The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth

Studies have suggested that diets rich in polyphenols Such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D I levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract. (c) 2005 Elsevier Inc. All rights reserved.

Recommending flavanols and procyanidins for cardiovascular health: current knowledge and future needs

Data on the potential health benefits of dietary flavanols and procyanidins, especially in the context of cardiovascular health, are considerable and continue to accumulate. Significant progress has been made in flavanol analytics and the creation of phytonutrient-content food databases, and novel data emanated from epidemiological investigations as well as dietary intervention studies. However, a comprehensive understanding of the pharmacological properties of flavanols and procyanidins, including their precise mechanisms of action in vivo, and a conclusive, consensus-based accreditation of a causal relationship between intake and health benefits in the context of primary and secondary cardiovascular disease prevention is still outstanding. Thus, the objective of this review is to identify and discuss key questions and gaps that will need to be addressed in order to conclusively demonstrate whether or not dietary flavanols and procyanidins have a role in preventing, delaying the onset of, or treating cardiovascular diseases, and thus improving human life expectancy and quality of life.

Prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study

BACKGROUND: The absorption of cocoa flavanols in the small intestine is limited, and the majority of the flavanols reach the large intestine where they may be metabolized by resident microbiota. OBJECTIVE: We assessed the prebiotic potential of cocoa flavanols in a randomized, double-blind, crossover, controlled intervention study. DESIGN: Twenty-two healthy human volunteers were randomly assigned to either a high-cocoa flavanol (HCF) group (494 mg cocoa flavanols/d) or a low-cocoa flavanol (LCF) group (23 mg cocoa flavanols/d) for 4 wk. This was followed by a 4-wk washout period before volunteers crossed to the alternant arm. Fecal samples were recovered before and after each intervention, and bacterial numbers were measured by fluorescence in situ hybridization. A number of other biochemical and physiologic markers were measured. RESULTS: Compared with the consumption of the LCF drink, the daily consumption of the HCF drink for 4 wk significantly increased the bifidobacterial (P < 0.01) and lactobacilli (P < 0.001) populations but significantly decreased clostridia counts (P < 0.001). These microbial changes were paralleled by significant reductions in plasma triacylglycerol (P < 0.05) and C-reactive protein (P < 0.05) concentrations. Furthermore, changes in C-reactive protein concentrations were linked to changes in lactobacilli counts (P < 0.05, R(2) = -0.33 for the model). These in vivo changes were closely paralleled by cocoa flavanol-induced bacterial changes in mixed-batch culture experiments. CONCLUSION: This study shows, for the first time to our knowledge, that consumption of cocoa flavanols can significantly affect the growth of select gut microflora in humans, which suggests the potential prebiotic benefits associated with the dietary inclusion of flavanol-rich foods. This trial was registered at clinicaltrials.gov as NCT01091922.

Fluorescence lifetime imaging microscopy (FLIM) to demonstrate the nuclear binding of flavanols and (--epigallocatechin gallate

The use of light microscopy and DMACA staining strongly suggested that plant and animal cell nuclei act as sinks for flavanols [1, 2]. Detailed uv-vis spectroscopic titration experiments indicated that histone proteins are the likely binding sites in the nucleus [2]. Here we report the development of a multi-photon excitation microscopy technique combined with fluorescent lifetime measurements of flavanols. Using this technique, (+) catechin, (-) epicatechin and (-) epigallocatechin gallate (EGCG) showed strikingly different excited state lifetimes in solution. Interaction of histone proteins with flavanols was indicated by the appearance of a significant τ2-component of 1.7 to 4.0ns. Tryptophan interference could be circumvented in the in vivo fluorescence lifetime imaging microscopy (FLIM) experiments with 2-photon excitation at 630nm. This enabled visualisation and semi-quantitative measurements that demonstrated unequivocally the absorption of (+)catechin, (-)epicatechin and EGCG by nuclei of onion cells. 3D FLIM revealed for the first time that externally added EGCG penetrated the whole nucleus in onion cells. The relative proportions of EGCG in cytoplasm: nucleus: nucleoli were ca. 1:10:100. FLIM experiments may therefore facilitate probing the health effects of EGCG, which is the major constituent of green tea.

