Oxidative Stress Protection by Canary Seed (Phalaris canariensis L.) Peptides in Caco-2 Cells and Caenorhabditis elegans

Abstract

During oxidative stress, degenerative diseases such as atherosclerosis, Alzheimer’s, and certain cancers are likely to develop. Recent research on canary seed (Phalaris canariensis) peptides has demonstrated the high in vitro antioxidant potential. Thus, this study aimed to assess the cellular and in vivo antioxidant capacity of a low-molecular-weight (<3 kDa) canary seed peptide fraction (CSPF) using Caco-2 cells and the Caenorhabditis elegans model. The results show that the CSPF had no cytotoxicity effect on Caco-2 cells at any tested concentration (0.3−2.5 mg/mL). Additionally, the cellular antioxidant activity (CAA) of the CSPF was concentration-dependent, and the highest activity achieved was 80% by the CSPF at 2.5 mg/mL. Similarly, incubation with the CSPF significantly mitigated the acute and chronic oxidative damage, extending the lifespan of the nematodes by 88 and 61%, respectively. Furthermore, it was demonstrated that the CSPF reduced the accumulation of reactive oxygen species (ROS) to safe levels after sub-lethal doses of pro-oxidant paraquat. Quantitative real-time PCR revealed that the CSPF increased the expression of oxidative-stress-response-related gene GST-4. Overall, these results show that the CSPFs relied on GST-4 upregulation and scavenging of free radicals to confer oxidative stress protection and suggest that a CSPF can be used as a natural antioxidant in foods for health applications.

Keywords: Caenorhabditis elegans; antioxidant potential; canary seed peptides; oxidative stress.

Figure 1

(A) Cell viability (%): MTT assay over canary seed peptide fraction (CSPF) at different protein concentrations, (B) intracellular antioxidant activity (%) of CSPF and ascorbic acid (ASC), and (C) inhibition of peroxyl-radical-induced DCFH oxidation to DCF by CSPF and ASC. Negative control: untreated cells. Bars and lines represent mean values of triplicate determinations ± standard deviation. Different letters (a–e) indicate statistical differences (p < 0.05) between samples.

Figure 2

In vivo antioxidant activity of canary seed peptide fraction (CSPF), using a C. elegans model. (A) Survival analysis of C. elegans pre-exposed to CSPF for 24 h followed by acute oxidative stress induction with tert-butyl hydrogen peroxide (t-BOOH). (B) Survival analysis of C. elegans pre-exposed to CSPF for 24 h followed by chronic oxidative stress induction with paraquat. (C) Intracellular accumulation of reactive oxygen species (ROS) in C. elegans after pre-exposure to CSPF for 24 h followed by paraquat for 48 h. (D) Relative mRNA levels of expression of DAF-16, SOD-3, SKN-1, GST-4, and GST-10 after 24 h exposure with 3 mg/mL of CSPF using ACT-1 as an internal control. Bars and lines represent mean values ± standard deviation. Three independent experiments were performed with at least 100 nematodes per treatment. Statistical differences are indicated as * p < 0.01, and ** p < 0.001 or different letters (a–c) p < 0.01.