Journal of Cereal Science 56 (2012) 410e417

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Journal of Cereal Science

journal homepage: www.elsevier.com/locate/jcs

Effect of germination on total phenolic compounds, total antioxidant capacity,

Maillard reaction products and oxidative stress markers in canihua
(Chenopodium pallidicaule)
F. Abderrahim a, E. Huanatico b, R. Repo-Carrasco-Valencia c, S.M. Arribas a, M.C. Gonzalez a,
L. Condezo-Hoyos a, *
a
Department of Physiology, Faculty of Medicine, Universidad Autnoma de Madrid, Arzobispo Morcillo 2, Madrid, Spain
b
Faculty of Agri-food Engineering, Universidad San Antonio Abad del Cusco, Cusco, Peru
c
Faculty of Food Science and Technology, Universidad Nacional Agraria La Molina, Av La Molina s/n, La Molina, Lima 100, Peru

a r t i c l e i n f o a b s t r a c t

Article history: Germination of cereals/pseudo-cereals has been suggested as an effective method to increase antioxidant
Received 10 January 2012 compounds. However, this process could also lead to high reducing sugar levels and subsequent Maillard
Received in revised form reaction products. The aim of this work was to determine the time course effect of canihua (Chenopodium
16 April 2012
pallidicaule) germination on: 1) antioxidant capacity, 2) extractable and non-extractable phenolic
Accepted 27 April 2012
compounds content, 3) Maillard reaction products and 4) oxidative stress markers. Germination
increased antioxidant capacity, phenolic compounds and Maillard reaction products, including advanced
Keywords:
glycated end products while it decreased oxidative stress markers. All parameters exhibited a similar
Antioxidant capacity
Canihua
time course pattern with a maximum at 72 h. In addition to the increase in phenolic compounds and
Germination antioxidant capacity, canihua germination produced advanced glycated end products. The impact on
Pseudo-cereal human health of these compounds in germinated seeds deserves future attention.
Maillard reaction products 2012 Elsevier Ltd. All rights reserved.
Oxidative stress markers

1. Introduction Maillard reaction, which is strongly dependent on the concentra-

Germination of cereals/pseudo-cereals has been suggested as an
inexpensive and effective method to enhance the antioxidant
capacity through the increase of low-molecular weight antioxi-
dants (Alvarez-Jubete et al., 2010). These improvements in antiox-
idant functional properties depend on cereal type and hence the
optimal conditions must be established for each seed (Gallegos-
Infante et al., 2010). In addition to antioxidant synthesis, other
biochemical reactions take place during germination. One of them
is the increase of the activities of cell wall hydrolases resulting from
the hydration of matrices (Nonogaki et al., 2010). These enzymes
are able to degrade carbohydrates into low-molecular weight
compounds, resulting in an increase of reducing sugars (Tian et al.,
2010). These compounds are implicated in the rst step of the
Abbreviations: AGEs, advanced glycated end products; TAC, total antioxidant
capacity.
* Corresponding author. Tel.: 34 91 497 5417; fax: 34 91 497 5788.
E-mail address: luisalberto.condezo@uam.es (L. Condezo-Hoyos).
tion of reducing sugars (Martins et al., 2000). Therefore, it is
plausible that Maillard reaction products, specically advanced
glycated endproducts (AGEs), might be inuenced by germination.
Serum levels of these compounds are inuenced by a diet con-
taining AGEs (Koschinsky et al., 1997), which has been described as
a risk factor in several diseases (Barlovic et al., 2011; Ko et al., 2010;
Park et al., 2011). Therefore, dietary recommendations have been
suggested to minimize risks induced by high-AGE intake.
