棉籽肽的制備及其對(duì)Ⅱ型糖尿病小鼠治療作用的研究
發(fā)布時(shí)間：2019-01-22 13:18
【摘要】：自由基是機(jī)體在正常代謝過(guò)程中不斷產(chǎn)生的代謝產(chǎn)物,自由基可對(duì)細(xì)胞造成損傷,進(jìn)而導(dǎo)致機(jī)體疾病的發(fā)生,有研究表明,Ⅱ型糖尿病的發(fā)生和發(fā)展與體內(nèi)自由基的產(chǎn)生有著密切聯(lián)系�？寡趸瘎┛梢郧宄w內(nèi)自由基,保護(hù)細(xì)胞和組織免受氧化損傷,進(jìn)而起到避免疾病發(fā)生和延緩衰老的作用。近年來(lái),天然抗氧化肽引起了人們的普遍關(guān)注,但對(duì)抗氧化肽的研究尚不夠深入,尤其是對(duì)抗氧化肽結(jié)構(gòu)的研究罕見(jiàn)報(bào)道。因此,本研究以棉籽蛋白為原料,對(duì)棉籽蛋白的酶解、分離純化、抗氧化活性、棉籽肽單體的結(jié)構(gòu)以及棉籽肽單體對(duì)Ⅱ型糖尿病小鼠的治療作用進(jìn)行研究,旨在制備出具有高抗氧化活性的棉籽肽單體,評(píng)價(jià)其對(duì)Ⅱ型糖尿病小鼠的治療效果,并試圖從抗氧化角度闡述棉籽肽單體在Ⅱ型糖尿病小鼠的治療過(guò)程中發(fā)揮的作用。本研究選取棉籽蛋白作為實(shí)驗(yàn)原料,采用蛋白酶進(jìn)行水解,水解液經(jīng)離心、過(guò)濾和超濾等粗分離后,得到分子量在3000 Da以下的棉籽蛋白水解產(chǎn)物。隨后,對(duì)此產(chǎn)物進(jìn)行DEAE Sephadex A-25離子交換層析和Sephadex G-10凝膠層析的進(jìn)一步純化,伴隨著純化過(guò)程,對(duì)層析得到的每一組分進(jìn)行體外抗氧化活性的檢測(cè),得到具有較高抗氧化活性的棉籽肽組分。棉籽肽經(jīng)液質(zhì)聯(lián)機(jī)系統(tǒng)(LC-MS/MS)的進(jìn)一步分離及氨基酸序列的鑒定,最終得到三種棉籽肽單體。此后,將棉籽肽單體作用于由高脂飲食聯(lián)合小劑量鏈脲佐菌素(STZ)誘導(dǎo)的Ⅱ型糖尿病小鼠模型中,檢測(cè)小鼠空腹血糖(FPG)以及血清胰島素(INS)、糖化血紅蛋白(Hb A1c)、甘油三酯(TG)、總膽固醇(TC)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽(GSH)水平。采用HE染色法觀察棉籽肽單體對(duì)Ⅱ型糖尿病小鼠胰腺和腎臟組織的病理形態(tài)學(xué)變化。研究結(jié)果表明,采用蛋白酶水解棉籽蛋白,其最適水解溫度為50℃,水解p H為8.0,水解時(shí)間100 min,水解度為23.6%。三種棉籽肽單體的結(jié)構(gòu):Thr-Asp-Gln-Leu(MW:475 Da),Gly-Asp-Leu-Leu(MW:416 Da),Leu-Leu-Glu-Pro-Ala(MW:541 Da)。建立的Ⅱ型糖尿病小鼠模型成模率較高(80%),成膜后具有高血糖、高血脂、胰島素分泌不足和胰島素抵抗等典型的Ⅱ型糖尿病臨床癥狀。棉籽肽單體可明顯降低Ⅱ型糖尿病小鼠的FPG和血清Hb A1c水平,提高血清INS水平,有效改善胰島素抵抗;棉籽肽單體可降低Ⅱ型糖尿病小鼠血清TG、TC水平,改善脂代謝紊亂;棉籽肽單體可明顯降低Ⅱ型糖尿病小鼠血清MDA水平,提高血清SOD和GSH水平,增強(qiáng)機(jī)體抗氧化能力,減少機(jī)體氧化應(yīng)激損傷;棉籽肽單體可使Ⅱ型糖尿病小鼠胰腺和腎臟組織病理學(xué)改變減輕,胰島體積變大,胰島細(xì)胞數(shù)目增多。綜上所述,本研究成功地采用酶法從棉籽蛋白中得到了三種高抗氧化活性棉籽肽單體。三種棉籽肽單體的結(jié)構(gòu):Thr-Asp-Gln-Leu(MW:475 Da),Gly-Asp-Leu-Leu(MW:416 Da),Leu-Leu-Glu-Pro-Ala(MW:541 Da)。棉籽肽單體可明顯降低Ⅱ型糖尿病小鼠的血糖、血脂水平,調(diào)節(jié)機(jī)體糖脂代謝,提高血清INS水平,改善胰島素抵抗。棉籽肽單體可在調(diào)節(jié)Ⅱ型糖尿病小鼠糖脂代謝的基礎(chǔ)上,增強(qiáng)機(jī)體抗氧化能力,降低脂質(zhì)過(guò)氧化反應(yīng)水平,減少機(jī)體氧自由基的產(chǎn)生,保護(hù)Ⅱ型糖尿病小鼠胰腺和腎臟組織。上述結(jié)果為棉籽肽單體用于Ⅱ型糖尿病的治療和更廣泛的應(yīng)用積累了重要資料。
[Abstract]:Free radicals are the metabolites produced by the body in the course of normal metabolism, free radicals can cause damage to the cells, which can lead to the occurrence of the diseases of the body, and the research shows that the occurrence and development of type II diabetes are closely related to the generation of free radicals in the body. The antioxidant can remove free radicals in the body, protect the cells and tissues from oxidative damage, and further play a role in preventing the occurrence of the disease and delaying the aging. In recent years, the natural antioxidant peptide has attracted the general interest of people, but the research on the anti-oxidation peptide is not deep enough, especially for the research of the anti-oxidation peptide structure. Therefore, the research on the enzymatic hydrolysis, separation and purification of the cottonseed protein, the anti-oxidation activity, the structure of the cottonseed peptide monomer and the therapeutic effect of the cottonseed peptide monomer on the type II diabetic mice by using the cottonseed protein as the raw material is to prepare the cottonseed peptide monomer with high antioxidant activity, To evaluate the therapeutic effect of the cotton seed peptide monomer in the treatment of type II diabetic mice, the effect of the cotton seed peptide monomer in the treatment of type II diabetic mice was evaluated. In this study, the cottonseed protein was selected as the raw material of the experiment, and the protease was used for the hydrolysis. The hydrolysate was separated by centrifugation, filtration and ultrafiltration to obtain the hydrolysate of the