Study On The Enzymatic Preparation Of Holotharia Tubulosa Hydrolysate And Its Hypoglycemic Effect

Diabetes is a chronic disease characterized by hyperglycaemia,which can cause serious complications and be harmful to people's health seriously.At present,a great variety of oral hypoglycemic drugs are used to treat diabetes,but they can cause many side effects.Therefore,it is particularly important to develop safe and effective food-derived hypoglycemic products.Sea cucumber is a kind of nutritional food,which has several bioactive ingredients with hypoglycemic effects.Protein is the main component of sea cucumber.However,few studies on the bioactive peptides from sea cucumber protein were reported.Therefore,in this study,the optimal species from different sea cucumbers was selected to prepare hypoglycemic peptides by comparing their basic nutrient composition and the hypoglycemic activities of their hydrolysates in vitro,and the preparation technology was optimized.Subsequently,the hypoglycemic activity of Holotharia tubulosa hydrolysate(HTH)in vivo was studied as well as the mechanism.Finally,the proective effects of repaired liver and kidney caused by diabetes mellitus were further explored.The main contents and results are as follows.(1)Firstly,the optimal species of sea cucumber was screened to prepare hypoglycemic peptides based on the basic nutrient composition of different sea cucumbers and dipeptidyl-peptidase IV(DPP-IV)inhibition of their hydrolysates.Furthermore,the preparation technology was optimized and DPP-IV inhibitors were identified in the hydrolysate.The results showed that the imported wild Holotharia tubulosa from the Mediterranean region was selected to prepare hypoglycemic peptide because its hydrolysate showed higher protein content and DPP-IV inhibition compared with other sea cucumber hydrolysates.The optimal enzymatic hydrolysis condition was as follows: the combination of papain with protamex;the total addition amount of protease,1%;hydrolysis time,4 h;p H 7,55?.Under the optimal condition,the DPP-IV inhibition was 66.97% at a final concentration of 2 mg/m L,and the protein recovery and degree of hydrolysis were 76.25%and 6.10%,respectively.In addition,more than 90% peptides in the hydrolysates was less than 5000 Da.Furthermore,combining UPLC-MS/MS with the typical feature of DPP-IV inhibitory,28 potent DPP-IV inhibitory peptides with Pro located at the second position of N-terminal were obtained such as APGPAGPA and GPAGLTG,and none of them was reported.(2)Subsequently,type 2 diabetic rats induced by HFD+STZ were used to evaluate the hypoglycemic effect of HTH and its regulation ability of lipid metabolism.The resultsshowed that HTH can significantly alleviate the symptoms of diabetes mellitus.After 8weeks,the fasting blood glucose of diabetes model group rats achieved 18.00 mmol/L.However,oral gavage of 200 mg/kg HTH could significantly(p<0.05)reduce the levels of glucose to 16.00 mmol/L.Particularly,oral gavage of 400 mg/kg HTH very significantly(p<0.01)decreased the glucose level to 13.60 mmol/L and significantly(p<0.05)maintain the body weight.Dose-effect relationship was observed.Additionally,high dose of HTH could alleviate glucose tolerance and decrease glycosylated hemoglobin levels.Furthermore,it could also significantly(p<0.05)reduce TC,TG and LDL levels.In addition,it was found that high-dose intake of HTH could significantly(p<0.05)increased the levels of HOMA-IR and increased ISI by analyzing the insulin levels in rats.Moreover,HTH could significantly(p<0.01)improve the levels of Akt and GSK-3? phosphorylation and GLUT4 protein expression in liver tissues.It suggested that HTH could enhance the blood glucose regulation ability in diabetic rats by regulating the PI3K-Akt signal path to alleviate liver insulin resistance.(3)Finally,the protective effects of HTH on liver and kidney tissue in type 2 diabetic rats were investigated by observing histological alteration and measuring the serum biochemical indicators and the levels of liver antioxidant and kidney inflammation.The results showed that high dose of HTH could significantly(p<0.01)reduce the levels of serum Urea and AST in diabetic rats and relieve liver and kidney tissue damage.It can also relieve oxidative stress and lipid peroxidation in the liver by improving the activity of GSH-Px significantly(p<0.05),indicating that it could repair the liver damage.Meanwhile,the high dose of HTH could relieve the inflammatory response by reducing the contents of TNF-?,TGF-?1 and IL-1? significantly(p<0.01)in the kidney tissues,suggesting that it could repair the kidney damage.Therefore,HTH could not only regulate the blood glucose and lipid levels in type 2 diabetes rats,but also repair the liver and kidney damage caused by diabetes.It can be used as a potential nutritional supplement to prevent diabetes and relieve and its complications.