基于多糖等成分转化-性味-药效相关联的关黄柏盐炙机理研究

Hellodendron amurense, bitter in flavor and cold in nature, possesses the properties of clearing heat with eliminating dampness and purging fire for removing toxin. The bitter and dryness of Hellodendron amurense can be reduce by salt processing which enhance the effect of reach kidney meridian, nourishing kidney yin, and clearing kidney fires at the same time. Researchers have been focusing on the chemical variances of alkaloids, volatile oil, and flavonoids from Hellodendron amurense. In contrast, however, the polysaccharides of Hellodendron amurense was found to change greatly after salt processing according to our previous researches, i.e. species of polysaccharides were reduced from 7 to 4 and their molecule weights lowered after salt processing by using the founded study model, including analysis methods of HPLC and UPLC-MS and structure identified methods of MS and NMR. Significant immunosuppressive activities of salt processed Hellodendron amurense were also found in previous researches. Asthenia heat of kidney meridian was mostly resulted from immunological reaction according to the experiences of traditional Chinese medical clinical. Thus, Hellodendron amurense polysaccharides probably performed its actions of entering kidney meridian and clearing kidney fire by immunosuppression. Too many processed methods resulted in the reduction of levels of small molecules like alkaloids in Hellodendron amurense, which is impossible to elucidated the mechanism of salt processed Hellodendron amurense as the exclusive therapeutic basis. Thus, we will study the relatives between salt processing, components of Hellodendron amurense, and pharmaceutical effects, which contributes to reveal the mechanism of salt processed Hellodendron amurense and discover safety and lower toxic polysaccharides immunosuppressive agents.

关黄柏味苦，性寒，功能清热燥湿、泻火解毒，盐炙可以缓和其苦燥之性，增强入肾经、滋肾阴及清肾火作用。一直以来，人们关注盐炙后关黄柏中生物碱、挥发油和黄酮成分的变化,而我们在前期的研究中，发现关黄柏富含多糖成分，盐炙后多糖的种类发生较大变化。在建立的以HPLC、UPLC-MS法分析均一多糖、以质谱、NMR等研究多糖的一级结构及以原子力显微镜观察多糖二维结构的研究模式下，发现盐炙后黄柏多糖由4种变为7种，糖链变短，且盐炙后的多糖具有显著的免疫抑制作用；依据中医临床经验总结，肾经虚热多为免疫反应发热，所以多糖极可能通过免疫抑制机制发挥入肾、清肾火作用。关黄柏中生物碱等小分子在使用各种炮制方法处理后含量均下降，如果作为唯一的药效物质，根本无法解释其盐炙炮制的意图。本研究对盐炙与多糖、生物碱等组分转化及缓和药性、清肾火相关性开展研究，以揭示盐炙关黄柏的炮制原理，并期待发现安全低毒的多糖类免疫抑制剂。

我们前期研究发现，关黄柏富含粘多糖组分，初步免疫抑制试验表明关黄柏多糖具有一定的免疫抑制作用，而且盐炙后的多糖作用强于生品关黄柏多糖，所以关黄柏多糖组分应当作为黄柏的重要药效物质成分。本项目完成了炮制前后关黄柏化学物质组成的拆分，并证明各拆分组分化学成分互不交叉；对拆分组分进行解热、抗炎及免疫抑制等药效作用考察，并建立寒、热证大鼠模型，观察各拆分组分对实验动物神经系统及能量代谢的影响，结果表明多糖组分、生物碱组分及乙酸乙酯组分为关黄柏发挥解热、抗炎、免疫作用的药效物质基础。且炮制后关黄柏生物碱组分作用降低，而多糖组分作用增强；同时表明关黄柏各性味拆分组分的药性应归属为寒性，且关黄柏经盐炙炮制后寒性增强。根据关黄柏的临床应用，建立实验性肾病大鼠模型、小鼠免疫性肝损伤模型、免疫性结肠炎大鼠模型，观察各拆分组分的药效，表明盐炙前后关黄柏多糖对上述疾病的治疗作用更加显著。完成了关黄柏及其盐炙品多糖的分离、纯化，制备出均一多糖，阐明了均一多糖的结构，建立了多糖化学研究方法学平台。确定多糖成分为关黄柏的重要药效物质基础，并具有免疫抑制作用，其可作用机制为抑制炎症因子IL-6、IL-8及IL-1β的释放且能下调MAPK家族蛋白和NF-κB蛋白的表达。发表相关学术论文10篇，其中SCI收录2篇，中文核心期刊4篇，一般论文4篇。培养博士毕业生1名，硕士毕业生2名。