基于体内代谢多源归一特性的青阳参活性成分发现和多成分药动-药效学研究

Qingyangshen (Cynanchum otophyllum, QYS) is frequently used to treat epilepsy as ethnic drug in Yunnan Province. However, its active compounds are still ambiguous. Moreover, owing to its in vivo accumulation, the treatment is abnormal. To study the in vivo metabolism of QYS is critical not only for screening the active compounds but also for an appropriate treatment. Based on the pre-test results and literature, we propose a hypothesis that several compounds with the same aglycone will transform into the same metabolites, which we call multilcomponent-produce-same-metabolite phenomenon (MPSM). In this project, we plan to discover the active compounds of QYS based on qualitative investigation of MPSM, and to illustrate mechanism of the abnormal treatment according to PK-PD study based on MPSM. The research will include (1) isolation the key metabolites guiding by serum pharmacochemistry; (2) qualitative illustration of MPSM according to metabolite identification by HPLC and UPLC-q TOFMS after oral administrate single compounds and the mixture respectively; (3) to evaluate the anticonvulsant effect of key metabolites with mice Maximal Electroshock Seizure test (MES); (4) to determinate the PKs of multicomponents and express the whole PK of QYS extract by the PK marker method, Total Moment method and BP net method; (5) to assess the ED50 value of QYS on anticonvulsant and pharmacodynamic effect, and (6) to establish the PK-PD relationship.

青阳参是云南民族药，治疗癫痫。然而其活性成分研究不透彻，且因在体内蓄积，故用法特殊。进行青阳参的体内代谢研究无论对寻找活性成分还是对合理用药均具有重要意义。根据预实验和文献，我们提出科学假设：青阳参中多个同苷元成分在体内代谢为相同的代谢产物，即体内代谢多源归一特性。本课题拟通过定性研究青阳参体内代谢多源归一特性，阐明其活性物质基础；通过基于多源归一的多成分PK-PD研究，解释其特殊用法。研究内容包括：用血清药物化学导向分离关键代谢产物；按“先单体后多个成分组合”给药，利用HPLC和UPLC-qTOFMS鉴定共同代谢产物，定性研究多源归一；用小鼠电惊厥（MES）模型测定代谢产物抗癫痫活性；采用LC-MS/MS测定多成分PK，用PK marker法、总量矩法和人工神经网络（BP模型）法表达青阳参的整体PK，定量阐明多源归一特性；用MES模型评价青阳参的ED50和时-效关系，构建PK-PD关系。

青阳参是云南民族药，治疗癫痫。然而其活性成分研究不透彻，且因在体内蓄积，故用法特殊。进行青阳参的体内代谢研究无论对寻找活性成分还是对合理用药均具有重要意义。本项目开展了基于青阳参体内代谢的抗癫痫活性成分定性研究和PK-PD定量研究，构建了一套新的“基于血清药物化学的天然药物有效成分组研究方法”,并用青阳参为例进行了实验验证。该方法从传统草药（包括中药复方）提取物提取分析开始，到蛋白分子机制的阐释结束，覆盖了现代天然药物研究的主要核心过程，可以快速、准确、简便地筛选出草药的有效成分；并组成中药有效成分组；进而采用网络药理学的协助，研究蛋白分子机制，较完整地阐明了“中药、民族药的多成分-多靶点”的机制。方法简单、高效、有较广阔的应用前景。本项目还建立了UPLC-SIM MS测定小鼠血浆、脑中青阳参有效成分组（青阳参皂苷M1和N）和苯巴比妥（PB）的定量分析方法，并应用于两轮次、18大组、1300多只小鼠的抗MES作用的PK-PD研究。本项研究发现了青阳参和PB相互影响，改变体内PK行为，药效有协同作用；并采用PK marker法、整合药代动力学法进行了整体药代动力学研究；对新的基于人工智能算法的PK-PD研究进行了初步的探索。