Canihua (Chenopodium pallidicaule), is an Andean annual crop
found in semidesert climate at 3600e4400 m altitude (Bolivia and
Peru) where it is grown as a secondary spontaneously seeding in
quinoa elds of the traditional agricultural system. Canihua usually
is referred to as pseudo-cereal since it is not a member of the grass
family, but produces seeds that can be milled into our and used
like a cereal crop. It is consumed as gruel-like food, beverage or
mixed with wheat for making breads and cakes of the local culinary
heritage. Canihua is exceptionally rich in avonoids, quercetin and
isorhamnetin being predominant (Repo-Carrasco-Valencia et al.,
2010). Despite having a high content of avonoids and other
phenolic compounds (Penarrieta et al., 2008), the applicability of
germination as a strategy to enhance this seeds nutritional and

0733-5210/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2012.04.013
 F. Abderrahim et al. / Journal of Cereal Science 56 (2012) 410e417
functional properties has not been reported. In addition to evalu-
ating the effect of germination on phenolic compounds content and
total antioxidant capacity, the aim of this study was to determine
the changes in Maillard reaction products as well as oxidative stress
markers in germinated canihua.
2. Experimental
2.1. Chemicals
2-Aminoethyldiphenylborinate, 2,2-azinobis(3-ethylbenzothia
zoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-
tetramethyl-chroman-2-caboxylic acid (Trolox), 2-mercap
toethanol, ()-catechin hydrate, chloramine T tryhydrate, couma-
ric acid, DMSO, gallic acid, glycine, hydroxymethyfurfural (HMF),
luminol, potassium iodide, potassium persulfate, SDS, sodium
nitrite, sulfanilamide, thiobarbituric and trichloroacetic acid were
obtained from SigmaeAldrich (Spain). Acetic acid, Folin Ciocalteu
phenol reagent and sodium carbonate were purchased from Merck
(Spain). Bio-Rad protein assay kit and PAGE reagents were acquired
from Bio-Rad S.A (Spain). Ethanol, hydrogen peroxide and methanol
were obtained from Panreac S.A. (Spain), acetone from Cofares
(Spain) and sulfuric acid from Probus S.A (Spain). MemCode
reversible protein stain kit was acquired from Pierce Biotechnology
(Rockford, USA). Primary antibody against 3-nitrotyrosine and anti-
mouse IgG-peroxidase conjugated were purchased from Santa Cruz
Biotechnology (Germany) and Amersham Bioscience (UK),
respectively.
2.2. Plant material
Canihua seeds (C. pallidicaule), cupi variety, were obtained from
Salcedo experimental station (Instituto Nacional de Investigacin
Agraria, INIA, Puno, Peru).
2.3. Canihua germination
Seeds were disinfected with 0.3% v/v sodium hypochlorite for
15 min and then rinsed four times with sterile water to remove any
remains of the disinfectant agent. Immediately after, the seeds were
hydrated at 20  C for 14 h to about 45 %humidity. Imbibited seeds
were dark-germinated in a chamber at 20  C for 48 h, 72 h or 96 h.
Germinated canihua was air-dried at 60  C for 12 h, milled and
sieved with No 100 mesh, packed in polyethylene bags and stored
at 20  C until analysis.
2.4. Total antioxidant capacity (TAC)
A direct measurement was used to assess the TAC of dormant
and germinated canihua (Serpen et al., 2008).
2.5. Extractable and non-extractable phenolic compounds
One hundred milligrams of the sample (dormant and germi-
nated) were placed in a capped centrifuge tube and 1 ml of acidic
methanol (HCl)/water solution (50:50 v/v, pH 2) was added. The
tube was shaken on a movil rod shaker (J.P. Selecta S.A, Barcelona,
Spain) at maximum speed for 1 h (room temperature) and then
centrifuged at 2100 g for 10 min (4  C) to obtain the acid methanolic
extract. Thereafter, 1 ml of acetone/water solution (70:30 v/v) was
added to the precipitate, followed by the shaking and centrifuga-
tion steps to obtain the acetonic extract. Residues obtained after
methanol/acetone extraction (which was namely extractable frac-
tion) were hydrolyzed with 1 ml of methanol/H2SO4 90:10 (v/v) at
85  C for 10 h to obtain non-extractable phenolic compounds
411
(Arranz and Saura Calixto, 2010; Hartzfeld et al., 2002). Aliquots of
all extracts were stored at 20  C until analysis of phenolic
compounds.
Total phenolic compounds were assessed by the Folin Ciocalteu
micro-method adapted to a microplate reader (Abderrahim et al.,
2011). A colorimetric assay to measure avonoids content, which
was adapted from Oomah and Mazza (1996). Flavan-3-ols content
was measured by the diazotized sulfanilamide-based specic
method (Singh et al., 1999) adapted to a microplate reader. Non-
extractable avonoids were directly assessed by 2-aminoethly
diphenylborinate and confocal laser scanning microscopy previ-
ously applied to avonoid tissue localization (Hutzler et al., 1998).
