Supplementation with cyanidin and delphinidin mitigates high fat diet-induced endotoxemia and associated liver inflammation in mice.

Consumption of high fat diets (HFD) and the associated metabolic endotoxemia can initiate liver inflammation and lipid deposition that with time can progress to non-alcoholic fatty liver disease (NAFLD). We previously observed that 14 weeks supplementation with the anthocyanidins cyanidin and delphinidin mitigated HFD-induced metabolic endotoxemia and liver insulin resistance, steatosis, inflammation and oxidative stress. This work investigated if a 4-week supplementation of mice with a cyanidin- and delphinidin-rich extract (CDRE) could mitigate or reverse HFD (60% calories from lard fat)-induced liver steatosis and inflammation. After a first 4-weeks period on the HFD, mice showed increased endotoxemia and activation of liver proinflammatory signaling cascades. Supplementation with CDRE between weeks 4 and 8 did not mitigate liver steatosis or the altered lipid and glucose plasma levels. However, CDRE supplementation reverted HFD-induced metabolic endotoxemia, in parallel with the mitigation of the overexpression of hepatic TLR2 and TLR4, and of the activation of: (i) NF-κB, (ii) AP-1 and upstream mitogen-activated kinases p38 and ERK1/2, and (iii) HIF-1. Thus, even a short-term consumption of cyanidin and delphinidin could help mitigate the adverse consequences, i.e. metabolic endotoxemia and associated liver inflammation, triggered by the regular consumption of diets rich in fat.

(-)-Epicatechin protects thoracic aortic perivascular adipose tissue from whitening in high-fat fed mice.

High adipose tissue (AT) accumulation in the body increases the risk for many metabolic and chronic diseases. This work investigated the capacity of the flavonoid (-)-epicatechin to prevent undesirable modifications of AT in mice fed a high-fat diet. Studies were focused on thoracic aorta perivascular AT (taPVAT), which is involved in the control of blood vessel tone, among other functions. Male C57BL/6J mice were fed for 15 weeks a high-fat diet with or without added (-)-epicatechin (20 mg per kg body weight per d). In high-fat diet fed mice, (-)-epicatechin supplementation: (i) prevented the expansion of taPVAT, (ii) attenuated the whitening of taPVAT (according to the adipocyte morphology, diameter, and uncoupling-protein 1 (UCP-1) levels) and (iii) blunted the increase in plasma glucose and cholesterol. The observed taPVAT modifications were not associated with alterations in the aorta wall thickness, aorta tumor necrosis factor-alpha (TNF-α) and NADPH-oxidase 2 (NOX2) expression, and endothelial nitric oxide synthase (eNOS) phosphorylation levels. In summary, our results indicate (-)-epicatechin as a relevant bioactive protecting from the slow and silent development of metabolic and chronic diseases as they are associated with excessive fat intake.

(-)-Epicatechin administration protects kidneys against modifications induced by short-term l-NAME treatment in rats.

The aim of this work was to evaluate the protective effects of (-)-epicatechin on the kidneys of NO-deprived rats. Male Sprague Dawley rats were divided into three groups: control (C), receiving water and standard diet; l-NAME (L), receiving a solution of N(ω)-nitro-l-arginine methyl ester (l-NAME) (360 mg l-1 in water) as a beverage and standard diet; and l-NAME-(-)-epicatechin (LE), receiving l-NAME solution as a beverage and standard diet supplemented with (-)-epicatechin (4 g kg-1 diet). The L-group showed altered kidney function parameters, evaluated based on plasma urea and creatinine. In parallel, kidney oxidative stress markers, i.e. superoxide anion production, malondialdehyde content, and 3-nitrotyrosine protein adducts, were significantly increased in the L group. In addition, l-NAME treatment induced modifications in kidney NO bioavailability determinants: increased expression of NOX subunits (p47phox, gp91phox, NOXO1, and NOX4) and lowered NOS activity. (-)-Epicatechin administration restored kidney function parameters, oxidative stress markers, expression of p47phox, gp91phox, and NOX4 and NOS activity to control values. These results indicate that (-)-epicatechin can mitigate NO-mediated impairment of kidney function, in part due to its capacity to modulate NOXs, NOSs, and consequently oxidative stress, and NO bioavailability.

