The Cardiovascular Effects of Cocoa Polyphenols-An Overview.

diseases Review

The Cardiovascular Effects of Cocoa Polyphenols—An Overview Ana Clara Aprotosoaie 1, *, Anca Miron 1 , Adriana Trifan 1 , Vlad Simon Luca 1 and Irina-Iuliana Costache 2 1

2

*

Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa Iasi, Universitatii Str. 16, 700115 Iasi, Romania; [email protected] (A.M.); [email protected] (A.T.); [email protected] (V.S.L.) Department of Cardiology, Faculty of Medicine, University of Medicine and Pharmacy Grigore T. Popa Iasi, Universitatii Str. 16, 700115 Iasi, Romania; [email protected] Correspondence: [email protected] or [email protected]; Tel.: +40-232-301-600

Academic Editor: Maurizio Battino Received: 12 October 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

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,

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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, Diseases 2016, 4, 39 2 of 25 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. clinical relevance, and the link between antioxidant activity and cardiovascular health. The limitations of these studies and future perspectives 2. Polyphenols Composition of Cocoa on cocoa polyphenols research are also presented.

Raw dried cocoa beans (Theobroma cacao, Malvaceae, cacao tree) contain high levels of 2. Polyphenols Composition of Cocoa polyphenols (12%–18% of whole beans dry weight). In addition, other important bioactive Raw dried cocoa beans (Theobroma cacao, Malvaceae, cacao tree) contain high levels of polyphenols phytochemicals cocoa beans methylxanthines (4%) with theobromine as the mainofcompound (12%–18% of of whole beans dry are weight). In addition, other important bioactive phytochemicals cocoa [4,5].beans The main categories of cocoa polyphenols are: are methylxanthines (4%) with theobromine as the main compound [4,5]. The main categories of (i)

cocoa polyphenols are:

Flavan-3-ols or catechins (29%–38% of total polyphenols) as monomers and polymers. The monomers include (−)-epicatechin, (+)-catechin, (+)-gallocatechin, and (−)-epigallocatechin (i) Flavan-3-ols or catechins (29%–38% of total polyphenols) as monomers and polymers. (Figure (−)-Epicatechin constitutes up(+)-catechin, to 35% of total polyphenolsand being the most abundant The1). monomers include (− )-epicatechin, (+)-gallocatechin, (−)-epigallocatechin polyphenol both in cocoa andconstitutes cocoa derived (chocolate) [4,6]. Flavanols commonly (Figure 1). (−)-Epicatechin up toproducts 35% of total polyphenols being the mostare abundant polyphenol both in cocoa[7]. and cocoa derived products (chocolate) [4,6]. Flavanols are commonly found in cocoa as R-forms found in cocoa as(58%–65% R-forms [7].of total polyphenols) are polymers of epicatechin and catechin (ii) Proanthocyanidins (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 molecules linked by 4→= 8 and 4→6found, bonds. and In cocoa, mainly oligomeric proanthocyanidins (degree of polymerization 10) are dimers to hexamers predominate. The most (degree of polymerization = 10) are found, and dimers to hexamers predominate. The most B2 important cocoa proathocyanidins are dimers: B1 [epicatechin-(4β→8)-catechin], important cocoa proathocyanidins are dimers: B1 [epicatechin-(4β→8)-catechin], B2 [epicatechin[epicatechin-(4β→8)-epicatechin] (Figure 2), B3 [catechin-(4α→8)-catechin], B4 [catechin(4β→8)-epicatechin] (Figure 2), B3 [catechin-(4α→8)-catechin], B4 [catechin-(4α→8)-epicatechin], (4α→8)-epicatechin], B5 [epicatechin-(4β→6)-epicatechin], and trimers: C1 [epicatechinB5 [epicatechin-(4β→6)-epicatechin], and trimers: C1 [epicatechin-(4β→8)2-epicatehin] (Figure 3) [4]. (4β→8)2-epicatehin] (Figure 3) [4]. (iii) Anthocyanins (4% of total polyphenols), such as: cyanidin-3α-L-arabinoside, cyanidin-3 (iii) Anthocyanins (4% [8], of total polyphenols), such cyanidin-3αL-arabinoside, cyanidin-3 β-Dβ-D-galactoside and leucoanthocyanins (L1, as: L2, L3, L4) [4]. galactoside [8], and leucoanthocyanins (L1, L2, L3, L4) [4].

Figure flavanols. Figure1.1.Cocoa Cocoa flavanols.

Other polyphenols from cocoa quercetin,luteolin, luteolin, kaempferol Other polyphenols from cocoaare: are:flavones flavones (apigenin, (apigenin, quercetin, kaempferol andand theirtheir glycosides), polyphenolic (caffeicacid, acid, chlorogenic acid, ferulic acid, coumaric acid, and glycosides), polyphenolicacids acids (caffeic chlorogenic acid, ferulic acid, coumaric acid, and syringic acid),acid), caffeoyl-conjugates (clovamide, deoxyclovamide), stilbens (trans-resveratrol and its glycoside, syringic caffeoyl-conjugates (clovamide, deoxyclovamide), stilbens (trans-resveratrol and its trans-piceid) [4,5]. The polyphenols profile inprofile cocoa varies depending of several factors, suchfactors, as: glycoside, trans-piceid) [4,5]. The polyphenols in cocoa varies depending of several plant genotype, geographic area, ripeness degree of beans, cocoa processing [4]. Alongside with ground such as: plant genotype, geographic area, ripeness degree of beans, cocoa processing [4]. Alongside green tea and wine, contains high amounts of polyphenols (460–610 mg/kg) [5,9]. with cocoa, ground cocoa, green teadark andchocolate wine, dark chocolate contains high amounts of polyphenols (460– 610 mg/kg) [5,9].

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Figure2.2.Major Major proanthocyanidins-dimers proanthocyanidins-dimers in Figure in cocoa. cocoa. Figure 2. Major proanthocyanidins-dimers in cocoa.

Figure 3. Proanthocyanidins-trimers in cocoa.

Figure Figure 3. 3. Proanthocyanidins-trimers Proanthocyanidins-trimers in in cocoa. cocoa.


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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].


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Figure 4. Pharmacokinetic profile of cocoa polyphenols. Figure 4. Pharmacokinetic profile of cocoa polyphenols.

4. Cardioprotective Effects of Cocoa Polyphenols 4. Cardioprotective Effects of Cocoa Polyphenols Numerous epidemiological studies, short-term human intervention studies, or small-cohort Numerous epidemiological human intervention studies, or small-cohort studies, showed that the intake studies, of cocoa short-term and polyphenols-rich cocoa products is correlated with studies, showed that the in intake of cocoa polyphenols-rich cocoa products isare correlated with cardiovascular benefits humans [16]. and Cocoa flavanols and proanthocyanidins pleiotropic cardiovascular benefits in humans [16]. Cocoa flavanols and proanthocyanidins are pleiotropic molecules that can influence various biomedical markers and clinical endpoints of cardiovascular molecules thatimprove can influence various function biomedical markers andendogenous clinical endpoints of cardiovascular health. They cardiovascular but also facilitate repair mechanisms [18]. health. They improve cardiovascular function also facilitate endogenous repair [18]. Experimental research and clinical trials have but investigated mainly the effects of cocoamechanisms products and Experimental researchonand clinicalstress trials and haveplasma investigated mainly the effects of cocoa products and cocoa polyphenols oxidative antioxidant capacity, endothelium-dependent cocoa polyphenols onand oxidative stressmediated and plasma antioxidant capacity, vasomotor function arterial flow dilatation (FMD), nitric oxideendothelium-dependent (NO) metabolism and activity, blood pressure, plateletflow function, lipiddilatation profile, and vascular An vasomotor function and arterial mediated (FMD), nitricinflammation oxide (NO) [5,10]. metabolism overview the most recent clinical trials (2014–2016) with cocoa and flavanols is shown in Table 1. The and activity,of blood pressure, platelet function, lipid profile, vascular inflammation [5,10]. cardioprotective properties of cocoa mainly antihypertensive, anti-atherogenic, and 1. Anpotential overview of the most recent clinical trialsinclude (2014–2016) with cocoa flavanols is shown in Table anti-inflammatory activities as well as inhibition of the platelet activation and aggregation, and The potential cardioprotective properties of cocoa include mainly antihypertensive, anti-atherogenic, attenuation of endothelial dysfunction (Figure 5). and anti-inflammatory activities as well as inhibition of the platelet activation and aggregation, and attenuation of endothelial dysfunction (Figure 5).


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Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products.

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Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. Year

2016

2016

2016

2015

2015

2015

2015

2015

2015

Study Design

Year

Aprox. No. of Mean No. Ageof Sex Aprox. Participants Diseases 2016, 4, 39 Study Design Mean Age (years) Participants

Duration Cocoa Product TF (mg/day) Comorbidities Product TF EC Duration Table 1. OutcomesIntervention of recent Cocoa (2014–2016) clinical trials of (mg/day) cocoaEC products. (weeks)

Sex (years)Aprox. No. of Mean Age Participants (years) Chronic

Comorbidities

Intervention

(mg/day)

(mg/day)

(weeks)

Outcomes

Outcomes

Ref.

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Ref.

