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Open AccessReview

The Cardiovascular Effects of Cocoa Polyphenols—An Overview

1
Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa Iasi, Universitatii Str. 16, 700115 Iasi, Romania
2
Department of Cardiology, Faculty of Medicine, University of Medicine and Pharmacy Grigore T. Popa Iasi, Universitatii Str. 16, 700115 Iasi, Romania
*
Author to whom correspondence should be addressed.
Academic Editor: Maurizio Battino
Diseases 2016, 4(4), 39; https://doi.org/10.3390/diseases4040039
Received: 12 October 2016 / Revised: 12 December 2016 / Accepted: 13 December 2016 / Published: 17 December 2016

Abstract

Cocoa is a rich source of high-quality antioxidant polyphenols. They comprise mainly catechins (29%–38% of total polyphenols), anthocyanins (4% of total polyphenols) and proanthocyanidins (58%–65% of total polyphenols). A growing body of experimental and epidemiological evidence highlights that the intake of cocoa polyphenols may reduce the risk of cardiovascular events. Beyond antioxidant properties, cocoa polyphenols exert blood pressure lowering activity, antiplatelet, anti-inflammatory, metabolic and anti-atherosclerotic effects, and also improve endothelial function. This paper reviews the role of cocoa polyphenols in cardiovascular protection, with a special focus on mechanisms of action, clinical relevance and correlation between antioxidant activity and cardiovascular health.
Keywords: cocoa; flavanols; proanthocyanidins; cardiovascular health cocoa; flavanols; proanthocyanidins; cardiovascular health

1. Introduction

Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity worldwide. Every year, about 17 million people die from CVDs, which represent almost one third of all deaths in the world [1]. By 2030, it is estimated that about 23.3 million people will die due to CVDs [2]. The major heart health issues are: high blood pressure (hypertension), heart ischemic and cerebrovascular diseases. Hypertension is the main risk factor for CVDs. It is a silent killer that affects one third of the worldwide adult population and contributes to nearly 45% of heart ischemic diseases and 51% of stroke cases around the globe [3]. It is considered that every 20-mmHg drop in systolic blood pressure (SBP) would reduce the relative risk of ischemic heart disease mortality by 50% [2]. Other significant behavioural risk factors of CVDs include: unhealthy diet, physical inactivity, smoking, alcohol, exposure to continuous stress, diabetes, obesity, hyperlipidemia. Change of lifestyle and diet is considered to be one of the main tools for the prevention of CVDs, particularly ischemic heart diseases, atherosclerosis, and arterial thrombosis. Epidemiological studies have shown that a diet rich in fruits and vegetables with a high intake of polyphenols have beneficial effects on human health; it can protect and limit the incidence of CVDs, cancer or age-related degenerative diseases. Cocoa is one of the most polyphenol-rich foods that is widely consumed in the world. In recent years it has received much attention due to its antioxidant, cardioprotective, neuroprotective, and chemopreventive properties [4].
In this review, we aim to describe the current evidence on the cardioprotective effects of cocoa polyphenols and cocoa derived products with a particular focus on the main mechanisms of action, clinical relevance, and the link between antioxidant activity and cardiovascular health. The limitations of these studies and future perspectives on cocoa polyphenols research are also presented.

2. Polyphenols Composition of Cocoa

Raw dried cocoa beans (Theobroma cacao, Malvaceae, cacao tree) contain high levels of polyphenols (12%–18% of whole beans dry weight). In addition, other important bioactive phytochemicals of cocoa beans are methylxanthines (4%) with theobromine as the main compound [4,5]. The main categories of cocoa polyphenols are:
(i)
Flavan-3-ols or catechins (29%–38% of total polyphenols) as monomers and polymers. The monomers include (−)-epicatechin, (+)-catechin, (+)-gallocatechin, and (−)-epigallocatechin (Figure 1). (−)-Epicatechin constitutes up to 35% of total polyphenols being the most abundant polyphenol both in cocoa and cocoa derived products (chocolate) [4,6]. Flavanols are commonly found in cocoa as R-forms [7].
(ii)
Proanthocyanidins (58%–65% of total polyphenols) are polymers of epicatechin and catechin molecules linked by 4→8 and 4→6 bonds. In cocoa, mainly oligomeric proanthocyanidins (degree of polymerization = 10) are found, and dimers to hexamers predominate. The most important cocoa proathocyanidins are dimers: B1 [epicatechin-(4β→8)-catechin], B2 [epicatechin-(4β→8)-epicatechin] (Figure 2), B3 [catechin-(4α→8)-catechin], B4 [catechin-(4α→8)-epicatechin], B5 [epicatechin-(4β→6)-epicatechin], and trimers: C1 [epicatechin-(4β→8)2-epicatehin] (Figure 3) [4].
(iii)
Anthocyanins (4% of total polyphenols), such as: cyanidin-3α-l-arabinoside, cyanidin-3 β-d-galactoside [8], and leucoanthocyanins (L1, L2, L3, L4) [4].
Other polyphenols from cocoa are: flavones (apigenin, quercetin, luteolin, kaempferol and their glycosides), polyphenolic acids (caffeic acid, chlorogenic acid, ferulic acid, coumaric acid, and syringic acid), caffeoyl-conjugates (clovamide, deoxyclovamide), stilbens (trans-resveratrol and its glycoside, trans-piceid) [4,5]. The polyphenols profile in cocoa varies depending of several factors, such as: plant genotype, geographic area, ripeness degree of beans, cocoa processing [4]. Alongside with ground cocoa, green tea and wine, dark chocolate contains high amounts of polyphenols (460–610 mg/kg) [5,9].

3. Bioavailability of Cocoa Polyphenols

Cocoa polyphenols have a relatively low bioavailability. They achieve low plasma Cmax, have a short half-life and present a rapid excretion [10]. The bioavailability is strongly influenced by molecular size; thus, compounds with low molecular weight like flavanol monomers can achieve higher concentrations in the blood and can reach the target organs in the body [10,11]. Monomers and dimeric or trimeric proanthocyanidins are quite stable in the gastric environment and they can travel to the small intestine where they could be absorbed (Figure 4) [6]. Monomers are able to cross the gut barrier and are absorbed, being largely distributed in the lymphoid organs (thymus, spleen, mesenteric lymphoid nodes), liver, or testes [12]. At two to three hours after ingestion of cocoa products, flavanols achieve the peak concentrations in the plasma and these are typically in the nanomolar or low micromolar range, depending on the administered dosage [8,12]. (−)-Epicatechin shows a higher bioavailability than other cocoa flavanols (catechins, galloylated derivatives) [13]. After the intake of a cocoa beverage that contains 323 mg of flavanols, the plasma epicatechin concentration was >10% of the peak plasma catechin level (5.92 μM compared to 0.16 μM) [14]. The different stereochemistry of epicatechin could allow a greater intestinal permeation of this flavanol [2]. Dimeric and trimeric proanthocyanidins are poorly absorbed through gastrointestinal tract (about 10–100 times less compared to epicatechin and catechin) [2]. In the acidic conditions of stomach some dimers can be degraded to epicatechin and undergo interflavan bond conversion. After the intake of cocoa with large amounts of flavanol monomers and dimers, the occurrence of B2 and B5 dimers in human plasma has been reported but in modest concentrations (41 nM and 55 nM, respectively) [14]. Oligomeric proanthocyanidins with degree of polymerization higher than three and polymeric flavanols are not absorbed in native forms [15]. In the colon, they are metabolized by the local microbiota within 48 h into various low-molecular weight phenolic compounds, such as hydroxyphenylvalerolactones, hydroxyphenylpropionic acids, hydroxyphenylacetic acids, hydroxycinnamic acids, and hydroxybenzoic acids [14,15]. These metabolites are more available, can be absorbed and reach the liver, and some of them show significant biological properties [12,15]. The mammalian metabolism of simple cocoa flavanols occurs in the liver and small intestine through glucuronidation, sulfation, and O-methylation [16], and the circulating flavanols are predominantly the conjugated forms [2]. The major metabolite of epicatechin in the plasma is 4’-O-methyl-epicatechin-7β-d-glucuronide [17]. Also, flavonoid metabolism can occur in the vascular endothelial cells. In human umbilical vein endothelial cells (HUVEC), (−)-epicatechin is converted via catechol-O-methyltransferase (COMT) to 3’-O-methyl-epicatechin and 4’-O-methyl-epicatechin, metabolites with physiological relevance. Although the endothelial concentrations of epicatechin are not known so far, it is appreciated that they are higher than the plasma concentrations [17]. Flavanol monomers have short-half lives in the body and are rapidly excreted in the urine or bile as conjugates [14]. After ingestion of 40 g chocolate bars, there has been reported a half-live of 1.9 h for (−)-epicatechin [2]. Some of flavanol metabolites such as glucuronides are no longer considered only excretion products. They appear to play an important role as transport metabolites in plasma to target cells [17]. The chirality of flavanol molecules and also cocoa-containing matrices may affect the bioavailability of polyphenols. (+)-Catechin enantiomer from cocoa beans is more bioavailable than (−)-catechin form, which is predominantly present in the commercial chocolates. Simultaneous use of carbohydrates increases significantly the absorption of polyphenols [8], while the lipids in cocoa matrix seem to stimulate the digestion of flavanols in the duodenum [2]. Conflicting data on the influence of milk on cocoa polyphenols bioavailability have been reported [10]. It has been suggested that milk proteins bind cocoa polyphenols, which causes a decrease of polyphenols absorption in the gastrointestinal tract, but this hypothesis has not been confirmed for (−)-epicatechin when cocoa was consumed with milk [10].

4. Cardioprotective Effects of Cocoa Polyphenols

Numerous epidemiological studies, short-term human intervention studies, or small-cohort studies, showed that the intake of cocoa and polyphenols-rich cocoa products is correlated with cardiovascular benefits in humans [16]. Cocoa flavanols and proanthocyanidins are pleiotropic molecules that can influence various biomedical markers and clinical endpoints of cardiovascular health. They improve cardiovascular function but also facilitate endogenous repair mechanisms [18]. Experimental research and clinical trials have investigated mainly the effects of cocoa products and cocoa polyphenols on oxidative stress and plasma antioxidant capacity, endothelium-dependent vasomotor function and arterial flow mediated dilatation (FMD), nitric oxide (NO) metabolism and activity, blood pressure, platelet function, lipid profile, and vascular inflammation [5,10]. An overview of the most recent clinical trials (2014–2016) with cocoa flavanols is shown in Table 1. The potential cardioprotective properties of cocoa include mainly antihypertensive, anti-atherogenic, and anti-inflammatory activities as well as inhibition of the platelet activation and aggregation, and attenuation of endothelial dysfunction (Figure 5).

