| In the experiment, bioactive peptides from protein hydrolysates of blood clam(Tegillarca granosa) muscle and Raja porosa cartilage were purified using ultrafiltration, anion-exchange chromatography, gel filtration chromatography and reversed phase high performance liquid chromatography(RP-HPLC). The amino acid sequences of peptides were determined using Procise Protein/Peptide Sequencer and electrospray ionization mass spectrometry(ESI-MS), and their antioxidant activities were evaluated by assays of radical scavenging and lipid peroxidation inhibition, and their antitumor activities were also measured.(1) Using ultrafiltration and consecutive chromatographic methods, three antioxidant peptides(DCPE-A, DCPE-B and DCPE-C) were isolated from the protein hydrolysate of R. porosa cartilage, and their amino acid sequences were identified as Phe-Ile-Met-Gly-Pro-Tyr(DCPE-A), Gly-Pro-Ala-Gly-Asp-Tyr(DCPE-B) and Ile-Val-Ala-Gly-Pro-Gln(DCPE-C)with molecμLar weights of 726.9, 578.58 and 583.69 Da, respectively. DCPE-A, DCPE-B and DCPE-C exhibited good scavenging activity on DPPH radical(EC50 3.83, 4.21 and 4.45 mg/l), hydroxyl radical(EC50 3.37, 3.92 and 4.23mg/m L), ABTS radical(EC50 0.77, 1.04 and 1.29mg/m L), and superoxide anion radical(EC50 1.61, 1.66 and 1.82 mg/m L). DCPE-A was also effectively against lipid peroxidation in a linoleic acid model system. In addition, DCPE-A displayed high anti-proliferation activity on He La cells by inducing apoptosis. The expression of the anti-apoptotic protein Bcl-2 was decreased and the expression of the apoptogenic protein Bax was increased when He La cells were treated by BCP-A for 24 h, which induced the increase of the Bax/Bcl-2 expression ratio. Moreover, the expression of caspase-3 in He La cells treated with BCP-A was upregμLated. Therefore, DCPE-A showed significantly antioxidant activity and anticancer properties.(2) Using similar separation method, two peptides isolated from protein hydrolysate of blood clam muscle were identified as Trp-Pro-Pro(BCP-A) and Gln-Pro(BCP-B) with molecular weights of 398.44 and 243.23 Da, respectively. BCP-A significantly inhibited lipid peroxidation and showed the highest radicals scavenging activities among the hydrolysate, fractions and purified peptides with EC50 of 1.388, 0.406, 0.536 and 2.75 mg/m L on DPPH radical, hydroxyl radical, superoxide anion radical and ABTS radical, respectively.Furthermore, BCP-A showed strong cytotoxicity to PC-3, DU-145, H-1299 and He La cell lines in a dose-dependent manner with IC50 of 1.99, 2.80, 3.3 and 2.54 mg/m L and significantly changed the morphology of PC-3 cells. The percentage of early stage of apoptotic PC-3 cells increased from 5.14% to 20.28% when they were treated with BCP-A at concentrations of 5 and 15 mg/m L for 24 h. The results indicated that BCP-A might be served as a natural antioxidant and anticancer agent for nutraceutical and pharmaceutical industries. |
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