Cocoa Product TF EC Duration Plasma NT-proBNP Plasma NT-proBNP (HFDC) vs. LFDC Year Study Design Sex Comorbidities Outcomes Ref. Intervention (mg/day) (mg/day) (weeks) (HFDC) vs. LFDC and Randomized, Diseases 2016, 4, 39 7 of 25De Palma heart failure 1064 (HFDC); and baseline; 32 70 M + F Dark chocolate ns 4 Table 1. Outcomes of recent (2014–2016) double-blind, 1064 trials of cocoa products. baseline; Plasma NT-proBNP (stable on GDMT) 88 (LFDC)clinical Diseases 2016, 4, 39 6 of 25et al. [19] DBP vs. LFDC and baseline; Chronic heart failure De Palma et al. placebo32 70 M + F Dark chocolate (HFDC); ns 4 DBP vs. LFDC and Randomized, vs.cocoa-free LFDC (stable on GDMT) (HFDC) No change in and platelet function. controlled, vs. control[19] Aprox. controlled, 88 (LFDC) baseline; No. of Cocoa Product TF EC Duration double-blind, 1064 baseline; crossover chocolate. Year Study Design Mean Age SexChronic heart Comorbidities Outcomes Ref. failure De Palma et al. crossover ♦ No change platelet Randomized, Intervention (HFDC);(mg/day) ns (mg/day) 4 (weeks) 2016 Diseases placebo32 Participants 70 M+F Dark chocolate DBPinvs. LFDC and 2016, 4, 39 7 of 25 Dower FMD, arteriolar and (years) (stable on GDMT) [19] FMD vs. control; function. 88 (LFDC) baseline; placebo controlled 20 controlled, 62 M None Table 1. Outcomes Dark chocolate 770 150 1 dose of recent (2014–2016) clinical trials of cocoa products. microvascular Plasma NT-proBNP et al. [20] AIx vs. control. Randomized, Randomized, crossover ♦ No change in platelet crossover vasodilator capacity, red Randomized, (HFDC) vs. LFDC and FMD vs. control; placebo controlled, vs. cocoa-free control double-blind, 22 young Aprox. function. 2016 20 62 M None Dark chocolate 770 150 1 dose Dower et al. [20] Heiss et al. [26] blood cell deformability 2015 22/60 M None Cocoa powder 900 128 2 No. of Product TF EC Duration double-blind, 1064 baseline; controlled AIx vs. control. Outcomes crossover chocolate. renal disease controlled, 20 elderly End-stage YearRandomized, Study Design Mean Age Sex Comorbidities Ref. et al. Chronic heart failure De Palma vs. baseline; Randomized, Participants Intervention (mg/day) (mg/day) (weeks) 2016 placebo32 70 M+F Dark chocolate (HFDC); ns 4 DBP vs. LFDC and and crossover parallel-group FMD, arteriolar (years) hemodialysis with chronic Rassaf FMD vs.peripheral control; (stable on GDMT) [19] FMD vs. control; placebo PWV, total controlled, 88 (LFDC) 20M + F 62 M End-stage renalNone Dark chocolate 770 150 1 dose Dower et al. [20] baseline; 65 Cocoa powder 900 ns 4 placebo-controlled, 2016 57 disease microvascular Plasma NT-proBNP controlled AIxresistance vs. control. (pharmacological et al. [21] DBP vs. control. Randomized, Randomized, vs. in baseline. crossover ♦ No change platelet double-blind with chronic vasodilator red crossover Randomized, (HFDC) vs.capacity, LFDC and FMD vs. control; placeboDiseases 2016, 4, 39 6 of 25 medication) double-blind, 22 young Randomized, Hypertension (grades I powder Dark chocolate900 + function. Rassaf et al. [21] 2016 57 65 M +F hemodialysis Cocoa ns Heiss al. [26] blood deformability 2015 22/60 M None Cocoa powder 900 1284 2 De JesúsetRomeroSBP,cell DBP, TG, hsCRP double-blind, 1064 baseline; End-stage renal disease controlled, controlled, 20 elderly 2015Randomized, controlled, blind 79 42 M +F and II) treated with dehydrated red apples + 425.8 ns 24 DBP vs. control. Chronic heart failure De Palma et al. Randomized, (pharmacological vs. baseline; Coronary heart disease, No evidence of beneficial effects of et al.7 [27] Prado vs. control 2016 placebo32 70 M + F Dark chocolate (HFDC); ns 4 with chronic DBP vs. LFDC and double-blind Diseases 2016, 4, 39 of 25 parallel-group open-label captopril or telmisartan green tea (stable on GDMT) [19] FMD vs. control; FMD vs. control; placeboplacebo medication) PWV,baseline; total peripheral controlled, Rassaf al. [21]et al. [20] 2016 65 hemodialysis Cocoa powder 900 88 (LFDC) ns 4 chocolate 2016 20 62 M + FDiabetes, M None Dark chocolate 770 150 1 dose Dower ENRICA 18–65 and 57 Hypertension, Balboa-Castillo regular consumption on et physical Diseases 2016, 4, 39 controlled, DBP, SBP vs. baseline; 7 of 25 controlled vs. control. AIx vs. 1272 M+F Chocolate ns ns 3 years No evidenceDBP of♦ beneficial resistance vs.control. baseline. crossover (pharmacological No change in platelet cohort study over Hypercholesterolemia, or mental components of Health-related et al. [22] TableCoronary 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. double-blind ♦ Improvement of crossover heart disease, effects of chocolate controlled, vs. cocoa-free control Randomized, Hypertension (grades I Dark chocolate + medication) function. StrokeHypertension, Quality ofBalboa-Castillo Life De Jesús RomeroSBP, DBP, TG, hsCRP subjects status from End-stage renal disease ENRICA cohort 18–65 and Diabetes, regular consumption on crossover chocolate. 2015 controlled, blind 79 42 M + F and II) treated with dehydrated red apples + 425.8 ns 24 controlled, Randomized, vs. cocoa-free control No evidence of beneficial Randomized, Aprox. 2015 1272 M+F ns ns 3 years vs. control toet al. [22] Prado et al. [27] hypertensive with chronic Chocolate No. of Cocoa Productgreen tea TF EC Duration study over Hypercholesterolemia, physical or mental FMD, arteriolar and Ref. open-label captopril or telmisartan crossover chocolate. HDLc vs. placebo; FMD vs.control; control; Coronary heart disease, effects of chocolate FMD vs. placeboplacebo Year Study Design Mean Age Sex Comorbidities Outcomes normotensive; Rassaf et al. al. [20] [21] 2016 57 65 MM+ FStroke hemodialysis Cocoa powder 900 ns 4 2016 20 62 None Dark chocolate 770 150 1components dose Dower et Participants Intervention (mg/day) (mg/day) (weeks) Diseases 2016, 4, 39 Randomized, 6 of 25 of Healthmicrovascular ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo DBP, SBP vs. baseline; controlled, controlled FMD, arteriolar and DBP vs. control. AIx vs. control. (years) Randomized, Serum insulin concentration Randomized, Koli et al. [28] controlled, 22 33–64 M + F M + F Mild hypertension Dark chocolate ns 8 ♦ No significant changes vs. placebo; 2015 2015 1272 Chocolate ns 602.70 ns 3 years related (pharmacological Quality of Life vasodilator capacity,of red et al. [22] study over Hypercholesterolemia, physical mental double-blind ♦or Improvement crossover microvascular Plasma NT-proBNP double-blind, Cocoa Basu Large artery elasticity vs. placebo; double-blind, 22 young crossover in CAVI vs. baseline; medication) Randomized, HDLc vs.blood placebo; 18 ns M +F Type22/60 2 diabetes, M Obese End-stage 480powder 40900 1128 dose Heiss et al. [26] cell deformability 2015 Cocoa 2 (HFDC) Stroke None components ofand Healthsubjects status from vasodilator capacity, red renal disease Randomized, vs. LFDC controlled, 20 elderly crossover, beverage ♦ No significant changes LDLc, TG, 7 of 25et al. [23] No effect on total cholesterol, No evidence of beneficial double-blind, 22 young Randomized, Diseases 2016, 4, 39 Serum insulin vs. baseline; related Quality of Life hypertensive to Heiss et al. [26] blood cell deformability 2015 22/60 M None Cocoa powder 900 128 2 with chronic double-blind, 1064 baseline; ineffects lipid profile, blood FMD vs.chocolate control; Coronary heart disease, of controlled, parallel-group 20 elderly placebopostprandial study glucose, hsCRP vs. placebo; Chronic heart failure De Palma et al. Table 1. of recent (2014–2016) clinical trials of cocoa 900 products. concentration vs.PWV, placebo; total peripheral Randomized, 32 baseline; Rassaf et al. [21] 2016 57 70 65 M Outcomes +F hemodialysis Cocoa powder ns 4 4 vs.DBP HDLc vs.normotensive; placebo; 2016 placebo-ENRICA M and +F Dark chocolate (HFDC); ns vs. regular LFDC and glucose, blood insulinon cohort 18–65 Diabetes, Balboa-Castillo parallel-group controlled, DBP vs. onHypertension, GDMT) [19] changes inconsumption SBP, DBP vs. placebo. Randomized, Large artery elasticity Koli et al. [28] 2015 controlled, 22 33–64 M Mild hypertension Dark chocolate 602.70 ns 8 No resistance vs. control. baseline. ♦ No significant changes 2015 1272 M+(stable +FF Chocolate ns ns 3PWV, years (pharmacological total peripheral Serum insulin controlled, controlled, 88 (LFDC) baseline; vs.physical cocoa-free control vs. baseline. Aprox. study over Hypercholesterolemia, or mental et al. [22] double-blind double-blind,Randomized, vs. placebo; crossover No. of ininCAVI vs. baseline; Hypertension (grades I Cocoa Product Dark chocolate + TF EC Duration resistance medication) vs. baseline. concentration vs. placebo; crossover ♦ No change platelet crossover chocolate. Randomized,2015Year crossover, ♦ No significant changes De Jesús RomeroStudy Design Mean Age M + FSex Type 2 diabetes, Comorbidities Outcomes Ref. SBP, DBP, TG, Stroke Cocoa beverage components ofhsCRP Health18 ns Obese 480 40 1 dose Basu et al. [23] ♦ No effect on total ♦ No significant changes 2015 controlled, blind 79 42 M + F and II) treated with dehydrated red apples + 425.8 ns 24 Participants Intervention (mg/day) (mg/day) (weeks) No evidence of beneficial Randomized, Randomized, Hypertension (grades I Dark chocolate + Large artery elasticity function. Randomized, in peripheral and central FMD, arteriolar and Prado et al. [27] (years) related Quality of Life vs. control controlled, Grassi FMD vs. control; De Jesús SBP, DBP, TG, hsCRP postprandial cholesterol, LDLc, TG,profile, lipid bloodRomerocaptopril orheart telmisartan green tea 425.8 Coronary disease, red apples effects of chocolate double-blind, 20 18–7079 ns 42 Cocoa powder 80–800 17–168 1 2015 controlled, blind open-label M + F None and II) treated with dehydrated + ns 24 vs.in placebo; Randomized, placeboblood pressure microvascular NT-proBNP HDLc vs. Prado placebo; et al.control [27] vs.Plasma control double-blind, et al. [24] SBP, DBP, PWV, ET1 vs. study glucose, hsCRP vs.SBP 1 dose; glucose, blood insulin Randomized, ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo DBP, vs. baseline; 2015 open-label crossover, 18 ns M + F Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] captopril or telmisartan green tea ♦ No effect on total FMD vs. control; placebo 2015 controlled, 20 40 18–43 None Cocoa extract 770 250 ns 1 dose vs. baseline; Massee et al. [29] vasodilator capacity, red et al. 1272 Mns+ F None Chocolate ns 150 3 years Randomized, vs. LFDC and Serum insulin 2016 Dark chocolate Dower [20] crossover placebo; 4 (HFDC) vs. baseline. double-blind, 22 young62 study overM Hypercholesterolemia, physical or mental et al. [22] postprandial ♦ Improvement of cholesterol, LDLc, TG, DBP, baseline; controlled AIxvs. vs. control. double-blind, ♦ No significant changes Heiss et al. [26] blood cell deformability 2015 22/60 M None Cocoa powder 1064 900 128 2 SBP double-blind, baseline; concentration vs.Healthplacebo; ♦ No changes in SBP, 20 elderly Diseases 2016, 4, controlled, 39 ♦ NohsCRP significant changes Stroke components of study subjects status from Chronic heart failure De Palma et al. 6 of 25 glucose, vs. ♦ Improvement of crossover parallel in central blood flow vs. baseline; Blinded, Diseases 2016 2016, 4, placebo70 M+F Dark chocolate (HFDC); ns 4 Randomized, 32 vs. LFDC and and Large artery elasticity DBPDBP vs. placebo. 39 parallel-group Randomized, inplacebo; peripheral central related Quality of Life hypertensive to (stablerenal on GDMT) [19]7 of 25 subjects status from End-stage disease vs. baseline. PWV, total peripheral function vs. controlled, 88 (LFDC) double-blind, baseline; randomized, Symptomatic peripheral No effect on microvascular Hammer vs. placebo; Randomized, Randomized, placeboblood pressure Randomized, HDLc placebo; ♦ No changes in vs. SBP, hypertensive tonormotensive; 21 ns ns 18 Dark chocolate ns beverage ns 480 1 dose with chronic FMD vs. control; 11dose; Haptoglobin, total and resistance vs. baseline. crossover 2015 crossover, ns M + F Type 2 diabetes, Obese Cocoa 40 dose Basu et [23] ♦ No change in platelet ♦ No effect on total FMD vs. control; placebocontrolled, 2016 Randomized, artery disease cocoa-free control chocolate. controlled, 2015 controlled, 57 40 18–43 None Cocoa extract 900 250 ns ns 4 vs. baseline; Massee etal. al. [29] et al. [25] Koli et al. [28] 2015 controlled, 22 33–64 M ns + F hemodialysis Mild hypertension Dark chocolate 602.70 ns 8 normotensive; ♦ No significant changes DBP vs. placebo. Serum insulin Rassaf et al. [21] 65 M + F Cocoa powder 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi et al. [24] 2015 SBP, DBP,function. PWV, ET1 controlled, control 4vs. cocoa-free proinflammatory Randomized, Hypertension (grades I Dark chocolate + postprandial cholesterol, LDLc, TG, controlled,double-blind, DBP vs. control. double-blind, crossover 2015 ♦in No significant changes crossover 22 CAVI vs. baseline; SBP, DBP, TG, hsCRP Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. Koli et al. [28]De Jesús Romerocontrolled, 33–64 M+F Mild(pharmacological hypertension Dark chocolate 602.70 ns 8 Randomized, ♦ No changes concentration vs. placebo; vs.chocolate. control crossover monocyte CD62L Diseases 2016, 4, 39 controlled, 6 of 25 2015 79 42 M+F and II) treated with dehydrated red apples + 425.8 ns 24significant studyblind FMDinvs. control; Randomized, glucose, hsCRP vs. double-blind crossover parallel central blood flow ♦ No significant changes Prado et al. [27] vs.artery control crossover Randomized, controlled, in CAVI vs.arteriolar baseline; Large elasticity medication) expression in obese FMD, and open-label captopril or telmisartan green tea FMD, arteriolar and microvascular FMD vs. control; placeboRandomized, 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi etMcFarlin al. [24] et al. 2015 Blinded, placebo; DBP, PWV, ET1blood Aprox. M Symptomatic peripheral ♦ NoSBP, effect on Hammer et al. vs. baseline. None/Overweight Dark chocolate in lipid profile, double-blind, 20 62 None 770 150 1 dose Dower et al. [20] double-blind, ♦ No significant changes Randomized,20152016 vs. placebo; No evidence of beneficial No. of24 ns Cocoa Product TF 640 EC 148 21 ns Dark chocolate ns dose Duration women vs.vs. baseline; 2015 22 F Cocoa powderns 4 vasodilator microvascular DBP, SBP baseline; controlled ♦ No changes inred SBP, AIxvs. vs. control. control capacity, blood Basu cell Year Study Design Mean Age Sex Comorbidities Outcomes Ref.[30] microvascular function [25] double-blind Obesity Obese glucose, bloodon insulin Haptoglobin, total and Randomized, 2015 crossover crossover, 18 ns Martery + F disease Type 2 diabetes, Cocoa beverage 480 40 1 dose et al. [23] in lipid profile, blood ♦ No effect total Coronary heart disease, effects of chocolate double-blind, 22randomized, young Heiss Participants Intervention (mg/day) (mg/day) (weeks) Endothelial vasodilator capacity, red ♦DBP Improvement of crossover 22/60 vs. placebo. (years) M None Cocoa powder 900 128 2 deformability vs. baseline; vs. baseline. double-blind, 22 young proinflammatory postprandial blood insulin Blinded, Symptomatic ♦deformability No effect on cholesterol, LDLc, TG,Hammer ENRICA cohort 18–65 Hypertension, Diabetes, regular consumption on Balboa-Castillo controlled, 2015 20 elderly et al. [26] Heiss et al. [26]et al. blood cell 22/60and M None Cocoa powder 128 2 1 dose glucose, microparticles obese subjects statusinfrom Table ofperipheral recent (2014–2016) clinical trials 900 of cocoa products. End-stage renal disease 21 ns ns 1. Outcomes Dark chocolate ns ns Randomized, 20152015 1272 M + F Chocolate ns ns 3 years Plasma NT-proBNP controlled, 20 elderly monocyte CD62L ♦ No significant changes PWV, total peripheral study vs. baseline. FMD vs. control; Randomized, randomized, artery disease microvascular function [25] hsCRP vs. et al. [22] study over Hypercholesterolemia, physical or glucose, mental vs. baseline; and overweight women hypertensive to parallel-group with chronic controlled, Randomized, (HFDC) vs.DBP, LFDC and parallel-group expression in obese Randomized, in vs. peripheral and central FMD control; ♦ No1components significant changes 20 Aprox. 18–70 ns None Cocoa powder 80–800 17–168 Grassi et al. [24] 2015placeboresistance vs. baseline. SBP, PWV, ET1 Stroke of HealthMcFarlin et al. None/Overweight vs.placebo; baseline. PWV, total peripheral Randomized, normotensive; Rassaf et al. [21] 2016 65 hemodialysis CocoaProduct powder 900 1064640 EC ns 4 double-blind, No.57of Cocoa double-blind, baseline; women vs. baseline; 2015 24 22 M + F F Cocoa powder TF 48Duration 4 related placeboblood pressure controlled, Randomized, in peripheral and central DBP vs. control. ♦ No changes in SBP, Quality of Life vs. control Year Study Design Mean Age 33–64 Sex Comorbidities Outcomes Ref. Chronic heart failure De Palma etal.al.[28] [30] double-blind Obesity 1 dose; ♦ No significant in BP, Koli et 2015 controlled, 22 M + F Mild hypertension Dark chocolate 602.70 ns 8 resistance vs. baseline. ♦ No significant changes (pharmacological crossover Dark chocolate Intervention 2016 placebo70 M + ns F Dark chocolate (HFDC); DBP vs.Endothelial LFDC and 2015 controlled,Participants3240 18–43 None Cocoa extract (mg/day) 250 (mg/day) nsns (weeks) 4 blood vs. baseline; Massee et al. [29] double-blind placebopressure DBP vs. placebo. HDLc vs. (years) Randomized, Hypertension (grades I (stable on GDMT) [19] 4 Randomized, platelet function, liver crossover inplacebo; CAVI vs. baseline; Randomized, Hypertension (grades I peripheral Dark chocolate + medication) 1 dose; Blinded, Symptomatic ♦ No effect on Hammer et al. De Jesús controlled, 88 (LFDC) baseline; double-blind, ♦ microparticles No significant changes + dehydrated in obese De Jesús SBP, DBP, TG, hsCRP Massee 2015 controlled, Cocoa extract Dark chocolate 250 vs. baseline; et al.Romero[29] 2015 ns ns None ns ns 1 dose Randomized, Serum insulin parallel-arm, panel (albumin, bilirubin, ♦NT-proBNP No significant changes controlled, blind 79 42 40 79 M + 18–43 F21 42 andnsII) with 425.8+ cocoa ns ns 24 2015 controlled, blind M treated +F and II) treated with dehydrated red apples 425.8 ns SBP, DBP, TG,in hsCRP vs. control 4 24 NoPlasma evidence of beneficial FMD vs. control; randomized, artery disease microvascular function [25]et al.Romero-Prado crossover parallel in central blood flow ♦ No change platelet Ottaviani Food-grade and overweight women Prado et al. [27] vs. control double-blind, ♦ No significant changes red apples + controlled, concentration vs. placebo; Randomized, LFDC andAlkPhos), 2015 double-masked, 46 35–55 MCoronary +F None 2000 220 12 ALT, AST, lipid profile, blood open-label captopril or heart telmisartan green teaCocoa powder disease, effectsvs. ofin chocolate 20 18–70or telmisartan ns None 80–800 17–168 1 (HFDC) Grassi[31] et al. [24] et al. [27] 2015 open-label captopril SBP, PWV, ET1 function. vs. baseline. extract vs.DBP, baseline. parallel in central blood flow double-blind, green tea Randomized, LargeSBP artery elasticity double-blind, 1064 baseline; controlled metabolic markers glucose, insulin ENRICA cohort dietary 18–65 and Hypertension, regular consumption on Balboa-Castillo DBP, vs. baseline; vs.blood control Chronic heart Diabetes, failure De Randomized, Haptoglobin, total ♦ No significant inand BP,Palma et al. vs. baseline. 2015 1272 M + F Chocolate ns ns 3 years crossover double-blind, vs. placebo; 2016 placebo70 M+F Dark chocolate (HFDC); ns 4 intervention 32 hemoglobin, et al. [22] DBP vs.(glucose, LFDC and vs.of baseline. studyplacebo over Hypercholesterolemia, or mental ♦ physical Improvement FMD vs. control; (stable on GDMT) [19] Hammer et al. Randomized, 18 platelet function, liver proinflammatory Haptoglobin, total and Symptomatic ♦ No effect on +, K+,et al. [23] 2015 2016 crossover, M+F 2 diabetes, Obese peripheral Cocoa beverage 480 40 ♦ No effect on total controlled, Blinded, hematocrit, urea,changes NaBasu 20 21 ns 62 ns MType Dark chocolate 150 ns1 dose 1 dose Dower et al. [20] baseline; ♦ No significant Stroke None components offrom Healthsubjects status 2015 ns Dark chocolate88 (LFDC)770 ns 1 dose controlled AIx vs. control. parallel-arm, panel (albumin, bilirubin, monocyte CD62L proinflammatory randomized, artery disease function [25] et al. postprandial cholesterol, LDLc, TG, crossover ♦ No change in to platelet Ottaviani Food-grade cocoa Randomized, inmicrovascular peripheral and central related Quality of Life hypertensive 2015study crossover double-masked, 46 35–55 M+F None 2000 220 12 ALT, AST, in AlkPhos), expression obese monocyte CD62L glucose, hsCRP vs. pressure function. [31]et al. extract McFarlin Randomized, None/Overweight placeboblood Randomized, normotensive; HDLc vs.metabolic placebo; End-stage renal disease controlled dietary markers women vs. baseline; 2015 24 22 F Cocoa powder 640 48 4 1expression dose; in obese placebo;changes Randomized, double-blind Obesity 2015 controlled, 22 40 33–64 18–43 ns None Cocoa extract 602.70 250 ns ns 8 vs. baseline; et al. [29] Randomized, Koli etetal. [28] [30] 2015 Randomized, controlled, M+F Mild hypertension Dark chocolate ♦ No significant McFarlin al.Massee None/Overweight Serum insulin with chronic Cocoa powder intervention (glucose, hemoglobin, 4♦ Novs. Endothelial women baseline; 2015 24 22 F 640 48 4 changes in SBP, FMD vs. control; placeboFMD control; double-blind, ♦vs. No significant changes placebo crossover in CAVI vs. baseline; vs. placebo; +[30] et al. [21] 2016 57 65 hemodialysis Cocoa powder ns , K+, Rassaf hematocrit, urea, NaDower 2016 double-blind 20 62 M M + F Obesity None Dark chocolate 770 900 150 1 dose 4 concentration et al. [20] microparticles in obese Endothelial DBP vs. placebo. controlled, parallel in central blood flow DBP vs. control. controlled AIx vs. control. ♦ No significant changes Randomized, Large artery elasticity women (pharmacological and overweight microparticles in obese Randomized, double-blind vs. baseline. crossover in lipid profile, blood double-blind, vs. placebo; FMD vs. control; medication) vs. baseline. and overweight women controlled, End-stage renal disease glucose, blood insulin Haptoglobin, total and 2015 crossover, 18 ns M + F Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] ♦ No effect on total 20 18–70 ns None Cocoa powder 80–800 17–168 1 2015 SBP, DBP, PWV, ET1 No evidence of beneficial Randomized, ♦ No significant inGrassi BP, et al. [24] vs. baseline. double-blind, with chronic vs. baseline. proinflammatory postprandial cholesterol, TG, vs. control Coronary heart disease, effects offunction, chocolate FMD vs.inLDLc, control; placeboRandomized, platelet liver ♦ No significant BP, crossover Rassaf et al. [21] 2016 57 65 M + F hemodialysis Cocoa powder 900 ns 4 monocyte CD62L ♦ Noglucose, significant changes study vs. bilirubin, ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo controlled, DBP vs.hsCRP control. (albumin, Randomized, platelet function, liver Blinded,parallel-arm, peripheral ♦ Nopanel effect on Hammer et al. 2015 1272 M +Symptomatic F (pharmacological Chocolate cocoa ns ns 3 years Ottaviani et al. Food-grade expression in obese Randomized, in peripheral and central placebo; 2015 parallel-arm, 21 ns ns Dark chocolate ns ns 1 dose panel study over Hypercholesterolemia, physical or mental et al. [22] et al. double-blind 2015 double-masked, 46 35–55 M + F None 2000 220 12 ALT, AST, AlkPhos), McFarlin Randomized, None/Overweight (albumin, bilirubin, randomized, disease function [25] medication) [31] extract women vs. baseline; 2015 24 22 F artery Cocoa powder 640 48 4microvascular placeboblood pressure Ottaviani et al. Food-grade cocoa ♦ No changes in SBP, Stroke components ofmarkers Healthcontrolled dietary metabolic [30] double-blind Obesity 1 dose; 20152015 double-masked, 46 35–55 M + F None 2000 220 12 ALT, AST, AlkPhos), No evidence of beneficial controlled, 40 18–43 ns None Cocoa extract 250 ns vs. baseline; Massee Endothelial [31] et al. [29] extract DBP vs. placebo. related Quality of Life (glucose, hemoglobin, 4 controlled dietaryintervention metabolic markers Coronary heart disease, ofmicroparticles chocolate double-blind, ♦ Noeffects significant changes in obese Randomized, hematocrit, Na+, K+, HDLc vs. urea, placebo; intervention (glucose, hemoglobin, FMD vs. control; ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo parallel in central blood flow and overweight women controlled, + + insulin 2015 1272 M+F Chocolate ns ns 3 years Serum

