基于代谢组学的骨疏丹多组分、多靶点整合分析方法研究

The anti-osteoporosis therapeutic basis of Gushudan and its action mechanism were further studied by the multi-constituents and multi-targets substances and metabolomics analysis, based on the preliminary researches. The various modern analytical technique , such as ultra high performance liquid chromatography tadem mass spectrometry (UPLC-MS/MS) and so on, has been applied to study the chemical.constituents of Gushudan in vitro and in vivo, and to investigated the change of endogenous metabolite profiles in rat plasma after administration of Gushudan in order to find some related biomarkers. The potential therapeutic basis of Gushudan will been screened out by investigating the pharmacokinetic characteristics of its multiple constituents in vivo with quantitative analysis method. In addition, the.anlysis method based on LC-MS which will be fully developed and validated to detetect all kinds of possible metabolites in vivo,which is the difficult problems existing in metabolomics study of tradition compound Chinese medicine. This study is a new fundamental work for investigating the therapeutic basis of the anti-osteoporotic tradition compound Chinese medicine, and provides a new techinuqe platform for the exploration and modernization of the study on the therapeutic basis and its action mechanism of traditional compound Chinese.medicine.

本项目以多组分、多靶点整合代谢组学策略深入研究骨疏丹在体内发挥精准医疗作用的物质基础和作用靶点：1.基于LC/MS技术的代谢组学检测方法研究：开发骨疏丹体内、外化学物质组复杂组分快速检出与鉴定的分析检测技术；采用亲水液相色谱法用于极性代谢物的检测，表面分子印迹技术结合固相萃取等样品前处理方法对低丰度的核苷类代谢物进行富集，提高低丰度代谢物的检测灵敏度。2.骨疏丹多组分、多靶点整合代谢组学研究：应用UPLC/MS等多种检测技术，深入研究骨疏丹体内、外物质组，进行体内多组分整合药代动力学研究；研究骨疏丹作用于模型动物后所引起的机体的内源性代谢物的变化，寻找相关的生物标记物；研究生物标记物定量变化与药效、体内物质组体内过程的相关性。本项目探索出一个由样品预处理技术及LC/MS、GC/MS等多种分析技术整合的代谢组学研究平台，为复方中药的代谢组学研究提供一个示范性的新分析检测技术体系。

本项目以多组分、多靶点整合代谢组学的研究策略，深入研究骨疏丹发挥抗骨质疏松作用的物质基础和作用机制：①建立UHPLC-FT-ICR-MS结合UHPLC-MS/MS法，发现了骨疏丹中73个黄酮苷类、香豆素类、色原酮类等化合物；②建立骨疏丹HPLC-DAD指纹图谱，采用一测多评法同时测定12种有效成分；③采用UHPLC-MS/MS法在大鼠含药血浆中检测到淫羊藿苷、蛇床子素、丹参酮IIA等29个原型成分和7个代谢物，进一步研究了9个入血移行成分在大鼠体内的动态变化规律；④采用网络药理学的方法构建了“骨疏丹中药复方-活性成分-靶点-通路”的加权网络；⑤分别建立了HILIC-UHPLC-MS/MS法用于大鼠血浆和尿液中19种氨基酸、大鼠脑组织中4种极性单胺类神经递质、大鼠尿液中牛磺酸、次黄嘌呤和胞嘧啶等5种极性潜在生物标志物的定量测定方法，应用于定量代谢组学研究中；⑥建立一种基于Fe3O4/GO/DMIPs磁固相萃取的MSPE-UHPLC-MS/MS法同时选择性分离检测大鼠脑组织中的多巴胺、肾上腺素和去甲肾上腺素；⑦分别对大鼠血浆、尿液、粪便和肾组织等不同生物样本，开展了基于UHPLC-MS、GC-MS、1H-NMR和ICP-MS不同检测技术的、骨疏丹抗骨质疏松作用的整合代谢组学研究，共发现了与GIOP相关的苯丙氨酸、牛磺酸、鞘氨醇、次黄嘌呤等100余种不同种类的潜在生物标志物，主要通过影响氨基酸、脂质、核苷酸、胆汁酸等代谢途径紊乱从而引发氧化应激失衡、能量代谢障碍、肠道菌群失调等而导致骨质疏松；骨疏丹能通过调节上述代谢通路起到预防糖皮质激素性骨质疏松的作用。还发现Zn、Mn、Se、Fe 4种微量金属元素与骨质疏松的发病关系密切。本项目充分发挥多种检测技术的优势与互补性，构建一个由样品预处理及LC-MS、GC-MS和NMR等多分析技术整合的研究平台，为复方中药药效物质基础和作用机制一体化研究提供示范作用。发表SCI论文8篇，北大核心期刊论文2篇，参加国内学术会议交流5次；培养博士研究生1名，硕士研究生9名。