4.1. Antioxidant Activity

Oxidative stress is actively involved in CVDs pathogenesis. The increase of reactive oxygen species (ROS) production, mainly of superoxide anion radical, affects NO metabolism and facilitates endothelial dysfunction [45]. Besides, lipid peroxidation contributes essentially to the initiation and progression of atherosclerosis. Cocoa is a rich source of antioxidants. The assessment of the antioxidant capacity of 1113 different foods showed that of 50 products exhibiting the highest activity, five products are cocoa-based [12]. Cocoa powder has a higher antioxidant activity than green tea (1000 ORAC units compared to 800 ORAC units), and the content of total polyphenols is positively correlated with ORAC value [14]. Cocoa polyphenols mainly flavanols and oligomeric proanthocyanidins have shown significant in vitro antioxidant properties. They act as scavengers of various radicals (DPPH, ABTS, superoxide anion, peroxynitrite, hypochloride), inhibit lipid peroxidation and chelate pro-oxidants metal ions (Fe2+) [4,46]. The main structural features of polyphenols that are responsible for their ROS quencher activity are catechol groups, phenolic quinoid tautomerism and delocalization of electrons [14]. Epicatechin enhances plasma antioxidant capacity and protects erythrocyte membrane from lipid peroxidation. Also, flavanol monomers and oligomeric proanthocyanidins protect against in vitro erythrocyte hemolysis induced by 2,2’-azobis (amidopropane)-dihydrochloride (AAHP) [14]. In animal experimental models, long-term feeding studies with flavanol-rich cocoa products showed an increase in total plasma antioxidant capacity [14]. In rat brain homogenates, cocoa polyphenolic extracts inhibit lipid peroxidation acting as chain-breaking antioxidants [11]. The suppressive effect of cocoa flavanols on lipid peroxidation has been suggested by some studies, which showed a decrease of the plasma level of F2 isoprostanes. However, many in vivo investigations did not identify similar results [17]. Also, some studies in healthy human subjects did not reveal changes of oxidative stress biomarkers consecutive to cocoa consumption. It is possible that the antioxidant effects of cocoa products could be better expressed in pathological conditions [4]. At the same time, it is difficult to translate in vivo antioxidant activity of cocoa polyphenols. The poor bioavailability and low plasma concentrations of cocoa polyphenols do not support a direct antioxidant activity. It is more likely that at low physiological levels reached by flavanols, the latter act indirectly as antioxidants by a specific interaction with lipids and enzymes associated with oxidant metabolism and cardiovascular diseases (NADPH oxidases, lipoxygenases, myeloperoxidase) [6,17,47]. Besides, cocoa polyphenols upregulate antioxidant defense response, such as the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, a master regulator of cellular resistance to oxidants [46]. Therefore, they can attenuate the rise of intracellular oxidants via NF-κB activation [48].

4.2. Modulation of Endothelium-Dependent Vasomotor Function

The endothelium participates critically to maintain vascular homeostasis. It plays a pivotal role in the regulation of vascular tone, vascular permeability, platelet activity and aggregation. Its effects are mediated by a complex network of molecules, such as NO, endothelin-1 (ET-1), prostacyclin, cell adhesion molecules [49], leukotrienes, prostaglandins, catecholamines, vasoactive peptides (angiotensin-II) [48]. The impairment of functional properties of endothelium is involved in the initiation and progression of atherosclerosis and other CVDs [9,47]. The undesirable phenotypic changes of endothelium, such as vasoconstriction, proliferation of arterial wall, inflammation, and thrombosis, occur with endothelial dysfunction [49]. The quantification of endothelial function in humans is accomplished by the use of brachial artery flow-mediated dilation technique (FMD). FMD value largely reflects NO-mediated arterial function (including that of coronary arteries) and low levels are associated with cardiovascular events and elevated risk factors for CVDs [47,49]. Most studies have shown that the acute or chronic (≥1 week) intake of cocoa/chocolate increases FMD value in healthy young and elderly subjects, smokers, obese, patients with coronary artery diseases and arterial hypertension, or end-stage renal diseases with chronic hemodialysis, and diabetics [21,49]. Acute ingestion of the medium and high dose of flavanols (321 mg of flavanols/dose, three doses/day) in elderly type 2 diabetic patients improves FMD value by over 40%, but the vascular response to oral treatment with nitroglycerin was not affected by the dietary intervention [50]. The intake for seven days of cocoa containing 74 mg of flavanols and 232 mg of procyanidins (three doses/day) improved significantly FMD in smokers, but after the cessation of cocoa consumption and a seven-day wash-out period, FMD level returned to preintervention values [16].
In addition, metabolites of epicatechin may have a relevant physiological activity. It was observed that the increase of FMD level correlates with plasma levels of epicatechin metabolites after cocoa ingestion [49]. Thus, FMD response follows a similar temporal pattern with the appearance of (−)-epicatechin metabolites in plasma, peak effects being observed two-hours after cocoa ingestion [17,49]. This short-time effect can be explained by the reduction of superoxide anion-mediated loss of NO and oxidative stress via inhibition of NADPH oxidase activity [17]. The O-methylated metabolites of (−)-epicatechin (3’-O-methyl epicatechin, 4’-O-methyl epicatechin) that occur in plasma in free or glucuronidated form significantly inhibit endothelial NADPH oxidase activity [13]. The increase of circulating bioactive NO pool (RXNO) plays an important role in the attenuation of vascular NO deficit in patients with risk cardiovascular factors and the restoration of endothelium-dependent vasodilation. In smokers, the consumption of a flavanol-rich cocoa beverage (176–185 mg/mL flavanols with 20–22 mg/mL epicatechin and 106–111 mg/mL proanthocyanidins) at 12 h after cessation of smoking and during abstinence dose-dependently increases FMD value by almost 50% and RXNO level by more than a third [51]. An improvement of endothelial function in healthy subjects was also observed after combined intake of low amounts of cocoa flavanols and nitrate-rich foods [32].
Repeated administration of high-flavanol cocoa produces a longer-term effect that is characterized by an increase in the baseline level of FMD. This effect can be mediated by changes in gene expression and protein synthesis (endothelial nitric oxide synthase, eNOS) [17]. Besides the improvement of NO bioavailability and bioactivity, the positive endothelial effects of cocoa can be correlated with other mechanisms, such as modulation of PGI2 and leukotrienes, reduction of xanthine oxidase and myeloperoxidase activities, suppression of the proinflammatory cytokines IL-1β, IL-2 and IL-8 production, inhibition of ET-1 release [52], decrease of the biomarkers associated with vascular damage like monocyte CD62L expression and the formation of elevated endothelial microparticles [30], and mobilization of functionally unaltered circulating angiogenic cells (EPCs) [18]. Also known as endothelial progenitor cells, EPCs originate from bone marrow cells and are capable to differentiate into mature endothelial cells with unaltered functional properties [53]. EPCs participate and significantly contribute to the endothelial reparative processes, neoangiogenesis tissue regeneration, and platelet function regulation via direct effects and indirect activities by production of paracrine/juxtacrine signals (proangiogenic cytokines, angiogenic growth factors) [53,54,55]. EPCs are currently considered potential biomarkers of cardiovascular risk. The number and functionality of EPCs are adversely affected in some conditions such as systemic hypertension, heart failure, coronary artery disease, stroke, atherosclerosis, diabetes mellitus, chronic kidney disease or chronic venous insufficiency, or in chronic inflammatory diseases (rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Kawasaki’s diseases). An increase in EPCs mobilization has been associated with acute ischemic events (acute myocardial infarction, unstable angina) [53,54,56].
Although the improvement of FMD induced by cocoa is supported by the preponderance of data, it is less clear what the minimal doses of cocoa/chocolate needed to be ingested to exert positive vascular effects are. Monahan [49] specifies that the minimal amount of cocoa that causes an increase in FMD value in healthy elderly individuals appears to be greater than 2 g and at most 5 g. Therefore, it seems that a pronounced and consistent increase in endothelial function occurs for large doses of about 900 mg of flavanols/day whereas low doses of 80 mg of flavanols/day do not produce a significant effect [34].
European Food Safety Authority (EFSA) [57] recommends 200 mg of cocoa polyphenols daily (provided by 2.5 g of polyphenol-rich cocoa powder or 10 g of polyphenol-rich dark chocolate) in order to obtain endothelium-dependent vasodilation in general population in the context of a balanced diet.

4.3. Effects on Blood Pressure

The research of cocoa effects on blood pressure (BP) was initiated by observations on Kuna Indians from the coast of the Panama islands. This population has very low levels of BP and incidence of hypertension and CVDs but the migration in the Panama urban continental areas leads to negative impact on the cardiovascular health. Subsequent investigations showed that the differences between the two populations are due mainly to the dietary habits. The high intake of flavanol-rich home-prepared cocoa in the island-dwelling Kuna in comparison to the mainland-dwelling Kuna (up to 10 times higher) appear to confer relevant beneficial effects [2,6,50]. Different epidemiological studies (Zutphen Elderly Study, Stockholm Heart Epidemiology Program) have reported a significant inverse relationship between the intake of cocoa/dark chocolate and cardiac mortality [58]. Many experimental studies and randomized intervention trials have shown BP-lowering effects of cocoa polyphenols and cocoa products. Single oral administration of flavonoid-enriched cocoa powder (128.9 mg/g of total procyanidins, 19.36 mg/g of epicatechin) at different doses (50, 100, 300, 600 mg/kg) clearly decreases BP in spontaneously hypertensive rats. A dose of 300 mg/kg cocoa powder produces the maximum effect, which is similar to that induced by captopril (50 mg/kg), a well-known antihypertensive drug [59]. Also, the presence of epicatehin in the diet of Sprague-Dawley rats (0.4 g epicatechin/100 g diet) for eight days prevents the increase of BP induced by NO deficiency associated to the NO-synthase inhibitor, nitro-l-arginine methyl ester (l-NAME) pretreatment [6]. It seems that epicatechin exerts the antihypertensive effects only in a pathological state. Besides, the epicatechin treatment limits infarct size in animal model of myocardial ischemia-reperfusion injury and after permanent coronary occlusion [60]. In humans, the BP-lowering properties of cocoa and cocoa polyphenols were assessed in studies with diverse design: various groups of subjects (young, elderly, overweight, obese, hypercholesterolemic, pre-hypertensive, hypertensive adults), different cocoa products and flavanols doses, and various duration of administration. A meta-analysis of 20 randomized controlled trials, which evaluated the cocoa polyphenols effects on BP, reported a small and significant reduction in both systolic blood pressure (SBP; −2.77 mmHg) and diastolic blood pressure (DBP; −2.20 mmHg) [2,61]. Other meta-analyses have identified large drops in both SBP and DBP: Desch 2010 (−4.52 mmHg SBP; −2.50 mmHg DSP) [62], Taubert 2007(−7.6 mmHg SBP; −4.8 mmHg DSB) [63] or Grassi 2005 (−11.30 mmHg SBP; −7.60 mmHg DSB) [2,64]. The study design but mostly the intake of various doses of cocoa flavanols (30–1080 mg) could explain this large variation of BP values.
Daily consumption of cocoa beverages containing high-flavanol (990 mg), intermediate-flavanol (520 mg) and low-flavanol (45 mg) in the elderly subjects, for 8 weeks, produces a significant reduction in SBP and DSP only for high and intermediate doses of flavanols [2,65]. The intake of cocoa containing 1052 mg of flavanols decreased significantly BP (−5.3 mmHg SBP; −3 mmHg DSP) in patients with untreated moderate hypertension [58].
The meta-analysis by Ried [61] revealed that the BP-lowering effects of cocoa were more pronounced in younger subjects, hypertensive patients, and in the studies with flavanol-free controls. Also, the change of BP was more significant in studies lasting more than two weeks. It seems that the high-flavanol cocoa produces a sustained reduction in patients with hypertension when treatment lasts for at least seven days, which suggests a time-dependent effect [60]. On the other hand, Hooper et al. [66] mentioned that the BP lowering effects of cocoa and chocolate appeared greater in studies with higher doses and shorter duration. These discrepancies could be due to differences in the backgrounds of cohorts and cocoa type products or absence of suitable placebos [52]. The amount of sugar associated with cocoa products may influence the BP lowering effects. Cocoa products with more than 10 g of sugar cause smaller reduction in SBP and DBP (−1.32 mmHg) in comparison with products containing less than 10 g of sugar (−2.52 mmHg SBP; −2.35 mmHg DBP) [2]. Vlachopoulos et al. [67] have found that high habitual cocoa consumption (≥4.63 g/day) decreases aortic stiffness and wave reflections that have a causative role in the pathogenesis of systolic hypertension.
The positive effects of cocoa on BP are supported mainly by the increase of endothelial bioavailability and bioactivity of NO and the inhibition of endothelial vasoconstriction. Up-regulation of NO is achieved by several mechanisms, such as: (i) the augmentation of eNOS activity, enzyme, which facilitates the production of NO from L-arginine. Cocoa flavanols such as epicatechin acutely enhances NO concentrations in rabbit aortic rings and in cultured human coronary arteries [2]; (ii) down-regulation of NADPH-oxidase and the reduction of superoxide anion and cellular oxidants levels. Superoxide anion is primarily generated from the electron transport chain but also through NADPH oxidase, xanthine oxidase and cytochrome P450 activities. It modulates NO availability in the smooth muscle cells. Superoxide anion directly reacts with NO producing peroxynitrite that enhances local oxidative stress. Cocoa flavanols prevent NO loss via superoxide anion reaction. In addition, they protect against the oxidative loss of tetrahydrobiopterin and prevent dysfunctional production of superoxide anion via eNOS uncoupling [6]; (iii) inhibition of arginase activity and the preservation of the intracellular arginine pool, which is the substrate for NO synthesis [6]. Epicatechin inhibits arginase-2 mRNA expression and activity in human umbilical vein endothelial cells [8], and flavanols-rich cocoa decreases the arginase activity in rat kidney and human erythrocytes [6]. Other anti-hypertensive mechanisms of cocoa polyphenols include: (i) inhibition of endothelin-1 (ET-1), an endothelium-derived peptide with vasoconstrictive effect. At low micromolar concentrations, cocoa flavanols inhibit transcription of ET-1 gene in endothelial cells andprevent ET-1 ROS production [2]; (ii) modulation of renin-angiotensin-aldosteron system. Cocoa flavanols and proanthocyanidins inhibit the renal angiotensin-converting enzyme (ACE) activity, which is involved in the regulation of BP by transforming angiotensin I in angiotensin II, a potent vasoactive and inflammatory peptide. In addition, cocoa polyphenols reduce the prooxidant effects of angiotensin II [58]; (iii) improvement of sympathovagal balance [34].

