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OBJECTIVES: Our goal was to test feasibility and efficacy of a dietary intervention based on daily intake of flavanol-containing cocoa for improving vascular function of medicated diabetic patients. BACKGROUND: Even in fully medicated diabetic patients, overall prognosis is unfavorable due to deteriorated cardiovascular function. Based on epidemiological data, diets rich in flavanols are associated with a reduced cardiovascular risk. METHODS: In a feasibility study with 10 diabetic patients, we assessed vascular function as flow-mediated dilation (FMD) of the brachial artery, plasma levels of flavanol metabolites, and tolerability after an acute, single-dose ingestion of cocoa, containing increasing concentrations of flavanols (75, 371, and 963 mg). In a subsequent efficacy study, changes in vascular function in 41 medicated diabetic patients were assessed after a 30-day, thrice-daily dietary intervention with either flavanol-rich cocoa (321 mg flavanols per dose) or a nutrient-matched control (25 mg flavanols per dose). Both studies were undertaken in a randomized, double-masked fashion. Primary and secondary outcome measures included changes in FMD and plasma flavanol metabolites, respectively. RESULTS: A single ingestion of flavanol-containing cocoa was dose-dependently associated with significant acute increases in circulating flavanols and FMD (at 2 h: from 3.7 +/- 0.2% to 5.5 +/- 0.4%, p < 0.001). A 30-day, thrice-daily consumption of flavanol-containing cocoa increased baseline FMD by 30% (p < 0.0001), while acute increases of FMD upon ingestion of flavanol-containing cocoa continued to be manifest throughout the study. Treatment was well tolerated without evidence of tachyphylaxia. Endothelium-independent responses, blood pressure, heart rate, and glycemic control were unaffected. CONCLUSIONS: Diets rich in flavanols reverse vascular dysfunction in diabetes, highlighting therapeutic potentials in cardiovascular disease.

Previous studies with plant sterols (PS) and cocoa flavanols (CF) provide support for their dietary use in maintaining cardiovascular health. This double-blind, placebo-controlled, cross-over study evaluated the efficacy of daily consumption of a cocoa flavanol-containing dark chocolate bar with added PS on serum lipids, blood pressure, and other circulating cardiovascular health markers in a population with elevated serum cholesterol. We recruited 49 adults (32 women, 17 men) with serum total cholesterol concentrations of 5.20-7.28 mmol/L and blood pressure of < or = 159/99 mm Hg. Following a 2-wk lead-in utilizing the AHA style diet, participants were randomized into 2 groups and instructed to consume 2 cocoa flavanol-containing dark chocolate bars per day with (1.1 g sterol esters per bar) or without PS. Each 419-kJ bar was nutrient-matched and contained approximately 180 mg CF. Participants consumed 1 bar 2 times per day for 4 wk then switched to the other bar for an additional 4 wk. Serum lipids and other cardiovascular markers were measured at baseline and after 4 and 8 wk. Blood pressure was measured every 2 wk. Regular consumption of the PS-containing chocolate bar resulted in reductions of 2.0 and 5.3% in serum total and LDL cholesterol (P < 0.05), respectively. Consumption of CF also reduced systolic blood pressure at 8 wk (-5.8 mm Hg; P < 0.05). Results indicate that regular consumption of chocolate bars containing PS and CF as part of a low-fat diet may support cardiovascular health by lowering cholesterol and improving blood pressure.

There has been growing interest in the potential cardiovascular benefits associated with cocoa consumption. As a result of accurate analytical methodologies, there is evidence to support that the flavanols in cocoa can be absorbed, are bioactive, and may be responsible for the cardiovascular benefits associated with regular cocoa consumption. The flavanols in cocoa exist in a multitude of different stereochemical configurations, thus giving rise to a unique and complex mixture of compounds. Given this complexity, the quantitative analysis of cocoa flavanols in foods can be challenging. While there are published methods suitable for the analysis of these compounds, these methods require sophisticated instrumentation and can be challenging to set up. As such, simpler techniques that measure such things as total phenolic content or antioxidant potential have been used as indicators of flavanol content. However, as these simpler assays are prone to interferences and are not specific for flavanols, these methods are not appropriate for use in studies that aim to examine the physiological effects of cocoa flavanols. It is only through the use of methods that can accurately quantify these flavanols that it will be possible to make meaningful dietary recommendations regarding the consumption of cocoa flavanol containing foods.

