Revista Mexicana

de Ingenierı́a Quı́mica
Academia Mexicana de Investigación y Docencia en Ingenierı́a Quı́mica, A.C.

Revista Mexicana de Ingeniería Química

Volumen 12, Número 1, Abril 2013

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

ISSN 1665-2738

CONTENIDO 1

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

COMPARATIVE STUDY OF ULTRASOUND AND MACERATION TECHNIQUES 1

FOR THE EXTRACTION OF POLYPHENOLS FROM COCOA

BEANS (Theobroma cacao L.)
213 Derivation and application of the Stefan-Maxwell equations
ESTUDIO COMPARATIVO ENTRE LAS TÉCNICAS DE ULTRASONIDO Y
MACERACI
(DesarrolloÓN PARAdeLA
y aplicación EXTRACCI
las ecuaciones ÓN DE POLIFENOLES DEL GRANO DE
de Stefan-Maxwell)
Stephen Whitaker
CACAO (Theobroma cacao L.)
C.N. Quiroz-Reyes1 , M.A. Aguilar-Méndez1∗ , M.E. Ramı́rez-Ortı́z2 y E. Ronquillo-De Jesús1
1
Centro de Investigación
Biotecnología en Ciencia Aplicada y Tecnologı́a Avanzada del Instituto Politécnico Nacional, Legaria
/ Biotechnology
694, Colonia Irrigación, 11500, Distrito Federal, México.
2
Facultad de
245 Modelado de Estudios Superiores
la biodegradación endebiorreactores
Cuautitlán, de
Universidad Nacional Autónoma
lodos de hidrocarburos de México.
totales del petróleo
Av. 1o. de Mayo s/n, col. Sta. Marı́a las Torres, 054740, Cuautitlán Izcalli, Estado de México, México.
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-Vázquez, A. Jiménez-
prevention. In this work, the phenolic content and antioxidant properties of cocoa extracts prepared by maceration and
ultrasound-assisted
González extraction were studied. The polyphenol content was determined using the Folin-Ciocalteu reagent and
y M. Gutiérrez-Rojas
the antioxidant activity was
259 Crecimiento, estimated byyDPPH
sobrevivencia (2,2-diphenyl-2-picrylhydrazyl)
adaptación de Bifidobacterium infantis and FRAP (Ferricácidas
a condiciones reducing/antioxidant power)
assays. The identification and quantification of (+)-catechin and (−)-epicatechin were performed using high-performance liquid
chromatography (HPLC).
(Growth, The and
survival results showedofthat
adaptation by using ultrasonic
Bifidobacterium radiation,
infantis to acidicitconditions)
was possible to obtain higher polyphenol
contents from both the husk and cotyledon of cocoa. Furthermore, extracts obtained by
L. Mayorga-Reyes, P. Bustamante-Camilo, A. Gutiérrez-Nava, E. Barranco-Florido sonication showed theAzaola-
y A. highest antioxidant
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 interés significativo en años recientes debido a su capacidad antioxidante y a su papel importante
(Optimización
en la prevención estadística
de enfermedades. En de
estelatrabajo,
fermentación etanólica
el contenido de Saccharomyces
fenólico cerevisiae
y las propiedades en presencia
antioxidantes de de cacao
de extractos
preparados por maceración
zeolita Valfor®yzeolite
extracción
NaA) asistida por ultrasonido, fueron estudiados. El contenido de polifenoles fue determinado
empleando el reactivo de Folin-Ciocalteu y la actividad
G. Inei-Shizukawa, H. A. Velasco-Bedrán, G. antioxidante fue estimada
F. Gutiérrez-López and H.por las técnicas de DPPH (2,2-diphenyl-
Hernández-Sánchez
2-picrylhydrazyl) y FRAP (Ferric reducing/antioxidant power). La identificación y cuantificación de (+)-catequina y (−)-
epicatequina fueron realizadas a través de la cromatografı́a de lı́quidos de alta resolución (HPLC). Los resultados mostraron que
mediante el uso dede
Ingeniería radiación
procesosultrasónica, fue posible extraer un mayor contenido de polifenoles tanto para cascarilla como para
/ Process engineering
cotiledón de cacao. Además, los extractos
271 Localización de una planta industrial: obtenidosRevisión
por sonicación
críticapresentaron
y adecuaciónla mayor
de losactividad
criterios antioxidante,
empleados en demostrando
ası́ que esta actividad depende directamente del contenido fenólico total. Los contenidos de (+)-catequina y (−)-epicatequina
fueron mayoresesta en
decisión
los extractos obtenidos del cotiledón con respecto a aquellos obtenidos de la cascarilla.
(Plant site selection: Critical review and adequation criteria used in this decision)
Palabras clave: cacao, polifenoles, extracción, ultrasonido, actividad antioxidante.
J.R. Medina, R.L. Romero y G.A. Pérez

∗ 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 Investigación y Docencia en Ingenierı́a Quı́mica A.C. 11

 Quiroz-Reyes
Quiroz-Reyes
Quiroz-Reyes etetet
al./
al./ Revista
al./
Revista Mexicana
Revista
Mexicana dede
Mexicana
de Ingenierı́a Quı́mica
Ingenierı́a
Ingenierı́a Vol.
Quı́mica
Quı́mica Vol.
Vol. 12,
12,
12, No.
No.
No. 11(2013)
1(2013) XXX-XXX
(2013) 11-18
XXX-XXX

