银莲花属植物共有抗炎活性成分及对人中性粒细胞氧化磷酸化的影响机理研究

Anemone is the biggest genus of Ranunculaceae and are rich in its many kinds of medicinal plants. Large amount of recoders in the ancient literatures and modern research have proved that most species of this genus have almost the same pharmacological action such as anti-inflamatory and antipyretic. In the past research work, we had deeply studied the rhizome of Anemone raddeana Regel which was included in Chinese Pharmacopeia for curing rheumatism and anti-inflamation. More than 30 compounds were isolated and identified from this herb medicine. We also set up the HPLC finger method for the quantitative and qualitative analysis of it and deeeply investigated the effects of the pure compounds on anti-inflamation and superoxide generation and phosphorylation in human neutrophils. Superoxide generation has been proved playing a important role in inflammatory reaction of body. Neutrophils' functions are essential for host denfence against injurious agents by phagocytosis. During phagocytosis, superoxide generation is stimulated that will cause inflammation. A variety of pro-inflammatory stimuli can prime neutrophils and result in one-electron reduction of molecular oxygen by NADPH oxidase leading to ``respiratory burst``. The catalytic core of the NADPH oxidase is cytochrome b558, an integral membrane protein consisting of gp91phox , p22phox and other subunits. The enzyme is inactive in the absence of three cytosol proteins p47phox, p67phox and Rac. Our research work has showed that some compounds from Anemone raddeana can strongly inhibited superoxide generation and phosphorylation in human neutrophils and possessed anti-inflamatory activity. They can dose-dependently inhibited some stimuli-induced tyrosyl phosphorylation of 45 kDa proteins in human neutrophil, and were parallel to their anti-inflamatory activity. In this work, we will use modern chromatograph methods to analyze some important and source-riched Anemone spieceis growing in northeast of China to decide the same and different components. Then to extract and isolate their chemical components, especially focus on the different components. By means of animal pharmacology test and in vitro work to find the compounds with more stronger anti-inflamatory activity, then to evalute the active compounds' effect on superoxide generation and phosphorylation in human neutrophils. Investigate the molecular mechanism of anti-inflamation especially the mechanism of translocation through cell membrane of some protein factor and G protein Rac2 which were the important part components of the NADPH oxidase， so as to clarify the relationship between inflammation and protein phosphorylation in neutrophils. These work can afford scientific data for the anti-inflamatory drug development from Anemone genus.

银莲花属（Anemone）是毛茛科中最大的一个属。历代本草记载及现代研究证实本属植物大多具有相近的抗炎，解热，镇痛等功效。本课题将首先运用现代色谱分析技术对银莲花属多种植物的化学成分进行定性、定量分析，在前期对多被银莲花研究工作的基础上确定本属植物的共有成分及不同成分。以抗炎活性为导向，对活性成分尤其是共有成分进行深入的分离纯化。并对活性成分进行构效关系分析，以筛选出活性更强的化合物及有效部位。采用与炎症密切相关的人中性粒细胞过氧化和蛋白络氨酸磷酸化实验结合整体动物药效试验来评价有效成分，活性部位（群）的抗炎作用分子机制，探讨活性成分对NADPH氧化酶中某些蛋白因子和小型G蛋白Rac2跨膜转运的影响机理。课题对阐明银莲花属药用植物资源共有的抗炎活性的物质基础，探讨炎症与人中性粒细胞蛋白磷酸化的关系具有重要意义。并可为银莲花属丰富的中药资源用于抗炎及相关药效药物的研发提供科学依据。

银莲花属（Anemone）药用植物大多具有相近的抗炎和抗肿瘤等作用。本课题以抗炎和抗肿瘤活性为导向，对共有活性成分进行了深入研究。采用与炎症密切相关的人中性粒细胞过氧化和蛋白络氨酸磷酸化及抗肿瘤等实验对所获得的单体化合物活性进行了确认。通过课题研究确定了银莲花属植物中齐墩果酸型皂苷元仅存在3位糖链时，尤其是具有3-O-α-L-rhamnopyranosyl-(1→2)-α-L- arabinopyranoside片段时往往具有显著的生物活性，这也是本属植物共有抗炎活性成分特征，而当齐墩果酸型皂苷元的28位再链接有糖链时其生物活性显著降低或者较弱。在此基础上，筛选出了具有显著抗炎和抗肿瘤活性的化合物22和两头尖次级总皂苷，并对它们的药动学行为和体外肠道菌代谢转化进行了研究。课题对阐明银莲花属药用植物资源共有的抗炎活性物质基础，探讨其抗炎物质基础与人中性粒细胞蛋白磷酸化的关系具有重要意义。并可为银莲花属丰富的中药资源用于抗炎及相关药效药物的研发提供科学依据。