A single-dose ingestion of flavanol-rich cocoa acutely reverses endothelial dysfunction. To investigate the time course of endothelial function during daily consumption of high-flavanol cocoa, we determined flow-mediated dilation (FMD) acutely (for up to 6 hours after single-dose ingestion) and chronically (administration for 7 days). The study population represented individuals with smoking-related endothelial dysfunction; in addition to FMD, plasma nitrite and nitrate were measured. The daily consumption of a flavanol-rich cocoa drink (3 x 306 mg flavanols/d) over 7 days (n=6) resulted in continual FMD increases at baseline (after overnight fast and before flavanol ingestion) and in sustained FMD augmentation at 2 hours after ingestion. Fasted FMD responses increased from 3.7 +/- 0.4% on day 1 to 5.2 +/- 0.6%, 6.1 +/- 0.6%, and 6.6 +/- 0.5% (each P < 0.05) on days 3, 5, and 8, respectively. FMD returned to 3.3 +/- 0.3% after a washout week of cocoa-free diet (day 15). Increases observed in circulating nitrite, but not in circulating nitrate, paralleled the observed FMD augmentations. The acute, single-dose consumption of cocoa drinks with 28 to 918 mg of flavanols led to dose-dependent increases in FMD and nitrite, with a maximal FMD at 2 hours after consumption. The dose to achieve a half-maximal FMD response was 616 mg (n=6). Generally applied biomarkers for oxidative stress (plasma, MDA, TEAC) and antioxidant status (plasma ascorbate, urate) remained unaffected by cocoa flavanol ingestion. The daily consumption of flavanol-rich cocoa has the potential to reverse endothelial dysfunction in a sustained and dose-dependent manner.

There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1beta, IL-6, IL-10, and TNF-alpha in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.

Substantial data suggest that flavonoid-rich food could help prevent cardiovascular disease and cancer. Cocoa is the richest source of flavonoids, but current processing reduces the content substantially. The Kuna living in the San Blas drink a flavanol-rich cocoa as their main beverage, contributing more than 900 mg/day and thus probably have the most flavonoid-rich diet of any population. We used diagnosis on death certificates to compare cause-specific death rates from year 2000 to 2004 in mainland and the San Blas islands where only Kuna live. Our hypothesis was that if the high flavanoid intake and consequent nitric oxide system activation were important the result would be a reduction in the frequency of ischemic heart disease, stroke, diabetes mellitus, and cancer--all nitric oxide sensitive processes. There were 77,375 deaths in mainland Panama and 558 deaths in the San Blas. In mainland Panama, as anticipated, cardiovascular disease was the leading cause of death (83.4 +/- 0.70 age adjusted deaths/100,000) and cancer was second (68.4 +/- 1.6). In contrast, the rate of CVD and cancer among island-dwelling Kuna was much lower (9.2 +/- 3.1) and (4.4 +/- 4.4) respectively. Similarly deaths due to diabetes mellitus were much more common in the mainland (24.1 +/- 0.74) than in the San Blas (6.6 +/- 1.94). This comparatively lower risk among Kuna in the San Blas from the most common causes of morbidity and mortality in much of the world, possibly reflects a very high flavanol intake and sustained nitric oxide synthesis activation. However, there are many risk factors and an observational study cannot provide definitive evidence.

