Antioxidants properties of chocolates sold in Peru

Abstract

The goals of this paper were to evaluate total polyphenol content (TPC), total flavonoid content (TFC), anti-oxidant capacity (AC) and reducing power (RP) of eight dark chocolates that are sold in Peru. Imported and domestic chocolate samples containing between 50% and 74% cacao were defatted. Defatted chocolate (DCh) was separated and treated to extract phenolic compounds. The TPC and AC were determined by using the Folin-Ciocalteu reagent and two in vitro models based on the free-radical capturing capacity DPPH and ABTS. The RP was evaluated by using the potassium pherricyanide method; in the TFCs assessments, catechin was used as the standard. Chocolate TPC varied between 1.69 ± 0.02 and 5.39 ± 0.17 mg gallic acid/g chocolate and AC (DPPH-IC50) varied between 52.97 ± 1.77 and 158.67 ± 2.04 μg/ml extract, and by means of ABTS the values were between 12.01 ± 0.18 and 32.74 ± 0.49 μmol TEAC/g chocolate. Chocolates with 71% and 72% cacao showed a greater antioxidant capacity, which was confirmed by the RP test. Chocolates showed different levels of TPC, TFC, AC and RP, depended on a large degree of the cacao percentage. However, in some cases, there was no direct relationship among results, most likely due to different technological and thermal processes as well as different biological nature of cacao beans.

Full text

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Acta Agron. (2018) 67 (4) p 479-485 ISSN 0120-2812 | e-ISSN 2323-0118 https://doi.org/10.15446/acag.v67n4.71357
Antioxidants properties of chocolates sold in Peru
Propiedades antioxidantes de chocolates comercializados en Perú
María Rosario Calixto-Cotos1, Gabriela Cristina Chire-Fajardo2*, Carmen Adela Orihuela-Rivera1
1.Facultad de Química e Ingeniería Química de la Universidad Nacional Mayor de San Marcos, Perú. 2.Facultad
de Industrias Alimentarias de la Universidad Nacional Agraria La Molina, Perú. *Author for correspondence:
gchire@lamolina.edu.pe
Rec.: 31.03.2018 Acep.: 24.01.2019
Abstract
The goals of this paper were to evaluate total polyphenol content (TPC), total flavonoid content (TFC), anti-oxidant
capacity (AC) and reducing power (RP) of eight dark chocolates that are sold in Peru. Imported and domestic
chocolate samples containing between 50% and 74% cacao were defatted. Defatted chocolate (DCh) was separated
and treated to extract phenolic compounds. The TPC and AC were determined by using the Folin-Ciocalteu reagent
and two in vitro models based on the free-radical capturing capacity DPPH and ABTS. The RP was evaluated
by using the potassium pherricyanide method; in the TFCs assessments, catechin was used as the standard.
Chocolate TPC varied between 1.69 ± 0.02 and 5.39 ± 0.17 mg gallic acid/g chocolate and AC (DPPH-IC50) varied
between 52.97 ± 1.77 and 158.67 ± 2.04 μg/ml extract, and by means of ABTS the values were between 12.01
± 0.18 and 32.74 ± 0.49 µmol TEAC/g chocolate. Chocolates with 71% and 72% cacao showed a greater anti-
oxidant capacity, which was confirmed by the RP test. Chocolates showed different levels of TPC, TFC, AC and
RP, depended on a large degree of the cacao percentage. However, in some cases there was no direct relationship
among results, most likely due to different technological and thermal processes as well as different biological
nature of cacao beans.
Key words: ABTS; cacao; defatted chocolate; DPPH; Folin-Ciocalteu; flavonoid; polyphenolic compounds; reducing
power.
