Objectives: 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 compounds 7 and 6-OHG were 81.16% and 86.94%, respectively, which were higher than those of rutin, the positive control. The removal rates of chemical compounds 3, 4, and 6 were all lower than 20%. Compared with the normal group, the cell viability of the hypoxia model group was significantly decreased (P<0.01). Compared with the hypoxia model group, compounds 3, 4, and 6 had no significant effect on cell viability under hypoxic conditions. At all experimental concentrations, the cell viability of the 6-OHG group was significantly higher than that of the hypoxia model group (all P<0.05). The cell viability of compound 7 group at 1×10-7 and 1×10-6 mol/L was significantly higher than that of the hypoxia model group (both P<0.05). The anti-hypoxia activity of 6-OHG and compound 7 was excellent, and the optimal drug concentration was 1×10-6 and 1×10-7 mol/L. After PC12 cells was treated with 6-OHG (1×10-6 mol/L) and compound 7 (1×10-7 mol/L), the cell damage was reduced, the apoptotic rate was significantly decreased (P<0.01), and the protein expression levels of HIF-1α and VEGF were significantly decreased in comparison with the hypoxia model group (both P<0.01).
Conclusions: The optimized synthesis route can increase the yield of 6-OHG and obtain 4 derivatives by methylation and selective demethylation. 6-OHG and compound 7 have excellent antioxidant and anti-hypoxia activities, which are related to the structure of the A-ring ortho-triphenol hydroxyl group in the molecule.
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对缺氧条件下的细胞活力无显著影响；在所有实验浓度下，6-OHG组的细胞活力均显著高于缺氧模型组(均P<0.05)；在给药浓度为1×10-7或1×10-6 mol/L时，化合物7组的细胞活力显著高于缺氧模型组(均P<0.05)。6-OHG和化合物7的抗缺氧活性优异，最佳药物浓度分别为1×10-6和1×10-7 mol/L。采用6-OHG(1×10-6 mol/L)和化合物7(1×10-7 mol/L)处理PC12细胞后，与缺氧模型组相比，细胞损伤明显减轻，细胞凋亡率显著下降(P<0.01)，HIF-1α和VEGF蛋白质的表达水平显著下调(均P<0.01)。结论: 优化后的合成路线可提高6-OHG的产率，通过甲基化和选择性去甲基化得到4个衍生物。6-OHG和其衍生物化合物7表现出优异的体外抗氧化和抗缺氧活性，该活性与其分子中存在的A环邻三酚羟基结构有关。.
Keywords: 6-hydroxygenistein; anti-hypoxia; antioxidant; chemical synthesis; methylated derivatives.