鼠曲草防治类风湿关节炎的药效物质基础及作用机制

Rheumatoid Arthritis(RA)is referred to a kind of auto-immune disease. There are approximate 4 million sufferers in China and the illness frequency rate is between 0.32%-0.4% in China. The cause of the illness is still unknown. Recently, medicines such as glucocorticoid nonsteroidal anti-inflammatory drugs, Syrrkptom modifying antirheumatic drugs, disease modifying antirheumatic drugs, biologicals were widely used for the treatment of RA. But these medicine or treatment often caused side reaction, untoward reaction and toxicity reaction to the patients, Therefore, alternative medicine which was based on complementary or altemative medicine is more likeable by patients now. Gnaphalium affine D. Don belongs to genus Gnaphalium, family compositae. Phytochemical and pharmacoligical investigations of this plant were rarely reported. In our previous studies, the crude extract of Gnaphalium affine D. Don exhibited strong complement modulation properties on the classical pathway of the complement system in vitro. In vivo, the crude extract of G. affine D. Don showed a significant inhibition of oedema on carrageenan-induced paw oedema in mice and on type II collagen-induced rheumatoid arthritis in rat. Therefore, this study provides theoretical foundation for investigating the active ingredients. However, the problem we are facing now is that we are ignorance about what truly works and what does not. In view of the rich resources of Gnaphalium affine in our country and the previous research work, our group decides to continue the isolation of the chemical constituents and set up the sample library composed of the small molecule compounds and effective parts for the activity screening using a variety of in vitro cellular and molecular model. The pharmacological effect and action mechanism would be further verified by the CIA rat. These results will provide the scientific basis for further development and utilization of Gnaphalium affine

鼠曲草为菊科鼠曲草属植物鼠曲草（Gnaphalium affine D.Don）的干燥全草，我国药食两用植物，国内对其药理作用的研究报道较少，化学成分方面的研究更是罕有报道。本课题组首次发现其提取物具有多途径、多靶点防治类风湿关节炎作用，可极显著抑制II型胶原关节炎大鼠足趾肿胀率，降低血清及关节浸液内TNFα和IL-1β水平，并具有显著抗补体活性。鉴于鼠曲草在我国资源较为丰富，课题组拟在前期工作基础上，对鼠曲草正丁醇部位化学成分进一步分离，构建小分子化合物及有效部位库，从机体免疫和抗炎两条途径，采用多种体外细胞和分子模型进行筛选评价，寻找具有重要价值的防治类风湿关节炎的活性成分群及单体化合物，在进行整体药效验证的基础上深入探讨作用机制，为进一步从天然产物中开发高效、低毒的新型药物提供科学依据，并为进一步开发利用我国鼠曲草这一植物资源提供资料。

鼠曲草为菊科鼠曲草属植物鼠曲草（Gnaphalium affine D.Don）的干燥全草，我国药食两用植物，国内对其药理作用的研究报道较少，本项目选择鼠曲草为研究对象，通过硅胶柱层析和葡聚糖凝胶柱层析等现代色谱技术和方法，从鼠曲草的乙醇提取物中共分离得到了67个化合物。根据理化性质和现代波谱技术(1H-NMR, 13C-NMR, 2D NMR, IR, ESI-MS, HRESI-MS)并辅以化学方法进行结构鉴定，发现5个新化合物和19个从鼠曲草属中首次分离得到的化合物。体内活性研究发现，鼠曲草总提物具有较好的预防角叉菜胶所致的小鼠急性炎症作用，对Ⅱ型胶原诱导的大鼠类风湿性关节炎具有显著的防治作用。进一步通过补体和抗炎信号通路研究发现，鼠曲草总提物及代表性的酰化黄酮苷类成分能够抑制NF-κB信号通路的激活和通过抑制C3，C4和C5发挥补体抑制作用。在明确鼠曲草活性成分的基础上，本研究选用HPLC-VWD方法进行了鼠曲草指纹图谱的研究，建立了指纹图谱检测方法。该方法操作简便，稳定性、重现性、精密度均较好，具有较好的适用性。同时，本研究还建立了多成分含量测定方法，考察发现不同产地的鼠曲草药材存在品质差异。为探究其化学多样性的原因，我们使用ISSR生物标记技术对不同产地的17份鼠曲草样品及2个近缘种进行了遗传多样性的研究。并对化学多样性和遗传多样性的UPGMA聚类进行了相关性分析，发现二者的聚类结果不完全一致。本研究为鼠曲草药材品质评价提供了依据。由前期药理活性研究结果可知，鼠曲草的黄酮类化合物尤其是酰化黄酮类化合物有着较为显著的抑制补体和抗炎活性，因此为了进一步对鼠曲草药材进行开发和利用，我们针对鼠曲草中活性较好的黄酮类成分进行相关制备研究，以6种黄酮类化合物的提取率为指标，经回流提取和大孔树脂纯化制备得到了黄酮部位，研究发现黄酮部位具有较强的抗炎和抗补体活性，机制研究发现黄酮部位主要是通过抑制IκBα的泛素化达到抗炎作用以及通过抑制C2,C3和C4表达实现补体抑制活性。