基于汞元素形态和circRNA芯片分析的伤科接骨片中朱砂、马钱子配伍相互减毒的机理研究

Shangkejiegupian is the most popular Chinese patent medicine in clinic for the bone fractures, in whose formula, two toxic medicines are included - cinnabar and strychni semen. However, there weren’t any reports about its adverse effects in clinic until now. In our previous pharmacological study, the medicine without cinnabar showed a great toxicity of central nerves induced by strychni semen and had a little bit discount in efficacy. Because the inorganic mercury ion of cinnabar is kind of metal complexing agent and it would react with alkaloids of strychni semen in human gastrointestinal tract, the content of the most toxic substances produced by cinnabar, methyl mercury and strychnine of strychni semen would decrease in human body, if they were taken simultaneously, suggesting that cinnabar and strychni semen might reduce the toxicity for each other. Besides, we found that a small number of mice, which were administrated with Shangkejiegupian in a big dosage, still suffered the toxicity of strychni, showing that the ratio of cinnabar and strychni semen should be optimized furthermore. Considering the toxicity of cinnabar varied with the forms of mercury in human body, we plan to establish the assay method for mercury forms and then explore the best ratio and mechanisms for their mutual influences between cinnabar and strychni semen to reduce their each toxicity. Our study will fatherly enrich the compatibility theory of traditional Chinese medicine and lay the foundation and provide a demonstration for reevaluations of all the medicines with cinnabar.

伤科接骨片是目前骨伤科销售量最大的中成药，含有两味有毒药材-朱砂和马钱子，该药在临床上使用多年，从未见不良反应报告。前期研究中，我们发现去除朱砂的处方有明显的马钱子神经毒性，且与朱砂比原处方的甲基汞量下降很多，推测朱砂的无机汞离子与马钱子共存于胃肠道时，可能生成络合物，从而减少朱砂生成毒性极强的甲基汞量以及马钱子生物碱溶出量，据此提出“朱砂与马钱子配伍相互减毒”这一新的配伍规律。此外毒理实验发现大剂量给予伤科接骨片，少数小鼠出现马钱子神经毒性，说明朱砂与马钱子在方中的配比有待于进一步优化。鉴于朱砂的毒性与在体内形成可溶性汞的形态直接相关，本研究拟建立汞元素形态分析方法，结合药理实验结果，阐明朱砂与马钱子配伍相互减毒的最佳配比及物质基础，从而丰富中医药传统配伍理论，为含朱砂制剂安全性再评价奠定基础并起到示范作用。

本研究研究了伤科接骨片中朱砂、马钱子配伍相互减毒的机理。取得成果如下：.1. 运用仿生提取后，采用测汞仪分别分析了朱砂、不含马钱子粉伤科接骨片、伤科接骨片的人工胃液和人工肠液中可溶性总汞含量。发现伤科接骨片中的马钱子粉可能影响朱砂中可溶性总汞的溶出。.采用液相原子荧光光谱仪联用技术分析了朱砂、朱砂-马钱子、伤科接骨片的人工胃液和人工肠液中不同形态汞的含量。发现伤科接骨片中马钱子粉可降低朱砂中无机汞的溶出。其后，探讨了朱砂配伍马钱子粉及伤科接骨片对小鼠体内汞蓄积的影响，结果表明，伤科接骨片中马钱子粉可能促进小鼠肝脏对无机汞的代谢来减少朱砂中汞蓄积。.2. 采用HPLC法测定马钱子、朱砂-马钱子、伤科接骨片人工胃液和人工肠液中马钱子生物碱（士的宁和马钱子碱）的含量。结果表明，伤科接骨片中朱砂不会影响马钱子粉毒性成分士的宁和马钱子碱的溶出。采用HPLC法测定马钱子粉和朱砂配伍马钱子粉经肝S9、肠S9孵育培养后士的宁和马钱子碱浓度，研究发现朱砂可能促进肝S9、肠S9对马钱子粉中士的宁和马钱子碱体外代谢。采用HPLC-MS/MS法测定了伤科接骨片中士的宁和马钱子碱在大鼠体内毒代动力学影响，发现朱砂能影响伤科接骨片中士的宁和马钱子碱毒代动力学。.3. 采用HPLC-ECD法研究配伍朱砂伤科接骨片对中枢神经系统内单胺类神经递质的影响，发现伤科接骨片中朱砂降低马钱子导致的神经毒性可能与纹状体中DA和海马中5-HT水平有关。.4. 通过研究急性毒性实验和镇痛药效实验，研究朱砂与马钱子的最佳配比，将伤科接骨片中朱砂与马钱子的配伍比例调整为2：1。.综上，本研究基本阐明了伤科接骨片中朱砂和马钱子配伍相互减毒的机理，马钱子降低朱砂中无机汞溶出及促进肝脏对无机汞的代谢来减小朱砂蓄积性毒性，朱砂通过影响士的宁和马钱子碱的毒代动力学来降低其毒性，并且是通过恢复纹状体中DA和海马中5-HT水平来降低马钱子导致的神经毒性，还确定伤科接骨片中朱砂与马钱子的最佳配伍比，进一步丰富了中药配伍理论。