枳壳中黄酮类化合物对活化的小胶质细胞抑制作用的构效关系研究

Structure-activity Relationship of Flavonoids from Fructusaurantii on Inhibiting Activation of Microglia Cells

目的从枳壳黄酮组分中分离纯化得到7种化合物,并探讨这7种化合物抑制脂多糖(LPS)诱导的原代小胶质细胞激活的构效关系。方法采用乙醇回流提取法提取及现代色谱技术获得橙皮素、红橘素、川陈皮素、柚皮素、橙皮苷、新橙皮苷、柚皮苷。采用MTT分析法检测小胶质细胞的细胞存活率,ELISA法检测LPS激活的小胶质细胞TNF-α释放的变化。结果在1~200μM浓度范围内,7种黄酮类化合物对小胶质细胞存活率无显著影响;预处理4 h后4种黄酮苷元具有明显抑制活化的小胶质细胞分泌TNF-α的作用,其作用顺序为川陈皮素=红橘素>橙皮素>柚皮素,而3种黄酮苷却未表现出明显的抑制作用;预处理24 h后4种黄酮苷元的生物活性变化不大,而3种黄酮苷的生物活性明显增加,其作用顺序为新橙皮苷>橙皮苷>柚皮苷。结论 7种黄酮类化合物均有不同程度的抑制作用。其中,黄酮类化合物的抑制活性强于二氢黄酮类,黄酮苷元的活性强于黄酮苷,极性低的黄酮苷元又强于极性较大的黄酮苷元。推测黄酮类化合物母核结构中C-2,3共轭双键可能是对抑制小胶质细胞激活作用的关键因素,其次是母核分子的取代类型,黄酮苷需要一定的反应时间才可能发挥生物活性。

Objective To evaluate the structure-activity relationship of the flavonoids of Fructusaurantiion inhibiting activation of lipopolysaccharide(LPS)-induced primary microglia cells. Methods Hesperetin,tangeretin, nobiletin, naringenin, hesperidin, neohesperidin and naringin were obtained from F. aurantii by chromatography. The microglia cells viability was detected by MTT assayand the change of TNF-α release in LPS-activated microglia cells was detected by ELISA. Results Seven flavonoids had no significant effect on microglia cells survival rate in the range of 1-200 μM. Four kinds of flavonoid aglycons had obvious inhibitory effect on TNF-α secretion in activated microglia cells with pretreatment 4 h, and the order of activity wasnobiletin=tangeretin>hesperetin>naringenin. The three flavonoid glycosides did not show significant inhibition. After pretreatment 24 h, the biological activities of the four flavonoid aglycons changed little, while the biological activities of the three flavonoid glycosides increased significantly, and the order of activity was neohesperidin>hesperidin>naringin. Conclusion The seven flavonoids had different degrees of inhibition. The activity of flavonoes is stronger than that of dihydroflavone, the activity of flavonoid aglycon is stronger than that of flavonoid glycosides, and the weak polar flavonoid aglycones is stronger than the strong polar flavonoid aglycones. C-2,3 double bond may be the key factor to restrain microglia cells activation, followed by substitution patterns of flavonoid molecules. Flavonoid glycosides require a certain reaction time to exert biological activity.