【作者】 周越；

【导师】 董英；

【作者基本信息】 江苏大学 ， 食品科学， 2013， 博士

【摘要】 贻贝是一种药食同源的海洋贝类,我国资源极其丰富。多肽与多糖是主要活性成分,可以改善或减缓年龄相关性疾病的发生,延长老年人的健康期,但其延缓衰老的作用及机制并不明确。鉴于此,本研究以紫贻贝为原料,制备贻贝肽和贻贝多糖,在初步证实了贻贝肽和贻贝多糖对衰老的干预作用的基础上,并从机体、组织器官、细胞以及分子等不同层次深入研究了贻贝肽和多糖的抗衰老机制,为高效利用贻贝资源以及抗衰老膳食补充剂或药物的开发提供实验依据和技术支撑。主要研究内容与结果如下：(1)采用碱提酸沉法提取贻贝蛋白,优化了酶解蛋白的工艺参数,并采用超滤分级制备贻贝寡肽。结果表明,在pH值为11.5时蛋白提取率可达76.1%；以中性蛋白酶和风味酶两种蛋白酶,复合酶法酶解贻贝蛋白,在酶用量各为4000U/g、温度50℃、pH值6.5、时间120min的条件下,酶解液对羟自由基清除率可达88%。采用3-10KDa的超滤膜对酶解液进行超滤分级,得到四种组分,其中分子量小于1kDa的贻贝肽占总量的77.1%,且抗氧化活性最强。(2)采用加酶辅助提取和热水浸提法相结合工艺,制备了贻贝多糖,并进行了初步纯化。结果表明,采用热水浸法提贻贝多糖时,较优的提取条件为料液比为1：40、浸提温度为60℃、浸提时间为6h；加酶辅助提取的总糖含量由8.8%增加到10.3%,且减少了脱蛋白次数；粗多糖经过脱蛋白后,经醇沉、透析等处理后,其特征吸收峰为196nm,贻贝多糖中含有的糖醛酸、硫酸基及糖胺聚糖的含量分别为9.77%、4.14%和10.2%,主要由鼠李糖、木糖、甘露糖、果糖、葡萄糖5种单糖组成,是一种潜在的活性功能多糖。(3)建立应激诱导的人二倍体细胞株MRC-5早衰细胞模型,系统地研究了贻贝肽与贻贝多糖对细胞早衰的影响。结果表明,200μmol/L的H202可诱导年轻的MRC-5细胞早衰；0.2mg/mL的贻贝肽、贻贝多糖对细胞早衰均有显著地干预作用,表现为细胞形态正常；细胞活力明显增加(p<0.01), SA-β-gal染色及SAHF阳性率显著降低(p<0.05)；并促使细胞增殖,缓解H202对线粒体膜电位的损伤,贻贝肽的干预作用更加显著,其机制可能与上调Sirtl及Nampt的mRNA的表达有关,同时使GSH含量上升,Prx1mRNA表达量上调,通过保持细胞较强的还原性,实现对细胞衰老的干预。(4)采用黑腹果蝇为模式生物,研究贻贝肽、多糖对雌雄果蝇的寿命和生命活力的影响。结果表明,0.3mg/mL以及3mg/mL的贻贝肽、贻贝多糖均可以延长果蝇平均寿命以及最高寿命,并显著提高果蝇的性活力(p<0.05);3mg/mL的贻贝肽、贻贝多糖还增强了SOD活性,并降低MDA的含量(p<0.05),减少果蝇体内的氧化损伤。(5)建立D-半乳糖致衰老小鼠模型,研究了贻贝肽、多糖对衰老小鼠脑退行性变化的保护作用及机制。结果表明,200mg/kg.d的D-半乳糖注射处理,可诱导ICR小鼠的学习记忆能力和运动平衡能力显著下降(p<0.05),1000mg/kg.d的贻贝肽、多糖均可以显著改善这些行为障碍(p<0.05);贻贝肽、多糖通过降低脑组织中乳酸水平、抑制iNOS活性,维持NO平衡,减少自由基损伤,促进神经细胞存活；贻贝肽还上调PI3K、Akt的蛋白表达、调控PI3K/Akt信号通路,从而改善小鼠的脑退行性变化。(6)采用D-半乳糖致衰老小鼠模型,研究了贻贝肽、多糖对衰老小鼠糖脂代谢的影响及其分子机制。结果表明,给小鼠灌胃1000mg/kg.d和200mg/kg.d贻贝肽、多糖,可缓解模型小鼠的衰老症状与腹腔脂肪堆积；高剂量的贻贝肽可使小鼠的血糖和TG水平显著降低(p<0.05),HDL-C水平的显著升高(p<0.05);同时,使肝脏组织中游离脂肪酸显著降低(p<0.05)、肝糖原含量升高；贻贝肽、多糖可使小鼠肝、肾组织中SOD活性和GSH含量均显著升高(p<0.05),MDA的含量均显著下降(p<0.05),减少了小鼠体内氧化损伤；同时使肝脏、脂肪组织的PPARa和PPARy蛋白表达增加,从而改善衰老小鼠的糖脂代谢。更多还原

