系列C20差向异构ocotillol型皂苷的合成、心肌缺血/再灌注损伤保护作用构效关系、ADME与作用机制研究

It has been shown that ginsenosides have widely beneficial myocardial protective effects. Our previous study results show that protective effects of series (20S, 24R)-ocotillol-type sapanions on myocardial ischemia injury are more significant than those of their associated C24 epimers. Their absorption and metabolism also have stereoselective differences. Based on our previous experiment results, twelve pairs of C20 epimeric ocotillol-type sapanions have been designed and to be synthesized in this study. Their structures are characterized by NMR, HRMS and X-ray single-crystal diffraction. The hypoxia/reoxygenation model based on in vitro cardiac muscle mitochondria and cardiomyocyte and the myocardial ischemia/reperfusion injury model based on vivi animal have been established. Their structure-activity relationships(SAR) of in vivo-in vitro myocardial ischemia/reperfusion injury protective effects will be studied. The relevance of their myocardial ischemia/reperfusion injury protection mechanism with oxidative stress state improvement and mitochondrion steady state protection will be elucidated. In order to further elucidate the correlathion between activities and ADME of C20 epimeric ocotillol-type sapanions, the stereoselective differences of ADME and occurrence mechanism of those C20 epimers which have significant different activites will be studied. Novel strategies and scientific evidences can be provided to elucidate the classic efficiency of Panax Ginseng and enhance R&D of novel myocardial anti-ischemia drug with independent intellectual property.

现有研究表明，人参皂苷具有广泛的心肌保护作用，我们前期研究发现，系列(20S,24R)-ocotillol型皂苷对心肌缺血损伤的保护作用明显优于其C24差向异构体，吸收与代谢亦存在显著立体差异。本项目基于前期研究基础，设计合成12对C20差向异构ocotillol型皂苷，采用NMR、HRMS和X-ray单晶衍射表征其结构；建立基于体外心肌线粒体和心肌细胞缺氧/复氧模型以及基于离体器官和整体动物的心肌缺血/再灌注损伤模型，研究其对体内外心肌缺血/再灌注损伤保护作用的构效关系，明确其对心肌缺血/再灌注损伤的保护作用机制与改善氧化胁迫状态、保护线粒体稳态的相关性。针对活性存在显著差异的C20差向异构体，研究其ADME的立体差异及其发生机制，进一步阐明C20差向异构ocotillol型皂苷活性差异与ADME的关联性。为阐释人参的经典功效和开发具有独立知识产权的抗心肌缺血新药提供新策略和科学依据。

用碱降解人参或西洋参茎叶总皂苷得到足量C20(S)-PPD、Rh2、Rg3、PPT、Rh1和Rg2，用酸降解上述总皂苷得到足量20(R)-PPD、Rh2、Rg3、PPT、Rh1和Rg2。合成出12对C20差向异构ocotillol型皂苷，采用1H NMR、13C NMR、HRMS、IT-TOFMS、ESI-MS、DEPT、HMBC、HSQC或HMQC确证结构。20(R)-PPD和源于20(S)-PPD、20(R)-PPD、20(S)-PPT、20(R)-PPT的4对C20差向异构ocotillol皂苷得到合适单晶，采用X-ray单晶衍射分析确证C20差向异构ocotillol型皂苷C20和C24构型。.通过细胞和离体心脏水平模拟缺血/再灌注损伤模型，评价了36个人参皂苷衍生物抗心肌缺血/再灌注损伤活性，实验结果如下：（1）二醇组皂苷较三醇组人参皂苷具有更优活性；（2）Ocotillol型皂苷较原人参二醇型皂苷活性更优；（3）Ocotillol型人参皂苷元C20构型不影响活性，C24为S构型为活性关键；（4）糖基侧链对人参皂苷抗心肌缺/再灌注损伤活性不利；（5）促进线粒体融合分裂过程，减少线粒体合成活性氧，维护线粒体功能稳态与人参皂苷心脏保护作用相关；（6）活化AMPK和AKT信号，抑制JNK信号与其抗心肌细胞凋亡相关；（7）人参皂苷或可通过干预炎症反应，抑制炎症信号通路以保护心肌缺血/再灌注损伤。.针对血清白蛋白（SA），以20(S)-Rh2和20(R)-Rh2模型分子，应用光谱分析和分子对接技术，探讨相互作用的立体选择性特征及其机制。结果表明，两个化合物在模拟生理条件下主要经氢键和疏水作用与SA自发形成1∶1稳定复合物，C20差向异构体在结合模式上存在明显差异，突出表现为结合区域、参与氢键和疏水作用基团、氨基酸残基数目及类别不同，可能是该类化合物与SA立体选择性相互作用的机制。在此基础上，进一步以血小板重要功能蛋白P2Y12受体为目标，通过构-效关系研究了ocotillol型皂苷生物学效应的立体选择性。结果发现，受试化合物均有明显的相互作用，但与靶蛋白相互作用模式和程度受立体构型影响，在作用力类型和形成氢键位点上呈现明显结构差异性，对血小板功能影响的立体选择性特征可能通过与关键氨基酸残基Thr213、Asn235、Val236及Ala243的氢键相互作用实现。