Characterisation and beneficial effects of a Lupinus angustifolius protein hydrolysate obtained by immobilisation of the enzyme alcalase®.

Bioactive peptides have been considered potential components for the future functional foods and nutraceuticals generation. The enzymatic method of hydrolysis has several advantages compared to those of chemical hydrolysis and fermentation. Despite this fact, the high cost of natural and commercial proteases limits the commercialization of hydrolysates in the food and pharmacological industries. For this reason, more efficient and economically interesting techniques, such as the immobilisation of the enzyme, are gaining attention. In the present study, a new protein hydrolysate from Lupinus angustifolius was generated by enzymatic hydrolysis through the immobilisation of the enzyme alcalase® (imLPH). After the chemical and nutritional characterization of the imLPH, an in vivo study was carried out in order to evaluate the effect of 12 weeks treatment with imLPH on the plasmatic lipid profile and antioxidant status in western-diet-fed apolipoprotein E knockout mice. The immobilisation of alcalase® generated an imLPH with a degree of hydrolysis of 29.71 ± 2.11%. The imLPH was mainly composed of protein (82.50 ± 0.88%) with a high content of glycine/glutamine, arginine, and aspartic acid/asparagine. The imLPH-treatment reduced the amount of abdominal white adipose tissue, total plasma cholesterol, LDL-C, and triglycerides, as well as the cardiovascular risk indexes (CRI) -I, CRI-II, and atherogenic index of plasma. The imLPH-treated mice also showed an increase in the plasma antioxidant capacity. For the first time, this study demonstrates the beneficial in vivo effect of a lupin protein hydrolysate obtained with the alcalase® immobilised and points out this approach as a possible cost-effective solution at the expensive generation of the hydrolysate through the traditional batch conditions with soluble enzymes.

A lupin protein hydrolysate protects the central nervous system from oxidative stress in WD-fed ApoE-/- mice.

Oxidative stress plays a crucial role in neurodegenerative diseases like Parkinson's and Alzheimer's. Studies indicate the relationship between oxidative stress and the brain damage caused by a high-fat diet. It is previously found that a lupin protein hydrolysate (LPH) has antioxidant effects on human leukocytes, as well as on the plasma and liver of Western diet (WD)-fed ApoE-/- mice. Additionally, LPH shows anxiolytic effects in these mice. Given the connection between oxidative stress and anxiety, this study aimed to investigate the antioxidant effects of LPH on the brain of WD-fed ApoE-/- mice. LPH (100 mg kg-1) or a vehicle is administered daily for 12 weeks. Peptide analysis of LPH identified 101 amino acid sequences (36.33%) with antioxidant motifs. Treatment with LPH palliated the decrease in total antioxidant activity caused by WD ingestion and regulated the nitric oxide synthesis pathway in the brain of the animals. Furthermore, LPH increased cerebral glutathione levels and the activity of catalase and glutathione reductase antioxidant enzymes and reduced the 8-hydroxy-2'-deoxyguanosine levels, a DNA damage marker. These findings, for the first time, highlight the antioxidant activity of LPH in the brain. This hydrolysate could potentially be used in future nutraceutical therapies for neurodegenerative diseases.

Mediterranean Diet and Melatonin: A Systematic Review.

The Mediterranean diet (MD) has beneficial effects on human health, which is evidenced by the observation of lower incidence rates of chronic diseases in Mediterranean countries. The MD dietary pattern is rich in antioxidants, such as melatonin, which is a hormone produced mainly by the pineal gland and controls several circadian rhythms. Additionally, melatonin is found in foods, such as fruit and vegetables. The purpose of this systematic review was to assess the melatonin content in Mediterranean foods and to evaluate the influence of the MD on melatonin levels in both humans and model organisms. A comprehensive search was conducted in four databases (PubMed, Scopus, Cochrane Library and Web of Science) and data were extracted. A total of 31 records were chosen. MD-related foods, such as tomatoes, olive oil, red wine, beer, nuts, and vegetables, showed high melatonin contents. The consumption of specific MD foods increases melatonin levels and improves the antioxidant status in plasma.

Bioactive Peptides from Lupin (Lupinus angustifolius) Prevent the Early Stages of Atherosclerosis in Western Diet-Fed ApoE-/- Mice.

