全谷豆包植物活性成分及其改善HepG2细胞胰岛素抵抗作用与AMPK通路机制探讨

发布时间：2018-05-15 10:44

本文选题：全谷豆包 + 植物化学物　；参考：《扬州大学》2013年硕士论文

【摘要】：目的：(1)提取熟化前后全谷豆包中膳食纤维(DF)、类黄酮(FN)、酚酸(PAD)和植物固醇(PS),测定其含量和体外抗氧化能力,了解全谷豆包中活性成分提取物产生生物学效应的物质基础； (2)观察全谷豆包中活性成分提取物对棕榈酸(PA)诱导的HepG2细胞胰岛素抵抗作用的影响,测定细胞对葡萄糖的消耗量与细胞中甘油三酯(TG)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)的含量,阐明不同种类活性成分提取物对HepG2细胞胰岛素抵抗作用的干预效果差异； (3)观察全谷豆包中活性成分提取物对胰岛素抵抗HepG2细胞中的腺苷酸活化蛋白激酶a2(AMPKa2)、乙酰辅酶A羟化酶(ACC1)、脂肪酸合成酶(FAS)、葡萄糖转运蛋白2(GLUT2)、过氧化物酶体增殖物活化受体γ协同激活因子-1(PGC-1)在mRNA水平上的影响,探讨全谷豆包中活性成分对胰岛素抵抗HepG2细胞的作用机制； (4)比较熟化对全谷豆包中活性物质及其改善HepG2细胞胰岛素抵抗作用的影响效果。方法：(1)用酶-化学法提取熟化前后全谷豆包中的膳食纤维,按国标法测定其含量；用浸提回流法提取熟化前后全谷豆包中的类黄酮,用超声波萃取法提取熟化前后全谷豆包中的酚酸和植物固醇,用比色法测定其含量。采用DPPH自由基清除法和T-AOC总抗氧化能力检测法测定不同活性成分提取物的体外抗氧化能力； (2)用棕榈酸诱导HepG2细胞建立体外胰岛素抵抗模型,通过MTT、油红O染色、葡萄糖氧化酶法检测细胞对葡萄糖的消耗量以及细胞中TG的含量,判定胰岛素抵抗细胞模型建立成功,确定棕榈酸最佳浓度； (3)棕榈酸诱导HepG2细胞胰岛素抵抗模型后,加入不同活性成分提取物,测定细胞对葡萄糖的消耗量与细胞中TG的含量和抗氧化指标MDA、SOD、GSH-PX的含量,半定量PCR测定AMPKα2、ACC1、GLUT2、FAS、PGC-1的基因表达。结果：(1)熟化前全谷豆包中膳食纤维的含量为17.5g/100g,类黄酮的含量为2.28g/100g,酚酸的含量为0.62g/100g,植物固醇的含量为4.60g/100g,熟化后全谷豆包中膳食纤维的含量为21.6g/100g,类黄酮的含量为3.51g/100g,酚酸的含量为0.52g/100g,植物固醇的含量为5.88g/100g;熟化前后全谷豆包中不同活性成分均具有很强的清除DPPH自由基能力和总抗氧化能力； (2)0.25mM的棕榈酸对细胞活性无显著影响,且细胞对葡萄糖的消耗量显著降低,细胞内TG显著升高,细胞内脂滴蓄积明显,是诱导HepG2细胞形成胰岛素抵抗的最佳浓度； (3)MTT实验中观察到,适当浓度(200μg/mL)的不同活性成分提取物对PA诱导的胰岛素抵抗HepG2细胞有一定的保护作用。与类黄酮组、植物固醇组和膳食纤维组相比,酚酸组的保护作用较弱。油红O染色结果也显示,PA可造成细胞内游离脂肪酸升高,而不同活性成分提取物处理后,可减少细胞内游离脂肪酸的形成。200μg/mL熟化前后全谷豆包中活性成分提取物提高了细胞对葡萄糖的摄取能力,与阴性对照组差异无统计学意义(P0.05),均明显低于PA模型组(P0.05)。与PA模型组相比,降低了细胞中TG、MDA含量,升高了SOD、GSH-PX含量(P0.05),表明熟化前后全谷豆包中活性成分对胰岛素抵抗均具有一定的改善作用； (4)与模型组相比,类黄酮组、酚酸组、植物固醇组和膳食纤维组细胞内AMPKa2、GLUT2和PGC-1mRNA表达显著增高,且ACC1和FAS mRNA表达显著降低(P0.05)。除酚酸组外,其余组的基因表达改善效果与阴性对照组差异无统计学意义(P0.05)。结论：(1)全谷豆包中富含膳食纤维、类黄酮、酚酸和植物固醇等抗氧化物质。这些活性成分具有很强的体外抗氧化能力； (2)0.25mM的棕榈酸与HepG2细胞孵育24小时,能使细胞对葡萄糖的消耗量显著降低,形成胰岛素抵抗细胞模型； (3)熟化前后全谷豆包中活性成分可以提高HepG2细胞内AMPKa2、GLUT2和PGC-1mRNA的表达,降低ACC1和FAS mRNA的表达,从而改善PA诱导的胰岛素抵抗状态。这可能是全谷豆包改善机体胰岛素抵抗的机制之一； (4)比较后发现,对HepG2细胞胰岛素抵抗状态的改善作用,类黄酮组效果最好,膳食纤维组和植物固醇组次之,而酚酸组效果不够明显,尤其是熟化后的酚酸组。可能原因是,酚酸是一类热不稳定物质,加热熟化会加速酚酸类物质的氧化分解。
[Abstract]:Objective: (1) to extract the dietary fiber (DF), flavonoid (FN), phenolic acid (PAD) and plant sterol (PS) in the whole valley bean bag before and after maturation, to determine its content and antioxidant capacity in vitro, and to understand the biological effect of the active ingredient extract from the whole valley bean bag.
(2) observe the effect of active ingredients extract from whole valley bean bag on the insulin resistance of HepG2 cells induced by palmitic acid (PA), determine the consumption of glucose and the content of triglyceride (TG), malondialdehyde (MDA), superoxide dismutase (SOD) and Gu Guang glypeptide peroxidase (GSH-PX) in cells, and clarify different kinds of active components The effect of intervention on insulin resistance in HepG2 cells was different.
(3) observe the effect of active component extracts from whole valley beans on adenylate activation protein kinase A2 (AMPKa2), acetyl coenzyme A hydroxylase (ACC1), fatty acid synthetase (FAS), glucose transporter 2 (GLUT2), and the activity of peroxisome activation receptor gamma coactivator -1 (PGC-1) in the level of mRNA in insulin resistant HepG2 cells. To explore the action mechanism of active ingredients in whole grain bean bag on insulin resistance HepG2 cells.
(4) to compare the effect of maturation on the active substance in whole grain soybean bag and its effect on improving insulin resistance in HepG2 cells.
Methods: (1) the dietary fiber in the whole valley bean bag before and after ripening was extracted by enzyme chemical method, and the content was determined according to the national standard. The flavonoid in the whole valley bean bag before and after curing was extracted by the reflux extraction method. The content of phenolic acid and plant solid alcohol in the whole valley bean bag before and after curing were extracted by ultrasonic extraction, and the content of DPPH free radical was determined by colorimetric method. The antioxidant capacity of the extracts from different active ingredients was determined by the clearance method and the total antioxidant capacity of T-AOC.
