(b)  

Cardiovascular diseases
Assays	Effects
Forty-two high-risk patients in a randomized crossover feeding trial for 4 weeks [32]	(i) 40 g of cocoa powder with 500 mL skim milk/day:  (a) ↓VLA-4, CD40, and CD36 in monocytes  (b) ↓ serum concentrations of P-selectin and ICAM-1
Resting human PBMCs from 13 healthy subjects treated with 25 μg/mL of procyanidin fractions isolated from cocoa [61]	(i) Individuals with low baseline levels of TGF-β 1: TGF-β 1 release was enhanced in the range of 16–66%. Low-molecular-weight fractions (≤ pentamers) were more effective than their larger counterparts (hexamer or higher), with the monomer and dimer inducing the greatest increases (66% and 68%, resp.) (ii) Individuals with high baseline levels of TGF-β 1: TGF-β 1 secretion was inhibited, being the inhibition most pronounced for trimmers through decamers (28–42%, resp.) and moderate for monomers to dimers (17–23%, resp.)
Twenty healthy subjects followed a balanced diet for 4 weeks; since day 14 to 27, they introduced daily 45 g of dark chocolate (860 mg of polyphenols, of which 58 mg were epicatechin) or white chocolate (5 mg polyphenols, undetectable epicatechin) [44]	(i) 2 h after dark chocolate intake (860 mg of polyphenols, of which 58 mg were epicatechin):   (a) detectable epicatechin levels were observed   (b) less DNA damage to mononuclear blood cells   (c) no effect on plasma total antioxidant activity (ii) Effects were no longer evident after 22 h: dark chocolate
Blinded parallel-design study with 32 healthy subjects consuming 234 mg cocoa phenolics a day for 28 days [63]	(i) ↑ Plasma epicatechin and catechin concentrations by 81% and 28%, respectively (ii) ↓ Platelet function
Double-blind, randomized study with 22 heart transplant recipients [64]	(i) 2 h after ingestion of 40 g of flavonoid-rich dark chocolate (0.27 mg/g of catechin and 0.9 mg/g of epicatechin, with a total polyphenol content of 15.6 mg of epicatechin equivalents per gram):  (a) ↑ coronary artery diameter  (b) improved endothelium-dependent coronary vasomotion  (c) ↓ platelet adhesion
Crossover, single blind study with 20 healthy subjects and 20 smokers who received 40 g of dark (cocoa > 85%) or milk (cocoa < 35%) chocolate [65]	(i) Smokers:   (a) ↑ platelet recruitment, platelet formation of ROS and eicosanoids, and NOX2 activation (ii) Smokers + dark chocolate group:   (a) ↓ platelet ROS, PGF2α, and NOX2 activation decreased significantly (iii) Healthy + dark chocolate:   (a) platelet variables did not change (iv) Milk chocolate (smokers and healthy):   (a) no changes detected in either of the groups treated with milk chocolate
Single oral administration of a natural flavonoid-enriched cocoa powder (50–600 mg/kg) in spontaneously hypertensive rats [67]	Antihypertensive effect in hypertensive rats without modifying the arterial blood pressure in normotensive rats. No dose-response effect was observed
Fifty male Kurosawa and Kusanagi-hypercholesterolemic rabbits received 100 g/day of standard diet or cacao liquor polyphenol diet [68]	(i) Polyphenol-treated group:   (a)↓ area of atherosclerotic lesions in the aortas of the polyphenol-treated group was significantly smaller than in the control group   (b) preserved parasympathetic nervous tone   (c) no differences in the plasma lipid concentrations
Six-month clinical trial with 36 prehypertensive healthy adult volunteers, at content levels of 120–139 and 80–89 mmHg [70]	(i) 50 g of dark chocolate/day:   (a) no significant differences were observed in the blood pressures of the treated and control groups
In vitro measurement of the kinetics of inhibition of purified angiotensin I converting enzyme from rabbit lung [72]	(i) Significantly inhibited the angiotensin I converting enzyme activity (ii) Monomeric flavan-3-ols: IC50 in the mM range (iii) Dimer and trimer: IC50 in the 100 μM range (iv) Larger procyanidins: IC50 in the 10 μM range