Revista Mexicana

de Ingeniera Qumica
Academia Mexicana de Investigaci
on y Docencia en Ingeniera Qumica, A.C.

Revista Mexicana de Ingeniera Qumica

Volumen 12, N
umero 1, Abril 2013

Vol. 12,
No. 1 (2013) 11-18
CONTENIDO

ISSN 1665-2738

Volumen 8, nmero 3, 2009 / Volume 8, number 3, 2009

COMPARATIVE STUDY OF ULTRASOUND AND MACERATION TECHNIQUES

FOR THE EXTRACTION OF POLYPHENOLS FROM COCOA
BEANS (Theobroma cacao L.)
1

213 Derivation and application of the Stefan-Maxwell equations

ESTUDIO COMPARATIVO ENTRE LAS TECNICAS

DE ULTRASONIDO Y

MACERACI
PARAdeLA
EXTRACCI
ON DE POLIFENOLES DEL GRANO DE
(DesarrolloON
y aplicacin
las ecuaciones
de Stefan-Maxwell)
CACAO (Theobroma cacao L.)
Stephen Whitaker
C.N. Quiroz-Reyes1 , M.A. Aguilar-Mendez1 , M.E. Ramrez-Ortz2 y E. Ronquillo-De Jesus1
1

Centro
de Investigaci
on en Ciencia Aplicada y Tecnologa Avanzada del Instituto Politecnico Nacional, Legaria
Biotecnologa
/ Biotechnology
694, Colonia Irrigacion, 11500, Distrito Federal, Mexico.
2
245 Modelado
de Estudios
la biodegradacin
endebiorreactores
lodos de hidrocarburos
petrleo
Facultad de
Superiores
Cuautitlan, de
Universidad
Nacional Autototales
noma del
de M
exico.
Av. 1o. de Mayo s/n, col. Sta. Mara las Torres, 054740, Cuautitlan Izcalli, Estado de Mexico, Mexico.
intemperizados en suelos y sedimentos

Received 23 of August 2012; Accepted 12 of December 2012

(Biodegradation modeling of sludge bioreactors of total petroleum hydrocarbons weathering in soil

Abstract and sediments)

Polyphenols have gained significant interest in recent years due to their antioxidant capacity and their important role in disease
S.A. Medina-Moreno, S. Huerta-Ochoa, C.A. Lucho-Constantino, L. Aguilera-Vzquez, A. Jimnezprevention. In this work, the phenolic content and antioxidant properties of cocoa extracts prepared by maceration and
Gonzlez
y M. Gutirrez-Rojas
ultrasound-assisted
extraction
were studied. The polyphenol content was determined using the Folin-Ciocalteu reagent and
the antioxidant
activity was
estimated byyDPPH
(2,2-diphenyl-2-picrylhydrazyl)
and
FRAP (Ferriccidas
reducing/antioxidant power)
259 Crecimiento,
sobrevivencia
adaptacin
de Bifidobacterium infantis
a condiciones
assays. The identification and quantification of (+)-catechin and ()-epicatechin were performed using high-performance liquid
(Growth,
survival
adaptation
Bifidobacterium
infantis
to acidicitconditions)
chromatography
(HPLC).
The and
results
showedofthat
by using ultrasonic
radiation,
was possible to obtain higher polyphenol
contents from
both
the
husk
and
cotyledon
of
cocoa.
Furthermore,
extracts
obtained
by
sonication showed
theAzaolahighest antioxidant
L. Mayorga-Reyes, P. Bustamante-Camilo, A. Gutirrez-Nava, E. Barranco-Florido
y A.
activity, thus proving that this activity depends directly on the total phenolic content. The contents of (+)-catechin and ()Espinosa
epicatechin were
higher in the cotyledon extracts compared to those of the husk.
265 Statistical approach to optimization of ethanol fermentation by Saccharomyces cerevisiae in the

Keywords: cocoa, polyphenols, extraction, ultrasound, antioxidant activity.

Resumen presence of Valfor zeolite NaA

Los polifenoles han ganado un interes significativo en anos recientes debido a su capacidad antioxidante y a su papel importante
(Optimizacin
estadstica
fermentacin
etanlica
cerevisiae
en presencia
de de cacao
en la prevenci
on de enfermedades.
En de
estelatrabajo,
el contenido
fenode
licoSaccharomyces
y las propiedades
antioxidantes
de extractos
preparados por
maceraci
on yzeolite
extracci
on asistida por ultrasonido, fueron estudiados. El contenido de polifenoles fue determinado
zeolita
Valfor
NaA)
empleando el
reactivo
de
Folin-Ciocalteu
y la actividad
fue estimada
las tecnicas de DPPH (2,2-diphenylG. Inei-Shizukawa, H. A. Velasco-Bedrn,
G. antioxidante
F. Gutirrez-Lpez
and H.por
Hernndez-Snchez
2-picrylhydrazyl) y FRAP (Ferric reducing/antioxidant power). La identificacion y cuantificacion de (+)-catequina y ()epicatequina fueron realizadas a traves de la cromatografa de lquidos de alta resolucion (HPLC). Los resultados mostraron que
mediante
el uso dede
radiaci
on ultras
onica, engineering
fue posible extraer un mayor contenido de polifenoles tanto para cascarilla como para
Ingeniera
procesos
/ Process
cotiledo271
n deLocalizacin
cacao. Adem
a
s,
los
extractos
obtenidosRevisin
por sonicaci
on presentaron
la mayor
demostrando
de una planta industrial:
crtica
y adecuacin
de losactividad
criterios antioxidante,
empleados en
as que esta actividad depende directamente del contenido fenolico total. Los contenidos de (+)-catequina y ()-epicatequina
esta en
decisin
fueron mayores
los extractos obtenidos del cotiledon con respecto a aquellos obtenidos de la cascarilla.
(Plant site selection: Critical review and adequation criteria used in this decision)

Palabras clave: cacao, polifenoles, extraccion, ultrasonido, actividad antioxidante.

J.R. Medina, R.L. Romero y G.A. Prez

Corresponding author. E-mail: maguilarme@ipn.mx

Tel. (+52) 55 57 29 63 00 ext. 67778, Fax (+52) 55 53 95 41 47

Publicado por la Academia Mexicana de Investigacion y Docencia en Ingeniera Qumica A.C.

11

Quiroz-Reyes
al./
Revista
Mexicana
dede
Ingenier
mica
Vol.
12,
No.
XXX-XXX
Quiroz-Reyes
al./
Revista
Mexicana
Ingenier
aQu
Qu
mica
Vol.
12,
No.
1(2013)
(2013)
11-18
Quiroz-Reyes
etetet
al./
Revista
Mexicana
de
Ingenier
aaQu
mica
Vol.
12,
No.
11(2013)
XXX-XXX
38
38

39
39
40
40
41
41
42
42
43
43
44
44
45
45
46
46
47
47
48
48
49
49
50
50
51
51
52
52
53
53
54
54
55
55
56
56
57
57
58
58
59
59
60
60
61
61
62
62
63
63
64
64
65
65
66
66
67
67
68
68
69
69
70
70
71
71
72
72
73
73
74
74
75
75
76
76
77
77
78
78
79
79
80
80
81
81
82
82
83
83
84
84
85
85
86
86
87
87
88
88

