Estudio de las propiedades antiinflamatorias de hidrolizados proteicos de altramuz (Lupinus angustifolius L.)

Programa de Doctorado en Biotecnología y Tecnología QuímicaLa inflamación crónica está implicada en el desarrollo de numerosas enfermedades como las cardiovasculares, cáncer, diabetes, obesidad, artritis, enfermedades neurológicas, pulmonares y autoinmunes. Se cree que la dieta es uno de los factores de riesgo en estas enfermedades, por lo que la modificación de la misma podría ayudar a prevenir o retrasarlas. En este trabajo se han obtenido hidrolizados proteicos de altramuz (Lupinus angustifolius L.) a partir de aislados proteicos usando dos proteasas Alcalase 2.4 L e Izyme AL. Su actividad antiinflamatoria fue evaluada in vitro sobre su capacidad para inhibir la actividad de diversas enzimas implicadas en el proceso inflamatorio y en un modelo de macrófagos mediante el estudio de la expresión y producción de diversos mediadores inflamatorios. A partir del hidrolizado que presentó mayor actividad, un péptido fue purificado por ultrafiltración y técnicas cromatográficas, y finalmente secuenciado e identificado como un fragmento presente en las proteínas de altramuz, siendo, hasta donde conocemos, el primer péptido con actividad antiinflamatoria purificado a partir de hidrolizados proteicos de altramuz.Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímic

A lupine (Lupinus angustifolious L.) peptide prevents non-alcoholic fatty liver disease in high-fat-diet-induced obese mice

Bioactive peptides are related to the prevention and treatment of many diseases. GPETAFLR is an octapeptide that has been isolated from lupine (Lupinus angustifolius L.) and shows anti-inflammatory properties. The aim of this study was to evaluate the potential activity of GPETAFLR to prevent non-alcoholic fatty liver disease (NAFLD) in high-fat-diet (HFD)-induced obese mice. C57BL/6J mice were fed a standard diet or HFD. Two of the groups fed the HFD diet were treated with GPETAFLR in drinking water at 0.5 mg kg-1 day-1 or 1 mg kg-1 day-1. To determine the ability of GPETAFLR to improve the onset and progression of non-alcoholic fatty liver disease, histological studies, hepatic enzyme profiles, inflammatory cytokine and lipid metabolism-related genes and proteins were analysed. Our results suggested that HFD-induced inflammatory metabolic disorders were alleviated by treatment with GPETAFLR. In conclusion, dietary lupine consumption can repair HFD-induced hepatic damage possibly via modifications of liver's lipid signalling pathways

Anti-inflammatory activity of lupine (Lupinus angustifolius L.) protein hydrolysates in THP-1-derived macrophages

The effect of two different lupine protein hydrolysates (LPHs) on in vitro macrophage activation in a THP-1-derived macrophage model was investigated. THP-1-derived macrophages were exposed to RPMI medium containing two LPHs obtained by enzymatic hydrolysis using two different proteases: Izyme AL and Alcalase 2.4 L. Cytokine's expression was measured by quantitative PCR. THP-1-derived macrophages exhibited attenuated expression of proinflammatory cytokines (tumor necrosis factor (TNF), IL-6, IL-1β) and increased expression of anti-inflammatory marker genes (chemokine (C-C motif) ligand 18 (CCL18)) relative to control without LPH. The anti-inflammatory effect of both hydrolysates favored M2 polarization by quenching C-C chemokine receptor type 2 (CCR2) expression and migratory capacity. Furthermore, LPHs significantly decreased nitric oxide production. Moreover, LPHs promoted the survival of human THP-1-derived macrophages. Therefore, inclusion of LPHs in foods may help to prevent chronic diseases associated with chronic inflammation.Ministerio de Economía y Competitividad AGL2012–40247-C02-0

Nutraceutical Extract from Dulse (Palmaria palmata L.) Inhibits Primary Human Neutrophil Activation

