Extraction And Purification Of Polypeptides From Arca Subcrenata And Preliminary Study Of Their Anti-oxidative Capacity And Antitumor Activity In Vitro

Arca subcrenata is one of important economic shellfish in China. Its meat istender and rich in nutrition; it also has many medicinal effects. At present, the studyof natural active constituents from the meat of Arca subcrenata is rare. For this reason,the extraction and application of activity constituents from Arca subcrenata wouldvery important; it would provide effective way to Arca subcrenata value-incrementand its medicinal prospects. In this paper, the proteins and peptides from Arcasubcrenata meat were extracted by solvent, gradient salting-out. Then they werepurified further by ultrafiltration and Sephadex gel chromatography. After purification,three groups of natural peptides from Arca subcrenata of different molecular weightrange were collected. Respectively, high performance liquid chromatography (HPLC)and Tricine-SDS-PAGE were taken to analyse the composition and molecular weightdistribution of the three groups of peptides. Finally, their anti-oxidative capacity andantitumor activity in vitro were preliminary investigated in this study.Firstly, three factors were discussed in the single-factor test of antioxidant activityprotein extraction, respectively are: the pH of PBS buffer, the NaCl concentration inthe buffer solution, the ratio of material to liquid. In order to the higher extraction andthe better antioxidant activity of proteins, the proper conditions as follows: the pH ofPBS buffer was7.0, the NaCl concentration in the buffer solution was0.05mol/L, andthe ratio of material to liquid was1:2.Secondly, the proteins extracted from Arca subcrenata were separated byammonium sulfate fractionation and ultrafiltration, then were desalted and purified bySephadex G-15gel chromatography.The result showed that the proteins salted out by30%~100%ammonium sulfate can be dissolved better and had a higher antioxidantactivity. After used by ultrafiltration with molecular weight cutoff（MWCO）of30kD、 10kD and3kD, in turn, they were separated into three groups of peptides of differentmolecular weight range. Then, the three groups of peptides were desalted and purifiedby Sephadex G-15gel column. The three groups of purified peptides werefreeze-dried to obtain component1, component2and component3.The molecular weight distribution of the three groups of peptides was analyzed.The results of HPLC showed that: The component1peptide composed of five parts,its molecular composition was440.59Da,721.98Da1252.45Da1734.17Da,3158.68Da, respectively; component2mainly consists of four parts, the molecularweight was approximately4776.48Da,5831.68Da,7478.94Da,9454.88Da,respectively. The result of Tricine-SDS-PAGE showed that the molecular weightdistribution of the protein component3ranged from10kD to20kD.Finally, preliminary study on antioxidant activity and inhibition ability of tumorcell proliferation in vitro of the purified natural peptides from Arca subcrenata wasinvestigated. Results showed that component1had good scavenging effect onsuperoxide anion radical and DPPH·radical. When the sample concentration was5.00mg/mL, scavenging rates of superoxide anion radical and DPPH·radical were40.54%and28.66%respectively. MTT assay results showed that component1had inhibitoryeffect on hepatoma carcinoma cell HepG2proliferation to a certain extent, component2potentially accelerated HepG2proliferation when specific concentration wasachieved and component3had no significant effect on HepG2proliferation. Theresults of in vitro activity studies indicated that the purified natural oligopeptides(MW <3kD) from the Arca subcrenata had good antioxidant activity and anti-tumoractivity, which demonstrated the great value and significance of further developmentand the potential of developing as a novel marine natural medicine.In summary, this paper for the first time separated and purified the naturallyoccurring small molecule peptides in Arca subcrenata and studied their biologicalactivity. Systematic steps of separation and purification on peptides from Arcasubcrenata were developed. Certain degrees of anti-oxidation and anti-tumor activityin the purified small molecule peptides were verified. Our results provided atheoretical foundation for the in-depth study of the clam polypeptides, and are expected to promote the development in the field of functional food and drugutilization of the Arca subcrenata.