Isolation,Purification,Identification And Activity Evaluation Of Hypoglycemic Peptides From Acaudina Molpadioides

Acaudina molpadioides is a kind of sea cucumber,belonging to Molpadidaes Family,Holothurioidea Class,Echinodermata Phylum,which is a kind of low-value Sea cucumbers,also known as Cucumaria.It is distributed all along the coast of China,and its resources are quite abundant.In addition to polysaccharides,saponins,collagen and other chemical components,the body wall of A.Molpadioides also contains a variety of active peptides,which have various physiological functions such as lowering blood pressure,preventing liver damage,lowering fatty liver and lowering blood sugar,and have good applications in food and medical fields,etc.At present,the research objects about sea cucumber mainly focus on species with high economic value such as Sashimi and Thelenota ananas,and there are not many domestic reports on the research of peptides of A.Molpadioides with low economic value.Therefore,in this project,the low value A.Molpadioides was used as the raw material for the separation and enrichment of A.Molpadioides hypoglycemic peptides using Aqueous two phase system based on Deep eutectic solvents,and the target peptides were further separated and purified by the combination of reverse-phase liquid chromatography and medium-pressure preparative liquid chromatography.The amino acid sequence of the target peptide was determined by Edman degradation method and synthesized by solid phase synthesis.The activity of A.Molpadioides hypoglycemic peptide was evaluated by α-amylase inhibition assay.and explore the structure-activity relationship.The main studies are as follows:(1)The effect of Deep eutectic solvents quality,peptide quality of A.Molpadioides,separation time,Potassium Phosphate Monobasic concentration,temperature and pH on the isolation and enrichment of hypoglycemic peptides from A.Molpadioides was evaluated byα-amylase activity inhibition assay,and the best isolation and enrichment conditions were obtained.The results showed that the optimal conditions for the Aqueous two phase system were Deep eutectic solvents mass of 14 g,a mass of 200 mg of A.Molpadioides peptide,concentration of 0.95 g/mL of Potassium Phosphate Monobasic,separation time of 150 min,no effect of pH,and temperature control of 15℃-40℃.(2)Isolation and purification of hypoglycemic peptides from A.Molpadioidesseparation and enrichment of hypoglycemic peptides from A.Molpadioides was carried out by Aqueous two-phase system based on a Deep eutectic solvent,and the enrichment was carried out by reversed-phase liquid chromatography and medium-pressure preparative liquid chromatography.The A.Molpadioides hypoglycemic peptide was separated and purified.After several iterations of isolation and purification,the target peptide: H11d-1 was finally obtained.(3)Identification of target peptide H11d-1The amino acid sequences of the target peptides were identified by Edman degradation method.The results showed that the amino acid sequence of the identified peptide was Gly-Pro-Ala-Gly-Pro-Thr/Ile-Gly-Ala-Gln,in which the amino acid at the sixth N-terminal site contained two amino acids Thr and Ile with low content at the same time,and the amino acid at the ninth N-terminal site was low considering possible modifications or other structures.The results of parameter optimization of the Aqueous two phase system based on Deep eutectic solvents were verified using high performance liquid chromatography,The results showed that the optimal conditions for the Aqueous two phase system were Deep eutectic solvents mass of 14 g,a mass of 200 mg of A.Molpadioides peptide,concentration of0.95 g/mL of Potassium Phosphate Monobasic,separation time of 150 min,no effect of pH,and temperature control of 15℃-40℃.and the results were consistent with the conclusions of(1)above.(4)Synthesis of target peptide H11d-1The H11d-1 peptide was synthesized using the solid-phase synthesis method,and the four peptides were synthesized according to the diversity of the identification results:P1:Gly-Pro-Ala-Gly-Pro-Thr-Gly-Ala-Gln P2:Gly-Pro-Ala-Gly-Pro-Thr-Gly-AlaP3:Gly-Pro-Ala-Gly-Pro-Iler-Gly-Ala-Gln P4:Gly-Pro-Ala-Gly-Pro-Iler-Gly-AlaThe four peptides were evaluated in α-amylase activity inhibition experiments.The results showed that P2,P3 and P4 had inhibitory activity on α-amylase and had hypoglycemic effect,and the average inhibition rate of α-amylase by the hypoglycemic peptides P2,P3 and P4 of A.Molpadioides reached about 50% when the concentration was 22 mg/mL.After conformational analysis,the amino acids in the amino acid sequence of the target peptide that inhibited α-amylase may be Gly,Pro,Ala,Ile.