Randomized, double-blind, placebo-controlled, 2016 crossover


7 of 25 Diseases 2016, 4, 39

6 of 25

Table 1. Cont. Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. Year

2015

Study Design

Aprox. No. of Mean Age Participants Year Study Design (years)

Randomized, 2016 controlled, 22 crossover Diseases 2016, 4, 39

Sex No. of Participants

Randomized, double-blind, placebo33–64 controlled, crossover Randomized, placebo

2015

Diseases 2016, 4, 39 2016 Randomized, controlled placebo-controlled, crossover 40 18–43 double-blind, No. of Randomized, Year Study Design parallel Participants

placebocontrolled, double-blindNo. of Randomized, Study Design Participants double-blind,

2015

2015

2015

2015

20

nsAprox.

70

M+F

Mild hypertension

62 Table

Cocoa Product Intervention Comorbidities

Chronic heart failure (stable on GDMT) Dark chocolate

TF (mg/day) Cocoa Product

EC (mg/day) TF

Intervention

(mg/day)

Dark chocolate

602.70

1064 (HFDC); ns 88 (LFDC)

M None(2014–2016) clinical Dark chocolate 770 1. Outcomes of recent trials of cocoa products.

None

Cocoa extract

250

ns

End-stage renal disease Cocoa Product TF EC Mean Age Sex Comorbidities chronic Intervention (mg/day) (mg/day) Table 1. Outcomes ofwith recent (2014–2016) clinical trials of cocoa products. (years) 57 65 M+F hemodialysis Cocoa powder 900 (pharmacological Aprox. Cocoa Product TF EC medication) Mean Age Sex Comorbidities None/Overweight Intervention (mg/day) (mg/day) 1064 (years) F Cocoa powder 640 48 Chronic heart failure 70 M + FObesity Dark chocolate (HFDC); ns (stable onCoronary GDMT) heart disease, 88 (LFDC) 18–65 and Hypertension, Diabetes, 1272 M+F Chocolate ns over Hypercholesterolemia, 1064 Chronic heart failure Stroke 70 M+F Dark chocolate (HFDC); ns (stable on GDMT) 88 (LFDC)

Duration

EC (weeks) Duration (mg/day) (weeks)

ns

8

150

1 dose; 4 Duration (weeks) ns

Outcomes

Outcomes

Ref.

Plasma NT-proBNP DBP, SBP vs. baseline; (HFDC) vs.of LFDC and status from Improvement subjects baseline; hypertensive to normotensive; De Palma et al. 4 DBP vs. LFDC and No significant changes in [19] baseline; CAVI vs. baseline; 6 of 25 ♦ No change in platelet No significant changes in lipid profile, function. blood glucose, blood insulin vs. baseline.

Koli et al. [28]

FMD vs. control;

6 of 25 al. [20] Dower etand significant changes in peripheral AIx vs. control. central blood pressure vs. baseline; No significant changes in central blood Outcomes Ref. flow vs. baseline. FMD vs. control;

1 dose No

Ref.

Massee et al. [29]