2.6. Reducing and free sugars
To assess reducing and free sugar contents, dormant and
germinated canihua (10 mg) were extracted with 1 ml 80% v/v
ethanol solution for 2 h on a movil rod at maximum speed and
room temperature. The supernatant, obtained by centrifugation at
2100 g for 30 min (room temperature), was vortex-mixed with PVP
solution to eliminate phenolic compounds (Tian et al., 2010) and
then centrifuged as described above. In this supernatant, reducing
and free sugar were assessed by 3, 5-dinitrosalicylic acid and
anthrone-sulfuric acid microtiter assays, respectively.
2.7. Maillard reaction products
Ten micrograms of dormant and germinated canihua were
placed in capped centrifuge tubes and 1 ml of aqueous SDS (6% w/v)
was added. The tubes were shaken on a movil rod at maximum
speed for 30 min (room temperature). The mixture was centrifuged
at 2100 g for 10 min (4  C). The supernatants obtained were used to
evaluate Maillard reaction products. Intermediate (FIC), advanced
(FAST index) and nal Maillard reaction products were assessed
according to previous reports (Michalska et al., 2008). A thio-
barbituric acid-based assay (Dolhofer and Wieland, 1981) was used
to measure AGEs content. Protein content was used to normalize all
parameters above.
2.8. Protein-bound carbonyls, 3-nitrotyrosine (NT) and advanced
oxidation protein products (AOPPs)
Whole canihua proteins were extracted following a proto-
col previously described (Branlard and Bancel, 2007). Protein-
bound carbonyls were assessed according to a 2,4-
dinitrophenylhydrazine-based assay (Hawkins et al., 2009). The
level of NT was evaluated by western blot. AOPPs content were
assessed by an iodide oxidation-based spectrophotometric method
(Witko-Sarsat et al., 1996).
2.9. Protein content
Protein content was assessed by Coomassie Blue-based micro-
titer plate assay according to the manufacturer procedure (Bio-Rad,
Spain).
All absorbance values were measured at 595 nm in a Synergy HT
Multi-Mode Microplate Reader (Biotek, Rochester, VT, USA).
2.10. Dry matter content
The dry matter content was determined in triplicate after
drying approximately 2 g of canihua sample at 100  C to constant
weight.
412 F. Abderrahim et al. / Journal of Cereal Science 56 (2012) 410e417
2.11. Statistical analysis
One-way analysis of variance (ANOVA) followed by a Bonferroni
post hoc test was performed with GraphPad Prism Software. A
value of p < 0.05 was considered statistically signicant.
3. Results and discussion
3.1. Total antioxidant capacity (TAC)
The TAC of dormant canihua, assessed by direct procedure, was
higher (70.95  1.7 mmol trolox/kg dry weight) than previously
reported (1.8e7.8 mmol trolox/kg dry weight) (Penarrieta et al.,
2008) (Fig. 1). It is likely related to the direct TAC assay used in
the present study, which allows assessment of both non-
extractable and extractable antioxidant compounds. It has been
well established that in many cereals, a high number of phenolic
compounds are covalently bound to macromolecules such as
proteins and carbohydrates and hence cannot be totally extracted
by solvents (Arranz et al., 2010; Chandrasekara and Shahidi, 2010),
where usually phenolic compounds are measured. Consequently, it
has been suggested that often phenolic compound content and, in
turn, cereal antioxidant capacity could be underestimated (Arranz
et al., 2010). Compared to cereals, TAC assessed in mmol trolox/kg
dry weight by this direct procedure, the value of dormant canihua
reported in the present study was higher than those found for
millet (Panicum miliaceum) (w10), wheat (Triticum aestivum L.)
(w35), oat (Avena sativa) (w30), barley (Hordeum vulgare) (w55)
and even comparable to wheat bran (60e80) (Serpen et al., 2008).
This result shows that canihua (C. pallidicaule) is a pseudo-cereal
rich in antioxidant compounds, something that may be linked to
its severe culture conditions such as high altitude, cold and
drought.
