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

12 páginas, 2 figuras.-- et al.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.This research was supported by national grants CICYT (AGL: 2004- 08378-C02-01/02, 2006-14228-C03-02/01 and 2009-13906-C02-01); CIBER 06/03 Fisiopatología de la Obesidad y la Nutrición is an initiative of the Instituto de Salud Carlos III, Spain; Centro Nacional de Investigaciones Cardiovasculares (CNIC-06) and Ingenio-CONSOLIDER program, Fun-cfood (CSD2007-063). M.U.-S. thanks the Sara Borrell postdoctoral program (CD09/00134), M.M. thanks the Ramon y Cajal program, R.Ll. thanks the Fondo de Investigación Sanitaria program (FIS, CD06/00161), and N.K. thanks the FPU fellowship program, all from the Ministry of Science and Innovation. M.R.-R. thanks the FI-DGR2010 fellowship program from the Generalitat de Catalunya. R.E. is recipient of a grant from FIS, Madrid, Spain.Peer reviewe

Metabolic fingerprint after acute and under sustained consumption of a functional beverage based on grape skin extract in healthy human subjects

Grape-derived polyphenols are considered to be one of the most promising ingredients for functional foods due to their health-promoting activities. We applied a HPLC-MS-based untargeted metabolomic approach in order to evaluate the impact of a functional food based on grape skin polyphenols on the urinary metabolome of healthy subjects. Thirty-one volunteers participated in two dietary crossover randomized intervention studies: with a single-dose intake (187 mL) and with a 15-day sustained consumption (twice per day, 187 mL per day in total) of a functional beverage (FB). Postprandial (4-hour) and 24-hour urine samples collected after acute consumption and on the last day of sustained FB consumption, respectively, were analysed using an untargeted HPLC-qTOF-MS approach. Multivariate modelling with subsequent application of an S-plot revealed differential mass features related to acute and prolonged consumption of FB. More than half of the mass features were shared between the two types of samples, among which several phase II metabolites of grape-derived polyphenols were identified at confidence level II. Prolonged consumption of FB was specifically reflected in urine metabolome by the presence of first-stage microbial metabolites of flavanols: hydroxyvaleric acid and hydroxyvalerolactone derivatives. Overall, several epicatechin and phenolic acid metabolites both of tissular and microbiota origin were the most representative markers of FB consumption. To our knowledge, this is one of the first studies where an untargeted LC-MS metabolomic approach has been applied in nutrition research on a grape-derived FB.This work has been supported by Spanish public grants AGL2006-14228-C03-02 and AGL2009-13906-C02-01 and the CONSOLIDER-INGENIO 2010 Programme, FUN-C-FOOD (CSD2007-063) from the Spanish Ministry of Economy and Competitiveness (MINECO), the PI13/01172 Project (Plan N de I+D+i 2013–2016) by ISCII-Subdirección General de Evaluación y Fomento de la Investigación as co-funded by FEDER (Fondo Europeo de Desarrollo Regional); the National Strategic Consortia for Technical Research Programme, in collaboration with the Bosch i Gimpera Foundation, FBG305132 and FBG305273; and Miguel Torres S.A. (Proyecto CENIT HIGEA CEN-20072003). We also thank the award of 2014SGR1566 from the Generalitat de Catalunya’s Agency AGAUR. The Juan de la Cierva postdoctoral fellowship contract from the Spanish government awarded by the Spanish Ministry to Olha Khymenets is acknowledged. Rosa Vazquez-Fresno, Rafael Llorach and Mireia Urpi-Sarda would like to thank, respectively, the FPI 2010 fellowship and the Ramon y Cajal programmes from MICINN and the European Social Funds.Peer Reviewe

Targeted and metabolomic study of biomarkers of cocoa powder consumption effects on inflammatory biomarkers in patients at high risk of cardiovascular disease