12
22

Introduction
11 Introduction

89
89

Oxidation reactions
reactions occur
occur naturally
naturally in
in the
the human
human
Oxidation
body
and
are
formed
as
byproducts
of
respiration
and
body and are formed as byproducts of respiration and
oxidative
metabolism
in
all
cells
of
aerobic
organisms.
oxidative metabolism in all cells of aerobic organisms.
Although oxygen
oxygen isis necessary
necessary for
for aerobic
aerobic cells
cells to
to
Although
generate
energy,
it
has
the
disadvantage
of
producing
generate energy, it has the disadvantage of producing
small amounts
amounts of
of reactive
reactive oxygen
oxygen species
species (ROS)
(ROS)
small
that
can
cause
damage
to
macromolecules.
This
that can cause damage to macromolecules. This isis
related to
to aa number
number of
of chronic
chronic degenerative
degenerative diseases
diseases
related
such
as
cancer,
rheumatoid
arthritis,
Alzheimers,
such as cancer, rheumatoid arthritis, Alzheimers,
atherosclerosis, emphysema,
emphysema, cirrhosis
cirrhosis and
and diabetes
diabetes
atherosclerosis,
among
others,
which
share
common
pathogenic
ROS
among others, which share common pathogenic ROS
as
a
key
factor
(Mora-Huerta
et
al.,
2010;
Yoshihara
as a key factor (Mora-Huerta et al., 2010; Yoshihara
al., 2010).
2010). Because
Because the
the body
body isis vulnerable
vulnerable to
to
etet al.,
these
radicals,
antioxidants
are
needed
to
decrease
these radicals, antioxidants are needed to decrease
the concentration
concentration of
of these
these reactive
reactive species
species to
to prevent
prevent
the
the
negative
effects
mentioned
above.
Antioxidants
the negative effects mentioned above. Antioxidants
in the
the body
body are
are primarily
primarily derived
derived from
from diet
diet and
and can
can
in
promote
good
health.
Thus,
due
to
their
significant
promote good health. Thus, due to their significant
rolein
indisease
diseaseprevention,
prevention,there
therehas
hasbeen
beenan
anincreasing
increasing
role
interest
in
recent
years
in
the
study
of
certain
fruits,
interest in recent years in the study of certain fruits,
vegetables
and
grains
with
high
antioxidant
contents
vegetables and grains with high antioxidant contents
toboost
boosttheir
theirconsumption
consumption(Wootton-Beard
(Wootton-Beardand
andRyan,
Ryan,
to
2011).
2011).
Cocoa isis recognized
recognized as
as aa major
major dietary
dietary source
source of
of
Cocoa
antioxidants
because
of
its
high
phenolic
compound
antioxidants because of its high phenolic compound
(procyanidins and
and flavonols
flavonols mainly)
mainly) content
content (Tom
(Tom
(procyanidins
aas-sBarber
a
n
et
al.,
2007).
It
has
even
been
observed
Barberan et al., 2007). It has even been observed
thatcocoa-based
cocoa-basedproducts
productscontain
containaahigher
higherantioxidant
antioxidant
that
capacity
and
greater
amounts
of
flavonoids
perserving
serving
capacity and greater amounts of flavonoids per
than
tea
or
red
wine
(Jonfia-Essien
et
al.,
2008).
than tea or red wine (Jonfia-Essien et al., 2008).
It
is
possible
to
distinguish
three
main
groups
of
It is possible to distinguish three main groups of
polyphenols
in
cocoa:
catechins
or
flavan-3-ols
polyphenols in cocoa: catechins or flavan-3-ols
(37%), anthocyanins
anthocyanins (4%)
(4%) and
and proanthocyanidins
proanthocyanidins
(37%),

(58%)
(Bel
s
c
ak
et
al.,
2009).
The
main catechin
catechin
(58%) (Belsc ak et al., 2009). The main
is
()-epicatechin,
which
constitutes
approximately
is ()-epicatechin, which constitutes approximately
35% of
of the
the total
total polyphenol
polyphenol content
content of
of cocoa.
cocoa. In
In
35%
addition
to
the
cotyledon,
the
husk
(a
byproduct
of
the
addition to the cotyledon, the husk (a byproduct of the
chocolateindustry)
industry)also
alsocontains
containsaasignificant
significantamount
amount
chocolate
of
phenolic
compounds
(Lecumberri
et
al.,
2006).
of phenolic compounds (Lecumberri et al., 2006).
The extraction
extraction of
of phenolic
phenolic compounds
compounds from
from
The
plant
materials
is
influenced
by
the
compounds
plant materials is influenced by the compounds
chemical nature,
nature, extraction
extraction method,
method, sample
sample size,
size,
chemical
time
and
storage
conditions
as
well
as
the
presence
time and storage conditions as well as the presence
of interfering
interfering substances
substances such
such as
as proteins
proteins and
and
of
carbohydrates
(Koffi
et
al.,
2010;
Garc

a-M
a
rquez
carbohydrates (Koffi et al., 2010; Garca-Marquez
al., 2012).
2012). ItIt has
has been
been reported
reported that
that the
the use
use of
of
etet al.,
aqueous
solutions
of
methanol,
ethanol
and
acetone
aqueous solutions of methanol, ethanol and acetone
dramatically improves
improves the
the extraction
extraction of
of polyphenols
polyphenols
dramatically
compared
to
a
single-compound
solvent
system
compared to a single-compound solvent system
(Yilmaz
and
Toledo,
2006).
The
most
reported
(Yilmaz and Toledo, 2006). The most reported

90
90
91
91
92
92
93
93
94
94
95
95
96
96
97
97
98
98
99
99
100
100
101
101
102
102
103
103
104
104
105
105
106
106
107
107
108
108
109
109
110
110
111
111
112
112
113
113
114
114
115
115
116
116
117
117
118
118
119
119
120
120
121
121
122
122
123
123
124
124
125
125
126
126
127
127
128
128
129
129
130
130
131
131
132
132
133
133
134
134
135
135
136
136

extraction methods
methods are
are maceration
maceration with
with solvents,
solvents,
extraction
hot-water
extraction,
alkaline
extraction,
resin-based
hot-water extraction, alkaline extraction, resin-based
extraction, enzyme-assisted
enzyme-assisted extraction,
extraction, extractions
extractions
extraction,
based
on
gamma
and
electron-beam
irradiation
and
based on gamma and electron-beam irradiation and
extraction
using
supercritical
fluids.
However,
some
extraction using supercritical fluids. However, some
of these
these methods
methods can
can cause
cause aa loss
loss of
of bioactive
bioactive
of
compounds
due
to
the
use
of
high
temperatures
and
compounds due to the use of high temperatures and
long
extraction
times;
or,
in
the
case
of
irradiation,
long extraction times; or, in the case of irradiation, itit
can represent
represent aa health
health risk
risk ifif the
the proper
proper care
care isis not
not
can
taken
(Liu
et
al.,
2005).
These
shortcomings
have
taken (Liu et al., 2005). These shortcomings have
led to
to the
the use
use of
of new
new sustainable
sustainable innovative
innovative green
green
led
techniques
that
increase
extraction
efficiency,
reduce
techniques that increase extraction efficiency, reduce
timeand
andenergy-consuming
energy-consumingprocedures
proceduresand
andcontribute
contribute
time
to
environmental
preservation
by
reducing
the use
use
to environmental preservation by reducing the
of
water
and
solvents,
fossil
energy
and
generation
of water and solvents, fossil energy and generation
of hazardous
hazardous substances,
substances, such
such as
as microwave
microwave and
and
of
ultrasound-assisted
extraction
(Chemat
et
al.,
2011).
ultrasound-assisted extraction (Chemat et al., 2011).
Theuse
useof
ofultrasonic
ultrasonicradiation
radiation(20-100
(20-100kHz)
kHz)to
toextract
extract
The
natural
compounds
provides
high
reproducibility,
easy
natural compounds provides high reproducibility, easy
handling,
low
solvent
and
energy
consumption,
lowhandling, low solvent and energy consumption, lowtemperature processing
processing and
and aa lower
lower loss
loss of
of bioactive
bioactive
temperature
compounds
(Pan
et
al.,
2011).
Compared
with
compounds (Pan et al., 2011). Compared with
other
novel
extraction
techniques
such
as
microwaveother novel extraction techniques such as microwaveassistedextraction,
extraction,the
theultrasound
ultrasoundapparatus
apparatusisischeaper
cheaper
assisted
and
its
operation
is
easier.
Furthermore,
the
and its operation is easier.
Furthermore, the
ultrasound-assisted
extraction,
like
Soxhlet
extraction,
ultrasound-assisted extraction, like Soxhlet extraction,
can be
be used
used with
with any
any solvent
solvent for
for extracting
extracting aa wide
wide
can
variety
of
natural
compounds
(Wang
and
Weller,
variety of natural compounds (Wang and Weller,
2006).
Ultrasound can
can facilitate
facilitate swelling
swelling and
and
2006).
Ultrasound
hydration
of
vegetal
tissue,
allowing
high
diffusion
hydration of vegetal tissue, allowing high diffusion
rates across
across the
the cell
cell wall
wall and
and enhancing
enhancing the
the mass
mass
rates
transfer.
On
the
other
hand,
cavitation
produced
transfer. On the other hand, cavitation produced
by ultrasonic
ultrasonic waves
waves can
can also
also disrupt
disrupt the
the cell
cell wall,
wall,
by
facilitating the
the release
release of
of contents
contents (Vinatoru,
(Vinatoru, 2001).
2001).
facilitating
Khan etet al.,
al., (2010)
(2010) reported
reported higher
higher extraction
extraction yields
yields
Khan
of
polyphenols
from
orange
peel
using
an
ultrasonic
of polyphenols from orange peel using an ultrasonic
processor operated
operated atat aa frequency
frequency of
of 25
25 kHz.
kHz.
processor
According
to
Jacques
et
al.
(2007),
sonication
According to Jacques et al. (2007), sonication isis
simpler, faster
faster and
and more
more effective
effective technique
technique than
than
aa simpler,
maceration
to
extract
organic
compounds
from
Ilex
maceration to extract organic compounds from Ilex
paraguariensis
leaves.
Currently,
there
are
no
reports
paraguariensis leaves. Currently, there are no reports
inthe
theliterature
literatureon
onthe
theextraction
extractionof
ofpolyphenols
polyphenolsfrom
from
in
cocoa
beans
using
ultrasonic
radiation.
Therefore,
the
cocoa beans using ultrasonic radiation. Therefore, the
objective
of
this
study
was
to
evaluate
the
effect
of
objective of this study was to evaluate the effect of
ultrasonic
treatment
on
the
total
phenolic
content
and
ultrasonic treatment on the total phenolic content and
antioxidant activity
activity of
of extracts
extracts from
from cocoa
cocoa husk
husk and
and
antioxidant
cotyledon.
In
addition,
a
comparison
was
made
with
cotyledon. In addition, a comparison was made with
respectto
tothe
thetraditional
traditionalmethod.
method.
respect

www.rmiq.org
www.rmiq.org
www.rmiq.org

Quiroz-Reyes
et et
al./ Revista
Mexicana
dede
Ingenier
a Qu
mica
Vol.
12,12,
No.
1 (2013)
XXX-XXX
Quiroz-Reyes
Revista
Mexicana
Ingenier
a Qu
mica
Vol.
No.
1 (2013)
11-18
Quiroz-Reyes
et al./al./
Revista
Mexicana
de Ingenier
a Qu
mica
Vol.
12, No.
1 (2013)
XXX-XXX
137
137