Effects of quercetin on heart nitric oxide metabolism in l-NAME treated rats.

This study investigated the effects of a quercetin-supplemented diet on the biochemical changes installed in the heart of NO-deficient rats in terms of oxidants production and NO bioavailability determinants. Sprague-Dawley rats were subjected to Nω-nitro-l-arginine methyl ester (l-NAME) treatment (360 mg/L l-NAME in the drinking water, 4 d) with or without supplementation with quercetin (4 g/kg diet). l-NAME administration led to increased blood pressure (BP) (30%), decreased nitric oxide synthase (NOS) activity (50%), and increases in NADPH oxidase (NOX)-dependent superoxide anion production (60%) and p47phox protein level (65%). The co-administration of quercetin prevented the increase in BP and the activation of NOX but did not modify the decrease in NOS activity caused by l-NAME. In addition, quercetin affected oxidative stress parameters as glutathione oxidation, and the activities of oxidant detoxifying enzymes superoxide dismutase, glutathione peroxidase, and catalase. Thus, quercetin administration counteracts l-NAME effects on NO bioavailability determinants in vivo, essentially through controlling NOX-mediated superoxide anion production.

Research trends in flavonoids and health.

Herein we describe, based on some bibliometric data, how the field of research on flavonoids has evolved over the last 25 years. The number of papers on flavonoids has risen in an exponential manner over these years, much faster than other fields on food constituents. This increase appears to be related to the contemporary explosion of interest in healthy foods, supplements and nutraceuticals. It was also probably triggered by large epidemiological studies on fruits and vegetables, and particularly on flavonoids, consumption and incidence of cancer, stroke and coronary heart disease. The widely distributed flavonols constitute the flavonoid subgroup upon which the greatest interest has been focused, followed by flavanols and more recently by anthocyanidins and other related polyphenols such as resveratrol. Research on isoflavones rapidly emerged in the 1990s but plateaued in the 2000s. In the 1990s flavonoids were mainly considered as the active components of medicinal plants, while from 2000 onward, they switched to be mainly regarded as bioactive food ingredients. We envision a continuation in the growth of research for the coming decade focused on clearly demonstrating the importance of flavonoids for human health.

Anthocyanins inhibit tumor necrosis alpha-induced loss of Caco-2 cell barrier integrity.

An increased permeability of the intestinal barrier is proposed as a major event in the pathophysiology of conditions characterized by chronic gut inflammation. This study investigated the capacity of pure anthocyanins (AC), and berry and rice extracts containing different types and amounts of AC, to inhibit tumor necrosis alpha (TNFα)-induced permeabilization of Caco-2 cell monolayers. Caco-2 cells differentiated into intestinal epithelial cell monolayers were incubated in the absence/presence of TNFα, with or without the addition of AC or AC-rich plant extracts (ACRE). AC and ACRE inhibited TNFα-induced loss of monolayer permeability as assessed by changes in transepithelial electrical resistance (TEER) and paracellular transport of FITC-dextran. In the range of concentrations tested (0.25-1 µM), O-glucosides of cyanidin, and delphinidin, but not those of malvidin, peonidin and petunidin protected the monolayer from TNFα-induced decrease of TEER and increase of FITC-dextran permeability. Cyanidin and delphinidin acted by mitigating TNFα-triggered activation of transcription factor NF-κB, and downstream phosphorylation of myosin light chain (MLC). The protective actions of the ACRE on TNFα-induced TEER increase was positively correlated with the sum of cyanidins and delphinidins (r2 = 0.83) content in the ACRE. However, no correlation was observed between TEER and ACRE total AC, malvidin, or peonidin content. Results support a particular capacity of cyanidins and delphinidins in the protection of the intestinal barrier against inflammation-induced permeabilization, in part through the inhibition of the NF-κB pathway.

Dietary (-)-epicatechin mitigates oxidative stress, NO metabolism alterations, and inflammation in renal cortex from fructose-fed rats.

