黄根化合物的分离及对脂多糖诱导RAW264.7细胞炎症的影响  

Isolation of Prismatomeris Connata Y.Z.Ruan compounds and their effects on LPS-induced inflammation in RAW264.7 cells

作　　者：姜昕 汤锦一 付志攀 王博雅 王昱萱 代现平[1] JIANG Xin;TANG Jinyi;FU Zhipan;WANG Boya;WANG Yuxuan;DAI Xianping(School of Pharmacy,Binzhou Medical University,Yantai 264003,Shandong,P.R.China)

机构地区：[1]滨州医学院药学院,山东烟台264003

出　　处：《滨州医学院学报》2024年第4期241-247,259,共8页Journal of Binzhou Medical University

基　　金：国家自然科学基金(31170320)。

摘　　要：目的研究黄根乙醇提取物对脂多糖(LPS)诱导RAW264.7细胞炎症模型的作用,并对其具有活性的乙酸乙酯组分的化学成分进行分离纯化。方法利用CCK-8法检测黄根化学成分对RAW264.7细胞活性的影响。利用浓度为500 ng/mL的LPS诱导RAW264.7细胞,以建立细胞炎症模型,Griess检测细胞上清液中一氧化氮(NO)水平,ELISA法检测细胞上清液IL-1β、TNF-α的含量,DCFH-DA荧光探针检测细胞内ROS水平。再通过溶剂提取法、硅胶色谱柱、高效液相色谱法等方法对黄根乙酸乙酯组分分离纯化。结果质量浓度在100μg/mL及以下的黄根醇提物、石油醚组分、乙酸乙酯组分、正丁醇组分和水组分对RAW264.7细胞无毒性。与模型组比较,黄根醇提物、乙酸乙酯组分对细胞中NO、TNF-α、IL-1β和ROS的表达水平具有下调抑制作用。根据理化性质和波谱资料确定化合物结构,从黄根乙酸乙酯部位分离鉴别了4个化合物,根据理化性质和波谱资料确定化合物结构为甲基异茜草素-1-甲醚(1)、1,3-二羟基-2-乙氧甲基蒽醌(2)、邻苯二甲酸二(2-乙基己)酯(3)、对苯二甲酸二(2-乙基己)酯(4)及实验证明分离得到单体不具抗炎活性在此不做赘述。结论黄根醇提物组、乙酸乙酯组可拮抗LPS诱导的小鼠巨噬细胞炎症反应,抑制炎症相关因子的表达。邻苯二甲酸二(2-乙基己)酯和对苯二甲酸二(2-乙基己)酯为首次从该植物中分离。Objective To study the effect of Prismatomeris Connata Y.Z.Ruan extract on the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model,and to isolate and purify the active ethyl acetate fraction.Methods CCK-8 method was used to detect the effects of chemical components of Prismatomeris Connata Y.Z.Ruan root on RAW264.7 cell activity.RAW264.7 cells induced with LPS at a concentration of 500 ng/mL were used to establish a cell inflammation model.Griess assay was used to detect the level of NO in the cell supernatant;ELISA was used to detect the content of IL-1β and TNF-α in the cell supernatant;DCFH-DA fluorescent probe was used to detect the level of ROS in the cells.The ethyl acetate fraction of Prismatomeris Connata Y.Z.Ruan root was isolated and purified by using solvent extraction,silica gel column chromatography,and high-performance liquid chromatography(HPLC)methods.Results Prismatomeris Connata Y.Z.Ruan extract,petroleum ether fraction,ethyl acetate fraction,n-butanol fraction,and water fraction at a mass concentration of 100μg/mL and below showed no toxicity to RAW264.7 cells.Compared with the model group,Prismatomeris Connata Y.Z.Ruan extract and ethyl acetate fraction had a down-regulating inhibitory effect on the expression levels of NO,TNF-α,IL-1β and ROS in the cells.Based on the physicochemical properties and spectroscopic data,four compounds were isolated and identified from the ethyl acetate fraction of Prismatomeris Connata Y.Z.Ruan extract which were methyl isalizarin-1-methyl ether(1),1,3-dihydroxy-2-ethoxymethylanthraquinone(2),di(2-ethylhexyl)phthalate(3),di(2-ethylhexyl)terephthalate(4).We did not discuss the isolated monomer which did not have anti-inflammatory activity.Conclusion The ethyl acetate fraction and the Prismatomeris Connata Y.Z.Ruan extract can antagonize the LPS-induced inflammatory response in mouse macrophages and inhibit the expression of inflammatory factors.Di(2-ethylhexyl)phthalate and di(2-ethylhexyl)terephthalate were isolated from this plant for the first

关 键 词：黄根 脂多糖 RAW264.7细胞 炎症 一氧化氮 活性氧 炎症因子 

分 类 号：R285.5[医药卫生—中药学]