Resumen
En muestras de chocolate oscuro comercializados en Lima (Perú), se evaluaron el contenido de polifenoles
(CPT), flavonoides totales (CFT), capacidad anti-oxidante (CA) y poder reductor (PR). Las muestras importadas
y nacionales que contenían entre 50 y 74% de cacao fueron desengrasadas. Así se obtuvieron los chocolates
desengrasados (ChD), que fueron tratados para extraer compuestos fenólicos. El CPT y CA fueron determinados
usando el reactivo de Folin-Ciocalteu y dos modelos in vitro basados en la capacidad captadora del radical libre
(DPPH) y del catión radical (ABTS), respectivamente. El PR fue medido usando el método del ferricianuro de
potasio; en la determinación de los CFT se usó la catequina como estándar. El CPT en los chocolates varió entre
1.69 ± 0.02 y 5.39 ± 0.17 mg equivalente de ácido gálico/g chocolate y la CA (DPPH-IC50) varió entre 52.97 ±
1.77 y 158.67 ± 2.04 μg/ml extracto. La evaluación utilizando el modelo ABTS arrojó valores entre 12.01 ± 0.18
y 32.74 ± 0.49 µmoles TEAC/g chocolate. Los chocolates con 71 y 72% de cacao presentaron mayor capacidad
anti-oxidante, lo cual fue confirmado con la prueba de PR. Los chocolates mostraron diferentes grados de CPT,
CFT, CA y PR, y dependieron en gran medida del porcentaje de cacao. No obstante, en algunos casos no se
encontró dependencia debido a que el grano de cacao pasa por diversos procesos tecnológicos térmicos, además
de la naturaleza biológica propia del grano.
Palabras clave: ABTS; cacao; compuestos polifenólicos; chocolate desengrasado; DPPH; flavonoide; Folin-Ciocalteu;
poder reductor.

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Acta Agronómica. 67 (4) 2018, p 479-485
Introduction
Many studies have reported that cacao beans
contain abundant amount of flavan-3-ols as
major compounds, these are identified as
catechin and epicatechin (Donovan et al., 2012).
In 2012, a scientific book stated five important
cacao effects on: (1) vascular endothelial
function and platelet activity, (2) antioxidant
and anti-inflammatory, (3) lipid and lipoprotein
metabolism, (4) insulin sensitivity, (5) cerebral
blood flow and neurocognitive functioning
(Donovan et al., 2012).
Dark chocolates contain more cacao and
show a higher anti-oxidant capacity, a bigger
phenolic content and contain more monomeric
procyanidins than milk chocolate (Da Silva et
al., 2015). Vinson y Motisi (2015) discovered
a significant linear relationship between the
percentage of cacao solids and the anti-oxidants.
Even though there were individual differences in
the polyphenol content in samples of the same
chocolate category, a good correlation between
the anti-oxidant potency and the declared cacao
content was observed. Therefore, the information
about cacao content can be a reliable indicator of
the anti-oxidant potency of chocolates produced
in Serbia (Todorovic et al., 2015).
Currently there is little information about the
anti-oxidant properties of the dark chocolates
that are sold in Lima (Peru). That is why the
goal of this paper was the evaluation of the total
polyphenol content (TPC), the total flavonoid
content (TFC), the anti-oxidant capacity (AC) and
the reducing power (RP) of eight dark chocolates
that are sold in Lima, as well as to relate the cacao
content to the anti-oxidant properties.
Materials and methods
Population and sample size. Random dark
chocolate samples supplied by different chocolate
companies were purchased from local market in
Lima (Peru) in 3 months period from March to May
2015. The percentage and origin of cacao, either
domestic or imported were printed on the chocolate´s
labels. Eight random samples were selected,
from different large-scale chocolate companies,
two of domestic origin and six of imported origin.
The samples had different cacao contents (Chire,
Valdivia, Orihuela and Ureña, 2017).
Sample treatment. The chocolate samples
were dried using the 931.04 method (Horwitz and
Latimer, 2016) and then they were defatted with
petroleum ether using the Soxhlet 963.15 method
(Horwitz and Latimer, 2016). The fat was removed
and defatted chocolate (DCh) were obtained (Hu
et al., 2016). The solvent excess was removed
at room temperature. DCh showed light brown
color, they were packaged in polyethylene bags
and stored in refrigeration until the extraction
process was conducted.
Preparation of lyophilized extracts from
DCh. The extraction was carried out using basically
the method described by Belščak et al. (2009), only
slight modifications were introduced (Table 1). For
the first extraction, 80% methanol (v/v) was used
for six days. The sample was protected from light
at 4 ºC and then it was placed in an ultrasound
bath (BRANSON 1510) for 10 minutes at room
temperature, only once. After this, it was filtered
(Whatman paper No. 4) and centrifuged (SORWALL)
at 5000 r.p.m. for 10 minutes. For the second
extraction with 50% methanol was carried out for
24 hours and for the third extraction the same
quantity of methanol was used for only one hour
under the same conditions. The filtrates from the
three extractions were combined, then centrifuged
at 7500 r.p.m. and 5 ºC. After this the extracts were
concentrated under reduced pressure in a rotary
evaporator (BOECO) at 540 r.p.m. and 40 °C, then
the extracts were lyophilized (RIFICOR) (Figure 1).