Cocoa and its derived products have received considerable attention in the last decade due to their numerous health benefits, 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 high polyphenol content, in particular of flavan-3-ols. Bioavailability is a key issue in order for cocoa polyphenols to exert their biological activities. In the case of flavan-3-ols, bioavailability is dependent on their degree of polymerization, with the colon being an active site for the metabolism of high molecular weight flavan-3-ols or procyanidins. 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 health effects in vivo. In this area, modern analytical techniques based on mass spectrometry are leading to considerable advances in targeted metabolite analysis and particularly in global metabolite analysis or metabolomics. In the current paper we have summarized the most relevant results of our recent research conducted on the bioavailability of cocoa polyphenols and inflammatory biomarkers of atherosclerosis in patients at high risk of cardiovascular disease (CHD), including the application of both targeted analysis and a metabolomic approach for estimating polyphenol bioavailability and biomarkers of exposure.Peer Reviewe

Metabolomic fingerprint in patients at high risk of cardiovascular disease by cocoa intervention

[Scope]: Metabolomics approach is focused on identifying all metabolites present in a biological sample (metabolome). Consumption of cocoa products has been related to health benefits including positive effect on cardiovascular health. [Methods and results]: Twenty volunteers were included in this randomized, crossover, and controlled clinical trial. After a 2-wk washout period, subjects received 40 g/day of cocoa powder with 500 mL skimmed milk (cocoa with skimmed milk intervention) or 500 mL/day of skimmed milk (skimmed milk intervention) for 4-wk. Urine (24 h) samples were collected at baseline and after each intervention and were analyzed by HPLC-hybrid quadrupole TOF in negative and positive ionization modes followed by multivariate analysis. This analysis revealed a marked separation between the cocoa with skimmed milk intervention and skimmed milk intervention and baseline periods. Thirty-nine compounds linked with cocoa intake, including alkaloid metabolites, polyphenol host and gut microbial metabolites (hydroxyphenylvalerolactones and hydroxyphenylvaleric acids), diketopiperazines and N-phenylpropenoyl-l-amino acids were identified. In the case of endogenous metabolites, putative identifications suggested that metabolites linked with carnitine metabolism and sulfation of tyrosine were decreased by the consumption of cocoa. [Conclusion]: LC-MS metabolomics strategy allows the defining of a complex metabolic profile derived from cocoa phytochemicals. Likewise, the identification of endogenous markers could lead to new hypotheses to unravel the relationship between cocoa intake and cardiovascular diseases. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Funded by: CICYT. Grant Numbers: AGL2009–13906-C02–01, AGL2010–10084-E.Peer Reviewe

Cocoa polyphenols and inflammatory markers of cardiovascular disease

This is an open access article distributed under the Creative Commons Attribution License.-- This article belongs to the Special Issue Chocolate and Cocoa in Human Health.Epidemiological studies have demonstrated the beneficial effect of plant-derived food intake in reducing the risk of cardiovascular disease (CVD). The potential bioactivity of cocoa and its polyphenolic components in modulating cardiovascular health is now being studied worldwide and continues to grow at a rapid pace. In fact, the high polyphenol content of cocoa is of particular interest from the nutritional and pharmacological viewpoints. Cocoa polyphenols are shown to possess a range of cardiovascular-protective properties, and can play a meaningful role through modulating different inflammatory markers involved in atherosclerosis. Accumulated evidence on related anti-inflammatory effects of cocoa polyphenols is summarized in the present review. © 2014 by the authors; licensee MDPI, Basel, Switzerland.The authors are grateful for the support granted by the following Spanish government programmes: CICYT AGL2009-13906-C02-01, AGL2010-10084-E and FUN-C Food CSD2007-063 from the Ingenio-CONSOLIDER programme of the Spanish Government. The postdoctoral contract fellowship, awarded by the Ministry of Innovation and Science to Olha Khymenets and Sara Tulipani (Juan de la Cierva Program), and the predoctoral fellowships awarded by the Generalitat de Catalunya (FI-DRG) to Ximena Mora-Cubillos and Mar Garcia-Aloy are acknowledged. Mireia Urpí-Sardà and Rafael Llorach would like to thank Ramon y Cajal Program of the Spanish Ministry (MINECO) and the Fondo Social Europeo.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Metabolomics study of human urinary metabolome modifications after intake of almond (Prunus dulcis (Mill.) D.A. Webb) skin polyphenols

