基于二维"组分-成分-效应关系"的蒙药苦地丁体内外抗炎物质基础及作用机制研究

Corydalis bungeana is a commonly used medicinal material of Mongolian medicine, which has the effect of heat-clearing and detoxifying as well as dissipating binds and dispersing swelling. Although it had been used in many compound preparations, its anti-inflammatory material basis and mechanism of action were not clear. In this work we will study the anti-inflammatory material basis of Corydalis bungeana in vivo/in vitro and its mechanism of action systematically in interdisciplinary perspective way. This project is aiming to: (1) Prepare flavonoids and alkaloids components by middle pressure preparative system and auto-purification system; study the mechanism of anti-inflammatory effect of Corydalis bungeana and its components using the cells in vivo/in vitro model and the whole animal model. That is called the one-dimensional component-effect relation.(2) On the basis of the one-dimensional component-effect relation, investigate chemical components and their anti-inflammatory effect mechanism; analyze the impact of mRNA and protein level on the expression of iNOS as well as the impact of active ingredients on NF-kB signal channel. Therefore, we can study the anti-inflammatory effect mechanism of Corydalis bungeana's active ingredients at the cellular and molecular levels; reveal the two-dimensional ingredient-effect relations; speculate the in vitro anti-inflammatory material basis can be illuminated.(3) Analyze the metabolites of animals after administration by UPLC-PDA-QTOF-MS and investigate chemical compositions in vivo and the metabolic pathway; explore the main law of action of Corydalis bungeana's active ingredients and clarify the in vivo anti-inflammatory material basis combining pharmacokinetics.

苦地丁为蒙医常用药材，可清热解毒，散结消肿，虽在多种复方制剂中有应用，但其抗炎物质基础和作用机制还模糊不清，尚待研究。本项目拟采用学科交叉的方法对苦地丁的体内外抗炎药效物质基础及机制进行系统性研究。采用中压制备和自动纯化系统制备黄酮类和生物碱类两类组分，借助体内外细胞模型和整体动物模型研究苦地丁及其组分的抗炎药效机制，此为一维组分-效应关系；在此基础上，进一步研究化学成分及其抗炎药效机制、分析mRNA和蛋白水平对iNOS表达及活性成分对NF-kB信号通路的影响，从而在细胞和分子水平上研究苦地丁活性成分的抗炎机制，揭示二维成分-效应关系，阐明苦地丁体外抗炎物质基础；采用UPLC-PDA-QTOF-MS分析动物给药后体内代谢产物，研究体内化学成分群组成和代谢途径，结合药代动力学研究，探究活性成分的主要作用规律，明确体内抗炎作用物质基础。

苦地丁为蒙医常用药材，可清热解毒，散结消肿，虽在多种复方制剂中有应用，但其抗炎物质基础和作用机制还不清楚，本项目研究清楚了苦地丁的抗炎物质基础和作用机制。化学成分为抗炎物质基础作用机制的研究奠定了基础。首先，建立了苦地丁的 UPLC 指纹图谱分析方法，以柱色谱技术分离制备了苦地丁中的7种化学成分单体；再次，以分离得到的化学成分为基础，采用LC-MS分析并鉴定苦地丁中化学成分，共鉴定了18个化学成分，揭示了苦地丁的体外化学成分群组成；进而以LC-MS分析鉴定了大鼠给予苦地丁药材后，血、胆汁、尿和粪便中化学成分的原型和代谢产物，揭示苦地丁体内代谢特征及体内化学成分群的组成。.对于苦地丁的抗炎药效及作用机制，首先，以脂多糖诱导的RAW264.7小鼠巨噬细胞和小鼠内毒素休克模型研究了苦地丁提取物的抗炎作用及机制，苦地丁的提取物对脂多糖诱导的 RAW 264.7 小鼠巨噬细胞产生的炎症介质和炎症因子具有抑制作用，其抗炎作用的分子机制与抑制NF-κB信号通路的活化有关系，进一步采用LPS诱导的小鼠内毒素休克模型验证了苦地丁的抗炎作用和机制。其次，以自动纯化系统制备了苦地丁中的生物碱组分和含黄酮的其它组分，进行质谱鉴定，进而以RAW264.7小鼠巨噬细胞研究组分的抗炎药效，结果表明生物碱组分是苦地丁的主要抗炎药效活性组分，进一步在炎症信号通路水平上研究了生物碱组分的抗炎作用机制。最后采用RAW264.7小鼠巨噬细胞研究苦地丁中化学成分单体的抗炎药效及作用机制，体外实验表明紫堇灵和二氢血根碱具有一定的抗炎药效，紫堇灵和二氢血根碱能显著抑制炎症上游通路TLR4、TLR2的蛋白表达水平（对TLR4表现出更强的抑制效果），及下游NF-κB信号通路IκBα、p65的磷酸化水平，抑制作用与给药剂量呈正相关。