JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x

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Sunlight exposure increases the phenolic content in

postharvested white grapes. An evaluation of their
antioxidant activity in Saccharomyces cerevisiae

José Peinadoa,b,*, Nieves López de Lermac, Angela Peralbo-Molinab,d,

Feliciano Priego-Capoteb,d, Cristina de Castroe, Brian McDonagha,f
a
Department of Biochemistry and Molecular Biology, University of Córdoba, E-14071 Córdoba, Spain
b
Institute of Biomedical Research Maimónides (IMIBIC), ReinaSofı́a Hospital, University of Córdoba, E-14071 Córdoba, Spain
c
Department of Agricultural Chemistry, University of Córdoba, E-14071 Córdoba, Spain
d
Department of Analytical Chemistry, University of Córdoba, E-14071 Córdoba, Spain
e
Department of Molecular Biology, University of León, E-24071 León, Spain
f
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease (IACD), University of Liverpool, L69 3GA, United Kingdom

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

Article history: Sunlight exposure is a traditional way to dry postharvested white Pedro Ximenez grapes,
Received 10 February 2013 and the dried grapes are the basis of a sweet fortified wine elaborated in the South of Spain.
Received in revised form In this paper, we have studied the effect of seven days of exposure of grapes to sunlight.
13 June 2013 The phenolic content in the skins and flesh has been determined. The transient induction
Accepted 20 June 2013 of phenolics has been detected and identified in skins by HPLC and MS/MS. Maximum
Available online xxxx induction was after 2 days exposure with an increase in the levels of quercetin-3-O-gluco-
side, engeletin (dihydrokaempferol-3-O-rhamnoside) and astilbin (taxifolin 3-O-rhamno-
Keywords: side). We have evaluated the antioxidant and protective effects of the phenolic extracts
Phenolics of these grapes in Saccharomyces cerevisiae after exposure to H2O2. Phenolic extracts reduced
Sundried grapes the basal intracellular level of peroxides in a concentration dependent manner. There was a
Protein oxidation
corresponding significant reduction in carbonylated proteins in treated and control cells
Cell survival
and increased survival of yeast cells exposed to H2O2. Our results indicate that sundried
grapes display a high antioxidant capacity resulting in a decrease in the oxidative cellular
environment.
 2013 Elsevier Ltd. All rights reserved.

1. Introduction such as cancer, cardiovascular disease, atherosclerosis, and

type 2 diabetes (Spormann et al., 2008). A common phenom-
Epidemiologic studies provide convincing evidence that con- enon in these pathologies is an imbalance between the rates
sumption of diets rich in fruits and vegetables is associated of production and elimination of reactive oxygen species
with the prevention or delay of chronic degenerative diseases (ROS) (Ulrich-Merzenich, Zeitler, Vetter, & Kraft, 2009).

* Corresponding author at: Department of Biochemistry and Molecular Biology, University of Córdoba, E-14071 Córdoba, Spain. Tel.: +34
957218317; fax: +34 957218592.
E-mail address: bb1pepej@uco.es (J. Peinado).
Abbreviations: BSA, bovine serum albumin; DCFDA, 2 0 7 0 -dichlorofluorescein diacetate; EDTA, ethylenediaminetetraacetic acid; PEDG,
phenolics extracts from dried grapes; PMSF, phenylmethanesulfonyl fluoride
1756-4646/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.06.007

Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
2 JOURNAL OF FUNCTIONAL FOODS
Increased uptake of food-based antioxidants is a promising
alternative measure to reduce oxidative cell damage and the
stress response (Spormann et al., 2008; Xia, Deng, Guo, & Li,
2010). In addition to vitamins, many fruits and vegetables
are rich in polyphenols, acting as powerful antioxidants
in vitro and in vivo that can scavenge diverse ROS, inhibit their
formation and the oxidation of biomolecules (Dani et al.,
2008a; Frankel & Finley, 2008). Grapes are a rich source of phe-
nolic compounds, and intake of grape products such as juice,
wine and grape diet supplements have recognized health
benefits (Williamson & Carughi, 2010; Xia et al., 2010). Raisins,
sultanas and currants, although with differences in the vari-
ety or color of grape used, refer to dried grapes. Dried grapes
are a favorite dried fruit throughout the world because of
their high nutritional value, and are obtained by dehydrating
grapes, usually with sun exposure or tunnel drying (Bennett
et al., 2011; Chiou et al., 2007; Meng et al., 2010; Peinado, López
de Lerma, Moreno, & Peinado, 2009). Typically, antioxidant
capacity is higher in raisins on a per-weight basis than fresh
grapes because of the concentration of antioxidants during
dehydration (Williamson & Carughi, 2010). The penolic com-
position of these dried grapes is variable and depends on
the grape variety. However, 3,4-dihydroxybenzoic or proto-
catechuic acid, caffeic acid, quercetin and catechin are the
most commonly detected polyphenols, independent of the
grape type (Chiou et al., 2007; Meng et al., 2010; Peinado, Ló-
pez de Lerma, & Peinado, 2010; Peinado et al., 2009; Serratosa,
López-Toledano, Mérida, & Medina, 2008). The induction of
resveratrol has been described in skins of red and white
grapes after UV-C irradiation pulses (Cantos, Espı́n, & Tom-
ás-Barberán, 2002). Additionally, dehydration of grapes and
temperature are factors that induce the synthesis of resvera-
trol and other phenolics. Aleatico postharvested grapes kept
in laboratory tunnels for 10–20 days reached 40% dehydration
between 20 and 30 C. Under these conditions, time and tem-
perature are critical factors in the induction of two represen-
tative phenolics, quercetin and resveratrol. Quercetin was
induced earlier than resveratrol, while the induction of resve-
ratrol was more dependent on temperature, and no increase
was detected at 30 C (Antelmi, Bellincontro, Mencarelli, Nic-
oletti, & Corradini, 2010; Mencarelli et al., 2010).
Pedro Ximenez (PX) wines are fortified sweet wines pre-
pared in the Montilla–Moriles winemaking region (southern
Spain), from white dried Pedro Ximenez grapes. The har-
vested bunches of grapes are exposed to sunlight for up to
7 days, and they lose 40% weight by dehydration, and the con-
centration of sugars in these grapes can reach up to 400 g/L.
The daily temperatures range from 15 to 20 C in the night
and up to 40 C during the day. An alternative to the tradi-
tional sun-drying is the chamber-drying at controlled temper-
ature of 40 or 50 C, shortening the drying-time to only 3 days
(Serratosa et al., 2008a). In comparison, raisins or sultanas
can be exposed to sunlight for 2–3 weeks (Williamson & Caru-
ghi, 2010).
The juice of dried grapes Pedro Ximenez and their pheno-
lic extracts were highly efficient in the inhibition of the in vitro
oxidation of DNA, sugars and lipids (Peinado et al., 2010). Juice
from dried grapes was also effective against the glycation of
proteins, an assay used to evaluate in vitro the complications
of diabetes (Cervantes-Laurean et al., 2006). The beneficial
Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
x x x ( 2 0 1 3 ) x x x –x x x
effects of sultanas have also been studied, the polar methanol
extracts from sultanas exerted antiradical activity in periphe-
ral blood mononuclear cells and cancer preventive efficacy in
gastric cancer cell growth (Kaliora, Kountouri, & Karathanos,
2009; Kaliora et al., 2008). Intervention studies with raisins
have shown various effects, modulation of glucose uptake,
decrease in cholesterol levels and effects on satiety-related
hormones. The effects reported were from raisins as a whole
food, and so, any effects may or may not be due to the poly-
phenol content (Williamson & Carughi, 2010).
In this study we have used Saccharomyces cerevisiae, a use-
ful model to screen for natural antioxidants in vivo (Dani et al.,
2008a). S. cerevisiae has similar antioxidant responses to
mammals and 30% of known genes involved in human dis-
ease have yeast orthologues, thus functional homologues
(Mager & Winderickx, 2005). Furthermore, it has been used
as a eukaryotic model to study the molecular mechanisms
associated with cell ageing and defense mechanisms after
exposure to oxidative stress (McDonagh, Ogueta, Lasarte,
Padilla, & Bárcena, 2009). In addition, using this microorgan-
ism to study antioxidant capacity allows neglecting differ-
ences in the bioavailability of polyphenols (Baroni, Di Paola
Naranjo, Garcı́a-Ferreira, Otaiza, & Wunderlin, 2012). Assays
measuring intracellular peroxide levels, protein carbonylation
and yeast survival have been carried out. Previously, it was
demonstrated that yeast cells exposed to millimolar H2O2 dis-
played a marked cell survival ratio and an increase in protein
carbonylation could be clearly ascertained (McDonagh et al.,
2009). The results presented in this article suggest that, both
the juice of dried grapes and phenolics extracted from dried
grapes (PEDG) effectively protect yeast cells from oxidative
stress.
The aim of this work was to determine the changes in the
content of phenolics in skins and flesh of postharvested Pedro
Ximenez (PX) grapes dried by sunlight. In addition we sought
to evaluate the in vivo the antioxidant activity of PX dried