22 Methodology
Methodology

138
138

2.1
2.1 Materials
Materials and
and reagents
reagents

139
139
140
140
141
141
142
142
143
143
144
144
145
145
146
146
147
147

The cocoa beans were purchased at a local market

The cocoa beans were purchased at a local market
in Mexico City.
The reagents 2,2-diphenylin Mexico City.
The reagents 2,2-diphenyl1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent,
1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent,
gallic acid, Tris-HCl buffer, 2,4,6-Tris (2-pyridyl)-sgallic acid, Tris-HCl buffer, 2,4,6-Tris (2-pyridyl)-striazine (TPTZ) and ferric chloride hexahydrate were
triazine (TPTZ) and ferric chloride hexahydrate were
purchased from Sigma-Aldrich (USA). Acetic acid,
purchased from Sigma-Aldrich (USA). Acetic acid,
ascorbic acid and sodium acetate were obtained from
ascorbic acid and sodium acetate were obtained from
JT Baker (Mexico). Standards and solvents for HPLC
JT Baker (Mexico). Standards and solvents for HPLC
analysis were provided by Sigma-Aldrich.
analysis were provided by Sigma-Aldrich.

148
148

2.2
2.2 Preparation
Preparation of
of extracts
extracts

149
149
150
150
151
151
152
152
153
153
154
154
155
155
156
156
157
157
158
158
159
159
160
160
161
161
162
162
163
163
164
164
165
165
166
166
167
167
168
168
169
169
170
170
171
171
172
172
173
173
174
174
175
175
176
176

Cocoa beans were husked manually and fractions

Cocoa beans were husked manually and fractions
(husk and cotyledon) ground separately in a disc mill
(husk and cotyledon) ground separately in a disc mill
(148-2, The Bauer Bros Co., USA). In the case of
(148-2, The Bauer Bros Co., USA). In the case of
cotyledon, fat was removed from the material by
cotyledon, fat was removed from the material by
soaking in hexane for 24 h at room temperature.
soaking in hexane for 24 h at room temperature.
The extraction of phenolic compounds from both
The extraction of phenolic compounds from both
fractions was performed by maceration and ultrasound
fractions was performed by maceration and ultrasound
application. Maceration: the plant material was
application. Maceration: the plant material was
subjected to a first extraction using a water-methanol
subjected to a first extraction using a water-methanol
solution (1:1 ratio) for 2 h at room temperature
solution (1:1 ratio) for 2 h at room temperature
and under constant stirring. Then, the mixture was
and under constant stirring. Then, the mixture was
centrifuged (3,000 rpm, 15 min) and filtered. The
centrifuged (3,000 rpm, 15 min) and filtered. The
residue was recovered for a second extraction (2 h)
residue was recovered for a second extraction (2 h)
with an acetone-water solution (70:30 ratio), repeating
with an acetone-water solution (70:30 ratio), repeating
the centrifugation and combining the supernatants
the centrifugation and combining the supernatants
with those obtained previously. Ultrasound: the
with those obtained previously. Ultrasound: the
process was similar to that described above except
process was similar to that described above except
that instead of soaking for 2 h, the mixture of plantthat instead of soaking for 2 h, the mixture of plantsolvent material was subjected to two 30-min periods
solvent material was subjected to two 30-min periods
of ultrasonic radiation (25 kHz) in an ultrasonic bath
of ultrasonic radiation (25 kHz) in an ultrasonic bath
(TI-H-5, Elma, Germany) using the same solvent
(TI-H-5, Elma, Germany) using the same solvent
systems. The extraction conditions used in both
systems. The extraction conditions used in both
methods were established after preliminary tests.
methods were established after preliminary tests.
In all the extractions the ratio sample-solvent was
In all the extractions the ratio sample-solvent was
1:20. Finally, the cocoa extracts were concentrated
1:20. Finally, the cocoa extracts were concentrated
in a rotary evaporator (40-60 rpm, 50 C) (RE-500,
in a rotary evaporator (40-60 rpm, 50 C) (RE-500,
Yamato, Japan) and dried under vacuum for 24 h at
Yamato,
Japan) and dried under vacuum for 24 h at
30 C.
30 C.

177
177

2.3
2.3 Determination
Determination of
of total
total phenol
phenol content
content

178
178
179
179
180
180
181
181
182
182

The total phenolic content was calculated from the

The total phenolic content was calculated from the
reduction capacity of Folin-Ciocolteau using gallic
reduction capacity of Folin-Ciocolteau using gallic
acid as a standard (Waterhouse 2002). A sample
acid as a standard (Waterhouse 2002). A sample
volume of 100 L was added to 7 mL distilled water,
volume of 100 L was added to 7 mL distilled water,
followed by the addition of 500 L of Folin-Ciocalteu
followed by the addition of 500 L of Folin-Ciocalteu

183
183
184
184
185
185
186
186
187
187
188
188
189
189

reagent (2N). The final solution was allowed to stand

reagent (2N). The final solution was allowed to stand
for 3 min at room temperature. Thereafter, 1.5 mL
for 3 min at room temperature. Thereafter, 1.5 mL
of a solution of sodium carbonate was added (20%
of a solution of sodium carbonate was added (20%
w/v). After 90 min of rest in the dark, the absorbance
w/v). After 90 min of rest in the dark, the absorbance
was determined at a wavelength of 760 nm (Cary 50,
was determined at a wavelength of 760 nm (Cary 50,
Varian, USA). The results were expressed as milligram
Varian, USA). The results were expressed as milligram
equivalents of gallic acid g1
of dry matter.
equivalents of gallic acid g1 of dry matter.

190
190

2.4
2.4 DPPH
DPPH radical
radical scavenging
scavenging capacity
capacity

191
191
192
192
193
193
194
194
195
195
196
196
197
197
198
198
199
199

200
200
201
201
202
202
203
203
204
204
205
205

The antiradical capacity was determined by the DPPH

The antiradical capacity was determined by the DPPH
assay following the methodology proposed by Othman
assay following the methodology proposed by Othman
et al. (2007), with some modifications. A 2-mL
et al. (2007), with some modifications. A 2-mL
aliquot of extract was mixed with 500 L of 0.1M
aliquot of extract was mixed with 500 L of 0.1M
Tris-HCl Buffer by vortex mixing for 5 s. To this
Tris-HCl Buffer by vortex mixing for 5 s. To this
solution, 2 mL of a 200-M solution of DPPH were
solution, 2 mL of a 200-M solution of DPPH were
added. After 30 min, the absorbance was determined
added. After 30 min, the absorbance was determined
at 517 nm. The percentage of DPPH reduction was
at 517 nm. The percentage of DPPH reduction was
calculated using the Eq. (1).
calculated using the Eq. (1).
!
Absorbance o f sample !
Inhibition (%) = 1 Absorbance o f sample 100
Inhibition (%) = 1 Absorbance o f control 100
Absorbance o f control
(1)
(1)
The EC50 value was determined from the data in
The EC50 value was determined from the data in
the graph of the DPPH reduction effect against the
the graph of the DPPH reduction effect against the
extract concentration. The EC50 was determined as the
extract concentration. The EC50 was determined as the
necessary amount of the extract studied to reduce the
necessary amount of the extract studied to reduce the
concentration of DPPH by 50%, using ascorbic acid as
concentration of DPPH by 50%, using ascorbic acid as
a control.
a control.