Germination has been described as a strategy to improve the
nutritional properties of cereals. This process, among others, allows
increase of phenolic compound content and antioxidant capacity
(Alvarez-Jubete et al., 2010). However, the effect depends on cereal
type, crop variety as well as of germination conditions (Gallegos-
Fig. 1. Total antioxidant capacity expressed as mmol of trolox per kilogram dry weight
in dormant (DC) and germinated (GC) during 48 h, 72 h or 96 h canihua seeds. Values
are expressed as the mean  standard deviation, n 3; p < 0.01 when compared to
DC, **p < 0.01 and ***p < 0.001 when compared to the other GC groups as shown in
graph.
Infante et al., 2010). In this study the optimal time germination
was established in order to increase antioxidant capacity of cani-
hua. TAC of canihua was found enhanced at 72 h of germination,
reaching a value of 100.94  5.94 mmol trolox/kg dry weight
(Fig. 1), but not at lower or higher germination times. This
improvement was even comparable to buckwheat (Fagopyrum
esculentum Mench), reported as the best cereal from the view-
point of antioxidant capacity (100e120 mmol trolox/kg dry weight)
(Penarrieta et al., 2008). Germination time to reach an increase in
TAC depends on the type of cereal. For instance, in amaranth, the
germination during 96 h was able to increase the antioxidant
activity measured in acid methanolic/acetonic extracts, but no
change was observed in quinoa (Chenopodium quinoa) at this
germination time (Pasko et al., 2009). On the other hand, meth-
anolic extracts from oat germinated during 24 h already increase
twofold the antioxidant activity against DPPH (Xu et al., 2009). It is
clear that optimal conditions of germination to enhance antioxi-
dant activity depend on the cereal/pseudo-cereal, but the under-
lying mechanisms that control these changes remain unclear.
3.2. Extractable and non-extractable phenolic compounds,
avonoids and avan-3-ols
The increase of cereal antioxidant capacity induced by germi-
nation has been associated with enhancements in phenolic
compound content, which can proceed from either biosynthesis de
novo or release of these compounds by induced enzymatic hydro-
lysis. In order to explain the mechanism through which the TAC of
canihua was increased at 72 h of germination (Fig. 1), in the present
study in addition to extractable phenolic compounds, the non-
extractable fraction was also measured in all samples as
a strategy for establishing the underlying mechanism. No change in
non-extractable fraction will reect that the observed increase
would possibly be associated with de novo biosynthesis induced by
germination.
Enhancements in extractable phenolic compound content were
observed in germinated canihua compared to dormant control,
reaching its maximum value at 72 h of germination. In agreement
with a previous report on oat (A. sativa) (Tian et al., 2010), TAC of
canihua observed at 72 h of germination is likely associated with
extractable phenolic compound content. The changes in extractable
phenolic compound content induced by germination are strongly
dependent on cereal type, variety and processing conditions and
the solvent used for extraction (Alvarez-Jubete et al., 2010;
Gallegos-Infante et al., 2010). A decrease to nearly one-fth and
one-third in extractable fraction was observed in acid methanolic
plus acetonic extracts, similar to that used in the present study,
from quinoa (C. quinoa) and amaranth (Amarantus cruentus Aztek
and Rawa varieties) after 96 h of germination at 20  2  C,
respectively (Pasko et al., 2009). An increase in this fraction, two
and fourfold, in quinoa (C. quinoa) and amaranth (Amarantus cau-
datus) germinated at 10  C during 98 h and 82 h, respectively has
been reported (Alvarez-Jubete et al., 2010).
There was not any difference in non-extractable phenolic
compound content between dormant and germinated canihua
(Fig. 2A). Similar data have been reported in oat after 24 h and 36 h
of germination, where an increase in extractable fraction without
changes in non-extractable fraction was observed (Xu et al., 2009).
These results might reect that germination induced biosynthesis
of phenolic compounds de novo in canihua. Non-extractable frac-
tions represented around twofold extractable fractions in accor-
dance with those reported for cereal (Arranz et al., 2010).
Finally, non-germinated canihua showed a higher total phenolic
compound content (extractable plus non-extractable, near
1000 mg GAE/100 g dry weight) compared to those previously
 F. Abderrahim et al. / Journal of Cereal Science 56 (2012) 410e417
Fig. 2. Content of total phenolic compounds (A), avonoids (B) and avan-3-ols(C) in
canihua seeds, dormant (DC) or germinated (GC) during 48 h, 72 h or 96 h. GAE, gallic
acid equivalents; QE, quercetin equivalents and CE, catechin equivalents. Values are
expressed as the mean  standard deviation, n 3; p < 0.05, p < 0.01
and p < 0.001 when compared to DC; *p < 0.05, **p < 0.01 and ***p < 0.001 when
compared to the other GC groups as shown in the graph.
reported (413 mg GAE/100 g dry weight) (Penarrieta et al., 2008),
given that a large number of phenolic compounds in this pseudo-
cereal are non-extractable (Arranz et al., 2010; Chandrasekara and
Shahidi, 2010).
Extractable avonoids content was increased in germinated
canihua at 96 h of germination (Fig. 2B). However, it was not related
with an improvement of total phenolic compound content and
TAC value observed (Figs. 2A and 1). In cereals/pseudo-cereals,
413
germination is able to enhance extractable individual avonoids
(Pradeep and Guha, 2010). So, quercetin, kaempferol, catechin, and
apigenin were slightly increased in germinated little millet
(Panicum sumatrense) (Pradeep and Guha, 2010). A moderate
improvement in quercetin and kaempferol glycosides content in
germinated quinoa has been reported, as well as a considerable
enhancement in luteolin and apigenin glycosides level in buck-
wheat (F. esculentum Mench) germinated (Alvarez-Jubete et al.,
2010). In addition, germination during 8 days signicantly
increased rutin and quercetin content in germinated buckwheat
(F. esculentum Mench) (Lin et al., 2008). In all previous reports,
a parallel increase in both phenolic compounds and scavenging
capacity toward free radicals was also described. The fact that direct
total antioxidant capacity of germinated canihua at 96 h of
germination does not match with extractable avonoids, suggests
that other antioxidant compounds, among them phenolic
compounds, could be induced by germination of canihua, as has
previously been reported for other cereals/pseudo-cereals
(Alvarez-Jubete et al., 2010; Pasko et al., 2009; Sharma and Gujral,
2010).
The present study demonstrates the possibility to assess non-
extractable avonoids fraction by assay previously used for
tissue localization of phenolic compounds (Hutzler et al., 1998).
This novel direct laser scanning confocal microscopy technique
with 2-aminoethyl diphenylborinate as specic uorescent probe
allows detection of these compounds in non-germinated and
germinated canihua (Fig. 3B) without previous solvent extraction.
Acid hydrolysis for 10 h at 95  C, used to release non-extractable
phenolic compounds, degraded and therefore it was not possible
to quantify avonoids in any food samples. Non-extractable avo-
noids were relatively increased in germinated canihua at 72 h of
germination (Fig. 3A), which is an unusual nding since a decrease
rather than an increase in macromolecule-bound avonoids would
be expected. It could be a particular in vivo mechanism of defence
against well-known oxidative stress-induced germination in cani-
hua (for full discussion see below).
Although catechin and catechin gallate have been found in ten
samples of canihua (Penarrieta et al., 2008), extractable avan-3-
ols content of canihua was not modied by the germination
contrarily to what was obtained in buckwheat (F. esculentum
Mench), in which germination induced an increase in catechin
content from 40.2  3.5 to 68.2  0.2 mmol/100 g dry weight
(Alvarez-Jubete et al., 2010). Likewise to that obtained for non-
extractable avonoids, non-extractable avan-3-ol compound
was increased at 96 h of germination (Fig. 2C); however, how and
why these compounds are produced in vivo still remained unclear.
In addition, for both dormant and germinated canihua, the amount
of avan-3-ols was around vefold lower in the extractable fraction
compared to non-extractable fraction (Fig. 2C), conrming that in
this Andean pseudo-cereal the non-extractable fraction signi-
cantly contributes to its high antioxidant capacity.
