6-羟基染料木素及其甲基化衍生物的合成和抗氧化与抗缺氧活性  

Synthesis,antioxidant and anti-hypoxia activities of 6-hydroxygenistein and its methylated derivatives

作　　者：张朋朋 张洁 田贻婷 石志群 马慧萍 景临林 ZHANG Pengpeng;ZHANG Jie;TIAN Yiting;SHI Zhiqun;MA Huiping;JING Linlin(Department of Pharmacy,First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061;Department of Pharmacy,940th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army,Lanzhou 730050;College of Pharmacy,Gansu University of Chinese Medicine,Lanzhou 730020,China)

机构地区：[1]西安交通大学第一附属医院药学部,西安710061 [2]中国人民解放军联勤保障部队第九四〇医院药剂科,兰州730050 [3]甘肃中医药大学药学院,兰州730020

出　　处：《中南大学学报（医学版）》2024年第2期236-246,共11页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81872796,81571847);军队后勤科研计划(CWH17J010);甘肃省自然科学基金(18JR3RA408);甘肃省卫生行业科研计划(GSWSKY-2019-62)。

摘　　要：目的:缺氧是一种常见的病理现象,通常由机体组织供氧不足或无法有效利用氧导致。羟基化及甲氧基化黄酮类化合物具有显著的抗缺氧活性。本研究旨在探索6-羟基染料木素(6-hydroxygenistein,6-OHG)及其甲基化衍生物的合成方法和抗氧化与抗缺氧活性。方法:以鹰嘴豆芽素A为原料,经甲基化反应、溴化反应、甲氧基化反应及去甲基化反应得到6-OHG及其4个甲基化衍生物[4’,6,7-三甲氧基-5-羟基异黄酮(化合物3)、4’,5,6,7-四甲氧基异黄酮(化合物4)、4’,6-二甲氧基-5,7-二羟基异黄酮(化合物6)、4’-甲氧基-5,6,7-三羟基异黄酮(化合物7)]。采用氢-1核磁共振波谱法(1H-nuclear magnetic resonance spectroscopy,1H-NMR)和质谱法(mass spectrometry,MS)表征产物结构;高压液相色谱法检测化合物的纯度;1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除实验检测化合物的体外抗氧化活性。将PC12细胞分为正常组、缺氧模型组、芦丁组(1×10-9~1×10^(-5)mol/L),以及常氧和缺氧条件下的目标化合物组(1×10-9~1×10^(-5)mol/L),采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测细胞活力,筛选得到抗缺氧活性优异的目标化合物及其最佳抗缺氧活性时的药物浓度。分别用抗缺氧活性优异的目标化合物、芦丁的最佳药物浓度处理PC12细胞后,在光镜下观察细胞形态,采用流式细胞术测定细胞凋亡率,蛋白质印迹法检测缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的蛋白质表达水平。结果:6-OHG及其4个甲基化衍生物的结构无误,纯度均>97%。当浓度为4 mmol/L时,化合物7和6-OHG的DPPH自由基清除率分别为81.16%和86.94%,均高于阳性对照芦丁,而化合物3、4、6的清除率均低于20%。与正常组相比,缺氧模型组的细胞活力显著下降(P<0.01);与缺氧模型组相比,化合物3、4、6对缺氧Objective:Hypoxia is a common pathological phenomenon,usually caused by insufficient oxygen supply or inability to use oxygen effectively.Hydroxylated and methoxylated flavonoids have significant anti-hypoxia activity.This study aims to explore the synthesis,antioxidant and anti-hypoxia activities of 6-hydroxygenistein(6-OHG)and its methoxylated derivatives.Methods:The 6-OHG and its methoxylated derivatives,including 4',6,7-trimethoxy-5-hydroxyisoflavone(compound 3),4',5,6,7-tetramethoxyisoflavone(compound 4),4',6-imethoxy-5,7-dihydroxyisoflavone(compound 6),and 4'-methoxy-5,6,7-trihydroxyisoflavone(compound 7),were synthesized by methylation,bromination,methoxylation,and demethylation using biochanin A as raw material.The structure of these products were characterized by 1hydrogen-nuclear magnetic resonance spectroscopy(1H-NMR)and mass spectrometry(MS).The purity of these compounds was detected by high pressure chromatography(HPLC).The antioxidant activity in vitro was investigated by 1,1-diphenyl-2-picrylhydrazyl radical(DPPH)free radical scavenging assay.PC12 cells were divided into a normal group,a hypoxia model group,rutin(1×10−9−1×10^(−5)mol/L)groups,and target compounds(1×10−9−1×10^(−5)mol/L)groups under normal and hypoxic conditions.Cell viability was detected by cell counting kit-8(CCK-8)assay,the target compounds with excellent anti-hypoxia activity and the drug concentration at the maximum anti-hypoxia activity were screened.PC12 cells were treated with the optimal concentration of the target compound or rutin with excellent anti-hypoxia activity,and the cell morphology was observed under light microscope.The apoptotic rate was determined by flow cytometry,and the expressions of hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)were detected by Western blotting.Results:The structure of 6-OHG and its 4 methylated derivatives were correct,and the purity was all more than 97%.When the concentration was 4 mmol/L,the DPPH free radical removal rates of chemical

关 键 词：6-羟基染料木素 甲基化衍生物 化学合成 抗氧化 抗缺氧 

分 类 号：R285[医药卫生—中药学]