207
207

2.5
2.5 Ferric
Ferric reducing/antioxidant
reducing/antioxidant
(FRAP)
assay
(FRAP) assay

208
208
209
209
210
210
211
211
212
212
213
213
214
214
215
215
216
216
217
217
218
218
219
219

The antioxidant capacity was determined using the

The antioxidant capacity was determined using the
FRAP test (Thaipong et al., 2006). This method
FRAP test (Thaipong et al., 2006). This method
determines the antioxidant capacity of polyphenols to
determines the antioxidant capacity of polyphenols to
reduce TPTZ-Fe3+
complex. The FRAP reagent was
reduce TPTZ-Fe3+ complex. The FRAP reagent was
prepared by mixing 25 mL of a 0.3 M acetate buffer
prepared by mixing 25 mL of a 0.3 M acetate buffer
(pH 3.6), 2.5 mL of TPTZ solution (0.01M) and 2.5
(pH 3.6), 2.5 mL of TPTZ solution (0.01M) and 2.5
mL of a solution of FeCl3 6H2 O (0.02M) at 37 C.
mL of a solution of FeCl3 6H2 O (0.02M) at 37 C.
A 150-L extract sample was mixed with 2850 L of
A 150-L extract sample was mixed with 2850 L of
FRAP solution and allowed to stand for 30 min in the
FRAP solution and allowed to stand for 30 min in the
dark. The absorbance was read at a wavelength of 593
dark. The absorbance was read at a wavelength of 593
nm. The results were reported in M ascorbic acid
nm. The results were reported in M ascorbic acid
equivalents.
equivalents.

220
220

2.6
2.6 HPLC
HPLC analysis
analysis of
of cocoa
cocoa extracts
extracts

221
221
222
222
223
223
224
224

The HPLC-analyses were carried out in an

The HPLC-analyses were carried out in an
Agilent 1200 chromatograph (Agilent Technologies,
Agilent 1200 chromatograph (Agilent Technologies,
Germany) equipped with a quaternary pump and
Germany) equipped with a quaternary pump and
a multiple wavelength detector coupled to an HP
a multiple wavelength detector coupled to an HP

206
206

www.rmiq.org
www.rmiq.org
www.rmiq.org

power
power

1333

Quiroz-Reyes et al./ Revista Mexicana de Ingeniera Qumica Vol. 12, No. 1 (2013) XXX-XXX
Quiroz-Reyes
Revista
Mexicana
Ingenier
a Qu
mica
Vol.
No.
1 (2013)
11-18
Quiroz-Reyes
et et
al./al./
Revista
Mexicana
dede
Ingenier
a Qu
mica
Vol.
12,12,
No.
1 (2013)
XXX-XXX
Chem Station (rev. B.04.01) data-processing station. 272
226
Separations were conducted on a Zorbax Eclipse
225
Chem Station
(rev.dimensions
B.04.01) data-processing
227
XDB-C18
with
4.6x150 mm station.
and 5 272
226
Separations
were
conducted
on
a
Zorbax
Eclipse
228
m particle size.
The mobile phase consisted
227
XDB-C18
with acid
dimensions
4.6x150
mm A
andand5
229
of
water/formic
(99.9/0.1)
as eluent
228
m
particle
size.
The
mobile
phase
consisted
230
methanol/acetonitrile (50/50) as eluent B. The system
229
of water/formic
acid (99.9/0.1)
eluent Ainand
231
was
run with a gradient
program:as 10-60%B
15
230
methanol/acetonitrile
(50/50)
as
eluent
TheBsystem
232
min, followed by isocratic elution withB.60%
for 5
231
was run
with temperature
a gradient program:
15
233
min.
Column
was set at10-60%B
30 C, flowinrate
1isocratic elution with 60% B for 5
232
min,
followed
by
234
was 400 l min and the injection volume was 5
233
min.Samples
Columnwere
temperature
wasdissolved
set at 30in
C,aflow
rate
235
L.
previously
mixture
1
234
was
400
l
min
and
the
injection
volume
was
236
of water/methanol (70%) and filtered through a 0.455
235
L. membrane
Samples were
previously
dissolved
in a mixture
237
m
filter.
The peaks
of (+)-catechin
and
236
of
water/methanol
(70%)
and
filtered
through
a 0.45
238
()-epicatechin were identified by comparing
the
237
m membrane
The peaks
(+)-catechin
and 273

239
retention
times filter.
of samples
with of
those
of standards.
238
()-epicatechin were
were recorded
identifiedat by
the 274 Fig. 1. Total phenolic content of cocoa extracts. Values
240
Chromatograms
280comparing
nm. Standard

239
retention times
of were
samples
those and
of standards.
273
241
calibration
curves
alsowith
prepared
used for 275
are expressed as meansd (n = 3). Means with
240
Chromatograms
were recorded at 280 nm. Standard 276
274
Fig.
phenolic
content of cocoa
extracts.
Fig.
242
quantitative
analysis.
different
letters
were significantly
different
(P <Values
0.05).
1.1.Total
241
calibration curves were also prepared and used for 275 are expressed as meansd (n = 3). Means with
Fig. 1. letters were significantly different (P < 0.05).
242
quantitative analysis.
276
different
277
for cocoa husk and cotyledon (Tomas-Barberan et
243
2.7 Statistical analysis
2007; Lecumberri et al., 2006).
278
al.,
However,
244
All
were performed in triplicate and 279
277
for iscocoa
husk and
cotyledon
(Tom
as-Barber
an of
et
243
2.7 measurements
Statistical analysis
it
important
to
emphasize
that
the
use

245
the results analyzed by ANOVA. Differences between 280
278
al.,
2007;radiation
Lecumberri
etimproved
al., 2006).
However,
ultrasonic
greatly
the
extraction
of
244
All measurements
performed
in triplicate
and 279 it is important to emphasize that the use of
246
means
were detectedwere
by Duncans
multiple
range test.

281
polyphenols,
especially in the case of cocoa cotyledon.
245
the results analyzed
by ANOVA.
Differences
247
Significant
differences
were considered
at a between
level of 282
280
ultrasonic
radiation greatly
extraction
of
The
big
difference
in theimproved
phenolic the
content
values
246
means
wereLinear
detected
by Duncans
range test.

248
P < 0.05.
regression
tests multiple
were performed
to 283
281
polyphenols,
especially
in the
case
of can
cocoa
cotyledon.
obtained
for
both
fractions
of
cocoa
be
attributed
247
Significantcorrelations
differences between
were considered
at a level of 282 The big difference in the phenolic content values
249
determine
data.
mainly
to the fact that the amount of polyphenols is

248
P < 0.05. Linear regression tests were performed to 284
283
obtained
for both
cocoaofcan
attributed
285
not
identical
in thefractions
differentofparts
the be
cocoa
bean.
249
determine correlations between data.

284
mainly
to the
themoisture
fact thatand
the particle
amountsize
of polyphenols
is
286
However,
of
the
samples
250
3 Results and discussion
285
not two
identical
in the
different
parts
of the influence
cocoa bean.
287
are
features
that
have
an
important
on

286
However,
the moisture
and particle
the samples
288
the
efficiency
of extraction
(Wang size
and of
Weller
2006).
250
3
Results
and
discussion
251
3.1 Total phenol content
287
are two
features
thatparticle
have ansize
important
influence
on
289
The
reduction
in the
of the plant
material
288
the efficiency
of extraction
(Wang
and Weller
2006).
290
will
increase
the
number
of
cells
directly
exposed
to
252
Figure
1
shows
the
total
phenolic
content
of
cocoa
251
3.1 Total phenol content

289
The
reduction
in the particle
size of cavitation
the plant material
extraction
by solvent
and ultrasonic
(Vilkhu
253
extracts obtained by ultrasound and maceration 291
290
will
increase
of cells
exposed
to
252
Figure 1 shows
total phenolic
content of
cocoa
al.,
et
2008).theInnumber
this case,
the directly
cotyledon
samples
254
techniques.
In thethe
extraction
of polyphenols
from
the 292
291
extraction higher
by solvent
and ultrasonic
cavitation
(Vilkhu
253
extracts
ultrasound
and maceration
presented
moisture
content and
lower values
of
255
husk,
the obtained
sonicationby
allowed
the extraction
of higher 293
292
et al., 2008).
this samples,
case, thewhich
cotyledon
254
techniques.
In the (25.341.82
extraction of mg
polyphenols
from the
particle
size thanInhusk
wouldsamples
explain
256
phenolic
contents
g1 ) compared
to 294
293
presented
higher
moisture
content
and
lower
values
of
255
husk,
the sonication
the extraction
of higher
the
greater
efficiency
in
polyphenol
release.
257
the
content
extractedallowed
by maceration
(17.851.33
mg 295

1
256
contentsthere
(25.341.82
mg g1 ) compared
to 294 particle size than husk samples, which would explain
258
gphenolic
). However,
were no significant
differences
257
the content
maceration
(17.851.33
mg 295 the greater efficiency in polyphenol release.
259
(P
> 0.05)extracted
betweenbyboth
methods.
In the case
296
3.2 Antiradical capacity (DPPH assay)
1
258
g the
). However,
no significant
differences
260
of
cotyledon,there
the were
phenolic
content results
were

259
(P > 0.05) different
between both
In the case
Figure
2 shows the capacity
scavenging(DPPH
activityassay)
of extracts
261
significantly
(P <methods.
0.05) between
the 297
296
3.2 Antiradical
260
of theextraction
cotyledon,
the phenolic
results
were 298 from

the husk obtained using the proposed methods.