Diet patterns are widely recognized as contributors to hypertension. Widely studied potential contributors include intake of sodium, potassium, magnesium, calcium, soluble fiber, ω-3 fatty acids, alcohol, protein, and calories. We add to that list the effect of dietary flavanols present in certain cocoas, which have sufficient activity on vascular nitric oxide to influence blood pressure control. Kuna Indians who live on islands near Panama have little age-related rise in blood pressure or hypertension. On migration to Panama City, blood pressure rises with age, and the frequency of essential hypertension matches urban levels elsewhere. We have identified a specific food that probably makes an important contribution to cardiovascular status. Island-dwelling Kuna drink more than 5 cups of flavanol-rich cocoa per day and incorporate that cocoa into many recipes. Mainland Kuna ingest little cocoa, and what they take is commercially available and flavanol-poor. The flavanol-rich cocoa activates nitric oxide synthase in vitro and in intact humans in the doses that the Kuna employ. Vasodilator responses to flavonoid-rich cocoa are prevented or reversed by the arginine analog, N-nitro-L-arginine methyl ester. Island-dwelling Kuna have a 3-fold larger urinary nitrate:nitrite than do Mainland dwellers. As endothelial dysfunction is central to current thinking on cardiovascular pathophysiology, a food that enhances endothelial function could have broad implications. The list of candidate conditions that might be influenced is impressive, ranging from atherosclerosis and diabetes mellitus to hypertension and preeclampsia, to vascular dementias and end-stage renal disease. The next decade will be interesting.

Flavanols are the main flavonoids found in cocoa and chocolate, and can be especially abundant in certain cocoas. Research over the past decade has identified flavanols as showing diverse beneficial physiologic and antioxidant effects, particularly in context of vascular function. The present study employed functional magnetic resonance imaging based on blood oxygenation level-dependent (BOLD) contrast to explore the effect of flavanols on the human brain. Magnetic resonance imaging was used to measure BOLD responses to a cognitive task in 16 healthy young subjects. The data presented show an increase in the BOLD signal intensity in response to a cognitive task following ingestion of flavanol-rich cocoa (5 days of 150 mg of cocoa flavanols). This may arise either as a result of altered neuronal activity, or a change in vascular responsiveness, or both--the net effect then being dependent on which of the two effects is dominant. No significant effects were evident in behavioral reaction times, switch cost, and heart rate after consumption of this moderate dose of cocoa flavanols. A pilot study evaluated the relationship between cerebral blood flow and a single acute dose (450 mg flavanols) of flavanol-rich cocoa and showed that flavanol-rich cocoa can increase the cerebral blood flow to gray matter, suggesting the potential of cocoa flavanols for treatment of vascular impairment, including dementia and strokes, and thus for maintaining cardiovascular health.

Flavanols and their related oligomeric compounds, the procyanidins, have received increased attention during the past decade due to their reported health benefits. On the basis of compelling data published during the past decade demonstrating that the consumption of certain flavanol-rich foods can improve markers of cardiovascular health, additional clinical, and epidemiological research is clearly warranted to establish appropriate public health recommendations. However, recommendations on the consumption of these foods appropriate for use by health professionals can only be made on the basis of clinical investigations that accurately identify and quantify--through proper analytical measurement systems--the flavanols in the foods used in these investigations. This manuscript provides an overview of the strengths, weaknesses, and limitations of commonly used analytical methods to characterize the content of flavanols in foods. Two nonspecific measurements widely used by investigators, the Folin-Ciocalteu assay and the Oxygen Radical Absorbance Capacity (ORAC) measurement, are discussed in this context, as is the use of various high-performance liquid chromatography methods that provide more specific data related to the content of flavanols in foods. A comparison of the data obtained from these analytical methods to those of the more rigorous high-performance liquid chromatography analyses demonstrates that these nonspecific methods are ill-suited for providing unequivocal data necessary to evaluate the importance of dietary flavanols in the context of improving cardiovascular health. Meaningful dietary recommendations for the consumption of flavanol-rich foods will only be made possible by additional well-designed clinical and epidemiological studies enabled by detailed compositional data obtained through use of appropriate analytical methods.