The lyophilized extracts (LE) showed a color that
varied between brown and yellowish brown and had
a soft touch consistency. The yield was obtained by
weight difference and was stored in refrigeration.
The chocolates’ characteristics and the extracts’
details are described in Table 1. The lyophilized
extracts (LE) were expressed based on one gram
of chocolate (Ch).
Figure 1. Experimental scheme for the assessment
of the anti-oxidant properties of chocolate.
Source: Own elaboration

481
Total polyphenol content (TPC).- The
analysis was carried out according to the
technique described by Singleton, Orthofer,
Lamela-Raventos (1999), only slight modifications
were introduced. LE were weighed and dissolved
in bidistilled water to find a compatible dilution
with the absorbances of the calibration curve.
The aqueous solutions for each of the extracts
were prepared from 0.35 to 0.91 mg/mL. A 100
μL of the extract solution was mixed with 150 μL
of diluted Folin-Ciocalteu reagent, five minutes
later, 150 μL of 20% sodium carbonate was
added and it was filled with bidistilled water to
complete an amount of 1 mL. After 30 minutes
in the dark, spectrophotometer readings were
recorded (Spectroquant, Pharo 300 Merck) at
760 nm. Each of the eight LE was analyzed
by triplicate. A standard calibration curve was
made (gallic acid of 1.0-7.5 μg/mL, y = 0.0848x
+ 0.0291 and R2 = 0.9928) and the concentration
was expressed in mg Gallic Acid Equivalent in
one gram of chocolate (mg GAE/g).
Total flavonoid content (TFC).- The method of
Lee, Kim, Lee and Lee (2003) was used to determine
the TFC, only some modifications were introduced.
The LE were dissolved in a mixture of ethanol and
water at 50% v/v and from each sample 0.2 mL
were measured, then 0.06 mL of 5% NaNO3 were
added. Six minutes later 0.06 mL of 10% AlCl3.6H2O
were added. Again, six minutes later 0.8 mL of 1M
NaOH were added. The samples were stirred for 30
seconds each time a reagent was added. Finally,
the mixture was diluted to 2 mL and two minutes
later the absorbance was measured at 510 nm. A
calibration curve was made with catechin (2.5 – 17.0
μg/mL, y = 0.0301x + 0.0038 y R2 = 0.9907). The
results were expressed as mg Catechin Equivalent
in one gram of chocolate (mg CE/g).
Determination of anti-oxidant capacity
Capture capacity of the radical DPPH. The
anti-oxidant capacity was determined by the
method described by Brand-Williams, Cuvelier and
Berset (1995). A methanolic solution of the 0.1
mM 1,1-diphenyl-2-picrylhydraz (DPPH) radical
was prepared. Each of the eight LE were used
to prepare five aqueous solutions in increasing
concentrations. The reaction tubes contained
0.40 mL of the LE in aqueous solution with 0.800
mL of the methanolic DPPH solution and were
stirred for 30 seconds. After 30 minutes in the
dark at room temperature, the absorbance was
recorded at 517 nm (A extr spectrophotometer).
In parallel, a control tube was prepared; another
blank sample was used to correct the absorbances
of the extracts, the tests were run in triplicate.
Simultaneously, a calibration curve was made
using 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox) (Aref), its concentrations
were in the range of 1.8 - 5.4 μg/mL. The results
were expressed as IC50, these values were
obtained by designing the linear equation in the
graph, for the percentage of the capture capacity
of the DPPH radical (PCC-DPPH) versus the extract
concentration of each sample (µg/mL extract).
The same procedure was used for the trolox (y =
18.275x – 6.4 y R2
= 0.9906). El PCC-DPPH was obtained by the
following relationship:
Capture capacity of the radical ABTS. The test
was performed as described by Re et al. (1999). The
first step was to generate the ABTS radical cation
[2,2’-Azino-bis (3-ethylbenzthiazoline-6-sulfonic
acid)]; 7 mM of the radical were prepared, this was
kept in the dark at room temperature for 16 hours
and then it reacted with potassium persulfate at
2.45 mM. As a next step, the solution was adjusted
with water at 0.7 ± 0.05 absorbance at 734 nm. The
reaction mixture consisted of 0.98 mL of the ABTS
solution with 0.02 mL of the extracts dissolved in
water with concentrations from 25 to 400 μg/mL.