Rassaf et al. [21] 4 DBPtotal vs. control. Plasma NT-proBNP Haptoglobin, and proinflammatory (HFDC) vs. LFDC and monocyte Year OutcomesCD62L expression Ref. in obese Randomized, McFarlin baseline; 24 22 4 No evidence beneficial womenofvs. baseline; De Palma et al. double-blind et al. [30] 2016 placebo32 4 DBP vs. LFDC andof chocolate effects Plasma NT-proBNP [19] Endothelial microparticles in obese and controlled, baseline; ENRICA cohort on baseline. Balboa-Castillo Randomized, (HFDC) vs.regular LFDC consumption and overweight vs. 2015 ns 3 years crossover ♦ No change inphysical plateletwomen or mental et al. [22] double-blind, study baseline; De Palma et al. function. components HealthDBP Novs. significant platelet function, 2016 placebo32 4 LFDC and inofBP, Randomized, [19] Randomized, related Quality of Life controlled, liver baseline; panel (albumin, bilirubin, ALT, AST, FMD vs. control; placebo parallel-arm, crossover vs. placebo; ♦ No change inHDLc platelet 2016 20 62 M None Dark chocolate 770 150 1 dose Dower et al.(glucose, [20] Food-grade AlkPhos), metabolic markers Diseases 2016, 4, 39 6 of 25 Ottaviani controlled AIx vs. control. double-masked, 46 35–55 M+F None 2000 220 12 function. insulin urea, Na+ , K+ , cocoa extract hemoglobin,Serum hematocrit, crossover Diseases 2016, 4, 39 7 of 25 et al. [31] controlled dietary concentration vs. placebo; Randomized, 2+ Ca , Cl , leucocytes, erythrocytes) 6 of 25 Diseases 2016, 4, 39 End-stage renal disease FMD vs. Large control; placebo Randomized, intervention artery elasticity Randomized, 2016 20 62 M Dark chocolate 770 150 1 dose Dower etcontrol al. [20] withNone chronic vs. cocoa flavanols-free controlled FMD control; AIx vs. control. placebo; control placebodouble-blind, controlled, vs.vs. cocoa-free Table 1. Outcomes of recent clinical trials of cocoa products. Rassaf et al. [21] 2016 M hemodialysis Cocoa(2014–2016) powder crossover, 57 18 65 ns + F M+F Type 2 diabetes, Obese Cocoa beverage 900 480 ns 40 4 1 dose DBP vs. control. Basu et al. [23] crossover ♦ No effect on total controlled, chocolate. Double-blind, Diseases2015 2016, 4, 39 crossover 6 of 25 (pharmacological Rodriguez-Mateos postprandial cholesterol, LDLc, TG, End-stage renal disease double-blind Aprox. None FMD, and randomized, 15 18–35 Mof Cocoaofpowder 1.4–10.9 /kg trials 0.37–1.5 /kg products. 1 dose Duration FMDarteriolar vs. baseline Randomized, medication) No. Cocoa Product TF EC Table 1. Outcomes recent (2014–2016) clinical of cocoa study et al. [32] glucose, hsCRP vs. Year Study Design Mean Age Sex with chronic Comorbidities Outcomes Ref. microvascular FMD vs. placebo- Randomized, crossover 2016 Participants Intervention (mg/day) (mg/day) (weeks) No evidence ofcontrol; beneficial placebo; Rassaf et al. [21] 57 65 M + F hemodialysis Cocoa powder 900 ns 4 (years) vasodilator capacity, red Aprox. controlled, double-blind, DBPofvs. control. Coronary heart disease, effects chocolate 22 of young No. Cocoa Product TF EC Duration ♦ No changes in SBP, (pharmacological Plasma NT-proBNP FBG, SBP, DBP, hsCRP, Heiss blood cell deformability 2015 cohort None (2014–2016) Cocoa powder 900 128 2 consumption Year Study Design Age Table Sex Comorbidities Outcomes Ref.et al. [26] 1.MOutcomes of recent clinical trials of cocoa products. double-blind ENRICA and Mean 22/60 Hypertension, Diabetes, regular on Balboa-Castillo Randomized, controlled, 2018–65 elderly (mg/day) (mg/day) (weeks) medication) DBP vs. 2015 1272 Participants M +2F diabetes and ChocolateIntervention ns ns 3 years Randomized, (HFDC) vs. placebo. LFDC (years) Type Rostami apolipoprotein Band vs. baseline; study Hypercholesterolemia, physical or mental baseline; et al. [22] parallel-group placebo-controlled, 60 57 M + FoverAprox. Dark chocolate 450 ns 8 Randomized, No evidence of beneficial double-blind, 1064 baseline; PWV, total peripheral Plasma NT-proBNP FMD SBP, vs. control; et al. [33] Hypertension Stroke of HealthFBG, DBP in dark Chronic heart failure De Palma et al. No. of Cocoa Product TF EC Duration components controlled, Coronary heart disease, chocolate double-blind Year 2016 placebo32 70 +F Dark chocolate (HFDC); ns 4 effects of (HFDC) DBP vs. LFDC and Design Mean Age Sex M Comorbidities Outcomes Ref. resistance vs. baseline. vs. LFDC and 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi et al. [24] 2015Study Randomized, related Quality of Life SBP, DBP, PWV, ET1 (stable on GDMT) chocolate group vs. control. 7[19] Participants 18–65 and Intervention (mg/day) (mg/day) (weeks) regular consumption double-blind, ENRICA cohort Hypertension, Diabetes, on Balboa-Castillo Diseases 2016, 4, 39 of 25 controlled, 88 1064 (LFDC) baseline; (years) M + F Randomized, Hypertension (grades I Dark chocolatens + double-blind, baseline; 2015 1272 Chocolate ns 3 years vs. control HDLc vs. placebo; JesúsetRomeroSBP, DBP, hsCRP Chronic heart failure DeDe Palma al. study crossover over Hypercholesterolemia, physical orNo mental et al. [22] crossover ♦ change in TG, platelet Plasma NT-proBNP 2015 controlled, and II) treated with dehydrated red apples + (HFDC); 425.8 Platelet aggregation 2016 placebo- blind 32 79 70 42 M +MF + F Dark chocolate ns ns 4 24 Serum DBP vs. LFDC and induced by insulin Prado vs. control (stable on GDMT) [19] etetal. Stroke components of HealthBlinded, Symptomatic peripheral ♦ No effect on Hammer al.[27] function. Randomized, (HFDC) vs. LFDC and open-label captopril or telmisartan green tea controlled, 88 (LFDC) baseline; 2015 controlled, 21 ns ns Dark chocolate ns ns 1concentration dose vs.thrombin-receptor activation, vs. placebo; cocoa-free control related Quality of Life randomized, artery disease microvascular function [25] Randomized, double-blind, 1064 crossover DBP, SBP baseline; ♦baseline; No change invs. platelet Randomized, crossover chocolate. ADP vs. baseline; Large artery elasticity Chronic heart failure De Palma et al. HDLc vs. placebo; vs.and control; of placebo Randomized, 2016 double-blind, placebo32 70 M+F Dark chocolate (HFDC); ns 4 vs.FMD LFDC function. ♦ Improvement 2016 20 62 M None Dark chocolate 150 1 dose vs. DBP Dower placebo; FMD, arteriolar and (stable on GDMT) [19] et al. [20] Pre-hypertension, 1064 (HFDC); 88 (LFDC)770 Rull Heart rate (LFDC) vs. baseline; Serum insulin controlled AIx vs. control. baseline; double-blind, 32controlled, 45–7018 M ns Dark chocolate ns 40 6 statusBasu fromet al. [23] Randomized, 2015 crossover, M + Fhypertension Type 2 diabetes, Obese Cocoa beverage 1 dose ♦No effect on subjects totalin total microvascular Mild 88 (LFDC) 480 et al. [34] No changes cholesterol, LDLc, concentration vs. placebo; crossover crossover Randomized, ♦ No change in vs. platelet hypertensive to FMD control; placebo-controlled postprandial placebo cholesterol, LDLc, TG, vasodilator capacity, red 2016 20 62 M Dark chocolate 770 150 1 dose Dower et al. [20] Randomized, HDLc, TG vs. Baseline Large artery elasticity End-stageNone renal disease double-blind, function. Randomized, 22 young normotensive; controlled AIx vs. control. study glucose, vs. Randomized, Heiss et al. [26] bloodhsCRP cell deformability 2015 double-blind, 22/60 M None Cocoa powder 900 128 2 vs. placebo; with controlled, significant changes in SBP, Koli DBPet al. [28] Randomized, 2015 controlled, 20 elderly 22 33–64 M+F Mildchronic hypertension Dark chocolate 602.70 ns 8No No significant changes crossover FMD vs. control; placebo; placebovs.♦baseline; 2015 crossover, 18 ns M + F Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] et al. [21] effect total Rassaf 2016 parallel-group 57 65 M+F hemodialysis Cocoa powder 900 ns 4 ♦ No (HFDC FMDon vs. control; placebo crossover in vs. baseline; )CAVI vs. or LFDC. End-stage renal disease Dark chocolate No changes in SBP, controlled, DBP vs. baseline control. PWV, total peripheral 2016 postprandial 20 62 M None 770 150 1 dose ♦ Dower et al. [20] Randomized, cholesterol, LDLc, TG, (pharmacological controlled control. ♦vs. No significant changes with chronic DBPresistance vs.AIx placebo. double-blind vs.vs. baseline. FMD vs. control; placebostudy medication) crossover in lipid profile, blood Rassaf et al. [21] 2016 Randomized, 57 65 M + F Hypertension hemodialysis Cocoa powder 900 ns 4 glucose, hsCRP Randomized, (grades I Dark chocolate + controlled, DBP vs.hsCRP control. placebo; FMD vs. control; NoDBP, evidence of beneficial De Jesús RomeroSBP, TG, glucose, blood insulin End-stage renal disease (pharmacological controlled, 2015 controlled, blind 79 42 M + F and II) treated with dehydrated red apples + 425.8 ns 24 Randomized, double-blind ♦SBP, No DBP, changes in SBP, 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi Prado et al. [24] 2015 Coronary heart disease, effects of chocolate PWV, ET1 et al. [27] vs. control with chronic vs. baseline. double-blind, open-label captopril ormedication) telmisartan green tea FMD vs. control; placeboDBP vs. placebo. ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on RassafBalboa-Castillo et al. [21] 2016 2015 57 1272 65 M + FM + F hemodialysis Cocoa powder 900 ns ns 4 3 years vs. control No significant changes No ♦ evidence of beneficial crossover Chocolate ns controlled, DBP vs.vs. control. DBP, SBP baseline; Randomized, study over Hypercholesterolemia, physical or mental (pharmacological Randomized, in on peripheral and central et al.et al. [22] Coronary heart disease, effects of chocolate FMD vs. control; Blinded, Symptomatic peripheral ♦ No Hammer double-blind ♦effect Improvement of Healthcontrolled, StrokeDiabetes, components of 2015 ns ns Dark chocolate ns ns 1 dose medication) placeboblood pressure ENRICA cohort 21 Hypertension, regular consumption on[25] 20 18–70 18–65 and ns None Cocoa powder 80–800 17–168 1 Grassi et al.Balboa-Castillo [24] 2015 SBP, DBP, PWV, ET1from randomized, artery disease microvascular function 1 dose; subjects status 2015 1272 M + F Chocolate ns ns 3 years double-blind, related of Life No evidence of Quality beneficial 2015 controlled, 40 18–43 ns None Cocoa extract 250 ns vs.orbaseline; Massee et al. [29] study over Hypercholesterolemia, physical mental et al. [22] control 4 vs. hypertensive to crossover double-blind, HDLc vs. of placebo; ♦of No significant changes Coronary heartStroke disease, effects chocolate components HealthRandomized, normotensive; Blinded, Symptomatic peripheral ♦ No effect on Hammer et al. Serum insulin parallel 21 in central ENRICA cohort 18–65 and ns Hypertension, Diabetes, regular consumption on Balboa-Castillo related Qualityblood of Lifeflow 2015 2015 ns Dark chocolate ns 602.70 ns ns 1 dose Koli et al. [28] 2015 randomized, controlled, 22 33–64 M++FF Mild hypertension Dark chocolate ns 8 1272 M Chocolate ns 3 years ♦ No significant changes artery disease microvascular function concentration vs. placebo;[25] study over Hypercholesterolemia, physical or mental et al. [22] vs. vs.baseline. placebo; crossover in CAVIHDLc vs. baseline; Randomized, Stroke components of HealthLarge artery elasticity Haptoglobin, total and Serum insulin ♦ No significant changes double-blind, related Quality of Life vs. placebo; proinflammatory concentration vs. placebo; in lipid profile, blood 2015 crossover, 18 ns M+F Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] ♦ vs. Nomonocyte effect on total HDLc placebo; CD62L Randomized, Large artery elasticity glucose, blood insulin postprandial cholesterol, LDLc, TG, expression Serum insulin double-blind, vs. placebo;in obese vs. baseline. McFarlin et al. Randomized, None/Overweight study glucose, hsCRP vs. women vs. 2015 Cocoa powder vs. placebo; 2015 crossover, 18 24 ns 22 M + FF Type 2 diabetes, Obese Cocoa beverage 480640 40 48 1 dose4concentration Basu et al. [23] ♦ No effect onbaseline; total ♦ No significant changes [30] double-blind Obesity placebo; Randomized, Endothelial Large artery elasticity postprandial cholesterol, LDLc, TG, Randomized, in peripheral and central ♦glucose, No changes in SBP, double-blind, microparticles in obese study vs. placebo; hsCRP vs. placeboblood pressure 2016

2015

32

M+F

Aprox. Comorbidities Mean Age Sex (years)

Duration (weeks)


8 of 25



Year

Aprox. No. of Mean Age Study DesignDiseases 2016, 4, 39 Participants controlled, (years)

2015

2015

2014

2014

2014

2014

2014

Sex

Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. Duration Cocoa Product TF (mg/day) EC (mg/day) Comorbidities (weeks) Intervention Aprox.

7 of 25

Outcomes

6 of 25

Ref.