High fructose consumption has been associated to deleterious metabolic conditions. In the kidney, high fructose causes renal alterations that contribute to the development of chronic kidney disease. Evidence suggests that dietary flavonoids have the ability to prevent/attenuate risk factors of chronic diseases. This work investigated the capacity of (-)-epicatechin to prevent the renal damage induced by high fructose consumption in rats. Male Sprague Dawley rats received 10% (w/v) fructose in the drinking water for 8 weeks, with or without supplementation with (-)-epicatechin (20mg/kg body weight/d) in the rat chow diet. Results showed that, in the presence of mild proteinuria, the renal cortex from fructose-fed rats exhibited fibrosis and decreases in nephrin, synaptopodin, and WT1, all indicators of podocyte function in association with: (i) increased markers of oxidative stress; (ii) modifications in the determinants of NO bioavailability, i.e., NO synthase (NOS) activity and expression; and (iii) development of a pro-inflammatory condition, manifested as NF-κB activation, and associated with high expression of TNFα, iNOS, and IL-6. Dietary supplementation with (-)-epicatechin prevented or ameliorated the adverse effects of high fructose consumption. These results suggest that (-)-epicatechin ingestion would benefit when renal alterations occur associated with inflammation or metabolic diseases.

(-)-Epicatechin reduces blood pressure increase in high-fructose-fed rats: effects on the determinants of nitric oxide bioavailability.

This work investigated the blood pressure (BP)-lowering effect of the flavanol (-)-epicatechin in a model of metabolic syndrome. Rats were fed a regular chow diet without (Control) or with 10% (w/v) fructose in the drinking water (high fructose, HF) for 8 weeks. A subgroup of the HF-fed rats was supplemented with (-)-epicatechin 20 mg/kg body weight (HF-EC). Dietary (-)-epicatechin reverted the increase in BP caused by the fructose treatment. In aorta, superoxide anion production and the expression of the NADPH oxidase (NOX) subunits p47(phox) and p22(phox) were enhanced in the HF-fed rats. The increase was prevented by (-)-epicatechin. Similar profile was observed for NOX4 expression. The activity of aorta nitric oxide synthase (NOS) was increased in the HF group and was even higher in the HF-EC rats. These effects were paralleled by increased endothelial NOS phosphorylation at the activation site Ser1177. Among the more relevant mitogen-activated protein kinase pathways in vascular tissue, c-Jun-N-terminal kinase was shown to be activated in the aorta of the HF-fed rats, and (-)-epicatechin supplementation mitigated this activation. Thus, the results suggest that dietary (-)-epicatechin supplementation prevented hypertension in HF-fed rats, decreasing superoxide anion production and elevating NOS activity, favoring an increase in NO bioavailability.

(-)-Epicatechin prevents alterations in the metabolism of superoxide anion and nitric oxide in the hearts of L-NAME-treated rats.

The aim of this work was to evaluate the effects of (-)-epicatechin administration in the heart of a rat model with reduced NO production that follows a short-term treatment with L-NAME. Sprague-Dawley rats were treated for 4 d with L-NAME in the absence or presence of (-)-epicatechin in the diet. The redox status in cardiac tissue was improved by (-)-epicatechin administration. L-NAME treatment induced a decrease in NO synthase activity (-62%, p<0.05) and an increase in NADPH-dependent superoxide anion production (+300%, p<0.05) that were totally prevented by (-)-epicatechin administration. These effects of (-)-epicatechin were associated with a higher endothelial NO synthase phosphorylation at an activation site and a reduced expression of the regulatory subunit, p47(phox), suggesting the involvement of posttranslational mechanisms in (-)-epicatechin action. Thus, the (-)-epicatechin treatment would restore NO steady state levels in vivo through effects on both, its synthesis and degradation via the reaction with superoxide anion. The fact that (-)-epicatechin is commonly present in human diet makes this compound a reasonable explanation for the positive cardiovascular effects of a high consumption of fruits and vegetables.

(-)-Epicatechin mitigates high-fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress.