The mixture was stirred and incubated for seven
minutes in the dark at room temperature and
then the absorbance was measured at 734 nm.
Trolox (1-4 μg/mL) was used as reference standard.
The results were expressed in terms μmol Trolox
Equivalent Anti-Oxidant Capacity (TEAC) in one
gram of chocolate (μmol TEAC/g).
Reducing power (RP). The reducing power
was evaluated as described by Beyhan, Elmastas
and Gedikli (2010), some modifications were
introduced. The “A” mixture was prepared, it
contained the aqueous dilution of the LE in
different concentrations that varied between
0.039 and 0.515 μg/mL. From each one 0.5
mL were measured, then 0.5 mL of phosphate
buffer (0.2 M, pH 6.6) and 0.5 mL of potassium
ferricyanide 1% were added, the mixture was
stirred and incubated at 50 ºC for 20 minutes.
Then it was allowed to cool and 0.5 mL of 10%
trichloroacetic acid were added. After that,
0.5 mL were measured and taken from each
“A” mixture and placed in another tube with
0.5 mL of bidistilled water plus 0.1 mL of 0.1%
ferric chloride; 30 minutes later the results were
read at 700 nm. The absorbances of each of
the eight LE were compared with a calibration
curve (Padilla, Rincon and Bou-Rached, 2008) of
L-ascorbic acid (0.2 – 10.0 μg/mL, y = 0.082x +
0.0688, R2 = 0.998), the results were expressed
in mg Ascorbic Acid Equivalent in one gram of
chocolate (mg AAE/g).
Antioxidants properties of chocolates sold in Peru

482
Statistical analysis. Eight different samples
of LE corresponding from each type of dark
chocolate were used, each trial was carried
out in triplicate. A Completely Randomized
Design (CRD) was applied (p ≤ 0.05), the
results were expressed as the mean ± standard
deviation. Tukey reports and Pearson correlation
coefficients were done between treatments. The
SAS® program 9.1 version, was used to perform
the respective statistical analysis.
Results and discussion
As it is stated on the packages’ labels, the cacao
content of the eight samples of dark chocolate
varied between 50 and 74% of cacao with different
fat content (Table 1). The weight of DCh used to
carry out the aqueous methanol extraction was
between 2.88 and 10.95 g. DCh were used to
make three successive extractions. The yield of
lyophilized extracts ranged from 0.90 to 3.29 g.
Table 1. Characteristics and extraction yields of
chocolate traded in Peru.
Code
Origin
Cacao
%
Fat
%
DCh (g) for
extraction
Yield
Extract
(g)
27 Imported 50 35.11 10.04 3.29
29 Imported 50 33.15 4.54 1.05
8 National 52 32.00 10.72 3.03
36 Imported 62 38.66 10.95 3.09
34 National 65 33.83 2.88 0.90
30 Imported 71 43.68 8.91 2.36
24 Imported 72 45.18 10.13 2.29
33 Imported 74 36.93 10.43 2.85
DCh. = Defatted chocolate.
Total polyphenol content (TPC). The TPC
varied from 1.69 ± 0.02 to 5.39 ± 0.17 mg
GAE/g Ch for chocolate containing 50%, 52%,
62%, 65%, 71%, 72% and 74% of cacao (Table
2), in this regard, Todorovic et al. (2015) found
between 7.21 ± 0.49 and 12.65 ± 0.45 mg GAE/g
in five chocolate samples whose cacao content
was between 65% and 75%. These results were
obtained in a study by Grassi et al. (2005),
where 5 mg of polyphenols correspond to 1 g of
chocolate, whereas Vinson et al. (2006) found
that 23.9 mg of polyphenols correspond to 1 g
of chocolate. In both studies the polyphenol
standard used is not mentioned. However, in our
study the gallic-acid standard was used.
There is an increase in TPC in chocolates with
52%, 62%, 71% and 72% cacao, according to
Belščak et al. (2009), who state that the TPC de-
pends on the content of cacao solids in the product.