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ∼1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime (τ2 = 1.9–3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there is significant nuclear absorption of flavanols. This advanced imaging using two-photon excitation and biophysical techniques described here will prove valuable for probing the intracellular trafficking and functions of flavanols, such as EGCG, which is the major flavanol of green tea.

Influence of sugar type on the bioavailability of cocoa flavanols.

The beneficial effects of cocoa on vascular function are mediated by the absorption of monomeric flavanols into the circulation from the small intestine. As such, an understanding of the impact of the food matrix on the delivery of flavanols to the circulation is critical in assessing the potential vascular impact of a food. In the present study, we investigated the impact of carbohydrate type on flavanol absorption and metabolism from chocolate. A randomised, double-blind, three-arm cross-over study was conducted, where fifteen volunteers were randomly assigned to either a high-flavanol (266 mg) chocolate containing maltitol, a high-flavanol (251 mg) chocolate with sucrose or a low-flavanol (48 mg) chocolate with sucrose. Test chocolates were matched for micro- and macronutrients, including the alkaloids theobromine and caffeine, and were similar in taste and appearance. Total flavanol absorption was lower after consumption of the maltitol-containing test chocolate compared with following consumption of its sucrose-containing equivalent (P = 0·002). Although the O-methylation pattern observed for absorbed flavanols was unaffected by sugar type, individual levels of unmethylated ( - )-epicatechin metabolites, 3'-O-methyl-epicatechin and 4'-O-methyl-epicatechin metabolites were lower for the maltitol-containing test chocolate compared with the sucrose-containing equivalent. Despite a reduction in the total plasma pool of flavanols, the maximum time (T max) was unaffected. The present data indicate that full assessment of intervention treatments is vital in future intervention trials with flavanols and that carbohydrate content is an important determinant for the optimal delivery of flavanols to the circulation.

Estimated dietary intakes and sources of flavanols in the German population (German National Nutrition Survey II)

Background/Objectives Data from intervention studies suggest a beneficial effect of flavanols on vascular health. However, insufficient data on their intake have delayed the assessment of their health benefits. The aim of this study was to estimate intake of flavanols and their main sources among people living in Germany. Subjects/Methods Data from diet history interviews of the German National Nutrition Survey II for 15,371 people across Germany aged 14–80 years were analyzed. The FLAVIOLA Flavanol Food Composition Database was compiled using the latest US Department of Agriculture and Phenol-Explorer Databases and expanded to include recipes and retention factors. Results Mean intake of total flavanols, flavan-3-ol monomers, proanthocyanidins (PA), and theaflavins in Germany was 386, 120, 196, and 70 mg/day, respectively. Women had higher intakes of total flavanols (399 mg/day) than men (372 mg/day) in all age groups, with the exception of the elderly. Similar results were observed for monomers (108 mg/day for men, 131 mg/day for women) and PA (190 mg/day; 203 mg/day), although intake of theaflavins was higher in men (74 mg/day; 66 mg/day). There was an age gradient with an increase in total flavanols, monomers, and theaflavins across the age groups. The major contributor of total flavanols in all subjects was pome fruits (27 %) followed by black tea (25 %). Conclusions This study demonstrated age- and sex-related variations in the intake and sources of dietary flavanols in Germany. The current analysis will provide a valuable tool in clarifying and confirming the potential health benefits of flavanols.

Interactions between cocoa flavanols and inorganic nitrate: additive effects on endothelial function at achievable dietary amounts

Dietary intervention studies have shown that flavanols and inorganic nitrate can improve vascular function, suggesting that these two bioactives may be responsible for beneficial health effects of diets rich in fruits and vegetables. We aimed to study interactions between cocoa flavanols (CF) and nitrate, focusing on absorption, bioavailability, excretion, and efficacy to increase endothelial function. In a double-blind randomized, dose-response crossover study, flow-mediated dilation (FMD) was measured in 15 healthy subjects before and at 1, 2, 3, and 4 h after consumption of CF (1.4-10.9 mg/kg bw) or nitrate (0.1-10 mg/kg bw). To study flavanol-nitrate interactions, an additional intervention trial was performed with nitrate and CF taken in sequence at low and high amounts. FMD was measured before (0 h) and at 1h after ingestion of nitrate (3 or 8.5 mg/kg bw) or water. Then subjects received a CF drink (2.7 or 10.9 mg/kg bw) or a micro- and macronutrient-matched CF-free drink. FMD was measured at 1, 2, and 4 h thereafter. Blood and urine samples were collected and assessed for CF and nitric oxide (NO) metabolites with HPLC and gas-phase reductive chemiluminescence. Finally, intragastric formation of NO after CF and nitrate consumption was investigated. Both CF and nitrate induced similar intake-dependent increases in FMD. Maximal values were achieved at 1 h postingestion and gradually decreased to reach baseline values at 4 h. These effects were additive at low intake levels, whereas CF did not further increase FMD after high nitrate intake. Nitrate did not affect flavanol absorption, bioavailability, or excretion, but CF enhanced nitrate-related gastric NO formation and attenuated the increase in plasma nitrite after nitrate intake. Both flavanols and inorganic nitrate can improve endothelial function in healthy subjects at intake amounts that are achievable with a normal diet. Even low dietary intake of these bioactives may exert relevant effects on endothelial function when ingested together.

