基于“组分-代谢标志物-效应”关系研究回药蜜煎菖蒲方配伍规律

The traditional Chinese Medicine and minority medicine, for more than a millennium, have been extensively used to alleviate various symptoms of diseases by our Chinese medicine practitioner based on the theory of TCM. “Mi Jian Chang Pu”, a classic Hui Medicine formula consisting of only two medicinal herbs including Crocus sativus L. and Acorus tatarinowii Schott, was recorded in 《Hui Hui Medicine Formulary》and used to treat cerebral infarction for many years, and this traditional medicinal use was also proved by the clinical trial data and our previously experimental animal results. However, the total chemical constituents of the formula and the relationship between the TCM and endogenous metabolites are not clear. In the present study, the compatibility mechanism of “Mi Jian Chang Pu” will estimate by techniques of pharmacokinetics and metabonomics based on the modern clinical drug trials in humans and our previous experiments in rats. Instruments such as LC-QTOF-MS and GC-MS will be utilized to identify the chemical constituents of Crocus sativus L. and Acorus tatarinowii Schott as well as their main metabolites in the blood and brain before and after combination. In pharmacolinetics study, both vivo and vitro methods will be used to observe the kinetics of main bioactive components and medicinal plant before and after combination: in vitro experiment, Caco-2 absorption model and blood-brain barrier model will be applied to evaluate the transport ability, and rat liver microsome to analyze the metabolism enzymes of the main components; in vivo study, the pharmacokinetics characters of the main constituents will be analyze. In pharmacodynamics study, the middle cerebral artery occlusion (MCAO)/reperfusion of the rat will be employed to compare the neuroprotective activities of “Mi Jian Chang Pu” before and after combination; meanwhile, the metabonomics will be applied to find out the possible compatibility mechanism of “Bai Mi Decoction” by discerning the endogenous metabolites in rats and ascertaining the neuroprotective effect of the formula on their metabolism, a “component/formula-metabolic markers-effect” network diagram will be established to find the possible neuroprotective mechanism of “Mi Jian Chang Pu”. Taken together, the compatibility mechanism of “Mi Jian Chang Pu” will be clarified by the results of phytochemistry, in vitro and in vivo pharmacolinetics, pharmacodynamics and metabonomics. We believe that our project will supply a novel idea and method to elucidate the compatibility of minority medicine of Ningxia.

蜜煎菖蒲方是《回回药方》治疗脑中风的经典复方，由“撒法郎（西红花）”和“石菖蒲”组成，经临床和课题组前期动物试验证实治疗脑卒中有效，但这两味药配伍治疗脑卒中的物质基础未见报道，其配伍规律尚不明确。因此，本研究拟以复方化学物质基础为切入点，利用LC-QTOF-MS和GC-MS对石菖蒲和西红花配伍前后原型、入血和入脑成分及代谢物进行整体辨识，明确配伍的化学物质基础；以体外透膜吸收、透血脑屏障和代谢途径分析结合体内药代动力学参数，进一步明确配伍活性物质基础，解析配伍代谢动力学机制；以大鼠MCAO致缺血性脑卒中为模型，以LC-MS代谢组学为手段，考察配伍药效作用和对缺血性脑卒中生物标志物的调控作用和途径。综合以上化学物质基础、体内外代谢动力学、整体动物药效学和代谢组学结果，构建蜜煎菖蒲方主要成分和药味的“组分-代谢标志物-效应”关系图，阐明其配伍规律，为宁夏民族药复方配伍规律的研究提供思路和方法。

项目以蜜煎菖蒲方为研究对象，围绕西红花和石菖蒲配伍前后体内外化学成分、药代动力学和代谢物特点，研究其配伍规律。采用LC-MS/MS结合自建数据库（收录西红花和石菖蒲化学成分310个）分析复方配伍前后体内外化学成分，在西红花、石菖蒲和蜜煎菖蒲方中分别鉴定了11、29和24个化学成分，在脑卒中模型鼠入血成分中分别鉴定了10、16和12个，在模型鼠入脑成分中分别鉴定了7、9和16个，除确证了复方在正常和模型鼠体内的成分组成不同外，还发现原本在西红花单味药模型大鼠脑组织中检测不到的5种成分，包括西红花酸、克罗西汀-甲基酯、番红花I、番红花醛和3-羟基-4-甲氧基苯甲酸，在复方入脑成分中检测到了，推测复方配伍后能够促进西红花中的极性较大的成分透过血脑屏障。紧接着，采用体外Caco-2细胞模型分析指标成分透膜吸收能力，发现西红花苷Ⅰ的透膜吸收率较低，β-细辛醚较高，两者合用时西红花苷Ⅰ的透膜吸收率明显增加，同时复方较单味药更能促进成分的透膜吸收率和吸收量。同步地，采用线栓法制备大脑中动脉闭塞（MCAO）脑卒中模型大鼠进行体内药代动力学研究，发现西红花苷Ⅰ和β-细辛醚合用后各自的血药浓度显著高于单独给药的血药浓度，同时复方较单味药显著提高了西红花苷Ⅰ和β-细辛醚的血药浓度。进一步地，借助LC-MS代谢组学技术分别在MCAO脑卒中模型大鼠血清和脑组织中分别筛选到了40和21种差异性代谢物，它们主要参与泛酸盐和辅酶A生物合成、氨基酸代谢、酮体的合成与降解等通路；多元统计结果显示模型鼠给予复方后的差异性代谢物较单体成分和单味药更趋近于假手术组，即石菖蒲和西红花配伍后能够协同调控代谢标志物使之趋近于正常组水平，从而达到协同增效的作用。综上，蜜煎菖蒲方配伍后促进了有效成分的入血和入脑，同时能够协同调控差异性代谢物，从而达到协同增效的作用。通过本课题研究，已发表SCI收录论文4篇，授权专利2项，登记软件著作权1项，培养研究生2名和青年骨干教师1名。