基于药物代谢动力学和网络药理学的回药复方白蜜汤配伍机制研究

“Bai Mi Decoction” (BMD), a characteristic Hui aroma medicine formula consisting of four medicinal herbs including Crocus sativus, Eugenia caryophyllata, Myristica fragrans and artificial Moschus, was used to treat cerebral infarction recorded specifically in ancient Hui Herbal Classic “Hui Hui Medicine Formulary”; and this traditional medicinal use was also proved by the clinical trial data of Ningxia Hui Hospital and our previously experimental animal results. In present study, the method of pharmacokinetic and network pharmacology will be used to analyze the compatibility mechanism of the BMD: LC-MS and GC-MS will be applied to determine the chemical constituents, blood and brain components of BMD before and after combination. The middle cerebral artery occlusion (MCAO)/reperfusion of the rat will be employed to compare the neuroprotective activities of BMD before and after combination. In pharmacokinetics study, both vivo and vitro methods will be used to observe the kinetics of main bioactive components: 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 these bioactive; in vivo study, the pharmacokinetics characters of the main active constituents will be analyze. Then, network pharmacology and molecular docking technology will be employed to identify the targets of the main active constituents of BMD on cardiovascular disease, a “component-target-effect” network diagram will be established to find the possible neuroprotective mechanism of BMD, and proper molecular biology techniques including western-blot and Elisa will be used to validate the targets. Finally, the compatibility mechanism of the BMD will be clarified by the results of phytochemistry, pharmacology, pharmacokinetics and network pharmacology.

白蜜汤是《回回药方》中明确记载用于治疗脑中风的回药特色香药复方，主要由“撒法郎（西红花）、丁香、肉豆蔻、桑吉木宝乞（麝香，临床用人工麝香）”四味药组成，经宁夏回医医院临床试验和课题组前期动物试验证实此方防治脑卒中有效。本课题中，我们以代谢动力学和网络药理学为切入点解析白蜜汤的配伍机制：采用LC-MS和GC-MS分析白蜜汤组方药材配伍前后化学成分、入血成分和入脑成分；以MCAO致大鼠缺血性脑卒中为模型比较配伍前后药效学的差异；体外分析组方药材有效成分透膜吸收能力、透血脑屏障能力和代谢途径及代谢产物的差异，体内考察主要有效成分药代动力学特点；网络药理学和分子对接技术虚拟筛选有效成分对应心脑血管疾病的作用靶点，构建白蜜汤“组分-靶标-效应”网络图，并通过分子生物学技术对关键靶点蛋白进行验证；综合以上化学物质基础、整体动物药效学、体内外代谢动力学和网络药理学结果，阐明白蜜汤的配伍规律和作用机制。

白蜜汤是《回回药方》中明确记载的用于治疗脑中风的回药特色香药复方，前期动物试验和临床试验证实防治脑卒中有效。本课题中，第一部分采用LC-MS和GC-MS分析白蜜汤组方药材配伍前后化学成分、入血成分和入脑成分，在单味药材丁香、肉豆蔻、麝香、西红花和复方白蜜汤提取物中分别鉴别了23、19、18、13和55个化学成分，模型鼠入血成分分别鉴定了4、12、5、8和27个，模型鼠入脑成分分别鉴定了7、12、6、5和23个，且正常和模型鼠入血及入脑成分有差异。白蜜汤入脑成分主要为2-Aminopyrimidine-1-oxide，缬氨酸，烟酰胺，尿嘧啶，反式-对-羟基-肉桂酸，苯丙氨酸，丁香苷Ⅰ，西红花酸，乙酰丁香酚，meso-monomethyldihydroguaiaretic acid，榄香素，丁香酚，fragransin C1，5'-methoxydehydrodiisoeugenol，肉豆蔻醚，3-羟基-β-紫罗兰酮，dihydrocarinatidin，麝香酮，茴香脑，西红花酸单酯，十八酰胺，樟脑。第二部分以MCAO致大鼠缺血性脑卒中为模型比较白蜜汤配伍前后药效学的差异，结果显示白蜜汤各组方单味药材能够明显减少脑梗死体积，减轻缺血性大鼠大脑皮质层神经细胞的损伤，保留较完整的神经元数目，并且复方白蜜汤组的染色结果优于其他单味药和有效成分组，趋于正常组，体现了白蜜汤对神经细胞具有保护和修复作用，初步说明了4味中药配伍之后具有协同增效的作用。第三部分体外分析了组方药材有效成分透膜吸收能力，结果显示除西红花苷Ⅰ外，其余3种成分榄香素、麝香酮和丁香酚具有良好的透膜吸收能力，且成分配伍后挥发性成分榄香素、麝香酮和丁香酚对西红花苷Ⅰ具有促透膜作用，西红花、麝香、丁香和肉豆寇4味中药合用药时的透膜能力强于单体成分合用之后的透膜能力，由此猜测可能是由于药材中其他成分也能够促进其吸收；体内药代动学结果显示配伍给药后能显著的提高其单独给药后的血药浓度，即具有协同作用。第四部分针对白蜜汤的23个入脑成分进行了心脑血管靶点蛋白的筛选，筛选到25个靶点分别作用于血栓形成过程，脂质代谢 (HMGCR，ADRB)，血压等，并且西红花主要通过脂质代谢和凝血系统作用于HMGCR，ADRB和凝血因子等，23个成分在通过不同的通路起协同增效作用。