We have previously reported the in vitro hypocholesterolemic, anti-inflammatory, and antioxidant effects of Alcalase-generated lupin protein hydrolysate (LPH). Given that lipoprotein deposition, oxidative stress, and inflammation are the main components of atherogenesis, we characterized the LPH composition, in silico identified LPH-peptides with activities related to atherosclerosis, and evaluated the in vivo LPH effects on atherosclerosis risk factors in a mouse model of atherosclerosis. After 15 min of Alcalase hydrolysis, peptides smaller than 8 kDa were obtained, and 259 peptides out of 278 peptides found showed biological activities related to atherosclerosis risk factors. Furthermore, LPH administration for 12 weeks reduced the plasma lipids, as well as the cardiovascular and atherogenic risk indexes. LPH also increased the total antioxidant capacity, decreased endothelial permeability, inflammatory response, and atherogenic markers. Therefore, this study describes for the first time that LPH prevents the early stages of atherosclerosis.

A Lupinus angustifolius protein hydrolysate exerts hypocholesterolemic effects in Western diet-fed ApoE-/- mice through the modulation of LDLR and PCSK9 pathways.

Lupin protein hydrolysates (LPHs) are gaining attention in the food and nutraceutical industries due to their several beneficial health effects. Recently, we have shown that LPH treatment reduces liver cholesterol and triglyceride levels in hypercholesterolemic mice. The aim of this study was to elucidate the effects of LPH treatment on the molecular mechanism underlying liver cholesterol metabolism in ApoE-/- mice fed the Western diet. After identifying the composition of the peptide within the LPH mixture and determining its ability to reduce HMGCoAR activity in vitro, its effect on the LDLR and PCSK9 pathways was measured in liver tissue from the same mice. Thus, the LPH reduced the protein levels of HMGCoAR and increased the phosphorylated inactive form of HMGCoAR and the pHMGCoAR/HMGCoAR ratio, which led to the deactivation of de novo cholesterol synthesis. Furthermore, the LPH decreased the protein levels of SREBP2, a key upstream transcription factor involved in the expression of HMGCoAR and LDLR. Consequently, LDLR protein levels decreased in the liver of LPH-treated animals. Interestingly, the LPH also increased the protein levels of pAMPK responsible for HMGCoAR phosphorylation. Furthermore, the LPH controlled the PSCK9 signal pathway by decreasing its transcription factor, the HNF1-α protein. Consequently, lower PSCK9 protein levels were found in the liver of LPH-treated mice. This is the first study elucidating the molecular mechanism at the basis of the hypocholesterolemic effects exerted by the LPH in an in vivo model. All these findings point out LPHs as a future lipid-lowering ingredient to develop new functional foods.

GPETAFLR, a peptide from Lupinus angustifolius L. prevents inflammation in microglial cells and confers neuroprotection in brain.

OBJECTIVES: Neuroinflammation is a complex inflammatory process in the central nervous system (CNS) where microglia may play a critical role. GPETAFLR is a peptide isolated from Lupinus angustifolius L. protein hydrolysates with functional activity in mononuclear phagocytes. However, it is unknown whether GPETAFLR has neuroprotective effects. METHODS: We analysed the potential anti-neuroinflammatory activity of GPETAFLR by using two different models of neuroinflammation: BV-2 microglial cells and mice with high-fat diet (HFD)-induced obesity. RESULTS: GPETAFLR hampered LPS-induced upregulation of pro-inflammatory and M1 marker genes in BV-2 cells. This effect was accompanied by an unchanged expression of anti-inflammatory IL-10 gene and by an increased expression of M2 marker genes. GPETAFLR also increased the transcriptional activity of M2 marker genes, while the microglia population remained unchanged in number and M1/M2 status in brain of mice with high-fat diet (HFD)-induced obesity. Furthermore, GPETAFLR counteracted HFD-induced downregulation of IL-10 and upregulation of pro-inflammatory markers in the mouse brain, both at gene and protein levels. DISCUSSION: This is the first report describing that a peptide from plant origin robustly restrained the pro-inflammatory activation of microglial cells in cultures and in brain. Our data suggest that GPETAFLR might be instrumental in maintaining CNS homeostasis by inhibiting neuroinflammation.

Antihypertensive and Antioxidant Activity of Chia Protein Techno-Functional Extensive Hydrolysates.