4.4. Antiplatelet Effects

Platelets activation and aggregation have a crucial importance in the ethiology and pathogenesis of CDVs and cerebrovascular diseases. Several studies reported that the intake of cocoa and dark chocolate in moderate amounts acutely or chronically determines a significant inhibition of platelet aggregation and adhesion in healthy volunteers, smokers or people who suffered a heart transplant [9]. The consumption of flavanol-rich cocoa beverages (600 and 900 mg of flavanols) causes a significant inhibition of platelet activation and aggregation, and inhibits platelet-monocyte conjugate and platelet-neutrophil conjugate formation [68]. The administration of high (897 mg) and moderate (220 mg) doses of cocoa flavanols reduces platelet aggregation induced by ADP + collagen, as well as by epinephrine + collagen [14]. Catechin and epicatechin, as well as their methylated metabolites, inhibit the formation of A2 thromboxane, a potent vasoconstrictor and platelet aggregator, and they also inhibit ADP-induced aggregation but only at supra-physiological doses [52]. Cocoa attenuates platelet aggregation induced by ADP and thyrombin-receptor activation (thrombin receptor activating peptide SFLLRNamide, TRAP6), but it does not influence the one induced by collagen and thromboxane analogue U46619 [34]. Theobromine, the major methylxanthine in cocoa, also inhibits platelet aggregation induced by ADP and TRAP6, the effects being mediated by PDE inhibition and increase of cAMP [34]. Significant antiplatelet properties of theobromine explain the more complex effects obtained from the use of cocoa products in comparison to those of flavanols alone. It is estimated that the intake of 100 g of dark chocolate with 70% cocoa solids produces an effect on platelets comparable to that induced by a standard dose of aspirin (80 mg), a classic anti-aggregant agent [47].
In contrast to the aforementioned data, some studies on the chronic intake of cocoa polyphenols did not find a significant effect on platelet function [68,69]. Ottaviani et al. [31] showed that the daily consumption of up to 2000 mg of cocoa flavanols for 12 weeks did not affect platelet function in humans. Also, a daily intake of up to 1000 mg of cocoa flavanols for two weeks was not associated with changes in platelet function. The authors state that the investigations were performed in healthy subjects and the usual approach to define the health status of participants do not allow a differentiated assessment of the individual health. The divergent or inconsistent results related to the antiplatelet efficacy of cocoa polyphenols could be explained considering the following variables:
(i)
Health status/gender of participants. The different basal level of platelet function may result in different responses after the exposure to compounds with antiplatelet potential. In a randomized controlled human intervention trial, Ostertag et al. [70] showed gender-dependent antiplatelet effects of dark chocolate. Acute intake of flavan-3-ol-enriched dark chocolate (907.4 mg flavanols/60 g chocolate) significantly reduces ADP-induced platelet aggregation and P-selectin expression in men and decreases thrombin receptor-activating peptide (TRAP)-induced platelet aggregation in women. The platelets from men are more sensitive to activation via adrenergic and serotoninergic pathways and show more intense thromboxane A2 receptor-related aggregation responses. The women’s platelets have a lower amount of thromboxane A2 receptors, which would explain the inhibitory effect of flavanol-enriched dark chocolate on TRAP-induced aggregation. Also, the use of oral contraceptives and menstrual phase may influence the effects on platelet function. In a critical review on antiplatelet effects of dietary polyphenols, Ostertag et al. [69] noted that the most significant changes on platelet functions have been reported for subjects with single or multiple cardiovascular risk factors. Also, acute intake of dark chocolate (40 g, cocoa > 85%) reduces platelet activation via antioxidant mechanisms only in smokers who have a higher baseline generation of oxidative stress compared to healthy subjects [71];
(ii)
Acute or chronic intake. The antiplatelet effects of cocoa flavanols appear to be more intense and meaningful in the case of acute intake. Even a modest amount of flavanols may modulate platelet reactivity in acute studies. The metabolism of cocoa flavanols, bioactivity of metabolites and their persistence could contribute to these findings. The existence of possible different mechanisms for acute and chronic administration does not allow a direct comparison of results from such studies. A better assessment of cocoa flavanols antiplatelet activity should measure both acute and chronic effects in the same study [69];
(iii)
Methodology. The assessment of antiplatelet effects of cocoa polyphenols was performed by various experimental approaches that differ in terms of principle, sensitivity, evaluated markers or functions. Besides, the correlations between methods are low. Bleeding time (BT) assesses primary hemostasis by in vivo measurement of bleeding block. Although BT is a simple and quick method, it has the disadvantage that is poorly standardized and is influenced by many variables (skin thickness, temperature among patients). Light transmission platelet aggregometry on platelet-rich plasma (PRP)-LTA is a standard test that evaluates various platelet functions such as platelet activation under action of different agonists (ADP, AA, collagen, and epinephrine, TRAP, thromboxane A2 mimetic U46619) and platelet-to-platelet clump formation in a glycoprotein (GP) IIb/IIIa-dependent manner. The preanalytical conditions (type of anticoagulant, lipid plasma, hemolysis, or low platelet count) as well as procedural conditions (manual sample processing, PRP preparation, use of different concentrations of agonists) may alter the final outcomes [72]. Besides, LTA is a relative non-physiological method, and platelets are not subjected to intense shear conditions [69]. The Platelet Function Analyzer—PFA-100 assesses platelet function in whole blood at the point-of-care under shear stress using collagen (C) plus ADP or collagen plus epinephrine as stimulators of hemostasis. The method presents some limitations such as platelet count-hematocrit-dependence and insensitivity to platelet secretion defects [72]. Platelet analysis based on flow cytometry provides information on platelet functional status in vivo, and includes different methods such as the assessment of platelet activation biomarkers, leukocyte–platelet aggregates or platelet-derived microparticles. However, the preanalytical phase may induce errors, and the measurement of circulating monocyte–platelet is performed under low shear conditions that do not accurately reproduce in vivo processes [69,72];
(iv)
Small size of subject lots and different populations [73].
Considering the heterogeneity of studies and the aforementioned aspects, the antiplatelet activity of cocoa polyphenols still remains a topic in debate. Several mechanisms have been suggested but they should be considered from the perspective of previous comments. Also, theobromine may be an important modulator of antiplatelet effects of cocoa products. Such antiplatelet mechanisms of cocoa polyphenols are: (i) modulation of eicosanoids metabolism involved in regulation of vascular homeostasis (stimulation of PGI2 synthesis, inhibition of leukotrienes production) [74,75]; (ii) increase of NO bioavailability, decrease of platelet reactivity [9] and down-regulation of NADPH oxidase and inhibition of platelet isoprostanes [71]; (iii) changes in membrane fluidity [9]; (iv) reduction of the ADP-induced expression of the activated conformation of glycoprotein IIb/IIIa surface proteins and the modulation of platelet integrins that are necessary for platelet-platelet and platelet-endothelial leukocyte interactions [14,76]; (v) inhibition of platelet lipoxygenase; (vi) antagonism of TxA2 platelet receptors [48]; (vii) decrease of phospholipase C activity, an enzyme involved in thrombin-induced platelet activation; (viii) regulation of genes involved in cell adhesion and trans-endothelial migration by acting on the NF-κB and MAPK signaling pathways. These effects are attributed to metabolites of epicatechin and they occur at low, physiological concentrations [52].
The well-controlled intervention studies with a large sample size and populations with cardiovascular risk factors along with a uniform methodology are needed to better assess the effects on platelet function exerted by cocoa flavanols.

4.5. Modulation of Lipid Profile

Cocoa polyphenols favorably influence the lipid profile and promote antiaterogenic effects. In vitro studies and studies on cell cultures showed an inhibition of the oxidation of low-density lipoproteins (LDL) and a reduction in the LDL oxidative susceptibility [8]. A diet with various concentrations of cocoa powder (0.5%–10%) or cocoa extract (600 mg/kg per day) for four weeks triggers a reduction in the levels of LDL and triglycerides (TG), a reduction of the LDL oxidability, an increase of the high-density lipoproteins (HDL) and of the plasma antioxidant capacity in normal rats and hypercholesterolemic rabbits [8]. Furthermore, in the case of rabbits with a hypercholesterolemic diet, chronic administration of cocoa proanthocyanidins reduces the levels of plasma lipid hydroperoxides and increases the plasma antioxidant capacity [74]. Numerous clinical trials with varied designs in normocholesterolemic, healthy, mildly hypercholesterolemic subjects, with glucose intolerance or hypertension have demonstrated positive effects on the lipid profile by reducing the plasma levels of LDL, and TG and LDL oxidation, increasing the plasma level of HDL and plasma antioxidant status, reducing the level of several lipid peroxidation markers (TBARS, F2-isoprostanes) [8] and apolipoprotein B (Table 1) [15]. Also, cocoa supplementation may offer protection against postprandial dyslipidemia [23]. Randomized double-blind studies highlighted the HDL enhancing effect of cocoa by the increase of the apolipoprotein A level [52]. The intensity of the effects as well as their profile varies among studies. Some short-term randomized and controlled investigations reported modest changes of the total cholesterol, and LDL, without influencing HDL in patients with cardiovascular risk, and the effects do not seem to be dose-dependent [52]. Other studies indicated that the effects of the cocoa and dark chocolate are higher in patients with cardiovascular risk, in short-term studies and for daily doses of 500 mg polyphenols [77]. The differences between the studies could be explained by the extremely varied designs in terms of the pathophysiologic status of the subjects, the initial lipid profile, the type of cocoa products, the length of the study (2–12 weeks), and the concentration of the polyphenols (187–657 mg proanthocyanidins/day; 46–377 mg epicatechin/day) [15]. At the same time, we must take into account the fact that in the case of cocoa products, other components can also modulate the lipid metabolism.
The positive effects of the cocoa polyphenols on the lipid profile could involve several mechanisms such as: (i) inhibition of cholesterol absorption in the digestive tract; (ii) inhibition of the hepatic biosynthesis of the cholesterol; (iii) the reduction of the susceptibility of LDL oxidation through changes on their surface [48]; (iv) increase of expression of scavenger receptor B type I, sterol regulatory element binding proteins, ATP binding cassette transporter A1 and apolipoprotein A1 [36].

