Characterization and analysis of antioxidant activity of walnut-derived pentapeptide PW5 via nuclear magnetic resonance spectroscopy

The antioxidative activity of natural products has commonly been studied by free radical scavenging methods. However, the mechanisms by which antioxidation is explored by free radical scavenging methods remain largely unknown. This study analyzed the composition of walnut-derived pentapeptides PW5 with potential biological activity and its oxidation reaction products in 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) test by nuclear magnetic resonance (NMR) spectroscopy. The amino acid sequence of PW5 peptide successfully characterized as Proline-Proline-Lysine-Asparagine-Tryptophan exhibited significant antioxidant activity with lower IC₅₀ value (0.2210 ± 0.0032 mM) compared to glutathione (GSH, 0.2567 ± 0.0023 mM, p < 0.001). Furthermore, we found that the tryptophan residue was the only residue in PW5 with obvious alteration after treatment with ABTS free radicals, which was linked to its potential antioxidant properties. These findings revealed how NMR-characterized structures and oxidation reaction products may be used to explore the antioxidative mechanisms of food-derived peptides as well as other natural products.

Keywords: ABTS test; Antioxidant capacity; Antioxidant mechanism; Characterization; Free radical scavenging methods; Nuclear magnetic resonance spectroscopy; PW5; Walnut-derived peptides.