vs. cocoa-free control chocolate. Outcomes Ref. 7 of 25 FMD, Randomized, FMD,arteriolar HDLcand vs. control; Sansone microvascular Plasma NT-proBNP double-blind, 100 Randomized, 44 M+F None Cocoa powder 450 64 4 SBP, DPB, PWV, total and Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. et al. [35] controlled, vs. cocoa-free control vasodilator capacity, red Randomized, (HFDC) vs. LFDC and controlled LDLc vs. control. double-blind, 22 young crossover chocolate. Heiss et al. [26] blood cell deformability 2015 M None Cocoa powder 900 128 2 double-blind, 1064 baseline; Aprox. 22/60 controlled, Chronic heart failure De Palma et al. No. of20 elderly Cocoa Product TF EC Duration arteriolar and 2nd vs. HDLc after placebo32 Dark chocolate (HFDC); ns 4 Year 2016 Study Design Mean Age70 Sex M + F Comorbidities Outcomes Ref. DBPFMD, vs.baseline; LFDC andthe parallel-group Participants (stable on GDMT) [19] Intervention (mg/day) (mg/day) (weeks) microvascular PWV, total peripheral controlled, 88 (LFDC) (years) baseline; Two consecutive Cocoa powder intervention vs. baseline; Randomized, None/Mild Sarriá vasodilator red vs. baseline. crossover ♦ resistance No change incapacity, platelet Plasma NT-proBNP controlled, 44 29 M +F rich in dietary 43.8/45.3 9.3/18.9 4 Plasma glucose and IL-1β in cocoa double-blind, 22 young hypercholesterolemia et al. [36] Heiss et al. [26] blood celland deformability 2015 22/60 M None Cocoa powder 900 128 2 (HFDC) vs. Hypertension (grades I Dark chocolate + function. controlled, 20 elderly crossover fibers/polyphenols product rich in dietary fiber Diseases 2016, 4,Randomized, 39 Randomized, 6 Jesús of 25RomeroDe SBP,LFDC DBP, TG, hsCRP vs. baseline; 2015double-blind, controlled, blind 79 42 M+F and II) treated with dehydrated red apples + 1064 425.8 ns 24 Randomized, baseline; parallel-group Prado et al. [27] vs. control group vs.peripheral baseline. Chronic heart failure De Palma et al. PWV, open-label captopril or telmisartan Dark chocolate green tea vs.total control; placebo 2016 placebo32 70 M+F (HFDC); ns 4 DBP vs.FMD LFDC and 2016 20 62 M None Dark chocolate 770 150 1 dose (stable on GDMT) [19] et al. [20] resistance baseline. Dower DBP, vs.vs. baseline; controlled controlled, AIxSBP vs. control. baseline; 1 mg/kg bw 88 (LFDC) Diseases 2016, 4, 39 6 of 25 Randomized, Pure flavanols Glucuronidated, sulfated, methylated Randomized, Hypertension (grades I Dark chocolate + ♦ Improvement of crossover crossover ♦ No change in platelet (EC, PCB1); Wiese De Jesús RomeroSBP, DBP, TG, hsCRP Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. double-blind, 7 24–31 M79 (monomers Single dose (-)EC and DHPV are predominant 2015 controlled, blind 42 None M + F End-stage and II) treated with dehydrated red apples + 425.8 ns 24 subjects status from renal disease function. Prado 6etof al.25 [27] vs. control 2 mg/kg et al. [37] Diseases 2016,Randomized, 4, 39 open-label captopril or telmisartan green tea hypertensive to crossover and polymers) metabolites in blood and urine. with chronic Randomized, Aprox. FMD vs. control; placebobw PPC TF Cocoa Product EC Randomized, No. of DBP, SBP vs. baseline; normotensive; 2016 57 Mean Age 65 Sex M + F Comorbidities hemodialysis Cocoa powder 900 ns Duration 4 FMD vs. control; Year Study placebo Design Outcomes Ref.Rassaf et al. [21] 1. Outcomes of recent (2014–2016) clinical trials cocoa products. controlled,Participants DBP vs. control. Dower 2016 2015 20 62 33–64 M Table None Dark chocolate 770 of602.70 150 1 dose 8 etKoli al. [20] (mg/day) et al. [28] controlled, 22 (years) M + Fand (pharmacological Mild hypertension Intervention Dark chocolate(mg/day) ns(weeks) ♦ Improvement of ♦ No significant changes controlled AIx vs. control. Randomised, None (young double-blind Cocoa Diseases 2016, 4, 39 crossover subjects in CAVI vs. status baseline; medication) Aprox. + EMP, +from − vs. baseline 6 of 25 Horn Plasma NT-proBNP double-blind, 40crossover 64/27/60 No.Mof+ F old)/Coronary 375 59 4 CD144 CD31 /412 Cocoa Product EC Duration Table 1. Outcomes beverage of recent (2014–2016) clinical trials ofTFcocoa products. ♦ No significant changes hypertensive to et al. [38] Year Study Design Mean Age Sex Comorbidities Outcomes Ref. No evidence of beneficial End-stage renal disease Randomized, crossover artery disease Participants Intervention (mg/day) (mg/day) (weeks)(HFDC) vs. LFDC and Randomized, in lipidnormotensive; blood (years) Coronary heart disease, effects ofprofile, chocolate Diseases 2016, 4,Randomized, 39 6 of 25 with chronic double-blind, 1064 baseline; Aprox. FMD vs. control; placeboChronic heart Mild failure De PalmaBalboa-Castillo etal. al. No. of 22 Cocoa Product Duration8 Koli 2015 ENRICA 33–64 M + F hemodialysis hypertension Dark chocolate 900 TF 602.70 ns EC ns glucose, blood insulin ♦ Noconsumption significant changes Diseases 4,cohort 39 7 of 25 18–65Age and Hypertension, Diabetes,Dark regular onvs. Plasma NT-proBNP Rassaf et [21] 2016 Cocoa powder Randomised, Oxidized LDLc control Diseases 4,2016, 39 controlled, 6 et ofal. 25[28] 2016 placebo32 57 1272 70 65 M +MF + F M chocolate (HFDC); ns 4 4 3 years DBP vs. LFDC and Year Study Design Mean Sex Outcomes Ref. 20152016, + F(stable on Comorbidities Chocolate ns ns controlled, DBPphysical vs. control. Ibero-Baibar [19] et al. [22] Participants Intervention (mg/day) (mg/day) (weeks) crossover invs. CAVI vs. baseline; 1.GDMT) Outcomes of recent (2014–2016) trials of cocoa products. study over Hypercholesterolemia, or mental (pharmacological (HFDC) vs.baseline. LFDC and controlled, 88 (LFDC) (years) baseline; parallel and 50 Randomized, 57 M+F None Table Cocoa powder 414.26 clinical 153.44 4 and vs. baseline; double-blind ♦ No significant changes et al. [39] ♦ No significant changes Stroke components of Healthmedication) double-blind, 1064 baseline; crossover ♦ No change in platelet Plasma NT-proBNP double-blind Diseases 2016, MPO, ICAM1 vs. baseline. 4, 39 6 of 25 controlled, vs.lipid cocoa-free control Chronic heart failure Aprox. in profile, blood De Palma et al. Randomized, inrelated peripheral and central Quality of Life 2016 placebo32 No. of 70 MTable +F Dark chocolate (HFDC); ns EC 4 Duration No evidence ofvs. beneficial DBP LFDC Cocoaclinical Product trials TF products. 1. Outcomes of recent (2014–2016) of cocoa function. (HFDC) vs. LFDCand and crossover chocolate. (stable on GDMT) [19] Year Study Design Mean Age Sex Comorbidities Outcomes Ref. Diseases 2016, 4, Randomized, 39 6 of 25 glucose, blood insulin placeboblood HDLc vs.pressure placebo; controlled, 88 1064 (LFDC) heart disease, effects of chocolate baseline; Non-randomized, Participants (mg/day) (weeks) Randomized, Table Coronary 1. Outcomes of recent (2014–2016) Intervention clinical trials of cocoa products.(mg/day) 1 dose; double-blind, baseline; FMD, arteriolar and De (years) None/ HDLc vs. baseline; 2016, 4, 39 crossover 6 of et 25 vs. baseline. Chronic Diabetes, heart Palma etal.al.[29] Martinez-López 2015 controlled, 40 18–43 nsHypertension, Nonefailure Cocoa extract 250 ns vs. baseline; Massee Aprox. ENRICA cohort consumption on Serum insulin ♦vs. No change in platelet FMD control; controlled,Diseases 44 29 1272 No. M + F18–65 and Cocoa powder Chocolate 45.3 18.9 ns nsEC 43 years placebo32 70 Dark chocolate 4 4 regular DBP vs. LFDC and Balboa-Castillo of Cocoa Product 770 ns(HFDC); TF Duration 2015 2016 placebo M + FM + F microvascular Plasma NT-proBNP 2016 20 62 M None Dark chocolate 150 1 dose Dower et al. al. [22] [20] ♦ No significant changes Hypercholesterolemia et al. [40] (stable on GDMT) [19] double-blind, ♦ No significant changes IL 10 vs. baseline. Year Study Design Mean Age Sex Hypercholesterolemia, Comorbidities Outcomes Ref. study over physical orfunction. mental et Aprox. concentration vs. placebo; Randomized, controlled AIx vs. control. controlled, 88 (LFDC) baseline; crossover Participants Intervention (mg/day) (mg/day) (weeks) Cocoa Product EC Duration Table 1. Outcomes of recent (2014–2016) clinical trials ofTF cocoa products. vasodilator red Randomized, No. of22 youngMean(years) (HFDC) vs.capacity, LFDC and parallel in central blood flow Randomized, in peripheral and central Randomized, Stroke components of HealthYear 2016, Study Design Age Sex Comorbidities Outcomes Ref.6 of 25 Diseases 4, Randomized, 39 double-blind, Large artery elasticity crossover ♦ No change in platelet Participants Intervention (mg/day) (mg/day) (weeks) Heiss et al. [26] blood cell deformability 2015 crossover 22/60 M None Cocoa powder 900 128 2 double-blind, baseline; Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa 1064 products. placeboblood pressure vs. baseline. Plasma NT-proBNP of Life double-blind, (years) FMD vs. control; controlled, 20 elderlyAprox. placebo SBP, DBP (HF, IF) vs. LF;De Palma et al. vs. placebo; heart failure End-stage renalChronic disease 1 dose;related Quality function. vs. baseline; baseline; 2016 62 18–43 None Dark chocolate 1 dose et al. et [20] 2016 Randomized, placebo32 M Dark chocolate ns Duration DBP vs. LFDC and Dower Randomized, 2015 controlled, ns+ F Type 2of None Cocoa extract 250 vs. Massee al. [29] No. of20 Cocoa Product TF 770 EC 150 TableMM 1. Outcomes recent (2014–2016) clinical trials of cocoa products. Haptoglobin, and crossover, 18 40 ns 70 diabetes, Cocoa beverage 480(HFDC); 40 ns 1 dose 44 HDLc Basu [23] (HFDC) vs. LFDC and parallel-group controlled vs. placebo; ♦ No effect ontotal total AIxNT-proBNP vs. control. Plasma (stable onObese GDMT) Plasma glucose concentrations chronic Year2015 Study Design Mean Age Sex + F with Comorbidities Outcomes Ref. et al. [19] Randomized, Aprox. PWV, total peripheral controlled, 88 (LFDC) FMD vs. control; baseline; placebodouble-blind, ♦ No significant changes Diabetes, Participants Intervention (mg/day) (mg/day) (weeks) proinflammatory postprandial double-blind, baseline; No. of Cocoa Product TF 1064 ns EC Duration crossover cholesterol, LDLc, TG, Serum insulin Randomized, (HFDC) vs. LFDC and Rassaf et al. [21] 2016Year 57 65 M + F hemodialysis Cocoa powder 900 4 (years) FMD vs. control; placebo (HF, IF) vs. LF; Chronic heart failure De Palma et al. Study Design Mean Age Sex Comorbidities Outcomes Ref. resistance vs. in baseline. Aprox. crossover ♦ No change platelet controlled, parallel Participants in central blood flow DBP vs. control. 2016 20 62 M None Dark chocolate 770 150 1 dose Dower et al. [20] monocyte CD62L Hypercholesterolemia study 2016 placebo32 70 M + F Dark chocolate (HFDC); ns 4 Intervention (mg/day) (mg/day) (weeks) DBP vs. LFDCvs. and glucose, hsCRP End-stage renal disease double-blind, baseline; No. of Cocoa Product EC Duration Table 1.(pharmacological Outcomes of (2014–2016) clinical trials TF of 1064 cocoa products. concentration vs. placebo; controlled AIx vs. Plasma NT-proBNP (stable onrecent GDMT) (years) Double-blind, 993 (HF); 185 (HF); Insulin resistance (HF,DeRef. IF) vs. LF; Diseases 4, 39Design 6 of[19] Randomized, Chronic heart failure Palma et25al. Year 2016, Study Mean Age Sex Comorbidities Outcomes Randomized, Hypertension (grades I Dark chocolate + function. double-blind vs.control. baseline. expression in obese controlled, 88 (LFDC) baseline; placebo; Mastroiacovo Hypertension with chronic Randomized, 2016 placebo32 70 M+F Dark chocolate (HFDC); ns Participants Intervention (mg/day) (mg/day) (weeks)4 De JesúsetRomeroSBP, DBP, TG, hsCRP McFarlin DBP vs. LFDC and Large artery elasticity medication) crossover Randomized, None/Overweight Randomized, (HFDC) vs. LFDC and Diseases 2016, 4, 39 61–85 7al.of 25 FMD vs. control; NT-proBNP placeboon GDMT) [19] 2015 controlled, blind and II) treated with dehydrated red apples + 95 425.8 controlled, 90 M Cocoa drinks 520 (IF); (IF); LDLc, TG, total cholesterol vs. LF; Randomized, Haptoglobin, total and Rassaf women vs. baseline; 2015 24+79F(years) 22 42 FFM + F (stable Cocoa powder 640 48 ns8 4 24 Plasma Aprox. 65(pharmacological crossover ♦ No change in platelet ♦ No changes in SBP, et al. et [21] 2016 57 M + hemodialysis Cocoa powder 900 ns 4 double-blind, controlled, 88 (LFDC) Prado al. [27] vs. control baseline; vs. placebo; et al. [41] of beneficial [30] double-blind Obesity End-stage renalordisease double-blind, 1064 baseline; No. of Cocoa Product TF EC Duration No evidence controlled, Randomized, (HFDC) vs. LFDC and DBP vs. control. open-label captopril telmisartan green tea Plasma NT-proBNP FMD vs. control; placebo parallel-arm 2015 48 (LF) 5 (LF) Plasma total 8-iso-prostaglandin F2α proinflammatory Endothelial Randomized, heart failure De Palma et al. Year Study Design Sex Comorbidities Ref. function. DBP vs.intotal placebo. (pharmacological crossover, 1832 ns70Age treatments), MM + +F F Coronary Type 2heart diabetes, Obese Cocoa beverage 480 40ns 1 dose Basu et al.Dower [23] crossover ♦No No change platelet ♦ effect on 2016 20 Mean 62 MChronic None Dark chocolate(mg/day) 770(mg/day) 150(weeks) 1 dose et al. [20] disease, effects ofOutcomes chocolate with chronic 2016 Diseases placeboDark chocolate (HFDC); 4 Participants Intervention DBP vs. LFDC and double-blind double-blind, 1064 baseline; DBP, SBP vs. baseline; Randomized, (HFDC) vs. LFDC and controlled 2016, 4, 39 7 of 25 AIx vs. control. monocyte CD62L FMD vs. control; placebomicroparticles in obese (stable on GDMT) [19] (years) (HF, LF)control vs. LF; Randomized, medication) Chronic hemodialysis heart failure De Palma etetal. Randomized, postprandial controlled, vs. cocoa-free function. cholesterol, LDLc, TG, ENRICA cohort 18–65 and Diabetes, consumption Balboa-Castillo al. [21] + FOutcomes powder 900products. 4 regular controlled, 88 (LFDC) baseline; smokers FMD vs.on control; placebo70 65Former DarkCocoa chocolate (HFDC); DBP vs. LFDC and 1.Hypertension, ofGDMT) recent (2014–2016) clinical trials of cocoa 1064 baseline; ♦ vs. Improvement of Rassaf crossover1272 32 57 20152016 2016double-blind, MTable +M F + FM Chocolate ns ns ns ns 3 years4 expression in obese controlled, overweight women control. Plasma NT-proBNP FMD vs. control; placebo (stable onNone/Overweight [19]et study controlled, Noand evidence of beneficial crossover chocolate. Circulating insulin (HF, vs. LF; glucose, hsCRP vs. Randomized, Chronic heart failure De al. study Hypercholesterolemia, orDBP mental et Palma al. IF) [22] McFarlin et al. Randomized, (pharmacological crossover ♦ No change in platelet 2016 20 24 over 62 22 M None renal disease Dark chocolate 770640 ns 17–168 150 48 4 1 dose Dower al.