4.6. Anti-Inflammatory Activity

Cardiovascular diseases are currently considered inflammatory diseases; the damage of various cells that participate in cardiovascular functions involves inflammatory and immune responses [12]. Cocoa polyphenols act on several inflammatory mediators and signaling pathways in patients with an increased risk of cardiovascular diseases [74]. On cell cultures, cocoa extracts with 5–100 μg/mL total polyphenols, as well as monomers and oligomeric proanthocyanidins (25 μg/mL) inhibit the production of inflammatory cytokines (IL-1β, IL-2, IL-6, TNF-α), but also the genic expression of iNOS through NF-κB and AP1 pathways. At the same time, in blood mononuclear cells, cocoa polyphenols (monomers to decamers) inhibit the expression of IL-1β. Dose-dependently, they decrease the activity of 15-LOX, 12-LOX and 5-LOX [8]. In mice with a high-fat diet, cacao supplementation (eight percent) for 10 weeks, reduced plasma levels of IL-6 and the TNF-α, IL-6, iNOS and NF-κB expression in adipose tissue. Also, cocoa flavanols can influence other markers of vascular inflammation as soluble adhesion molecules. Thus, the anti-inflammatory effects of cocoa polyphenols contribute not only to the alleviation of endothelial dysfunction, and arterial function and prevention of atherosclerosis, but they also provide benefits in atherothrombotic clinical syndromes. A reduction in mRNA expression of IL-1β, IL-6, E-selectin and vascular cell adhesion molecule (VCAM-1) was also observed in an experimental model of myocarditis in mice [78]. The proanthocyanidins-rich fractions (1–3 μg/mL) and B2 dimer (1.3 μM) reduce the vascular smooth muscle cells inflammatory phenomena by inhibiting the proMMP-2 expression and the MMP-2 activity, an enzyme involved in the degradation of extracellular matrix and the occurrence of atherothrombotic syndromes [78,79]. The intake of flavanol-rich cocoa products (446 mg of flavanols/day) significantly reduces the expression of VCAM-1 in women with postmenopausal hypercholesterolemia. Also, in patients with high cardiovascular risk, the intake of chocolate rich in polyphenols (495 mg of total polyphenols) decreases the levels of intercellular adhesion molecule 1 (ICAM-1). The use of a controlled diet that included 300–900 mg of flavanols/day did not alter ICAM-1 and VCAM-1 levels in obese adults at risk for insulin resistance or in patients with normal/hyper-cholesterolemia. These contradictory data could be explained by different polyphenol intake and extremely diverse pathophysiological status of patients, including the inflammatory picture. Epicatechin and type B proanthocyanidin dimers and their metabolites could be responsible for the effects on soluble adhesion molecules via NF-κB pathway inhibition [78].

4.7. Is (−)-Epicatechin the Main Compound Responsible for the Cardioprotective Properties of Cocoa Products?

A very wide range of data from in vitro and animal studies support the cardioprotective potential of (−)-epicatechin. As mentioned in the previous paragraphs, epicatechin has antioxidant properties, alleviates oxidative stress, diminishes ROS-mediated NO inactivation, up-regulates eNOS and increases the bioavailability of NO inducing endothelium-dependent relaxation in animals. Also, it stimulates Nrf2/ARE pathway and inhibits the vascular expression of some proinflammatory and proatherogenic markers (IL-1β, ICAM-1, and TNFα). Epicatechin improves some markers of endothelial function in ApoE knockout mice, a model of atherosclerosis. It reduces plasma ET-1 levels in the apolipoprotein E (ApoE) (−/−) gene-knockout mouse and in Deoxycorticosterone acetate (DOCA)-salt hypertensive rats most probably via Akt-regulation of the ET-1 promoter. The chronic administration of epicatechin prevents the progressive increase in SBP, the proteinuria, and the endothelial dysfunction in uninephrectomized rats chronically exposed to DOCA-salt [80]. In rats after permanent coronary occlusion, the chronic treatment with epicatechin protects against myocardial ischemic injury and preserves left ventricular structure and function [60]. Despite of these promising data, the studies with pure epicatechin in humans are scarce, and the results on endothelial function are conflictual and inconclusive (Table 2).
Schroeter et al. [87] showed that the oral administration of pure flavanol (−)-epicatechin (1 or 2 mg/kg body weight) to healthy subjects increases FMD and mimics some of acute vascular effects of cocoa, accounting, at least in part, for cocoa beneficial activity. Also, Loke et al. [88] demonstrated that the acute oral treatment with epicatechin (200 mg) modulates some important endothelial markers in healthy subjects. It significantly reduces plasma ET-1 concentration and increases circulating concentrations of vasoactive NO products most probably via eNOs activation and inhibition of NADPH oxidase. On the contrary, Dower et al. [84] did not identify a significant change of FMD or a reduction of BP following acute or chronic epicatechin administration (100 mg/day, four weeks) in apparently healthy older adults but the authors do not exclude the contribution of epicatechin to cardioprotective effects of cocoa. They found that epicatechin negatively modulates fasting plasma glucose and insulin resistance, which are closely related to the endothelial dysfunction. Also, in another interventional study in healthy (pre)hypertensive men and women, they showed that the treatment with epicatechin (100 mg/day, four weeks) causes a decrease of seven percent in sE-selectin, a marker of endothelial dysfunction that is inversely associated with FMD [83]. Barnett et al. [82] showed that a multi-dose intake of epicatechin (50 mg × 2/day, five days) in healthy adults improves mitochondrial enzyme function and increases plasma follistatin levels, an indicator of muscle growth. A careful analysis of the exposed studies reveals several limitations of these interventions, some of them even mentioned by the authors themselves. The main limitations are:
(i)
Small number of subjects and statistical underpowered trials [87];
(ii)
Large heterogeneity of the study population (40–80 years) and biological variations among subjects [84];
(iii)
Dose of epicatechin. As we already noted, EFSA recommends 200 mg of cocoa polyphenols daily for a beneficial effect on endothelial function. Although, Dower et al. [83,84] chosed the dosage of epicatechin in line with the amount of epicatechin present in previous cocoa/chocolate intervention studies (46–107 mg/day); in those studies, the level of total polyphenols was significantly higher (more than 200 mg and even higher than 800 mg). In cocoa products, the effects of epicatechin can be boosted by the pharmacokinetic and pharmacological interactions with other cocoa flavonoids and compounds. The activity of a compound within the natural phytocomplexes may be different as intensity or even sense from that of pure compound. The interactions between compounds in the phytocomplex affect their solubility, bioavailability or bioactivity and lead to a nuanced expression of biological response. In fact, even the authors mentioned that the dose of 100 mg epicatechin is likely to be too low to exert an effect on NO metabolism, the main target of vasodilatory mechanisms. Moreover, using a nonlinear meta-regression model with a Bayesian approach, Ellinger et al. (2012) [88] showed that the dose of ingested epicatechin influences the mean treatment effect. In this respect, authors showed that the daily intake of 25 mg epicatechin via cocoa consumption (but not as pure compound) can reduce BP through an increased availability of NO. In the study of Schroeter et al. [87], the dosage of epicatechin can be even lower or higher than 100 mg, depending on the body weight of subjects. In this context, the occurrence of the effects at low doses is difficult to explain. Maybe the fact that the subjects are more age-homogenous (25–32 years) exerts a positive influence;
(iv)
Health status of subjects. Baseline values of cardiovascular status and metabolism are different for young adults (25–32 years) and older adults (over 50 years), the last category being included in the studies of Dower et al. [83,84]. The type of treatment with epicatechin (acute vs. acute-on-chronic effect) may influence the final outcome. Dower et al. [84] showed that it is possible to reach a plateau regarding the effect on the endothelial function after 4 weeks of epicatechin administration, and one additional acute dose could not elicit more effect on FMD. The present data do not allow to define the cardiovascular profile of epicatechin in humans. The topic remains in discussion, and further long-term well-designed studies, with a larger number of subjects and appropriate methodology, are needed to gain more insight into the cardioprotective potential of epicatechin.

5. Safety of Cocoa Polyphenols

Daily intake of cocoa flavanols in amounts up to 2000 mg was well tolerated in healthy adults; only mild gastrointestinal effects have been reported [31]. Cocoa polyphenols may reduce iron absorption. A cocoa beverage with 100–400 mg of polyphenols per serving could decrease iron absorption by about 70% [89]. Also, the administration of cocoa products with other caffeine-containing foods and drinks may enhance the side effects of caffeine.

6. Limitations of Cocoa Studies

Many of the current clinical studies using cocoa products show some shortcomings that should be corrected to allow a better assessment of cocoa’s cardioprotective effects. The major critical points are: (i) a large heterogeneity in terms of cocoa products type (cocoa powder, dark chocolate, milk chocolate, cocoa beverages, semi-sweet chocolate baking bits), dose of flavanols, control, pathophysiological status and age of subjects; (ii) human intervention trials with a small number of subjects; (iii) few cross-over designed studies; (iv) lack of data related to polyphenolic profile of cocoa products (type and proportions of polyphenols as monomers, oligomers and polymers); (v) short-term studies (less than two weeks) and poor controls [8,58]; (vi) lack of data regarding plasma polyphenol concentrations [8]; (vii) numerous studies including only healthy subjects; (viii) lack of randomized controlled trials concerning the effects of cocoa on pivotal cardiovascular outcomes as cardiovascular mortality, myocardial infarction or stroke [49]. Therefore, the design of future clinical studies should comply with the following guidelines: (i) randomized, controlled, cross-over, multi-dose trials [10]; (ii) long-term trials in larger cohortes [34]; (iii) analytically well-characterized and standardized cocoa products in terms of flavanol and procyanidin content and profile and specification of the concentrations of fats, sugars, milk proteins [2,10,16]; (iv) flavanol-free controls [2]; (v) well-characterized participant population (life-style and diet habits, medical status) and the inclusion of subjects with elevated cardiovascular risk factors [16]; (vi) quantification of circulating flavanol levels and assessment of characteristic flavanol metabolites [16]; (vii) use of a dose of cocoa product, which could be included in the daily diet [10].

7. Future Perspectives

Future research on cardiovascular effects of cocoa polyphenols should consider the following topics: (i) clinical trials on individual polyphenols of cocoa: flavanol-monomers and proanthocyanidins [52]; (ii) assessment of cardiovascular activity of conjugates metabolites of cocoa polyphenols (mainly epicatechin metabolites) [2]; (iii) identification of the minimal doses of cocoa/chocolate that need to be ingested to exert cardioprotective effects [49]; (iv) investigation of molecular mechanisms of cocoa flavanols [52]; (v) investigation of polyphenol bioavailability from different cocoa-containing matrices [10].