A very specific case is the chocolate with the lowest
percentage of cacao (50%) and with different TPC
values, one with 5.39 ± 0.17 mg GAE/g Ch (code
27, 35.11% fat, imported) and the other with 2.74 ±
0.10 mg GAE/g Ch (code 29, 33.15% fat, imported).
In the first case its increase is significant,because
on the packages´ label said “chocolate with at least
50% of cacao content”. There are several reasons
that explain these differences: (1) If the cacao beans
were roasted at low temperatures, (2) If the pro-
duced chocolate contains exogenous polyphenol as
an additive, as it is the case with the vanilla (Sun,
Da Silva and Spranger, 1998) which is usually used
in chocolate production for mass consumption, (3)
It may also be due to the presence of polyphenols
in the cacao butter. Although the chocolates were
defatted before determining the level of polyphe-
nols, there may also be vestiges of cacao butter (or
tocopherols, natural antioxidant) in the product,
which would impact the results, since cacao butter
has been found to be a source of anti-oxidants that
causes an increase in polyphenols (Hu et al., 2016).
Regarding chocolate containing 72% (45.18% fat,
imported) and 74% (36.93% fat, imported) cacao
with 4.57 ± 0.05 and 2,82 ± 0.08 mg GAE/g Ch,
respectively, there is no direct relationship be-
tween them. This can be explained by the reports
of Belščak et al. (2009) and Todorovic et al. (2015)
who indicated that variations in polyphenol levels
depend on the variety of cacao bean, geographic
origin, degree of ripeness and post-harvest condi-
tions (fermentation and drying), which as far as we
understand also affect manufacturing conditions
such as temperatures and the amount of time used
in the roasting and conching processes. In addition,
Perea-Villamil et al. (2009) attribute variations in
polyphenol content to roasting cacao beans in the
manufacturing processes. Likewise, Cooper et al.
(2008) report that the percentage of cacao that ap-
pears on the labels of chocolates cannot be used to
estimate the concentration of polyphenols.
Table 2. Total polyphenols and avonoid content of
chocolates traded in Peru.
Code Cacao Total polyphenols Total avonoids
(%) (mg GAE/g Ch) (mg CE/g Ch)
27 50 5.39 ± 0.17a* 1.99 ± 0.10a*
29 50 2.74 ± 0.10c* 1.04 ± 0.14bc*
8 52 2.18 ± 0.05d* 0.86 ± 0.18c*
36 62 2.33 ± 0.08d* 0.84 ± 0.27c*
34 65 1.69 ± 0.02e* 0.28 ± 0.03d*
30 71 4.34 ± 0.14b* 1.37 ± 0.05b*
24 72 4.57 ± 0.05b* 1.29 ± 0.12b*
33 74 2.82 ± 0.08c* 1.32 ± 0.16b*
*The values are expressed as mean ± standard deviation
(n = 3).
GAE. - Gallic acid equivalent. CE.- Catechin equivalent.
Acta Agronómica. 67 (4) 2018, p 479-485

483
Chocolates with a percentage of 65%, 52% and
62% of cacao (two of them domestic products) had
the lowest TPC values of the entire group, whereas
chocolates with a lower percentage of cacao
(50% code 27 and 29) have a higher TPC value.
This is explained by Todorovic et al. (2015) who
indicate that the quality of the raw material and
the production processes can have a significant
influence on the quality of the final product.
All samples assessed were different in TPC (p
value ≤ 0.05). Tukey results report first chocolate
containing 50% cacao (code 27, 35.11% fat,
imported) had high level of TPC and last level
was chocolate contained 65% cacao (domestic
product, 33.83% fat).
Total flavonoid content (TFC). The TFC
varied from 0.28 ± 0.03 to 1.99 ± 0.10 mg CE/g
Ch for chocolate between 50 and 74% cacao and
Meng et al. (2009) found 0.28 mg CE/g defatted
dark chocolate. Chocolate samples (Table 2) with a
50% (code 27), 71% and 74% cacao content show
higher TFC values, whereas those that contain
52%, 62% and 65% cacao have lower ones. These
results are quite different from those reported by
Hu et al. (2016) who found values ranging from
0.02 to 1.16 mg catechin/g defatted chocolate
and 0.02 to 6.01 mg epicatechin/g defatted
chocolate. The differences could stem from the
different evaluation techniques that were used.