【Abstract】 Mussels are nutritional and medicinal food from marine shellfish, which are abundant natural resources in our country. The polypeptides and polysaccharides are the main bioactive components in mussels. All of them can relieve or slow down age-related diseases, resulted the prolonged healthy stage. However, the anti-aging functions and mechanisms of mussel polypeptides and polysaccharides are still not well known. In view of the facts, the polypeptides and polysaccharides were prepared from the raw material of mussel(Mytilus edulis). In this study, firstly, effetlive intervention function of polypeptides and polysaccharides on anti-ageing was preliminary confirmed. Furthermore, the anti-ageing mechanisms of mussel polypeptides and polysaccharides were studied on individual, histological, cellular, and molecular levels. The research results will provide experimental foundations and technical supports for efficient utilization of mussel resources and development of dietary supplements or anti-ageing drugs. The main research contents and results are as follows:(1) Alkaline extraction and acid precipitation were employed to prepare proteins from mussels. The process parameters in enzymolysis mussel protein were optimized. And the mussel polypeptides were prepared by multi stage ultrafiltration method. The results show that the extraction rate of mussel protein can reach77.1%in the pH value of11.5. The optimal conditions of enzymolysis mussel protein are:using complex containing4000U/g of neutral protease and flavourzyme respectively,50℃, pH6.5, incubate120min. The free radical scavenging rate of the protein enzymatic hydrolysate is88%. Then the protein enzymatic hydrolysate was separated by multi stage ultrafiltration method using3～10KDa ultrafiltration membranes. Four protein components were obtained, and the mussel polypeptides which molecular weight less than1KDa accounted for77.1%of all components and possessed the strongest antioxidant activity.(2) Enzymatic-associated extraction and hot water extraction technology were combined to preparation and preliminary purification mussel polysaccharides. The optimal process conditions of mussel polysaccharides by hot water extraction were as following:1:40for the ratio of solid to liquid,60℃water bath for6h. The total sugar content of extraction increased from8.8%to10.3%and reduced the times of removing proteins by using enzyme assisted extraction method. After removing proteins, alcohol precipitation, and dialysis, the mussel polysaccharides has characteristic absorption peak at wave length196nm. The mussel polysaccharide is a potential bio-activity polysaccharide containing9.77%uronic acids,4.14%sulfate group and10.2%glycosaminoglycan, which mainly belonging to rhamnose, xylose, mannose, fructose, and glucose.(3) The anti-aging activities of mussel peptides and polysaccharides were evaluated using H2O2-induced prematurely senescent MRC-5fibroblasts. Young MRC-5cell can be induced prematurely senescent by200μmol/L H2O2.0.2mg/ml of mussel peptides and polysaccharides could evidently protect against cellular senescence induced by H2O2. The protective performances were maintaining normal cell morphology, increasing cell viability significantly (p<0.01), reducing SA-β-gal activity and SAHF positive cells significantly (p<0.05), promoting cell proliferation, alleviating the damage of mitochondrial transmembrane potential caused by H2O2. The intervention effect of mussel peptides is more significant than polysaccharides. The mechanisms of protective effect maybe rely in up-regulation mRNA expression of Sirtland Nampt. Meantime, cells maintain strong reducibility rely on increased GSH content, elevated Prx1mRNA expression, then achieve intervention on senescent cells.(4) The effects of mussel peptides and polysaccharides on life span and vitality were studied using Drosophila melanogaster as a model. The results show that either0.3mg/ml or3mg/ml of mussel peptides and polysaccharides can prolong the average life-span and maximum life, and can significantly elevate sexual vitality of D. melanogaster.3mg/mL of mussel peptides and polysaccharides can also enhance SOD activity and decrease MDA content (p<0.05), and reduce the oxidative damage in D. melanogaster. (5) The protective effects of mussel peptides and polysaccharides on degenerative brain and its mechanisms were investigated in ageing mice caused by D-galactosidase. The passive learning, spatial learning and memory ability, and balance ability decreased significantly (p<0.05) in200mg/kg.d D-galactosidase treated mice.1000mg/kg.d of mussel peptides or polysaccharides can improve these behavior disorders (p<0.05). Mussel peptides and polysaccharides promote neurocyte survival by reducing lactic acid level, inhibiting the activity of iNOS, maintaining NO balance, reducing free radical damage in brain tissue. Mussel peptides also up-regulate the protein expression of PI3K and Akt, thereby improve the mouse brain degenerative changes.(6) The effects and molecular mechanisms of mussel peptides and polysaccharides on glucose and lipid metabolism were investigated in D-galactosidase induced mice. The ageing symptoms and abdominal fat accumulation can be lessened in mice by gavage mussel peptides and polysaccharides with1000mg/kg.d or200mg/kg.d. In gavage mice with high dose mussel peptides, the blood sugar and TG decreased significantly (p<0.05), HDL-C level increased significantly (p<0.05); Meantime, in liver tissues, the free fatty acids decreased significantly (p<0.05) and hepatic glycogen level elevated. Mussel peptides and polysaccharides can significantly increase the SOD activity and GSH content (p<0.05) and decrease MDA content (p<0.05) in tissues of mice liver and kidney. All these enhance the anti-oxidative enzyme system activity in mice. At the same time, the expression levels of PPARa and PPARy proteins increased in liver and fat tissues, the glucose and lipid metabolism also improved.更多还原