Studies On The Antioxidant Activity Of Oligopeptide Isolated From Ruditapes Philippinarum Hydrolysate

Oxidation is an essential biological process in many living organisms. The radicals,products from oxidation, are very unstable and can lead to DNA and protein damage, andmay contribute to many diseases including cancer, Alzheimer’s disease, and otherneurodegenerative diseases. Although synthetic antioxidants (BHT, BHA, TBHQ and so on)have strong antioxidant activities, consumers show increasing resistance to them due topotential adverse effect on human health, and toxicity toward humans. As a result, there isa great interest in exploring new antioxidants from natural sources.This study, we use enzymolysis approach to extract peptide from short-necked clam,Enzymolysis condition is optimized according to the orthogonal test, and the crude extractis got under the optimum condition.Through the separation purification, amino acid test, structureCharacterization, the oligopeptide is purified. Studies are researched in vitro and vivoon the antioxidant of oligopeptide isolated from Ruditapes philippinarum hydrolysate. Theresearch includes the following aspects:Part1, the production of oligopeptides from Short-necked clamShort-necked clam Ruditapes philippinarum (Adams et Reeve,1850) muscle was homogenized andhydrolyzed with trypsin, and the optimum hydrolysis condition was determined using an orthogonalarray design according to the1,1-dipheny-l-2-picrylhydrazyl (DPPH), reducing power. The optimumhydrolysis condition was substrate concentration1:2, enzyme content dosage of2000U/g, pH8.0,temperature40℃, time duration12h. Under the optimum hydrolysis condition, the hydrolysate wasdivided into four parts according to the molecular weight, and the group with best antioxidant activitywas collected, concentrated and freeze-dried. The hydrolysate was fractionated by chromatographicmethods using Sephadex G-25gel filtration to get the best antioxidant activity parts which wascontinued to be concentrated and be subdivided and the structure of the amino acid sequence of thepurified antioxidant oligopeptide was found to be Gly-Asp-Gln-Gln-Lys, the molecular weight was575.45Da.Part2, the antioxidant activity of oligopeptides from Short-nected clam in vitro1. The scavenging activity and reducing power of ABTS,O2-·,·OH and metal ion,schelating ability are tested to find that oligopeptides from Short-necked clam have thesignificant ABTS （EC500.204g.L-1）,·OH (EC500.815g.L-1), O2-·(EC500.066g.L-1)scavenging activity, reducing power and the metal ion,s chelating ability. 2. The test of protective effect of the purified peptide against hydroxyl-radical-inducedDNA damage.The result shows that, compared with the blank, a the supercoiled (SC) DNAform of the control almost disappeared indicating a complete was completely conversion toan open circular (OC) form.While only a little of DNA was damaged into an open circular(OC) form in the sample group. The purified oligopeptide had a protective effect againstDNA damage induced by hydroxyl radical with increasing concentration of the purifiedoligopeptide.3、The anti-prostatic cancer DU-145activity was evaluated by MTT assay. The resultsshowed that oligopeptides can significantly restrain the proliferation of anti-prostaticcancer DU-145, and have a good dose-effect relationship. And the cytotoxicity test ofnormal prostate cells RWPE-1shows that oligopeptides are virulent to the RWPE-1cells.The third part, to study the antioxidation effect in vivo of oligopeptides fromShort-necked clam,ICR mice were randomly divided into4group (stomach irritation with high/middle/low oligopeptides and saline only for30days), being collected blood from heartand sample from organization. Enzyme activities and content of superoxide dismutase(SOD) and glutathione peroxidase (GSH-Px) in the serum, liver/brain and enzymeactivities were measured by reagent box. The results showed that oligopeptides fromShort-necked clam can significantly increase the activity of SOD/GSH PX and decreasethe contents of MDA in the serum and the organs. Oligopeptides from Short-necked clamcan improve antioxidation ability in vivo and balance the state of antioxidation/oxidation.