多毛橐吾总黄酮的提取及其抗炎机制研究

Extraction and purification of total flavonoids from hairy Ligularia and its anti-inflammatory mechanism

【目的】提取并纯化多毛橐吾总黄酮,并对其抗炎活性及相关作用机制进行探究。【方法】采用超声波法提取多毛橐吾总黄酮,用AB-8大孔吸附树脂对其进行纯化,考察上样质量浓度、上样量、上样流速、上样液pH、解吸液中乙醇体积分数、乙醇用量6个因素对纯化效果的影响。用脂多糖(LPS)刺激RAW264.7巨噬细胞建立体外炎症模型,再用多毛橐吾总黄酮对其进行处理,RT-PCR检测炎症细胞因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)mRNA的表达水平,ELISA测定IL-6和TNF-α的分泌量,Western blot检测IκB-α蛋白磷酸化的表达水平,研究多毛橐吾总黄酮抗炎及其对NF-κB信号转导通路的影响。【结果】多毛橐吾总黄酮纯化的最优工艺条件为:上样质量浓度0.8mg/mL、上样量30mL、上样流速1mL/min、上样液pH 4、乙醇体积分数90%、乙醇用量40mL,在此条件下得到的总黄酮纯度为67.3%。多毛橐吾总黄酮能显著抑制IL-6和TNF-α的分泌以及IκB-α蛋白的磷酸化表达,且随总黄酮质量浓度的增加呈剂量依赖性。其中在总黄酮质量浓度为25,50,100μg/mL时,TNF-α分泌量分别下降了37.4%,57.5%和79.1%,IL-6分泌量分别下降了57.7%,77.1%和88.3%,磷酸化IκB-α蛋白的表达量分别下降了10.7%,42.5%和48.8%。【结论】筛选出了纯化多毛橐吾总黄酮的最优工艺条件,获得了纯度为67.3%的多毛橐吾总黄酮;多毛橐吾总黄酮可以抑制NF-κB信号途径的活化,有显著的抗炎效果。

【Objective】This study aimed to extract and purify total flavonoids from hairy Ligulariaand explore its anti-inflammatory activity and related mechanism.【Method】Total flavonoids from hairy Ligularia were extracted by ultrasonic and purified by AB-8 macroplume adsorption resin.The purification effect of 6 factors including sample concentration,sample volume,sample flow rate,sample pH,ethanol concentration,and ethanol volume were investigated,and lipopolyssacharide（LPS）-activated macrophages was used to establish vitro models of inflammation.Total flavonoids were used to treat the RAW264.7 macrophages.Then,its effects on LPS-induced inflammatory cytokines TNF-α,and IL-6 mRNA were detected by RT-PCR,the effects on their secretion levels were detected by ELISA,and the effects on IκB-αexpression was detected by Western blot to research the inhibition and the effect on NF-κB signal transduc-tion pathway.【Result】The optimal conditions for the purification of total flavonoids from hairy Ligularia included sample concentration of 0.8 mg/mL,sample volume of 30 mL,sample flow rate of 1 mL/min,sample pH of 4.0,ethanol concentration of 90%,and ethanol volume of 40 mL,under which the purity of total flavonoids was 67.3%.Total flavonoids significantly inhibited production of TNF-αand IL-6 and phosphorylation of IκB-αprotein expression in a dose-dependent manner.At concentration of 25,50,and 100μg/mL,TNF-αlevel was decreased by 37.4%,57.5%,and 79.1%,IL-6 was decreased by 57.7%,77.1%,and 88.3%,IκB-αprotein phosphorylation level was decreased by 10.7%,42.5%,and 48.8%,respectively.【Conclusion】This paper selected the optimal purification condition of total flavonoids from hairy Ligularia with the purity of 67.3%.The activation of NF-κB signaling pathway was inhibited by flavonoids from hairy Ligulariaand total flavonoids had significant anti-inflammatory effect.