华中枸骨叶总黄酮纯化及抗RA滑膜炎症活性评价

Purification of total flavonoids from the leaves of Ilex centrochinensisand their anti-rheumatoid arthritis synovial inflammation

以总黄酮含量和转移率为评价指标,采用多种方法提取纯化华中枸骨叶总黄酮(ICTF),并以TNF-α诱导人关节滑膜成纤维细胞MH7A为滑膜炎症细胞模型评价其抗RA滑膜炎症活性,试剂盒测定炎症介质NO含量,RT-qPCR检测炎症因子IL-1β、IL-6、IL-8 mRNA的转录水平.结果表明,华中枸骨叶乙醇总提取物经石油醚脱脂后,经D101大孔吸附树脂吸附,50%乙醇洗脱5 BV,再经葡聚糖凝胶Sephadex LH-20吸附后甲醇洗脱5 BV后,所得总黄酮含量达53.91%,转移率为39.53%;所得ICTF能剂量依赖性降低滑膜炎症模型细胞NO水平以及IL-1βmRNA的转录水平,200μg/mL剂量可显著降低IL-6和IL-8 mRNA的水平.该纯化方法稳定可行,所得ICTF具有抑制RA滑膜炎症潜力.

Taking the purity and yield of total flavonoids as the evaluation indexes,various methods were used to purify the total flavonoids from the leaves of Ilex centrochinensis,and their anti-rheumatoid arthritis synovial inflammation activities in vitro were determined by TNF-αinduced human synovial fibroblasts MH7A.The content of NO was assayed by the Griess kit and the expression of interleukin-1β(IL-1β),interleukin-6(IL-6)and interleukin-8(IL-8)mRNA were detected by RT-qPCR.The results indicated that the total ethanol extract from the leaves of Ilex centrochinensis was extracted by petroleum ether,then purified by D101 macroporous adsorption resin using 5 times of the column volume 50%ethanol,and finally purified by Sephadex LH-20 column chromatography with methanol elution,the first to fifth colume vilume eluent was collected,concentrated and dried to obtained the total flavonoids with the purity of 53.91%and the yield of 39.53%.The total flavonoids significantly decreased the content of NO and the expression of IL-1βmRNA in TNF-αinduced MH7A cells in a dose-dependent manner,IL-6 and IL-8 mRNA expression levels were also significantly decreased at 200μg/mL.The results all above showed that the optimized purificition method was stable and could be used to purify total flavonoids from the leaves of Ilex centrochinensis,which can inhibit rheumatoid arthritis synovial inflammation.