| 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. |
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