262
two
methods.
The content
polyphenol
content
297
Figure
the scavenging
of extracts
261
significantly
different (Pto 135.923.77
< 0.05) between
It
can 2beshows
seen that
the activityactivity
increased
rapidly
263
varied
from 91.060.86
mg g1 the
for 299
298
from
husk obtainedrange
usingofthe0.04-0.1
proposedmg
methods.
262
two extractionand
methods.
Themethods
polyphenol
content 300
in
thetheconcentration
mL1 ,
264
conventional
ultrasound
respectively.
1
299
It
can
be
seen
that
the
activity
increased
rapidly
263
varied
from
91.060.86
to
135.923.77
mg
g
for
265
According to Pan et al. (2011), the mechanical effects 301 remaining constant at higher concentrations. The
300
in the concentration
0.04-0.1 application
mg mL1 ,
264
conventional
and ultrasound
respectively.
compounds
extractedrange
by of
ultrasound
266
of
sonication allow
for a greatermethods
penetration
of solvent 302
301
remaining
constant
at
higher
concentrations.
The
265
According
to
Pan
et
al.
(2011),
the
mechanical
effects
267
into the cells, enhancing mass transfer. In the process 303 showed greater inhibition activity toward the DPPH
302
compounds
extracted
by
ultrasound
application
266
of
sonication
allow
for
a
greater
penetration
of
solvent
268
of extraction, ultrasonic radiation can also break cell 304 radical compared to that toward the extracts obtained
303
showed
greater inhibition
activity
toward
the DPPH
267
into thefacilitating
cells, enhancing
massoftransfer.
In the process 305
by
the conventional
method.
As for
the compounds
269
walls,
the release
the compounds.
304
radical
compared
to
that
toward
the
extracts
obtained
268
of
extraction,
ultrasonic
radiation
can
also
break
cell
270
The results obtained by the traditional method 306 extracted from the cotyledon (Fig. 3.), the antiradical
305
by
the
conventional
method.
As
for
the
compounds
269
walls,
facilitating
the
release
of
the
compounds.
271
are consistent with those reported in the literature 307 activity was greater in the range of 0.01-0.04 mg
270
The results obtained by the traditional method 306 extracted from the cotyledon (Fig. 3.), the antiradical
307
activity was greater in the range of 0.01-0.04 mg
271
are
consistent with those reported in the literature
4
www.rmiq.org
225

14
4

www.rmiq.org
www.rmiq.org

Quiroz-Reyes et al./ Revista Mexicana de Ingeniera Qumica Vol. 12, No. 1 (2013) XXX-XXX
Quiroz-Reyesetetal./
al./Revista
RevistaMexicana
Mexicanade
deIngenier
Ingenier
Qu
micaVol.
Vol.12,
12,No.
No.1 1(2013)
(2013)XXX-XXX
11-18
Quiroz-Reyes
aaQu
mica
different extracts. In the case of the husk, there were
different
extracts.
InInthe
ofofthe
there
were
different
extracts.
thecase
case
thehusk,
husk,
there
were
no
significant
differences
between
the
EC
50 values.
no
significant
differences
between
the
EC
values.
50
332
no significant
differences
between
the EC
333
However,
the control
had an
EC50 value
significantly
50 values.
control
had
EC
value
5050of
333 However,
However,
control
had
EC
value
significantly
334
lower
(P the
<the
0.05)
than
theanan
values
the significantly
husk
fraction
lower
(P
<
0.05)
than
the
values
of
the
fraction
334
lower
(P
<
0.05)
than
the
values
of
thehusk
husk
fraction
335
extracts. On the other hand, the extracts
of cotyledon
extracts.
On
the
other
hand,
the
extracts
of
cotyledon
335
extracts.
On
the
other
hand,
the
extracts
of
cotyledon
336
had EC50 values lower than those obtained for the
those
obtained
for
5050values
336 had
hadEC
EC
lowerthan
than
those
obtained
forthe
the
337
control.
Invalues
thislower
fraction,
the
extract
obtained
via
control.
In
this
fraction,
the
extract
obtained
via
337
control.
In
this
fraction,
the
extract
obtained
via
338
ultrasound showed the lowest mean EC50 value, which
showed
which
5050value,
338 ultrasound
ultrasound
showedthe
thelowest
lowestmean
EC
value,
which
339
was
significantly
different
(Pmean
<EC
0.05)
from
that
was
significantly
different
(P
<
0.05)
from
that
339
was
significantly
different
(P
<
0.05)
from
that
340
obtained by the maceration method. The analysis
of
obtained
by
the
maceration
method.
The
analysis
340
obtained
by
the
maceration
method.
The
analysis
of
341
variance revealed statistical differences between of
the
variance
revealed
statistical
differences
between
the
341
variance
revealed
statistical
differences
between
the
342
EC50 values of the husk and cotyledon extracts. The
the
The
342 EC
EC
values
thehusk
huskand
andcotyledon
cotyledon
extracts.
The
50 50values
343
results
showofof
good
correlation
(R2 2 = extracts.
0.72)
between
2 = 0.72) between
show
good
correlation
(R
343 results
results
show
good
correlation
(R
=
0.72)
between
344
the phenolic content and the antiradical activity of
content
the
344 the
thephenolic
phenolic
content
and
theantiradical
antiradicalactivity
activityofof
345
cocoa
extracts.
The and
EC
50 results determined in this
309
extracts.
The
EC
results
determined
ininthis
345 cocoa
cocoa
extracts.
The
EC
results
determined
this
50
50

346
research are considerably lower than those reported
309
310
Fig.
2. Scavenging effect of cocoa husk extracts on 346 research
are
considerably
lower
than
those
reported

research
are
considerably
lower
than
those
reported
347
by Othman et al. (2007), who published EC50 values
310 Fig. 2. Scavenging effect of cocoa husk extracts on
311
DPPH radicals. Values are expressed as meansd 347 bybyOthman
1
etet
al.al.
(2007),
who
published
EC
values
Othman
(2007),
who
published
EC
values
5050extracts
348
in the
range
of
1.2-1.5
mg
mL1
for ethanol

311
radicals. acid
Values
as meansd
1for ethanol extracts
312
(nDPPH
= 3). Ascorbic
was are
usedexpressed
as the control.
the
range
of
1.2-1.5
mg
mL
Fig.
348 inin
the
range
of
1.2-1.5
mg
mL
for
ethanol
extracts
349
of cocoa. These differences can be attributed to the
2. 3). Ascorbic acid was used as the control.
312
(n =
cocoa.
can
bebeattributed
totothe
349 ofof
cocoa.
Thesedifferences
differences
canproduction
attributed
the
Fig.
2.
350
variety
ofThese
cocoa
species
used,
area
and

of
cocoa
species
used,
production
area
and
350 variety
variety
of
cocoa
species
used,
production
area
and
351
even the methodology used by the authors.

351 even
eventhe
themethodology
methodologyused
usedbybythe
theauthors.
authors.

Table 1. Scavenging activity (EC50 ) of cocoa extracts

Table 1. Scavenging
activity
(EC50 ) of cocoa extracts

on DPPH
radicals.
Table 1. Scavenging
activity
(EC50) of cocoa extracts

on DPPH
radicals.
1
on
DPPH
radicals.
Extraction
method
EC
50 (DPPH) mg mL 1

Extraction
method
EC
(DPPH)
mg
mL
50

Husk
Extraction method
EC50 (DPPH)Cotyledon
mg mL1
Husk
Cotyledon

Husk a 0.01640.0012
Cotyledonc

Maceration
0.05330.0022
352
Maceration
0.05330.0022aa a 0.01240.0017
0.01640.0012dc c

352 Ultrasound
0.04860.0018
Maceration
0.0533 0.0022
0.0164 0.0012
a
d

Ultrasound
0.04860.0018
b a 0.01240.0017b
d
Control
0.02430.0009
0.02430.0009

Ultrasound
0.0486

0.0018
0.0124

0.0017
b
Control
0.02430.0009 b 0.02430.0009b b

Means Control
with different 0.0243
letters were
significantly
0.0009
0.0243 0.0009
308

331

308

331
332

313
313
314
314
315
315
316
316

317
317
318
318
319
319
320
320
321
321
322
322
323
323
324
324
325
325
326
326
327
327
328
328
329
329
330
330

Fig.
3.3. Scavenging effect of cocoa cotyledon extracts
Fig.
Fig.3.3. Scavenging effect of cocoa cotyledon extracts
Fig.
on
DPPH radicals. Values are expressed as meansd

radicals. Values are expressed as meansd

(non= DPPH
3). Ascorbic acid was used as the control.
(n = 3). Ascorbic acid was used as the control.

