圆齿野鸦椿抗炎活性成分色原酮碳苷的发现及构效关系研究

Euscaphis fukienensis，belonged to Staphyleaceae family, was a tradition Chinese medicine, named Yeyachun, which distributed in south of China. We have confirmed that the extract of pericarps of E. fukienensis was able to decreased the dimethylbenzene-induced ear edema in rats, which was similar to the inhibitory activity of dexamethasone. Thirty-one compounds, including chromone C-glycosides (CCGs), flavonoids, triterpenes, have been isolated from the extract and CCGs were exhibited appropriate activity. Biflorin, a representative CCGs, has been shown significant anti-inflammation activity and good dose-effect relationship in vivo and in vitro. More impressive, its anti-inflammation activity was seven times better than the effect of ibuprofen. Therefore, the research project, the discovery and Structure-activity relationship (SAR) of the CCGs for anti-inflammation from E. fukienensis, would be put into practice. HPLC-DAD-MS and NMR technologies would be used for quickly finding the CCGs. Plenty of CCGs, especially microconstituents, would been isolated by kinds of chromatographic technique and identificated by spectroscopy technology from the extract of E. fukienensis. Further quantity CCGs would derive from the modification of biflorin. All of the CCGs would be tested their anti-inflammation activities. If the research project implemented successfully, it would be contributed to the illumination of SAR for CCGs, development and utilization of E. fukienensis.

圆齿野鸦椿是省沽油科野鸦椿属植物，资源丰富。前期发现其果皮提取物对二甲苯致小鼠耳肿胀有明显改善作用，活性与地塞米松相当；活性追踪并分离化合物31个，色原酮碳苷类成分抗炎活性好，代表性成分biflorin体内外抗炎活性显著，是阳性对照药布洛芬的7倍，并呈现良好剂量关系。因此，本项目将以色原酮碳苷类的发现及抗炎活性为中心，通过增加圆齿野鸦椿果皮的投入量，运用HPLC-DAD-MS及NMR技术快速富集色原酮碳苷类成分，采用各种色谱方法和光谱学技术分离、鉴定色原酮碳苷类成分（特别是微量成分），并对 biflorin进行结构修饰，构建色原酮碳苷化合物库；结合脂多糖（LPS）诱导小鼠巨噬细胞RAW264.7炎症模型进行抗炎活性评价，阐明色原酮碳苷类成分抗炎构效关系，并优选2-3个结构新颖、活性较强的化合物进行体内药效学验证。本项目的顺利实施，将为圆齿野鸦椿药用植物资源开发和利用提供重要的理论依据。

圆齿野鸦椿隶属省沽油科野鸦椿属，是福建特色珍稀药赏两用植物，资源丰富。其提取物对二甲苯致小鼠耳肿胀有明显改善作用，活性与地塞米松相当，色原酮碳苷类成分biflorin体内外抗炎活性显著，呈现良好剂量关系。因此，本项目对圆齿野鸦椿的化学成分及生物活性展开了深入的研究。.化学成分研究方面，以色原酮碳苷类成分及生物活性为中心，运用HPLC-DAD-MS及NMR技术快速富集色原酮碳苷类成分，采用各种色谱方法和光谱学技术从圆齿野鸦椿中分离、鉴定80余个化合物，其中，新化合物4个，70个成分为首次从该植物中分离得到，并构建了野鸦椿化合物库；开发了biflorin、isobiflorin等在内的10个单体的制备工艺及产品，优化了野鸦椿类花青素、籽油、黄酮等生产工艺；构建了野鸦椿化学指纹图谱和HPLC含量测定方法。.生物活性研究方面，发现biflorin和isobiflorin对DPPH自由基、ABTS自由基、超氧阴离子具有清除作用，并能还原Fe³+,且isobiflorin的还原能力强于阳性药BHT。2个化合物在0-100mg/mL范围内能明显抑制H2O2导致L02细胞活力、提高ROS清除能力，具有剂量依赖性，并能提高细胞内抗氧化因子HO-1、PPAR-γ、Nrf2、NQO-1的 mRNA表达水平升高并增加其蛋白表达量，提示他们具有良好的抗氧化活性，对过氧化氢诱导肝细胞损伤的保护作用，可能与其激活抗氧化信号通路相关蛋白和基因的表达相关。EA在野鸦椿中含量大，且能抑制HepG2细胞增殖、诱导其细胞凋亡及引起细胞G0/G1期阻滞，且随EA浓度的递增，HepG2细胞划痕的愈合能力逐渐减弱，穿过小室的细胞数目也逐渐减少，而增大EA的给药浓度，可逐渐上调HepG2细胞中E-Cadherin的蛋白和mRNA表达，而下调N-Cadherin、Vimentin、MMP-2和MMP-9蛋白和mRNA的表达，提示EA可显著抑制肝癌细胞的侵袭和迁移，且与抑制HepG2的EMT进程，降低金属基质蛋白酶MMP的表达有关。.截止目前，本项目已按原定计划顺利完成目标，发表中文核心以上论文21篇，其中,SCI论文11篇，CSCD论文9篇；申请国家发明专利5项，授权2项，转让2项，为圆齿野鸦椿药用植物资源开发和利用提供重要的理论依据。