基于“结构相似性分类-质谱可视化解析”的甘草制吴茱萸物质基础及减毒机理研究

Process technique is a unique characteristic of Chinese Materia Medica (CMM) applied in clinics, usually toxic CMM can reduce toxicity and remain efficacy through these processing techniques. In our previous researches, we have investigated the processing procedure, HPLC determination before and after process, HPLC-MS identification, structurally similar classification MS data analysis method and preliminary liver injury pathology study of Evodia Fructus. In this project, the plant origins will be investigated by UPLC/GC fingerprint method, mass untargeted MS data will be fully elucidated by "structural similar classification – MS data visualization strategy" and to obtain the multiple dimensions of vitro &vivo bioactive components. “Components-toxicity-efficacy” relations were fully explored, in order to illustrate the underlining relation of toxic and effective components. Biological and molecular techniques, as well as pharmacology method, were developed to explain the toxicity and detoxification principles at whole-cell-molecular levels, and the comprehensive research system was constructed during this procedure. The scientific significance of processing detoxification was addressed, as well as glycyrrhiza reducing toxicity and heat of Evodia Fructus. This project will provide strong supports for the processing practice, quality control, clinical reasonable medication, and may provide a new methodology for the safety evaluation and toxic-effect investigation of toxic CMM.

中药炮制是中医药临床用药的特色，有毒中药经过炮制后可以减毒增效。在前期吴茱萸炮制工艺摸索、炮制前后HPLC液相色谱对比、液相质谱联用考察、结构相似性分类质谱解析方法及肝损伤药理研究基础上，采用超高效液相色谱/气相指纹图谱方法评价吴茱萸不同产地药材属性；基于“结构相似性分类-质谱可视化解析”策略，全谱、非靶向、高效解析液质联用质谱数据，获得体内外物质基础的多维度信息；分离分析毒性-药效作用部分，进行“物-毒-效”关联分析，探究毒性与药效作用的物质基础；结合药理与分子生物学技术手段，探索整体动物-细胞-分子水平毒性和炮制减毒机理，构建吴茱萸炮制前后物质基础-药效毒理-炮制机理综合研究体系，阐释炮制减毒的现代科学意义，阐明甘草制“缓和药性、降低毒性”的科学内涵，为炮制工艺规范、质量标准制定、临床安全合理用药提供科学支持，为有毒中药的安全性评价及毒性效用研究提供新思路。

本课题以小毒中药吴茱萸为例，建立了吴茱萸UPLC指纹图谱定性定量方法，对多产地药材的吴茱萸碱、吴茱萸次碱、柠檬苦素进行了精确定量，从药典指标评价各产地吴茱萸药材质量特点；同时，对挥发油进行了定性定量分析，发现商品规格、产地、采收年份对挥发油含量均有影响。吴茱萸经甘草制后，挥发油含量显著降低，同时挥发油、水提物、70%醇提物LD50尤其是LD5甘草制后显著升高，阐释了吴茱萸经甘草制后，“缓和燥性、降低毒性”的科学内涵。随后展开的三种提取物药效研究表明，甘草制后醇提物的抗炎镇痛效果显著增强。在此基础上，以药效、毒性兼有的醇提物开展吴茱萸亚急性毒性考察。结果表明，吴茱萸有确切的肝脏毒性，28天生品给药后高剂量组雌雄鼠均有肝脏指数显著升高（P<0.001），AST显著升高，伴有血糖降低，血清TG显著降低；病理结果显示，吴茱萸生品给药后有局部肝细胞坏死、炎性细胞浸润、胆管增生、部分肝细胞空泡样病变；并且吴茱萸经甘草炮制后，肝毒性有显著改善。炮制后安全性提高，同时药效也有一定程度的提高，在临床指导剂量下，具有安全合理性，体现吴茱萸经甘草炮制后，增效减毒的科学内涵。. 根据植物内源性成分的结构内在关联，构建了适合中药复杂成分非靶标分析的“结构相似性分类-质谱可视化解析”分析策略，编译成分聚类、热图、云图模块，从吴茱萸中共鉴定出89种成分。采用“物-毒-效”策略，关联体内药效、毒性结果，从中筛选出毒性成分、药效成分、炮制前后变化差异成分。代谢水平发现，大鼠28天生品给药后，有明显脂质代谢紊乱，并呈现清晰的剂量效应和时序累积效应，且高剂量组停药后经过14天恢复期后，基本恢复至正常代谢水平，与肝脏指数、血清AST变化趋势相一致。从剂量梯度和时序代谢角度，研究吴茱萸生品及制品给药对机体代谢的扰动，阐释了吴茱萸炮制减毒的现代科学意义。