Twelve high-quality chia protein hydrolysates (CPHs) were produced from chia protein isolate (CPI) in a pilot plant of vegetable proteins. To obtain functional hydrolysate, four CPHs were hydrolyzed by the action of Alcalase, an endoprotease, and the other eight CPHs were hydrolyzed by the action of Flavourzyme, an exoprotease. Alcalase-obtained CPHs showed significant antihypertensive properties particularly, the CPH obtained after 15 min of hydrolysis with Alcalase (CPH15A), which showed a 36.2% hydrolysis degree. In addition, CPH15A increased the antioxidant capacity compared to CPI. The CPH15A physicochemical composition was characterized and compared to chia defatted flour (CDF) and CPI, and its techno-functional properties were determined by in vitro experiments through the analysis of its oil absorption capacity, as well as the capacity and stability of foaming and emulsifying, resulting in an emulsifier and stabilizer better than the intact protein. Therefore, the present study revealed that CPH15A has potent antihypertensive and antioxidant properties and can constitute an effective alternative to other plant protein ingredients sources that are being used in the food industry.

Lupinus angustifolius Protein Hydrolysates Reduce Abdominal Adiposity and Ameliorate Metabolic Associated Fatty Liver Disease (MAFLD) in Western Diet Fed-ApoE-/- Mice.

Metabolic-associated fatty liver disease (MAFLD) is the most important cause of liver disease worldwide. It is characterized by the accumulation of fat in the liver and is closely associated with abdominal obesity. In addition, oxidative stress and inflammation are significant features involved in MAFLD. Recently, our group demonstrated that lupin protein hydrolysates (LPHs) had lipid lowering, antioxidant, and anti-inflammatory effects. Sixty male mice fed with a Western diet were intragastrically treated with LPHs (or vehicle) for 12 weeks. Liver and adipose tissue lipid accumulation and hepatic inflammatory and oxidant status were evaluated. A significant decrease in steatosis was observed in LPHs-treated mice, which presented a decreased gene expression of CD36 and LDL-R, crucial markers in MAFLD. In addition, LPHs increased the hepatic total antioxidant capacity and reduced the hepatic inflammatory status. Moreover, LPHs-treated mice showed a significant reduction in abdominal adiposity. This is the first study to show that the supplementation with LPHs markedly ameliorates the generation of the steatotic liver caused by the intake of a Western diet and reduces abdominal obesity in ApoE-/- mice. Future clinical trials should shed light on the effects of LPHs on MAFLD.

Anti-Inflammatory and Antioxidant Activity of Hydroxytyrosol and 3,4-Dihydroxyphenyglycol Purified from Table Olive Effluents.

New liquid effluents based on the use of acetic acid in the table olive industry make it easier to extract bioactive compounds to be used for food, cosmetic, and pharmaceutical purposes. The use of water acidified with acetic acid or in brine with or without acetic acid for storing the table olive enhances the extraction of two more active phenolic compounds: hydroxytyrosol (HT) and 3,4-dihydroxyphenylglycol (DHPG). This work has two aims: (1) measure the solubilization of phenolics controlled for two years using more than thirty olive varieties with different ripeness index as a potential source of HT and DHPG, and (2) evaluate the anti-inflammatory activity of the purified phenolics. The effluents with a higher concentration of phenolics were used for the extraction of HT and DHPG in order to evaluate its antioxidant and anti-inflammatory activity in vitro by the determination of pro-inflammatory cytokines such as Human Tumor Necrosis Factor-α (TNF), Interleukin-6 (IL-6), and Interleukin-1ß (Il-1ß). The anti-inflammatory activity of these phenolic extracts was demonstrated by studying the expression of cytokines by qPCR and the levels of these proteins by enzyme-linked immunosorbent assay (ELISA).

Actividad antioxidante y antiinflamatoria in vitro de extractos de chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) / In vitro antioxidant and anti-inflammatory activity of chaya extracts (Cnidoscolus aconitifolius (Mill.) I.M. Johnst)

