Effect of cocoa powder on the modulation of inflammatory biomarkers in patients at high risk of cardiovascular disease

Background: Epidemiologic studies have suggested that flavonoid intake plays a critical role in the prevention of coronary heart disease. Because atherosclerosis is considered a low-grade inflammatory disease, some feeding trials have analyzed the effects of cocoa (an important source of flavonoids) on inflammatory biomarkers, but the results have been controversial. Objective: The objective was to evaluate the effects of chronic cocoa consumption on cellular and serum biomarkers related to atherosclerosis in high-risk patients. Design: Forty-two high-risk volunteers (19 men and 23 women; mean 6 SD age: 69.7 6 11.5 y) were included in a randomized crossover feeding trial. All subjects received 40 g cocoa powder with 500 mL skim milk/d (C+M) or only 500 mL skim milk/d (M) for 4 wk. Before and after each intervention period, cellular and serum inflammatory biomarkers related to atherosclerosis were evaluated. Results: Adherence to the dietary protocol was excellent. No significant changes in the expression of adhesion molecules on T lymphocyte surfaces were found between the C+M and M groups. However, in monocytes, the expression of VLA-4, CD40, and CD36 was significantly lower (P = 0.005, 0.028, and 0.001, respectively) after C+M intake than after M intake. In addition, serum concentrations of the soluble endothelium-derived adhesion molecules P-selectin and intercellular adhesion molecule-1 were significantly lower (both P = 0.007) after C+M intake than after M intake. Conclusions: These results suggest that the intake of cocoa polyphenols may modulate inflammatory mediators in patients at high risk of cardiovascular disease. These antiinflammatory effects may contribute to the overall benefits of cocoa consumption against atherosclerosis. This trial was registered in the Current Controlled Trials at London, International Standard Randomized Controlled Trial Number, at controlled-trials.com as ISRCTN75176807.

Flavonoids affect host-microbiota crosstalk through TLR modulation

Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs) in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases.

Flavonoids affect host-microbiota crosstalk through TLR modulation

Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs) in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases.

Cocoa flavonoid-enriched diet modulates systemic and intestinal immunoglobulin synthesis in adult Lewis rats

Previous studies have reported that a diet containing 10% cocoa, a rich source of flavonoids, has immunomodulatory effects on rats and, among others effects, is able to attenuate the immunoglobulin (Ig) synthesis in both systemic and intestinal compartments. The purpose of the present study was focused on investigating whether these effects were attributed exclusively to the flavonoid content or to other compounds present in cocoa. To this end, eight-week-old Lewis rats were fed, for two weeks, either a standard diet or three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols from conventional defatted cocoa, and two others with 0.4% and 0.8% polyphenols, respectively, from non-fermented cocoa. Diet intake and body weight were monitored and fecal samples were obtained throughout the study to determine fecal pH, IgA, bacteria proportions, and IgA-coated bacteria. Moreover, IgG and IgM concentrations in serum samples collected during the study were quantified. At the end of the dietary intervention no clear changes of serum IgG or IgM concentrations were quantified, showing few effects of cocoa polyphenol diets at the systemic level. However, in the intestine, all cocoa polyphenol-enriched diets attenuated the age-related increase of both fecal IgA and IgA-coated bacteria, as well as the proportion of bacteria in feces. As these effects were not dependent on the dose of polyphenol present in the diets, other compounds and/or the precise polyphenol composition present in cocoa raw material used for the diets could be key factors in this effect.

Simultaneous Evaluation of Intact Glucosinolates and Phenolic Compounds by UPLC-DAD-MS/MS in Brassica oleracea L. var. botrytis

A method for the simultaneous determination of intact glucosinolates and main phenolic compounds (flavonoids and sinapic acid derivatives) in Brassica oleracea L. var. botrytis was proposed. A simplified sample extraction procedure and a UPLC separation were carried out to reduce the total time of analysis. Brassica oleracea samples were added with internal standards (glucotropaeolin and rutin), and extracted with boiling methanol. Crude extracts were evaporated under nitrogen, redissolved in mobile phase and analyzed by UPLC with double detection (ESI--MRM for glucosinolates and flavonoids, and DAD for main sinapic acid derivatives). The proposed method allowed a satisfactory quantification of main native sinapic acid derivatives, flavonoids and glucosinolates with a reduced time of analysis.