38
38 11 Introduction
Introduction 89
89
extraction methods
extraction methods are are maceration
maceration with with solvents,
solvents,
90
90
hot-water extraction, alkaline extraction,
hot-water extraction, alkaline extraction, resin-based resin-based
39
39
Oxidation reactions
Oxidation reactions occur occur naturally
naturally in in thethe human
human 91
91
extraction, enzyme-assisted
extraction, enzyme-assisted extraction, extraction, extractions
extractions
40
40
body and are formed as byproducts
body and are formed as byproducts of respiration and of respiration and 92
92
based on gamma and electron-beam
based on gamma and electron-beam irradiation and irradiation and
41
41
oxidative metabolism in all cells of aerobic
oxidative metabolism in all cells of aerobic organisms. organisms. 93
93
extraction using supercritical fluids.
extraction using supercritical fluids. However, some However, some
42
42
Although oxygen
Although oxygen isis necessary
necessary for for aerobic
aerobic cells cells toto 94
94
of these
of these methods
methods can can cause
cause aa loss loss of of bioactive
bioactive
43
43
generate energy, it has the disadvantage
generate energy, it has the disadvantage of producing of producing 95
95
compounds due to the use of
compounds due to the use of high temperatures andhigh temperatures and
44
44
small amounts
small amounts of of reactive
reactive oxygen
oxygen speciesspecies (ROS)(ROS) 96
96 long extraction times; or, in the case of irradiation, itit
long extraction times; or, in the case of irradiation,
45
45 that can cause damage to macromolecules. This isis
that can cause damage to macromolecules. This 97
97
can represent
can represent aa healthhealth riskrisk ifif the
the proper
proper carecare isis not
not
46
46
related to
related to aa number
number of of chronic
chronic degenerative
degenerative diseasesdiseases 98
98
taken (Liu et al., 2005). These
taken (Liu et al., 2005). These shortcomings have shortcomings have
47
47
such as cancer, rheumatoid arthritis,
such as cancer, rheumatoid arthritis, Alzheimer’s, Alzheimer’s, 99
99
led to
led to the
the use
use of of new
new sustainable
sustainable innovative
innovative greengreen
48
48
atherosclerosis, emphysema,
atherosclerosis, emphysema, cirrhosis cirrhosis and and diabetes
diabetes 100
100
techniques that increase extraction
techniques that increase extraction efficiency, reduce efficiency, reduce
49
49
among others, which share common
among others, which share common pathogenic ROS pathogenic ROS 101
101
timeand
time andenergy-consuming
energy-consumingprocedures proceduresand andcontribute
contribute
50
50
as a key factor (Mora-Huerta et
as a key factor (Mora-Huerta et al., 2010; Yoshiharaal., 2010; Yoshihara 102
102
to environmental preservation
to environmental preservation by reducing the by reducing the use
use
51
51 etet al.,
al., 2010).
2010). BecauseBecause the the body
body isis vulnerable
vulnerable to to 103
103
of water and solvents, fossil energy
of water and solvents, fossil energy and generation and generation
52
52
these radicals, antioxidants are
these radicals, antioxidants are needed to decrease needed to decrease 104
104
of hazardous
of hazardous substances,
substances, such such as as microwave
microwave and and
53
53
the concentration
the concentration of of these
these reactive
reactive species
species to to prevent
prevent 105
105
ultrasound-assisted extraction (Chemat
ultrasound-assisted extraction (Chemat et al., 2011). et al., 2011).
54
54
the negative effects mentioned
the negative effects mentioned above. Antioxidants above. Antioxidants 106
106
Theuse
The useofofultrasonic
ultrasonicradiation
radiation(20-100
(20-100kHz) kHz)to toextract
extract
55
55
in the
in the body
body are are primarily
primarily derived
derived fromfrom dietdiet andand can
can 107
107
natural compounds provides high reproducibility,
natural compounds provides high reproducibility, easy easy
56
56
promote good health. Thus, due
promote good health. Thus, due to their significant to their significant 108
108
handling, low solvent and energy
handling, low solvent and energy consumption, low- consumption, low-
57
57
rolein
role indisease
diseaseprevention,
prevention,there therehashasbeen
beenan anincreasing
increasing 109
109
temperature processing
temperature processing and and aa lower
lower loss
loss ofof bioactive
bioactive
58
58
interest in recent years in the study
interest in recent years in the study of certain fruits, of certain fruits, 110
110
compounds (Pan et al.,
compounds (Pan et al., 2011). Compared with 2011). Compared with
59
59
vegetables and grains with high
vegetables and grains with high antioxidant contentsantioxidant contents 111
111
other novel extraction techniques
other novel extraction techniques such as microwave- such as microwave-
60
60
toboost
to boosttheir
theirconsumption
consumption(Wootton-Beard
(Wootton-Beardand andRyan,
Ryan, 112
112
assistedextraction,
assisted extraction,the theultrasound
ultrasoundapparatus
apparatusisischeaper
cheaper
61
61
2011).
2011). 113
113
and its operation
and its operation is easier. is easier. Furthermore,
Furthermore, the the
Cocoa isis recognized
recognized as as aa major
major dietary
dietary source
source of of 114 ultrasound-assisted extraction, like Soxhlet
ultrasound-assisted extraction, like Soxhlet extraction, extraction,
62
62 Cocoa 114

antioxidants because of its high phenolic compound 115 can be

can be used
used with
with any any solvent
solvent for for extracting
extracting aa wide wide
63
63 antioxidants because of its high phenolic compound 115

(procyanidins and and flavonols

flavonols mainly)
mainly) content
content (Tomás-
(Tomás- 116 variety of natural compounds
variety of natural compounds (Wang and Weller, (Wang and Weller,
64
64 (procyanidins 116

Barberán et al., 2007). It has even been observed 117 2006).

2006). Ultrasound can
Ultrasound can facilitate
facilitate swelling
swelling and and
65
65 Barberán et al., 2007). It has even been observed 117

thatcocoa-based
cocoa-basedproductsproductscontaincontainaahigher
higherantioxidant
antioxidant 118 hydration of vegetal tissue, allowing
hydration of vegetal tissue, allowing high diffusion high diffusion
66
66 that 118

capacity and greater amounts of flavonoids perserving

serving 119 rates across
rates across the the cellcell wall
wall and and enhancing
enhancing the the mass
mass
67
67 capacity and greater amounts of flavonoids per 119

than tea or red wine (Jonfia-Essien et al., 2008). 120 transfer. On the other hand,
transfer. On the other hand, cavitation produced cavitation produced
68
68 than tea or red wine (Jonfia-Essien et al., 2008). 120

It is possible to distinguish three main groups of 121 by ultrasonic

by ultrasonic waveswaves can can alsoalso disrupt
disrupt thethe cell
cell wall,
wall,
69
69 It is possible to distinguish three main groups of 121

polyphenols in cocoa: catechins or flavan-3-ols 122 facilitating the

facilitating the release
release of of contents
contents (Vinatoru,
(Vinatoru, 2001).
2001).
70
70 polyphenols in cocoa: catechins or flavan-3-ols 122

(37%), anthocyanins
anthocyanins (4%) (4%) and and proanthocyanidins
proanthocyanidins 123 Khan etet al.,
Khan al., (2010)
(2010) reported
reported higherhigher extraction
extraction yields
yields
71
71 (37%), 123

(58%) (Bels̆c̆ak et al., 2009). The main catechin

catechin 124 of polyphenols from orange peel
of polyphenols from orange peel using an ultrasonic using an ultrasonic
72
72 (58%) (Bels̆c̆ak et al., 2009). The main 124

is (−)-epicatechin, which constitutes approximately 125 processor operated

processor operated atat aa frequency frequency of of 25 25 kHz.kHz.
73
73 is (−)-epicatechin, which constitutes approximately 125

74
74
35% of
35% of the
the total
total polyphenol
polyphenol content content of of cocoa.
cocoa. In In 126
126 According to Jacques et al. (2007), sonication isis
According to Jacques et al. (2007), sonication
75
75
addition to the cotyledon, the husk (a
addition to the cotyledon, the husk (a byproduct of the byproduct of the 127
127 aa simpler,
simpler, faster
faster and and moremore effective
effective technique
technique than than
chocolateindustry)
industry)also alsocontains
containsaasignificant
significantamount amount 128 maceration to extract organic
maceration to extract organic compounds from Ilex compounds from Ilex
76
76 chocolate 128

of phenolic compounds (Lecumberri et al., 2006). 129 paraguariensis leaves. Currently, there
paraguariensis leaves. Currently, there are no reports are no reports
77
77 of phenolic compounds (Lecumberri et al., 2006). 129

