胡芦巴叶黄酮组分的制备、表征及其对肝细胞氧化损伤的保护作用

Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes

研究胡芦巴叶黄酮组分(fenugreek leaf flavonoids,FLFs)中的主要成分及其抗氧化活性。利用溶剂萃取法对胡芦巴叶中的黄酮组分进行富集制备,采用高效液相色谱-四极杆飞行时间串联质谱(HPLC-Q-TOF-MS/MS)对其中的黄酮类化合物进行表征;同时考察FLFs对H;O;诱导的L02肝细胞应激损伤的保护作用,首先采用MTT法检测L02细胞活力;H;O;诱导L02细胞建立氧化应激损伤模型;试剂盒检测乳酸脱氢酶(lactic dehydrogenase,LDH)的泄漏量、还原型谷胱甘肽(glutathione,GSH)和丙二醛(malondialdehyde,MDA)含量以及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的活力;荧光染色法观察细胞凋亡情况;Western bolt法检测c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK1/2)、核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)、血红素加氧酶1(heme oxygenase 1,HO-1)等蛋白及其磷酸化蛋白的表达水平。结合MS碎片离子信息,经数据库检索和人工解析,FLFs中含有以槲皮素和山柰酚为苷元的8种黄酮类化合物;活性研究发现,与H;O;处理组相比,3.125~25μg·mL;的FLFs能够提高损伤后L02细胞存活率,并且能浓度依赖性地降低L02细胞中LDH泄漏量和MDA的含量,增强GSH、SOD和CAT等酶的活性;抑制JNK和ERK1/2蛋白磷酸化水平,促进Nrf2和HO-1蛋白水平。荧光染色结果显示,H;O;组细胞核呈浓缩致密的强蓝色荧光,FLFs组的蓝色荧光强度明显降低。FLFs对H;O;所致的L02细胞氧化损伤具有保护作用,作用机制与其激活MAPKs/Nrf2/HO-1信号通路从而增强细胞清除自由基能力和改善细胞凋亡2个方面因素有关,胡芦巴叶抗氧化作用与其中含有丰富的黄酮类化合物有关。

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.)leaf flavonoids(FLFs)and their antioxidant activity.FLFs were prepared and enriched by solvent extraction,and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS).The protective effect of FLFs against H;O;-induced stress damage to L02 hepatocytes was also investigated.Firstly,the cell viability was measured by MTT assay.The oxidative stress injury model was induced by H;O;in L02 cells.The release of lactate dehydrogenase(LDH),the content of reduced glutathione(GSH)and malondialdehyde(MDA),and the activities of superoxide dismutase(SOD)and catalase(CAT)were measured by assay kits.Hoechst fluorescence staining was performed to observe the cell apoptosis.The expression levels of c-Jun N-terminal kinase(JNK),extracellular signal-regulated kinase 1/2(ERK1/2),nuclear factor erythroid-2 related factor 2(Nrf2),heme oxygenase 1(HO-1),and their phosphorylated proteins were detected by Western blot.Based on the MS fragment ion information and data in databases,FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons.The cell viabi-lity assay revealed that as compared with the conditions in the H;O;treatment group,3.125-25μg·mL;FLFs could increase the viability of L02 cells,reduce LDH release and MDA content in a dose-dependent manner,potentiate the activities of SOD,CAT,and GSH,decrease the phosphorylation of JNK and ERK1/2 proteins,and up-regulate the expression of Nrf2 and HO-1.The results of fluorescence staining showed that the nucleus of the H;O;treatment group showed concentrated and dense strong blue fluorescence,while the blue fluorescence intensity of the FLFs group decreased significantly.FLFs showed a protective effect against H;O;-induced oxidative damage in L02 cells,and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway.The antioxidant effect of fenugreek leaf is related to its rich flavonoids.