Means (P
with
different letters were significantly
different
< 0.05).
Means (P
with
different letters were significantly
different
< 0.05).
Ascorbic
acid
was
used as a control
different
(P
<
0.05).
Ascorbic acid was used as a control
Ascorbic acid was used as a control

1
mL . The extracts obtained by sonication had higher
mL1 . The extracts obtained by sonication had higher

antiradical
activity than those obtained by maceration.
antiradical activity than those obtained by maceration.
It is noteworthy that the DPPH radical scavenging
It is noteworthy that the DPPH radical scavenging
activity of the phenolic extracts obtained from the
activity of the phenolic extracts obtained from the

cotyledon
was significantly higher (P < 0.05) than the

cotyledon was significantly higher (P < 0.05) than the

activity exhibited by extracts obtained from the husk.

activity exhibited by extracts obtained from the husk.

EC50 is defined as the amount of antioxidant

EC50 is defined as the amount of antioxidant

necessary
to decrease the initial concentration of
necessary
to decrease the initial concentration of
DPPH radical
50%. The lower the EC value is
DPPH radicalby
by 50%. The lower the EC5050 value is
the
greater
the
activity
extracts as DPPH radical
the greater the activityof
of extracts as DPPH radical

scavengers
(Wootton-Beard and Ryan, 2011). The
scavengers (Wootton-Beard and Ryan, 2011). The
EC
EC
50 value was determined by plotting the inhibition
50 value was determined by plotting the inhibition
percentage
percentageofofDPPH
DPPHradical
radicalagainst
againstthe
theconcentration
concentration

ofofthe
extract.
Table
1
shows
the
EC
the extract. Table 1 shows the EC50 values
valuesfor
forthe
the

50

353
353
354
354
355
355
356
356
357
357
358
358

Fig.
4. Antioxidant capacity of cocoa extracts

Fig.
4. Antioxidant capacity of cocoa extracts

measured
by FRAP assay. Concentration of sample
measured by FRAP
assay. Concentration of sample
was
0.10
mg mL1
. Values are expressed as meansd
Fig.
4.
was
Fig.0.10
4. mg mL1 . Values are expressed as meansd
(n
= 3). Means
(n
Meanswith
with different
different letters
letters were
were significantly
significantly
= 3). (P
different
<
0.05).

different (P < 0.05).

www.rmiq.org
www.rmiq.org

www.rmiq.org

5
155

Quiroz-Reyes et al./ Revista Mexicana de Ingeniera Qumica Vol. 12, No. 1 (2013) XXX-XXX
Quiroz-Reyes
aaQu
mica
Quiroz-Reyesetetal./
al./Revista
RevistaMexicana
Mexicanade
deIngenier
Ingenier
Qu
micaVol.
Vol.12,
12,No.
No.11(2013)
(2013)XXX-XXX
11-18

359
359
360
360

361
361
362
362
363
363
364
364
365
365
366
366
367
367
368
368
369
369
370
370
371
371
372
372
373
373
374
374
375
375
376
376
377
377
378
378
379
379

5. Typical chromatograms of cocoa extracts obtained by ultrasound-assisted extraction.

Fig.
5. Typical chromatograms of cocoa extracts obtained by ultrasound-assisted extraction.
Fig.

Fig. 5.
Fig. 5.
3.3 Antioxidant activity (FRAP assay)
3.3 Antioxidant activity (FRAP assay)

FRAP determination is based on the reduction of Fe3+ FRAPcomplex

determination
based complex.
on the reduction
of Fe3+ TPTZ
to Fe2+is-TPTZ
The reducing
2+
TPTZ complex
to Fe -TPTZ
complex.
Thepresence
reducing
properties
are generally
associated
with the
properties
are
generally
associated
with
the
presence
of reducing agents, which have been shown to
exert
of
reducing
agents,
which
have
been
shown
exert
antioxidant action by breaking the free radicaltochain
antioxidant
action
by
breaking
the
free
radical
chain
by donating a hydrogen atom. The FRAP results (Fig.
a hydrogen
atom. The
FRAPobtained
results (Fig.
4.)by donating
indicate that
the cotyledon
extract
by
4.)
indicate
that
the
cotyledon
extract
obtained
ultrasound application had the highest antioxidantby
ultrasound
application
the highest
activity,
reaching
a valuehad
of 35015
MEantioxidant
ascorbic
1 reaching a value of 35015 ME ascorbic
activity,
acid L . However, no significant differences between
acid
L1 . However,
no detected.
significantOn
differences
the
methods
used were
the otherbetween
hand,
the
methods
used
were
detected.
On
theinother
hand,
the FRAP values for the husk were located
the range
1
the
FRAP
values
for
the
husk
were
located
in
the
range
of 141-148 ME of ascorbic acid L . These values
of significantly
141-148 ME
of ascorbic
acid than
L1 . that
These
values
are
lower
(P < 0.05)
reported
are
significantly
lower
(P
<
0.05)
than
that
reported
for cotyledon. The FRAP results correlate well with
fortotal
cotyledon.
FRAP
well
the
phenolic The
content
(R2 results
= 0.78)correlate
and DPPH
(R2with
=
2
the
total
phenolic
content
(R
=
0.78)
and
DPPH
(R2 =
0.98). Othman et al. (2007) also reported a positive
0.98). Othman
et al.
(2007) also
reported
a positive
correlation
between
antioxidant
activity
(FRAP)
and
correlation between antioxidant activity (FRAP) and

6
166

Table 2. Concentration of (+)-catechin and

()-epicatechin in cocoa fractions.
Table
2.
Concentration
of (+)-catechin
and
Treatment/cocoa
Content
(g mg1 dry
extract)
Table
2.
Concentration
of
(+)-catechin
and
()-epicatechin
in
cocoa
fractions.
fraction
(+)-Catechin
()-Epicatechin
()-epicatechin in cocoa fractions.
Treatment/cocoa
Content (g mg1 dry extract)
Maceration
1
Treatment/cocoa
Content
(gamg
dry extract)
fraction
()-Epicatechin
Cotyledon (+)-Catechin
4.62
0.047
132.88
0.245a
fraction
(+)-Catechin
()-Epicatechin
b
Husk
0.28

0.046
2.64

0.018b
Maceration
a
Maceration
Ultrasound
Cotyledon
4.620.047
132.880.245a
381
ba a
b ac
Cotyledon
4.620.047
132.880.245
144 4.850
Cotyledon
4.26

0.025
Husk
0.280.046
2.640.018
381
b b
b b
Husk
0.280.046
2.640.018
Husk
0.32 0.083
2.77 0.340
Ultrasound
a
c
Ultrasound
Cotyledon
4.260.025
Means
with different
letters within the1444.850
same column are
c
ba
Cotyledon
4.260.025
1444.850
significantly
(p < 0.05).
2.770.340b
Husk different
0.320.083
b
Husk
0.320.083
2.770.340b

380

380

Means with different letters within the same column are

Means
withdifferent
different
withinextracts.
the same column are
significantly
(p letters
< 0.05).
the
polyphenol
content
of cocoa
significantly different (p < 0.05).

382
382

the polyphenol content of cocoa extracts.

the polyphenol content of cocoa extracts.

www.rmiq.org
www.rmiq.org
www.rmiq.org

Quiroz-Reyes
et
Revista
Mexicana
Ingenier
Qu
a Qu
mica
Vol.
No.
1 (2013)
11-18
Quiroz-Reyes
al./al./
Revista
Mexicana
dede
Ingenier
mica
Vol.
12,12,
No.
(2013)
XXX-XXX
Quiroz-Reyes
etetal./
Revista
Mexicana
de
Ingenier
aaQu
mica
Vol.
12,
No.
11(2013)
XXX-XXX
383
383
384
384

385
385
386
386
387
387
388
388
389
389
390
390
391
391
392
392
393
393
394
394
395
395
396
396
397
397
398
398
399
399
400
400
401
401
402
402

3.4 Identification
Identification
3.4
(HPLC)
(HPLC)

and
and

quantification
quantification

Using HPLC
HPLC analysis
analysis itit was
was possible
possible to
to identify
identify
Using
the
presence
of
(+)-catechin
and
()-epicatechin
the presence of (+)-catechin and ()-epicatechin
in the
the cotyledon
cotyledon and
and husk
husk samples
samples of
of cocoa.
cocoa.
in
Typical
chromatograms
of
cocoa
extracts
obtained
by
Typical chromatograms of cocoa extracts obtained by
sonication
are
shown
in
Fig.
5.
The
(+)-catechin
sonication are shown in Fig. 5. The (+)-catechin
and ()-epicatechin
()-epicatechin contents
contents were
were significantly
significantly
and
higher
(P
<
0.05)
in
cotyledon
than
in husk
husk for
for
higher (P < 0.05) in cotyledon than in
both
extraction
procedures
(Table
2).
Only
in
both extraction procedures (Table 2).
Only in
the
case
of
()-epicatechin,
the
ANOVA
showed
the case of ()-epicatechin, the ANOVA showed
significant differences
differences between
between ultrasound
ultrasound and
and
significant
maceration
methods.
These
results
are
compatible
maceration methods. These results are compatible
with total
total polyphenol
polyphenol quantification
quantification using
using the
the FolinFolinwith
Ciocalteu
method,
and
explain
why
the
cotyledon
Ciocalteu method, and explain why the cotyledon
presents greater
greater efficiency
efficiency in
in tests
tests to
to determine
determine freefreepresents
radical
scavenging
activity
and
antioxidant
capacity.
radical scavenging activity and antioxidant capacity.
According to
to Ortega
Ortega etet al.
al. (2008),
(2008), polyphenols
polyphenols
According
belonging
to
the
catechin
group
are
mainly
responsible
belonging to the catechin group are mainly responsible
for
the
antioxidant
properties
of
cocoa.
for the antioxidant properties of cocoa.