[24] [20] controlled, 88 (LFDC) 20 None Cocoa powder 80–800 1 4physical etetal. SBP, DBP, PWV, ET1 baseline; 2016 2015 placebo32 70 18–70 M + F ns Dark chocolate subjects status from Grassi DBP vs. LFDC and End-stage women vs. baseline; 2015 F Coronary Cocoa powder(HFDC); double-blind vs. baseline. Randomized, (HFDC) vs. LFDC and controlled double-blind, AIx vs. control. heart disease, effects of chocolate FMD vs. control; placebo; FMD, arteriolar and placebo (stable on GDMT) [19] Strokemedication) componentsfunction. ofvs. HealthRandomized, HDLc (HF,control IF) vs. LF.et al. [20] [30] double-blind Obesity controlled, cocoa-free Aprox.62 crossover ♦ No baseline; change in platelet vs. control 2016 20 M Nonewith chronic Cocoa Dark chocolate 770 1 dose controlled, 88 (LFDC) hypertensive to Dower Endothelial ♦ Novs. significant inon BP, Product TF EC 150 Duration double-blind, 1064 baseline; crossover crossover ENRICA cohort No. of 18–65 and Hypertension, Diabetes, regular consumption Balboa-Castillo ♦ No changes in SBP, controlled microvascular related Quality of Life AIx control. FMD vs. control; placebocrossover chocolate. evidence of beneficial De Ref. Year Study Design Mean Age Sex M + FM Chronic Outcomes Randomized, heart hemodialysis failure Palma Rassaf et al. et al. [21] 1272 57 Chocolate ns 900 ns ns 3 years ♦4 No No function. Randomized, crossover normotensive; Randomized, +Comorbidities F Symptomatic Cocoa powder change in platelet microparticles in obese Randomized, platelet function, liver Randomized, 2016 2015 2016 Participants (mg/day) (mg/day) (weeks) placebo32 70 over 65M + F Dark chocolate (HFDC); ns 4 DBP vs. LFDC and End-stage renal disease Intervention study Hypercholesterolemia, physical or mental et al. [22] Blinded, peripheral ♦placebo. No effect oncontrol. Hammer et al. DBP vs. crossover vasodilator capacity, red [19] controlled, DBP vs. vs. placebo; heart disease, effects of chocolate FMD, arteriolar and (years) ns 33–64 vs. control; placebo Sarría onMild GDMT) HDLc vs. baseline; Randomized, 20152015 21young ns M + (stable DarkDark chocolate ns 602.70 ns ns 1 dose 8 HDLcFMD Randomized, Koli et al. [28] controlled, F Coronary hypertension chocolate88 (LFDC) double-blind, ♦ No significant changes (pharmacological function. and overweight women parallel-arm, panel (albumin, bilirubin, 2016 2015 20 22 62 M None chocolate 770 9.3 150 Dower et al. [20] controlled, with chronic Stroke components of Healthbaseline; controlled, 44 29 M +22F None/Hypercholeste-romia Cocoa powderDarkFood-grade 44.1 randomized, artery disease microvascular function [25] Randomized, Heiss et al. [26] blood cell deformability 22/60 M End-stage None Cocoa powder 900 128 4 1 dose 2 Plasma renal disease double-blind Serum insulin ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo Ottaviani et al. cocoa microvascular controlled FMD vs. control; AIx vs. control. NT-proBNP placeboFMD control; placebo crossover et al. [42] FMD vs. control; Glucose, and IL-10 vs. baseline. inIL-1β CAVI vs. baseline; controlled, 2057 elderly Randomized, medication) Randomized, vs. baseline. 2015 1272 M + F Chocolate ns ns 3 years 2015 double-masked, 46 35–55 M + F None 2000 220 12 ALT, AST, AlkPhos), Randomized, crossover Rassaf et al. 2016 65 M + F hemodialysis Cocoa powder 900 ns 4 related Quality of Life ♦ No change in platelet 2016 20 62 M None Dark chocolate 770 150 1 dose Dower et al. [20] vs.orbaseline; controlled, with chronic crossover 2015 study over Hypercholesterolemia, physical mental etal.al.[24] [22] [21] [31] extract concentration vs. placebo; vasodilator capacity, red crossover controlled, Randomized, (HFDC) vs. LFDC and DBP vs. control. controlled FMD vs. control; ♦ No significant changes 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi et AIx vs. control. parallel-group placeboSBP, DBP, PWV, ET1 FMDfunction. vs. control; No evidence of beneficial placebo controlled dietary metabolic markers ♦ No significant in BP, double-blind, (pharmacological HDLc vs. placebo; double-blind, Rassaf al. [21] hemodialysis powder PWV, total peripheral 20162016 Randomized, 20 57 22 young62 65 MM + F None DarkCocoa chocolate 770 900 1 dose 4 Dower et al.etHeiss [20] Stroke components of HealthLarge artery elasticity et al. [26] blood cell deformability 2015 22/60 M None Cocoa powder 1064 900 150 ns 128 2 End-stage renal disease double-blind double-blind, baseline; crossover in lipid profile, blood controlled, DBP vs. control. vs. Coronary effects of chocolate controlled AIx vs.control control. Randomized, platelet function, liver intervention (glucose, hemoglobin, controlled, 20 elderly Randomized, medication) Chronic heart failure heart disease, De Palma et al. Randomized, crossover (pharmacological Serum insulin resistance vs.ofbaseline. double-blind, related Quality Lifeinsulin vs. baseline; vs. placebo; withHypertension, chronic 2016 placebo32 70 M +and F Dark chocolate (HFDC); ns 4 DBP vs. LFDC and glucose, blood +, K+, on End-stage renal disease Diabetes, double-blind ENRICA cohort 18–65 regular consumption Balboa-Castillo crossover parallel-arm, panel (albumin, bilirubin, hematocrit, urea, Na parallel-group FMD vs. control; placebo(stable on GDMT) [19] FMD vs. control; No evidence of beneficial placebo Randomized, medication) Blinded, Symptomatic peripheral ♦ No effect onvs. Hammer et al. concentration placebo; 2015 1272ns 62 M + F Chocolate ns ns 3 years Randomized, 18 20 Hypertension (grades I Dark chocolate + 20152016 crossover, M + FM Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] Ottaviani et al. Food-grade cocoa ♦ No effect on total HDLc vs. placebo; PWV, total peripheral Rassaf et al. [21] 2016 57 65 + F hemodialysis Cocoa powder 900 ns 4 controlled, 88 (LFDC) M None Dark chocolate chocolate 770 dose Dower et [20] baseline; vs. baseline. with chronic 2015 21 ns Dark ns ns study overns Hypercholesterolemia, physical or mental et al. [22] Deal. Jesús RomeroSBP, DBP, TG, hsCRP End-stage renal disease 2015 double-masked, 46 35–55 M + F artery None 2000 150 220 11 dose 12microvascular ALT, AST, AlkPhos), controlled, DBP vs.control. control. Coronary disease, effects of chocolate controlled vs. control; AIx vs. placeboRandomized, randomized, disease function [25] No FMD evidence of beneficial Large artery elasticity 2015 controlled, blind 79 42 M + F (pharmacological and II)heart treated with dehydrated red apples + 425.8 ns 24 Randomized, [31] extract cholesterol, LDLc, TG, resistance vs. of baseline. insulin crossover ♦ No change in platelet Rassaf etPrado al. [21] 2016 postprandial 65 M+F hemodialysis Cocoa powder 900 ns 4 ♦Serum No significant changes components Healthet al. [27] vs. control with chronic Stroke controlled dietary 57 metabolic markers double-blind ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo crossover controlled, double-blind, DBP vs. control. Coronary heart disease, effects of chocolate open-label captopril or telmisartan green tea FMD vs. control; vs. placebo; placebo- Randomized, hsCRP vs. vs. medication) Hypertension (grades I Dark chocolate + 900 concentration placebo; 2015 study 1272 M + F (pharmacological Chocolate ns ns 3 yearsglucose, function. Randomized, in(glucose, peripheral andof central related Quality Life et al.De Rassaf 2016 2015 57 hemodialysis Cocoa powder ns intervention hemoglobin, Jesús RomeroSBP, DBP, TG, study over M + F M + F End-stage Hypercholesterolemia, physical oron mental renal disease double-blind crossover, 18 79 65 ns 2 and diabetes, Obese Cocoa beverage 40 ns 4 1 dose 24No Basuet[21] etal. al.[22] [23] ENRICA cohort blind 18–65 and Diabetes, regular consumption on hsCRP Balboa-Castillo ♦vs. No effect total controlled, DBP control. DBP, SBP vs. baseline; placebo; Randomized, 2015 controlled, 42 M + F M (pharmacological +Hypertension, F Type II) treated with dehydrated red apples + ns480 425.8 evidence of beneficial Large artery elasticity Randomized, medication) placeboblood pressure 2015 Randomized, 1272 Chocolate ns 3 years HDLc vs. placebo; hematocrit, urea, Na+, K+, et al.Prado vs. control Stroke components of Healthwith chronic postprandial 1♦ dose; studyopen-label over Hypercholesterolemia, physical or mental [22] et al. [27] cholesterol, LDLc, TG, double-blind ♦ Improvement of No changes in SBP, double-blind, captopril or telmisartan green tea Coronary heart disease, effects of chocolate FMD vs. placebo; vs.vs. control; placebo- controlled, placebo 2015 40 18–43 ns medication) None Cocoa extract 250 ns vs. baseline; Massee et al. [29] NoFMD evidence ofcontrol; beneficial Serum insulin related Quality of Life Rassaf et al. [21] 2016 2015 57 65 M + F hemodialysis Cocoa powder 900 ns 4 2016 20 62 M None Dark chocolate 770 150 1 dose Dower et al. [20] study 4DBP components of Healthglucose, hsCRP vs.from subjects status vs.♦ placebo. crossover, ns M + F Coronary Typeheart 2Stroke diabetes, Obese Cocoa beverage 480 40 1 dose No Basu et al. [23] ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo No effect on vs. total DBP, SBP baseline; controlled, controlled DBP vs. control. double-blind, 1272 18 ♦ No significant changes AIx vs. control. disease, effects of chocolate evidence of beneficial 2015 M+F Chocolate ns ns 3 years (pharmacological HDLc vs.ofplacebo; relatedconcentration Quality Lifevs.toplacebo; placebo; Randomized, postprandial study over Hypercholesterolemia, physical or mental et al. [22] LDLc, double-blind ♦central Improvement of crossover parallel inhypertensive blood flow ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption onTG, Balboa-Castillo Coronary heart disease, effects of chocolate Randomized, FMDcholesterol, vs. control; Large artery elasticity medication) 2015 1272 M + F Chocolate ns ns 3 years Serum insulin ♦ No changes in SBP, HDLc vs. placebo; Randomized, normotensive; controlled, study Stroke components ofvs. Healthglucose, hsCRP vs.Grassi subjects status from baseline. study over Hypercholesterolemia, physical or mental et al. al. [24] [22] End-stage renal disease ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo double-blind, 20 18–70 ns None Cocoa powder 80–800 17–168 1 et 2015 DBP, PWV, ET1 vs. placebo; No related evidence beneficial Randomized, concentration DBP vs. placebo. Koli et al. [28] 2015 controlled, 1272 22 33–64 M + F with chronic Mild hypertension Dark chocolate ns 602.70 ns ns 3 years 8 SBP, 2015 M+F Chocolate Serum insulin ♦ Noof significant changes double-blind, Quality of vs. Lifeplacebo; placebo; hypertensive to and Stroke components Healthtotal study Hypercholesterolemia, physical orHaptoglobin, mental et al. [22] Basu et al. [23] 2015 crossover, 18 over ns M +F Typedisease, 2 diabetes, Obese Cocoa beverage 480 40 1 dose ♦of No effect on total vs. control FMD vs. Coronary heart effects of chocolate placeboRandomized, Large artery elasticity Randomized, crossover incontrol; CAVI vs. baseline; concentration vs. placebo; crossover ♦ No changes in SBP, Randomized, HDLc vs. placebo; normotensive; related Quality of Life Rassaf et al. [21] 2016 57 65 M + F hemodialysis Cocoa powder 900 ns 4 FMD vs. control; Stroke Diabetes, components ofproinflammatory Healthpostprandial cholesterol, LDLc, TG, ENRICA cohort 18–65 and Hypertension, regular consumption on Balboa-Castillo controlled, double-blind, DBP vs. control. vs. placebo; controlled, Randomized, ♦DBP No significant changes Large artery elasticity Blinded, Symptomatic peripheral effect on Hammer et al.Koli vs. placebo. al. [28] 2015 controlled, 33–64 M(pharmacological +F Mild hypertension Dark chocolate 602.70 ns 17–168 ns 3 years 1 8 ♦ No ♦ No changes Chocolate Serum insulin HDLc vs. placebo; monocyte CD62L ns None Cocoa powder et al.et[24] Quality ofsignificant Life SBP, DBP, PWV, ET1 study 21 1272 2018 22 ns over18–70 glucose, hsCRP vs. etGrassi 20152015 2015 nsM + F M Dark chocolate ns ns 80–800 ns 1 dose 1 dose related study Hypercholesterolemia, physical or mental al. [22] double-blind 2015 crossover, ns + F Type 2 diabetes, Obese Cocoa beverage 480 40 Basu et al. [23] ♦ No effect on total double-blind, double-blind, in lipid profile, blood vs. in placebo; randomized, artery disease microvascular function crossover CAVI vs. in baseline; Randomized, medication) concentration vs. placebo; expression obese [25] Serum insulin vs. HDLc vs. placebo; placebo; FMD vs. control; Stroke components ofcontrol Healthpostprandial McFarlin et al. Randomized, None/Overweight cholesterol, LDLc, TG, crossover 2015 crossover, 18 ns M + F Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] glucose, blood insulin ♦ No effect on total ♦ofNo significant changes controlled, Randomized, No evidence beneficial Large artery elasticity women vs. baseline; F powder 4concentration vs. placebo; ♦ No changes in SBP, Grassi Serum insulin 20 24 18–70 22 ns None CocoaCocoa powder 80–800 640 17–168 48 1 et al. [24] 2015 2015postprandial related Quality of Life SBP, DBP, PWV, study double-blind Obesity glucose, hsCRP vs. Blinded, Symptomatic peripheral ♦ No effect on al. vs. baseline. cholesterol, LDLc, TG,ET1 in lipid profile, blood Hammer et[30] double-blind, double-blind, effects ofartery chocolate vs. placebo; Endothelial 2015 Randomized, 21 ns ns Coronary heart disease, Dark chocolate ns ns 1 doseconcentration Large elasticity DBP vs. placebo. vs. placebo; vs. control HDLc vs. placebo; study randomized, artery disease microvascular function ♦effect No significant hsCRP vs. changes glucose, blood insulin crossover 2015 ENRICA crossover, 18 ns M + F Hypertension, Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al.[25] [23] cohort 18–65 and Diabetes, regular consumption on Balboa-Castillo ♦glucose, No on total double-blind, microparticles in obese vs. placebo; Randomized, Randomized, Large artery elasticity 2015 M+F Chocolate ns ns 3 years ♦ No changes in SBP, Serum insulin Randomized, 1272 inor peripheral and central placebo; FMD vs. vs. baseline. postprandial Blinded, Symptomatic peripheral Cocoa beverage ♦ No effect on control; study overns physical mental et al.Hammer [22] cholesterol, LDLc, TG, 2015 2015double-blind, crossover, 18 21 M + F ns Hypercholesterolemia, Type 2 diabetes, Obese 480 ns 40 ns 1 dose Basu et al. [23]et al. and overweight women ♦ vs. Noplacebo; effect on total controlled, ns 18–70 Dark chocolate 1 dose concentration DBP vs. placebo. vs. placebo; placeboblood pressure ♦ No changes in SBP, 20 ns None Cocoa powder 80–800 17–168 1 Grassi 2015 SBP, PWV, ET1 ♦ of No significant changes study randomized, arteryObese disease microvascular function [25] et al. [24] Stroke components Healthglucose, hsCRP vs. postprandial vs.DBP, baseline. cholesterol, LDLc, TG, 2015 crossover, ns M+F Type 2 diabetes, Cocoa beverage 480 40 1 dose 1 dose; Basu et al. [23] double-blind, 18 ♦ No effect on total Randomized, Randomized, Large artery elasticity 2015 controlled, 40 18–43 ns None Cocoa extract 250 ns vs. baseline; Massee et al. [29] DBP vs. placebo. vs. control in♦ peripheral and central related Quality of Life placebo; FMD vs. control; study Randomized, 4 cholesterol, No significant in BP, glucose, hsCRP vs. postprandial crossover LDLc, TG, double-blind, controlled, double-blind, ♦ No significant changes vs. placebo; Randomized, placeboblood pressure ♦ No changes in SBP, HDLc vs. placebo; 20 18–70 ns None peripheral Cocoa powder 80–800 17–168 11 dose; et al. [24] 2015 study SBP, DBP, PWV, Randomized, platelet function, liver Grassi placebo; FMD vs. Blinded, Symptomatic ♦control; No effectET1 on Hammer et al. glucose, hsCRP vs. 2015 crossover, 18 ns Type Cocoa beverage 480 Basu et al. [23] et al. double-blind, parallel in central blood flow ♦ No effect on total controlled, 2015 21 nsM + F ns 2 diabetes, Obese Dark ns 40 ns 1 dose 1 dose controlled, 40 ns None Cocoachocolate extract vs. baseline; Massee [29] DBP vs. placebo. Serum insulin parallel-arm, panel (albumin, vs. control ♦ No changes in SBP, 20 18–70 18–43 ns None Cocoa powder 80–800 250 17–168 ns 1 Grassi et al. [24] 2015 2015 SBP, DBP, PWV, ET1bilirubin, randomized, artery disease microvascular function [25] et al. placebo; 4 cholesterol, postprandial crossover Ottaviani Food-grade cocoa vs. baseline. LDLc, TG, crossover Year Design Diseases Study 2016, 4, 39

