Studies On Active Peptides From Marine Organism—Arca Subcrenata Lischke

Marine organisms possess species diversity and hold immense potential as a source of new drugs. Bioactive peptides are an important class of compounds in marine active materials. This thesis reports the studies on the isolation, purification, chromatographic fingerprint, and anti-tumor activity of bioactive peptides from Arca subcrenata Lischke, as well as on the isolation, purification, and antitumor and antioxidant activities of bioactive peptides from hydrolysates of A. subcrenata Lischke.The thesis consists of two sections. In the first section, the extraction of A. subcrenata was investigated through L8(4×24) orthogonal array design, and the maximum response was achieved at a3-min homogenizing time with double volume10mmol/L sodium phosphate buffer (pH8.0) added to the sample. Furthermore, two different fractionation methods (ultrafiltration and ammonium sulfate precipitation) were employed. Different fractions from A. subcrenata were evaluated in vitro for anti-tumor activities against eight tumor cell lines, namely, oral epithelium cancer cell (KB), uterine cervix cancer cell (Hela), lung cancer cell (A549), hepatoma cell (BEL-7404), prostatic carcinoma cell (PC-3), nasopharyngeal carcinoma cell (CNE), and two different kinds of leukemia cells (HL-60and P-388). The fraction from A. subcrenata with70-100%ammonium sulfate saturation was strongly active against Hela, HL-60and KB (IC50=6,24and76μg/mL, respectively).The fraction from A. subcrenata with70-100%ammonium sulfate saturation was analyzed by Reversed-Phase High Performance Liquid Chromatography (RP-HPLC), and the best condition to separate unknown composition was developed as follows:temperature:30℃, wavelength:280nm, flow rate:1.0mL/min, the concentration of mobile phase A from50%to70%as that of mobile phase B from50%to30%(mobile phase A:80%acetonitrile including0.1%TFA, mobile phase B:0.1%TFA). Under these conditions, the chromatographic fingerprints of ten batch samples were analyzed.The purified polypeptides G-2, G-3, G-4-2, G-6-1and G-6-2were obtained from A. subcrenata by ion-exchange chromatography and gel filtration chromatography. The purity of G-3and G-4-2was more than96%, as measured by RP-HPLC. The molecular weights of G-2, G-3, G-4-2, G-6-1and G-6-2were determined to be14,665.1Da,8,250.4Da,15,970.8Da,24,051.6Da and20,099.8Da, respectively, by SDS-PAGE and IEF-PAGE, and isoelectric points to be7.0,6.6,6.1,5.6and5.3, respectively. Amino acid constituents of G-3and G-4-2were also determined, and phenol-vitriol test revealed that saccharides exist in G-3and G-6-2.In the second section, the optimal conditions of hydrolysis of A. subcrenata by neutrase, alcalase and papain were studied through orthogonal experiments according to the degree of hydrolysis (DH). As a result, the DH of alcalase was found to be higher than those of other two proteases.The results from the analysis of gel filtration chromatography showed that the papain hydrolysate consisted of higher proportion of components with large molecular masses, while the alcalase hydrolysate consisted of higher proportion of components with lower molecular masses. In addition, amino acids of hydrolysates were analyzed by ion chromatography, and the results indicated that the content of peptide of three hydrolysates followed the order as alcalase> papain> neutrase.The antioxidant activities of three hydrolysates of A. subcrenata were determined in vitro. All the hydrolysates had the abilities to scavenge DPPH free radicals and H2O2. The alcalase hydrolysate exhibited the best activity among them. Moreover, the reducing powers of three hydrolysates were found to be similar.Based on the effect of hydrolysis and antioxidant activity, the alcalase was chosen as the optimal protease for hydrolysis of A. subcrenata. The peptides with DPPH scavenging ability were isolated and purified from AS-A. The hydrolysate was fractionated by ion exchange column, and three peaks were named A-A, A-B and A-C. The portion A-B showed the strongest scavenging ability against DPPH free radicals, with the scavenging rate of67.64%(5mg/mL). This portion was loaded onto Sephadex G-25gel filtration column, and eleven peaks were eluted. The seventh and eighth peaks were named peaks g (A-Bg) and h (A-Bh), with the scavenging rates of32.89%(2mg/mL) and28.08%(2mg/mL), respectively. The peak g was further purified by RE-HPLC, and two single peaks were obtained. The peak A-Bgl had higher scavenging activity than peak A-Bg2.The amino acid sequences of N-terminal end of A-Bg1and of A-Bh were determined:FCCC was the sequence for A-Bgl and WWW for A-Bh.