8. Conclusions

Cocoa and cocoa polyphenols appear to exert promising cardioprotective effects in humans. Their clinical use depends largely on the clarification of the issues related to the pharmacokinetic and pharmacological properties as well as the interactions between polyphenols and other compounds of cocoa in well-designed studies.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. World Health Organization. Cardiovascular Diseases. Available online: http://www.who.int/mediacentre/factsheets/fs317/en/ (accessed on 10 July 2016).
  2. Field, D.; Newton, G. The antihypertensive actions of cocoa polyphenols—A review. Curr. Top. Nutraceuticals Res. 2013, 1, 113–128. [Google Scholar]
  3. World Health Organization. A Global Brief on Hypertension-Silent Killer, Global Public Health Crisis; WHO Press: Geneva, Switzerland, 2013; pp. 7–12. [Google Scholar]
  4. Aprotosoaie, A.C.; Luca, S.V.; Miron, A. Flavor chemistry of cocoa and cocoa products—An overview. Compr. Rev. Food Sci. Food Saf. 2016, 15, 73–91. [Google Scholar] [CrossRef]
  5. Jalil, A.M.M.; Ismail, A. Polyphenols in cocoa and cocoa products: Is there a link between antioxidant properties and health. Molecules 2008, 13, 2190–2219. [Google Scholar] [CrossRef] [PubMed]
  6. Fraga, C.G.; Litterio, M.C.; Prince, P.D.; Calabro, V.; Piotrkowski, B.; Galleano, M. Cocoa flavanols: Effects on vascular nitric oxide and blood pressure. J. Clin. Biochem. Nutr. 2010, 48, 63–67. [Google Scholar] [CrossRef] [PubMed]
  7. Keen, C.L. Chocolate: Food as medicine/medicine as food. J. Am. Coll. Nutr. 2001, 20, 436S–439S. [Google Scholar] [CrossRef] [PubMed]
  8. Rimbach, G.; Melchin, M.; Moehring, J.; Wagner, A.E. Polyphenols from cocoa and vascular health—A critical review. Int. J. Mol. Sci. 2009, 10, 4290–4309. [Google Scholar] [CrossRef] [PubMed]
  9. Sudano, I.; Flammer, A.J.; Noll, G.; Corti, R. Vascular and platelet effects of cocoa. In Chocolate and Health; Paoletti, R., Poli, A., Conti, A., Visioli, F., Eds.; Springer: Milan, Italy, 2012; pp. 103–111. [Google Scholar]
  10. Cooper, K.A.; Donovan, J.L.; Waterhouse, A.L.; Williamson, G. Cocoa and health: A decade of research. Br. J. Nutr. 2008, 99, 1–11. [Google Scholar] [CrossRef] [PubMed]
  11. Schinella, G.; Mosca, S.; Cienfuegos-Jovellanos, E.; Pasamar, M.A.; Miguerza, B.; Ramón, D.; Rios, J.L. Antioxidant properties of polyphenol-rich cocoa products industrially processed. Food Res. Int. 2010, 43, 1614–1623. [Google Scholar] [CrossRef]
  12. Goya, L.; Martin, M.A.; Sarriá, B.; Ramos, S.; Mateos, R.; Bravo, L. Effect of cocoa and its flavonoids on biomarkers of inflammation: Studies of cell culture, animals and humans. Nutrients 2016, 8, 212. [Google Scholar] [CrossRef] [PubMed]
  13. Steffen, Y.; Gruber, C.; Schewe, T.; Sies, H. Mono-O-methylated flavanols and other flavonoids as inhibitors of endothelial NADPH oxidase. Arch. Biochem. Biophys. 2008, 469, 209–219. [Google Scholar] [CrossRef] [PubMed]
  14. Keen, C.L.; Holt, R.R.; Polagruto, J.A.; Wang, J.F.; Schmitz, H.H. Cocoa flavanols and cardiovascular health. Phytochem. Rev. 2002, 1, 231–240. [Google Scholar] [CrossRef]
  15. Khan, N.; Monagas, M.; Andres-Lacueva, C.; Casas, R.; Urpi-Sardà, M.; Lamuela-Raventos, R.M.; Estruch, R. Regular consumption of cocoa powder with milk increases HDL cholesterol and reduces oxidized LDL levels in subjects at high-risk of cardiovascular disease. Nutr. Metab. Cardiovasc. 2012, 22, 1046–1053. [Google Scholar] [CrossRef] [PubMed]
  16. Schroeter, H.; Heiss, C.; Spencer, J.P.E.; Keen, C.L.; Lupton, J.R.; Schmitz, H.H. Recommending flavanols and procyanidins for cardiovascular health: Current knowledge and future needs. Mol. Aspects Med. 2010, 31, 546–557. [Google Scholar] [CrossRef] [PubMed]
  17. Schewe, T.; Steffen, Y.; Sies, H. How do dietary flavanols improve vascular function? A position paper. Arch. Biochem. Biophys. 2008, 476, 102–106. [Google Scholar] [CrossRef] [PubMed]
  18. Heiss, C.; Jahn, S.; Taylor, M.; Real, W.M.; Angeli, F.S.; Wong, M.L.; Amabile, N.; Prasad, M.; Rassaf, T.; Ottaviani, J.I.; et al. Improvement of endothelial function with dietary flavanols is associated with mobilization of circulating angiogenic cells in patients with coronary artery diseases. J. Am. Coll. Cardiol. 2010, 56, 218–224. [Google Scholar] [CrossRef] [PubMed]
  19. De Palma, R.; Sotto, I.; Wood, E.G.; khan, N.Q.; Butler, J.; Johnston, A.; Rothman, M.T.; Corder, R. Cocoa flavanols reduce N-terminal pro-B-type natriuretic peptide in patients with chronic heart failure. ESC Heart Fail. 2016, 3, 97–106. [Google Scholar] [CrossRef] [PubMed]
  20. Dower, J.I.; Geleijnse, J.M.; Kroon, P.A.; Philo, M.; Mensink, M.; Kromhout, D.; Hollman, P.C.H. Does epicatechin contribute to the acute vascular function effects of dark chocolate? A randomized, crossover study. Mol. Nutr. Food Res. 2016, 60, 2379–2386. [Google Scholar] [CrossRef] [PubMed]
  21. Rassaf, T.; Rammos, C.; Hendgen-Cotta, U.B.; Heiss, C.; Kleophas, W.; Dellanna, F.; Floege, J.; Hetzel, G.R.; Kelm, M. Vasculoprotective effects of dietary cocoa flavanols in patients on hemodialysis: A double-blind, randomized, placebo-controlled trial. Clin. J. Am. Soc. Nephrol. 2016, 11, 108–118. [Google Scholar] [CrossRef] [PubMed]
  22. Balboa-Castillo, T.; López-García, E.; León-Muñoz, L.M.; Pérez-Tasigchana, R.F.; Banegas, J.R.; Rodríguez-Artaleji, F.; Guallar-Castillón, P. Chocolate and health-related quality of life: A prospective study. PLoS ONE 2015, 10, e0123161. [Google Scholar] [CrossRef] [PubMed]
  23. Basu, A.; Betts, N.M.; Leyva, M.J.; Fu, D.; Aston, C.E.; Lyons, T.J. Acute cocoa supplementation increases postprandial HDL cholesterol and insulin in obese adults with type 2 diabetes after consumption of a high-fat breakfast. J. Nutr. 2015, 145, 2325–2332. [Google Scholar] [CrossRef] [PubMed]
  24. Grassi, D.; Desideri, G.; Necozione, S.; di Giosia, P.; Barnabei, R.; Allegaert, L.; Bernaert, H.; Bernaert, H.; Ferri, C. Cocoa consumption dose-dependently improves flow-mediated dilation and arterial stiffness decreasing blood pressure in healthy individuals. J. Hypertens. 2015, 33, 294–303. [Google Scholar] [CrossRef] [PubMed]
  25. Hammer, A.; Koppensteiner, R.; Steiner, S.; Niessner, A.; Goliasch, G.; Gschwandtner, M.; Hoke, M. Dark chocolate and vascular function in patients with peripheral artery disease: A randomized, controlled cross-over trial. Clin. Hemorrheol. Microcirc. 2015, 59, 145–153. [Google Scholar]
  26. Heiss, C.; Sansone, R.; Karimi, H.; Krabbe, M.; Schuler, D.; Rodriguez-Mateos, A.; Kraemer, T.; Cortese-Krott, M.M.; Kuhnle, G.G.; Spencer, J.P.; et al. Impact of cocoa flavanol intake on age-dependent vascular stiffness in healthy men: A randomized, controlled, double-masked trial. Age 2015, 37, 9794. [Google Scholar] [CrossRef] [PubMed]
  27. De Jesús Romero-Prado, M.M.; Curiel-Beltrán, J.A.; Miramontes-Espino, M.V.; Cardona-Muñoz, E.G.; Rios-Arellano, A.; Balam-Salazar, L.B. Dietary flavonoids added to pharmacological antihypertensive therapy are effective in improving blood pressure. Basic Clin. Pharmacol. Toxicol. 2015, 117, 57–64. [Google Scholar] [CrossRef] [PubMed]
  28. Koli, R.; Köhler, K.; Tonteri, E.; Peltonen, J.; Tikkanen, H.; Fogelholm, M. Dark chocolate and reduced snack consumption in midly hypertensive adults: An intervention study. Nutr. J. 2015, 14, 84. [Google Scholar] [CrossRef] [PubMed]
  29. Massee, L.A.; Ried, K.; Pase, M.; Travica, N.; Yoganathan, J.; Scholey, A.; Macpherson, H.; Kennedy, G.; Sali, A.; Pipingas, A. The acute and sub-chronic effects of cocoa flavanols on mood, cognitive and cardiovascular health in young healthy adults: A randomized, controlled trial. Front. Pharmacol. 2015, 6, 93. [Google Scholar] [CrossRef] [PubMed]
  30. McFarlin, B.K.; Venable, A.S.; Henning, A.L.; Prado, E.A.; Sampson, J.N.B.; Vingren, J.L.; Hill, D.W. Natural cocoa consumption: Potential to reduce atherogenic factors? J. Nutr. Biochem. 2015, 26, 626–632. [Google Scholar] [CrossRef] [PubMed]
  31. Ottaviani, J.I.; Balz, M.; Kimball, J.; Ensunsa, J.L.; Fong, R.; Momma, T.Y.; Kwik-Uribe, C.; Schroeter, H.; Keen, C.L. Safety and efficacy of cocoa flavanol intake in healthy adults: A randomized, controlled, double-masked trial. Am. J. Clin. Nutr. 2015, 102, 1425–1435. [Google Scholar] [CrossRef] [PubMed]
  32. Rodriguez-Mateos, A.; Hezel, M.; Aydin, H.; Kelm, M.; Lundberg, J.O.; Weitzberg, E.; Spencer, J.P.; Heiss, C. Interactions between cocoa flavanols and inorganic nitrate: Additive effects on endothelial function at achievable dietary amounts. Free Radic. Biol. Med. 2015, 80, 121–128. [Google Scholar] [CrossRef] [PubMed]
  33. Rostami, A.; Khalili, M.; Haghighat, N.; Eghtesadi, S.; Shidfar, F.; Heidari, I.; Ebrahimpour-Koujan, S.; Eghtesadi, M. High-cocoa polyphenol-rich chocolate improves blood pressure in patients with diabetes and hypertension. ARYA Atheroscler. 2015, 11, 21–29. [Google Scholar] [PubMed]
  34. Rull, G.; Mohd-Zain, Z.N.; Shiel, J.; Lundberg, M.H.; Collier, D.J.; Johnston, A.; Warner, T.D.; Corder, R. Effects of high flavanol dark chocolate on cardiovascular function and platelet aggregation. Vascul. Pharmacol. 2015, 71, 70–78. [Google Scholar] [CrossRef] [PubMed][Green Version]