2015

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Table 1. Cont.

No. of Participants

Mean Age (years)

Sex

Comorbidities

Cocoa Product Intervention

TF (mg/day)

EC (mg/day)

Duration (weeks)


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Table 1. Cont. Diseases 2016, 4, 39

Year

Study Design

No. of Participants controlled,

Aprox. Mean Age (years)

7 of 25

Sex

Comorbidities

Cocoa Product Intervention

TF (mg/day)

EC (mg/day)

Duration (weeks)

Outcomes

Ref.

vs. cocoa-free control 7 of 25 chocolate. FMD, and of the retinal venules in Meanarteriolar dilatation Randomized, age-matched controlled, vs. microvascular cocoa-free control controls, but not in single-blinded,DiseasesRandomized, Terai vasodilator capacity, red 2016, 7 of 25 crossover chocolate. 2014 60 4, 39 65 ns None/Glaucoma Dark chocolate ns ns 1 dose glaucoma patients; double-blind, 22 young prospective2015 et al. [43] Heiss et al. [26] blood cell deformability 22/60 M None Cocoa powder 900 128 2 FMD, arteriolar and controlled, 20 elderly Venous vasodilatation in control group, vs. baseline; placebo-controlled microvascular parallel-group but not incontrol the glaucoma group. controlled, vs. cocoa-free Randomized, PWV, total peripheral vasodilator chocolate. capacity, red crossover double-blind, 22 young resistance vs. baseline. Randomized,2015 Dark chocolate Heiss et al. [26] blood cell deformability 22/60 M None Cocoa powder 900 128 2 FMD, and basal and peak Overweight, West Basalarteriolar blood flow, controlled, 20 elderly Randomized, Hypertension (grades I Dark chocolate + baseline; 2014 double-blind, 2 30 52 M+F and sugar-free 814 73.6 4 De Jesús RomeroSBP,vs. DBP, TG, hsCRP microvascular parallel-group Moderate obesity et al. [44] diameter of the brachial artery, vs. baseline 2015 controlled, blind 79 42 M + F and II) treated with dehydrated red apples + 425.8 ns 24 Randomized, PWV, total peripheral Prado et al. [27] vs. control period, crossover cocoa powder vasodilator capacity, red open-label captopril or telmisartan green tea double-blind, 22 young resistance vs. baseline. Heiss et al. [26] blood 2015 22/60 M None Cocoa powder 900 128 2 DBP, SBPcell vs. deformability baseline; controlled, index; 20 elderly Abbreviations: Aix, augmentation ALT, alanine aminotransferase; AST, Iaspartate aminotransferase; AlkPhos, alkaline phosphatase; CAVI, cardio-ankle vascular index; Randomized, Hypertension (grades Dark chocolate + vs.TG, baseline; De Jesús RomeroSBP, DBP, hsCRP ♦ Improvement of parallel-group 2015 controlled, blind 79 42 M + F and II) treated with dehydrated red apples + 425.8 ns 24 CHD, chronic heart disease; DBP, diastolic blood pressure; DHPV, 5-(3’,4’-dihydroxyphenyl)-valerolactone; EC, epicatechin; EMP, endothelial microparticles; ET1, endothelin 1; PWV, totalfrom peripheral Prado et al. [27] vs. status control subjects open-label captopril or telmisartan green tea resistance vs.tobaseline. F, females; FBG, fasting blood glucose; FMD, flow-mediated dilation; GDMT, guideline-directed medical therapy; HDLc, high density lipoprotein-cholesterol; HFDC, high-flavanol hypertensive DBP, SBP vs. baseline; Randomized, Hypertension (grades I Dark chocolate + Randomized, De intermediate Jesús RomeroSBP,interleukin; DBP, TG, dark chocolate; HF, high flavanol intake;79hsCRP, highly sensitive C-reactive protein; ICAM-1, intercellular adhesion molecule 1;♦normotensive; IL, flavanol Improvement of hsCRP IT, 2015 controlled, blind 42 M + F and II) treated with dehydrated red apples + 425.8 ns 24 KoliPrado et al. [28] 2015 controlled, 22 33–64 M+F Mild hypertension Dark chocolate 602.70 ns 8 ♦ No significant changes et al. [27] vs. control subjects status from intake; LDLc, low density lipoprotein-cholesterol; LF, low flavanol intake; LFDC, low-flavanol dark chocolate; M, males; MPO, myeloperoxidase; NF-κB, nuclear factor open-label captopril or telmisartan green tea crossover in CAVI vs. baseline; hypertensive to DBP, SBP vs. baseline; ♦ No significant kappa-light-chain-enhancer of activated B cells; ns, not specified; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PCB1, procyanidin B1;changes PPC, pure polymeric procyanidin Randomized, normotensive; Improvement in lipid♦profile, blood of fraction from cocoa; pulse wave velocity; SPB, systolicM blood pressure; TF, total flavanols. Koli et al. [28] 2015 PWV, controlled, 22 33–64 +F Mild hypertension Dark chocolate 602.70 ns 8 ♦ No significant changes subjects glucose, bloodstatus insulinfrom crossover in CAVI vs. baseline; to vs. hypertensive baseline. ♦ No significant changes Randomized, normotensive; ♦ No significant changes in lipid profile, blood Koli et al. [28] 2015 Randomized, controlled, 22 33–64 M+F Mild hypertension Dark chocolate 602.70 ns 8 ♦ No significant changes in peripheral and central glucose, blood insulin crossover in CAVI vs. baseline; placeboblood pressure vs. baseline. 1 dose; ♦ No significant changes 2015 controlled, 40 18–43 ns None Cocoa extract 250 ns vs. baseline; Massee et al. [29] ♦ No significant changes 4 in lipid profile, blood double-blind, ♦ No significant changes Randomized, in peripheral and central glucose, blood insulin parallel in central blood flow placebobloodvs. pressure baseline. 1 dose; vs. baseline. 2015 controlled, 40 18–43 ns None Cocoa extract 250 ns vs. baseline; Massee et al. [29] ♦ No significant changes 4 Haptoglobin, total and double-blind, ♦ No significant changes Randomized, in peripheral and central parallel inproinflammatory central blood flow placeboblood pressure monocyte CD62L 1 dose; vs. baseline. 2015 controlled, 40 18–43 ns None Cocoa extract 250 ns vs.in baseline; Massee et al. [29] expression obese 4 Haptoglobin, total and McFarlin et al. Randomized, None/Overweight double-blind, ♦ No significant women vs. baseline;changes 2015 24 22 F Cocoa powder 640 48 4 proinflammatory [30] double-blind Obesity parallel in central blood flow Endothelial monocyte CD62L vs. baseline. microparticles in obese expression in obese Haptoglobin, total and McFarlin et al. Randomized, None/Overweight and overweight women women vs. baseline; 2015 24 22 F Cocoa powder 640 48 4 [30] double-blind Obesity vs.proinflammatory baseline. Endothelial monocyte CD62L ♦ No significant in BP, microparticles in obese expression in obese Randomized, platelet function, liver McFarlin et al. Randomized, None/Overweight and overweight women women vs. baseline; 2015 parallel-arm, 24 22 F Cocoa powder 640 48 4 panel (albumin, bilirubin, [30] double-blind Obesity vs. baseline. Ottaviani et al. Food-grade cocoa 2015 double-masked, 46 35–55 M+F None 2000 220 12 ALT, AST,Endothelial AlkPhos), ♦ No significant in in BP, [31] extract microparticles controlled dietary metabolic markers obese Randomized, platelet function, liver and overweight women intervention (glucose, hemoglobin, parallel-arm, panel (albumin, bilirubin, baseline. Ottaviani et al. Food-grade cocoa hematocrit,vs. urea, Na+, K+, 2015 double-masked, 46 35–55 M+F None 2000 220 12 ALT, AST, AlkPhos), ♦ No significant in BP, [31] extract controlled dietary metabolic markers Randomized, platelet function, liver intervention (glucose, hemoglobin, parallel-arm, panel (albumin, bilirubin, Ottaviani et al. Food-grade cocoa hematocrit, urea, Na+, K+, 2015 double-masked, 46 35–55 M+F None 2000 220 12 ALT, AST, AlkPhos), [31] extract controlled dietary metabolic markers intervention (glucose, hemoglobin, hematocrit, urea, Na+, K+,

Diseases 2016,crossover 4, 39

Diseases 2016, 2016, 4, 4, 39 x Diseases

10 of of 25 25 10

Figure 5.5.MainMain cardioprotective properties of cocoaof polyphenols. Abbreviations: ACE, angiotensinFigure cardioprotective properties cocoa polyphenols. Abbreviations: ACE, converting enzyme; EPCs, endothelial progenitor cells; progenitor ET-1, endothelin FMD, flow-mediated angiotensin-converting enzyme; EPCs, endothelial cells; 1; ET-1, endothelin 1; dilation; HDLc, high-density lipoprotein-cholesterol; ICAM, intercellular adhesion molecule; FMD, flow-mediated dilation; HDLc, high-density lipoprotein-cholesterol; ICAM, intercellular adhesion IL, interleukin; LDLc, low-density lipoprotein-cholesterol; LOX, lipooxygenase; LTs, leukotrienes; molecule; IL, interleukin; LDLc, low-density lipoprotein-cholesterol; LOX, lipooxygenase; LTs, MMP-2, matrix metalloproteinase 2; MPO, myeloperoxidase; NADPH, NADPH, reduced nicotinamide adenine leukotrienes; MMP-2, matrix metalloproteinase 2; MPO, myeloperoxidase; reduced nicotinamide dinucleotide phosphate; NF-κB, nuclear of activated B cells; adenine dinucleotide phosphate; NF-κB, factor nuclearkappa-light-chain-enhancer factor kappa-light-chain-enhancer of activated NO, nitric oxide; Nrf2, nuclear factor erythroid-related factor 2; PGI2, prostaglandin I2; 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 PLC, phospholipase C; TG, triglycerides; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; XO, XO, xanthine xanthine oxidase. oxidase. molecule;

4.1. Antioxidant Antioxidant Activity Activity 4.1. Oxidative stress stress is is actively actively involved involved in in CVDs CVDs pathogenesis. pathogenesis. The The increase increase of of reactive reactive oxygen oxygen Oxidative species (ROS) production, mainly of superoxide anion radical, affects NO metabolism and facilitates species (ROS) production, mainly of superoxide anion radical, affects NO metabolism and facilitates endothelial dysfunction dysfunction [45]. [45]. Besides, Besides, lipid lipid peroxidation peroxidation contributes contributes essentially essentially to to the the initiation initiation and and endothelial progressionofofatherosclerosis. atherosclerosis. Cocoa is asource rich source of antioxidants. The assessment of the progression Cocoa is a rich of antioxidants. The assessment of the antioxidant antioxidant capacity of 1113 different foods showed that of 50 products exhibiting the highest activity, capacity of 1113 different foods showed that of 50 products exhibiting the highest activity, five products fivecocoa-based products are[12]. cocoa-based [12]. Cocoa has a higheractivity antioxidant activity green tea are Cocoa powder has a powder higher antioxidant than green teathan (1000 ORAC (1000compared ORAC units compared to 800 and ORAC the content of total polyphenols is positively units to 800 ORAC units), the units), contentand of total polyphenols is positively correlated with correlated with ORAC value [14]. Cocoa polyphenols mainly flavanols and oligomeric ORAC value [14]. Cocoa polyphenols mainly flavanols and oligomeric proanthocyanidins have shown proanthocyanidins have shownproperties. significantThey in vitro properties. They act as(DPPH, scavengers of significant in vitro antioxidant act antioxidant as scavengers of various radicals ABTS, various radicals (DPPH, ABTS, superoxide anion, peroxynitrite, hypochloride), inhibit lipid superoxide anion, peroxynitrite, hypochloride), inhibit lipid peroxidation and chelate pro-oxidants 2+) [4,46]. The main structural features of peroxidation chelate pro-oxidants metalfeatures ions (Fe 2+ ) [4,46]. metal ions (Feand The main structural of polyphenols that are responsible for their polyphenols are responsible their ROS quencher activity are catechol groups, phenolic ROS quencherthat activity are catecholfor groups, phenolic quinoid tautomerism and delocalization of quinoid tautomerism and delocalization of electrons [14]. Epicatechin enhances plasma antioxidant electrons [14]. Epicatechin enhances plasma antioxidant capacity and protects erythrocyte membrane capacity and protects erythrocyte membrane from lipid peroxidation. Also, flavanol monomers and from lipid peroxidation. Also, flavanol monomers and oligomeric proanthocyanidins protect against oligomeric proanthocyanidins protect against in vitro erythrocyte hemolysis induced by 2,2’-azobis in vitro erythrocyte hemolysis induced by 2,2’-azobis (amidopropane)-dihydrochloride (AAHP) [14].


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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)


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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) [53–55]. 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


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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


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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


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(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].


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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


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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).


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Table 2. Outcomes of clinical trials of epicatechin (2016–2006). Year

Year

2016

No. of Aprox. Mean Aprox. ParticipantsNo. of Age (years)

Study Design

Study Design

Randomized, multicenter, Randomized, 30 placebo-controlled, double-blinddouble-blind, 2016

Participants

placebocontrolled, crossover


2015

Mean Age (years)

18–55 32

Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. Form of EC Duration Comorbidities EC (mg/day) Intervention Cocoa Product TF EC(weeks)Duration

Sex

Sex

M+F 70

M+F

Comorbidities

Hypertriglyceridemia Chronic heart failure (stable on GDMT)

Intervention

(mg/day)

Capsules

100

Dark chocolate

1064 (HFDC); 88 (LFDC)

(mg/day)

(weeks)

4 ns

4

9 23–68 ↑ Single-dose; Non-randomized (single-dose); (single-dose); Dissolved in ns None 50; 100 multiple-dose, Randomized, open label 8 22–45 Table 1. Outcomes of recent (2014–2016)water clinical trials of cocoa products. 5 days placebo 20164, 39 62 M None Dark chocolate 770 150 1 dose (multiple-dose)20 (multiple-dose) Diseases 2016, controlled Aprox. Mean Age (years)

Ref. Ref.