We investigated the capacity of dietary (-)-epicatechin (EC) to mitigate insulin resistance through the modulation of redox-regulated mechanisms in a rat model of metabolic syndrome. Adolescent rats were fed a regular chow diet without or with high fructose (HFr; 10% w/v) in drinking water for 8 weeks, and a group of HFr-fed rats was supplemented with EC in the diet. HFr-fed rats developed insulin resistance, which was mitigated by EC supplementation. Accordingly, the activation of components of the insulin signaling cascade (insulin receptor, IRS1, Akt, and ERK1/2) was impaired, whereas negative regulators (PKC, IKK, JNK, and PTP1B) were upregulated in the liver and adipose tissue of HFr rats. These alterations were partially or totally prevented by EC supplementation. In addition, EC inhibited events that contribute to insulin resistance: HFr-associated increased expression and activity of NADPH oxidase, activation of redox-sensitive signals, expression of NF-κB-regulated proinflammatory cytokines and chemokines, and some sub-arms of endoplasmic reticulum stress signaling. Collectively, these findings indicate that EC supplementation can mitigate HFr-induced insulin resistance and are relevant for defining interventions that can prevent/mitigate MetS-associated insulin resistance.

Exploring the benefits and challenges of establishing a DRI-like process for bioactives.

Bioactives can be defined as: "Constituents in foods or dietary supplements, other than those needed to meet basic human nutritional needs, which are responsible for changes in health status" (Office of Disease Prevention and Health Promotion, Office of Public Health and Science, Department of Health and Human Services in Fed Reg 69:55821-55822, 2004). Although traditional nutrients, such as vitamins, minerals, protein, essential fatty acids and essential amino acids, have dietary reference intake (DRI) values, there is no such evaluative process for bioactives. For certain classes of bioactives, substantial scientific evidence exists to validate a relationship between their intake and enhanced health conditions or reduced risk of disease. In addition, the study of bioactives and their relationship to disease risk is a growing area of research supported by government, academic institutions, and food and supplement manufacturers. Importantly, consumers are purchasing foods containing bioactives, yet there is no evaluative process in place to let the public know how strong the science is behind the benefits or the quantitative amounts needed to achieve these beneficial health effects. This conference, Bioactives: Qualitative Nutrient Reference Values for Life-stage Groups?, explored why it is important to have a DRI-like process for bioactives and challenges for establishing such a process.

Blood pressure-lowering effect of dietary (-)-epicatechin administration in L-NAME-treated rats is associated with restored nitric oxide levels.

Epidemiological and intervention studies have shown that the intake of certain chocolates or cocoa products decreases blood pressure (BP) in humans. (-)-Epicatechin is the most abundant flavanol present in cocoa seeds and its derived foods. This work investigates the effects of dietary (-)-epicatechin on BP in rats that received N(ω)-nitro-l-arginine methyl ester (L-NAME) for 4 days. (-)-Epicatechin administration prevented the 42mm Hg increase in BP associated with the inhibition of NO production in a dose-dependent manner (0.2-4.0g/kg diet). This BP effect was associated with a reduction in L-NAME-mediated increase in the indexes of oxidative stress (plasma TBARS and GSSG/GSH(2) ratio) and with a restoration of the NO concentration. At the vascular level, none of the treatments modified NOS expression, but (-)-epicatechin administration avoided the L-NAME-mediated decrease in eNOS activity and increase in both superoxide anion production and NOX subunit p47(phox) expression. In summary, (-)-epicatechin was able to prevent the increase in BP and in oxidative stress and restored NO bioavailability. The fact that (-)-epicatechin is present in several plants usually consumed by humans gives the possibility of developing diets rich in those plants or pharmacological strategies using that flavonoid to diminish BP in hypertensive subjects.

Flavonoids and metabolic syndrome.

Increasing evidence indicates that several mechanisms, associated or not with antioxidant actions, are involved in the effects of flavonoids on health. Flavonoid-rich beverages, foods, and extracts, as well as pure flavonoids are studied for the prevention and/or amelioration of metabolic syndrome (MS) and MS-associated diseases. We summarize evidence linking flavonoid consumption with the risk factors defining MS: obesity, hypertriglyceridemia, hypercholesterolemia, hypertension, and insulin resistance. Nevertheless, a number of molecular mechanisms have been identified; the effects of flavonoids modifying major endpoints of MS are still inconclusive. These difficulties are explained by the complex relationships among the risk factors defining MS, the multiple biological targets controlling these risk factors, and the high number of flavonoids (including their metabolites) present in the diet and potentially responsible for the in vivo effects. Consequently, extensive basic and clinical research is warranted to assess the final relevance of flavonoids for MS.