The extraction
extraction of of phenolic
phenolic compounds
compounds from from 130 inthe
in theliterature
literatureon onthetheextraction
extractionof ofpolyphenols
polyphenolsfrom from
78
78 The 130
cocoa beans using ultrasonic radiation. Therefore, the
79
79
plant materials is influenced
plant materials is influenced by the compounds’ by the compounds’ 131
131 cocoa beans using ultrasonic radiation. Therefore, the
chemical nature, nature, extraction
extraction method,method, samplesample size, size, 132 objective of this study was to evaluate
objective of this study was to evaluate the effect of the effect of
80
80 chemical 132
ultrasonic treatment on the total phenolic content and
81
81
time and storage conditions as
time and storage conditions as well as the presencewell as the presence 133
133 ultrasonic treatment on the total phenolic content and
of interfering
interfering substances
substances such such as as proteins
proteins and and 134 antioxidant activity
antioxidant activity of of extracts
extracts fromfrom cocoa
cocoa huskhusk andand
82
82 of 134
cotyledon. In addition, a comparison was made with
83
83
carbohydrates (Koffi et al., 2010;
carbohydrates (Koffi et al., 2010; Garcı́a-Márquez Garcı́a-Márquez 135
135 cotyledon. In addition, a comparison was made with
respectto tothe
thetraditional
traditionalmethod.
method.
etet al.,
al., 2012).
2012). ItIt has has been
been reported
reported that that thethe use
use ofof respect
136
84 136
84
85
85
aqueous solutions of methanol,
aqueous solutions of methanol, ethanol and acetone ethanol and acetone
86
86
dramatically improves
dramatically improves the the extraction
extraction of of polyphenols
polyphenols
87
87
compared to a single-compound
compared to a single-compound solvent system solvent system
88
88
(Yilmaz and Toledo, 2006).
(Yilmaz and Toledo, 2006). The most reported The most reported

22
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 Quiroz-Reyes
Quiroz-Reyeset et
al./ Revista Mexicana dede
Ingenierı́a Quı́mica Vol. 12,12,
No. 1 (2013) XXX-XXX
Quiroz-Reyes et al./al./ Revista
Revista Mexicana
Mexicana Ingenierı́a
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Quı́mica Vol.
Vol. 12, No.No. 1 (2013)
1 (2013) 11-18
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137
137
22 Methodology
Methodology 183
183
reagent (2N). The final solution was allowed to stand
reagent (2N). The final solution was allowed to stand
184 for 3 min at room temperature. Thereafter, 1.5 mL
184 for 3 min at room temperature. Thereafter, 1.5 mL
138
138
2.1
2.1 Materials
Materials and
and reagents
reagents
185
185
of a solution of sodium carbonate was added (20%
of a solution of sodium carbonate was added (20%
186 w/v). After 90 min of rest in the dark, the absorbance
The cocoa beans were purchased at a local market 186 w/v). After 90 min of rest in the dark, the absorbance
139
The cocoa beans were purchased at a local market 187 was determined at a wavelength of 760 nm (Cary 50,
139
in Mexico City. The reagents 2,2-diphenyl- 187 was determined at a wavelength of 760 nm (Cary 50,
140
in Mexico City. The reagents 2,2-diphenyl- 188 Varian, USA). The results were expressed as milligram
140
1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, 188 Varian, USA). The results were expressed as milligram
141
1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, 189 equivalents of gallic acid g−1 of dry matter.
141
142 gallic acid, Tris-HCl buffer, 2,4,6-Tris (2-pyridyl)-s- 189 equivalents of gallic acid g−1 of dry matter.
142 gallic acid, Tris-HCl buffer, 2,4,6-Tris (2-pyridyl)-s-
143 triazine (TPTZ) and ferric chloride hexahydrate were
triazine (TPTZ) and ferric chloride hexahydrate were 2.4
2.4 DPPH
DPPH radical
radical scavenging
scavenging capacity
143
144 purchased from Sigma-Aldrich (USA). Acetic acid, 190
capacity
144 purchased from Sigma-Aldrich (USA). Acetic acid, 190

145 ascorbic acid and sodium acetate were obtained from The antiradical capacity was determined by the DPPH
145 ascorbic acid and sodium acetate were obtained from 191
The antiradical capacity was determined by the DPPH
146 JT Baker (Mexico). Standards and solvents for HPLC 191
assay following the methodology proposed by Othman
146 JT Baker (Mexico). Standards and solvents for HPLC 192
assay following the methodology proposed by Othman
147 analysis were provided by Sigma-Aldrich. 192
et al. (2007), with some modifications. A 2-mL
147 analysis were provided by Sigma-Aldrich. 193
193 et al. (2007), with some modifications. A 2-mL
194 aliquot of extract was mixed with 500 µL of 0.1M
194 aliquot of extract was mixed with 500 µL of 0.1M
2.2 Tris-HCl Buffer by vortex mixing for 5 s. To this
2.2 Preparation
Preparation of
of extracts
195
148
extracts 195 Tris-HCl Buffer by vortex mixing for 5 s. To this
148
196 solution, 2 mL of a 200-µM solution of DPPH were
196 solution, 2 mL of a 200-µM solution of DPPH were
149 Cocoa beans were husked manually and fractions added. After 30 min, the absorbance was determined
149 Cocoa beans were husked manually and fractions 197
197 added. After 30 min, the absorbance was determined
150 (husk and cotyledon) ground separately in a disc mill at 517 nm. The percentage of DPPH reduction was
150 (husk and cotyledon) ground separately in a disc mill 198
198 at 517 nm. The percentage of DPPH reduction was
151 (148-2, The Bauer Bros Co., USA). In the case of calculated using the Eq. (1).
151 (148-2, The Bauer Bros Co., USA). In the case of 199
199 calculated using the Eq. (1).
152 cotyledon, fat was removed from the material by !
152 cotyledon, fat was removed from the material by
soaking in hexane for 24 h at room temperature. Absorbance o f sample !
Inhibition (%) = 1 − Absorbance o f sample × 100
153
153 soaking in hexane for 24 h at room temperature.
154 The extraction of phenolic compounds from both Inhibition (%) = 1 − Absorbance o f control × 100
154 The extraction of phenolic compounds from both Absorbance o f control
fractions was performed by maceration and ultrasound (1)
155
155 fractions was performed by maceration and ultrasound (1)
application. Maceration: the plant material was 200 The EC50 value was determined from the data in
156
156 application. Maceration: the plant material was 200 The EC50 value was determined from the data in
subjected to a first extraction using a water-methanol 201 the graph of the DPPH reduction effect against the
157
157 subjected to a first extraction using a water-methanol 201 the graph of the DPPH reduction effect against the
solution (1:1 ratio) for 2 h at room temperature 202 extract concentration. The EC50 was determined as the
158
158 solution (1:1 ratio) for 2 h at room temperature 202 extract concentration. The EC50 was determined as the
and under constant stirring. Then, the mixture was 203 necessary amount of the extract studied to reduce the
159
159 and under constant stirring. Then, the mixture was 203 necessary amount of the extract studied to reduce the
centrifuged (3,000 rpm, 15 min) and filtered. The 204 concentration of DPPH by 50%, using ascorbic acid as
160
160 centrifuged (3,000 rpm, 15 min) and filtered. The 204 concentration of DPPH by 50%, using ascorbic acid as
residue was recovered for a second extraction (2 h) 205 a control.
161
161 residue was recovered for a second extraction (2 h) 205 a control.
162 with an acetone-water solution (70:30 ratio), repeating
162 with an acetone-water solution (70:30 ratio), repeating
the centrifugation and combining the supernatants 2.5
2.5 Ferric
Ferric reducing/antioxidant power
163
163 the centrifugation and combining the supernatants 206
reducing/antioxidant power
164 with those obtained previously. Ultrasound: the 206
164
165
with those obtained previously. Ultrasound: the
process was similar to that described above except
207
207
(FRAP) assay
(FRAP) assay
165 process was similar to that described above except
166 that instead of soaking for 2 h, the mixture of plant- The antioxidant capacity was determined using the
166 that instead of soaking for 2 h, the mixture of plant- 208
208 The antioxidant capacity was determined using the
167 solvent material was subjected to two 30-min periods FRAP test (Thaipong et al., 2006). This method
167 solvent material was subjected to two 30-min periods 209
209 FRAP test (Thaipong et al., 2006). This method
168 of ultrasonic radiation (25 kHz) in an ultrasonic bath determines the antioxidant capacity of polyphenols to
168 of ultrasonic radiation (25 kHz) in an ultrasonic bath 210
210 determines the antioxidant capacity of polyphenols to
169 (TI-H-5, Elma, Germany) using the same solvent reduce TPTZ-Fe3+ complex. The FRAP reagent was
169 (TI-H-5, Elma, Germany) using the same solvent 211
211 reduce TPTZ-Fe3+ complex. The FRAP reagent was
170 systems. The extraction conditions used in both prepared by mixing 25 mL of a 0.3 M acetate buffer
170 systems. The extraction conditions used in both 212
212 prepared by mixing 25 mL of a 0.3 M acetate buffer
171 methods were established after preliminary tests. (pH 3.6), 2.5 mL of TPTZ solution (0.01M) and 2.5
171 methods were established after preliminary tests. 213
213 (pH 3.6), 2.5 mL of TPTZ solution (0.01M) and ◦2.5
172 In all the extractions the ratio sample-solvent was mL of a solution of FeCl3 · 6H2 O (0.02M) at 37◦ C.
172 In all the extractions the ratio sample-solvent was 214
214 mL of a solution of FeCl3 · 6H2 O (0.02M) at 37 C.
173 1:20. Finally, the cocoa extracts were concentrated A 150-µL extract sample was mixed with 2850 µL of
173 1:20. Finally, the cocoa extracts were concentrated 215
A 150-µL extract sample was mixed with 2850 µL of
174 in a rotary evaporator (40-60 rpm, 50◦◦ C) (RE-500, 215
FRAP solution and allowed to stand for 30 min in the
174 in a rotary evaporator (40-60 rpm, 50 C) (RE-500, 216
216 FRAP solution and allowed to stand for 30 min in the
175 Yamato, Japan) and dried under vacuum for 24 h at dark. The absorbance was read at a wavelength of 593
175 Yamato, Japan) and dried under vacuum for 24 h at 217
dark. The absorbance was read at a wavelength of 593
176 30◦◦ C. 217
nm. The results were reported in µM ascorbic acid
176 30 C. 218
218 nm. The results were reported in µM ascorbic acid
219 equivalents.
219 equivalents.
177
177
2.3
2.3 Determination
Determination of
of total
total phenol
phenol content
content
220 2.6
2.6 HPLC
HPLC analysis
analysis of
of cocoa
cocoa extracts
extracts
178 The total phenolic content was calculated from the 220
178 The total phenolic content was calculated from the
179 reduction capacity of Folin-Ciocolteau using gallic 221 The HPLC-analyses were carried out in an
179 reduction capacity of Folin-Ciocolteau using gallic 221 The HPLC-analyses were carried out in an
180 acid as a standard (Waterhouse 2002). A sample 222 Agilent 1200 chromatograph (Agilent Technologies,
180 acid as a standard (Waterhouse 2002). A sample 222 Agilent 1200 chromatograph (Agilent Technologies,
181 volume of 100 µL was added to 7 mL distilled water, 223 Germany) equipped with a quaternary pump and
181 volume of 100 µL was added to 7 mL distilled water, 223 Germany) equipped with a quaternary pump and
182 followed by the addition of 500 µL of Folin-Ciocalteu 224 a multiple wavelength detector coupled to an HP
182 followed by the addition of 500 µL of Folin-Ciocalteu 224 a multiple wavelength detector coupled to an HP

