Effects Of Oyster And Its Enzymatic Hydrolysate On Anti-photoaging

Hong Kong oyster(Crassostrea hongkongensis)is an economic shellfish widely cultivated along the coast of southern China.It is rich in protein and the polypeptides extracted from it have good activity.It has great potential for the development of functional foods."Marine milk",made from oysters,is popular with consumers.The "Compendium of Materia Medica" records: " Oyster meat is good for whitening and smoothing skin ",indicating that oysters have the effect of beauty and skin care.In recent years,with the in-depth research on skin aging,it has been found that ultraviolet rays are the most important factor leading to skin aging.Therefore,it is of great significance to explore the anti-skin photoaging activity of oyster peptides.In this paper,the active peptides from Hong Kong oysters were prepared by enzymatic hydrolysis,and they were separated and purified.The anti-photoaging activity of active peptides from Hong Kong oysters was evaluated through the mice skin photoaging model and the photoaging HaCaT cell model.The main results are as follows:(1)The protein concentration of oyster meat freeze-dried powder(OM)was 605.2g/kg,and the protein concentration of oyster enzyme freeze-dried dry powder(OH)was 599.1 g/kg.The photoaging model of mice skin was constructed by UVA+UVB combined irradiation,and the anti-photoaging activity of OM and OH was explored.The results showed that both OM and OH reduced skin wrinkles in mice,improve skin elasticity,reduced the atrophy of subcutaneous blood vessels,and prevented the increase in vascular permeability;HE staining results showed that OM and OH slowed down hyperkeratosis and thickening of the epidermis;The results of Masson staining and fuchsin staining showed that OM and OH reduced the curling and degradation of collagen fibers,avoid the abnormal proliferation of elastic fibers,and restore the arrangement of the dermal extracellular matrix network;compared with the model group,OM and OH made the activity of SOD and GSH-Px in skin increased significantly,while the concentrations of MDA and 8-OHDG were reduced;compared with the model group,medium and high doses of OM and OH inhibited the expression of MMP-3 and MMP-9,reduced the expression of MMP-3 and MMP-9 in the dermis significantly,and it reduced the degradation of Hyp in dermis significantly.Compared with the model group,high-dose OH reduced the expression of IL-6 and TNF-? in the skin,and increased the expression of TGF-?.In summary,OM and OH had anti-oxidation,inhibition of skin matrix degradation and anti-inflammatory activities,both of them exhibited anti-skin photoaging activity,and OH was better than OM at the same dose.Therefore,the separation and purification of OH was continued with the mouse photoaging skin model in this study,and the corresponding biochemical indexes were added to screen the optimal components of anti-photoaging.(2)OH was filtered through ceramic membrane and ultrafiltration membrane and three components of > 5 ku,3-5 ku and <3 ku were obtained.The protein concentration were 654.9 g/kg,674.5 g/kg and 672.7 g/kg.The same animal model and gavage dose as in the previous chapter were used to explore the anti-photoaging activity of each component.The results showed that both the >5 ku component and the 3-5 ku component reduced the wrinkles of the photo-aged skin,prevent the skin from thickening and increase the skin elasticity,while the <3 ku component had no obvious effect.The 3-5 ku component improved the condition of capillary atrophy and increased permeability.The results of HE staining and Masson staining showed that the 3-5 ku component reduced the thickening of the epidermal layer,slowed down the twisting and aggregation of collagen fibers in the skin,and made them regular and evenly distributed.Compared with the model group and other dose groups,the 3-5 ku component had the most significant effect in increasing the content of SOD and GSH-Px in the skin,reduced the content of MDA and 8-OHDG in the skin.Compared with the model group,the content of MMP-1 and MMP-9 was reduced in the >5 ku high-dose group and 3-5 ku all dose groups.The 3-5 ku middle-dose group can significantly reduce the accumulation of MMP-1 in the dermis.The filter component had no significant effect on MMP-3.The 3-5 ku high-dose group increased the Hyp content in the skin most significantly,which was close to the blank group.The 3-5 ku high-dose group had good anti-inflammatory ability.The content of IL-6 and TNF-? was close to the blank group,and there was no significant difference.All ultrafiltration components had no significant effect on TGF-? in the skin.In summary,the 3-5 ku component had the best anti-photoaging effect,and it was further isolated and purified.(3)Sephadex-G25 gel column chromatography was used to separate and purify the3-5 ku components to obtain seven components(OP1-OP7).The results showed that the protein content of OP5 and OP7 was more than 95 %,the seven components were all mainly peptides,and the total sugar content was low.The total antioxidant capacity of OP5,OP6 and OP7 was better than OP1,OP2,OP3 and OP4.Infrared spectrum analysis showed that OP3 contained a small amount of polysaccharides,OP5 had carboxylic acid functional groups,and the molecular structure was asymmetric.The results of mass spectrometry and molecular weight analysis showed that the three main protein sources of OP3 were glyceraldehyde 3 phosphate dehydrogenase,type I actin(from oyster)and cytoplasmic heat shock protein 70;the three main protein sources of OP5 were II Type actin(oyster source),ATP synthase FO6 subunit and glyceraldehyde 3 phosphate dehydrogenase.The molecular weight of OP3 was about2843 k Da-2876 k Da,and the molecular weight of OP5 was about 2474 k Da-2524 k Da.(4)The effect of OP3,OP4 and OP5 on the proliferation of HaCaT cells was studied.The results showed that OP3 and OP5 promoted the proliferation of HaCaT cells at the concentration of 25-200 ?g/m L;OP4 had no cell proliferation promotion effect,and had no cell proliferation promotion effect.The concentration of 400 ?g/m L caused cell apoptosis significantly.The photoaging HaCaT cell model of UVA+UVB combined irradiation was used to explore the anti-photoaging activity of OP3 and OP5.The results showed that both OP3 and OP5 reduced the content of ROS in HaCaT cells,and the optimal concentration was 25 ?g/m L and 100 ?g/m L.Both OP3 and OP5 reduced the content of MMP-1 and MMP-9 secreted by HaCaT cells due to UV stimulation,and increased the expression of PINP.The optimal concentration was25 ?g/m L.OP3 was superior to OP5 in reducing the content of MMPs.