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

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 (\u3c3 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

Enzyme kinetics, molecular docking, and in silico characterization of canary seed (Phalaris canariensis L.) peptides with ACE and pancreatic lipase inhibitory activity

The bioactivity of canary seed peptides (CSP) towards metabolism-regulating enzymes was evaluated. Peptides with angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPP-IV), and pancreatic lipase activity remained stable (p \u3c 0.05) to simulated gastrointestinal digestion (SGD). CSP-SGD were transported efficiently (\u3e10%) through the Caco-2 monolayer, indicating absorption through the intestinal epithelium. Lineweaver-Burk plots demonstrated that CSP-SGD act as mixed-type inhibitors for DPP-IV and α-glucosidase. Furthermore, CSP-SGD were potent as antihypertensive and antiobesity agents. Molecular docking and in silico analyses were targeted to understand CSP-SGD interactions with ACE and pancreatic lipase. ACE-inhibitory peptides (LHPQ, QTPHQ, KPVPR, and ELHPQ) acted as non-competitive inhibitors by destabilization of the transition state and Zn(II) coordination in ACE. The uncompetitive inhibition of pancreatic lipase by peptides (VPPR, LADR, LSPR, and TVGPR) destabilized the open-lid conformation of pancreatic lipase. The results of this study showed that canary seed proteins could serve as a source of biologically active peptides

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

During oxidative stress, degenerative diseases such as atherosclerosis, Alzheimer\u27s, 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 (\u3c3 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

Improvement of Functional and Bioactive Properties of Chia Seed (Salvia Hispanica) Protein Hydrolysates and Development of Biodegradable Films Using Chia Seed Mucilage

Chia seed (Salvia hispanica) has shown potential as an alternative source of nutrients with a high content of fiber (36 %), protein (25%), and fat (25%). Unfortunately, the presence of a viscous biopolymer (mucilage), surrounding the chia seed (CS), limits the accessibility of the protein and other nutrients. Nevertheless, this biopolymer’s chemical composition makes it suitable for the development of biodegradable films. Regarding CS protein, disulfide bonding, and nonproteinprotein interactions often frequent in plant protein, have limited its technological application in food matrices. Therefore, scientists have pointed at processing methods involving enzymatic proteolysis to improve the functionality of plant protein ingredients. The objective of this study was to establish processing techniques to exploit the functionality, extraction, and health benefits of chia seed components. First, ultrasonication followed by vacuum-filtration was used to separate mucilage from CS prior to fat extraction by oil press. Mucilage-free and defatted CS were treated using conventional (enzymatic hydrolysis with alcalase) or sequential (enzymatic hydrolysis with alcalase+flavourzyme), and under water bath or microwave-assisted hydrolysis. Chia seed protein hydrolysates (CSPH) derived from the sequential hydrolysis with microwave treatment showed superior (p\u3c0.05) in vitro antioxidant activity. The highest (p\u3c0.05) cellular antioxidant activity was achieved by the sequential (94.76%) and conventional (93.13%) hydrolysis with microwave. Dipeptidyl peptidase-V inhibition was higher (p\u3c0.05) for sequential hydrolysis with water bath, while Angiotensin-Converting Enzyme (ACE) inhibition activity increased (p\u3c0.05) with hydrolysis for all treatments compared to the control. Regarding functionality, sequential hydrolysis with microwave showed higher (p\u3c0.05) solubility at lower pH (3 and 5), while conventional hydrolysis with microwave was better at pH 7 and 9. Emulsification properties and foaming capacity were also higher in conventional hydrolysis with microwave, but conventional hydrolysis with water bath was more stable for foaming properties only. In terms of mucilage applicability, biodegradable films were developed by casting technique where CS mucilage was plasticized with different polyol mixtures (sorbitol and glycerol). CS mucilage films with higher sorbitol content showed superior tensile strength (3.23 N/mm2 ), and lower water vapor permeability (1.3*109 g/ m*s*Pa) but had poor flexibility compared to other treatments. Conversely, films with high glycerol content showed high elongation at break (67.55%) and solubility (22.75%), but reduced water vapor permeability and tensile strength. The hydrophobicity, measured as water contact angle, was higher (p\u3c0.05) for mixtures containing equal amounts of polyols. Lastly, Raman Spectroscopy analysis showed shifts from 854 to 872 cm-1 and 1061 to 1076 cm-1, which corresponded to β(CCO) modes. These shifts represent an increase in hydrogen bonding, responsible for the high tensile strength and decreased water vapor permeability. This study demonstrated that ultrasonication followed by vacuum filtration can successfully separate mucilage from chia seeds; microwave-assisted and enzymatic hydrolysis generated protein hydrolysates with improved bioactivity and functionality. Finally, chia seed mucilage was able to form films with potential to be used in drug delivery and edible food coating applications

Canary Seed (Phalaris canariensis L.) Peptides Prevent Obesity and Glucose Intolerance in Mice Fed a Western Diet

Previous research showed that canary seed (Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger