Effects of Dark Chocolate on NOX-2-generated Oxidative Stress in Patients With Non-alcoholic Steatohepatitis

L. Loffredo; M. Del Ben; L. Perri; R. Carnevale; C. Nocella; E. Catasca; F. Baratta; F. Ceci; L. Polimeni; P. Gozzo; F. Violi; F. Angelico


Aliment Pharmacol Ther. 2016;44(3):279-286. 

In This Article

Abstract and Introduction


Background Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is considered a pathogenetic mechanism determining fibrosis and disease progression in non-alcoholic steatohepatitis (NASH). Polyphenols exert antioxidant action and inhibit NADPH oxidase in humans.

Aim To analyse the effect of cocoa polyphenols on NADPH oxidase isoform 2 (NOX2) activation, oxidative stress and hepatocyte apoptosis in a population affected by NASH.

Methods In a cross-sectional study comparing 19 NASH and 19 controls, oxidative stress, as assessed by serum NOX2 activity and F2-isoprostanes, and hepatocyte apoptosis, as assessed by serum cytokeratin-18 (CK-18) levels, were measured. Furthermore, the 19 NASH patients were randomly allocated in a crossover design to 40 g/day of dark chocolate (>85% cocoa) or 40 g/day of milk chocolate (<35% cocoa), for 2 weeks. sNOX2-dp, serum isoprostanes and CK-18 were assessed at baseline and after 2 weeks of chocolate intake.

Results Compared to controls, NASH patients had higher sNOX2-dp, serum isoprostanes and CK-18 levels. A significant difference for treatments was found in subjects with respect to sNOX2-dp, serum isoprostanes and serum CK-18. The pairwise comparisons showed that, compared to baseline, after 14 days of dark chocolate intake, a significant reduction in sNOX2-dp serum isoprostanes and CK-18 M30 was found. No change was observed after milk chocolate ingestion. A simple linear regression analysis showed that Δ of sNOX2-dp was associated with Δ of serum isoprostanes.

Conclusion Cocoa polyphenols exert an antioxidant activity via NOX2 down-regulation in NASH patients.


Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic disease worldwide, with a prevalence of approximately 30% in the common population and of 70–80% in particular subsets of patients, such as diabetic or obese subjects.[1] NAFLD represents a wide spectrum of conditions, including simple fatty liver but also non-alcoholic steatohepatitis (NASH) which is characterised by hepatic necro-inflammation with a potential of evolution to fibrosis and even to cirrhosis.[2–4]

Oxidative stress is considered of primary importance in the progression from simple steatosis to NASH, representing one of the most plausible mechanisms involved in the progression of liver damage.[5–7]

Nicotinamide adenine dinucleotide phosphate (NOX) oxidase is considered the major cellular reactive oxygen species (ROS) source in humans[8] and its activation has been associated with liver damages.[9] In a recent study of our group we found a correlation between soluble NOX2-derivative peptide (sNOX2-dp), a marker of NOX2 activation,[10] serum 8-iso-prostaglandin F2 alpha (PGF2 α) currently accepted as one of the best indicator of oxidative stress in vivo,[11] and cytokeratin-18 (CK-18), a validated marker of hepatocyte apoptosis[12] in patients with NAFLD.

Modulation of oxidative stress by antioxidant agents could represent a novel therapy for NASH. Vitamin E has been recommended for nondiabetic patients with NASH.[13,14] Other nutraceutical supplementations have shown promising results, especially those with polypenols.[15] Cocoa has high content of polyphenols and exerts antioxidant properties;[16–19] in particular, dark chocolate is able to reduce oxidative stress via lowering activation of NOX isoform 2.[20,21]

Recent animal studies suggest that cocoa might help avert alcoholic fatty liver disease development by oxidative stress reduction.[22] Experimental studies showed that NOX plays a pivotal role in the development of NASH[9] and that cocoa supplementation reduces liver damage by NOX inhibition.[23] To the best of our knowledge, studies have not analysed (i) the relationship between NOX2 activation and NASH, and (ii) the effect of cocoa on NOX2-derived oxidative stress and liver damage in patients with NASH.