發(fā)布時間：2018-05-15 10:44

  本文選題：全谷豆包 + 植物化學(xué)物��； 參考：《揚州大學(xué)》2013年碩士論文

【摘要】：目的：(1)提取熟化前后全谷豆包中膳食纖維(DF)、類黃酮(FN)、酚酸(PAD)和植物固醇(PS),測定其含量和體外抗氧化能力,了解全谷豆包中活性成分提取物產(chǎn)生生物學(xué)效應(yīng)的物質(zhì)基礎(chǔ)； (2)觀察全谷豆包中活性成分提取物對棕櫚酸(PA)誘導(dǎo)的HepG2細(xì)胞胰島素抵抗作用的影響,測定細(xì)胞對葡萄糖的消耗量與細(xì)胞中甘油三酯(TG)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽過氧化物酶(GSH-PX)的含量,闡明不同種類活性成分提取物對HepG2細(xì)胞胰島素抵抗作用的干預(yù)效果差異； (3)觀察全谷豆包中活性成分提取物對胰島素抵抗HepG2細(xì)胞中的腺苷酸活化蛋白激酶a2(AMPKa2)、乙酰輔酶A羥化酶(ACC1)、脂肪酸合成酶(FAS)、葡萄糖轉(zhuǎn)運蛋白2(GLUT2)、過氧化物酶體增殖物活化受體γ協(xié)同激活因子-1(PGC-1)在mRNA水平上的影響,探討全谷豆包中活性成分對胰島素抵抗HepG2細(xì)胞的作用機(jī)制； (4)比較熟化對全谷豆包中活性物質(zhì)及其改善HepG2細(xì)胞胰島素抵抗作用的影響效果。 方法：(1)用酶-化學(xué)法提取熟化前后全谷豆包中的膳食纖維,按國標(biāo)法測定其含量；用浸提回流法提取熟化前后全谷豆包中的類黃酮,用超聲波萃取法提取熟化前后全谷豆包中的酚酸和植物固醇,用比色法測定其含量。采用DPPH自由基清除法和T-AOC總抗氧化能力檢測法測定不同活性成分提取物的體外抗氧化能力； (2)用棕櫚酸誘導(dǎo)HepG2細(xì)胞建立體外胰島素抵抗模型,通過MTT、油紅O染色、葡萄糖氧化酶法檢測細(xì)胞對葡萄糖的消耗量以及細(xì)胞中TG的含量,判定胰島素抵抗細(xì)胞模型建立成功,確定棕櫚酸最佳濃度； (3)棕櫚酸誘導(dǎo)HepG2細(xì)胞胰島素抵抗模型后,加入不同活性成分提取物,測定細(xì)胞對葡萄糖的消耗量與細(xì)胞中TG的含量和抗氧化指標(biāo)MDA、SOD、GSH-PX的含量,半定量PCR測定AMPKα2、ACC1、GLUT2、FAS、PGC-1的基因表達(dá)。 結(jié)果：(1)熟化前全谷豆包中膳食纖維的含量為17.5g/100g,類黃酮的含量為2.28g/100g,酚酸的含量為0.62g/100g,植物固醇的含量為4.60g/100g,熟化后全谷豆包中膳食纖維的含量為21.6g/100g,類黃酮的含量為3.51g/100g,酚酸的含量為0.52g/100g,植物固醇的含量為5.88g/100g;熟化前后全谷豆包中不同活性成分均具有很強(qiáng)的清除DPPH自由基能力和總抗氧化能力； (2)0.25mM的棕櫚酸對細(xì)胞活性無顯著影響,且細(xì)胞對葡萄糖的消耗量顯著降低,細(xì)胞內(nèi)TG顯著升高,細(xì)胞內(nèi)脂滴蓄積明顯,是誘導(dǎo)HepG2細(xì)胞形成胰島素抵抗的最佳濃度； (3)MTT實驗中觀察到,適當(dāng)濃度(200μg/mL)的不同活性成分提取物對PA誘導(dǎo)的胰島素抵抗HepG2細(xì)胞有一定的保護(hù)作用。與類黃酮組、植物固醇組和膳食纖維組相比,酚酸組的保護(hù)作用較弱。油紅O染色結(jié)果也顯示,PA可造成細(xì)胞內(nèi)游離脂肪酸升高,而不同活性成分提取物處理后,可減少細(xì)胞內(nèi)游離脂肪酸的形成。200μg/mL熟化前后全谷豆包中活性成分提取物提高了細(xì)胞對葡萄糖的攝取能力,與陰性對照組差異無統(tǒng)計學(xué)意義(P0.05),均明顯低于PA模型組(P0.05)。與PA模型組相比,降低了細(xì)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對胰島素抵抗均具有一定的改善作用； (4)與模型組相比,類黃酮組、酚酸組、植物固醇組和膳食纖維組細(xì)胞內(nèi)AMPKa2、GLUT2和PGC-1mRNA表達(dá)顯著增高,且ACC1和FAS mRNA表達(dá)顯著降低(P0.05)。除酚酸組外,其余組的基因表達(dá)改善效果與陰性對照組差異無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：(1)全谷豆包中富含膳食纖維、類黃酮、酚酸和植物固醇等抗氧化物質(zhì)。這些活性成分具有很強(qiáng)的體外抗氧化能力； (2)0.25mM的棕櫚酸與HepG2細(xì)胞孵育24小時,能使細(xì)胞對葡萄糖的消耗量顯著降低,形成胰島素抵抗細(xì)胞模型； (3)熟化前后全谷豆包中活性成分可以提高HepG2細(xì)胞內(nèi)AMPKa2、GLUT2和PGC-1mRNA的表達(dá),降低ACC1和FAS mRNA的表達(dá),從而改善PA誘導(dǎo)的胰島素抵抗?fàn)顟B(tài)。這可能是全谷豆包改善機(jī)體胰島素抵抗的機(jī)制之一； (4)比較后發(fā)現(xiàn),對HepG2細(xì)胞胰島素抵抗?fàn)顟B(tài)的改善作用,類黃酮組效果最好,膳食纖維組和植物固醇組次之,而酚酸組效果不夠明顯,尤其是熟化后的酚酸組�？赡茉蚴�,酚酸是一類熱不穩(wěn)定物質(zhì),加熱熟化會加速酚酸類物質(zhì)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.

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R151.2

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