Hypertension, nitric oxide, oxidants, and dietary plant polyphenols.

Fruits and vegetables are key foods whose high ingestion is associated with the improvement of numerous pathological conditions, including hypertension. Such health promoting actions have been increasingly ascribed to the antioxidant characteristics of different polyphenols in fruits and vegetables. Consequently, based on this assumption, many beverages and foods rich in polyphenols, grape, tea, cocoa, and soy products and many of their chemical constituents purified, are being studied both, as antioxidants and antihypertensive agents. This paper reviews the current evidence linking high polyphenol consumption with reductions in blood pressure. Basic chemical aspects of flavanols, flavonols, isoflavones and stilbenes, as possible responsible for the observed effects of those foods on blood pressure are included. Human interventions studies by using grapes and wine, cocoa and chocolate, black and green tea, soy products, and purified compounds ((+)-catequin, quercetin, (-)-epigallocatechin gallate) are summarized. The discussed hypothesis, strongly supported by experimental data in animals, is that by regulating nitric oxide bioavailability, polyphenols present in fruits and vegetables affect endothelial function and as a consequence, blood pressure. Even when data are not definitive and many questions remain open, the whole evidence is encouraging to start considering diets that can provide a benefit to hypertensive subjects, and those benefits will be more significant in people that do not have controlled his/her elevated blood pressure.

Basic biochemical mechanisms behind the health benefits of polyphenols.

Polyphenols and consequently many flavonoids have several beneficial actions on human health. However, the actual molecular interactions of polyphenols with biological systems remain mostly speculative. This review addresses the potential mechanisms of action that have been so far identified, as well as the feasibility that they could occur in vivo. Those mechanisms include: i) non specific actions, based on chemical features common to most polyphenols, e.g. the presence of a phenol group to scavenge free radicals; and ii) specific mechanisms; based on particular structural and conformational characteristics of select polyphenols and the biological target, e.g. proteins, or defined membrane domains. A better knowledge about the nature and biological consequences of polyphenol interactions with cell components will certainly contribute to develop nutritional and pharmacological strategies oriented to prevent the onset and/or the consequences of human disease.

Inhibition of angiotensin converting enzyme activity by flavanol-rich foods.

Angiotensin converting enzyme (ACE) activity was evaluated in the presence of flavanol-rich foods, i.e., wines, chocolates, and teas, and of purified flavonoids. All foods assayed inhibited ACE activity, red wines being more effective than white wine, and green tea more effective than black tea. The inhibition of ACE activity was associated with both phenolic and flavanol content in the foods. When isolated polyphenols were assayed, procyanidins (dimer and hexamer) and epigallocatechin significantly inhibited enzyme activity; similar concentrations of (+)-catechin, (-)-epicatechin, gallic acid, chlorogenic acid, caffeic acid, quercetin, kaempferol, and resveratrol were ineffective. When ACE activity was assayed in rat kidney membranes in the presence of chocolate extracts or purified procyanidins, it was observed that the inhibition depended on the chocolate content of flavanols and the number of flavanol units constituting the procyanidin. These experiments demonstrate that flavanols either isolated or present in foods could inhibit ACE activity. The occurrence of such inhibition in vivo needs to be determined, although is supported by the association between the consumption of flavanol-rich foods and reductions in blood pressure observed in several experimental models.

Relevance, essentiality and toxicity of trace elements in human health.

The metals Mn, Fe, Cu, and Zn, and the non-metal Se are considered "trace elements" (TE) because of their essentiality and very limited quantity in humans. The biological activities of Cu, Fe, Mn, and Se are strongly associated with the presence of unpaired electrons that allow their participation in redox reactions. In biological systems these metals are mostly bound to proteins, forming metalloproteins. Many of the metals in metalloproteins are part of enzymatic systems, have structural and storage functions, or use the protein to be transported to their target site in the organism. In humans Mn, Fe, Cu, Zn, and Se accomplish decisive functions to maintain human health. Deficiency in any of these TE leads to undesirable pathological conditions that can be prevented or reversed by adequate supplementation. In sufficiently nourished persons, supplementation should be carefully controlled, given the toxic effects ascribed to TE when present in quantities exceeding those required for accomplishing their biological functions. The dietary reference intakes provided by national regulatory agencies are guides to define intake, supplementation and toxicity of Mn, Fe, Cu, Zn, and Se, as well other elements considered micronutrients for humans.

