基于体内过程分析的肉苁蓉苯乙醇苷成分肝保护作用药效物质研究

Herba Cistanche had been used for many years in China and other countries, as it had the effect of nourishing the liver and kidney, benefiting essence-blood, relieving constipation, and so on. The main chemical constituents of Herba Cistanche were phenylethanoid glycosides, which had antibacterial, anti-inflammaory, hepatoprotective, antioxidant, immunomodulative, enhancing memory, anti-aging effects. In our previous study, acteoside and echinacoside were the prominent compounds of Herba Cistanche, and they had the strong hepatoprotective activity. The pharmacokinetic and metabolic processes in vivo of echinacoside were reported that echinacoside was converted to acteoside in intestinal by desugarization, which implied that acteoside and its metabolites were the effective substances of hepatoprotective activity of echinacoside. However, the reports on the analysis in vivo of acteoside had not been seen. In our issue, these studies will be carried as follow. Firstly, the metabolites will be isolated from rat urinary sample of rat orally given acteoside and their structures will be identified. Secondly, the tissue distribution, metabolism and excretion of acteoside will be systematically study by HPLC-UV and HPLC-MS. Finally, the pharmacodynamics of main metabolites will be carried. The purpose of our research is the explaintion of metabolism in vivo and the effective substances of hepatoprotective activity of phenylethanoid glycosides.

中药肉苁蓉药用历史悠久，具有滋肝养肾，益精血，润肠通便等功效，是我国西部地区的名贵药材，素有"沙漠人参"的美誉。苯乙醇苷为肉苁蓉中主要化学成分，具有抗菌、抗炎、保肝、抗氧化、免疫调节、增强记忆力、抗衰老等作用。本项目组前期研究表明，毛蕊花糖苷和松果菊苷是肉苁蓉苯乙醇苷的主要成分，且体内、外保肝活性最强。文献研究表明，松果菊苷在大肠中经脱糖反应转化成毛蕊花糖苷，提示毛蕊花糖苷或其代谢产物可能是松果菊苷真正发挥药效的活性物质，然而目前有关毛蕊花糖苷的内体分析研究未见报导。因此，本项目对毛蕊花糖苷的大鼠尿中代谢产物进行分离纯化和结构鉴定；采用HPLC-UV和HPLC-MS等分析方法对毛蕊花糖苷的体内组织分布、代谢、排泄进行系统研究；并对主要代谢产物进行药效学实验。力求以毛蕊花糖苷为代表阐明肉苁蓉苯乙醇苷成分体内代谢规律和保肝作用的真正药效物质基础，为发现活性代谢产物及开发新药奠定实验和理论基础。

项目背景：毛蕊花糖苷和松果菊苷是肉苁蓉苯乙醇苷的主要药效成分，也是药典中肉苁蓉药材含量测定的指标成分，约占苯乙醇苷成分含量的70%，在肉苁蓉的药理活性中起到决定性作用。松果菊苷比毛蕊花糖苷多一分子葡萄糖，推测松果菊苷在大肠中脱糖转化成毛蕊花糖苷。在灌胃松果菊苷的大鼠粪便中检测到毛蕊花糖苷、去咖啡酰基毛蕊花糖苷等，推测毛蕊花糖苷或其代谢产物是松果菊苷体内真正发挥药效的活性物质，然而有关毛蕊花糖苷的体内分析研究未见报导。.主要研究内容：系统研究毛蕊花糖苷和松果菊苷的体内代谢，对毛蕊花糖苷大鼠尿中代谢产物分离纯化和结构鉴定；分析毛蕊花糖苷和松果菊苷的大鼠血、尿、胆汁、粪便中代谢产物；研究人和大鼠肠道菌及大鼠肠道酶对毛蕊花糖苷和松果菊苷的影响；主要代谢产物药效学实验；研究肉苁蓉复方制剂的质量控制。.重要结果、关键数据及科学意义：1.建立毛蕊花糖苷和松果菊苷的制备工艺，制得毛蕊花糖苷12g、松果菊苷15g，为体内分析研究提供原料物质，为苯乙醇苷成分制备提供方法学参考。2.从毛蕊花糖苷大鼠尿中分离得到7个代谢产物，鉴定出4个化合物的结构，为研究毛蕊花糖苷及苯乙醇苷类成分的体内药效物质奠定化学物质基础。3.采用UPLC-QTOFMS法，系统研究毛蕊花糖苷和松果菊苷的血、尿、胆汁、粪便中代谢产物，检测到35个毛蕊花糖苷代谢物，28个松果菊苷代谢产物。二者共有代谢产物18个，说明他们的体内代谢和排泄过程相似，证实了毛蕊花糖苷或其代谢产物是松果菊苷体内药效物质的推测，对揭示毛蕊花糖苷及苯乙醇苷成分的体内药效物质具有重要意义。4.在人和大鼠肠道菌群孵育的毛蕊花糖苷和松果菊苷中检测到13个化合物，归纳总结了毛蕊花糖苷和松果菊苷在肠道菌作用下的代谢途径。5.在大鼠肠道酶孵育物中检测到13个化合物，代谢产物含量测定结果表明肠道酶对毛蕊花糖苷和松果菊苷的影响弱于肠道菌。6.毛蕊花糖苷、松果菊苷及主要代谢产物对肝损伤模型小鼠的ALT和AST升高均有抑制作用。7.建立抗骨增生片中肉苁蓉的TLC鉴别和HPLC含量测定法，并对市售制剂进行测定，为提高抗骨增生片的质量标准奠定实验基础，且为含有肉苁蓉药材的复方制剂质量控制研究提供实验参考。.本项目阐明了毛蕊花糖苷和松果菊苷体内代谢规律和药效物质，对揭示肉苁蓉苯乙醇苷成分的体内药效物质基础具有重要意义，同时也为发现活性代谢产物及开发新药奠定实验和理论基础。