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Quiroz-Reyes
Quiroz-Reyes et et
al./al./ Revista
Revista Mexicana
Mexicana dede Ingenierı́a
Ingenierı́a Quı́mica
Quı́mica Vol.
Vol. 12,12,
No.No. 1 (2013)
1 (2013) 11-18
XXX-XXX
225 Chem Station (rev. B.04.01) data-processing station. 272
226 Separations were conducted on a Zorbax Eclipse
225
227
Chem Station
XDB-C18 with(rev.dimensions
B.04.01) data-processing
4.6x150 mm station. and 5 272
226
228
Separations
µm particle size. were conducted on a Zorbax
The mobile phase consisted Eclipse
227
229
XDB-C18
of with acid
water/formic dimensions
(99.9/0.1) 4.6x150 mm A
as eluent andand5
228
230
µm particle size. The mobile
methanol/acetonitrile (50/50) as eluent B. The systemphase consisted
229
231
of water/formic
was acid (99.9/0.1)
run with a gradient program:as 10-60%B eluent Ainand 15
230
232 min, followed by isocratic elution withB.60%
methanol/acetonitrile (50/50) as eluent TheBsystem
for 5
231
233
was run
min. with temperature
Column a gradient program:
was set at10-60%B30◦ C, flowinrate 15
232
234
min, followed by−1isocratic elution with 60% B for 5
was 400 µl min and the injection volume was 5
233
235
min.Samples
µL. Columnwere temperature
previously set at 30◦in
wasdissolved C,aflow rate
mixture
−1
234
236 of water/methanol (70%) and filtered through a 0.455
was 400 µl min and the injection volume was
235
237 µm Samples were
µL. membrane previously
filter. The peaks dissolved in a mixture
of (+)-catechin and
236
238
of water/methanol (70%) and
(−)-epicatechin were identified by comparingfiltered through a 0.45
the
237
239
µm membrane
retention times filter. The peaks
of samples with of (+)-catechin
those of standards.and 273  
238
240
(−)-epicatechin were
Chromatograms were recorded
identifiedat by 280comparing
nm. Standard the 274 Fig. 1. Total phenolic content of cocoa extracts. Values
 
239
241
retention times
calibration curvesof were
samples alsowith those and
prepared of standards.
used for 275 273
are expressed as mean±sd (n = 3). Means with  
240
242
Chromatograms
quantitative were recorded at 280 nm. Standard 276
analysis. 274 Fig.  1.1.Total
Fig.
different phenolic
letters content of cocoa
were significantly extracts.
different (P <Values
0.05).
241 calibration curves were also prepared and used for 275 are expressed as mean±sd (n = 3). Means with
242 quantitative analysis.  Fig. 1. letters were significantly different (P < 0.05).
different
2.7 Statistical analysis
276
243 277 for cocoa husk and cotyledon (Tomás-Barberán et
278 al.,
   2007; Lecumberri et al., 2006). However,
244
243
All
2.7 measurements were performed in triplicate and 279
Statistical analysis 277 for iscocoa
it husk and
important to cotyledon
emphasize (Tomás-Barberán
that the use ofet
245 the results analyzed by ANOVA. Differences between 280 278
  
al.,
ultrasonic2007;radiation
Lecumberri
greatly etimproved
al., 2006). the However,
extraction of
244 All measurements
means were detectedwere performed
by Duncan’s in triplicate
multiple and 279 it is important to emphasize that the use of
range test.
246
the results analyzed by ANOVA. Differences 281 polyphenols,
   especially in the case of cocoa cotyledon.
245
247 Significant differences were considered at a between
level of 282280 ultrasonic
The big radiation greatly
difference in theimproved
phenolic the extraction
content of
values
246 means
P < 0.05.wereLinear
detected by Duncan’s
regression tests multiple range test.
were performed to 283 polyphenols,
   especially in the case of can
cocoa cotyledon.
248
Significantcorrelations
differences between
were considered
281
obtained for both fractions
at a level of 282 The big difference in the phenolic content values of cocoa be attributed
247
249 determine data. mainly to the fact that the amount of polyphenols is
P < 0.05. Linear regression tests were performed to 284   
248
283
285
obtained
not identicalfor both
in thefractions
differentofparts
cocoaofcan the be attributed
cocoa bean.
249 determine correlations between data. mainly
   to the
themoisture
fact thatand the particle
amountsize of polyphenols is
3 Results and discussion
284
286 However, of the samples
250
285
287
not two
are identical
featuresin the
thatdifferent
have anparts of the influence
important cocoa bean.on
  