HPLC analyses of flavanols and phenolic acids in the fresh young shoots of tea (Camellia sinensis) grown in Australia

As part of a 4-year project to study phenolic compounds in tea shoots over the growing seasons and during black tea processing in Australia, an HPLC method was developed and optimised for the identification and quantification of phenolic compounds, mainly flavanols and phenolic acids, in fresh tea shoots. Methanol proved to be the most suitable solvent for extracting the phenolic compounds, compared with chloroform, ethyl acetate and water. Immediate analysis, by HPLC, of the methanol extract showed higher separation efficiency than analyses after being dried and redissolved. This method exhibited good repeatability (CV 3-9%) and recovery rate (88-116%). Epigallocatechin gallate alone constituted up to 115 mg/g, on a dry basis, in the single sample of Australian fresh tea shoots examined. Four catechins (catechin, gallocatechin, epicatechin and epigallocatechin) and six catechin gallates (epigallocatechin gallate, catechin gallate, epicatechin gallate, gallocatechin gallate, epicatechin digallate and epigallocatechin digallate) have been identified and quantified by this HPLC method. In addition, two major tea alkaloids, caffeine and theobromine, have been quantified, while five flavonol glycosides and six phenolic acids, including quinic acids and esters, were identified and quantified. (C) 2003 Elsevier Ltd. All rights reserved.

Phenolic compounds and antioxidant activity of seed and skin extracts of red grape (Vitis vinifera and Vitis labrusca) pomace from Brazilian winemaking

The phenolic compounds content and antioxidant activity of seed and skin of pomace from the vinification of grape varieties widely produced in Brazil were investigated with a view to their exploitation as a potential source of natural antioxidants. There was a greater concentration of phenolic compounds in the seeds (2128 to 16,518 mg of catechin equivalents (CE)/100 g) than in the skins (660 to 1839 mg CE/100 g). The highest antioxidant activity values determined as DPPH radical-scavenging ability and ferric reducing-antioxidant power (FRAP) were found for the seeds of the Pinot Noir variety (16,925 mu mol Trolox equivalents (TE)/100 g and 21,492 mu mol Fe(2+)/100 g, respectively) and in the skin extracts of the Isabel variety (3640 mu mol TE/100 g and 4362 mu mol Fe(2+)/100 g, respectively). The skin of Cabernet Sauvignon and Primitivo varieties had the highest contents of anthocyanins (935 and 832 mg/100 g, respectively). The grape seed extracts were rich in oligomeric and polymeric flavanols. The data suggested that grape seed and skin extracts may be exploited as antioxidant agents. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of cocoa powder in the prevention of cardiovascular disease: biological, consumption and inflammatory biomarkers. A metabolomic approach