Another reason is the origin of the samples: the
50% (code 27), 71% and 74% cacao samples were
imported, whereas the chocolate samples having
52% and 65% cacao were domestic products. It
should be noted that for a long time domestic
chocolate companies processed cacao paste (the
raw material used to produce chocolate) under
high temperatures, confirmed by results of Perea-
Villamil et al. (2009). Todorovic et al., (2015)
reported the TFC in dark chocolates. Thus, with
samples containing 75% cacao they obtained 24.4
± 0.6 μmol CE/g. In our study, using 74% cacao
samples we obtained 1.32 ± 0.16 mg CE/g Ch.
Several other studies suggest that epimerization
reactions occur during the manufacturing of
chocolate, where (-)-epicatechin is transformed
into (-)–catechin (Lambert, 2017). This compound
is not found naturally in cacao seeds (Gotti et al.,
2006). A correlation (r = 0.85937) between TPC
and TFC was observed (Meng et al. 2009).
All samples assessed were different in TFC
(p value ≤ 0.05). Tukey results report the first
chocolate sample containing 50% cacao (Code
27, imported) had high level of TFC and last level
was chocolate contained 65% cacao (domestic
product).
Anti-oxidant capacity (AC). Table 3 show the
results of the capturing capacity of the free radical
DPPH and the radical cation ABTS. Todorovic et
al. (2015) recommend that at least two methods
be used to determine the anti-oxidant capacity
in vitro since they offer us information about
the total anti-oxidant capacity of food products.
According to this study, the authors found
between 63.0 ± 4.2 and 92.2 ± 1.8 μM TEAC/g
in dark chocolates with 70% to 75% cacao
produced in Serbia, while our study on ABTS
obtained values of 27.87 ± 0.65 to 24.13 ± 0.08
μmolTEAC/g Ch in chocolate samples containing
71% to 74% cacao respectively, thereby indicating
that our samples had a lower anti-oxidant
capacity. Muñoz et al., (2002) grouped IC50 values
and classified the degree of anti-oxidant capacity
obtained by the DPPH radical technique. The
values of IC50 obtained by the DPPH technique
ranged from 52.97 ± 1.77 to 158.67 ± 2.04 μg/
mL extract (Table 3). Chocolates with 72% and
71% of cacao had a lower IC50 value with respect
to the DPPH radical and this indicates a greater
anti-oxidant capacity (Sánchez-Moreno, Larrauri
and Saura-Calixto, 1998). There is a relationship
between the TPC and TFC levels (r = 0.85937),
such as five samples of chocolate with IC50 values
have the following levels: 52.97 ± 1.77 (71%
cacao); 53.19 ± 0.79 (72% cacao); 61.99 ± 1.60
(50% cacao, code 27); 73.39 ± 0.17 (50% cacao,
code 29); and 75.83 ± 1.90 (74% cacao) μg/mL
extract (Table 3). These values are considered to
correspond to a moderate anti-oxidant capacity
(IC50 ≥ 50μg/mL and IC50 <100μg/mL). And when
the IC50 values are 129.06 ± 2.49 (62% cacao);
133.73 ± 1.17 (52% cacao) and 158.67 ± 2.04
(65% cacao) μg/mL extract, they show a low
anti-oxidant capacity (IC50 ≥100 μg/mL and IC50
<200 μg/mL) (Muñoz et al., 2002). Therefore,
chocolates with 52%, 62% and 65% of cacao (two
of them domestic products) showed low anti-
oxidant capacity due to the reduced polyphenols
and flavonoid content unlike the other sample.
Hu et al. (2016) reported that chocolate
manufacturers use a variety of cacao cultivars,
as well as processing and storage parameters, all
of which impact the anti-oxidant capacity and
phenolic profiles of the final products.
All assessed samples were different in AC (p
value ≤ 0.05). Tukey results report first chocolate
sample containing 50% cacao (code 27, imported)
had high level of AC in ABTS and the last chocolate
sample contained 65% cacao (domestic product).
Test for DPPH (IC50) was inverse than ABTS (r =
-0.89835), and ABTS has good correlation with
TPC (r = 0.93934).
Reducing power (RP). Finally, Table 3 shows
the reducing power (RP) data in dark chocolate.
Different studies have indicated that the ability
to donate electrons reflects the reducing power
(RP) of bioactive compounds (Beyhan et al., 2010).