Absorption and pharmacokinetics of grapefruit flavanones in beagles

The present study evaluated the pharmacokinetics of three different grapefruit flavanone forms in dog plasma and demonstrated their absorption after an oral intake of a grapefruit extract; pharmacokinetic parameters of these forms were also determined. Ten healthy beagles were administered 70 mg citrus flavonoids as a grapefruit extract contained in capsules, while two additional dogs were used as controls and given an excipient. The grapefruit flavanone naringin, along with its metabolites naringenin and naringenin glucuronide, was detected in dog plasma. Blood samples were collected between 0 and 24 h after administration of the extract. Naringin reached its maximun plasma concentration at around 80 min, whereas naringenin and naringenin glucuronide reached their maximun plasma concentrations at around 20 and 30 min, respectively. Maximum plasma concentrations of naringin, naringenin and naringenin glucuronide (medians and ranges) were 0·24 (0·05 2·08), 0·021 (0·001 0·3) and 0·09 (0·034 0·12) mmol/l, respectively. The areas under the curves were 23·16 l (14·04 70·62) min £ mmol/for nariningin, 1·78 (0·09 4·95) min £ mmol/l for naringenin and 22·5 (2·74 99·23) min £ mmol/l for naringenin glucuronide. The median and range values for mean residence time were 3·3 (1·5 9·3), 2·8 (0·8 11·2) and 8·0 (2·3 13·1) h for naringin, naringenin and naringenin glucuronide, respectively. The results of the present study demonstrate the absorption of grapefruit flavanones via the presence of their metabolites in plasma, thus making an important contribution to the field since the biological activities ascribed to these compounds rely on their specific forms of absorption.

Absorption and pharmacokinetics of green tea catechins in beagles

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12·35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 02, 26, 68 and 824 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for (2)-epigallocatechin-3-gallate (EGCG), (2)-epicatechin-3-gallate (ECG), (2)-epigallocatechin glucuronide (EGCglucuronide), (2)-epicatechin glucuronide (EC-glucuronide), (2)-epicatechin sulphate (EC sulphate) were 0·3 (range 0·11·9), 0·1 (range 00·4), 0·8 (range 0·23·9), 0·2 (range 0·1 1·7) and 1 (range 0·33·4) mmol/l, respectively. The areas under the plasma concentration v. time curves (AUC0!24) were 427 (range 1021185) mmol/l £ min for EGC-glucuronide, 112 (range 53919) mmol/l £ min for EC-sulphate, 71 (range 26306) mmol/l £ min for EGCG, 40 (range 12258) mmol/l £ min for EC-glucuronide and 14 (range 0·1124) mmol/l £ min for ECG. The values of mean residence time (MRT0!24) were 5 (range 216), 2 (range 111), 10 (range 213), 3 (range 216) and 2·4 (range 118) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.

Absorption and pharmacokinetics of grapefruit flavanones in beagles

The present study evaluated the pharmacokinetics of three different grapefruit flavanone forms in dog plasma and demonstrated their absorption after an oral intake of a grapefruit extract; pharmacokinetic parameters of these forms were also determined. Ten healthy beagles were administered 70 mg citrus flavonoids as a grapefruit extract contained in capsules, while two additional dogs were used as controls and given an excipient. The grapefruit flavanone naringin, along with its metabolites naringenin and naringenin glucuronide, was detected in dog plasma. Blood samples were collected between 0 and 24 h after administration of the extract. Naringin reached its maximun plasma concentration at around 80 min, whereas naringenin and naringenin glucuronide reached their maximun plasma concentrations at around 20 and 30 min, respectively. Maximum plasma concentrations of naringin, naringenin and naringenin glucuronide (medians and ranges) were 0·24 (0·05 2·08), 0·021 (0·001 0·3) and 0·09 (0·034 0·12) mmol/l, respectively. The areas under the curves were 23·16 l (14·04 70·62) min £ mmol/for nariningin, 1·78 (0·09 4·95) min £ mmol/l for naringenin and 22·5 (2·74 99·23) min £ mmol/l for naringenin glucuronide. The median and range values for mean residence time were 3·3 (1·5 9·3), 2·8 (0·8 11·2) and 8·0 (2·3 13·1) h for naringin, naringenin and naringenin glucuronide, respectively. The results of the present study demonstrate the absorption of grapefruit flavanones via the presence of their metabolites in plasma, thus making an important contribution to the field since the biological activities ascribed to these compounds rely on their specific forms of absorption.

The effects of milk as a food matrix for polyphenols on the excretion profile of cocoa (-)-Epicatechin metabolites in healthy humans subjects

The effect of different food matrices on the metabolism and excretion of polyphenols is uncertain. The objective of the study was to evaluate the possible effect of milk on the excretion of (2)-epicatechin metabolites from cocoa powder after its ingestion with and without milk. Twenty-one volunteers received the following three test meals each in a randomised cross-over design with a 1-week interval between meals: (1) 250 ml whole milk as a control; (2) 40 g cocoa powder dissolved in 250 ml whole milk (CC-M); (3) 40 g cocoa powder dissolved in 250 ml water (CC-W). Urine was collected before consumption and during the 0-6, 6-12 and 12-24 h periods after consumption. (2)-Epicatechin metabolite excretion was measured using liquid chromatography-MS. One (2)-epicatechin glucuronide and three (2)-epicatechin sulfates were detected in urine excreted after the intake of the two cocoa beverages (CC-M and CC-W). The results show that milk does not significantly affect the total amount of metabolites excreted in urine. However, differences in metabolite excretion profiles were observed; there were changes in the glucuronide and sulfate excretion rates, and the sulfation position between the period of excretion and the matrix. The matrix in which polyphenols are consumed can affect their metabolism and excretion, and this may affect their biological activity. Thus, more studies are needed to evaluate the effect of these different metabolite profiles on the body.