3 Results and discussion 286 However,
the efficiencythe moisture and particle
of extraction (Wang size
and of the samples
Weller 2006).
250
251 3.1 Total phenol content 288
are   two features thatparticle
have ansize important influence on
287
289 The reduction in the of the plant material
Figure 1 shows the total phenolic content of cocoa the efficiency
will increase of extraction
the number of (Wang
cells and Weller
directly 2006).
exposed to
252
251 3.1 Total phenol content 288
290
  
253 extracts obtained by ultrasound and maceration 291 289 The reduction
extraction in the particle
by solvent size of cavitation
and ultrasonic the plant material
(Vilkhu
252
254
Figure 1 shows
techniques. In thethe total phenolic
extraction content of
of polyphenols fromcocoa
the 292
290 will
et increase
   al., 2008).theInnumber this case,of cells
the directly
cotyledon exposed
samplesto
253
255
extracts
husk, the obtained
sonicationby ultrasound
allowed and maceration
the extraction of higher 293 291 extraction higher
presented by solvent and ultrasonic
moisture content and cavitation (Vilkhu
lower values of
254
256
techniques.
phenolic In the (25.34±1.82
contents extraction of mg polyphenols from the
g−1 ) compared to 294
292 et    al., 2008).
particle size thanInhusk this samples,
case, thewhichcotyledonwouldsamples
explain
255
257
husk,
the the sonication
content extractedallowed the extraction
by maceration of higher
(17.85±1.33 mg 295
293 presented
the   greater higher moisture
efficiency in content
polyphenol and lower
release. values of
256
258 gphenolic
−1 contentsthere
). However, (25.34±1.82 mg g−1 ) compared
were no significant differences to 294 particle size than husk samples, which would explain
257 the content
(P > 0.05)extracted
betweenbyboth maceration
methods. (17.85±1.33
In the case mg 295 the   greater efficiency in polyphenol release.
259
−1
g the
). However, 3.2 Antiradical capacity (DPPH assay)
cotyledon,there the were no significant differences 296
258
260 of phenolic content results were   
(P > 0.05) different
significantly between both (P <methods. In the case
0.05) between the 297 Figure 2 shows the capacity scavenging(DPPH activityassay)of extracts
259
261 296 3.2 Antiradical
260
262
of theextraction
two cotyledon, the phenolic
methods. The content
polyphenol results were 298 from
content   the husk obtained using the proposed methods.
261
263
significantly
varied different (Pto 135.92±3.77
from 91.06±0.86 < 0.05) between mg g−1 the for 299
297 Figure
It can 2beshows the scavenging
seen that the activityactivity
increased of extracts
rapidly
262
264
two extractionand
conventional methods.
ultrasound Themethods
polyphenol content 300
respectively. 298 from
in thetheconcentration
husk obtainedrange usingofthe0.04-0.1
proposedmg methods.
mL−1 ,
−1
263
265
varied from 91.06±0.86 to 135.92±3.77 mg g for It can be seen
According to Pan et al. (2011), the mechanical effects 301 remaining constant at higher concentrations. The
299 that the activity increased rapidly
264
266
conventional
of and ultrasound
sonication allow for a greatermethods
penetration respectively.
of solvent 302300 in the concentration
compounds extractedrange by of ultrasound mg mL−1 ,
0.04-0.1 application
265
267
According to Pan et al. (2011), the mechanical effects remaining constant
into the cells, enhancing mass transfer. In the process 303 showed greater inhibition activity toward the DPPH
301 at higher concentrations. The
266
268
of sonication allow for a greater penetration of solvent compounds extracted
of extraction, ultrasonic radiation can also break cell 304 radical compared to that toward the extracts obtained
302 by ultrasound application
267
269
into thefacilitating
walls, cells, enhancing
the releasemassoftransfer. In the process 305
the compounds. 303 showed
by greater inhibition
the conventional method. activity
As fortoward the DPPH
the compounds
268
270
of extraction, ultrasonic radiation can also break cell radical compared
The results obtained by the traditional method 306 extracted from the cotyledon (Fig. 3.), the antiradical
304 to that toward the extracts obtained
269
271
walls, facilitating the release of the compounds. by the conventional
are consistent with those reported in the literature 307 activity was greater in the range of 0.01-0.04 mg
305 method. As for the compounds
270 The results obtained by the traditional method 306 extracted from the cotyledon (Fig. 3.), the antiradical
271
4 are consistent with those reported in the literature www.rmiq.org
307 activity was greater in the range of 0.01-0.04 mg

14
4 www.rmiq.org
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 Quiroz-Reyes et al./ Revista Mexicana de Ingenierı́a Quı́mica Vol. 12, No. 1 (2013) XXX-XXX
Quiroz-Reyesetetal./
Quiroz-Reyes al./Revista
RevistaMexicana
Mexicanade
deIngenierı́a
Ingenierı́aQuı́mica
Quı́micaVol.
Vol.12,
12,No.
No.1 1(2013)
(2013)XXX-XXX
11-18
308 different extracts. In the case of the husk, there were
331
different
different
no extracts.
significantextracts. InInthe
differences thecase
case ofofthe
between thehusk,
husk,
the ECthere
there were
were
308 331
332 50 values.
332
333
no significant
no significant
However, differences
differences
the control had an between
between
EC50 valuethe EC
the EC values.
50 values.
significantly
50
333 However,
334 However,
lower (P the
<the control
control
0.05) than had theanan
had EC
valuesEC value
5050of the significantly
value significantly
husk fraction
334
335
lower
lower (P (P< 0.05)
0.05) than
than thethe
extracts. On the other hand, the extracts
< values
values ofofthethehusk
husk fraction
fraction
of cotyledon
335
336
extracts.
extracts. OnOn thetheother
other hand,
hand, the
had EC50 values lower than those obtained for the theextracts
extracts ofofcotyledon
cotyledon
336 had
337 hadEC
control. EC5050values thislower
Invalues lowerthan
fraction, than those
the those obtained
extractobtained
obtainedfor
forthethe
via
337
338
control.
control. In In this
this fraction,
fraction, the the
ultrasound showed the lowest mean EC50 value, which extract
extract obtained
obtained via
via
338 ultrasound
339 ultrasound
was showed
showedthe
significantly thelowest
lowestmean
different (Pmean <EC EC 5050value,
0.05) value,
from which
which
that
339
340
was
was significantly
significantly different
different
obtained by the maceration method. The analysis(P (P < < 0.05)
0.05) from
from that
that
of
340
341
obtained
variance revealed statistical differences between of
obtained byby thethe maceration
maceration method.
method. The
The analysis
analysis of
the
341
342
variance
variance revealed
revealed statistical
statistical differences
EC50 values of the husk and cotyledon extracts. The differences between
between the
the
342 EC 50 50values
343
EC
results showofof
values the
goodthehuskhuskand andcotyledon
correlation (R2 2 = extracts.
cotyledon extracts.
0.72) The
betweenThe
343 results show good correlation (R 2 = 0.72) between
344
results show good correlation
the phenolic content and the antiradical activity of (R = 0.72) between
344 the
thephenolic content
345 cocoa phenolic
extracts. The and
content ECand thetheantiradical
antiradicalactivity
50 results determined in this
activityofof
345 cocoa
cocoa extracts.
extracts. TheThe EC EC results
results determined
determined ininthis
this
309
  346 research are considerably lower than those reported
5050
309
310 Fig. 2. Scavenging effect of cocoa husk extracts on 346 research
research are
are considerably
considerably lower
lower than
than those
those reported
reported
 
