Abstract
Peptides derived from food proteins after enzymatic treatment and/or processing, are known to be bioactive in both biological and food systems; for this reason fish muscle peptides were investigated for their antihypertensive and antioxidant activity. Atlantic mackerel muscle proteins were hydrolysed with pepsin and pancreatine and the resultant hydrolysate was sequentially fractionated on 2 kDa membrane ultrafilters and further by gel filtration, ion exchange and high performance liquid chromatography and the resultant peptide fraction contained the amino acids histidine, proline, tyrosine, methionine, leucine, tryptophan and lysine. For ACE inhibitory activity, the peptide fraction (MFPH-V-JPA) had an inhibitory concentration (IC50) of 0.15 mg/ml and showed competitive inhibition for ACE with an inhibition constant (Ki) of 0.32 mg/ml. In terms of antioxidant activity, the HPLC isolated peptide fraction (LC1-Z) contained the amino acids serine, histidine, tyrosine, phenylalanine, tryptophan and lysine. It inhibited the formation of both peroxides and malonaldehyde in a linoleic acid model emulsion in a dose dependent manner with lipid oxidation inhibitory concentration (IC50) of 1.80 mg/ml. At concentration of 8 mg/ml, the inhibition of linoleic acid oxidation was more than that of 0.01 % butylated hydroxytoluene (BHT) and trolox (p<0.001). The mechanism of antioxidant activity of the peptide (LC1-Z) was by carbon centered radical scavenging (5.34 %), hydroxyly radical scavenging (IC50 value of 1.60 mg/ml), metal chelating (5.72 %) and reducing ability. In caco-2 cells, 1 mg/ml of the peptide (LC1-Z) was not toxic to the cells seeded at 2 x 104 cells/well. Proxidant tBHP (2.5 mM) reduced cell viability significantly (79.3 %) but this increased to 94.7 % in the presence of the peptide or trolox. The peptide (1 mg/ml) also reduced TBARS formation (33.18 mug/ml) in cells compared to cells treated with tBHP alone (38.18 mug/ml). The activity of caspases-3 and -7, was higher in caco-2 cells treated with tBHP only (157.5 +/- 7.99 %) compared with those treated with the peptide (25.7 + 3.92 %). Morphological modification of the caco-2 cells treated with tBHP was evident as the cells appeared detached from the flask surface compared to those treated with the peptide (LC1-Z) which were healthy and attached to the flask surface. In Ea.hy 926 cells, reactive oxygen species were reduced by 26 % and 39 % in the lucigenin-chemiluminscence and flourescence methods respectively in cells treated with the peptide. In a caco-2 cell monolayer, transepithelial transport of the peptide was observed in both directions with a basolateral to apical apparent permeability of 0.95 +/- 0.12 cm-1 and apical to basolateral flux of 0.74 + 0.20 cm-1. HPLC chromatograms of the buffer solution taken from the apical and basolateral side showed the presence of the peptide in both sides i.e. 11.5% for apical to basolateral flux and 12.2 % for basolateral to apical. These results demonstrate that peptides with antihypertensive and antioxidant activity can be derived from Atlantic mackerel muscle proteins, with potential for neutraceutical applications.