Journal of Functional Foods 23 (2016) 177–187

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Cocoa polyphenols exhibit antioxidant, anti-

inflammatory, anticancerogenic, and anti-
necrotic activity in carbon tetrachloride-
intoxicated mice

Jasminka Giacometti a,*, Damir Muhvić b, Adriano Pavletić a,

Luka Ðudarić c
a
Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, Rijeka, Croatia
b
Department of Physiology and Immunology, Faculty of Medicine, University of Rijeka, Braće Branchetta 20,
Rijeka, Croatia
c
Department of Anatomy, Faculty of Medicine, University of Rijeka, Braće Branchetta 20, Rijeka, Croatia

A R T I C L E I N F O A B S T R A C T

Article history: The therapeutic effects of cocoa polyphenols on acute carbon tetrachloride (CCl 4 )-
Received 6 November 2015 intoxicated mice were investigated. After determining polyphenols profile in the cocoa extract
Received in revised form 17 (CE), CE at a dose of 34.5 mg/kg and epicatechin (EC) at a dose of 2.24 mg/kg were intra-
February 2016 peritoneally (i.p.) administered to BALB/cN mice following CCl4 treatment. CE and EC post-
Accepted 18 February 2016 treatments suppressed hepatotoxicity and inflammation, ameliorated serum markers of liver
Available online 31 March 2016 injury, returned blood lactate and glucose levels to normal, stimulated synthesis and storage
of liver glycogen, and modulated glucose metabolism imbalance induced by CCl4. Cocoa poly-
Keywords: phenols also exhibited early stage antifibrotic and anticarcinogenic effects. They affected
Cocoa polyphenols beneficially the mitochondrial functionality acting on dysfunction in mitochondrial respi-
Epicatechin ration caused by CCl4 and antioxidants regulation at subcellular levels.
Liver damage © 2016 Elsevier Ltd. All rights reserved.
Antioxidants
Inflammation
Oxidative stress

High amounts of flavonoids were found in cocoa, mainly

1. Introduction
Cocoa and its derivatives are widely consumed worldwide due
to its highly attractive sensory characteristics. In the study of
50 foods with the highest antioxidant capacity, five were based
on cocoa (Halvorsen et al., 2006).
(−)-epicatechin (EC), (+)–catechin, (C) and their dimers
procyanidins B2 (PB2) and B1 (PB1) (Sánchez-Rabaneda et al.,
2003).
Cocoa flavonoids have been reported to affect many im-
portant biological functions in vitro and in vivo (reviewed in
Andújar, Recio, Giner, & Ríos, 2012). The protective effects of

* Corresponding author. Department of Biotechnology, University of Rijeka, Radmile Matejčić 2, HR 51000 Rijeka, Croatia. Tel.: +385 51
584 557; fax: +385 51 584 599.
E-mail address: jgiacometti@biotech.uniri.hr (J. Giacometti).
http://dx.doi.org/10.1016/j.jff.2016.02.036
1756-4646/© 2016 Elsevier Ltd. All rights reserved.
178 Journal of Functional Foods 23 (2016) 177–187
cocoa phenolics in heart disease are partially due to their
antioxidant, antiradical (Andújar et al., 2012) and antiplatelet
effects (Peluso, Palmery, & Serafini, 2015). Furthermore, they
are able to modulate the immune response (Becker, Geisler,
Ueberall, Fuchs, & Gostner, 2013; Pérez-Cano, Massot-Cladera,
Franch, Castellote, & Castell, 2013), anti-inflammatory (Monagas
et al., 2009) and anticarcinogenic properties (Ren, Qiao, Wang,
Zhu, & Zhang, 2003). Cocoa has been shown to exert anti-
diabetic effects by lowering glucose levels (Cordero-Herrera
et al., 2015; Dorenkott et al., 2014; Martín, Cordero-Herrera,
Bravo, Ramos, & Goya, 2014) and protecting the diabetic
retina from the insult (Duarte et al., 2015). Ulcerative colitis
was inhibited by administering a polyphenol-enriched cocoa
extract (Andújar et al., 2011). Cocoa is also able to prevent
liver damage by direct radical scavenging or stimulating
apoptosis, inhibition of inflammation, cell proliferation and
angiogenesis through the regulation of several signal trans-
duction pathways (Andújar et al., 2012; Suzuku et al.,
2015).
Health effects derived from cocoa flavonoids also depend
on their chemical structure and especially their bioavailability
(absorption, distribution, metabolism, and elimination)
(Urpi-Sarda et al., 2009).
Carbon tetrachloride (CCl4)-induced hepatotoxicity is a
well-known experimental model to induce both acute and
chronic liver injury. CCl4 toxicity is a complex process that is
used for the comprehensive examination of the pathogen-
esis of liver injuries, such as centrilobular steatosis,
inflammation, apoptosis and necrosis (Wang, 2014), as a
model for investigating a number of liver diseases, such as
liver steatosis, cirrhosis, hepatocellular death and carcinoge-
nicity. This process leads to the formation of CCl4-derived
free radicals, lipid peroxidation, covalent binding to macro-
molecules, loss of calcium homeostasis, nucleic acid
hypomethylation, and inflammatory cytokines (Weber, Boll,
& Stampfl, 2003).
CCl4 is activated by the hepatic microsomal cytochrome
P450 oxygenase system, mainly as (CYP) 2E1, to form
trichloromethyl radical (•CCl3) and marginally as (CYP) 2B and
(CYP) 3A, which react with oxygen and form trichloroperoxyl
radical (CCl3OO•) (Jia et al., 2007). Therefore, the permeability
of the plasma, lysosomal, endoplasmic reticulum and mito-
chondrial membranes has been altered, leading to subsequent
cell injury (Weber et al., 2003). Oxidative stress and mem-
brane damage in hepatocytes, mainly caused via CYP2E1
(Manibusan, Odin, & Eastmond, 2007), also alter the antioxi-
dant profile of the liver by the reaction of sulfhydryl groups
of glutathione (GSH) and protein thiols, including the antioxi-
dant enzymes as superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GPx), glutathione reductase (GR), and
glutathione transferase (GST) (Knockaert et al., 2012; Yang,
Zhang, Guan, & Hua, 2015).
With regard to cocoa and cocoa products belonging to func-
tional foods, our intention was to investigate some biological
actions related to the hepatoprotective effect of cocoa and
suggest it for daily consumption in the prevention and treat-
ment of liver diseases. Based on the above-mentioned, in the
present study, we investigated the effect of the cocoa poly-
phenols extract and epicatechin on acute liver injury in carbon
tetrachloride (CCl4)-intoxicated mice.
2. Materials and methods
2.1. Cocoa sample, chemicals, assay kits and antibodies
Alkalised cocoa cake sample, Madagascar origin, was sup-
plied by the leading Croatian chocolate manufacturer.
All chemicals and solvents were of HPLC-grade or analyti-
cal grade purity. Hexane, formic acid, methanol, hydrochloric
acid (37%) and acetonitrile (HPLC grade) were purchased from
J. T. Baker (Avantor Performance Materials B.V., Deventer, The
Netherlands). Carbon tetrachloride (CCl4), hydrogen peroxide
(H2O2) and sodium hydroxide (NaOH) were purchased from
Kemika (Zagreb, Croatia). 2,2′-Azino-bis(3-ethylbenzothiazoline-
6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-
picryl-hydrazyl (DPPH), potassium persulphate (K2S2O8), Trolox,
aluminum chloride hexahydrate (AlCl3 · 6H2O), sodium nitrite
(NaNO2), sodium carbonate (anhydrous) (Na2CO3), gallic acid
monohydrate, vanillin, 4-(dimethylamino)cinnamaldehyde
(DMAC), paraformaldehyde, 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB), ethylenediaminetetraacetic acid (EDTA), Brilliant Blue
R250, Bovine Serum Albumin (BSA), sodium azide (NaN3), BioStab
general proteolysis inhibitor and Folin–Ciocalteu phenol reagent
were purchased from Sigma–Aldrich (Taufkirchen, Germany).
Tris(hydroxymethyl) aminomethane (Tris), ammonium sul-
phate ((NH4)2SO4)), sodium chloride (NaCl) were purchased from
Prolabo (WVR International, Dublin, Ireland). D(−)-mannitol, D(+)-
sucrose, monopotassium phosphate (KH2PO4) and disodium
phosphate (Na2HPO4) were purchased from Prolabo (WVR In-
ternational, Dublin, Ireland). Triton X-100 was purchased from
Merck, KGaA (Darmstadt, Germany).
Vanillin, (−)-epigallocatechin (EGC), (+)-catechin (C), (−)-
epicatechin (EC), (−)-epigallocatechin gallate (EGCG), (−)-
gallocatechin gallate (GCG), (−)-epicatechin gallate (ECG) and
quercetin were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). p-coumaric acids and m-coumaric acid were pur-
chased from Extrasynthese (Genay Cedex, France).
NuPAGE® Novex® Bis-Tris Mini Gels (4–12% Bis-Tris),
NuPAGE® MES SDS Running Buffer (20X), NuPAGE® LDS
Sample Buffer (4X), NuPAGE® Sample Reducing Agent (10X)
and NuPAGE® Antioxidant were purchased from Novex (Grand
Island, NY, USA). PageRuler™ Plus Prestained Protein Ladder,
10 to 250 kDa and Pierce™ BCA Protein Assay Kit were
purchased from Thermo Fisher Scientific (Waltham, MA,
USA).
RANSOD kit for superoxide dismutase (SOD) activity deter-
mination was obtained from Randox Laboratories Ltd. (Crumlin,
UK).
Rabbit polyclonal antibodies to hemopexin (Hpx)
(AP17484PU-N), heat shock protein 70 (Hspa4) (21206-1-AP),
heat shock protein (Hsp90) (10979-1-AP), mitogen-activated
protein kinase-2 (Pkm2) (15822-1-AP), alpha-actinin-4 (Actn4)
(GTX101669) as well as HRP Anti-IgG secondary antibody
were purchased from Acris Antibodies GmbH (Herford,
Germany). Polyvinylidene difluoride (PVDF) membrane and
blocking reagent were obtained from Roche Diagnostics
GmbH (Mannheim, Germany). Chemiluminescent substrate
Roti-Lumin was purchased from Carlo Roth (Karlsruhe,
Germany).
Olive oil was supplied by Agroproduct (Vodnjan, Croatia).
 Journal of Functional Foods 23 (2016) 177–187 179

2.2. Cocoa extract ministration at the temperature (22 ± 1 °C) maintained at 12 h

In brief, cocoa extract was obtained after extraction proce-
dure from cocoa cake of Madagascar origin and characterised
by the determination of total phenolics (TP), total flavonoids
(TF) and flavan-3-ols before its administration in mice (Di
Stefano, Cravero, & Gentilini, 1989; Rashed, Potočnjak,
Giacometti, Škoda, & Domitrović, 2014).
The antioxidant activities of the cocoa extract were deter-
mined using the DPPH• radical scavenging assay and the ABTS•+
decolourisation assay described previously (Rashed et al., 2014).
The concentration of individual phenolics in cocoa extract
was determined by ultra-performance liquid chromatogra-
phy (UPLC) on the Agilent 1290 Infinity LC system (Agilent
Technologies, Palo Alto, CA, USA) equipped with ChemStation
software and a diode array detection system using Zorbax RRHD
SB-C18 column (2.1 mm × 50 mm, 1.8 µm particle size) using
“in-house” method. Each compound was identified based on
retention time, UV spectrum and already published identifi-
cation of compounds from the global cocoa (Patras, Milev,
Vrancken, & Kuhnert, 2014; Tomas-Barberan et al., 2007) and
quantified by calibration curve using (−)-EC as an external
standard.
The results of the analysed extract were presented in Table 1.
2.3. Animals
light/dark cycle, at constant temperature and humidity (50 ± 5%).
Before each treatment throughout the study, mice were
weighted.
All experimental procedures were performed in compli-
ance with the Declaration of Helsinki and approved by the
Ethical Committee of the Faculty of Medicine, University of
Rijeka.
2.4. Experimental design
The experimental mice were divided into four groups of eight
animals each. Saline solution (0.9% NaCl) was given orally by
gavage in the mice of the control group. The CCl4-intoxicated
group was administered CCl4 dissolved in olive oil (1:1, v/v) twice
a week intraperitoneally (1 mL/kg body weight). The post-
treated group with CE received i.p. 34.5 mg/kg cocoa extract
(CE) and group EC received 2.51 mg/kg of (−)-epicatechin, both
dissolved in saline, twice weekly for 2 weeks, after CCl4 pre-
treatment. The selected doses were previously optimised (data
not shown). Twenty-four hours after the last dose of CCl4, CE,
EC or saline, mice were sacrificed. The blood was collected pre-
viously from the orbital sinus, and plasma and serum were
separated. The liver was removed, washed with saline, blotted
dry and divided into three samples of each. One part of the
liver sample was immersed in 4% paraformaldehyde for his-
tology. The remaining two samples were stored at −80 °C.

Female BALB/cN mice from our breeding colony, 2–3 months
old, weighing 20–25 g, were divided into groups as described
in the Experimental design section. Mice were fed a standard
rodent diet (pellet, type 4RF21 GLP, Mucedola, Italy) and water
ad libitum. The animals were acclimated for 1 week before ad-
2.5. Blood biochemistry
Immediately after blood samples were taken, the concentra-
tion of glucose (Glc), triglycerides (TAG), cholesterol (CHO), and
lactate (Lac) were measured using the corresponding assay
strips (Accutrend Plus, Roche, Mannheim, Germany).

2.6. Assessment of liver damage

Table 1 – Cocoa extract phenolic content and their
bioactivities. Serum levels of ALT, AST, and the ALP as markers of hepatic
Cocoa extract function were measured using commercially available test kits
(Randox, UK) by using a Bio-Tek EL808 Microplate Reader (BioTek
TP (mg GAE/g NFCS) 3.08 ± 0.18
TF (mg ECE/g NFCS) 23.85 ± 2.03
Instruments, Winooski, VT, USA) according to the manufac-
Flavan-3-ols (mg catechin/g NFCS) turer’s instructions.
DMAC method 5.30 ± 0.20 Histopathological assessment of liver damage was done by
Vanillin method 8.83 ± 1.31 studying haematoxylin and eosin (H&E) stained slides of liver
Antioxidant activity (mM TEAC/g NFCS) tissue.
DPPH method 22.52 ± 0.01
To observe the location and quantity of hepatic glycogen,
ABTS method 20.10 ± 2.02
the PAS method (Periodic acid–Schiff base) was used.
Individual phenolics in cocoa extract (mg/g of DM)
2.7. ELISA assay
Gallic acid 1.15
Procyanidin B1 (PB1) 1.63
(−)-epigallocatechin (EGC) 13.46 Serum levels of tumour necrosis factor alpha (TNF-α) and
(+)-catechin 0.61 interferon-gamma (IFN-γ) in mice were determined by com-
Procyanidin B2 (PB2) 10.51 mercially available enzyme-linked immunosorbent assay (ELISA)
(−)-epicatechin (EC) 17.38 kit (Biovendor Laboratory, Inc., Brno, Czech Republic) accord-
(−)-epigallocatechin gallate (EGCG) 4.56
ing to manufacturer’s instructions. These ELISA assays were
Vanillin 0.21
performed in duplicate with detection limits 3 pg/mL for TNF-α
p-coumaric acid 3.81
m-coumaric acid 1.05 and 5.3 pg/mL for IFN-γ, respectively.
(−)-epicatechin gallate (ECG) 2.25
Quercetin 0.24
2.8. Liver tissue fractionation

TP, total phenolics; TF, total flavonoids; NFCS, nonfat cocoa solids;
A fraction of cytosol (Cyt), plasma membranes (PM) and crude
DM, dry matter.
mitochondrial fractions (Mt) was isolated from the liver tissue,
180 Journal of Functional Foods 23 (2016) 177–187
according to the modified protocol by Wieckowski, Giorgi,
Lebiedzinska, Duszynski, and Pinton (2009) (see supplementary
Fig. S1).
In brief, the liver was cut into small pieces and manually
homogenised in a Potter–Elvehjem homogeniser with a Teflon
pestle in 1:4 (weight: volume) solution I (225 mM mannitol,
75 mM sucrose, 0.1 mM EDTA, 0.5% BSA and 30 mM Tris-HCl,
pH 7.4), solution II (225 mM mannitol, 75 mM sucrose, 0.5% BSA
and 30 mM Tris-HCl, pH, 7.4) and solution III (225 mM manni-
tol, 75 mM sucrose and 30 mM Tris-HCl, pH 7.4) on ice by using
differential centrifugation protocol at 4 °C (for 5 min at 740 g,
for 10 min at 9000 g and for 10 min at 10,000 g). The crude mi-
tochondrial pellet was resuspended in 225 mM mannitol, 75 mM
sucrose, 0.5% Triton-100 and 30 mM Tris-HCl, pH 7.4 under
sonification during 15 sec, and kept on ice until the fractions
were performed.
Protein concentrations were determined in each fraction
using a BCA protein assay kit (Pierce, Thermo Scientific, Rock-
ford, IL, USA).
2.9. SDS-PAGE
About 15–20 µg liver protein of each sample was solubilised in
NuPAGE sample buffer (Invitrogen, Carlsbad, CA, USA) and
heated at 95 °C for 5 min. The electrophoretic separation was
performed with precast 4–12% Bis–Tris gels in an OmniPAGE
mini, a vertical system (Cleaver Scientific Ltd, Rugby, Warwick-
shire, UK) according to the manufacturer’s procedure. The gels
were stained with colloidal Coomassie Blue G-250 stain and
visualised by a VersaDoc Imaging System (BioRad, Hercules, CA,
USA).
2.10. Western blot analysis
The liver protein expression of hemopexin (Hpx), heat shock
protein 70 (Hspa4), heat shock protein (Hsp90), mitogen-
activated protein kinase-2 (Pkm2) and alpha-actinin-4 (Actn4)
in liver homogenate was studied by Western blot analysis.
Liver homogenates were prepared in PBS buffer (pH 7.4) con-
taining 0.5% Triton X-100, 0.05% sodium azide and Biostab
protease inhibitor. After protein determination with BCA
method, 50 µg protein was subjected to SDS-PAGE and trans-
ferred to a PVDF membrane. The membrane was blocked in
TBST with a 1% BSA, incubated with primary antibody at 4 °C
overnight with agitation, and after that washed with TBST three
times for 5 min with agitation and incubated with appropri-
ate HRP-secondary antibody for 2 h at room temperature (with
agitation). Then, it was washed again with TBST, three times
for 5 min at room temperature and incubated with chemilu-
minescence substrate for 30 min at 56 °C. Protein band intensity
was visualised and captured in the VersaDoc Imaging System.
2.11. Assessment of antioxidative enzymes in subcellular
fractions
Catalase activity (CAT) was assayed at 25 °C by the method of
Claiborne (1985). Briefly, the assay mixture consisted of 1.95 mL
phosphate buffer (0.05 M, pH 7.0), 1.0 mL hydrogen peroxide
(0.019 M), and 0.05 mL of sample (Cyt fraction) in a total volume
of 3.0 mL. Changes in absorbance were measured at 240 nm
by an ultraviolet/visible (UV/VIS) spectrophotometer Specord
50 with WinASPECT software (Analytik Jena AG, Jena, Germany).
CAT activity was calculated in terms of units/mg protein/min.
Superoxide dismutase activity (SOD) was assessed in frac-
tion Mt using a commercially available test kit (Sigma, St. Louis,
MO, USA) at 440 nm by the same spectrophotometer as pre-
viously. SOD activity was calculated in terms of units/mg
protein/min.
The total protein thiols (TPT) assay was based on the prin-
ciple of formation of relatively stable yellow colour by sulfhydryl
groups with DTNB (Moron, Depierre, & Mannervik, 1979). Briefly,
0.25 mL of Cyt and dissolved Mt fraction were mixed with 1 mL
of 0.25 M Tris-20 mM EDTA buffer (pH 8.2) and this mixture was
incubated for 10 min at 37 °C. Reactions were initiated with
0.125 mL of 10 mM DTNB in methanol. The absorbance was
measured at 412 nm against appropriate blanks (without DTNB).
TPT was calculated in terms of µg/mg protein. All measure-
ments were performed in triplicate and data expressed as the
mean ± SD.
2.12. Statistical analysis
The data were analysed using StatSoft STATISTICA version 12
software. Differences between two groups were assessed by a
nonparametric Mann–Whitney U-test and between all groups
by Kruskal–Wallis test. Data were expressed as the mean ± SD.
Differences with P < 0.05 were considered to be statistically
significant.
3. Results and discussion
Currently, there are many challenges to be solved in the in-
vestigation of the possible application of “Superfoods” in the
prevention and treatment of communicable and non-
communicable diseases. It is considered that cocoa and its
products are very powerful “Superfoods.” The consumption of
cocoa products and chocolate contributes to human nutri-
tion, and importantly intake of potent antioxidants such as
polyphenols. In the last few years, many health studies have
been conducted utilising different cocoa products; however, al-
though cacao and cocoa products are functional foods, there
are still no official recommendations of related daily consump-
tion or dose-dependent intake in the prevention and treatment
of certain diseases. This study strengthens the fact and con-
firms the hypothesis about cocoa as a functional food by
presenting new knowledge about the effectiveness of dietary
polyphenols on liver disease.
Oxidative stress and inflammation are considered to play
a central role in the pathophysiology of many diseases, in-
cluding acute liver damage induced by toxic compounds. In
this paper, we demonstrated that cocoa significantly sup-
pressed oxidative liver damage in acute CCl4-intoxicated mice,
modulated carbohydrate metabolism, changed the expres-
sion of several proteins involved in main metabolic processes,
changed the level of glucose and liver enzymes, lowered the
production of pro-inflammatory cytokines, changed the level
of antioxidative enzymes and total protein thiols, and conse-
quently effected the restoration and the function of the liver.
 Journal of Functional Foods 23 (2016) 177–187 181

Table 2 – Body weight, blood lipids, glucose and lactate levels, serum transaminases and phosphatase in the control,
CCl4-treated, EC and CE-post-treated female BALB/cN mice.
Control CCl4-intoxicated Epicatechin Cocoa extract
group post-treated group post-treated group
Body weight (g) 20.06 ± 1.36 20.46 ± 1.77 21.34 ± 0.70 21.06 ± 2.50
Glucose* (mmol/L) 9.83 ± 2.29 5.67 ± 0.15a 10.57 ± 0.77b 11.13 ± 1.64b
Lactate* (mmol/L) 4.08 ± 0.55 5.75 ± 0.50a 3.98 ± 0.70b 3.69 ± 0.53b
Triglyceride (mmol/L) 2.44 ± 0.35 3.95 ± 0.40 2.66 ± 0.49 2.15 ± 0.26
Cholesterol (mmol/L) 4.43 ± 0.34 3.22 ± 0.24 5.18 ± 1.30 4.61 ± 0.26
AST* (U/L) 27.38 ± 2.59 249 ± 19.21a 56.62 ± 3.93b 45.29 ± 3.65b,c
ALT* (U/L) 22.30 ± 3.25 229.96 ± 10.91a 38.64 ± 4.20b 30.48 ± 6.24b
ALP* (U/L) 68.64 ± 5.29 94.17 ± 8.94a 71.90 ± 4.37b 65.42 ± 4.12b,c
* Significant difference between all groups (Kruskal–Wallis ANOVA by ranks).
a
Significant difference between control and CCl4-intoxicated group.
b
Significant difference between CCl4-intoxicated group and EC and CE post-treated groups.
c
Significant difference between post-treated groups. The difference was significant at P < 0.05.