Cocoa flavanols and procyanidins have numerous biological activities. It is known that (-)-epicatechin, (+)-catechin, epicatechin-(4beta-8)-epicatechin (dimer B2), and epicatechin-(4beta-6)-epicatechin (dimer B5) are unstable at physiologic pH, degrading almost completely within several hours, whereas they are relatively stable at pH 5.0. The present study investigated the effects of ascorbic and citric acid on the stability of monomers and dimers in simulated intestinal juice (pH 8.5) and in sodium phosphate buffer (pH 7.4). The addition of ascorbic acid to the incubation mixture significantly increased the stability of the monomers and dimers, whereas the addition of citric acid provided no protective effects. LC-MS showed that with the degradation of dimer B2 and dimer B5, doubly linked A-type dimers were formed. The present results, although not directly transferable to in vivo conditions, suggest that ascorbic acid may stabilize cocoa flavanols and procyanidins in the intestine where the pH is neutral, or alkaline, before absorption.

BACKGROUND: Polyphenolic procyanidins are abundant flavonoid polymers in Western diets. In vitro biological activity has been reported for these compounds, but activity in vivo depends on the amount and chemical nature of the flavonoids reaching the gastrointestinal tract. Degradation of procyanidins under simulated gastric conditions at pH 2.0 has been reported in vitro. OBJECTIVE: The objective was to examine whether depolymerization of procyanidins occurs in the stomach of human subjects in vivo. DESIGN: After an overnight fast, 6 healthy subjects (3 men and 3 women) consumed 500 mL of a cocoa beverage containing 733 mg procyanidin polymers and 351 mg structurally related flavanol monomers. With the use of a nasogastric tube, stomach contents were collected every 10 min after beverage ingestion until the stomach was emptied. Flavanols and procyanidins (up to pentamers) were quantified by normal and reversed-phase HPLC. RESULTS: In all subjects, gastric transit lasted approximately 50-60 min. No change in the HPLC profile of procyanidins was observed during this period, showing that procyanidins were remarkably stable in the stomach environment. CONCLUSION: The results suggest that most ingested procyanidins reach the small intestine intact and are available for absorption or metabolism.

Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-alpha (TNF-alpha) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-alpha released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3-4 fold increase in TNF-alpha relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-alpha secretion in the range of 48-128% relative to the PHA control. The monomers and dimers were slightly inhibitory (-1.5 and -15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-alpha levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.

Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-alpha (TNF-alpha) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-alpha released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3-4 fold increase in TNF-alpha relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-alpha secretion in the range of 48-128% relative to the PHA control. The monomers and dimers were slightly inhibitory (-1.5 and -15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-alpha levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.

Cocoa and chocolate foods produced by appropriate methods can contribute significant amounts of heart-healthy flavanols to the diet. These flavanols may enhance cardiovascular health by delaying blood clotting, improving vascular endothelial function, and helping to moderate inflammation. The benefits of chocolate can be enjoyed without guilt as part of a healthful balanced diet.

Procyanidins are a subclass of flavonoids found in commonly consumed foods that have attracted increasing attention due to their potential health benefits. However, little is known regarding their dietary intake levels because detailed quantitative information on the procyanidin profiles present in many food products is lacking. Therefore, the procyanidin content of red wine, chocolate, cranberry juice and four varieties of apples has been determined. On average, chocolate and apples contained the largest procyanidin content per serving (164.7 and 147.1 mg, respectively) compared with red wine and cranberry juice (22.0 and 31.9 mg, respectively). However, the procyanidin content varied greatly between apple samples (12.3-252.4 mg/serving) with the highest amounts on average observed for the Red Delicious (207.7 mg/serving) and Granny Smith (183.3 mg/serving) varieties and the lowest amounts in the Golden Delicious (92.5 mg/serving) and McIntosh (105.0 mg/serving) varieties. The compositional data reported herein are important for the initial understanding of which foods contribute most to the dietary intake of procyanidins and may be used to compile a database necessary to infer epidemiological relationships to health and disease.

