摘要
目的:从射干中分离制备高纯度的鸢尾甲苷A和鸢尾甲苷B,并探究其体外抗炎活性及机制。方法:采用聚酰胺柱色谱-重结晶法相结合从射干中分离纯化制备鸢尾甲苷A及鸢尾甲苷B单体;采用改良的Ellman比色法测定鸢尾甲苷A和鸢尾甲苷B对乙酰胆碱酯酶(acetylcholinesterase,AChE)活性的影响;采用CCK-8法测定不同浓度的鸢尾甲苷A和鸢尾甲苷B对细胞活力的影响,确定给药范围;以脂多糖(lipopolysaccharide,LPS)诱导小鼠RAW264.7巨噬细胞为炎症模型,采用Griess法检测细胞上清液中一氧化氮(nitric oxide,NO)的含量,Elisa法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-β)和IL-6含量;Western blot法检测细胞环氧合酶-2(cyclooxygenase-2,COX-2)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7nAChR)蛋白表达。结果:聚酰胺对鸢尾甲苷A及鸢尾甲苷B有良好的分离纯化效果,并通过重结晶得到鸢尾甲苷A和鸢尾甲苷B单体,经HPLC峰面积归一化法计算其纯度均>98%;鸢尾甲苷A和鸢尾甲苷B对AChE具有一定的抑制作用;鸢尾甲苷A和鸢尾甲苷B给药浓度在12.5~200μg·mL^(-1)范围内对RAW 264.7细胞无抑制作用;与LPS模型组比较,鸢尾甲苷A和鸢尾甲苷B均能抑制细胞中NO,TNF-α,IL-β,IL-6的释放(P<0.05),且呈剂量关系,并能上调胆碱能抗炎通路α7nAChR蛋白表达(P<0.05)及下调花生四烯酸通路中COX-2蛋白和NO通路中iNOS蛋白的表达(P<0.05)。结论:鸢尾甲苷A和鸢尾甲苷B具有一定的体外抗炎活性,其抗炎机制是通过调节胆碱能抗炎通路、花生四烯酸代谢通路及NO通路,减少巨噬细胞中NO,TNF-α,IL-β和IL-6等炎性介质的释放,实现多途径抗炎作用。
Objective:To isolate and prepare high-purity iristectorin A and iristectorin B from Belamcanda chinensis,and explore their anti-inflammatory activity and mechanism in vitro.Methods:The iristectorin A and iristectorin B monomers were isolated and purified from Belamcanda chinensis by polyamide column chromatographyrecrystallization method.The effects of iristectorin A and iristectorin B on acetylcholinesterase(AChE)activity were determined by modified Ellman colorimetric method.The effects of different concentrations of iristectorin A and iristectorin B on cell viability were determined by CCK-8 method,and the range of administration was determined.Lipopolysaccharide(LPS)-induced mouse RAW264.7 macrophages were used as an inflammatory model.The content of nitric oxide(NO)in the cell supernatant was detected by Griess method,and the contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 were detected by ELISA.The protein expressions of cyclooxygenase-2(COX-2),inducible nitric oxide synthase(iNOS)andα7 nicotinic acetylcholine receptor(α7nAChR)were detected by Western blot.Results:Polyamide showed good separation and purification effect for iristectorin A and iristectorin B,and iristectorin A and iristectorin B monomers were obtained by recrystallization,and their purity was greater than 98%as calculated by HPLC peak area normalization method.Iristectorin A and iristectorin B had certain inhibitory effect on acetylcholinesterase;iristectorin A and iristectorin B had no inhibitory effect on RAW 264.7 cells in the range of 12.5~200μg·mL^(-1).Compared with LPS model group,iristectorin A and iristectorin B could inhibit the release of NO,TNF-α,IL-1βand IL-6 in cells in a dose-dependent manner(P<0.05),up-regulate the expression ofα7nAChR protein in cholinergic anti-inflammatory pathway(P<0.05),and down-regulate the expression of COX-2 protein in arachidonic acid pathway and iNOS protein in NO pathway(P<0.05).Conclusion:Iristectorin A and iristectorin B have certain anti-inflammatory activity i
作者
郭文慧
高萌
杨元丰
熊豪
陈海芳
朱文杰
杨武亮
GUO Wen-hui;GAO Meng;YANG Yuan-feng;XIONG Hao;CHEN Hai-fang;ZHU Wen-jie;YANG Wu-liang(Key Laboratory of Modern Preparation of Traditional Chinese Medicine,Ministry of Education,Jiangxi University of Chinese Medicine,Nangchang 330004,China;Jiangzhong Pharmaceutical Co.,Ltd.,Nangchang 330004,China)
出处
《中国新药杂志》
CAS
CSCD
北大核心
2024年第18期1943-1951,共9页
Chinese Journal of New Drugs
基金
江西中医药大学中药炮制技术继承与创新团队项目(CXT22003)
江西中医药大学博士启动基金课题项目(2021WBZR003)。