3.3. Reducing and free sugar content
An increase in activity of enzymes involved in the induced
hydrolysis of carbohydrates has been reported, which led to
conversion of these macromolecules to compounds with lower
molecular weight such as sugars and/or oligosaccharides (Nirmala
et al., 2000). In agreement with the above, reducing sugar and free
sugar contents increased considerably in germinated canihua,
reaching maximum levels at 72 h of germination (6.3  0.3 and
16.2  0.4 g glucose/100 g dry weight, respectively; Fig. 4) in
parallel with the improvement in direct total antioxidant capacity
(Fig. 1). However, both reducing and free sugars at 96 h of germi-
nation were decreased in comparison to the value at 72 h of
414 F. Abderrahim et al. / Journal of Cereal Science 56 (2012) 410e417
Fig. 3. Semi-quantitative analysis (A) and representative images (B) of non-extractable
avonoid content estimated from confocal microscopy images from canihua seeds
dormant (DC) or germinated (GC) during 48 h, 72 h or 96 h and incubated with the
uorescent probe 2-aminoethly diphenylborinate. A Leica TCS SP2 confocal microscope
was used to capture serial 1 mm thick images at 488 nm excitation and 497e547 nm
emission wavelengths. Values are expressed as the mean  standard deviation,
n 3; p < 0.001 when compared to DC; ***p < 0.001 when compared to the other
GC groups as shown in graph.
germination. These patterns are compatible with the increase of
amylase and glycosidase activities described in early degradation of
starch (Saman et al., 2008). One rational explanation is that sugars
released from carbohydrates by enzymatic hydrolysis during
germination of canihua might be used as a substrate in other
metabolic pathways that could be induced by germination, as
previously reported in rice (Oryza sativa) (Saman et al., 2008). For
instance, glucose can be used as a substrate in biosynthesis of
secondary metabolites such as avonoids in yellow lupine (Lupinus
luteus L. cv. Polo) (Morkunas et al., 2011).
Fig. 4. Reducing (A) and free (B) sugar content in dormant (DC) and germinated (GC)
during 48 h, 72 h or 96 h canihua seeds. Values are expressed as the mean  standard
deviation, n 3; p < 0.001 when compared to DC; *p < 0.05 and ***p < 0.001
when compared to the other GC groups as shown in graph.
3.4. Maillard reaction products and oxidative stress markers
Despite reducing and free sugars being signicantly increased in
germinated canihua, the protein-bound carbonyls were only
markedly elevated at 72 h (Fig. 5A). Incubation of bovine albumin
serum with high glucose concentration (200 mM) during 5 days at
37  C can greatly increase protein-bound carbonyls nearly fourfold
relative to the control without sugar (Liggins and Furth, 1997),
although glucose glycation activity in vivo has been found to be low
(Negre-Salvayre et al., 2009). Therefore, an increase in reducing
sugars, which might be associated with glucose content in germi-
nated canihua, does not necessarily match the protein-bound
carbonyl levels (Figs. 4 and 5A). Protein-bound carbonyl has been
identied as an intermediate product of the Maillard reaction,
 F. Abderrahim et al. / Journal of Cereal Science 56 (2012) 410e417 415

Fig. 5. Protein-bound carbonyls (A), FIC value (B), FAST index (C) and AGEs (D) content in dormant (DC) and germinated (GC) during 48 h, 72 h or 96 h canihua seeds. Values are
expressed as the mean  standard deviation, n 3; p < 0.01 and p < 0.001 when compared to DC; *p < 0.05 and ***p < 0.001 when compared to the other GC groups as
shown in graph.

generated in the second step. Despite protein-bound products
associated with glycoxidation or sugar auto-oxidation reactions
(Liggins and Furth, 1997) it remains unclear whether they might be
directly inducing the biosynthesis of phenolic compounds and
consequently increasing direct TAC in germinated canihua as
a defence mechanism against an oxidative stress condition.
