黑果枸杞果实中抗炎活性成分的筛选及作用机制研究

Research on the Screening and Mechanism of Active Ingredients for Anti-Inflammatory in Lycium ruthenicum Murr.Fruit

目的:对黑果枸杞果实中的抗炎活性成分进行筛选和评价,同时对其作用机制进行研究。方法:利用甲醇对黑果枸杞果实进行提取,并分别在微孔树脂(MCI)和大孔树脂柱上进一步分离。利用脂多糖诱导小鼠单核巨噬细胞RAW 264.7构建炎症模型,对获得的组分进行筛选和评价。采用MTT法检测细胞活力、比色法检测NO水平、ELISA法检测IL-6、IL-1β、TNF-α等炎症因子水平,Western blot检测COX-2、IκBα等蛋白及其磷酸化的表达水平。结果:初步筛选发现经MCI处理后的3个组分中,Fr1表现出较好的抑制NO生成的能力。对Fr1进一步分离获得Fr1-1和Fr1-2组分。Fr1-2在抑制NO生成、炎症因子水平等方面均优于Fr1。对其作用机制进一步研究发现Fr1-2能抑制COX-2、p-IκBα蛋白表达水平从而发挥抗炎作用。对Fr1-2活性部位中的主要成分进一步分离制备,发现其主要成分为5-羟甲基糠醛,5-羟甲基糠醛在所选浓度范围内对细胞活力无显著影响,且其可以抑制炎症模型NO生成,具有一定的抗炎作用。结论:黑果枸杞果实提取物具有一定的抗炎作用,其机制可能与抑制COX-2蛋白的表达、IκBα蛋白的磷酸化水平,从而降低炎症因子的表达有关,其作用的物质基础可能与5-羟甲基糠醛有关。

Objective:The anti-inflammatory active components in the fruit of Lycium ruthenicum Murr.were screened and evaluated,and the mechanism was studied.Methods:The fruits of Lycium ruthenicum were extracted with methanol,the extracts was further separated on MCI and macroporous resin columns.The inflammatory model of mouse monocyte macrophage cell RAW264.7 induced by lipopolysaccharide(LPS)was established to screen and evaluate the inflammatory function of the obtained fraction.MTT as-say was used to detect cell viability and NO level was detected by colorimetric detection.ELISA was used to measure the inflammatory factor levels of IL-6、IL-1β、TNF-α.The protein expression levels of COX-2、IκBαand its phosphorylation were detected by western blot.Results:Among the three compo-nents treated with MCI,Fr1 exhibited the best inhibition on the production of NO.Fr1 was further sepa-rated to obtain Fr1-1 and Fr1-2 components.And Fr1-2 displayed better function than Fr1 in inhibiting NO production and inflammatory factor level.Further study on its mechanism found that Fr1-2 could in-hibit COX-2 and p-IκBα/IκBαprotein expression level.The main component of Fr1-2 was further sepa-rated,prepared and identified as 5-hydroxymethylfurfural.5-hydroxymethylfurfural had no significant ef-fect on cell viability in the selected concentration range,and it could inhibit NO production in inflamma-tory models,and had a certain anti-inflammatory effect.Conclusion:The extract of Lycium ruthenicum fruit exhibited certain anti-inflammatory effect,and the mechanism might be related to inhibition of the expression of COX-2 and IκBαprotein phosphorylation level,and the reduction of the inflammatory fac-tors level.The material basis for its anti-inflammatory effect might be related to the component 5-hy-droxymethylfurfural.