  35. Sansone, R.; Rodriguez-Mateos, A.; Heuel, J.; Falk, D.; Schuler, D.; Wagstaff, R.; Kuhnle, G.G.C.; Spencer, J.P.; Schroeter, H.; Merx, M.W.; et al. Cocoa flavanol intake improves endothelial function and Framingham Risk Score in healthy men and women: A randomised, controlled, double-masked trial: The Flaviola Health Study. Br. J. Nutr. 2015, 114, 1246–1255. [Google Scholar] [CrossRef] [PubMed]
  36. Sarriá, B.; Martínez-López, S.; Sierra-Cinos, J.L.; Garcia-Diz, L.; Mateos, R.; Bravo, L. Regular consumption of a cocoa product improves the cardiometabolic profile in healthy and moderately hypercholesterolaemic adults. Br. J. Nutr. 2014, 111, 122–134. [Google Scholar] [CrossRef] [PubMed]
  37. Wiese, S.; Esatbeyoglu, T.; Winterhalter, P.; Kruse, H.P.; Winkler, S.; Bub, A.; Kulling, S.E. Comparative biokinetics and metabolism of pure monomeric, dimeric, and polymeric flavan-3-ols: A randomized cross-over study in humans. Mol. Nutr. Food Res. 2015, 59, 610–621. [Google Scholar] [CrossRef] [PubMed]
  38. Horn, P.; Amabile, N.; Angeli, F.S.; Sansone, R.; Stegemann, B.; Kelm, M.; Springer, M.L.; Yeghiazarianis, Y.; Schroeter, H.; Heiss, C. Dietary flavanol intervention lowers the levels of endothelial microparticles in coronary artery disease patients. Br. J. Nutr. 2014, 111, 1245–1252. [Google Scholar] [CrossRef] [PubMed]
  39. Ibero-Baraibar, I.; Abete, I.; Navas-Carretero, S.; Massias-Zaid, A.; Martinez, J.A.; Zulet, M.A. Oxidised LDL levels decreases after the consumption of ready-to-eat meals supplemented with cocoa extract within a hypocaloric diet. Nutr. Metab. Cardiovasc. Dis. 2014, 24, 416–422. [Google Scholar] [CrossRef] [PubMed]
  40. Martínez-López, S.; Sarriá, B.; Sierra-Cinos, J.L.; Goya, L.; Mateos, R.; Bravo, L. Realistic intake of a flavanol-rich soluble cocoa produt increases HDL-cholesterol without inducing anthropometric changes in healthy and moderately hypercholesterolemic subjects. Food Funct. 2014, 5, 364–374. [Google Scholar] [CrossRef] [PubMed]
  41. Mastroiacovo, D.; Kwik-Uribe, C.; Grassi, D.; Necozione, S.; Raffaele, A.; Pistacchio, L.; Righetti, R.; Bocale, R.; Lechiara, M.C.; Marini, C.; et al. Cocoa flavanol consumption improve cognitive function, blood pressure control, and metabolic profile in elderly subjects: The Cocoa, Cognition, and Aging (CoCoA) Study—A randomized controlled trial. Am. J. Clin. Nutr. 2015, 101, 538–548. [Google Scholar] [CrossRef] [PubMed]
  42. Sarriá, B.; Martínez-López, S.; Sierra-Cinos, J.L.; Garcia-Diz, L.; Goya, L.; Mateos, R.; Bravo, L. Effects of bioactive constituents in functional cocoa products on cardiovascular health in humans. Food Chem. 2015, 174, 214–218. [Google Scholar] [CrossRef] [PubMed]
  43. Terai, N.; Gedenk, A.; Spoerl, E.; Pillunat, L.E.; Stodtmeister, R. The short-term effect of flavonoid-rich dark chocolate on retinal vessel diameter in glaucoma patients and age-matched controls. Acta Ophthalmol. 2014, 92, e341–e345. [Google Scholar] [CrossRef] [PubMed]
  44. West, S.G.; McIntyre, M.D.; Piotrowski, M.J.; Poupin, N.; Miller, D.I.; Preston, A.G.; Wagner, P.; Groves, L.F.; Skulas-Ray, A.C. Effects of dark chocolate and cocoa consumption on endothelial function and arterial stiffness in overweight adults. Br. J. Nutr. 2014, 111, 653–661. [Google Scholar] [CrossRef] [PubMed]
  45. Franzini, L.; Ardigò, D.; Valtueňa, S.; Pellegrini, N.; del Rio, D.; Bianchi, M.A.; Scazzina, F.; Piatti, P.M.; Brighenti, F.; Zavaroni, I. Food selection based on high total antioxidant capacity improves endothelial function in a low cardiovascular risk population. Nutr. Metab. Cardiovasc. 2012, 22, 50–57. [Google Scholar] [CrossRef] [PubMed]
  46. Scapagnini, G.; Davinelli, S.; di Renzo, L.; de Lorenzo, A.; Olarte, H.H.; Micali, G.; Cicero, A.F.; Gonzalez, S. Cocoa Bioactive Compounds: Significance and Potential for the Maintenance of Skin Health. Nutrients 2014, 6, 3202–3213. [Google Scholar] [CrossRef] [PubMed]
  47. Habauzit, V.; Morand, C. Evidence for a protective effects of polyphenols-containing foods on cardiovascular health: An update for clinicians. Ther. Adv. Chronic Dis. 2012, 3, 87–106. [Google Scholar] [CrossRef] [PubMed]
  48. Galleano, M.; Oteiza, P.I.; Fraga, C. Cocoa, chocolate, and cardiovascular disease. J. Cardiovasc. Pharmacol. 2009, 54, 483–490. [Google Scholar] [CrossRef] [PubMed]
  49. Monahan, K.D. Effect of cocoa/chocolate ingestion on brachial artery flow-mediated dilation and its relevance to cardiovascular health and disease in humans. Arch. Biochem. Biophys. 2012, 527, 90–94. [Google Scholar] [CrossRef] [PubMed]
  50. Campia, U.; Panza, J.A. Flavanol-rich cocoa—A promising new dietary intervention to reduce cardiovascular risk in type 2 diabetes? J. Am. Coll. Cardiol. 2008, 51, 2150–2152. [Google Scholar] [CrossRef] [PubMed]
  51. Heiss, C.; Kleinbongard, P.; Dejam, A.; Perré, S.; Schroeter, H.; Sies, H.; Kelm, M. Acute consumption of flavanol-rich cocoa and the reversal on endothelial dysfunction in smokers. J. Am. Coll. Cardiol. 2005, 46, 1276–1283. [Google Scholar] [CrossRef] [PubMed]
  52. Kerimi, A.; Williamson, G. The cardiovascular benefits of dark chocolate. Vascul. Pharmacol. 2015, 71, 11–15. [Google Scholar] [CrossRef] [PubMed]
  53. Liu, H.-B.; Gong, Y.-F.; Yu, C.-J.; Sun, Y.-Y.; Li, X.-Y.; Zhao, D.; Zhang, Z.-R. Endothelial progenitor cells in cardiovascular diseases: From biomarker to therapeutic agent. Regen. Med. Res. 2013, 1, 9. [Google Scholar] [CrossRef] [PubMed]
  54. Alexandru, N.; Badila, E.; Georgescu, A. The Role of Endothelial Progenitor Cells in the Cardiovascular Disease Pathogenesis. J. Stem Cells Res. Rev. Rep. 2014, 1, 1006. [Google Scholar]
  55. Balaji, S.; King, A.; Crombleholme, T.M.; Keswani, S.G. The role of endothelial progenitor cells in postnatal vasculogenesis: Implications for therapeutic neovascularization and wound healing. Adv. Wound Care 2013, 2, 283–295. [Google Scholar] [CrossRef] [PubMed]
  56. Grisar, J.C.; Haddad, F.; Gomari, F.A.; Wu, J.C. Endothelial progenitor cells in cardiovascular disease and chronic inflammation: From biomarker to therapeutic agent. Biomark. Med. 2011, 5, 731–744. [Google Scholar] [CrossRef] [PubMed]
  57. European Food Safety Authority (EFSA). Scientific opinion on the substantiation of a health claim related to cocoa flavanols and maintenance of normal endothelium-dependent vasodilatation pursuant to Article 13 (5) of Regulation (EC) No 1924/20061. EFSA J. 2012, 10, 2809–2830. [Google Scholar]
  58. Grassi, D.; Desideri, G.; Ferri, C. Blood pressure and cardiovascular risk: What about cocoa and chocolate? Arch. Biochem. Biophys. 2010, 501, 112–115. [Google Scholar] [CrossRef] [PubMed]
  59. Cienfuegos-Jovellanos, E.; del Mar Quiñones, M.; Muguerza, B.; Moulay, L.; Miguel, M.; Aleixandre, A. Antihypertensive effect of a polyphenol-rich cocoa powder industrially processed to preserve the original flavonoids of the cocoa beans. J. Agric. Food Chem. 2009, 57, 6156–6162. [Google Scholar] [CrossRef] [PubMed]
  60. Yamazaki, K.G.; Taub, P.R.; Barraza-Hidalgo, M.; Rivas, M.M.; Zambon, A.C.; Ceballos, G.; Villarreal, F.J. Effects of (−)-epicatechin on myocardial infarct size and left ventricular remodeling after permanent coronary occlusion. J. Am. Coll. Cardiol. 2010, 55, 2869–2876. [Google Scholar] [CrossRef] [PubMed]
  61. Ried, K.; Sullivan, T.R.; Fakler, P.; Frank, O.R.; Stocks, N.P. Effect of cocoa on blood pressure. Cochrane Database Syst. Rev. 2012, 15, CD008893. [Google Scholar]
  62. Desch, S.; Schmidt, J.; Kobler, D.; Sonnabend, M.; Eitel, I.; Sareban, M.; Rahimi, K.; Schuler, G.; Thiele, H. Effect of cocoa products on blood pressure: Systematic review and meta-analysis. Am. J. Hypertens. 2010, 23, 97–103. [Google Scholar] [CrossRef] [PubMed]
  63. Taubert, D.; Roesen, R.; Schomig, E. Effect of cocoa and tea intake on blood pressure: A meta-analysis. Arch. Intern. Med. 2007, 167, 626–634. [Google Scholar] [CrossRef] [PubMed]
  64. Grassi, D.; Necozione, S.; Lippi, C.; Croce, G.; Valeri, L.; Pasqualetti, P.; Desideri, G.; Blumberg, J.B.; Ferri, C. Cocoa reduces blood pressure and insulin resistance and improves endothelium-dependent vasodilation in hypertensives. Hypertension 2005, 46, 398–405. [Google Scholar] [CrossRef] [PubMed]
  65. Desideri, G.; Kwik-Uribe, C.; Grassi, D.; Necozione, S.; Ghiadoni, L.; Mastroiacovo, D.; Raffaele, A.; Ferri, L.; Bocale, R.; Lechiara, M.C.; et al. Benefits in cognitive function, blood pressure, and insulin resistance through cocoa flavanol consumption in elderly subjects with mild cognitive impairment: The Cocoa, Cognition, and Aging (CoCoA) study. Hypertension 2012, 60, 794–801. [Google Scholar] [CrossRef] [PubMed]
  66. Hooper, L.; Kroon, P.A.; Rimm, E.B.; Cohn, J.S.; Harvey, I.; le Cornu, K.A.; Ryder, J.J.; Hall, W.L.; Cassidy, A. Flavonoids, flavonoid-rich foods and cardiovascular risk: A meta-analysis of randomized controlled trials. Am. J. Clin. Nutr. 2008, 88, 38–50. [Google Scholar] [PubMed]
  67. Vlachopoulos, C.V.; Alexopoulos, N.A.; Aznaouridis, K.A.; Ioakeimidis, N.C.; Dima, I.A.; Dagre, A.; Vasiliadou, C.; Stefanadi, E.C.; Stefanadis, C.I. Relation of habitual cocoa consumption to aortic stiffness and wave reflections, and to central hemodynamics in healthy individuals. Am. J. Cardiol. 2007, 99, 1473–1475. [Google Scholar] [CrossRef] [PubMed]
  68. Arranz, S.; Valdera-Martinez, P.; Chiva-Blanch, G.; Casas, R.; Urpi-Sarda, M.; Lamuela-Raventos, R.M.; Estruch, R. Cardioprotective effects of cocoa: Clinical evidence from randomized clinical intervention trials in humans. Mol. Nutr. Food Res. 2013, 57, 936–947. [Google Scholar] [CrossRef] [PubMed]