Almond, as a part of the nut family, is an important source of biological compounds, and specifically, almond skins have been considered an important source of polyphenols, including flavan-3-ols and flavonols. Polyphenol metabolism may produce several classes of metabolites that could often be more biologically active than their dietary precursor and could also become a robust new biomarker of almond polyphenol intake. In order to study urinary metabolome modifications during the 24 h after a single dose of almond skin extract, 24 volunteers (n = 24), who followed a polyphenol-free diet for 48 h before and during the study, ingested a dietary supplement of almond skin phenolic compounds (n = 12) or a placebo (n = 12). Urine samples were collected before ((-2)-0 h) and after (0-2 h, 2-6 h, 6-10 h, and 10-24 h) the intake and were analyzed by liquid chromatography-mass spectrometry (LC-q-TOF) and multivariate statistical analysis (principal component analysis (PCA) and orthogonal projection to latent structures (OPLS)). Putative identification of relevant biomarkers revealed a total of 34 metabolites associated with the single dose of almond extract, including host and, in particular, microbiota metabolites. As far as we know, this is the first time that conjugates of hydroxyphenylvaleric, hydroxyphenylpropionic, and hydroxyphenylacetic acids have been identified in human samples after the consumption of flavan-3-ols through a metabolomic approach. The results showed that this non-targeted approach could provide new intake biomarkers, contributing to the development of the food metabolome as an important part of the human urinary metabolome. © 2010 American Chemical Society.Peer Reviewe

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: From chemical characterization to targeted analysis of phenolic metabolites in humans

10 páginas, 3 figuras, 3 tablas.-- El pdf del artículo es la versión de autor.In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism.This work has received financial support from the CONSOLIDER INGENIO 2010 Programme: FUN-C-FOOD CSD2007- 063 from the Spanish Ministry of Science and Innovation (MICINN). I.G. was the recipient of a fellowship from the I3P Program funding by the European Social Fund. M.U.-S. thanks the FPI fellowship from MICINN. M.M. thanks the ‘‘Ramón y Cajal” post-doctoral program. R.L.L. thanks the ‘‘Fondo de Investigación Sanitaria” postdoctoral program (F.I.S. CD06/00161) from MICINN.Peer reviewe

Profile of plasma and urine metabolites after the intake of almond [Prunus dulcis (Mill.) D.A. Webb] polyphenols in humans

Nut skins are considered to be a rich source of polyphenols and may be partially responsible for the numerous health effects associated with nut consumption. However, more bioavailability studies of nut skin polyphenols are needed to understand the health effects derived from nut consumption. The aim of the present study was to determine the profiles of both phase II and microbial-derived phenolic metabolites in plasma and urine samples before and after the intake of almond skin polyphenols by healthy human subjects (n = 2). Glucuronide, O-methyl glucuronide, sulfate, and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and glucuronide and sulfate conjugates of isorhamnetin, were detected in plasma and urine samples after consumption of almond skin polyphenols. The main microbial-derived metabolites of flavanols, such as 5-(dihydroxyphenyl)-γ-valerolactone and 5-(hydroxymethoxyphenyl)-γ-valerolactone, were also detected in their glucuronide and sulfate forms. In addition, numerous metabolites derived from further microbial degradation of hydroxyphenylvalerolactones, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic, and hydroxyhippuric acids, registered major changes in urine after the consumption of almond skin polyphenols. The urinary excretion of these microbial metabolites was estimated to account for a larger proportion of the total polyphenol ingested than phase II metabolites of (epi)catechin, indicating the important role of intestinal bacteria in the metabolism of highly polymerized almond skin polyphenols. To the authors' knowledge this study constitutes the most complete report of the absorption of almond skin polyphenols in humans.I.G. was the recipient of a fellowship from the I3P Program funding by the European Social Fund. M.U.-S. and A.M.-R. gratefully acknowledge the FPI fellowship from MICINN and FI program for the training of researchers from the Generalitat of Catalonia, respectively. M.M. thanks the “Juan de la Cierva” postdoctoral program. This work has received financial support from AGL2004-07075-C02-02, AGL2004-08378-C02-01, AGL2006-14228-C03-02, and the CONSOLIDER INGENIO 2010 Program: FUN-C-FOOD CSD2007-063 from the Spanish Ministry of Science and Innovation (MICINN).Peer reviewe