310   Fig. 2. Scavenging effect of cocoa husk extracts on
347 by Othman et al. (2007), who published EC50 values
311 DPPH radicals. Values are expressed as mean±sd 347 bybyOthman Othman etetal.al.(2007),
(2007), whowho published
published
−1 ECEC values
values
in the range of 1.2-1.5 mg mL−1 for ethanol 5050extracts
(nDPPH radicals. acid
Values
was are
usedexpressed as mean±sd 348
311   = 3). Ascorbic as the control. 348 inin
the range of 1.2-1.5 mg mL −1for ethanol extracts
312  Fig. 2. 3). Ascorbic acid was used as the control. the range of 1.2-1.5 mg mL
of cocoa. These differences can be attributed to the for ethanol extracts
 (n =
349
312
349 ofof
cocoa.
Fig. 2. 350
cocoa.
variety ofThese
Thesedifferences
cocoa differences
species can
used, bebeattributed
canproductionattributed totothe
area the
and
350 variety
variety ofofcocoa
cocoa species
species used,
used, production
production area
area and
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 on DPPHactivity (EC50 ) of cocoa extracts
radicals.
 
Table 1. Scavenging on DPPHactivity (EC50) of cocoa extracts
radicals.
on DPPH radicals. −1
  Extraction method EC 50 (DPPH) mg mL −1
Extraction method EC
Husk (DPPH) mg mL
EC50 (DPPH)Cotyledon
  50
 
Extraction method Husk mg mL−1
Cotyledon
  Maceration 0.0533±0.0022Husk a 0.0164±0.0012 Cotyledonc
0.0533±0.0022aa a 0.0124±0.0017 0.0164±0.0012dc c
352
  Maceration
352 Ultrasound 0.0486±0.0018
Maceration
Ultrasound 0.0533 ± 0.0022
0.0486±0.0018 a 0.0164 ± 0.0012 d
b a 0.0124±0.0017b
 
  Control
Ultrasound 0.0243±0.0009
0.0486 ± 0.0018 0.0243±0.0009
0.0124 ± 0.0017 d
  Control 0.0243±0.0009 b 0.0243±0.0009b b
b
Means Control
with different 0.0243 letters ±were 0.0009 0.0243 ± 0.0009
significantly
  Means (P
different with different letters were significantly
< 0.05).
  Means (P
different with different letters were significantly
< 0.05).
  Ascorbic
differentacid
(P <was used as a control
0.05).
  Ascorbic acid was used as a control
313
 
  Ascorbic acid was used as a control
313  
314 Fig.
  3.3. Scavenging effect of cocoa cotyledon extracts
Fig.
314
  on
Fig.3.3. Scavenging effect of cocoa cotyledon extracts
315 Fig.DPPH radicals. Values are expressed as mean±sd
315
316
 (non= DPPH
  radicals. Values are expressed as mean±sd
3). Ascorbic acid was used as the control.
316    (n = 3). Ascorbic acid was used as the control.
  
    −1
317 mL . The extracts obtained by sonication had higher
317     mL−1 . The extracts obtained by sonication had higher
318 antiradical
  activity than those obtained by maceration.
318 antiradical activity than those obtained by maceration.
 It is noteworthy that the DPPH radical scavenging
319
319  It is noteworthy that the DPPH radical scavenging
320 activity of the phenolic extracts obtained from the
  activity of the phenolic extracts obtained from the
320
321 cotyledon
  was significantly higher (P < 0.05) than the
321 cotyledon was significantly higher (P < 0.05) than the
 activity exhibited by extracts obtained from the husk.
322
322  activity exhibited by extracts obtained from the husk.
323 EC50 is defined as the amount of antioxidant
323
  EC50 is defined as the amount of antioxidant
324 necessary
  to decrease the initial concentration of
324 necessary
 DPPH radical to decrease the initial concentration of
325
325
 DPPH radicalby 50%. The lower the EC value is
by 50%. The lower the EC5050 value is
353
353
 
 
326
326
  the greater the activityof
the greater the activity extracts as DPPH radical
of extracts as DPPH radical
354
354
Fig.
Fig.
 
4. Antioxidant capacity of cocoa extracts
4. Antioxidant capacity of cocoa extracts
 
327 scavengers (Wootton-Beard and Ryan, 2011). The 355 measured
  by FRAP assay. Concentration of sample
327 scavengers (Wootton-Beard and Ryan, 2011). The 355 measured by FRAP assay. Concentration of sample
 EC
50 value was determined by plotting the inhibition was
Fig. 0.10
4. mg mL−1 . Values are expressed as mean±sd
4. mg mL−1 . Values are expressed as mean±sd
328  EC 356
328 50 value was determined by plotting the inhibition 356 was
Fig.0.10
329   percentageofofDPPH
percentage DPPHradical
radicalagainst
againstthe
theconcentration
concentration 357 (n
(n = 3). Means
Meanswith
with different
different letters
letters were
were significantly
329  
ofofthe
357
  = 3). (P significantly
330
330
  the extract. Table 1 shows the EC50 values
extract. Table 1 shows the EC valuesfor
50 forthe
the 358
358
different
  < 0.05).
different (P < 0.05).
   
 
www.rmiq.org
www.rmiq.org 5
www.rmiq.org
 
  155
 
 
  

  
 Quiroz-Reyes et al./ Revista Mexicana de Ingenierı́a Quı́mica Vol. 12, No. 1 (2013) XXX-XXX
Quiroz-Reyes
Quiroz-Reyesetetal./
al./Revista
RevistaMexicana
MexicanadedeIngenierı́a
Ingenierı́aQuı́mica
Quı́micaVol.
Vol.12,
12,No.
No.11(2013)
(2013)XXX-XXX
11-18

 
 
