基于“反向溯源”分析的川芎对药治疗偏头痛药效物质基础及川芎“引药上行”作用研究

Ligusticum Chuanxiong, an upstream leader according to Traditional Chinese Medicine theory, plays an important role on the treatment of headache. Some herb pairs, such as Dachuanxiong formula, Xiongzhi San, the experience formula of Ligusticum Chuanxiong-Paeonia lactiflora, showed significant effect on the treatment of migraine. Due to the complexity and extreme low concentration, the components in brain from these medicines are quite difficult to be detected. Therefore, the pharmaceutical substances of Ligusticum Chuanxiong herb pairs are unclear until now, and the action of “lead-in upstream” has not yet been elucidated. In this study, ultra-high sensitivity detection methods such as supercritical chromatography-mass spectrometry (including chemical derivatization sensitization) and fluorescence detection will be established for the detection of small molecular chemicals in brain (including prototypes and metabolites). And the pharmaceutical substances from these medicines will be screened and analyzed systematically. The pharmaceutical substances will be determined on the basis of the “reverse tracing” analysis of target from organ, plasma to original herbs, computer docking screening and bioactivity evaluation. Furthermore, the comparative analysis of the contents of effective components in the brain and the MDCK-MDR1 cell model assessment of the intracerebral blood-brain barrier will be investigated. On the basis of all above results, the blood-brain barrier changes and mechanisms related to Ligusticum Chuanxiong application will be clarified, which might explain its role of “lead-in upstream”. These researches will provide new ideas and methods for in vivo drug analysis, pharmaceutical substance discovery, rational application of Ligusticum Chuanxiong, theoretical explanation of traditional Chinese medicine.

川芎为治疗头痛圣药，上行头目，川芎对药（大川芎方、芎芷散、经验方）治疗偏头痛疗效显著，但限于脑部药效成分检测浓度低和成分间相互作用复杂，川芎对药药效物质及如何“引药上行”尚未阐明。本项目针对入脑川芎对药药源小分子化合物（包括原型及代谢物）检测难点，建立超临界色谱-质谱联用（包括化学衍生增敏）及荧光检测等超高灵敏检测方法，系统筛查、定量测定入脑药源成分，突破入脑药效物质检测难点，进一步进行靶器官-血浆-药材“反向溯源”分析，并结合分子对接虚拟筛选和活性验证确定川芎对药治疗偏头痛的药效物质基础。在此基础上，结合脑组织内效应成分分析结果和体外拟血脑屏障MDCK-MDR1等细胞模型研究，弄清川芎促进对药效应成分的数量、含量变化规律及配伍川芎效应成分引起透过血脑屏障变化及机制，阐明川芎“引药上行”科学内涵。研究成果将为体内药物分析、药效物质发现、川芎合理应用、中药用药理论阐释提供新思路及方法。

川芎为治疗头痛圣药，从对药（大川芎、芎芷、芎芍）角度研究其药效物质及增效机制，弄清其含成分体内暴露量、活性及成分间“引药上行”作用具有应用及理论价值，本项目进展如下：①建立了拟靶向鉴定、荧光检测、超临界流体色谱等新方法，鉴定大川芎218个化学成分，含量高的成分依次为天麻素、洋川芎内酯I、阿魏酸、巴利森苷、洋川芎内酯A等；芎芷62个香豆素类化合物，欧前胡素含量最高，氧化前胡素、异欧前胡素、佛手柑内酯等次之；芎芍中82个化合物，主要为芍药苷类和鞣质类成分。②通过示差扫描识别发现大川芎入血成分90个、入脑19个，阿魏酸、洋川芎内酯A、正丁基苯酞、藁本内酯、洋川芎内酯I暴露量较高，为潜在起效成分。苯酞类成分可转化为羟基化苯酞类，易发生氧化、脱氢、谷胱甘肽、半胱氨酸等代谢；芎芷入血成分95个、入脑51个，欧前胡素、氧化欧前胡素等暴露量较高，香豆素类成分主要以原型入血；白芍入血成分12个、入脑成分未检测到，芍药苷类及鞣质类在胃肠道易发生酸、碱、酶水解代谢。③通过网络药理学和分子对接筛选到大川芎17个活性成分，涉及神经调节、血管作用、抗炎等14条通路；芎芷41个活性成分，涉及血液循环、化学突触传递等20个通路；芎芍32个活性成分，涉及氮素代谢、TNF信号等20个通路；实验验证了大川芎方中藁本内酯、欧当归内酯A等活性成分对神经和内皮细胞具有保护作用。④从药动学行为比较发现，配伍川芎后，白芷入脑的香豆素类成分水合氧化前胡素、佛手柑内酯、欧前胡素及异欧前胡素的入脑药峰浓度Cmax分别增加43.85%、51.33%、81.83%和95.15%，AUC也分别增加107.35%、75.50%、186.26%和78.81%，川芎可促进白芷中香豆素类化合物透过血脑屏障的吸收速度和吸收程度发挥“引药上行”作用。通过小鼠脑微血管内皮细胞体外血脑屏障细胞模型通透性研究发现，川芎中主要活性成分（藁本内酯+洋川芎内酯A、洋川芎内酯I+H）可促进天麻素透过血脑屏障。本项目共发表文章9篇，其中2篇SCI论文，7篇中文核心期刊论文；培养博士后1名，博士1名，硕士6名。