Absorption and pharmacokinetics of green tea catechins in beagles

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12·35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 0-2, 2-6, 6-8 and 8-24 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for (2)-epigallocatechin-3-gallate (EGCG), (2)-epicatechin-3-gallate (ECG), (2)-epigallocatechin glucuronide (EGCglucuronide), (2)-epicatechin glucuronide (EC-glucuronide), (2)-epicatechin sulphate (EC sulphate) were 0·3 (range 0·1-1·9), 0·1 (range 0-0·4), 0·8 (range 0·2-3·9), 0·2 (range 0·1 1·7) and 1 (range 0·3-3·4) mmol/l, respectively. The areas under the plasma concentration v. time curves (AUC0!24) were 427 (range 102-1185) mmol/l £ min for EGC-glucuronide, 112 (range 53-919) mmol/l £ min for EC-sulphate, 71 (range 26-306) mmol/l £ min for EGCG, 40 (range 12-258) mmol/l £ min for EC-glucuronide and 14 (range 0·1-124) mmol/l £ min for ECG. The values of mean residence time (MRT0!24) were 5 (range 2-16), 2 (range 1-11), 10 (range 2-13), 3 (range 2-16) and 2·4 (range 1-18) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.

Absorption and pharmacokinetics of green tea catechins in beagles

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12·35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 0-2, 2-6, 6-8 and 8-24 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for (2)-epigallocatechin-3-gallate (EGCG), (2)-epicatechin-3-gallate (ECG), (2)-epigallocatechin glucuronide (EGCglucuronide), (2)-epicatechin glucuronide (EC-glucuronide), (2)-epicatechin sulphate (EC sulphate) were 0·3 (range 0·1-1·9), 0·1 (range 0-0·4), 0·8 (range 0·2-3·9), 0·2 (range 0·1 1·7) and 1 (range 0·3-3·4) mmol/l, respectively. The areas under the plasma concentration v. time curves (AUC0!24) were 427 (range 102-1185) mmol/l £ min for EGC-glucuronide, 112 (range 53-919) mmol/l £ min for EC-sulphate, 71 (range 26-306) mmol/l £ min for EGCG, 40 (range 12-258) mmol/l £ min for EC-glucuronide and 14 (range 0·1-124) mmol/l £ min for ECG. The values of mean residence time (MRT0!24) were 5 (range 2-16), 2 (range 1-11), 10 (range 2-13), 3 (range 2-16) and 2·4 (range 1-18) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RAR¿ and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL¿6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RAR¿ and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RAR¿ and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.

Polyphenol intake and mortality risk: a re-analysis of the PREDIMED trial

Background: Polyphenols may lower the risk of cardiovascular disease (CVD) and other chronic diseases due to their antioxidant and anti-inflammatory properties, as well as their beneficial effects on blood pressure, lipids and insulin resistance. However, no previous epidemiological studies have evaluated the relationship between the intake of total polyphenols intake and polyphenol subclasses with overall mortality. Our aim was to evaluate whether polyphenol intake is associated with all-cause mortality in subjects at high cardiovascular risk. Methods: We used data from the PREDIMED study, a 7,447-participant, parallel-group, randomized, multicenter, controlled five-year feeding trial aimed at assessing the effects of the Mediterranean Diet in primary prevention of cardiovascular disease. Polyphenol intake was calculated by matching food consumption data from repeated food frequency questionnaires (FFQ) with the Phenol-Explorer database on the polyphenol content of each reported food. Hazard ratios (HR) and 95% confidence intervals (CI) between polyphenol intake and mortality were estimated using time-dependent Cox proportional hazard models. Results: Over an average of 4.8 years of follow-up, we observed 327 deaths. After multivariate adjustment, we found a 37% relative reduction in all-cause mortality comparing the highest versus the lowest quintiles of total polyphenol intake (hazard ratio (HR) = 0.63; 95% CI 0.41 to 0.97; P for trend = 0.12). Among the polyphenol subclasses, stilbenes and lignans were significantly associated with reduced all-cause mortality (HR =0.48; 95% CI 0.25 to 0.91; P for trend = 0.04 and HR = 0.60; 95% CI 0.37 to 0.97; P for trend = 0.03, respectively), with no significant associations apparent in the rest (flavonoids or phenolic acids). Conclusions: Among high-risk subjects, those who reported a high polyphenol intake, especially of stilbenes and lignans, showed a reduced risk of overall mortality compared to those with lower intakes. These results may be useful to determine optimal polyphenol intake or specific food sources of polyphenols that may reduce the risk of all-cause mortality.