Recent data has demonstrated that cacao liquor polyphenols (procyanidins) have antioxidant activity, inhibit mRNA expression of interleukin-2 and are potent inhibitors of acute inflammation. Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the pro-inflammatory cytokine, interleukin-1 beta. Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the levels of transcription and protein secretion. Individual cocoa fractions were shown to augment constitutive IL-1 beta gene expression, although values varied between subjects. Interestingly, the smaller fractions of cocoa (monomer-tetramer) consistently reduced IL-1 beta expression of PHA-stimulated cells by 1-15%, while the larger oligomers (pentamer-decamer) increased expression by 4-52%. These data, observed at the transcription level, were reflected in protein levels in PHA-induced PBMC. The presence or absence of PHA did not alter the effects of the cocoa procyanidins with the exception of the pentamer. This study offers additional data for the consideration of the health-benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

Recent epidemiological research indicates that diets rich in flavonoid-containing foods may be associated with a reduced risk for cardiovascular disease. This protective effect is attributed, in part, to the ability of flavonoids to act as antioxidants. Certain chocolates and cocoas contain substantial amounts of procyanidins, and thus belong in the category of flavonoid-rich foods. Recent advancements in the identification and isolation of procyanidins, especially oligomeric procyanidins, from chocolate and cocoa have facilitated the investigation of individual procyanidin fractions with regard to their potential cardiovascular health benefits. In the following paper, we report on the antioxidant capacity of a cocoa as determined by the Oxygen Radical Absorbance Capacity (ORAC) assay, and the ability of individual procyanidin fractions from this same cocoa to inhibit low-density lipoprotein (LDL) oxidation in vitro. In addition, mechanisms are discussed by which flavonoids in chocolate and cocoa may enhance cardiovascular health.

Given the widespread ingetion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the antiinflammatory cytokine, interleukin (IL-4).  Both resting and phytohemagluttinin (PHA)- stimulated peripheral mononuclear blood cells (PMBC) were investigated at the protein secretion level.  The smaller-sized cocoa fractions (tetramer or less) were unable to induce an IL-4 response (i.e. values fell below the detection limit of 0.25 pg/ml).  The larger oligomeric procyanidines (penatmer or greater) stimulated secretion of IL-4 in resting PBMC by as much as 1.42 pg/ml, as shown by the nonamer.  However only the hexameric, heptameric and decameric fractions proved to be statistically significant.  Cells co-incubated with PBA showed an immense increase in IL-4 (21.1 ± 1.1 pg/ml). Only the monomeric fraction was able to induce the PHA-induced secretion of 48%.  The other procyanidin oligomers supressed IL-4 production; in particular the hexameric, heptameric and octameric fractions significantly inhibited mitogen-stimulated secretion of IL-4 by 55%, 61% and 71%, respectively.  This study offers additional data for the consideration of the health benefits of polyphenols from a wide variety of foods, including those benefits associated specifically  with cooca and chocolate consumption. 

Sir Ilya Arts and colleagues (Aug 7, p 488) report that chocolate and tea may contribute significantly to total dietary catechin intake (20% and 55%, respectively).

However, their methods only took into account the monomeric catechins and neglected the more abundant oligomers found in chocolate,2 which are present in only minor concentrations in tea.
Indeed, Arts and colleagues' method to determine catechin content illustrates a commonly encountered difficulty when trying to assess total dietary intake of flavonoids. The commonly used reverse-phase high-performance liquid chromatography techniques are superior for the separation of simple flavonoids, such as those found in tea. However, they are insufficient for analysis of larger oligomeric procyanidins, such as those found in cocoa and chocolate, and normal-phase chromatography is better suited.3 Adamson and colleagues have shown that the monomers (−)-epicatechin and (+)-catechin, are only a fraction of the total quantifiable procyanidins in cocoa and chocolate.4 Hence, the total concentrations of procyanidins in chocolate may have been substantially underestimated by Arts and colleagues.