3.1. Cocoa polyphenols suppress hepatotoxicity 3.2. Cocoa polyphenols affect glucose metabolism after
CCl4-induced liver injury
CCl4 treatment induced a strong increase of liver enzyme serum
levels (see Table 2), changed the liver histological morphology Disturbance in carbohydrate metabolism is one of the fea-
and reduced liver enzyme activity in the damaged pericentral tures of liver damage involved in CCl4 toxicity. Subchronic
area and visualisation of apoptotic cell nuclei. exposure to CCl4 resulted in a significant loss of blood glucose
Mice intoxication with CCl4 elevated AST (9.1-fold vs. Control) level, producing hypoglycaemia, which might be due to de-
while post-treatments with EC and CE reduced the elevation clining in hepatic glycogen (Fig. 2). Blood glucose (P = 0.0281)
of AST (4.4-fold and 5.5-fold vs. CCl4 group). Likewise, the el- was significantly decreased and lactate (P = 0.0281) increased
evation of ALT and ALP (10.3-fold and 1.4-fold vs. Control) was by CCl4 intoxication while CE and EC post-treatment showed
reduced (6-fold and 7.5-fold, apropos 1.3-fold and 1.4-fold, vs. restored blood glucose and reduced lactate (P = 0.0226 and
CCl 4 group). McKim et al. (2002) also reported that P = 0.0281, respectively).
coadministration of the cocoa flavonoid extract decreases ALT Blood glucose levels connected with intoxication with CCl4
level and protects against early alcohol-induced liver injury in are quite oppositely reported in some studies. The previous op-
rats. posite results also have shown that cocoa exerts anti-diabetic
The areas of necrosis are present mainly around blood effects by lowering glucose levels (Cordero-Herrera et al., 2015).
vessels while in EC post-treated group there is no necrosis
around blood vessels (Fig. 1). Our results suggest that the main
way of spreading i.p. given CCl4 after its peritoneal absorp-
tion is vascular. Cocoa polyphenols can modify NO availability
and contribute better endothelial function (Andújar et al., 2012).
The size of nuclei was larger in post-treated CE group
(Fig. 1D) in comparison with post-treated EC (Fig. 1C) and CCl4-
treated group (Fig. 1B), indicating that CE prevents nuclear
damage provoked by CCl4 treatment and has the better pro-
tective effect of nucleus than EC treated group. Also, CCl4 treated
group showed mild lipid accumulation (Fig. 1B) while it was
not presented in EC and CE-treated groups (Fig. 1C and 1D).
The level of blood triacylglycerols (TAG) increased in the CCl4-
treated group (see Table 2). Post-treatment with EC and CE
lowered TAG levels most in CE post-treated group. Choles-
terol (CHO) was lowered in the group treated with CCl4; however,
post-treatments with EC and CE lowered total CHO level,
whereby the CE-post-treated group achieved normalised levels.
Lipid accumulation is a marker for hepatotoxicity, which means Fig. 1 – Liver histology after administration of 1 mL/kg CCl4:
that EC and CE prevented lipid accumulation in hepatocytes, (A) control mice liver, (B) CCl4-treated mouse liver, (C)
thus showing a protective effect. It is in accordance with the epicatechin post-treated mouse liver, and (D) cocoa extract
results that CE significantly decreases gene expression levels post-treated mouse liver. Magnification ×40 and 100. CV–
of lipogenic enzymes, while increases the mRNA levels re- central vein; black arrow shows hepatocytes, yellow arrow
sponsible for lipolysis enzymes and shows that CE shows sinusoids with Kupffer cells, red arrow shows pro-
administration differentially regulates gene expression in- inflammatory cells, and blue arrows shows the lipid
volved in lipid metabolism (Ali, Ismail, Esa, & Pei, 2015). droplet.
182 Journal of Functional Foods 23 (2016) 177–187
Fig. 2 – PAS staining of glycogen in the liver (40×): (A)
control mice liver, (B) liver of treated mice with 1 mL/kg
CCl4, (C) epicatechin post-treated mouse liver, and (D) cocoa
extract post-treated mouse liver. The centrilobular areas
exhibit some small patches of round cell infiltration and
decreased glycogen staining in the cytoplasm after
hepatocytes but little necrosis (B).
However, our results showed that CE and EC stimulated the
production of glucose, significantly decreased lactate and ame-
liorated acidosis provoked by CCl 4 treatment, indicating
protective effects of CE and EC post-treatment. Lactate has a
key role in the transport of oxide substrates between organs.
In the liver, they are the basis for gluconeogenesis and they
are the product of glycolysis. It is thought that high activity
of lactate dehydrogenase (LDH) has an effect of higher per-
meability of sinusoidal membranes and hepatocyte membranes
(Cordero-Herrera et al., 2015), allowing the transmigration of
inflammatory cells through sinusoids. Therefore, we pro-
posed the hypothesis that lactate acidosis provoked hypoxia,
hypoxia-induced mitochondrial toxicity, while mitochondrial
toxicity caused liver damage in the CCl4-treated mice.
Histochemical observations in Fig. 2 demonstrated that the
glycogen content was reduced in CCl4-treated mice, indicat-
ing reduced glycogen synthesis and storage. The amount of
glycogen differs with the significantly reduced content in de-
generated and necrotic cells. Considering a significant decrease
in hepatocellular glycogen in the degenerated and a necrotic
centrilobular zone in CCl4-intoxicated mice, the excess of gly-
cogen in the individual vacuolated cells over normal ones
existed. The liver intoxicated with CCl4 (Fig. 2B) showed an in-
crease in the content of glycogen around necrotic areas, while
CE and EC post-treated livers decreased glycogen, which is a
mark for disturbance of glucose homeostasis.
Some studies have demonstrated decreased hepatic glyco-
gen content after treatment with CCl 4 and decreased
gluconeogenesis in the liver (Muriel, Alba, Pérez-Alvarez,
Shibayama, & Tsutsumi, 2001). Ghafoory et al. (2013) ex-
plained these opposites by two glycogen synthesis routes that
take place within different metabolic zones of the liver and fur-
thermore in the production of lactate by glycolysis, which is
Fig. 3 – Effect of EC and CE-post-treatments on pro-
inflammatory cytokines, TNF-α and IFN-γ, after the
previous CCl4 treatment. Data are expressed as mean ± SD
(N = 8). aSignificant difference between control and CCl4-
intoxicated group; bsignificant difference between CCl4-
intoxicated group and EC and CE post-treated groups. The
difference was significant at P < 0.05.
released by pericentral hepatocytes and at least partly reab-
sorbed and used for gluconeogenesis by periportal hepatocytes.
Moreover, disruption of glycogen storage is associated with the
increase of the necrotic areas in the liver, but also due to in-
hibition of key enzymes involved in carbohydrate metabolism
(Ghafoory et al., 2013). Increased activity of LDH is directly linked
with increased necrosis and lactate and glucose levels in CCl4-
treated mice. In the glycolytic process, pyruvates are reduced
by LDH to lactate in the cytosol or enter into the mitochon-