(2) HepG2 cells were induced by palmitic acid to establish an in vitro insulin resistance model. By MTT, oil red O staining, the glucose oxidase method was used to detect the consumption of glucose and the content of TG in the cells. It was determined that the insulin resistance cell model was established successfully and the optimum concentration of palmitic acid was determined.
(3) after inducing the insulin resistance model of HepG2 cells by palmitic acid, the extracts of different active components were added to determine the consumption of glucose and the content of TG in cells and the content of MDA, SOD, GSH-PX, and the expression of AMPK alpha 2, ACC1, GLUT2, FAS and PGC-1 by semi quantitative PCR.
Results: (1) the content of dietary fiber in whole valley bean bag before maturation is 17.5g/100g, the content of flavonoids is 2.28g/100g, the content of phenolic acid is 0.62g/100g, the content of plant sterol is 4.60g/100g, the content of dietary fiber in whole valley bean bag after maturation is 21.6g/100g, the content of flavonoid is 3.51g/100g, the content of phenolic acid is 0.52g/100g, plant sterol The content of 5.88g/100g in the whole grain bean cake before and after ripening had strong scavenging ability of DPPH free radical and total antioxidant capacity.
(2) the palmitic acid of 0.25mM had no significant effect on cell activity, and the consumption of glucose decreased significantly, the intracellular TG increased significantly, and the accumulation of lipid droplets in the cells was obvious, which was the best concentration to induce HepG2 cells to form insulin resistance.
(3) in the MTT experiment, it was observed that the different active components of the appropriate concentration (200 mu) had certain protective effects on PA induced insulin resistance to HepG2 cells. Compared with the flavonoid group, the plant sterol group and the dietary fiber group, the protective effect of the phenolic acid group was weak. The result of oil red O staining also showed that PA could cause intracellular free fatty acid rise. The extracts of different active components could reduce the formation of intracellular free fatty acids and the activity of the active components in the whole valley bean bag before and after the.200 micron g/mL maturation increased the cell uptake of glucose. There was no significant difference from the negative control group (P0.05), all obviously lower than the PA model group (P0.05). Compared with the PA model group, the decrease was lower than that of the PA model group. The content of TG and MDA in the cells increased the content of SOD and GSH-PX (P0.05), which showed that the active components in the whole valley bean bag before and after maturation had a certain effect on insulin resistance.
(4) compared with the model group, the expression of AMPKa2, GLUT2 and PGC-1mRNA in the flavonoid group, the phenolic acid group, the plant sterol group and the dietary fiber group increased significantly, and the expression of ACC1 and FAS mRNA decreased significantly (P0.05). The improvement effect of the gene expression in the other groups, except the phenolic acid group, was not statistically significant (P0.05).
Conclusions: (1) the whole valley bean bags are rich in dietary fiber, flavonoids, phenolic acids and phytosterols. These active ingredients have strong antioxidant capacity in vitro.
(2) 0.25mM palmitic acid incubated with HepG2 cells for 24 hours could reduce the consumption of glucose and form an insulin resistance cell model.
(3) the active ingredients in the whole valley bean bag before and after maturation can improve the expression of AMPKa2, GLUT2 and PGC-1mRNA in HepG2 cells, reduce the expression of ACC1 and FAS mRNA, thus improve the insulin resistance induced by PA, which may be one of the mechanisms of whole valley bean bag to improve the body's insulin resistance.
(4) after comparison, it was found that the effect of the flavonoid group was the best for the improvement of the insulin resistance in HepG2 cells, the dietary fiber group and the plant sterol group, and the phenolic acid group was not obvious, especially after the curing of the phenolic acid group. The possible reason is that the phenolic acid is a kind of thermal unstable substance, and the heating and curing will accelerate the oxidation decomposition of phenolic acids.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R151.2

【参考文献】