359
359
360
  5. Typical chromatograms of cocoa extracts obtained by ultrasound-assisted extraction.
Fig.
360
  5. Typical chromatograms of cocoa extracts obtained by ultrasound-assisted extraction.
Fig.
Fig. 5.
Fig. 5. Table 2. Concentration of (+)-catechin and
361 3.3 Antioxidant activity (FRAP assay) 380
361 3.3 Antioxidant activity (FRAP assay) 380 (−)-epicatechin in cocoa fractions.
362 FRAP determination is based on the reduction of Fe3+ - Table 2. Concentration
Treatment/cocoa of (+)-catechin
Content (µg mg−1 dry andextract)
FRAPcomplex
determination of Fe3+ - Table 2. Concentration of (+)-catechin and
362
363 TPTZ to Fe2+is-TPTZ
based complex.
on the reduction
The reducing (−)-epicatechin
fraction in cocoa
(+)-Catechin fractions.
(−)-Epicatechin
TPTZ complex 2+
to Fe -TPTZ complex. Thepresence
reducing (−)-epicatechin in cocoa fractions.
363
364 properties are generally associated with the Treatment/cocoa
Maceration Content (µg mg−1 dry extract)
properties are generally associated
of reducing agents, which have been shown to with the presence
exert Treatment/cocoa
fraction Content (µgamg −1
dry extract)
Cotyledon (+)-Catechin (−)-Epicatechin
364
365
4.62 ± 0.047 132.88 ± 0.245a
365
366 antioxidant action by breaking the free radicaltochain
of reducing agents, which have been shown exert fraction
Husk
Maceration
(+)-Catechin
0.28 ± 0.046 b (−)-Epicatechin
2.64 ± 0.018b
366 antioxidant action by breaking the free
by donating a hydrogen atom. The FRAP results (Fig. radical chain Maceration
367
Ultrasound
Cotyledon 4.62±0.047 a
132.88±0.245a
367
368 4.)by donating a hydrogen
indicate that atom. The
the cotyledon FRAPobtained
extract results (Fig.
by 381
Cotyledon
Cotyledon
Husk 4.62±0.047
4.26
0.28±0.046± ba a
0.025 132.88±0.245 b ac
144 ± 4.850
2.64±0.018
ultrasound application had the highest antioxidantby
4.) indicate that the cotyledon extract obtained 381
368 b b b b
369 Husk
Husk
Ultrasound 0.28±0.046
0.32 ± 0.083 2.64±0.018
2.77 ± 0.340
369
370
ultrasound
activity, application
reaching a valuehad the highest
of 350±15 µMEantioxidant
ascorbic Ultrasound a c
activity,
−1 reaching a value of 350±15 µME ascorbic
Cotyledon
Means 4.26±0.025
with different letters within the144±4.850
same column are
370
371 acid L . However, no significant differences between Cotyledon
Husk different
significantly 4.26±0.025
0.32±0.083
(p < 0.05).ba 144±4.850
2.77±0.340b
c
371
372
acid
the L−1 . However,
methods used were no detected.
significantOndifferences
the otherbetween
hand, Husk 0.32±0.083 b
2.77±0.340b
the methods used were detected. On theinother hand, Means with different letters within the same column are
372
373 the FRAP values for the husk were located the range Means withdifferent
different
the FRAP values for the husk were located
−1 in the range the polyphenol
significantly (p letters
content withinextracts.
of cocoa
< 0.05). the same column are
373
374 of 141-148 µME of ascorbic acid L . These values significantly different (p < 0.05).
374
375
of significantly
are 141-148 µME of ascorbic
lower (P < 0.05) L−1 . that
acid than These values
reported
375
376
are significantly lower (P < 0.05) than
for cotyledon. The FRAP results correlate well with that reported 382 the polyphenol content of cocoa extracts.
fortotal
cotyledon. the polyphenol content of cocoa extracts.
phenolic The FRAP (R2 results
= 0.78)correlate well
(R2with
382
376
377 the content and DPPH =
2
377
378
the total phenolic content (R = 0.78) and
0.98). Othman et al. (2007) also reported a positive DPPH (R2 =
378
379
0.98). Othman
correlation between et al. (2007) also
antioxidant reported
activity (FRAP)a positive
and
379 correlation between antioxidant activity (FRAP) and
6 www.rmiq.org
166 www.rmiq.org
www.rmiq.org
 Quiroz-Reyes
Quiroz-Reyes
Quiroz-Reyes et
al./al./
etetal./ Revista
Revista
Revista Mexicana
Mexicana
Mexicana dede
de Ingenierı́a
Ingenierı́a
Ingenierı́a Quı́mica
Quı́mica
Quı́mica Vol.
Vol.
Vol. 12,12,
12, No.
No.No. 1 (2013)
11(2013)
(2013) 11-18
XXX-XXX
XXX-XXX

383
383
3.4 Identification
3.4 Identification and
and quantification
quantification 427
427
References
References
384
384
(HPLC)
(HPLC)
428
428
Bels̆c̆ak, A.,
Bels̆c̆ak, A., Komes,
Komes, D.,
D., Horz̆ić,
Horz̆ić, D.,
D., Ganić,
Ganić, K.K.
K.K.
385
385
Using HPLC
Using HPLC analysis
analysis itit was
was possible
possible to to identify
identify 429
429
and Karlović, D. (2009). Comparative study
and Karlović, D. (2009). Comparative study of of
386
386
the presence of (+)-catechin and
the presence of (+)-catechin and (−)-epicatechin (−)-epicatechin 430
430
commercially available cocoa products in
commercially available cocoa products in terms terms
387
387
in the
in the cotyledon
cotyledon and and husk
husk samples
samples of of cocoa.
cocoa. 431
431
of their
of their bioactive
bioactive composition.
composition. Food
Food Research
Research
388
388
Typical chromatograms of cocoa extracts
Typical chromatograms of cocoa extracts obtained by obtained by 432
432
International 42, 707-716.
International 42, 707-716.
389
389
sonication are shown in Fig. 5. The
sonication are shown in Fig. 5. The (+)-catechin (+)-catechin
390
390
and (−)-epicatechin
and (−)-epicatechin contents
contents were were significantly
significantly 433
433
Chemat, F.,
Chemat, F., Zill-e-Huma
Zill-e-Huma and and Khan,
Khan, M.K.
M.K. (2011).
(2011).
391
391
higher (P < 0.05) in cotyledon
higher (P < 0.05) in cotyledon than in than in husk
husk for
for 434
434
Applications of
Applications of ultrasound
ultrasound in
in food
food technology:
technology:
392
392
both extraction procedures
both extraction procedures (Table 2). (Table 2). Only
Only in in 435
435
Processing, preservation
Processing, preservation and and extraction.
extraction.
393
393
the case of (−)-epicatechin, the
the case of (−)-epicatechin, the ANOVA showed ANOVA showed 436
436
UltrasonicsSonochemistry
Ultrasonics Sonochemistry18, 18,813-835.
813-835.
394
394
significant differences
significant differences between
between ultrasound
ultrasound and and
maceration methods. These results are compatible 437 Garcı́a-Márquez, E.,
Garcı́a-Márquez, E., Román-Guerrero,
Román-Guerrero, A., A., Pérez-
Pérez-
395
395 maceration methods. These results are compatible 437

with total
total polyphenol
polyphenol quantification
quantification using
using thethe Folin-
Folin- 438 Alonso, C., Cruz-Sosa, F., Jiménez-Alvarado,
Alonso, C., Cruz-Sosa, F., Jiménez-Alvarado,
396
396 with 438

