棉籽肽的制备及其对Ⅱ型糖尿病小鼠治疗作用的研究
发布时间：2019-01-22 13:18
【摘要】：自由基是机体在正常代谢过程中不断产生的代谢产物,自由基可对细胞造成损伤,进而导致机体疾病的发生,有研究表明,Ⅱ型糖尿病的发生和发展与体内自由基的产生有着密切联系。抗氧化剂可以清除体内自由基,保护细胞和组织免受氧化损伤,进而起到避免疾病发生和延缓衰老的作用。近年来,天然抗氧化肽引起了人们的普遍关注,但对抗氧化肽的研究尚不够深入,尤其是对抗氧化肽结构的研究罕见报道。因此,本研究以棉籽蛋白为原料,对棉籽蛋白的酶解、分离纯化、抗氧化活性、棉籽肽单体的结构以及棉籽肽单体对Ⅱ型糖尿病小鼠的治疗作用进行研究,旨在制备出具有高抗氧化活性的棉籽肽单体,评价其对Ⅱ型糖尿病小鼠的治疗效果,并试图从抗氧化角度阐述棉籽肽单体在Ⅱ型糖尿病小鼠的治疗过程中发挥的作用。本研究选取棉籽蛋白作为实验原料,采用蛋白酶进行水解,水解液经离心、过滤和超滤等粗分离后,得到分子量在3000 Da以下的棉籽蛋白水解产物。随后,对此产物进行DEAE Sephadex A-25离子交换层析和Sephadex G-10凝胶层析的进一步纯化,伴随着纯化过程,对层析得到的每一组分进行体外抗氧化活性的检测,得到具有较高抗氧化活性的棉籽肽组分。棉籽肽经液质联机系统(LC-MS/MS)的进一步分离及氨基酸序列的鉴定,最终得到三种棉籽肽单体。此后,将棉籽肽单体作用于由高脂饮食联合小剂量链脲佐菌素(STZ)诱导的Ⅱ型糖尿病小鼠模型中,检测小鼠空腹血糖(FPG)以及血清胰岛素(INS)、糖化血红蛋白(Hb A1c)、甘油三酯(TG)、总胆固醇(TC)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽(GSH)水平。采用HE染色法观察棉籽肽单体对Ⅱ型糖尿病小鼠胰腺和肾脏组织的病理形态学变化。研究结果表明,采用蛋白酶水解棉籽蛋白,其最适水解温度为50℃,水解p H为8.0,水解时间100 min,水解度为23.6%。三种棉籽肽单体的结构: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较高(80%),成膜后具有高血糖、高血脂、胰岛素分泌不足和胰岛素抵抗等典型的Ⅱ型糖尿病临床症状。棉籽肽单体可明显降低Ⅱ型糖尿病小鼠的FPG和血清Hb A1c水平,提高血清INS水平,有效改善胰岛素抵抗;棉籽肽单体可降低Ⅱ型糖尿病小鼠血清TG、TC水平,改善脂代谢紊乱;棉籽肽单体可明显降低Ⅱ型糖尿病小鼠血清MDA水平,提高血清SOD和GSH水平,增强机体抗氧化能力,减少机体氧化应激损伤;棉籽肽单体可使Ⅱ型糖尿病小鼠胰腺和肾脏组织病理学改变减轻,胰岛体积变大,胰岛细胞数目增多。综上所述,本研究成功地采用酶法从棉籽蛋白中得到了三种高抗氧化活性棉籽肽单体。三种棉籽肽单体的结构:Thr-Asp-Gln-Leu(MW:475 Da),Gly-Asp-Leu-Leu(MW:416 Da),Leu-Leu-Glu-Pro-Ala(MW:541 Da)。棉籽肽单体可明显降低Ⅱ型糖尿病小鼠的血糖、血脂水平,调节机体糖脂代谢,提高血清INS水平,改善胰岛素抵抗。棉籽肽单体可在调节Ⅱ型糖尿病小鼠糖脂代谢的基础上,增强机体抗氧化能力,降低脂质过氧化反应水平,减少机体氧自由基的产生,保护Ⅱ型糖尿病小鼠胰腺和肾脏组织。上述结果为棉籽肽单体用于Ⅱ型糖尿病的治疗和更广泛的应用积累了重要资料。
[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，
本文编号：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，
本文编号：2413244
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