Cocoa Polyphenols and Their Potential Benefits for Human Health
Table 1
Effects of cocoa phenolics on metabolism, cardiovascular diseases, inflammation, and cancer prevention.
(a)
Metabolic and endocrine disorders
Assays
Effects
In vitro
Pancreatic α-amylase, pancreatic lipase, and secreted PLA2 inhibitions [45]
(i) Cocoa extracts and procyanidins dose-dependently inhibited pancreatic α-amylase, pancreatic lipase, and secreted PLA2, showing greater inhibitory activity against PLA2. The inhibitory potency increases with the degree of polymerization
(i) (+)-catechin, (−)-epicatechin, procyanidin B2, procyanidin C1, and cinnamtannin A2 (10 μM): ApoA1 ApoB due to upregulation of SREBPs and increased LDL receptor activity
In vivo
Randomized crossover trial with 19 hypertensive patients with impaired glucose tolerance [47]
(i) Flavanol-rich dark chocolate (100 g/day for 15 days): (a) insulin resistance, systolic and diastolic blood pressure, total cholesterol, and LDL-cholesterol (b) insulin sensitivity, β-cell function, and flow-mediated dilation
Randomized, crossover feeding trial in 42 high-risk volunteers [51]
(i) Chronic cocoa consumption (42 g/day for 4 weeks): (a) significant increases of phase II metabolites (glucuronide, sulfate conjugates) in 24 h urine (b) HDL-cholesterol (c) LDL-cholesterol
Meta-analysis of 8 randomized controlled trials involving 215 participants [52]
(i) Short-term cocoa consumption: (a) LDL and total cholesterol (changes depended on the amount of cocoa consumed and the health status of participants)
Randomized, single-blind, crossover study with 14 overweight/obese subjects [53]
(i) 20 g of dark chocolate with 500 mg of polyphenols and then 20 g of dark chocolate with 1000 mg of polyphenols or vice versa for 2 weeks separated by a 1-week washout period: (a) both doses were equally effective in reducing fasting blood glucose levels, systolic blood pressure, and diastolic blood pressure
(i) High polyphenol chocolate (16.6 mg of epicatechins in 45 g): (a) HDL-cholesterol (b) LDL-cholesterol, total cholesterol/HDL ratio High-polyphenol chocolate is effective in improving the atherosclerotic cholesterol profile in patients with diabetes
(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-: TGF- 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-: TGF- 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, , 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
(i) Monomers-pentamers: of LPS-induced synthesis of IL-1β, IL-6, IL-10, and TNF-α (ii) Long- and short-chain flavanol fractions: the production of GM-CSF in the absence of LPS and the expression of CD69 and CD83
Polyphenol extract of cocoa (50 μmol of gallic acid/L); 4 h treatment
(i) In the absence of proinflammatory stimuli, the polyphenol extract of cocoa induces a basal PGE2 synthesis due to COX-1 induction (ii) After 24 or 48 h stimulation: it decreased PGE2 synthesis 4.6- and 2.2-fold, respectively
In vivo/Ex vivo
Polyphenol
Effect
DSS-induced ulcerative colitis in Balb/C mice [27]
Cocoa polyphenol-enriched extract (500 mg/kg)
(i) In vivo: weight loss, improves normal stool consistency, visible blood in feces, neutrophil infiltration, NO production, COX-2 expression, STAT3 and STAT1α phosphorylation (ii) Ex vivo in peritoneal macrophages: IL-6, IL-1β, and TNF-α (iii) In vitro in LPS-stimulated RAW 264.7 cells: inhibits NF-κB
(i) After incubation with 250 ng/μL of cocoa extract, 7 genes out of 84 were overexpressed and 1 was underexpressed in MCF-7 cells, whereas 9 genes were overexpressed in SKBR3 cells (ii) CYP1A1 mRNA, protein levels, and enzymatic activity increased (iii) The combination of polyphenol cocoa extract + tamoxifen caused a synergistic cytotoxicity
(i) Cocoa polyphenol extracts dose-dependently (10–100 μM) attenuated in vitro hydrogen peroxide-induced (a) inhibition of GJIC (b) phosphorylation and internalization of connexin 43 (c) accumulation of ROS and activation of ERK (ii) Ex vivo in RLE cell lysates (a) inhibits hydrogen peroxide-induced MAPK/ MEK1 activity
In vivo
Animals
Effects
Wistar Han rats (5 weeks old) pretreated for 2 weeks with a cocoa-rich diet and injected with azoxymethane once a week for 2 weeks [82]
(i) The cocoa-rich diet (1 g of polyphenol/kg of diet): (a) antiproliferative effects in azoxymethane-induced colon cancer: ERK, Akt, and cyclin D1 (b) proapoptotic effects: Bcl-XL levels and levels of Bax and caspase-3 activity
(i) Treatment of cells with 50 μg/mL of procyanidin-enriched extracts: (a) inhibits cell growth by 70%, blocking the cell cycle in the G2/M phase (b) activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, and therefore, the intracellular pool of polyamines
(i) Cocoa procyanidin fraction (1 and 5 μg/mL) and procyanidin B2 (1 and 5 μM): (a) cell death attenuating the hydrogen peroxide-induced fragmentation of the nucleus and DNA PARP cleavage, increased Bcl-XL and Bcl-2 expression, and also inhibited activation of caspase-3 by hydrogen peroxide while attenuating the phosphorylation of JNK and MAPK
(i) Pretreatment of cells subjected to oxidative stress with 0.05–50 μg/mL of cocoa polyphenol extract for 2 or 20 h: (a) completely prevented cell damage and enhanced the activity of antioxidant enzymes (b) recovered levels of GSH (c) prevented in a dose-dependent fashion the increase in ROS
Salmonella typhimurium strain TA 98 and TA 102 [86]
(i) Treatment with benzo[a]pyrene (a) white chocolate did not modulate the number of revertant colonies produced by treatment (b) milk chocolate and cocoa powder extracts did (ii) Treatment with -butyl hydroperoxide (a) none of the cocoa products tested affected the number of revertant colonies (b) 13.25 mg cocoa powder/mL reduced ethoxyresorufin -deethylase activity to 17.4% suggesting that whole cocoa products inhibit CYP1A activity
(i) After incubation with 250 ng/μL of cocoa extract, 7 genes out of 84 were overexpressed and 1 was underexpressed in MCF-7 cells, whereas 9 genes were overexpressed in SKBR3 cells (ii) CYP1A1 mRNA, protein levels, and enzymatic activity increased (iii) The combination of polyphenol cocoa extract + tamoxifen caused a synergistic cytotoxicity
(i) Cocoa polyphenol extracts dose-dependently (10–100 μM) attenuated in vitro hydrogen peroxide-induced (a) inhibition of GJIC (b) phosphorylation and internalization of connexin 43 (c) accumulation of ROS and activation of ERK (ii) Ex vivo in RLE cell lysates (a) inhibits hydrogen peroxide-induced MAPK/MEK1 activity
In vivo
Animals
Effects
Wistar Han rats (5 weeks old) pretreated for 2 weeks with a cocoa-rich diet and injected with azoxymethane once a week for 2 weeks [82]
(i) The cocoa-rich diet (1 g of polyphenol/kg of diet): (a) antiproliferative effects in azoxymethane-induced colon cancer: ERK, Akt, and cyclin D1 (b) proapoptotic effects: Bcl-XL levels and levels of Bax and caspase-3 activity