  69. Ostertag, L.M.; O’Kennedy, N.; Kroon, P.A.; Duthie, G.G.; de Roos, B. Impact of dietary polyphenols on human platelet function—A critical review of controlled dietary intervention studies. Mol. Nutr. Food Res. 2010, 54, 60–81. [Google Scholar] [CrossRef] [PubMed]
  70. Ostertag, L.M.; Kroon, P.A.; Wood, S.; Horgan, G.H.; Cienfuegos-Jovellanos, E.; Saha, S.; Duthie, G.G.; de Roos, B. Flavan-3-ol-enriched dark chocolate and white chocolate improve acute measures of platelet function in a gender-specific way—A randomized-controlled human intervention trial. Mol. Nutr. Food Res. 2013, 57, 191–202. [Google Scholar] [CrossRef] [PubMed]
  71. Carnevale, R.; Loffredo, L.; Pignatelli, P.; Nocella, C.; Bartimoccia, S.; di Santo, S.; Martino, F.; Catasca, E.; Perri, L.; Violi, F. Dark chocolate inhibits platelet isoprostanes via NOX2 down-regulation in smokers. J. Thromb. Haemost. 2012, 10, 125–132. [Google Scholar] [CrossRef] [PubMed]
  72. Paniccia, R.; Priora, R.; Liotta, A.A.; Abbate, R. Platelet function tests: A comparative review. Vasc. Health Risk Manag. 2015, 11, 133–148. [Google Scholar] [CrossRef] [PubMed]
  73. Loffredo, L.; Perri, L.; Nocella, C.; Violi, F. Antioxidant and antiplatelet activity by polyphenol-rich nutrients: Focus on extra virgin olive and cocoa. Br. J. Clin. Pharmacol. 2016. [Google Scholar] [CrossRef] [PubMed]
  74. Quiñones, M.; Miguel, M.; Aleixandre, A. Beneficial effects of polyphenols on cardiovasculare disease. Pharmacol. Res. 2013, 68, 125–131. [Google Scholar] [CrossRef] [PubMed]
  75. Di Castelnuovo, A.; di Giuseppe, R.; Iacoviello, L.; de Gaetano, G. Consumption of cocoa, tea and coffee and risk of cardiovascular disease. Eur. J. Intern. Med. 2012, 23, 15–25. [Google Scholar] [CrossRef] [PubMed]
  76. Holt, R.R.; Actis-Goretta, L.; Momma, T.Y.; Keen, C.L. Dietary flavanols and platelet reactivity. J. Cardiovasc. Pharmacol. 2006, 47, S187–S193. [Google Scholar] [CrossRef] [PubMed]
  77. Tokede, O.A.; Gaziano, J.M.; Djousse, L. Effects of cocoa products/dark chocolate on serum lipids: A meta-analysis. Eur. J. Clin. Nutr. 2011, 65, 879–886. [Google Scholar] [CrossRef] [PubMed]
  78. Lee, K.W.; Kang, N.J.; Oak, M.-H.; Hwang, M.K.; Kim, J.H.; Schini-Kerth, V.B.; Lee, H.J. Cocoa procyanidins inhibit expression and activation of MMP-2 in vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP activities. Cardiovasc. Res. 2008, 79, 34–41. [Google Scholar] [CrossRef] [PubMed]
  79. Paramo, J.A. A new favourable effect of cocoa on atherosclerosis? Cardiovasc. Res. 2008, 79, 3–4. [Google Scholar] [CrossRef] [PubMed]
  80. Jiménez, R.; Duarte, J.; Perez-Vizcaino, F. Epicatechin: Endothelial function and blood pressure. J. Agric. Food Chem. 2012, 60, 8823–8830. [Google Scholar] [CrossRef] [PubMed]
  81. Gutiérrez-Salméan, G.; Meaney, E.; Lanaspa, M.A.; Cicerchi, C.; Johnson, R.J.; Dugar, S.; Taub, P.; Ramírez-Sánchez, I.; Villarreal, F. A randomized, placebo-controlled, double-blind study on the effects of (−)-epicatechin on the triglyceride/HDLc ratio and cardiometabolic profile of subjects with hypertriglyceridemia: Unique in vitro effects. Int. J. Cardiol. 2016, 223, 500–506. [Google Scholar] [CrossRef] [PubMed]
  82. Barnett, C.F.; Moreno-Ulloa, A.; Shiva, S.; Ramirez-Sanchez, I.; Taub, P.R.; Su, Y.; Ceballos, G.; Dugar, S.; Schreiner, G.; Villarreal, F. Pharmacokinetic, partial pharmacodynamics and initial safety analysis of (−)-epicatechin in healthy volunteers. Food Funct. 2015, 6, 824–833. [Google Scholar] [CrossRef] [PubMed]
  83. Dower, J.I.; Geleijnse, J.M.; Gijsbers, L.; Schalkwijk, C.; Kromhout, D.; Hollman, P.C. Supplementation of the pure flavonoids epicatechin and quercetin effects some biomarkers of endothelial dysfunction and inflammation in (pre)hypertensive adults: A randomized double-blind, placebo-controlled, crossover trial. J. Nutr. 2015, 145, 1459–1463. [Google Scholar] [CrossRef] [PubMed]
  84. Dower, J.I.; Geleijnse, J.M.; Gijsbers, L.; Zock, P.L.; Kromhout, D.; Hollman, P.C.H. Effects of the pure flavonoids epicatechin and quercetin on vascular function and cardiometabolic health: A randomized, double-blind, placebo-controlled, crossover trial. Am. J. Clin. Nutr. 2015, 101, 914–921. [Google Scholar] [CrossRef] [PubMed]
  85. Gutiérrez-Salméan, G.; Ortiz-Vilchis, P.; Vacaseydel, C.M.; Rubio-Gayosso, I.; Meaney, E.; Villareal, F.; Ramírez-Sánchez, I.; Ceballos, G. Acute effects of an oral supplement of (−)-epicatechin on postprandial fat and carbohydrate metabolism in normal and overweight subjects. Food Funct. 2014, 5, 521–527. [Google Scholar]
  86. Loke, W.M.; Hodgson, J.M.; Proudfoot, J.M.; McKinley, A.J.; Puddey, I.B.; Croft, K.D. Pure dietary flavonoids quercetin and (−)-epicatechin augment nitric oxide products and reduce endothelin-1 acutely in healthy men. Am. J. Clin. Nutr. 2008, 88, 1018–1025. [Google Scholar] [PubMed]
  87. Schroeter, H.; Heiss, C.; Balzer, J.; Kleinbongard, P.; Keen, C.L.; Hollenberg, N.K.; Sies, H.; Kwik-Uribe, C.; Schmitz, H.H.; Kelm, M. (−)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. Proc. Natl. Acad. Sci. USA 2006, 103, 1024–1029. [Google Scholar] [CrossRef] [PubMed]
  88. Ellinger, S.; Reusch, A.; Stehle, P.; Helfrich, H.-P. Epicatechin ingested via cocoa products reduces blood pressure in humans: A nonlinear regression model with a Bayesian approach. Am. J. Clin. Nutr. 2012, 95, 1365–1377. [Google Scholar] [CrossRef] [PubMed]
  89. Braun, L.; Cohen, M. Herbs and Natural Supplements, an Evidence-Based Guide, 4th ed.; Elsevier Churchill Livingstone: Sydney, Australia, 2015; Volume 2, pp. 202–214. [Google Scholar]
Figure 1. Cocoa flavanols.
Figure 1. Cocoa flavanols.
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Figure 2. Major proanthocyanidins-dimers in cocoa.
Figure 2. Major proanthocyanidins-dimers in cocoa.
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Figure 3. Proanthocyanidins-trimers in cocoa.
Figure 3. Proanthocyanidins-trimers in cocoa.
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Figure 4. Pharmacokinetic profile of cocoa polyphenols.
Figure 4. Pharmacokinetic profile of cocoa polyphenols.
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Figure 5. Main cardioprotective properties of cocoa polyphenols. Abbreviations: ACE, angiotensin-converting enzyme; EPCs, endothelial progenitor cells; ET-1, endothelin 1; FMD, flow-mediated dilation; HDLc, high-density lipoprotein-cholesterol; ICAM, intercellular adhesion molecule; IL, interleukin; LDLc, low-density lipoprotein-cholesterol; LOX, lipooxygenase; LTs, leukotrienes; MMP-2, matrix metalloproteinase 2; MPO, myeloperoxidase; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; Nrf2, nuclear factor erythroid-related factor 2; PGI2, prostaglandin I2; PLC, phospholipase C; TG, triglycerides; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; XO, xanthine oxidase.
Figure 5. Main cardioprotective properties of cocoa polyphenols. Abbreviations: ACE, angiotensin-converting enzyme; EPCs, endothelial progenitor cells; ET-1, endothelin 1; FMD, flow-mediated dilation; HDLc, high-density lipoprotein-cholesterol; ICAM, intercellular adhesion molecule; IL, interleukin; LDLc, low-density lipoprotein-cholesterol; LOX, lipooxygenase; LTs, leukotrienes; MMP-2, matrix metalloproteinase 2; MPO, myeloperoxidase; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; Nrf2, nuclear factor erythroid-related factor 2; PGI2, prostaglandin I2; PLC, phospholipase C; TG, triglycerides; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; XO, xanthine oxidase.
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Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products.
Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products.
YearStudy DesignNo. of ParticipantsAprox. Mean Age (years)SexComorbiditiesCocoa Product InterventionTF (mg/day)EC (mg/day)Duration (weeks)OutcomesRef.
2016Randomized, double-blind, placebo-controlled, crossover 3270M + FChronic heart failure (stable on GDMT)Dark chocolate1064 (HFDC);
88 (LFDC)
ns4 Diseases 04 00039 i001 Plasma NT-proBNP (HFDC) vs. LFDC and baseline;
Diseases 04 00039 i001 DBP vs. LFDC and baseline;
♦ No change in platelet function.
De Palma et al. [19]
2016Randomized, placebo controlled crossover 2062MNoneDark chocolate7701501 dose Diseases 04 00039 i002 FMD vs. control;
Diseases 04 00039 i001 AIx vs. control.
Dower et al. [20]
2016Randomized, placebo-controlled, double-blind5765M + FEnd-stage renal disease with chronic hemodialysis (pharmacological medication)Cocoa powder900ns4 Diseases 04 00039 i002 FMD vs. control;
Diseases 04 00039 i001 DBP vs. control.
Rassaf et al. [21]
2015ENRICA cohort study127218–65 and overM + FCoronary heart disease, Hypertension, Diabetes, Hypercholesterolemia, StrokeChocolatensns3 yearsNo evidence of beneficial effects of chocolate regular consumption on physical or mental components of Health-related Quality of LifeBalboa-Castillo et al. [22]
2015Randomized, double-blind, crossover, postprandial study18nsM + FType 2 diabetes, ObeseCocoa beverage480401 dose Diseases 04 00039 i002 HDLc vs. placebo;
Diseases 04 00039 i002 Serum insulin concentration vs. placebo;
Diseases 04 00039 i001 Large artery elasticity vs. placebo;
♦ No effect on total cholesterol, LDLc, TG, glucose, hsCRP vs. placebo;