Introducción: las enfermedades crónicas no transmisibles (ECNT) son la principal causa de muerte en todo el mundo. Los metabolitos secundarios provenientes de fuentes vegetales como Cnidoscolus aconitifolius pueden usarse como coadyuvante en la prevención de las enfermedades relacionadas con el estrés oxidativo y la inflamación, tales como las ECNT. Objetivo: se evaluó la actividad antioxidante y antiinflamatoria in vitro de los compuestos biológicamente activos de extractos de C. aconitifolius. Métodos: se determinó el contenido de fenoles, flavonoides, flavanonas e hidroflavonoles. El potencial antioxidante se determinó con los ensayos de 1,1-difenil-2-picrilhidrazilo (DPPH), ácido 2,2'-azinobis (3-etilbenzotiazolina-6-sulfónico) (ABTS) y la actividad inhibitoria de la enzima convertidora de la angiotensina (ACE). Para la actividad antiinflamatoria se utilizaron la reacción en cadena de la polimerasa (PCR) cuantitativa en tiempo real y el ensayo por inmunoabsorción ligado a enzimas (ELISA) en macrófagos diferenciados de monocitos THP-1 y estimulados con lipopolisacárido (LPS). Resultados: el extracto acuoso registró el mayor contenido de fenoles (70,61  0,07 g/100 g de extracto) y el extracto etanólico registró el mayor contenido de flavonoides (47,76  4,84 g/100 g de extracto), flavanonas y dihidroflavonoles (70,10  7,29 g/100 g de extracto). El extracto acetónico registró la mayor inhibición del radical DPPH (49,85 ± 5,30 %) mientras que el etanólico presentó la mayor inhibición del radical ABTS (41,01 ± 3,81 %). Los extractos etanólico y acuoso inhibieron la ECA. El extracto etanólico tuvo la mayor actividad antiinflamatoria al reducir la expresión génica de TNF- en un 39,78 % y la de IL-6 en un 97,81 %, y su producción en un 46 % y un 48,38 %, respectivamente. Conclusiones: los extractos mostraron in vitro su potencial antioxidante y antiinflamatorio por su contenido en compuestos bioactivos

Introduction: noncommunicable diseases (NCDs) are the main cause of death worldwide. Secondary metabolites from plant sources such as Cnidoscolus aconitifolius may be used as adjuvants in the prevention of diseases related to oxidative stress and inflammation such as NCDs. Objective: the in vitro antioxidant and anti-inflammatory activities associated with biologically active compounds in C. aconitifolius extracts were evaluated. Methods: the contents of phenols, flavonoids, flavonones and hydroflavonoles were determined. The potential antioxidant activity was determined with 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, and angiotensin-converting enzyme (ACE) activity. For anti-inflammatory activity quantitative PCR and enzyme-linked immunosorbent assay (ELISA) tests were used in macrophages derived from THP-1 monocytes and stimulated with LPS. Results: the aqueous extract recorded the highest phenolic content (70.61  0.07 g/100 g of extract), and the ethanolic extract registered the highest content in flavonoids (47.76  4.84 g/100 g of extract), flavonones and dihydroflavonoles (70.10  7.29 g/100 g of extract). The acetone extract obtained the highest DPPH inhibition (49.85 ± 5.30 %), while the ethanolic extract showed the highest ABTS inhibition (41.01 ± 3.81 %). The etanolic and aqueous extracts had the highest ACE inhibition. The ethanolic extract had the highest anti-inflammatory activity, decreasing gene expression for TNF- by 39.78 % and for IL-6 by 97.81 %, and their production by 46 % and 48.38 %, respectively, in macrophages stimulated with LPS. Conclusions: these extracts demonstrated in vitro their antioxidant and anti-inflammatory potential due to their content of bioactive compounds

[In vitro antioxidant and anti-inflammatory activity of chaya extracts (Cnidoscolus aconitifolius (Mill.) I.M. Johnst)]. / Actividad antioxidante y antinflamatoria in vitro de extractos de chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst).

INTRODUCTION: Introduction: noncommunicable diseases (NCDs) are the main cause of death worldwide. Secondary metabolites from plant sources such as Cnidoscolus aconitifolius may be used as adjuvants in the prevention of diseases related to oxidative stress and inflammation such as NCDs. Objective: the in vitro antioxidant and anti-inflammatory activities associated with biologically active compounds in C. aconitifolius extracts were evaluated. Method: the contents of phenols, flavonoids, flavonones and hydroflavonoles were determined. The potential antioxidant activity was determined with 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, and angiotensin-converting enzyme (ACE) activity. For anti-inflammatory activity quantitative PCR and enzyme-linked immunosorbent assay (ELISA) tests were used in macrophages derived from THP-1 monocytes and stimulated with LPS. Results: the aqueous extract recorded the highest phenolic content (70.61  0.07 g/100 g of extract), and the ethanolic extract registered the highest content in flavonoids (47.76  4.84 g/100 g of extract), flavonones and dihydroflavonoles (70.10  7.29 g/100 g of extract). The acetone extract obtained the highest DPPH inhibition (49.85 ± 5.30%), while the ethanolic extract showed the highest ABTS inhibition (41.01 ± 3.81%). The etanolic and aqueous extracts had the highest ACE inhibition. The ethanolic extract had the highest anti-inflammatory activity, decreasing gene expression for TNF- by 39.78% and for IL-6 by 97.81%, and their production by 46% and 48.38%, respectively, in macrophages stimulated with LPS. Conclusion: these extracts demonstrated in vitro their antioxidant and anti-inflammatory potential due to their content of bioactive compounds.

