基于HPLC-TPMT EAD-MS联用技术的天芪降糖胶囊中TPMT酶亲和活性成分研究

Tianqi Jiangtang (TQ) capsule, a traditional Chinese medicine (TCM) preparation containing ten herbal medicines, has been commonly utilized for early type 2 diabetes and its complications treatment in China. Our previous work indicated that the thiopurine S-methyltransferase (TPMT) gene was significantly associated with the anti-diabetic effect of the drug. TPMT is an enzyme that catalyzes S-methylation of thiopurine drugs. TPMT activity is associated with the clinical efficacy and excessive toxicity of these drugs. However, there is no relevant report about TPMT, metabolism of traditional Chinese medicines (TCMs) and diabetes mechanism research. In the present study, a new HPLC-TPMT EAD-MS hyphenated technique will be established for rapid screening and identification of TPMT substrates in order to quickly define the active constituents in TQ capsule. HSCCC will be applied for separation of the active constituents of TQ capsule. A quickly screening and preparation method will be developed for TPMT substrates in TCM prescription based on HPLC-TPMT EAD-MS/HSCCC. Modern spectroscopy and chemical method will also be used to identify the structure of the active compounds. Establishing a rapid screening and analyzing approach for complex material base research of TCM basing on targeted bioactive compounds and metabolite analysis.

天芪降糖胶囊（TQ）由黄芪、天花粉等十味中药组成，主要用于2型糖尿病早期及并发症的干预治疗。我们前期对其进行临床基因芯片研究表明，药物代谢酶硫代嘌呤甲基转移酶（TPMT）与该药抗糖尿病作用有关。TPMT是一种催化硫嘌啉类化合物甲基化的酶，参与硫嘌啉类药物的体内代谢，TPMT的活性与这类药物的临床疗效和毒性密切相关。但目前尚无TPMT与中药成分代谢及糖尿病机制研究的相关报道。本项目拟建立快速筛选和识别复杂体系中TPMT酶亲和活性成分的联用新技术：HPLC-TPMT EAD-MS，对天芪降糖胶囊的体内体外活性成分进行靶向筛选及分析，并采用HSCCC导向制备该类化合物。建立一套基于HPLC-TPMT EAD-MS/HSCCC技术的中药复方中TPMT酶亲和底物的快速筛选和制备方法，并应用现代光谱学和化学方法鉴定活性成分结构。建立基于靶标活性成分及体内代谢物的快速筛选、适合中药复杂成分的研究方法。

天芪降糖胶囊是治疗2型糖尿病前期的中药复方制剂，可显著降低糖耐量受损患者向二型糖尿病发展的风险、预防糖尿病发生、有效改善患者的临床不适症状。本课题利用糖尿病动物模型，结合液质联用、分子生物学、代谢组学及肠道菌群测序等技术，探究天芪降糖胶囊及其主要成分小檗碱治疗早期2型糖尿病的作用机制。首先利用超高液相色谱-双压线性离子阱高分辨率组合型质谱联用技术(UPLC-LTQOrbitrap Velos Pro ETD)，根据精确相对分子质量，二级质谱裂解碎片和色谱峰保留时间等信息结合对照品的碎片裂解规律和文献数据对天芪降糖胶囊化学成分进行分析和鉴定，共鉴定或推测出21个化合物；通过糖尿病动物模型研究TPMT酶在实验动物各组间活性及蛋白表达差异，与对照组比较，模型组动物红细胞裂解液TPMT活性高于对照组，且肝组织中TPMT蛋白表达水平亦高于对照组；小檗碱治疗组动物红细胞裂解液TPMT活性下降，但在肝组织的蛋白表达水平上调；代谢组学研究分析共找到39个与小檗碱作用相关的代谢标志物（肝组织8个，血浆10个，尿液21个）；肠道菌群测序找到16个差异菌属；关联分析结果找到药物对肠道菌群的调节作用与体内代谢水平的相互关系。综合上述结果，本研究为探讨天芪降糖胶囊及其主要成分小檗碱治疗早期2型糖尿病的作用机制提供了重要的研究基础。