基于生物碱成分转化与毒-效相关性的甘草汁蒸制高乌头“减毒存效”的机制研究

Aconitum sinomontanum Nakai is one of the widely used Chinese medicine in the mid-west. It can dispel wind, remove damp and regulate qi to alleviate pain. It is effective to put into clinic, but low-security for toxicity of the crude without toxic diester diterpenoid alkaloids. At present, it is not clear whether influence of processing on toxicity and efficacy. Our previous study found that lappaconitine content is decreasing and total alkaloids are increasing, meanwhile, LD50 is increasing and efficacy remains after steamed by licorice. We speculate that the method of steamed by licorice can weaken toxicity and remain the same efficacy, and propose a hypothesis that toxicity reduced by processing is related to the decreasing content of toxic components; Efficacy existing is related to the increasing content of total alkaloids and the composition of dissolution; Efficacy existing is also related to the percentage of components which is changed by the toxic components converted active components. This subject is planned to study the change of chemical composition and toxicity and efficacy before and after processing by the method of chemistry and pharmacology to prove up the relationship between toxicity, efficacy and chemical composition. We study on the toxicity and efficacy of the different solvent extraction of the crude and the processed to verify toxicity and active groups by the method of pharmacology; We study on the chemical changes of toxicity and active groups of the crude and the processed to ascertain the toxic composition and active composition by using HPLC fingerprint, LC/MS and NMR. Meanwhile, we plan to explore the transformation mechanism of chemical compositions by simulating processing. The research will explore the processing mechanism of “toxicity reducing and efficacy existing ”,providing scientific basis for the clinical safety of medication.

高乌头是中西部地区民间习用中药，具祛风除湿、理气止痛之功效。临床疗效显著，但生品有毒且不含双酯型乌头碱。目前炮制对其毒效的影响还不清楚。前期研究发现，高乌头用甘草汁蒸制后，主要成分高乌甲素含量降低但总生物碱含量增加，同时LD50增大而疗效不变。我们推测经甘草汁蒸制可降低其毒性而保存药效，提出“炮制减毒与毒性成分含量降低有关，存效与总生物碱含量增加、促进有效成分溶出，从而引起成分比例变化有关”的假说。本课题拟采用化学结合药理学方法，研究高乌头炮制前后化学成分及毒效变化，探明毒性、药效与化学成分之关系。采用药理学方法考察生、制品不同溶剂萃取物的毒性与药效，探明其毒性与活性部位群；采用HPLC指纹图谱、LC/MS及NMR方法，研究生、制品毒性与活性部位群的化学成分变化，探明毒性与活性成分。并进行模拟炮制，揭示化学成分转化机制。本项目探究炮制对高乌头“减毒存效”的机制，为临床安全用药提供理论依据。

高乌头是中西部地区民间习用中药，具祛风除湿、理气止痛之功效。临床疗效显著，但生品有毒且不含双酯型乌头碱，其炮制原理与乌头不同。本项目探究高乌头经过甘草汁蒸制“减毒存效”的机制。采用化学结合药理学方法，研究了其炮制前后毒性、药效与化学成分的关系；采用药理学方法筛选生、制品的毒性、活性及“减毒存效”部位群；采用HPLC指纹图谱、LC/MS及NMR方法，探明毒性与活性成分；通过模拟炮制，最终揭示化学成分转化机制。研究发现，高乌头经甘草汁蒸制后确能达到“减毒存效”炮制目的。三氯甲烷部分既是毒性部位群（可能毒性成分为高乌甲素、冉乌头碱、8-o-acetylexcelsine），又是其镇痛抗炎的药效部位群，该部分炮制后毒性减低而药效保持，因此也是炮制“减毒存效”部位群。高乌头炮制后，物质基础发生了质变和量变，化学成分组成和比例发生了显著变化，主要生物碱发生了相互转化，其炮制原理为：（1）与炮制后毒性成分消失或含量下降密切相关：毒性成分8-o-acetylexcelsine消失；高乌甲素含量降低，并部分转化为毒性较小的刺乌宁、N-去乙酰高乌甲素、冉乌头碱、异刺乌头碱等；冉乌头碱含量降低，并部分转化为毒性较小的生物碱Ranaconidine及羟基化产物；还与“辛”味成分2,4-二叔丁基苯酚、正壬基琥珀酸酐、十五烷酸、十六酸乙酯、(Z,Z,Z),9,12,15-十八三烯酸、亚油酸乙酯、油酸乙酯、香紫苏醇含量下降密切相关；（2）与炮制后药效成分的含量增加密切相关：与新增成分5-HMF具镇痛抗炎作用有关；与“行气”止痛成分棕榈油酸、Z-11，十六烷酸、十六烷酸、(Z,Z)-9,12-十八二烯酸甲酯、(Z,Z),9,12-十八二烯酸等明显增加密切相关。本项目在国家自然科学基金的支持下，明确了高乌头炮制“减毒存效”的机制，为临床安全用药提供理论依据。