(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