Cocoa antioxidants and cardiovascular health.

An increasing body of epidemiologic evidence supports the concept that diets rich in fruits and vegetables can promote health and attenuate, or delay, the onset of various diseases. Epidemiologic data support the idea that these health benefits are causally linked to the consumption of certain flavonoids present in fruit and vegetables. In the context of cardiovascular health, a particular group of flavonoids, namely, the flavan-3-ols (flavanols), has received attention. Flavanol-rich, plant-derived foods and beverages include wine, tea, and various fruits and berries, as well as cocoa and cocoa products. Numerous dietary intervention studies in humans and animals indicate that flavanol-rich foods and beverages might exert cardioprotective effects with respect to vascular function and platelet reactivity. This review discusses the bioactivity of flavanols in the context of cardiovascular health, with respect to their bioavailability, their antioxidant properties, and their vascular effects.

The regular supplementation with an antioxidant mixture decreases oxidative stress in healthy humans. Gender effect.

BACKGROUND: Antioxidant consumption is claimed to be associated with improved antioxidant defenses and with the prevention of free radical-associated diseases. We evaluated if the regular supplementation with an antioxidant mixture modified oxidative stress parameters in healthy humans. METHODS: Malondialdehyde (MDA), vitamin E (alpha-tocopherol), beta-carotene, and ubiquinol-10 were determined in plasma by HPLC; plasma 2-thiobarbituric acid reactive substances (TBARS) were evaluated fluorometrically. The supplement contained 106 IU vitamin E, 10 mg beta-carotene, 60 mg coenzyme Q-10, and 40 microg selenium. RESULTS: After a 10-day wash out period, 16 healthy, free-living adults (31-48 years old; 9 women, 7 men) consumed the supplement daily during 30 days. At day 20, plasma concentration of vitamin E (alpha-tocopherol), beta-carotene, and ubiquinol-10 increased 45%, 66%, and 104%, respectively, over baseline values. Plasma MDA concentrations were reduced at days 20 (25%) and 30 (32%). Plasma TBARS concentrations decreased steadily during the treatment, reaching at day 30 an 11% decrease compared to baseline. Segregating by gender, both MDA and TBARS plasma concentrations were higher in men than in women at baseline. However, such differences disappeared after 30 days of supplementation. CONCLUSION: The observed modifications in variables associated with oxidative stress are indicative of an effective antioxidant action of the used mixture of lipid soluble compounds, and gender differences suggest that men should optimize their antioxidants defenses earlier in life than women.

Comparative study on the antioxidant capacity of wines and other plant-derived beverages.

Consistent epidemiological data point to a reduced morbidity and mortality from coronary heart disease and atherosclerosis in people consuming plant-derived beverages such as tea or wine. We studied the antioxidant capacity of three red wines (W) and compared it those of tea and herbal "mate" tea infusions. The antioxidant capacity was evaluated measuring: (1) the inhibition of the luminol-induced chemiluminescence assay (TRAP); (2) the inhibition of 2.2'-thiobarbituric-reactive substances (TBARS) formation in liposomes by fluorescence; (3) the protection of Jurkat cells from AMVN-induced oxidation, measuring the oxidation of 5-(and-6)-carboxy-2'7'-dichlorodihydrofluorescein diacetate to a fluorescent derivative. The polyphenolic content was estimated spectrophotometrically and by HPLC with electrochemical detection. All three beverages provided antioxidant protection in the three assays in a dose-dependent manner. Significant and positive correlations were found between antioxidant capacity and total polyphenol content, especially in the Jurkat cell oxidation assay (r: 0.96, p < 0.01). Results suggest that these dietary components could be a source of antioxidants that protect from oxidative stress. Further studies of absorption and metabolism of the active compounds will judge the physiological relevance of these results for human health.