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Peptide fractions extracted from Flathead by-products were evaluated for in vitro free radical scavenging and cancer cell cytotoxic activities. The degree of hydrolysis (DH), presence of protease and molecular weight (MW) influenced the 2,2-diphenyl-1-pycryl-hydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic (ABTS) radical scavenging activities (RSA) of Flathead peptide fraction. Low MW peptides (<3 kDa), obtained from overnight incubation with added an exogenous protease, significantly inhibited free radicals and showed the highest RSA of 94.03 and 82.89 % against DPPH and ABTS, respectively. The presence of bioactive peptides during HO exposure increased viability of T4056 normal cells. Furthermore, peptide fractions <3 kDa inhibited the growth of HT-29 colon cancer cells up to 91.04 %, although the activity was found to be non-selective. Further purification revealed a novel peptide, Met-Gly-Pro-Pro-Gly-Leu-Ala-Gly-Ala-Pro-Gly-Glu-Ala-Gly-Arg, with RSA properties. These results indicated that peptides extracted from Flathead by-products have a potential to be used as natural antioxidants and/or chemo-protective agents. [ABSTRACT FROM AUTHOR]