Antioxidants properties of chocolates sold in Peru

484
Table 3. Antioxidant capacity of chocolate traded in Peru with different cacao percentage.
Cacao DPPH IC 50 ABTS Reducing power
% (µg/mL extract) (µmol TEAC/g Ch) (mg AAE/g Ch)
50 61.99 ± 1.60e* 32.74 ± 0.49a* 2.43 ± 0.51ab*
50 73.39 ± 0.17d* 19.75 ± 0.86d* 1.35 ± 0.61c*
52 133.73 ± 1.17b* 13.94 ± 0.02e* 1.34 ± 0.06c*
62 129.06 ± 2.49c* 15.22 ± 0.16e* 1.18 ± 0.06c*
65 158.67 ± 2.04a* 12.01 ± 0.18f* 2.06 ± 0.19bc*
71 52.97 ± 1.77f* 27.87 ± 0.65b* 3.16 ± 0.45a*
72 53.19 ± 0.79f* 25.47 ± 1.01c* 3.18 ± 0.09a*
74 75.83 ± 1.90d* 24.13 ± 0.08c* 3.11 ± 0.48ab*
*The values are expressed as mean ± standard deviation (n = 3).
DPPH. - 1,1-diphenyl-2-picrylhydraz. ABTS. - 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid). IC50.-
The half maximal inhibitory concentration. TEAC.- Trolox equivalent anti-oxidant capacity.
Chocolates with 72%, 71% and 74% cacao contain
3.18 ± 0.09, 3.16 ± 0.45 and 3.11 ± 0.48 mg AAE/g
Ch, respectively, and these show greater reducing
power. In contrast, chocolates with 62%, 52% and
50% cacao (code 29) (one of them domestic product)
contain 1.18 ± 0.06, 1.34 ± 0.06 and 1.35 ± 0.61
mg AAE/g Ch, and these exhibit a lower reducing
power (Table 3). Padilla et al. (2008), studied the
seeds of Theobroma cacao with a TPC of 6.66 ± 0.04
g GAE/100g and an RP of 5,80 ± 0.08 g AAE/100g,
and found that the TPC level influences the RP
value. A similar effect was found in this study, with
the samples containing 71% and 72% cacao, for
which there was a slight increase in RP for each
increase of TPC. This may be due to the existence
of different chemical structures in the TPC that
react as electron donors, which will influence
the RP (Padilla et al., 2008). The results showed
that the methanol extracts of DCh are capable
of donating electrons that can react with free
radicals. The difference in the RP test is explained
by Todorovic et al. (2015) who demonstrate that
the quality of the raw material (liquor or cacao
paste) and the production processes of chocolate
can have a significant impact on the quality of the
final product.
All samples assessed were different in RP (p value
≤ 0.05). Tukey results report the first group
chocolate sample containing 72 and 71% cacao
had high level of RP and the last group chocolate
samples contained 50% cacao (code 29), 52 and
62% cacao. RP has not a good relationship with
others properties.
Conclusions
Dark chocolates marketed in Lima (Peru) had
total polyphenol contents (TPC) of between 1.69 ±
0.02 and 5.39 ± 0.17 mg GAE/g Ch, and a total
flavonoid content (TFC) of between 0.28 ± 0.03 and
1.99 ± 0.10 mg CE/g Ch. A relationship was found
to exist between the TPC and anti-oxidant capacity
(AC) (ABTS, r = 0.93934) of dark chocolate. For
IC50 values (DPPH) was inverse than TEAC (ABTS)
(r = -0.89835). Chocolate containing 71 and 72%
cacao had the highest values of reducing power
among the group of eight dark chocolate samples
due to polyphenol. Domestic dark chocolates had
low antioxidant properties, suggesting studies with
novel technologies.
The components investigated in chocolate are
largely dependent on the percentage of cacao and
it is worth noting that in some cases there was no
dependence because the cacao bean goes through
various technological processes, especially
thermal treatments (roasting, sterilization and
conching) that affect the TPC in chocolates. In
addition, the biological nature and the variability
of the cacao beans does not favor a direct
relationship between the TPC and the percentage
of cacao declared on the labels of the chocolates.
Acknowledgments
Dra. Bertha Jurado Teixeira of the Universidad
Nacional Mayor de San Marcos, Peru, for her
valuable contribution to this study.
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Antioxidants properties of chocolates sold in Peru