“甘草-马钱子“药对汤液相态差异与毒效消长相关性的研究

Licorice root - nux vomica is a typical compatibility combination of reducing poisonousness medicine pair. The preliminary study of the research group found that although the colloid phase has a large proportion in the decoction, the proportion of its toxic component is only 11%; while in the sediment phase containing more than 80%. Therefore, it is speculated that "the mechanism of attenuate poisonousness of the Licorice root - nux vomica is caused by the difference of phase distribution." This study intends to split the soup into four different phases: true solution, colloidal solution, suspension and sedimentation particles. The conversion of the materials in each phase are identified by HPLC-Q-TOF technique through particle size distribution and micro-morphology. Then giving experimental animals of different phase. The measure relationship between vivo absorption and organization distribution of the toxic substances are analyzed by HPLC-MS / MS. Finally, AChE and potential biomarkers are used as indicators to establish the PBTK / TD model, and the correlation between toxicity and effect are studied. In addition, the expression of different phase toxicity is revealed. So as to verify the correlation between different phase and the toxicity of growth. Its significance lies in: make the scientific connotation of the compatibility of licorice root and nux vomica clear, but also for compatibility of traditional Chinese medicine " detoxification and save effect " provide the scientific basis for the theory of Chinese medicine.

甘草-马钱子是一个经典配伍减毒药对，课题组前期研究发现其共煎液中胶体相态比重较大，但毒效成分仅占11%；而沉积物相态中却含近80%的毒效物质。因此，猜想“甘草-马钱子配伍减毒机制是由于相态分布的差异性而引起的”。本课题拟将该汤液拆分成真溶液、胶体溶液、混悬液及沉降颗粒等四个不同相态，以粒径分布和微观形态来表征拆分结果，采用HPLC-Q-TOF技术鉴定各相态中的物质转化；将各拆分相态给予实验动物，采用HPLC- MS/MS技术，分析毒效物质在体吸收和组织分布的量度关系；并以染毒剂量下血清中毒效物质成分、AChE及潜在生物标记物含量为指标，建立PBTK/TD模型，研究其毒性与效应之间的相关性，揭示不同相态毒性表达情况，从而验证该药对相态差异与毒效消长的相关性，其意义在于：合理阐释马钱子与甘草配伍后去毒存效、以毒取效的科学内涵，也为中药配伍“毒解而效不减”这一中医理论提供科学依据。

中药汤液是一个天然的共融体系，多种成分共同作用于汤液体系中，可使汤液形成不同的相态。甘草能缓和马钱子的毒性，已为一种共识，但其减毒的机制仍在不断的探索。课题组前期研究基础上，提出“甘草-马钱子配伍减毒机制是由于相态分布的差异性而引起的”假说，并对其进行系列研究，以期探讨相态与减毒之间的相关性。具体研究内容如下：.1.汤液相态的拆分、表征及鉴定研究.采用物理沉降、高速离心及透析等技术，将配伍汤液拆分成沉积物、混悬液、胶体及溶液4个相态；并对不同相态进行表征，结果配伍后出现大量白色絮状沉淀，微观结构为类似白色包裹物；对相态的成分分析发现，不同相态中成分具有差异性；对拆分相态中毒效物质含量进行测定，发现毒（效）成分主要聚集在沉积物相态。.2.物质组分吸收研究.进行毒效物质体内行为和分布特征研究，结果不同相态吸收和消除的速率不同，胶体组和溶液组快速吸收后快速消除；沉积物和混悬组在体内缓慢溶解，并出现二次吸收趋势，而其消除速度较快，加速体内毒物的代谢。组织分布结果显示，共煎液在组织中的毒效成分均有所下降，肝、肾、肺中毒效物质含量下降最为明显，可显著减轻代谢器官的毒性蓄积；各相态组织分布与毒代动力学血药浓度密切相关，且随着血药浓度的升高或下降，组织分布快速或缓慢，有一定的滞后效应。.3.PBTK/TD毒性表达研究.以AchE为TD指标，通过WinNonlin 软件进行PBTK-TD模型拟合，结果表明，马钱子抑制效应不仅与毒效物质血药浓度相关，还与其存在状态即汤液的物理特性有关。单煎液、胶体、溶液组在体内快速吸收，毒性效应较强；混悬组与共煎组中的大颗粒物质减缓了毒性效应；而沉积物组因毒性成分释放最为缓慢，其体内血药浓度低，达不到中毒浓度，且可长期发挥药效。.综上，本课题研究结果提示，甘草可影响马钱子毒效成分体、内外行为差异；汤液的不同相态与体内的物质吸收与毒性表达具有相关性；甘草解马钱子毒性的关键点在于配伍后沉积相态的形成。