♦ No changes in SBP, DBP vs. placebo.
Basu et al. [23]
2015Randomized, controlled, double-blind, crossover 2018–70nsNoneCocoa powder80–80017–1681 Diseases 04 00039 i002 FMD vs. control;
Diseases 04 00039 i001 SBP, DBP, PWV, ET1 vs. control
Grassi et al. [24]
2015Blinded, randomized, controlled, crossover 21nsnsSymptomatic peripheral artery diseaseDark chocolatensns1 dose♦ No effect on microvascular function vs. cocoa-free control chocolate.Hammer et al. [25]
2015Randomized, double-blind, controlled, parallel-group22 young
20 elderly
22/60MNoneCocoa powder9001282 Diseases 04 00039 i002 FMD, arteriolar and microvascular vasodilator capacity, red blood cell deformability vs. baseline;
Diseases 04 00039 i001 PWV, total peripheral resistance vs. baseline.
Heiss et al. [26]
2015Randomized, controlled, blind open-label7942M + FHypertension (grades I and II) treated with captopril or telmisartanDark chocolate + dehydrated red apples + green tea425.8ns24 Diseases 04 00039 i001 SBP, DBP, TG, hsCRP vs. controlDe Jesús Romero-Prado et al. [27]
2015Randomized, controlled, crossover 2233–64M + FMild hypertensionDark chocolate602.70ns8 Diseases 04 00039 i001 DBP, SBP vs. baseline;
♦ Improvement of subjects status from hypertensive to normotensive;
♦ No significant changes in CAVI vs. baseline;
♦ No significant changes in lipid profile, blood glucose, blood insulin vs. baseline.
Koli et al. [28]
2015Randomized, placebo-controlled, double-blind, parallel 4018–43nsNoneCocoa extract250ns1 dose; 4♦ No significant changes in peripheral and central blood pressure vs. baseline;
♦ No significant changes in central blood flow vs. baseline.
Massee et al. [29]
2015Randomized, double-blind2422FNone/Overweight ObesityCocoa powder640484 Diseases 04 00039 i001 Haptoglobin, total and proinflammatory monocyte CD62L expression in obese women vs. baseline;
Diseases 04 00039 i001 Endothelial microparticles in obese and overweight women vs. baseline.
McFarlin et al. [30]
2015Randomized, parallel-arm, double-masked, controlled dietary intervention4635–55M + FNoneFood-grade cocoa extract200022012♦ No significant in BP, platelet function, liver panel (albumin, bilirubin, ALT, AST, AlkPhos), metabolic markers (glucose, hemoglobin, hematocrit, urea, Na+, K+, Ca2+, Cl-, leucocytes, erythrocytes) vs. cocoa flavanols-free controlOttaviani et al. [31]
2015Double-blind, randomized, crossover1518–35MNoneCocoa powder1.4–10.9 /kg0.37–1.5 /kg1 dose Diseases 04 00039 i002 FMD vs. baselineRodriguez-Mateos et al. [32]
2015Randomized, placebo-controlled, double-blind 6057M + FType 2 diabetes and HypertensionDark chocolate 450 ns8 Diseases 04 00039 i001 FBG, SBP, DBP, hsCRP, apolipoprotein B vs. baseline;
Diseases 04 00039 i001 FBG, SBP, DBP in dark chocolate group vs. control.
Rostami et al. [33]
2015Randomized, double-blind, placebo-controlled 3245–70MPre-hypertension, Mild hypertensionDark chocolate1064 (HFDC);
88 (LFDC)
ns6 Diseases 04 00039 i001 Platelet aggregation induced by thrombin-receptor activation, ADP vs. baseline;
Diseases 04 00039 i002 Heart rate (LFDC) vs. baseline;
♦No changes in total cholesterol, LDLc, HDLc, TG vs. Baseline
♦No significant changes in SBP, DBP (HFDC ) vs. baseline or LFDC.
Rull et al. [34]
2015Randomized, double-blind, controlled 10044M + FNoneCocoa powder 450644 Diseases 04 00039 i002 FMD, HDLc vs. control;
Diseases 04 00039 i001 SBP, DPB, PWV, total and LDLc vs. control.
Sansone et al. [35]
2015Two consecutive controlled, crossover 4429M + FNone/Mild hypercholesterolemiaCocoa powder rich in dietary fibers/polyphenols43.8/45.3 9.3/18.94 Diseases 04 00039 i002 HDLc after the 2nd intervention vs. baseline;
Diseases 04 00039 i001 Plasma glucose and IL-1β in cocoa product rich in dietary fiber group vs. baseline.
Sarriá et al. [36]
2015Randomized, double-blind, crossover724–31MNonePure flavanols (monomers and polymers)1 mg/kg bw (EC, PCB1); 2 mg/kg bw PPC-Single doseGlucuronidated, sulfated, methylated (-)EC and DHPV are predominant metabolites in blood and urine. Wiese et al. [37]
2014Randomised, double-blind, crossover4064/27/60M + FNone (young and old)/Coronary artery diseaseCocoa beverage375594 Diseases 04 00039 i001 CD144+ EMP, CD31+/412 vs. baselineHorn et al. [38]
2014Randomised, parallel and double-blind5057M + FNoneCocoa powder414.26153.444 Diseases 04 00039 i001 Oxidized LDLc vs. control and vs. baseline;
Diseases 04 00039 i001 MPO, ICAM1 vs. baseline.
Ibero-Baibar et al. [39]
2014Non-randomized, controlled, crossover4429M + FNone/
Hypercholesterolemia
Cocoa powder45.318.94 Diseases 04 00039 i002 HDLc vs. baseline;
Diseases 04 00039 i001 IL 10 vs. baseline.
Martinez-López et al. [40]
2014Double-blind, controlled, parallel-arm 9061–85M + FDiabetes, Hypercholesterolemia
Hypertension (pharmacological treatments), Former smokers
Cocoa drinks993 (HF);
520 (IF);
48 (LF)
185 (HF);
95 (IF);
5 (LF)
8 Diseases 04 00039 i001 SBP, DBP (HF, IF) vs. LF;
Diseases 04 00039 i001 Plasma glucose concentrations (HF, IF) vs. LF;
Diseases 04 00039 i001 Insulin resistance (HF, IF) vs. LF;
Diseases 04 00039 i001 LDLc, TG, total cholesterol vs. LF;
Diseases 04 00039 i001 Plasma total 8-iso-prostaglandin F2α (HF, LF) vs. LF;
Diseases 04 00039 i001 Circulating insulin (HF, IF) vs. LF;
Diseases 04 00039 i002 HDLc (HF, IF) vs. LF.
Mastroiacovo et al. [41]
2014Randomized, controlled, crossover4429M + FNone/Hypercholeste-romiaCocoa powder44.19.34 Diseases 04 00039 i002 HDLc vs. baseline;
Diseases 04 00039 i001 Glucose, IL-1β and IL-10 vs. baseline.
Sarría et al. [42]
2014Randomized, single-blinded, prospective placebo-controlled6065nsNone/GlaucomaDark chocolatensns1 dose Diseases 04 00039 i002 Mean dilatation of the retinal venules in age-matched controls, but not in glaucoma patients;
Diseases 04 00039 i002 Venous vasodilatation in control group, but not in the glaucoma group.
Terai et al. [43]
2014Randomized, double-blind, 2 period, crossover3052M + FOverweight, Moderate obesityDark chocolate and sugar-free cocoa powder81473.64 Diseases 04 00039 i002 Basal blood flow, basal and peak diameter of the brachial artery, vs. baselineWest et al. [44]
Abbreviations: Aix, augmentation index; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AlkPhos, alkaline phosphatase; CAVI, cardio-ankle vascular index; CHD, chronic heart disease; DBP, diastolic blood pressure; DHPV, 5-(3’,4’-dihydroxyphenyl)-valerolactone; EC, epicatechin; EMP, endothelial microparticles; ET1, endothelin 1; F, females; FBG, fasting blood glucose; FMD, flow-mediated dilation; GDMT, guideline-directed medical therapy; HDLc, high density lipoprotein-cholesterol; HFDC, high-flavanol dark chocolate; HF, high flavanol intake; hsCRP, highly sensitive C-reactive protein; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; IT, intermediate flavanol intake; LDLc, low density lipoprotein-cholesterol; LF, low flavanol intake; LFDC, low-flavanol dark chocolate; M, males; MPO, myeloperoxidase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; ns, not specified; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PCB1, procyanidin B1; PPC, pure polymeric procyanidin fraction from cocoa; PWV, pulse wave velocity; SPB, systolic blood pressure; TF, total flavanols.
Table 2. Outcomes of clinical trials of epicatechin (2016–2006).
Table 2. Outcomes of clinical trials of epicatechin (2016–2006).
YearStudy DesignNo. of ParticipantsAprox. Mean Age (years)SexComorbiditiesForm of EC InterventionEC (mg/day)Duration (weeks)OutcomesRef.
2016Randomized, multicenter, placebo-controlled, double-blind3018–55M + FHypertriglyceridemiaCapsules1004 Diseases 04 00039 i001 TG, TG/HDLc ratio, hsCRP vs. baseline Gutiérrez-Salméan et al. [81]
2015Non-randomized open label9
(single-dose);
8
(multiple-dose)
23–68
(single-dose);
22–45
(multiple-dose)
nsNoneDissolved in water50; 100Single-dose; multiple-dose, 5 days↑ Plasma nitrite vs. baseline;
↑ Platelet mitochondria complexes I, IV, citrate synthase activities (multi-dose) vs. baseline;
↑ Plasma follistatin levels (multi-dose) vs. baseline.
Barnett et. al. [82]
2015Randomized, double blind, placebo-controlled crossover3540–80M + FPre-hypertensionCapsules1004 Diseases 04 00039 i001 Plasma soluble endothelial selectin vs. controlDower et al. [83]
2015Randomized, double-blind, placebo-controlled, crossover3340–80M + FNoneCapsules 1004 Diseases 04 00039 i001 Fasting plasma insulin and insulin resistance vs. controlDower et al. [84]
2014Pilot, open-labeled, crossover2028M + FNone/OverweightCapsules1 mg/kg1 dose Diseases 04 00039 i001 Blood glucose and TG after 2 h vs. controlGutiérrez-Salméan et al. [85]
2008Randomized, placebo-controlled, crossover1243MNoneDissolved in water2001 dose Diseases 04 00039 i002 Plasma S-nitrosothiols and nitrite after 2 h and urinary nitrate after 5 h vs. Baseline
Diseases 04 00039 i001 Plasma entothelin-1 after 2 h vs. baseline
Loke et al. [86]
2006Randomized, double-blind, crossover6 (3 + 3)25–32MNoneDissolved in water1 or 2 mg/kg1 dose Diseases 04 00039 i001 FMD and PAT responses after 2 h vs. baseline and vs. controlSchroeter et al. [87]
Abbreviations: EC, epicatechin; F, females; FMD, flow-mediated dilation; HDLc, high density lipoprotein-cholesterol; hsCRP, highly sensitive C-reactive protein; M, males; ns, not specified; PAT, peripheral arterial tonometry; TG, triglycerides.
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