dria to produce ATP through the tricarboxylic acid cycle (TCA).
Cancer cells are also able to deregulate pathways that enhance
glycolysis, and as a result stimulate glucose uptake and produce
higher levels of lactate.
3.3. Cocoa polyphenols suppress inflammation after
CCl4-induced liver damage
Plant-derived polyphenols modulate the production and ac-
tivity of inflammatory molecules directly (e.g. TNF-α) or
indirectly by induction of other inflammatory mediators in a
regulation of cell signalling, including e.g. cyclooxygenase 2
(COX-2) and inducible nitric oxide synthase (iNOS) (Fraga &
Oteiza, 2011; Gupta et al., 2014). Cocoa supplementation de-
creased mRNA levels of TNF-α, interleukin-6 (IL-6), and iNOS,
and this was accompanied by decreased nuclear protein levels
of nuclear factor-κB (NF-κB) (Mao, van de Water, Keen, Schmitz,
& Gershwin, 2002).
A shown in Fig. 3 cocoa and epicatechin markedly de-
creased pro-inflammatory cytokines after mice intoxication with
CCl4. Compared to the control, the level of TNF-α was signifi-
cantly increased in the CCl4 post-treated group (P = 0.0122). Post-
treatment with CE and EC significantly reduced serum TNF-α
level (P = 0.0058, and P = 0.0081, respectively). The levels of
another pro-inflammatory cytokine, IFN-γ, showed that CE sig-
nificantly decreased the elevation of IFN-gamma provoked by
CCl4 treatment (P = 0.0058) even better than EC (P = 0.0081).
 Journal of Functional Foods 23 (2016) 177–187
It is important to note that TNF-α, as one of the mediators
of hepatotoxicity, is able to contribute to the restoration of func-
tional liver mass through hepatocyte proliferation and liver
regeneration, thus showing a possibility of acting in opposite
ways as a hepatotoxic cytokine or as a stimulator of hepato-
cyte regeneration (Schwabe & Brenner, 2006).
CCl4 treatment stimulated outgoing of cellular elements in
tissue while it was not present in CE and EC post-treated group,
indicating that the number of inflammatory cells, which came
out of circulation, was lower in the CE and EC-treated group
and showing that CE has an anti-inflammatory capacity. That
is in accordance with results showing that the cocoa-rich diet
decreases the nuclear levels of NF-κB and the expression of
pro-inflammatory enzymes such as cyclo-oxygenase-2 (COX-
2) and iNOS induced by azoxymethane (AOM)-induced colon
carcinogenesis (Rodríguez-Ramiro et al., 2013). Additionally, the
experiments in Caco-2 cells, an in vitro model of experimen-
tally induced intestinal inflammation, confirm that cocoa
polyphenols effectively down-regulate the levels of inflam-
matory markers induced by TNF-α by inhibiting NF-κB
translocation and JNK phosphorylation (Rodríguez-Ramiro et al.,
2013).
The hepatoprotective effect of cocoa has been reported in
other animal models of liver injury. Coadministration of the
cocoa flavonoid extract (400 mg/kg per day during 4 weeks) in
rats decreased TNF-α levels, the accumulation of protein adducts
of 4-hydroxynonenal and lipid peroxidation, and thus can
prevent early alcohol-induced liver injury (McKim et al., 2002).
Cocoa-rich diet (16%) protected the liver against the oxida-
tive damage induced by N-nitrosodiethylamine (DEN) through
modulation of the activities of enzymes and antioxidants in-
volved in oxidative stress (catalase, glutathione peroxidase,
glutathione reductase, glutathione S-transferase, glutathi-
one) and exerted an anti-apoptotic effect that was associated
with prevention of both JNK and caspase-3 activation
(Granado-Serrano, Martín, Bravo, Goya, & Ramos, 2009). On this
way, cocoa-diet increase the survival/proliferation signal PI3K/
AKT as a key factor in the aggressiveness of hepatocellular
cancer.
Anti-inflammatory effect of CE and EC post-treated mice
can be compared with the effect of catechins from green tea.
It also shows that cocoa extract acts like green tea extract, which
attenuates CCl4-enhanced oxidative stress, levels of biochemi-
cal parameters, development of pathology, acute-phase protein
secretion and preserves antioxidant/antiapoptotic protein ex-
pression (Hung et al., 2012).
Altogether, these results indicated that cocoa polyphenols
exhibited antioxidant, anti-inflammatory, anti-necrotic and
anticarcinogenic activities in CCl4-intoxicated mice.
Besides that, CCl4 treatment causes obvious metabolic al-
terations in other important organs such as the kidney, spleen
and lung, while cocoa has a beneficial effect on the whole or-
ganism (Jiang, Huang, Wang, & Tang, 2012).
3.4. Protein candidates as biomarkers of CCl4-induced
liver injury
Based on the literature overview and our unpublished proteomic
results, we selected some proteins that can be involved as pos-
sible markers of oxidative stress, inflammation, liver fibrosis
183
Fig. 4 – SDS-PAGE and Western blot analysis: (A) SDS-PAGE
of whole proteins in the mouse liver, (B) Western blot
analysis of hemopexin (Hpx), pyruvate kinase isozyme M2
(Pkm2), heat shock protein 110 (Hspa4), endoplasmin
(Hsp90) and alpha actinin 4 (Actn4) levels in the liver
samples from control. Representative results from 8
similarly treated mice.
and carcinogenesis. The protein levels of targeted liver pro-
teins after induced CCl 4 injury and regeneration with
epicatechin and cocoa extract were analysed by western blot-
ting (Fig. 4B).
Most pathological states are characterised by increased oxi-
dative stress, which may then promote proteotoxic stress. Heat
shock protein (Hsp) genes are involved in a number of human
diseases and are essential for cellular survival under differ-
ent stressors exposure. The mechanisms of redox regulation
of chaperones allow new insights into these diseases and find-
ings on an efficient treatment (Lee et al., 2004).
Polyphenols are able to inhibit Hsp90 and have the effects
on lowering cytotoxic activity, and have anti-proliferative, anti-
inflammatory, and antiviral activities. EGCG, which is contained
in green tea and cocoa, binds Hsp90 and induces degradation
of Hsp90-dependent substrates (Li et al., 2012; Yin, Henry, &
Gasiewicz, 2009).
Fig. 4B presents the induction of Hsp90 in the CCl4-treated
group and attenuation in EC and CE post-treated groups when
compared with the control. Expression of Hsp90, which pro-
motes oncogenesis, was inhibited in CE and EC post-treated
animals. Hsp90 is particularly involved in the inflammatory re-
sponse while Hsp90 inhibitors down-regulate pro-inflammatory
proteins and display anti-inflammatory activity, which makes
them prominent in traditional medicine. To our knowledge, it
is the first report that CE inhibits Hsp90 and thus prevents car-
cinogenesis. Contrary to expectation, we did not find expression
of Hsp70 protein (Hspa4) in the CCl4-treated group. Hspa4 was
slightly suppressed in CCl4-treated group when compared with
CE and EC post-treated groups where normal levels were
observed.
After four post-treatments with cocoa polyphenols, pyru-
vate kinase isozyme isoform M2 (Pkm2) decreased both in the
184 Journal of Functional Foods 23 (2016) 177–187

