到了正己烷部位为最佳活性部位；b. 通过靶向追踪研究从正己烷中发现并分离得到了一个具有PTP1B抑制活性的化合物；c. 通过多种色谱方法联合使用，从正己烷部位分离得到了四个具有PTP1B抑制活性的化合物。本项目建立了龙须菜中蛋白酪氨酸磷酸酶抑制剂的靶向追踪研究方法，为龙须菜的进一步深入的结构与功能、分子机制研究奠定物质基础，也为龙须菜的进一步开发提供了理论依据。