427
427

428
428
429
429
430
430
431
431
432
432

433
433
434
434
435
435
436
436

437
437
438
438
439
439
440
440
441
441
442
442
443
443
444
444

445
445

403
403

404
404
405
405
406
406
407
407
408
408
409
409
410
410
411
411
412
412
413
413
414
414
415
415
416
416
417
417
418
418
419
419
420
420
421
421

422
422

423
423
424
424
425
425
426
426

446
446

Conclusion
Conclusion

447
447
448
448

The use
use of
of ultrasonic
ultrasonic radiation
radiation facilitated
facilitated the
the
The
extraction
of
polyphenols
from
cocoa
beans,
extraction of polyphenols from cocoa beans,
increasing the
the content
content by
by 50%
50% compared
compared to
to the
the
increasing
traditional
method.
In
addition,
the
antioxidant
traditional method. In addition, the antioxidant
activity measured
measured by
by DPPH
DPPH and
and FRAP
FRAP methods
methods was
was
activity
greater
in
the
compounds
obtained
by
sonication
for
greater in the compounds obtained by sonication for
husk
and
cotyledon
fractions.
The
total
phenolic
husk and cotyledon fractions. The total phenolic
contentextracted
extractedfrom
fromthe
thecotyledon
cotyledonwas
wassignificantly
significantly
content
greater
than
that
extracted
from
the
husk
fraction.
The
greater than that extracted from the husk fraction. The
results
demonstrate
a
positive
correlation
between
the
results demonstrate a positive correlation between the
total
phenolic
content
and
antioxidant
activity
of
cocoa
total phenolic content and antioxidant activity of cocoa
extracts. Cocoa
Cocoa cotyledon
cotyledon presented
presented significantly
significantly
extracts.
greater
quantities
of
(+)-catechin
and
()-epicatechin
greater quantities of (+)-catechin and ()-epicatechin
ascompared
comparedwith
withthe
thehusk.
husk. Finally,
Finally, we
wecan
canconclude
conclude
as
that the
the use
use of
of ultrasonic
ultrasonic radiation
radiation isis an
an excellent
excellent
that
method for
for the
the extraction
extraction of
of natural
natural antioxidants
antioxidants
method
sinceititprovides
providesaashort
shortextraction
extractiontime,
time,ititoffers
offershigh
high
since
reproducibilityand
andlow
lowloss
lossof
ofbioactive
bioactivecompounds.
compounds.
reproducibility

449
449
450
450

451
451
452
452
453
453
454
454

455
455
456
456
457
457
458
458
459
459

460
460
461
461
462
462
463
463
464
464

465
465

Acknowledgments
Acknowledgments

466
466

This work
work was
was financially
financially supported
supported by
by SIPSIPThis
IPN
(projects
20113551
and
20120422).
QuirozIPN (projects 20113551 and 20120422). QuirozReyes thanks
thanks COFAA-IPN
COFAA-IPN and
and CONACyT
CONACyT for
for the
the
Reyes
scholarships
received.
scholarships received.

467
467
468
468
469
469
470
470
471
471

References
References
Bel
ak, A.,
A., Komes,
Komes, D.,
D., Hor
Hor
D., Gani
Gani
K.K.
Bel
ssccak,
zzii
cc,, D.,
cc,, K.K.
and
Karlovi
c
,
D.
(2009).
Comparative
study
of
and Karlovic, D. (2009). Comparative study of
commercially
available
cocoa
products
in
terms
commercially available cocoa products in terms
of their
their bioactive
bioactive composition.
composition. Food
Food Research
Research
of
International
42,
707-716.
International 42, 707-716.
Chemat, F.,
F., Zill-e-Huma
Zill-e-Huma and
and Khan,
Khan, M.K.
M.K. (2011).
(2011).
Chemat,
Applications of
of ultrasound
ultrasound in
in food
food technology:
technology:
Applications
Processing, preservation
preservation and
and extraction.
extraction.
Processing,
UltrasonicsSonochemistry
Sonochemistry18,
18,813-835.
813-835.
Ultrasonics
Garc
a-M
rquez, E.,
E., Rom
Rom
n-Guerrero, A.,
A., P
P
rezGarc
a-M
aarquez,
aan-Guerrero,
eerezAlonso,
C.,
Cruz-Sosa,
F.,
Jim
e
nez-Alvarado,
Alonso, C., Cruz-Sosa, F., Jimenez-Alvarado,
R. and
and Vernon-Carter,
Vernon-Carter, E.J.
E.J. (2012).
(2012). Effect
Effect of
of
R.
solvent-temperature
extraction
conditions
on
the
solvent-temperature extraction conditions on the
initial antioxidant
antioxidant activity
activity and
and total
total phenolic
phenolic
initial
content
of
muitle
extracts
and
their
decay
upon
content of muitle extracts and their decay upon
storage
at
different
pH.
Revista
Mexicana
de
storage at different pH. Revista Mexicana de
Ingenier

a
Qu

mica
11,
1-10.
Ingeniera Qumica 11, 1-10.
Jacques,R.A.,
R.A.,Freitas,
Freitas,L.S.,
L.S.,Flores,
Flores,V.,
V.,Dariva,
Dariva,C.,
C.,de
de
Jacques,
Oliveira,
A.P.,
de
Oliveira,
J.V.,
Caram
a
o,
E.B.
Oliveira, A.P., de Oliveira, J.V., Caramao, E.B.
(2007). The
The use
use of
of ultrasound
ultrasound in
in the
the extraction
extraction
(2007).
of
Ilex
paraguariensis
leaves:
A
comparison
of Ilex paraguariensis leaves: A comparison
withmaceration.
maceration.Ultrasonics
UltrasonicsSonochemistry
Sonochemistry14,
14,
with
6-12.
6-12.
Jonfia-Essien, W.A.,
W.A., West,
West, G.,
G., Alderson,
Alderson, P.G.
P.G. and
and
Jonfia-Essien,
Tucker, G.
G. (2008).
(2008). Phenolic
Phenolic content
content and
and
Tucker,
antioxidant capacity
capacity of
of hybrid
hybrid variety
variety cocoa
cocoa
antioxidant
beans. Food
FoodChemistry
Chemistry108,
108,1155-1159.
1155-1159.
beans.
Khan, M.K.,
M.K., Abert-Vian,
Abert-Vian, M.,
M., Fabiano-Tixier,
Fabiano-Tixier, A.S.,
A.S.,
Khan,
Dangles,
O.
and
Chemat,
F.
(2010).
UltrasoundDangles, O. and Chemat, F. (2010). Ultrasoundassisted extraction
extraction of
of polyphenols
polyphenols (flavanone
(flavanone
assisted
glycosides)
from
orange
(Citrus
sinensis
L.)
glycosides) from orange (Citrus sinensis L.)
peel.
Food
Chemistry
119,
851-858.
peel. Food Chemistry 119, 851-858.
Koffi, E.,
E., Sea,
Sea, T.,
T., Dodehe,
Dodehe, Y.
Y. and
and Soro,
Soro, S.
S.
Koffi,
(2010). Effect
Effect of
of solvent
solvent type
type on
on extraction
extraction of
of
(2010).
polyphenols from
from twenty
twenty three
three Ivorian
Ivorian plants.
plants.
polyphenols
Journal of
of Animal
Animal and
and Plant
Plant Sciences
Sciences 5,5, 550550Journal
558.
558.
Lecumberri, E.,
E., Mateos,
Mateos, R.,
R., Ramos,
Ramos, S.,
S., Al
Al
Lecumberri,
a,a,
M.,
R
u
perez,
P.,
Goya,
L.,
Izquierdo-Pulido,
M., Ruperez, P., Goya, L., Izquierdo-Pulido,
M. and
and Bravo,
Bravo, L.
L. (2006).
(2006). Caracterizaci
Caracterizaci
M.
oonn
de
la
fibra
de
cacao
y
su
efecto
sobre
de la fibra de cacao y su efecto sobre lala
capacidadantioxidante
antioxidanteen
ensuero
suerode
deanimales
animalesde
de
capacidad
experimentaci
o
n.
Nutrici
o
n
Hospitalaria
21,
experimentacion. Nutricion Hospitalaria 21,
622-628.
622-628.