Numerous health benefits have been attributed to cocoa and its derived products in the last decade including antioxidant, anti-platelet and positive effects on lipid metabolism and vascular function. Inflammation plays a key role in the initiation and progression of atherosclerosis. However, cocoa feeding trials focused on inflammation are still rare and the results yielded are controversial. Health effects derived from cocoa consumption have been partly attributed to its polyphenol content, in particular of flavanols. Bioavailability is a key issue for cocoa polyphenols in order to be able to exert their biological activities. In the case of flavanols, bioavailability is strongly influenced by several factors, such as their degree of polymerization and the food matrix in which the polyphenols are delivered. Furthermore, gut has become an active site for the metabolism of procyanidins (oligomeric and polymeric flavanols). Estimation of polyphenol consumption or exposure is also a very challenging task in Food and Nutrition Science in order to correlate the intake of phytochemicals with in vivo health effects. In the area of nutrition, modern analytical techniques based on mass spectrometry are leading to considerable advances in targeted metabolite analysis and particularly in Metabolomics or global metabolite analysis. In this chapter we have summarized the most relevant results of our recent research on the bioavailability of cocoa polyphenols in humans and the effect of the matrix in which cocoa polyphenols are delivered considering both targeted analysis and a metabolomic approach. Furthermore, we have also summarized the effect of long-term consumption of cocoa powder in patients at high risk of cardiovascular disease (CVD) on the inflammatory biomarkers of atherosclerosis.

Effect of cocoa powder in the prevention of cardiovascular disease: biological, consumption and inflammatory biomarkers. A metabolomic approach

Numerous health benefits have been attributed to cocoa and its derived products in the last decade including antioxidant, anti-platelet and positive effects on lipid metabolism and vascular function. Inflammation plays a key role in the initiation and progression of atherosclerosis. However, cocoa feeding trials focused on inflammation are still rare and the results yielded are controversial. Health effects derived from cocoa consumption have been partly attributed to its polyphenol content, in particular of flavanols. Bioavailability is a key issue for cocoa polyphenols in order to be able to exert their biological activities. In the case of flavanols, bioavailability is strongly influenced by several factors, such as their degree of polymerization and the food matrix in which the polyphenols are delivered. Furthermore, gut has become an active site for the metabolism of procyanidins (oligomeric and polymeric flavanols). Estimation of polyphenol consumption or exposure is also a very challenging task in Food and Nutrition Science in order to correlate the intake of phytochemicals with in vivo health effects. In the area of nutrition, modern analytical techniques based on mass spectrometry are leading to considerable advances in targeted metabolite analysis and particularly in Metabolomics or global metabolite analysis. In this chapter we have summarized the most relevant results of our recent research on the bioavailability of cocoa polyphenols in humans and the effect of the matrix in which cocoa polyphenols are delivered considering both targeted analysis and a metabolomic approach. Furthermore, we have also summarized the effect of long-term consumption of cocoa powder in patients at high risk of cardiovascular disease (CVD) on the inflammatory biomarkers of atherosclerosis.

Enriquecimento de compostos fenólicos de folhas de Inga edulis por extração em fase sólida: quantificação de seus compostos majoritários e avaliação da capacidade antioxidante

A phenolic fraction was obtained from of the acetone-water-acetic acid extract of Inga edulis leaves, by liquid-liquid partition and SPE-C18 cartridges. This method provided an increase of 108, 66, 51, 50 and 36% of flavonols, proanthocyanidins, total polyphenols, gallotannins and flavanols, respectively. The major phenolics in purified fraction were procyanidin B2, catechin and myricetin-3-O-α-L-rhamnopyranoside, which achieved increases of 111, 47 and 45%, respectively, after SPE. Acid hydrolysis confirmed the presence of procyanidins, prodelphinidins and glycosylated flavonoids.

The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury

Neuroinflammation plays an integral role in the progression of neurodegeneration. In this study we investigated the anti-inflammatory effects of different classes of flavonoids (flavanones, flavanols and anthocyanidins) in primary mixed glial cells. We found that the flavanones naringenin and hesperetin and the flavols (+)-catechin and (-)-epicatechin, but not the anthocyanidins cyanidin and pelargonidin, attenuated LPS/IFN-gamma-induced TNF-alpha production in glial cells. Naringenin also inhibited LPS/IFN-gamma-induced iNOS expression and nitric oxide production in glial cells, thus showing the strongest antiinflammatory activity among all flavonoids tested. Moreover, naringenin protected against inflammatory-induced neuronal death in a primary neuronal-glial co-culture system. Naringenin also inhibited LPS/IFN-gamma-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and downstream signal transducer and activator of transcription-1 (STAT-1) in LPS/IFN-gamma stimulated primary mixed glial cells. Taken together, our results suggest that naringenin may produce an anti-inflammatory effect in LPS/IFN-gamma stimulated glial cells that may be due to its interaction with p38 signalling cascades and the STAT-I trascription factor. (C) 2009 Elseiver Inc. All rights reserved.