Ciocalteu method, and explain why the cotyledon 439

R.R. and
and Vernon-Carter,
Vernon-Carter, E.J.
E.J. (2012).
(2012). Effect
Effect of
of
397
397 Ciocalteu method, and explain why the cotyledon 439

presents greater
greater efficiency
efficiency inin tests
tests to
to determine
determine free-
free- 440 solvent-temperature extraction conditions
solvent-temperature extraction conditions on the on the
398
398 presents 440

radical scavenging activity and antioxidant capacity. 441 initial antioxidant

initial antioxidant activity
activity and
and total
total phenolic
phenolic
399
399 radical scavenging activity and antioxidant capacity. 441

According to to Ortega
Ortega etet al.
al. (2008),
(2008), polyphenols
polyphenols 442 content of muitle extracts and their decay
content of muitle extracts and their decay upon upon
400
400 According 442

belonging to the catechin group are mainly responsible 443 storage at different pH. Revista Mexicana
storage at different pH. Revista Mexicana de de
401
401 belonging to the catechin group are mainly responsible 443

for the antioxidant properties of cocoa. 444 Ingenierı́a Quı́mica 11, 1-10.
Ingenierı́a Quı́mica 11, 1-10.
402
402 for the antioxidant properties of cocoa. 444

445
445
Jacques,R.A.,
Jacques, R.A.,Freitas,
Freitas,L.S.,
L.S.,Flores,
Flores,V.,
V.,Dariva,
Dariva,C.,
C.,de
de
446
446
Oliveira, A.P., de Oliveira, J.V., Caramão,
Oliveira, A.P., de Oliveira, J.V., Caramão, E.B. E.B.
403
403
Conclusion
Conclusion 447
447
(2007). The
(2007). The use
use of
of ultrasound
ultrasound inin the
the extraction
extraction
448
448
of Ilex paraguariensis leaves: A
of Ilex paraguariensis leaves: A comparisoncomparison
The useuse of of ultrasonic
ultrasonic radiation
radiation facilitated
facilitated thethe 449 withmaceration.
with maceration.Ultrasonics
UltrasonicsSonochemistry
Sonochemistry14, 14,
404
404 The 449

extraction of polyphenols from cocoa beans, 450 6-12.

6-12.
405
405 extraction of polyphenols from cocoa beans, 450

406 increasing the

increasing the content
content by by 50% 50% compared
compared to to the
the
406
451 Jonfia-Essien, W.A.,
Jonfia-Essien, W.A., West,
West, G.,
G., Alderson,
Alderson, P.G.
P.G. and
and
407 traditional method. In addition,
traditional method. In addition, the antioxidant the antioxidant 451
407
452 Tucker, G.
Tucker, G. (2008).
(2008). Phenolic
Phenolic content
content and
and
408 activity measured
activity measured by by DPPH
DPPH and and FRAP
FRAP methods
methods waswas 452
408
453 antioxidant capacity
antioxidant capacity of
of hybrid
hybrid variety
variety cocoa
cocoa
409 greater in the compounds obtained by
greater in the compounds obtained by sonication for sonication for 453
409
454 beans. Food
beans. FoodChemistry
Chemistry108,
108,1155-1159.
1155-1159.
410
410
husk and cotyledon fractions. The
husk and cotyledon fractions. The total phenolic total phenolic 454

411 contentextracted
content extractedfrom
fromthethecotyledon
cotyledonwas wassignificantly
significantly
411
455 Khan, M.K.,
Khan, M.K., Abert-Vian,
Abert-Vian, M.,M., Fabiano-Tixier,
Fabiano-Tixier, A.S.,
A.S.,
412 greater than that extracted from the husk fraction.
greater than that extracted from the husk fraction. The The 455
412
456 Dangles, O. and Chemat, F. (2010). Ultrasound-
Dangles, O. and Chemat, F. (2010). Ultrasound-
413 results demonstrate a positive correlation
results demonstrate a positive correlation between the between the 456
413
457 assisted extraction
assisted extraction of
of polyphenols
polyphenols (flavanone
(flavanone
414 total phenolic content and antioxidant activity
total phenolic content and antioxidant activity of cocoa of cocoa 457
414
458 glycosides) from orange (Citrus sinensis
glycosides) from orange (Citrus sinensis L.) L.)
415 extracts. Cocoa
extracts. Cocoa cotyledon
cotyledon presented
presented significantly
significantly 458
415
459 peel. Food Chemistry 119, 851-858.
peel. Food Chemistry 119, 851-858.
416
416
greater quantities of (+)-catechin and (−)-epicatechin
greater quantities of (+)-catechin and (−)-epicatechin
459

417 ascompared
as comparedwith withthe
thehusk.
husk. Finally,
Finally, we
wecan canconclude
conclude Koffi, E.,E., Sea,
Sea, T.,T., Dodehe,
Dodehe, Y. Y. and
and Soro,
Soro, S.S.
417 460
460 Koffi,
418 that the
that the use
use ofof ultrasonic
ultrasonic radiation
radiation isis an an excellent
excellent (2010). Effect
Effect of
of solvent
solvent type
type on
on extraction
extraction of
of
418 461
461 (2010).
419 method for
method for the
the extraction
extraction of of natural
natural antioxidants
antioxidants polyphenols from
from twenty
twenty three
three Ivorian
Ivorian plants.
plants.
419 462
462 polyphenols
420 sinceititprovides
since providesaashort
shortextraction
extractiontime,time,ititoffers
offershigh
high Journal of
of Animal
Animal and and Plant
Plant Sciences
Sciences 5,5, 550-
550-
420 463
463 Journal
421 reproducibilityand
reproducibility andlow
lowloss
lossof ofbioactive
bioactivecompounds.
compounds. 558.
421 464
464 558.

465
465
Lecumberri, E.,
Lecumberri, E., Mateos,
Mateos, R.,R., Ramos,
Ramos, S.,
S., Alı́a,
Alı́a,
422
422
Acknowledgments
Acknowledgments 466
466
M., Rúperez, P., Goya, L., Izquierdo-Pulido,
M., Rúperez, P., Goya, L., Izquierdo-Pulido,
467
467
M. and
M. and Bravo,
Bravo, L. L. (2006).
(2006). Caracterización
Caracterización
423
423
This work
This work waswas financially
financially supported
supported by
by SIP-
SIP- 468
468
de la fibra de cacao y su efecto
de la fibra de cacao y su efecto sobre sobre lala
424
424
IPN (projects 20113551 and 20120422).
IPN (projects 20113551 and 20120422). Quiroz- Quiroz- 469
469
capacidad antioxidante en suero de animales
capacidad antioxidante en suero de animales de de
425
425
Reyes thanks
Reyes thanks COFAA-IPN
COFAA-IPN and and CONACyT
CONACyT for for the
the 470
470
experimentación. Nutrición Hospitalaria
experimentación. Nutrición Hospitalaria 21, 21,
426
426
scholarships received.
scholarships received. 471
471
622-628.
622-628.

www.rmiq.org
www.rmiq.org
www.rmiq.org 1777
 Quiroz-Reyes
Quiroz-Reyes
Quiroz-Reyes et Revista
etetal./
al./al./ Revista
Revista Mexicana
Mexicana
Mexicana de Ingenierı́a
deIngenierı́a
de Ingenierı́a Quı́mica
Quı́mica
Quı́mica Vol.Vol.
Vol. 12,
No.No.
12,No.
12, 1 (2013)
11(2013)
(2013) 11-18
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