Preparation Of Antihypertensive Peptides From Phascolosoma Esculenta And The Research Of Inhibition Mechanism

Phascolosoma esculenta is one kind of marine animal which grows in the coastal areas in China and the body contains rich protein and trace elements. We utilized Phascolosoma esculenta as as raw material, extracted its water-soluble protein, separated its proteins by2-D electrophoresis and identified by MS method. We hydrolyzed water soluble protein in different enzyme enzymolysis. After separation and purification of mixed peptides, antihypertensive peptides with high activity were acquired. In addition, the molecular docking method was used to study interaction mechanism between antihypertensive peptide and angiotensin converting enzyme (ACE). Therefore, the work was done as following,(1) Two-dimensional electrophoresis method was adopted to separated the water soluble protein of Phascolosoma esculenta, after in-gel digestion, we used MALDI-TOF/TOF and data base to identify and match the kind of proteins. Three kind of proteins were identified. Two were actins. One was NADH-quinone oxidoreductase. According to the amino acid sequence and in silico hydrolysis, we can refer that the protein of Phascolosoma esculenta contained potential antihypertensive peptides.(2) We used gastrointestinal proteases to hydrolyze the soluble protein of Phascolosoma esculenta, the hydrolysate was separated and purified, then the antihypertensive peptide was identified and the inhibition mode was studied. The results exhibited that the IC50value of hydrolysate was0.671mg/ml after hydrolysis by pepsin. And the hydrolysate was further hydrolyzed by trypsin and a-chymotrypsin, the activity of the hydrolysate increased and the IC50value was found to be0.240mg/ml. Then the hydrolysate was separated by the Sephadex G15. The fraction with the highest IC50value0.029mg/ml was collected, which was again separated by RP-HPLC and identified by MALDI-TOF/TOF. Three peptides were identified. The amino acid sequence were Tyr-Ala-Ser-Gly-Arg, Arg-Tyr-Asp-Phe, Gly-Asn-Gly-Ser-Gly-Tyr-Val-Ser-Arg, molecular weights were552.59,559.65and895.93KDa, IC50value were184,235and29μM, respectively. Through Lineweave-Burk plot, we found that the ACE inhibition pattern of three peptides were all uncompetitive.(3) We used molecular docking to study the interaction mechanism between antihypertensive peptides and ACE, which indicated that the antihypertensive peptide interacted with Zn2+and amino acids with activity close to it, therefore the inhibition of ACE was generated. When the interaction energy of ligand and receptor was lower, the interaction was stronger, the inhibition was more excellent.