Palmaria palmata L. (Palmariaceae), commonly known as "dulse", is a red alga that grows on the northern coasts of the Atlantic and Pacific oceans, and is widely used as source of fiber and protein. Dulse is reported to contain anti-inflammatory and antioxidant compounds, albeit no study has investigated these effects in primary human neutrophils. Implication strategies to diminish neutrophil activation have the potential to prevent pathological states. We evaluated the ability of a phenolic dulse extract (DULEXT) to modulate the lipopolysaccharide (LPS)-mediated activation of primary human neutrophils. Intracellular reactive oxygen species (ROS) were measured by fluorescence analysis and nitric oxide (NO) production using the Griess reaction. Inflammatory enzymes and cytokines were detected by ELISA and RT-qPCR. The results show that DULEXT diminished the neutrophil activation related to the down-regulation of TLR4 mRNA expression, deceased gene expression and the LPS-induced release of the chemoattractant mediator IL-8 and the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. ROS, NO, and myeloperoxidase (MPO) were also depressed. The data indicated that DULEXT has the potential to disrupt the activation of human primary neutrophils and the derived inflammatory and prooxidant conditions, and suggest a new role for Palmaria palmata L. in the regulation of the pathogenesis of health disorders in which neutrophils play a key role, including atherosclerosisSpanish Ministry of Science, Innovation and Universities grant CYTED-2019 119RT056

Antioxidant and anti-inflammatory properties of bioavailable protein hydrolysates from lupin-derived agri-waste

Agri-food industries generate several by-products, including protein-rich materials cur-rently treated as waste. Lupine species could be a sustainable alternative source of protein compared to other crops such as soybean or chickpea. Protein hydrolysates contain bioactive peptides that may act positively in disease prevention or treatment. Inflammatory responses and oxidative stress underlie many chronic pathologies and natural treatment approaches have gained attention as an alternative to synthetic pharmaceuticals. Recent studies have shown that lupin protein hydrolysates (LPHs) could be an important source of biopeptides, especially since they demonstrate anti-inflam-matory properties. However, due to their possible degradation by digestive and brush-border en-zymes, it is not clear whether these peptides can resist intestinal absorption and reach the blood-stream, where they may exert their biological effects. In this work, the in vitro cellular up-take/transport and the anti-inflammatory and antioxidant properties of LPH were investigated in a co-culture system with intestinal epithelial Caco-2 cells and THP-1-derived macrophages. The results indicate that the LPH crosses the human intestinal Caco-2 monolayer and exerts anti-inflam-matory activity in macrophages located in the basement area by decreasing mRNA levels and the production of pro-inflammatory cytokines. A remarkable reduction in nitric oxide and ROS in the cell-based system by peptides from LPH was also demonstrated. Our preliminary results point to underexplored protein hydrolysates from food production industries as a novel, natural source of high-value-added biopeptides.Ministerio de Ciencia, Innovación y Universidades CYTED-2019/119RT056

Wheat gluten protein hydrolysates anti-inflammatory study

Motivation: It is known that some peptides have bioactive properties, anticarcinogenic, anti-inflammatory or antimicrobial among others. Lately, researches made in this area have been focused on vegetable sources peptides, which have shown a great capacity of having anti-inflammatory effects. Therefore, the aim of this investigation was to check wheat gluten hydrolysates anti-inflammatory capacities.Methods: 1) Wheat gluten was used as starting material, which was hydrolysated with two different enzymes each on their own, Alcalse 2.4L and Izyme AL during 1 and 2 hours respectively, taking samples each 15 minutes (named A0, A15, A30, A45, A60; I0, I15, I30, I45, I60, I75, I90, I105, I120). The hydrolysis grade was obtained using 2,4,6-trinitrobenzene sulfonic acid (TNBS) method. 2) In order to determine their immunommodulatory capacity, the inhibitory effect of each sample was tested among chronic inflammatory diseases pathways common enzymes: thrombin and angiotensin-converting enzyme (ECA). 3) Blood cells, monocytes, submitted to lipopolysaccharide (LPS) infection, were incubated for 24 hours with A45 and I90 at different concentrations (10µg/mL and 5µg/mL) for the purpose of seeing IL-1β, TNF-α (pro-inflammatory proteins) and IL-10 (anti-inflammatory protein), cytokines genes expression change; studied by Q-PCR.Results & Conclusions: 1) The more was the time that were being hydrolysed, the bigger was the hydrolysis grade, reaching a maximum of 36% of it with Alcalase 2.4.L and a 11,869% with Izyme. 2) The hydrolysates named as A45 and I90 were the ones that shown the most effective inhibitory effect above inflammatory enzymes; their physicochemical features should be studied for understanding it. 3)The hydrolysates chosen in the enzymatic tests (A45 and I90) showed to have a anti-inflammatory activity potential on cells, since in most cases they tend to repress pro-inflammatory genes expression (TNF-α and IL-1β) and enhanced the expression of other anti-inflammatory cytokines such as IL-10 (in all cases). Therefore, this study reveals that the inclusion of wheat gluten protein hydrolysates on our diets could help in the treatment of inflammatory diseases