EC and CE post-treated group and return to the control (Fig. 4B).
Our results indicated a possible modulation of the constitu-
tive activation of PI3K/Akt and Erk1/2 pathways in the liver after
CCl4 treatment and post-treatment with EC and CE. As a gly-
colytic enzyme, Pkm2 is found as predominant in proliferation
and cancer cells. Higher Pkm2 expression rather than isoform
M1 (Pkm1) allows regulation of oncogene and tumour sup-
pressors in cancer cells (Wong, De Melo, & Tang, 2013).
Furthermore, we found higher expression of Pkm2 in CCl4-
treated group, indicating that CCl4 treatment directly stimulates
carcinogenesis in the liver of treated animals. The treatment
with CCl4 and further CE and EC post-treatments through Pkm2
expression suggests that carcinogenesis is early initiated, and
importantly post-treatments with CE and EC may have an
anticarcinogenic effect on this stage. The links found between
the lactate level and Pkm2 in the liver after CCl4 mice treat-
ment, and EC and CE post-treatments, are also important
knowledge on the possible development of carcinogenesis in
the liver. Glucose lowering was associated with a decrease in
liver glycogen and higher Pkm2 expression in the group treated
with CCl4. EC and CE post-treatments raised blood glucose levels,
affected the increase in glycogen storage in the liver and sup-
pressed Pkm2.
Fig. 4B shows the induction of Actn4 in the liver of CCl4-
intoxicated mice and slight suppression after CE and EC post-
treatments. As actin-binding protein with structural function,
Aktn4 is responsible for regulating several processes in the cell.
Due to their known effects on the cell motility and adhesion,
we believe that Aktn4 plays an important role in the develop-
ment of liver fibrogenesis. The application of cocoa polyphenols
in acute liver injury leads us to conclude that cocoa has an
antifibrogenic effect.
We found that Hpx was induced only in the CCl4-treated
group (Fig. 4B) where, due to CCl 4 -induced intravascular
haemolysis, prevented pro-oxidant and pro-inflammatory
effects of heme released from hepatocytes. Expression of Hpx,
an acute phase protein, was present only in CCl4-treated group,
indicating the presence of an inflammatory process, while it
was absent in CE and EC post-treated groups.
3.5. Redox state in subcellular organelles as a result of
oxidative stress modulation
To better understand the underlying mechanisms of damage
and regeneration of tissue, the impact of xenobiotics in oxi-
dative stress at the level of subcellular organelles has been
examined recently (Forbes-Hernández et al., 2014;
Sandoval-Acuña, Ferreira, & Speisky, 2014). The redox state of
subcellular organelles, especially mitochondria and peroxi-
somes, which are major sites of cellular ROS/RNS and
antioxidant production, can be modulated and may be asso-
ciated with their ability to act as antioxidants (Sandoval-Acuña
et al., 2014). The changes found in protein level in subcellular
protein fractions provoked the modulation of protein activi-
ties during chemical/antioxidants exposure and may have an
impact on the cellular redox state.
The responsive changes in the whole and the subcellular
mouse liver proteome were observed (Fig. 4A and 5B). The
protein pattern of the whole liver homogenates showed strong
alterations in protein expression between CCl4 exposed and
control mice (Fig. 4A). As a result of CCl4 exposure, some changes
were observed by the disappearance and attenuation of several
protein bands in the range >50 kDa. In the CCl4-intoxicated
group, observable changes of some protein bands in the ranges
of 50–70 kDa, 70–120 kDa and 140–160 kDa were found (Fig. 4A).
As shown in Fig. 5A and 5B, protein levels and subcellular
mice liver protein profiles differ between the examined groups
as well as subcellular fractions. The highest concentration of
protein was found in the fraction PM and the lowest in Cyt frac-
tion (see Fig. 5A). Significant differences in the protein
concentration between groups were found in the Mt fraction
(P = 0.0381). Compared to the control, the CCl4-treated group
showed a significantly lower protein concentration in the Mt
fraction. Also, in post-treated groups, markedly increased protein
concentration was observed in the Mt fraction when com-
pared with the CCl4-treated group.
Antioxidant enzymes, catalase (CAT) and superoxide
dismutase (SOD) were localised according to their higher ac-
tivity (Fig. 6).

