蒙药特色应用“诃子解草乌毒” 的减毒存效机理研究

The severe toxic reaction of Aconitum Kusnezoffii (AK) becomes the major obstacle for the marketization of Mongolian medicine. The major toxic component is diester aconitine in AK. The “Terminalia Chebula Retz (TC) detoxicates Aconitum Kusnezoffii” is a characteristic application of decreasing toxicity and maintain efficacy in Mongolian medicine. So, we hypothesis that it is the primitive application of controlled or slow release in Mongolian medicine. The project will validate the hypothesis and clarify this application systematically the pharmacological mechanism. The major pharmacological effect of AC will be compared, and the change of Ca2+-ATPase and RyR2 in myocardium will be examined in different groups. Moreover, we will examine the influence of TC on AK in acute cardiac toxicity and Long-term genotoxicity. The detection method in vivo of diester aconitine, which is based on UPLC/Q-Exactive/Ms technology, will be established. The change of toxicokinetic parameters of diester aconitine will be examined, after the TC is used together. The mechanism of parameters’ change will be studied, according to the process of medicine in vivo (absorption, distribution, metabolism, and excretion ). The TK-TD model will be established to study the influence of TC on the toxicity of AK. We will establish the metabonomics analysis methods for AK toxicity based on UPLC/Q-Exactive/Ms technology, and find the specific small molecule metabolites and particular metabolism for AK cardiotoxicity by data pretreatment and pattern recognition. Furthermore, we can clarify the metabonomics mechanism of the characteristic application. We expected to study the mechanism of the characteristic application “Terminalia Chebula Retz (TC) detoxicates Aconitum Kusnezoffii”, and provide the safety basis for Mongolian compound medicine contained AK.

草乌严重毒性反应是限制含草乌蒙药市场化主要障碍，双酯型乌头碱是主要毒性成分。“诃子解草乌毒”是蒙药特色减毒存效方法，我们提出这是蒙医中（缓）控释应用假说，课题验证该假说并阐明应用机理。比较诃子对草乌主要药效学影响，考察合用前后对于心肌Ca2+-ATP酶和RyR2影响；研究诃子对草乌心脏急性和长期遗传毒性影响；建立双酯型乌头碱高分辨质谱技术的体内检测方法，评价诃子对双酯型乌头碱毒代动力学参数影响，结合药物体内变化过程（吸收、分布、代谢、排泄）阐明应用致参数变化原理；在毒代动力学及毒效学基础上建立心脏毒性TK-TD模型，研究TK-TD模型变化；建立草乌血液及尿液小分子代谢物检测方法，通过数据预处理、模式识别并结合生物数据库发现草乌心脏毒性特异性小分子代谢物及心脏毒性的代谢途径，阐明该特色应用的代谢组学原理。通过课题的完成系统研究蒙药中该特色应用减毒存效原理，为含草乌蒙药安全应用提供研究依据。

“诃子解草乌毒”是蒙药特色配伍应用之一，课题中我们提出了“诃子解草乌毒”的“减毒存效”假说、通过对于草乌在体内药动学变化、草乌主要药效学的研究、代谢组学研究以及草乌心脏毒性作用影响，评价并验证蒙药配伍“诃子解草乌毒”的科学性，诃子与草乌配伍“减毒存效”假说的合理性。通过主要药效学研究发现，不同比例的诃子与草乌配伍后对于草乌镇痛和抗炎作用影响不显著，但是却可以显著的降低动物的死亡率。诃子与草乌配伍后可以显著的改善长期给予草乌后所致的心脏毒性，对于草乌所致的心律失常，心肌细胞病理学改变均有很好的缓解作用，草乌经诃子炮制后对于遗传毒性解毒作用更显著。诃子可以影响草乌中双酯型乌头碱的药动学过程，诃子单宁酸可以明显抑制乌头碱、新乌头碱和次乌头碱的吸收。三种乌头碱的血浆蛋白结合率均属于低血浆蛋白结合率，生物利用度较高，单宁酸在一定比例上可以显著降低3种乌头碱的蛋白结合率。诃子可以降低三种乌头碱在各组织的分布。单宁酸对三种乌头碱的代谢没有影响。三种乌头碱排泄方式以肾脏为主，排泄较快，诃子配伍后可以明显减少乌头碱的排泄量，新乌头碱与次乌头碱与之相反可能是发生了代谢方式内化代谢，诃子各配伍组的代谢半衰期延长，可以延长草乌在体内的作用时间。建立了生草乌整合TK-TD整合模型用于口服生草乌心脏毒性研究，但是诃子配伍后模型预测效果并不理想。代谢组学发现生草乌主要通过影响氨基酸代谢产生毒性作，如色氨酸代谢，精氨酸和脯氨酸代谢和谷氨酸代谢等相关代谢通路，推测生草乌的心脏、肝脏和肾脏毒性可能由其导致的氨基酸代谢紊乱所致致，诃子尤其是诃子制草乌可以影响上述代谢途径而降低生草乌的毒性反应，而起到“减毒存效”的作用。通过本课题完成，证明了蒙药经典配伍“诃子解草乌毒”的科学性和合理性，诃子通过缓控释等过程而对于草乌起到“减毒存效”的作用，蒙药中通过诃子与草乌配伍应用为含草乌类蒙药复方药物开发提供原理学依据。