Anti-inflammatory activity of Chia protein hydrolysates on primary human monocytes

Bakcground & Aims Chia (Salvia hispanica L.) has recently garnered interest in the scientific community and food industry owing to its high protein content and potential bioactive compounds. Chia protein hydrolysates (CPH) could prove an effective functional ingredient in a wide range of foods. The present study aimed to test whether peptides released from the enzymatic hydrolysis of chia protein may modulate the inflammatory responses on primary human monocytes

Nutritional composition, ultrastructural characterization, and peptidome profile of antioxidant hemp protein hydrolysates

Hemp is the term commonly used to refer to the variety of Cannabis sativa L. cultivated for industrial purposes. The seeds have gained interest in recent years as functional foods due to their nutritional composition and high content of protein and bioactive compounds. In this study, ten hemp protein hydrolysates (HPHs) were obtained by enzymatic hydrolysis with Alcalase and Flavourzyme from hemp protein isolate (HPI) and their antioxidant properties (DPPH radical scavenging activity, beta-carotene activity and ferric ion reducing antioxidant power (FRAP)) were evaluated. Shorter peptide sequences, mainly obtained with Flavourzyme, were found to react with free radicals more easily. The peptidome of all the hydrolysates was characterized, identifying, and quantifying the peptides. Furthermore, 19 unique peptides were assessed by in silico tools to hypothesize those that could be responsible of the bioactivity reported for the hydrolysates. From the identified peptides, based on the molecular features and the predictions, the peptides KNAIYTPH, EERPGHF, and KNGMMAPH, among others, are proposed to be highly contributing to the antioxidant activity of the hydrolysates.Junta de Andalucía US-138149

GPETAFLR, a biopeptide from Lupinus angustifolius L., protects against oxidative and inflammatory damage in retinal pigment epithelium cells

GPETAFLR, an octapeptide released from the enzymatic hydrolysis of lupine (Lupinus angustifolius L.) protein, has demonstrated anti-inflammatory effect in myeloid lineage. This work aims to evaluate in retinal pigment epithelium (RPE) cells the protective role of GPETAFLR on both oxidative and inflammatory markers known to be involved in age-related macular degeneration (AMD). In comparison with stimulated control cells, GPETAFLR increased glutathione production and diminished the secretion and gene expression of VEFG, IL-1β, IL-6, IFNγ, and TNF-α, as well as reactive oxygen species, and nitrite output. Our findings reveal that GPETAFLR, a novel plant peptide, is able to protect against RPE oxidative stress and inflammation. Taken together, these results strongly support innovative nutritional strategies considering Lupinus angustifolius L. as source of proteins to prevent the onset and progression of AMD. Practical applications: We reveal a novel nutraceutical impact of GPETAFLR peptide in human RPE cells to prevent oxidative and inflammatory mediators. Our results support that the intake of Lupine angustifolius L., proposed to be a reservoir of GPETAFLR, could lessen the functional decay of RPE cells, leading therefore to a slowdown of the progress of AMD during age. Not only this work, but also future simple clinical studies should raise new nutritional strategies focused on understanding the etiological role of the foods, nutrition, and metabolism in the pathogenesis of ocular disorders.Ministerio de Economía y Competitividad AGL2012‐40247‐C02‐0

Antioxidant and Immunomodulatory Properties of Chia Protein Hydrolysates in Primary Human Monocyte–Macrophage Plasticity

Chia (Salvia hispanica L.) seed has high potential in the development of functional food due to its protein content with a special amino acid profile. Among the hematopoietic-derived cells, monocytes are endowed with high plasticity, responsible for their pro- and anti-inflammatory function in M1 and M2 phenotype polarization, respectively. Indeed, monocytes are involved in several oxidative- and inflammatory-associated disorders such as cancer, obesity, and cardiovascular and neurodegenerative diseases. This study was designed to investigate the role of chia protein hydrolysates (CPHs) in primary human monocyte–macrophage plasticity response using biochemical, RT-qPCR, and ELISA assays. Our results showed that CPHs reduce ROS and nitrite output, as pro inflammatory cytokine secretion, and enhance the expression and release of anti-inflammatory cytokines. In addition, CPHs reverse LPS-associated M1 polarization into M2. These findings open new opportunities for developing nutritional strategies with chia as a dietary source of biopeptides to prevent the development and progression of oxidative- and inflammatory-related diseases.Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo 119RT056