INTRODUCCIÓN: Introducción: las enfermedades crónicas no transmisibles (ECNT) son la principal causa de muerte en todo el mundo. Los metabolitos secundarios provenientes de fuentes vegetales como Cnidoscolus aconitifolius pueden usarse como coadyuvante en la prevención de las enfermedades relacionadas con el estrés oxidativo y la inflamación, tales como las ECNT. Objetivo: se evaluó la actividad antioxidante y antinflamatoria in vitro de los compuestos biológicamente activos de extractos de C. aconitifolius. Métodos: se determinó el contenido de fenoles, flavonoides, flavanonas e hidroflavonoles. El potencial antioxidante se determinó con los ensayos de 1,1­difenil­2­picrilhidrazilo (DPPH), ácido 2,2'-azinobis (3-etilbenzotiazolina-6-sulfónico) (ABTS) y la actividad inhibitoria de la enzima convertidora de la angiotensina (ACE). Para la actividad antinflamatoria se utilizaron la reacción en cadena de la polimerasa (PCR) cuantitativa en tiempo real y el ensayo por inmunoabsorción ligado a enzimas (ELISA) en macrófagos diferenciados de monocitos THP-1 y estimulados con lipopolisacárido (LPS). Resultados: el extracto acuoso registró el mayor contenido de fenoles (70,61  0,07 g/100 g de extracto) y el extracto etanólico registró el mayor contenido de flavonoides (47,76  4,84 g/100 g de extracto), flavanonas y dihidroflavonoles (70,10  7,29 g/100 g de extracto). El extracto acetónico registró la mayor inhibición del radical DPPH (49,85 ± 5,30%) mientras que el etanólico presentó la mayor inhibición del radical ABTS (41,01 ± 3,81%). Los extractos etanólico y acuoso inhibieron la ECA. El extracto etanólico tuvo la mayor actividad antinflamatoria al reducir la expresión génica de TNF- en un 39,78% y la de IL-6 en un 97,81%, y su producción en un 46% y un 48,38%, respectivamente. Conclusiones: los extractos mostraron in vitro su potencial antioxidante y antinflamatorio por su contenido en compuestos bioactivos.

Lupine protein hydrolysates decrease the inflammatory response and improve the oxidative status in human peripheral lymphocytes.

Although cell-free systems and immortalized cell lines have been used to demonstrate the potential health benefits of lupine proteins and peptides, no study has examined the effects of lupine protein hydrolysates (LPHs) on the immune and oxidative responses of non-immortalized human cells. Therefore, the aims of this study were to evaluate the effects of the in vitro administration of LPHs from Lupinus angustifolius on the immunological and oxidative statuses of human peripheral blood mononuclear cells (PBMCs) from 53 healthy donors. LPHs reduced PBMCs proliferation and the levels of Th1, Th9 and Th17 pro-inflammatory cytokines without being cytotoxic. LPHs also skewed the pro-/anti-inflammatory balance towards a Th2 protective response. Additionally, LPHs increased superoxide dismutase and catalase activities, and the total antioxidant capacity (TAC). This study is the first to show that LPHs reduce T cell inflammatory responses and improve the anti-inflammatory/pro-inflammatory cytokine balance and the TAC by PBMCs. Thus, LPHs may represent an effective option for developing nutritional strategies to prevent pathologies with underlying inflammation and oxidative stress.

Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway.

Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hydrolysates obtained by hydrolysis with Izyme and subsequently with Alcalase, with more than 70% inhibition obtained in some cases. All of the hydrolysates tested inhibited more than 60% of the COX-2 activity. In no case did the percentage of thrombin activity inhibition exceed 40%. The best inhibitory activities were found in the LPH obtained after 15 min of hydrolysis with Alcalase and in the LPH obtained after 60 min of hydrolysis with Izyme followed by 15 min of hydrolysis with Alcalase. Enzyme kinetic analyses were conducted to determine the Km and Vmax parameters of these two hydrolysates using the Lineweaver-Burk equation. Both hydrolysates competitively inhibited the thrombin and PLA2 activities. In the case of COX-2 and TG, the inhibition appeared to be the mixed type.

Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates.

BACKGROUND: Some dietary proteins possess biological properties which make them potential ingredients of functional or health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme. RESULTS: All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea protein isolate. The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by 30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: ß-carotene bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant activity in all assays, especially reducing power and DPPH scavenging effect than chickpea protein isolate. CONCLUSION: The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients. The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time.