Abstract
The present study investigated the in vitro antioxidant and ACE-inhibitory activities of egg albumen hydrolysate (EAH) prepared with pepsin and pancreatin enzymes. The EAH and peptides were purified by ultrafiltration (UF), gel filtration (GF), and High Performance Liquid Chromatography (HPLC) and tested for antioxidant and ACE-inhibitory activities. Antioxidant activity was assessed by lipid peroxidation inhibition in a linoleic acid system using the ferric thiocyanate (FTC) and thiobarbituric acid reactive species (TBARS) methods. The EAH, and 2, 5 and 10 kDa UF fractions, as well as the GF26 peptide fractions (1 mg/ml) inhibited linoleic acid autoxidation, by 40, 76, 63, 53 and 79 % respectively, which was inversely related to peptide fraction size. However, 0. 01 % butylated hydroxytoluene (BHT) and 0. 01 % trolox had higher activity (95 and 82 %, respectively) compared with the peptide fractions (p<0. 05). Similarly, inhibition of TBARS was in the order 29, 39, 27, 17, 70 and 78 % for EAH, 2 kDa, 5 kDa, 10 kDa, trolox and BHT respectively. The putative antioxidant mechanism of EAH involved scavenging activity based on the 1, 1-diphenyl-2-picrylhydrazyl (DPPH·), hydroxyl (OH·) and superoxide anion (O2·- ) radical scavenging assays, and ferric (Fe3+) reducing and ferrous (Fe2+) ion chelating activities in a dose-dependent manner. All peptide fractions exhibited ACE-inhibitory activity, based on the ACE-catalyzed liberation of hippuric acid from the hippuryl-L-histidyl-L-leucine residue, which improved on further GF and HPLC purification. The 2, 5 and 10 kDa peptides exhibited % ACE inhibitory activities of 78, 71 and 62 %, respectively, compared to the positive control captopril (96 %). The 2, 5 and 10 kDa peptides had IC50 values of 6. 01, 6. 86 and 7. 93 mg/ml respectively. Further, GF and HPLC purification of the 2 kDa peptides improved the IC50 values to 5. 76 and 5. 13 mg/ml, respectively. Cell viability of human colon carcinoma mono- layer (caco-2) cell line, assayed by the tetrazolium dye (MTT) colorimetric assay confirmed that the 2 kDa peptides were not toxic. The 2 kDa peptides (0. 1 mg/ml) reduced most of the endogenous antioxidant enzyme activities in a dose-dependent manner, indicating scavenging of ROS. It was evident that a significant proportion of the 2 kDa peptides were resistant to cellular aminopeptidases present in caco-2 cell epithelium and were therefore transported in their intact forms across the caco-2 cell epithelium. In addition, The 2 kDa peptides exhibited significant ROS scavenging activity evidenced by enhanced viability of the Ea. hy926 (HUVECs) cell lines, using the lucigenin-enhanced chemiluminescent and fluorescence methods. The results confirm that bioactive peptides derived from the EAH have significant antioxidant and ACE-inhibitory activities and potentially useful neutraceutical applications.