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
	HepG2 and Caco2 [50]	(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
	Randomized, placebo-controlled double-blind crossover trial [31]	(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-𝛽1: TGF-𝛽1 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-𝛽1: TGF-𝛽1 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

(c)  

Anti-inflammatory properties
In vitro assays	Polyphenol	Effect
Unstimulated and LPS-stimulated PBMCs [20]	Different flavanol fractions (20 μg/mL)	(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
Macrophages [73]	(i) Epicatechin (58 μg/mL); (ii) Cocoa extract (50 μg/mL); (iii) Isoquercitrin (18.6 μg/mL)	(i) ↓ MCP-1 and TNF-α at the transcriptional level; (ii) ↓ TNF-α, IL-1α, and IL-6 mRNA levels; (iii) ↓ NO secretion
THP-1 [72]	Procyanidin dimer B2: pretreatment for 30 min with 50 μM	(i) ↓ Expression of COX-2; (ii) ↓ Activation of ERK, JNK, and p38 MAPK; (iii) Suppresses NF-κB activation through stabilization of IκB proteins
Caco-2 IL-1β-stimulated [77]	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
Human PBMCs from 18 healthy volunteers [78]	Cocoa powder (40 g) with either milk or water	(i) Cocoa + water: (a) ↓ NF-κB activation in leukocytes and adhesion molecules; (b) ↓ sICAM-1; (c)↓ E-selectin; (ii) Cocoa + milk: (a) no change in NF-κB activation; (b) ↓ sICAM-1; (iii) Milk: (a) ↑ NF-κB activation
MCF-7 and SKBR3 [87]	(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
RLE cells in vitro and ex vivo [84]	(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

(d)  

Cancer prevention
In vitro
Cell line	Effects
Caco-2 cells [85]	(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
PC12 rat pheochromocytoma cells [81]	(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; (b)↓ 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
HepG2 cells [83]	(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 𝑡-butyl hydroperoxide; (a) none of the cocoa products tested affected the number of revertant colonies; (b) 13.25 mg cocoa powder/mL reduced ethoxyresorufin 𝑂-deethylase activity to 17.4% suggesting that whole cocoa products inhibit CYP1A activity
JB6 P+ cells [88, 89]	(i) Cocoa procyanidin fraction (5 μg/mL) and procyanidin B2 (40 μM) inhibit; (a) TPA-induced neoplastic cell transformation at 47 and 93%, respectively; (b) phosphorylation of MEK, ERK, and p90 ribosomal s6 kinase; (c) COX-2 expression; (d) AP-1 and NF-κB activation, and the TPA induced; (ii) Cocoa polyphenol extract (5–20 μg/mL); (a) inhibits TNF-α-induced upregulation of VEGF by reducing TNF-α-induced activation of AP-1 and NF-κB; (b) inhibits TNF-α-induced phosphorylation of Akt and ERK; (c) suppresses PI3K activity by binding PI3K directly; (d) suppresses TNF-α-induced MEK1 activity
MCF-7 and SKBR3 [87]	(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
RLE cells in vitro and ex vivo [84]	(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