Plasma NT-proBNP ratio, Gutiérrez-Salméan TG, TG/HDLc (HFDC) vs. vs. LFDC and et al. [81] hsCRP baseline baseline; De Palma et al. DBP vs. LFDC and [19] 6 of 25 ↑ Plasmabaseline; nitrite vs. baseline; ♦ No change in platelet Platelet mitochondria complexes I, function. IV, citrate synthase activities Barnett

(multi-dose) vs. baseline; et. al. [82] FMD vs. control; Dower et al. [20] ↑ Plasma follistatin levels 6 of 25 AIx vs. control. (multi-dose) vs. baseline.

Cocoa Product TF EC Duration Comorbidities Outcomes Ref. Intervention (mg/day) (mg/day) (weeks) End-stage renal disease with chronic Plasma NT-proBNP FMD vs. control; Dower Plasma soluble endothelial of recent (2014–2016) Cocoa clinical trials of cocoa100 products. ns 4 Rassaf 6etof al.25 [21] 57 M++Outcomes F hemodialysis powder 900 4 2015 40–80 65 TableM1. F Pre-hypertension Capsules (HFDC) vs. LFDC andcontrol DBP vs. control. et al. [83] selectin vs. (pharmacological 1064 baseline; Aprox. Chronic heart failure De Palma et al. Cocoa Product TF EC Duration medication) placebo32 70 Dark chocolate (HFDC); ns 4 Year 2016 Study Design Mean Age Sex M + F Comorbidities Outcomes Ref. DBP vs. LFDC and (stable on GDMT) [19] Intervention (mg/day) (mg/day) (weeks) Participants No evidence of beneficial controlled, 88 (LFDC) (years) Randomized, baseline; Coronary heart disease, effects of chocolate Table 1. Outcomes of recent (2014–2016) clinical trials of cocoa products. crossover ♦ No change in platelet Plasma NT-proBNP double-blind, Dower Fasting plasma insulin and insulin ENRICA cohort 33 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo 2015 40–80 MM++ F None Capsules 100 4 function. 2015 1272 F Chocolate ns ns 3 years Randomized, (HFDC) vs. LFDC and vs. control placebo-controlled, et al. [84] resistance Aprox. study over Hypercholesterolemia, physical or mental et al. [22] No. of Cocoa Product TF EC Duration Randomized, double-blind, 1064 baseline; Diseases 2016, 4, Design 39 crossover Year Study Mean Age Sex ChronicComorbidities Outcomes of HealthStroke components heart failure De Palma Ref. et al. 6 of 25 Intervention (mg/day) Participants FMD vs. and control; 2016 2016,placebo32 70 M+F Dark chocolate (HFDC); ns(mg/day) 4(weeks) DBP vs. LFDC Diseases 4, 39placebo (years) related Quality of Life Dower 2016 20 62 M None Dark chocolate 770 150 1 dose et of al. 25 [20] (stable on GDMT) [19] 7 controlled controlled, 88 (LFDC) AIx vs. control. Pilot, baseline; Plasma NT-proBNP HDLc vs. placebo; Gutiérrez-Salméan Blood glucose and TG after crossover crossover ♦ No change in platelet 2014 open-labeled, 20 28 M + F None/Overweight Capsules 1 mg/kg 1 dose vs. LFDC and Serum et al. [85] Diseases 2016,controlled, 4,Randomized, 39 6 of 25 2control h vs.insulin control vs. (HFDC) cocoa-free End-stage renal disease function. crossover Table 1. Outcomes of recent (2014–2016) clinical trials1064 of cocoa products. Randomized, double-blind, baseline; vs. placebo; concentration crossover chocolate. withheart chronic Chronic failure De Palma et al. Randomized, FMD control; placebo2016 placebo32 70 M+F Dark chocolate (HFDC); ns 4 DBP vs.vs. LFDC Randomized, Large artery elasticity and FMD, andand Rassaf et al. [21] 2016 57 65 M+F hemodialysis Cocoa powder 900 ns 4 Plasma S-nitrosothiols nitrite (stable on GDMT) [19] FMD arteriolar vs. control; placebo controlled, controlled, 88 (LFDC) DBP vs. double-blind, 2016 20 No. of 62 Aprox. M None Dark chocolate 770 150 EC 1 dose baseline; vs. control. placebo; Dower et al. [20] Cocoa Product TF Duration microvascular (pharmacological controlled AIx vs. Outcomes control. Randomized, after 2 h and urinary nitrate Year Study Design Mean Age Sex Comorbidities Ref.et al. [23] Randomized, double-blind crossover 2015 crossover, 18 ns Table M +Outcomes F Type of 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu ♦ No change in platelet ♦capacity, No effect on total Dissolved in Loke Intervention (mg/day) (mg/day) (weeks) Participants 1. recent (2014–2016) clinical trials of cocoa products. vasodilator red medication) crossover (years) 12young 43 M None 200 1 dose 2008 placebo-controlled, after 5 h LDLc, vs. Baseline double-blind, postprandial 22 function. cholesterol, TG, water et al. [86] Heiss et al. [26] blood cell deformability 2015 22/60 M None Cocoa powder 900 128 2 No evidence of beneficial End-stage renal disease Plasma NT-proBNP controlled, 20 elderly crossover Plasma entothelin-1 after study Randomized, Randomized, glucose, hsCRP vs. Aprox. vs. baseline; Coronary heart disease, effects ofvs. chocolate with chronic No. of Cocoa Product TF EC Duration Randomized, (HFDC) LFDC and parallel-group FMD vs. control; FMD control; placebo2vs.h vs. baseline placebo placebo; Year Study Design Mean Ageand Sex ComorbiditiesDiabetes, Outcomes Ref. PWV, total peripheral ENRICA cohort Participants 18–65 Hypertension, regular consumption on Rassaf Balboa-Castillo et al. et [21] 2016 2016 57 20 65 M+FM hemodialysis Cocoa powder 900 770 ns 150 4 1 dose 62 None Dark chocolate Dower al. [20] Intervention (mg/day) (mg/day) (weeks) double-blind, 1064 baseline; 2015 controlled, 1272 M+F Chocolate ns ns 3 years controlled DBP vs. control. ♦ No changes in SBP, AIx vs. control. (years) Chronic heart failure DeetPalma resistance vs. baseline. study over Hypercholesterolemia, physical or mental al. [22]et al. (pharmacological Randomized, 2016 double-blind placebo32 70 M+F Dark chocolate (HFDC); ns 4 DBP vs. vs. LFDC and crossover DBP placebo. Plasma NT-proBNP Dissolved in Schroeter (stable on GDMT) FMD and PAT responses after[19] 2h Stroke components of HealthRandomized, (grades I Dark chocolate + medication) controlled, (LFDC) baseline; 2006 double-blind, 6 (3 + 3) 25–32 MHypertension None 188or 2 mg/kg 1 dose De Jesús RomeroSBP, DBP, TG, hsCRP End-stage renal disease dehydrated red apples Randomized, Randomized, vs. and related Quality ofand Life vs. control water+ et al. [87] 2015 controlled, blind 79 42 M+F and II) treated with 425.8 ns 24 vs. baseline No(HFDC) evidence ofLFDC beneficial Randomized, FMD vs. control; crossover ♦vs. Nocontrol change in platelet Prado et al. [27] crossover with chronic controlled, double-blind, 1064 baseline; captopril heart or telmisartan greenCocoa tea powder vs. placebo; FMD vs. control; Coronary disease, effectsHDLc of SBP, chocolate placebo20 18–70 ns None 80–800 17–168 1 Grassi et al. [24] 2015open-label DBP, PWV, ET1 heart failure De Rassaf Palma et function. et al. al. [21] 57 65 M + F Chronichemodialysis Cocoa powder 900 ns 4 double-blind, 20162016 ENRICA placebo32 70 M + F Dark chocolate (HFDC); ns 4 DBP vs. LFDC and DBP,consumption SBP vs.vs. baseline; cohort 18–65 and Hypertension, Diabetes, HDLc, high density lipoprotein-cholesterol; hsCRP, regular on C-reactive Balboa-Castillo controlled, Serum insulin DBP control. vs. control (stable ondilation; GDMT) [19] protein; M, males; Abbreviations: EC, epicatechin; highly sensitive Randomized, 2015 1272 F, females; FMD,Mflow-mediated +F Chocolate ns ns 3 years (pharmacological crossover controlled, 88 (LFDC) baseline; study over Hypercholesterolemia, physical or mental ♦ Improvement ofplacebo; et al. [22] double-blind concentration FMD vs.vs. control; placebo ns, not specified; PAT, peripheral arterial tonometry; TG, M triglycerides. medication) Blinded, Symptomatic peripheral ♦inNo effect on Hammer et al. crossover ♦ No change platelet 2016 20 62 None Dark chocolate 770 150 1 dose Dower et al. [20] Stroke of Healthsubjects status from Large artery elasticity 2015 Randomized, 21 ns ns Dark chocolate ns ns 1 dosecomponents controlled AIx vs. No evidence ofcontrol. beneficial microvascular function [25] randomized, artery disease function. related Quality of to Life hypertensive double-blind, vs. placebo; crossover Coronary heart disease, effects of chocolate Randomized, normotensive; 2015 Randomized, crossover, 18 ns M+F Type 2 diabetes, Obese Cocoa beverage 480 40 1 dose Basu et al. [23] HDLc vs.effect placebo; ♦ No on total End-stage renal disease ENRICA cohort 18–65 and Hypertension, Diabetes, regularvs. consumption on Balboa-Castillo control; placebo Randomized, Koli etetal.al.[28] 20152015 controlled, 22 1272 33–64 MM+M F +F Mild hypertension Dark chocolate chocolate 602.70 ns ns 83 years ♦ NoFMD significant changes postprandial Chocolate cholesterol, LDLc, TG, Dower Serum insulin 2016 20 62 over None Dark 770 ns 150 1 dose with chronic study Hypercholesterolemia, physical or mental et al. [20] [22] controlled AIx vs. control. FMD vs. control; placebocrossover in CAVI vs.vs. baseline; study glucose, hsCRP vs. placebo; Rassaf et al. [21] 2016 57 65 M+F hemodialysis Cocoa powder 900 ns 4 concentration Stroke components ofcontrol. Healthcrossover controlled, DBP vs. ♦ Large No significant changes placebo; Randomized, arteryQuality elasticity (pharmacological related of Life End-stage renal disease double-blind in lipid profile, blood ♦ changes in SBP, double-blind, vs.No placebo; Randomized, medication) HDLc vs.insulin placebo; chronic glucose, blood DBP vs. placebo. 2015 crossover, 18 ns M+F Type 2with diabetes, Obese Cocoa beverage 480 40 1 dose ♦ No effect on total FMD vs. control; placeboNo evidence of beneficialBasu et al. [23] Serum insulin Rassaf et al. [21] 2016 57 65 M+F hemodialysis Cocoa powder 900 ns 4 vs. baseline. postprandial Randomized, cholesterol, TG, controlled, DBP vs.LDLc, control. Coronary heart disease, effects of vs. chocolate FMD vs. control; concentration placebo; (pharmacological ♦ No significant changes study controlled, glucose, hsCRP vs. double-blind ENRICA cohort 18–65 and Hypertension, Diabetes, regular consumption on Balboa-Castillo 20 18–70 ns+ F None Cocoa powder 80–800 17–168 1 Grassi et al. [24] 2015 SBP, DBP, ET1 Randomized, Large artery elasticity medication) 2015 Randomized, 1272 M Chocolate ns ns 3 years in peripheral and PWV, central double-blind, placebo; study over Hypercholesterolemia, physical or mental et al. [22] double-blind, vs. control vs. placebo; No evidence of beneficial placeboblood pressure crossover ♦ No changes in SBP, Stroke components of Health1 dose; crossover, TypeNone 2heart diabetes, Obese Cocoa beverage 1 dose Basu et al. [23] ♦ No effect on total Massee disease, effects ofNo chocolate 20152015 controlled, 40 18 18–43ns nsM + F Coronary Cocoa extract 250 480 ns 40 vs. baseline; et al. [29] Blinded, Symptomatic peripheral ♦ effect on Hammer et al. DBP vs. placebo. related Quality of Life 4 1 dose regular 2015 ENRICA 21 ns ns Hypertension, Diabetes, Dark chocolate ns ns postprandial cholesterol, LDLc, cohort 18–65 and consumption onTG, Balboa-Castillo double-blind, ♦ Nomicrovascular significant changes randomized, artery disease function [25] Randomized, 2015 1272 M + F Chocolate ns ns 3 years HDLc vs. placebo; study FMD vs.blood glucose, hsCRP study over Hypercholesterolemia, orcontrol; mental et al. [22] parallel inphysical central flow vs. controlled, Serum insulin 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi et al. [24] 2015 placebo; SBP,vs. DBP, PWV, ET1 Stroke components of Healthbaseline. double-blind, concentration vs. placebo; ♦vs. No changes inand SBP, related Quality of Life control Haptoglobin, total crossover Randomized, Large artery elasticity DBP placebo. HDLc vs.vs. placebo; proinflammatory Blinded, Symptomatic peripheral ♦ No effect on Hammer et al. double-blind, vs. placebo; 2015 21 ns ns Dark chocolate ns ns 1 dose Randomized, Serum insulin monocyte CD62L FMD vs. control; function [25] artery 2015 randomized, crossover, 18 ns M+F Typedisease 2 diabetes, Obese Cocoa beverage 480 40 1 dose microvascular Basu et al. [23] ♦ No effect on total controlled, concentration vs. placebo; expression in obese 20 18–70 ns None Cocoa powder 80–800 17–168 1 Grassi et et al. al. [24] 2015 Randomized, SBP, DBP, PWV, postprandial None/Overweight cholesterol, LDLc,ET1 TG, McFarlin double-blind, Randomized, Large artery elasticity women vs. baseline; 2015 24 22 F Cocoa powder 640 48 4 vs. control study [30] double-blind Obesity glucose, hsCRP vs. crossover Year

Study crossover Design

No. of Participants

Outcomes Outcomes

Randomized,Randomized, double blind, placebo2016 35 Diseases 2016, 4, 39 Randomized, controlled, placebo-controlled double-blind, double-blind crossover No. of

Sex


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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) (ii)

Small number of subjects and statistical underpowered trials [87]; 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.


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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.

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