Fig. 5 – Subcellular fractionation: (A) differences in protein concentration in the cytosol (Cyt), plasma membrane (PM) and
crude mitochondria (Mt) in the liver; (B) SDS-PAGE of proteins in subcellular fractions (Cyt, PM and Mt) in the liver. Values
were expressed as mean ± SD (N = 8). Different letters above indicate statistical difference (P < 0.05). aSignificant difference
between control and CCl4-intoxicated group; bsignificant difference between CCl4-intoxicated group and EC and CE post-
treated groups.
 Journal of Functional Foods 23 (2016) 177–187 185

Fig. 6 – Activities of the antioxidant enzymes and total protein thiols in liver protein fractions: (A) catalase in the cytosol
(Cyt-CAT); (B) superoxide dismutase in crude mitochondria fraction (Mt-SOD); and (C) total protein thiols (TPT) in cytosol
and crude mitochondria fraction (Cyt-TPT, Mt-TPT). Values were expressed as mean ± SD (N = 8). Different letters above
indicate statistical difference (P < 0.05). aSignificant difference between control and CCl4-intoxicated group; bsignificant
difference between CCl4-intoxicated group and EC and CE post-treated groups; csignificant difference between EC and CE
post-treated groups.

Hydrogen peroxide (H2O2) is the main redox messenger mol-
ecule present both in the mitochondria and in peroxisomes.
However, in relation to the mitochondria, the peroxisomes
contain high amounts of H2O2. As a result of excessive accu-
mulation of subcellular H2O2 during CCl4 treatment, a marked
decrease in CAT activity was found in the liver Cyt fraction.
Also, a significant difference between CE and EC post-treated
group was found, indicating that CE increased CAT activity in
cytosol better than EC. However, Mt-SOD was elevated mark-
edly in CCl 4 -treated animals, while CE and EC showed a
tendency to increase the levels of Mt-SOD. We speculate that
this could be due to de novo synthesis of SOD in the mitochon-
dria due to CCl4 intoxication. Alteration of CAT activity reflected
on the cellular protein disulfide content, referred to as total
protein thiols (TPT), and enhanced inflammation through el-
evated TNF-α and IFN-γ (Figs. 3 and 6A and 6C). CE and EC post-
treatments significantly enhanced the CAT activity and TPT
levels when compared with CCl4-treated animals. It is impor-
tant to point out that CCl4 treatment per se decreased TPT both
in the cytosol and crude mitochondrial fraction, while it in-
creased significantly after CE and EC post-treatments.
Furthermore, we think that CE and EC increase the produc-
tion of TPT by their de novo synthesis. Anti-inflammatory activity
of CE and EC was shown by the significantly lower produc-
tion of two pro-inflammatory cytokines, TNF-α and IFN-γ, in
comparison to their production in CCl4-treated animals.
In many forms of experimental and clinical liver injury, in-
cluding alcoholic liver disease (ALD), these inflammatory
cytokines are increased and regulated by the transcription factor
NF-κB (Pena, Hill, & McClain, 1999). Considering those pa-
tients with cirrhosis have significantly lower glutathione (or
TPT, respectively) compared with healthy subjects, cocoa poly-
phenols may have important therapeutic implications for
multiple cytokine-mediated diseases.
The excess in the production of superoxide anion (O2•−) in
the peroxisomes leads to cellular lipoperoxidation and finally
results in an increase in mitochondrial production of H2O2 (Wang
et al., 2013). We found that the activity of peroxisomal CAT was
significantly lower (Fig. 6A) and Mt-SOD higher in the liver of
CCl4-treated animals (Fig. 6B). These results were explained by
CCl4-induced mitochondrial dysfunction (Knockaert et al., 2012),
but a possibility of relationships between the lower peroxi-
somal CAT activity and higher Mt-SOD activity may exist.
However, the relationship between mitochondrial activity and
peroxisomal functions is still unclear.
A major limitation of the effectiveness of polyphenols in
disease prevention is their bioavailability (Williamson & Manach,
2005) and dose effect with consideration of physiological
amounts in vivo. The identification and quantification of poly-
phenol metabolites focused on their potential biological activity
should be part of the personalised, functional food strategy in
disease prevention in the future.
4. Conclusions
Recent advances in the study of the effectiveness of dietary
polyphenols involve intake of functional foods in the preven-
tion, treatment and co-treatment in the control of several
diseases. Although the majority of studies were conducted in
vitro, confirmation in vivo is needed to report on their health
benefits.
Following oxidative stress upon CCl4 damage in the liver,
we observed that cocoa polyphenols had a beneficial effect on
cellular/subcellular redox state and on specific signalling
pathways.
Cocoa polyphenols decreased necrosis of hepatic cells, af-
fected glucose metabolisms through glycolytic and
gluconeogenic processes, and suppressed Pkm2. Post-treatments
with CE and EC influenced the increase of hepatic glycogen,
blood glucose was significantly increased and reached the
normal level, and lactate decreased after previous CCl 4
treatment.
Hepatotoxicity was reduced with cocoa polyphenols by de-
creasing serum transaminases and phosphatase activity (AST,
ALT and ALP). Cocoa caused down-regulation of Hsp90 acting
as the carcinogenic inhibitor, returned the expression of Hsp70
to normal levels after the decrease provoked by CCl4 treat-
ment, and thus improved cell survival. Furthermore, cocoa
polyphenols stimulated liver regeneration after intoxication with
CCl4 and had an anti-necrotic effect by decreasing necrosis and
186 Journal of Functional Foods 23 (2016) 177–187
increasing viability acting across Hsp70. Suppression of Aktn4
had an influence on the course of fibrogenesis while the re-
duction of Pkm2 expression may be associated with the
development of carcinogenesis. Furthermore, CE decreased in-
flammation by inhibiting acute phase protein Hpx and
decreasing the production of TNF-α and IFN-γ. Thus, CE has
numerous hepatoprotective effects, acting through the syn-
thesis of mitochondrial proteins and plasma membrane proteins
in hepatocytes. It protects the mitochondria from the damage
provoked by intoxication with hepatotoxins.
Localisation and alteration of cellular antioxidant status were
linked to the redox state of organelles through the produc-
tion of ROS and redox signalling between subcellular organelles.
Cocoa ameliorated CAT activity in the cytosol, modulated Mt-
SOD activity, and increased total protein thiols level.
Due to their palatability, cocoa products are desirable world-
wide, but due to their potent health benefits they can also be
recommended for the prevention and therapy of liver diseases.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgement
This work has been supported by the University of Rijeka under
Project Number 13.11.1.2.02.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.jff.2016.02.036.
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