It is well known that protein-bound carbonyls are precursors of
both uorescent and non-uorescent AGEs produced in the later
stage of the Maillard reactions (Liggins and Furth, 1997). In the case
of cereals, the FIC value has been dened as an index of interme-
diate Maillard reaction products (Michalska et al., 2008). In agree-
ment with the above protein-bound carbonyl levels, both FIC and
FAST index reached its maximum in germinated canihua at 72 h of
germination (Fig. 5B and C). Despite the FAST index being associ-
ated with advanced Maillard reaction product (Michalska et al.,
2008), it changed at 48 h and 96 h of germination although the
FIC, dened as intermediate products of Maillard, was not changed
(Fig. 5C). It likely can be explained by the fact that germination
favors the formation of advanced Maillard reaction products in
canihua from the intermediate precursor. Thus, the conversion of
uorescent product to AGE/pigments has been previously
described (Matiacevich et al., 2005). Finally, germination of canihua
was not able to increase the brown pigment product (data not
shown). It has been observed that the transformation of uorescent
product to brown pigments is a limiting step of the Maillard
reaction at the temperature usually employed in cereal germination
(Matiacevich et al., 2005).
In vitro AGEs formation can be assessed by FAST index and FIC
values (Wang et al., 2011). In accordance with FAST index value,
canihua AGEs content was only signicantly increased at 72 h of
germination (Fig. 5D) in parallel with TAC and non-extractable
avonoid content (Figs. 1 and 3). This observed association
suggests that AGE-induced germination might regulate pathways
related to biosynthesis of antioxidant compounds, increasing the
antioxidant activity in germinated grain as a response to stress
conditions. Thus, free radicals can be directly or indirectly gener-
ated during the early stage of glycation from Schiff bases and gly-
cated protein (Wu et al., 2011). It is likely that the increase in
phenolic compounds and non-extractable avonoids at 72 h of
germination occurs as a response mechanism against the oxidative
stress that might occur in canihua.
Advanced oxidation protein products (AOPPs) and 3-
nitrotyrosine (NT), well-established oxidative stress markers,
were signicantly increased by germination in comparison to
dormant canihua. AOPPs and NT (bands of 25e37, 37 and 50 kDa)
levels were signicantly larger at 48 h, followed by a decrease at
72 h and a further increase at 96 h of germination (Fig. 6A and B).
50 kDa proteins at 72 h of germination only showed a signicant
reduction relative to canihua germinated for 48 h and 96 h.
Oxidative stress markers exhibited an inverse relationship with
416 F. Abderrahim et al. / Journal of Cereal Science 56 (2012) 410e417
Fig. 6. Advanced oxidation protein products (AOPPs) (A) and 3-nitrotyrosine (NT)
content (B) in dormant (DC) and germinated (GC) during 48 h, 72 h or 96 h canihua
seeds. Values are expressed as the mean  standard deviation, n 3; p < 0.05
and p < 0.001 when compared to DC; *p < 0.05 and **p < 0.01 when compared to
the other GC groups as shown in graph.
AGE, i.e. they were signicantly decreased at 72 h. This reduction is
the result of an enhancement of canihua antioxidants, non-
extractable avonoids among others, produced during germina-
tion. Under natural conditions, release of dormancy seed generally
could occur during imbibitions at low temperature, which was
associated with a marked enhancement in the level of hydrogen
peroxide and superoxide anion (Oracz et al., 2007), which are
produced by NADPH oxidase (Kranner et al., 2010). This enzyme
in vivo can be inhibited by avonoids (Wu et al., 2009). The fact that
TAC did not show an increase at 48 h and 96 h of germination,
which are associated with the highest oxidative stress marker
levels, established that Maillard reactions, among them AGEs, could
be implicated in induction of biosynthesis of antioxidant
compounds in germinated canihua.
4. Conclusions
Germination is able to enhance direct total antioxidant capacity
in canihua, which includes both extractable and non-extractable
antioxidant compounds. Optimal germination time was estab-
lished at 72 h, which was associated with concomitant increase of
extractable total phenolic compounds and non-extractable avo-
noids. The improvement in total antioxidant capacity observed in
germinated caihua was matched with increased Maillard reaction
products, such as protein-bound carbonyl, FIC, FAST index and
advanced glycated end products (AGEs). It might represent
a common phenomenon in cereal germination; therefore, toxic
effect of AGEs-related to germination deserves further attention. An
inverse relationship between total antioxidant capacity and
oxidative stress parameter advanced oxidation protein products
and 3-nitrotyrosine was observed in germinated canihua.
Conict of interest
None declared.
Acknowledgments
Source of funding: This work was supported by MICINN, Spain
(FEM2009-13434-C02).
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jcs.2012.04.013.
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