www.rmiq.org
www.rmiq.org
www.rmiq.org

1777

Quiroz-Reyes
et Revista
al./
Revista
Mexicana
de Ingenier
amica
Qu
mica
12,
1 (2013)
11-18
Quiroz-Reyes
al./
Revista
Mexicana
deIngenier
Ingenier
Qu
mica
Vol.Vol.
12,No.
No.No.
(2013)
XXX-XXX
Quiroz-Reyes
etetal./
Mexicana
de
aaQu
Vol.
12,
11(2013)
XXX-XXX
472
472
473
473
474
474
475
475
476
476

477
477
478
478
479
479
480
480

481
481
482
482
483
483
484
484
485
485
486
486

487
487
488
488
489
489
490
490

491
491
492
492
493
493
494
494
495
495

496
496
497
497
498
498
499
499
500
500
501
501

502
502
503
503
504
504
505
505

Liu, C.,
C., Wu,
Wu, C.,
C., Weng,
Weng, Y.Y. and
and Tseng,
Tseng, C.
C. (2005).
(2005).
Liu,
Ultrasound-assisted
extraction
methodology
Ultrasound-assisted extraction methodology asas
tool toto improve
improve the
the antioxidant
antioxidant properties
properties
aa tool
herbaldrug
drugXiao-chia-hu-tang.
Xiao-chia-hu-tang. Journal
Journalofof
ofofherbal
Ethnopharmacology99,
99,293-300.
293-300.
Ethnopharmacology
Mora-Huerta, C.E.,
C.E., Fessi,
Fessi, H.
H. and
and Elaissari,
Elaissari, A.
A.
Mora-Huerta,
(2010).
Polymer-based nanocapsules
nanocapsules for
for
(2010).
Polymer-based
drug delivery.
delivery.
International Journal
Journal ofof
drug
International
Pharmaceutics385,
385,113-142.
113-142.
Pharmaceutics
Ortega, N.,
N., Romero,
Romero, M.P.,
M.P., Maci`
Maci`
A., Reguant,
Reguant,
Ortega,
aa, , A.,
Angl`
N., Morell
Morell
J.R. and
and Motilva,
Motilva,
J.,J., Angl`
ees,s, N.,
oo, , J.R.
M.J.(2008).
(2008). Obtention
Obtentionand
andcharacterization
characterizationofof
M.J.
phenolicextracts
extractsfrom
fromdifferent
differentcocoa
cocoasources.
sources.
phenolic
JournalofofAgricultural
Agriculturaland
andFood
FoodChemistry
Chemistry56,
56,
Journal
9621-9627.
9621-9627.
Othman, A.,
A., Ismail,
Ismail, A.,
A., Ghani,
Ghani, N.A.
N.A.and
andAdenan,
Adenan,
Othman,
(2007). Antioxidant
Antioxidant capacity
capacity and
and phenolic
phenolic
I.I. (2007).
contentofofcocoa
cocoabeans.
beans. Food
FoodChemistry
Chemistry100,
100,
content
1523-1530.
1523-1530.
Pan, Z.,
Z., Qu,
Qu, W.,
W., Mab,
Mab, H.,
H., Atungulu,
Atungulu, G.G.
G.G.
Pan,
and
McHugh,
T.H.
(2011).
Continuous
and McHugh, T.H. (2011).
Continuous
and pulsed
pulsed ultrasound-assisted
ultrasound-assisted extractions
extractions
and
of
antioxidants
from
pomegranate
peel.
of antioxidants from pomegranate peel.
Ultrasonics
Sonochemistry
18,
1249-1257.
Ultrasonics Sonochemistry 18, 1249-1257.
Thaipong, K.,
K., Boonprakob,
Boonprakob, U.,
U., Crosby,
Crosby, K.,
K.,
Thaipong,
Cisneros-Zevallos,
L.
and
Byrne,
D.H.
(2006).
Cisneros-Zevallos, L. and Byrne, D.H. (2006).
Comparison ofof ABTS,
ABTS, DPPH,
DPPH, FRAP,
FRAP, and
and
Comparison
ORAC
assays
for
estimating
antioxidant
activity
ORAC assays for estimating antioxidant activity
from guava
guava fruit
fruit extracts.
extracts. Journal
Journal ofof Food
Food
from
Composition
and
Analysis
19,
669-675.
Composition and Analysis 19, 669-675.
Tom
s-Barber
F.A., Cienfuegos-Jovellanos,
Cienfuegos-Jovellanos, E.,
E.,
Tom
aas-Barber
aan,n, F.A.,
Mar
n, A.,
A., Muguerza,
Muguerza, B.,
B., Gil-Izquierdo,
Gil-Izquierdo, A.,
A.,
Mar
n,
Cerd
B., Zafrilla,
Zafrilla, P.,
P., Morillas,
Morillas, J.,J., Mulero,
Mulero,
Cerd
aa, , B.,
Ibarra,A.,
A.,Pasamar,
Pasamar,M.A.,
M.A.,Ram
Ram
D.and
and
J.,J.,Ibarra,
oon,n,D.

8818

Esp
n,J.C.
J.C.(2007).
(2007). AAnew
newprocess
processtotodevelop
develop
Esp
n,
a
cocoa
powder
with
higher
flavonoid
monomer
a cocoa powder with higher flavonoid monomer
contentand
andenhanced
enhancedbioavailability
bioavailabilityininhealthy
healthy
content
humans. Journal
Journal ofof Agricultural
Agricultural and
and Food
Food
humans.
Chemistry55,
55,3926-3935.
3926-3935.
Chemistry

506
506
507
507
508
508
509
509
510
510

511
511
512
512
513
513
514
514
515
515

516
516
517
517
518
518
519
519

520
520
521
521
522
522

523
523
524
524
525
525
526
526

527
527
528
528
529
529
530
530

531
531
532
532
533
533
534
534
535
535

536
536
537
537
538
538

Vilkhu, K.,
K., Mawson,
Mawson, R.,
R., Simons,
Simons, L.,
L., Bates,
Bates,
Vilkhu,
D. (2008).
(2008). Applications
Applications and
and opportunities
opportunities
D.
for ultrasound
ultrasound assisted
assisted extraction
extraction inin the
the food
food
for
industry: AAreview.
review. Innovative
InnovativeFood
FoodScience
Science
industry:
andEmerging
EmergingTechnologies
Technologies9,9,161-169.
161-169.
and
Vinatoru, M.
M. (2001).
(2001).
An overview
overview ofof
Vinatoru,
An
the ultrasonically
ultrasonically assisted
assisted extraction
extraction ofof
the
bioactive principles
principles from
from herbs.
herbs. Ultrasonics
Ultrasonics
bioactive
Sonochemistry8,8,303-313.
303-313.
Sonochemistry
Wang,L.L.and
andWeller
WellerC.L.
C.L.(2006).
(2006).Recent
Recentadvances
advancesinin
Wang,
extractionofofnutraceuticals
nutraceuticalsfrom
fromplants.
plants.Trends
Trends
extraction
FoodScience
Scienceand
andTechnology
Technology17,
17,300-312.
300-312.
ininFood
Waterhouse, A.L.
A.L. (2002).
(2002). Determination
Determination ofof total
total
Waterhouse,
phenolics. In:
In: R.E.
R.E. Wrolstad
Wrolstad (Ed)
(Ed) Current
Current
phenolics.
ProtocolsininFood
FoodAnalytical
AnalyticalChemistry
Chemistry(Units
(Units
Protocols
I.1.1.1-I1.1.8).New
NewYork,
York,John
JohnWiley
Wileyand
andSons.
Sons.
I.1.1.1-I1.1.8).
Wootton-Beard,P.C.
P.C.and
andRyan,
Ryan,L.L.(2011).
(2011).Improving
Improving
Wootton-Beard,
public health?:
health?: The
The role
role ofof antioxidant-rich
antioxidant-rich
public
fruitand
andvegetable
vegetablebeverages.
beverages. Food
FoodResearch
Research
fruit
International44,
44,3135-3148.
3135-3148.
International
Yilmaz,Y.Y.and
andToledo,
Toledo,R.T.
R.T.(2006).
(2006). Oxygen
Oxygenradical
radical
Yilmaz,
absorbance capacities
capacities ofof grape/wine
grape/wine industry
industry
absorbance
byproducts and
and effect
effect ofof solvent
solvent type
type on
on
byproducts
extractionofofgrape
grapeseed
seedpolyphenols.
polyphenols. Journal
Journal
extraction
FoodComposition
Compositionand
andAnalysis
Analysis19,
19,41-48.
41-48.
ofofFood
Yoshihara,D.,
D.,Fujiwara,
Fujiwara,N.
N.and
andSuzuki,
Suzuki,K.
K.(2010).
(2010).
Yoshihara,
Antioxidants: Benefits
Benefitsand
andrisks
risksfor
forlong-term
long-term
Antioxidants:
health.Maturitas
Maturitas67,
67,103-107.
103-107.
health.

www.rmiq.org
www.rmiq.org