Targeted analysis of conjugated and microbial-derived phenolic metabolites in human urine after consumption of an almond skin phenolic extract

A single-blind, placebo-controlled, and randomized trial study was carried out with 16 healthy volunteers (7 men and 5 women). The test group ingested an encapsulated almond skin phenolic extract (884 mg of total polyphenols/dose) containing flavan-3-ols, flavonols, and flavanones, whereas the placebo group ingested microcrystalline cellulose. Our aim in this study was to determine changes in the urinary excretion of conjugated and microbial-derived phenolic metabolites before (-2 to 0 h) and after (0-2, 2-6, 6-10, and 10-24 h) intake of the almond polyphenols compared with the placebo group. For the test group, maximum urinary excretion of (epi)catechin and naringenin conjugates derived from phase II metabolism was attained at 2-6 h after consumption of the almond skin extract and excretions differed from the placebo group during this time period (P ≤ 0.0001). However, excretion of conjugated metabolites of isorhamnetin was highest at 10-24 h and did not differ from the placebo group during this time (P> 0.05). Hydroxyphenylvalerolactones reached maximum urinary levels at 6-10 h after consumption of almond polyphenols, and excretion differed from the placebo group during this time period (P = 0.0004). For the test group, excretions of phenolic acids (hydroxyphenylpropionic, hydroxyphenylacetic, hydroxybenzoic, and hydroxycinnamic acids) did not differ from the placebo group at any time period of urine collection (P> 0.05). The findings presented in this work provide evidence concerning the bioavailability of almond skin polyphenols considering the effects of both phase II and microbial metabolism. © 2010 American Society for Nutrition.Peer Reviewe

Effect of Milk on the Urinary Excretion of Microbial Phenolic Acids after Cocoa Powder Consumption in Humans

6 páginas, 1 figura, 1 tabla.Health effects of cocoa flavonols depend on their bioavailability, which is strongly influenced by the food matrix and the degree of flavanol polymerization. The effect of milk on the bioavailability of cocoa flavanoids considering phase II metabolites of epicatechin has been the subject of considerable debate. This work studies the effect of milk at the colonic microbial metabolism level of the nonabsorbed flavanol fraction that reaches the colon and is metabolized by the colonic microbiota into various phenolic acids. Twenty-one human volunteers followed a diet low in polyphenols for at least 48 h before taking, in a random order, 40 g of cocoa powder dissolved either in 250 mL of whole milk or in 250 mL of water. Urine samples were collected before the intake and during three different periods (0−6, 6−12, and 12−24 h). Phenolic acids were analyzed by LC-MS/MS after solid-phase extraction. Of the 15 metabolites assessed, the excretion of 9 phenolic acids was affected by the intake of milk. The urinary concentration of 3,4-dihydroxyphenylacetic, protocatechuic, 4-hydroxybenzoic, 4-hydroxyhippuric, hippuric, caffeic, and ferulic acids diminished after the intake of cocoa with milk, whereas urinary concentrations of vanillic and phenylacetic acids increased. In conclusion, milk partially affects the formation of microbial phenolic acids derived from the colonic degradation of procyanidins and other compounds present in cocoa powder.This research was supported by national grants CICYT (AGL: 2004-08378-C02-01/02 and 2006- 14228-C03-02/01); CIBER 06/03 Fisiopatologı´ a de la Obesidad y la Nutrici on is an initiative of the Instituto de Salud Carlos III, Spain; Centro Nacional de Investigaciones Cardiovasculares (CNIC-06) and Ingenio-CONSOLIDER program, Fun-c-food (CSD2007-063).M.U.-S. and N.K. thank the FPI and FPU fellowship programs, respectively, and M.M. and R.Ll., the postdoctoral programs Juan de la Cierva and Fondo de Investigaci on Sanitaria (FIS CD06/00161), respectively, all from the Ministry of Science and Innovation.M.R.-R. thanks the APIF fellowship from the University of Barcelona. R.E. is recipient of a grant from F.I.S., Madrid, Spain.Peer reviewe