cottonseed protein with a molecular weight of not less than 3000 Da. Then, the product was further purified by DEAE Sephadex A-25 ion exchange chromatography and Sephadex G-10 gel chromatography. The further separation of the cottonseed peptide and the identification of the amino acid sequence by the liquid-based on-line system (LC-MS/ MS) resulted in three cottonseed peptide monomers. Thereafter, the cottonseed peptide monomer is applied to a type II diabetic mouse model induced by a high-fat diet combined with a low-dose-chain cozizin (STZ) to detect a mouse fasting blood glucose (FPG) and a serum insulin (INS), a glycated hemoglobin (Hb), a triglyceride (TG), Total cholesterol (TC), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) level. The pathological changes of the pancreas and kidney of type II diabetic mice were observed by HE staining. The results showed that the optimal hydrolysis temperature of the cottonseed protein was 50 鈩，
本文編號(hào)：2413244
[Abstract]:Free radicals are the metabolites produced by the body in the course of normal metabolism, free radicals can cause damage to the cells, which can lead to the occurrence of the diseases of the body, and the research shows that the occurrence and development of type II diabetes are closely related to the generation of free radicals in the body. The antioxidant can remove free radicals in the body, protect the cells and tissues from oxidative damage, and further play a role in preventing the occurrence of the disease and delaying the aging. In recent years, the natural antioxidant peptide has attracted the general interest of people, but the research on the anti-oxidation peptide is not deep enough, especially for the research of the anti-oxidation peptide structure. Therefore, the research on the enzymatic hydrolysis, separation and purification of the cottonseed protein, the anti-oxidation activity, the structure of the cottonseed peptide monomer and the therapeutic effect of the cottonseed peptide monomer on the type II diabetic mice by using the cottonseed protein as the raw material is to prepare the cottonseed peptide monomer with high antioxidant activity, To evaluate the therapeutic effect of the cotton seed peptide monomer in the treatment of type II diabetic mice, the effect of the cotton seed peptide monomer in the treatment of type II diabetic mice was evaluated. In this study, the cottonseed protein was selected as the raw material of the experiment, and the protease was used for the hydrolysis. The hydrolysate was separated by centrifugation, filtration and ultrafiltration to obtain the hydrolysate of the cottonseed protein with a molecular weight of not less than 3000 Da. Then, the product was further purified by DEAE Sephadex A-25 ion exchange chromatography and Sephadex G-10 gel chromatography. The further separation of the cottonseed peptide and the identification of the amino acid sequence by the liquid-based on-line system (LC-MS/ MS) resulted in three cottonseed peptide monomers. Thereafter, the cottonseed peptide monomer is applied to a type II diabetic mouse model induced by a high-fat diet combined with a low-dose-chain cozizin (STZ) to detect a mouse fasting blood glucose (FPG) and a serum insulin (INS), a glycated hemoglobin (Hb), a triglyceride (TG), Total cholesterol (TC), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) level. The pathological changes of the pancreas and kidney of type II diabetic mice were observed by HE staining. The results showed that the optimal hydrolysis temperature of the cottonseed protein was 50 鈩，
本文編號(hào)：2413244
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