苗药消疤草抗肝纤维化活性成分研究

Study on Anti-Liver Fibrosis Active Constituents of Miao Medicine Corydalis sheareri

目的:明确苗药消疤草(地锦苗)抗肝纤维化的物质基础。方法:采用溶剂萃取结合酸碱处理法将消疤草提取物分为石油醚萃取(P)、乙酸乙酯并三氯甲烷萃取(C)、三氯甲烷萃取后乙酸乙酯部位(EE)以及正丁醇萃取(B)四个组分,以人肝星状细胞株HSC-LX2作为模型,用MTT法检测各组分对HSC-LX2细胞增殖的影响,筛选出活性组分;以硅胶、MCI、Sephadex LH-20柱层析和半制备HPLC等色谱技术对活性组分进行分离纯化,根据各种波谱学技术对化合物进行结构鉴定,并通过MTT法评价化合物的抗肝纤维化活性。结果:组分EE和C分别在31和15μg/mL时有显著的抗肝纤维化活性,从这两个组分中分离并鉴定了8个化合物,分别为:原阿片碱(1)、β-卡波林(2)、降氧化北美黄连次碱(3)、对乙氧基乙酰苯胺(4)、3-亚乙基-2-吡咯烷酮(5)、(6S,7E)-6-hydroxy-4,7-megastigmadien-3,9-dione(6)、macleayin E(7)、2-(4-羟基-3-甲氧苯基)-3-[N-2-(4-羟基苯基)乙基]氨基甲酰-5-[N-2-(4-羟基苯基)乙基]氨基甲酰乙烯基-7-甲氧基苯并二氢呋喃(8)。其中,化合物1、2、3、6、7和8具有抗肝纤维化活性,且化合物1、7、8在较低浓度下亦具有明显的活性。结论:组分EE和C是消疤草抗肝纤维化的有效组分;化合物2~8为首次从该植物中分离得到,化合物1、7、8是消疤草抗肝纤维化的主要物质基础。

Objective:To study the material basis of anti-liver fibrosis of Miao medicine Corydalis sheareri.Methods:Solvent extraction combined with acid-base treatment method was used to classify the extracts of Corydalis sheareri into four fractions:petroleum ether extraction(P),ethyl acetate and trichloromethane extraction(C),ethyl acetate fraction after trichloromethane extraction(EE)and n-butanol extraction(B).Human liver statin cell line HSC-LX2 was used as the model to detect the effects of various components on the proliferation of HSC-LX2 cells by MTT method and the active components were selected.The active components were isolated and purified by silica gel,MCI,Sephadex LH-20 column chromatography and semi-prepared HPLC.The structures of the compounds were identified according to various spectral techniques and the anti-liver fibrosis activities of the compounds were evaluated by MTT method.Results:Fractions EE and C showed significant anti-fibrosis effect at 31 and 15μg/mL,respectively.Eight compounds were isolated from the two active fractions and identified as protopine(1),β-carboline(2),noroxyhydrastinine(3),p-ethoxyacetanilide(4),3-athyliden-2-pyrrolidon(5),(6S,7E)-6-hydroxyl-4,7-megastigmadien-3,9-dione(6),macleayin E(7),2-(4-hydroxyl-3-methoxyphenyl)-3-[N-2-(4-hydroxyphenyl)ethyl]carbamoyl-5-[N-2-(4-hydroxyphenyl)ethyl]carbamoylethenyl-7-methoxybenzodihydrofuran(8).Among them,compounds 1,2,3,6,7 and 8 showed varying degrees of inhibition against the cell proliferation of HSC-LX2,while compounds 1,7 and 8 showed significant inhibition at lower concentrations.Conclusion:Fractions EE and C are the active fractions of Corydalis sheareri against liver fibrosis.Compounds 2 to 8 are obtained from this plant for the first time,compounds 1,7 and 8 are the main material basis of Corydalis sheareri against liver fibrosis.