grapes using S. cerevisiae.
2. Materials and methods
2.1. Preparation of samples
At least 10 different points were selected to prepare a com-
posite sample of 20 kg of grapes, and random samples of
white Pedro Ximenez grapes from the Montilla–Moriles wine-
making region were collected at 0, 1, 2, 4 and 7 days of drying
by direct exposure to sunlight in the pasera (drying site).
Bunches were turned every day. After collection of bunches,
they were kept for 24 h at 23 C in the dark and processed
for further studies.
2.2. Quantification of phenolics in skins
Grapes were peeled, skins and flesh were ground in liquid
nitrogen to homogenize the samples. Particle size distribution
was determined by sieving into three particle classes: (4–
2 mm), (2–1 mm) and (1–0.25 mm), and samples were stored
at 20 C until analyzed. Skin ranged from 11% of the total
fresh weight of grape berry (day 0) to 26% (day 7). Samples
 JOURNAL OF FUNCTIONAL FOODS
(1 g of skin or 2 g of flesh) were homogenized with 4 mL of
HPLC grade methanol:formic acid (97:3, v/v) according to
Selma et al. (2008). 20 lL of the clear filtered extract from
skins or flesh grapes were analyzed by HPLC (Beckman, Fuller-
ton CA, USA) equipped with a pump (125 Solvent module) and
a 168 photodiode array UV/vis detector. Separations were
achieved on a reversed-phase Supelcosil RP-18 column (Tek-
nokroma, Barcelona, Spain) (250 · 4 mm, 5 lm particle size).
The mobile phase and gradient conditions were as described
by Selma et al., 2008, except phase A was 0.1% formic acid.
The gradient method was as follows: 68% A for 30 min, from
68% to 60% A in 10 min, from 60% to 5% in 10 min, from 5%
to 98% in 5 min and kept for 5 min. The concentration of
flavonols was quantified at 360 nm using quercetin-3-O-
glucoside as a standard, and expressed as micrograms of
quercetin-3-O-glucoside/kg of grape. All samples were ana-
lyzed in triplicate.
2.3. LC–QqTOF MS/MS analysis and data processing
An injection volume of 5 lL and a flow rate of 1 mL/min were
used. The mobile phases were 0.1% (v/v) formic acid aqueous
solution (phase A) and pure acetonitrile (phase B). Separation
of the analytes was performed on a Supelcosil RP-18 column
(Teknokroma, Barcelona, Spain) (25 · 0.4 cm, 5 lm particle
size). The dual ESI source operated in negative ionization
mode using the following conditions: nebulizer gas at 35 psi,
drying gas flow rate and temperature at 10 L/min and
325 C, respectively. The capillary voltage was set at 3500 V,
while the fragmentor, skimmer and octapole voltages fixed
at 175, 65 and 750 V, respectively.
The data were acquired in centroid mode in the extended
dynamic range (2 GHz). Full scan was carried out at 1 spectrum
per s within the m/z range 100–1700 with subsequent activation
of the three most intense precursor ions (only allowed charge,
single or double) by MS/MS using a collision energy of 10, 20 and
40 eV at 1 spectrum per s within the m/z range 100–1700. An ac-
tive exclusion window was programmed after one spectrum
and released after 0.75 min to avoid repetitive fragmentation
of the most intense precursor ions and, in this way, increase
the detection coverage. Before the experiments, the instru-
ment reported mass detection resolution of 25,000 FWHM (Full
Width at Half Maximum) at m/z 112.985587, and 45,000 FWHM
at m/z 966.000725. To assure the desired mass accuracy of re-
corded ions, continuous internal calibration was performed
during analyses with the use of signals at m/z 119.0362 (proton
abstracted purine) and m/z 966.0007-formate adduct of
hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine.
MassHunter Workstation Data acquisition software (Agi-
lent Technologies, Santa Clara, CA, USA) was used to control
the instrument. Data were processed using MassHunter Qual-
itative Analysis software (Agilent Technologies, Santa Clara,
CA, USA). Extraction of unknown molecular features from
raw data was carried out by the Molecular Feature Extraction
(MFE) algorithm in MassHunter Qualitative analysis software.
The feature extraction algorithm took all ions into account
exceeding 500 counts with a charge state equal to or above
one and a feature had to be composed of two or more
ions to be valid (e.g. two ions in the isotope cluster). The
theoretical formula adjusted to the corresponding isotopic
Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
x x x ( 2 0 1 3 ) x x x –x x x 3
distribution of molecular features was generated with the
Molecular Formula Generation software (Agilent Technolo-
gies, Santa Clara, CA, USA). Analyses were processed using
MassHunter Qualitative software and compound identifica-
tion was performed using the METLIN Database (http://met-
lin.scripps.edu) and MetFrag (http://msbi.ipb-halle.de/
MetFrag/). The allowed negative precursor ions for identifica-
tion were formate adducts and deprotonated ions. Dehydra-
tion neutral losses were also allowed.
2.4. Quantification of phenolics in juice
Destemmed grapes were pressed and the resulting juice was
clarified by centrifugation at 15,000·g for 10 min, and pre-
served at 20 C. The HPLC analyses were performed using
a Supelcosil RP-18 column (250 · 4 mm, 5 lm particle size),
50 mM tartaric acid (solvent A) and HPLC grade methanol (sol-
vent B) at a flow rate of 0.8 mL min1, as previously reported
(Peinado et al., 2010). Phenolics were identified by comparison
of the retention time and spectra of standards and by co-
injection of samples containing a standard. trans-Caftaric
acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epi-
catechin, quercetin-3-O-glucoside and rutin were from Sig-
ma–Aldrich (Barcelona, Spain), and procyanidins B1 and B2
from Extrasynthese (Genay Cedex, France). cis-Caftaric acid
was obtained by UV exposure of trans-caftaric acid. All sam-
ples were analyzed in triplicate.
2.5. Assays in vivo of the antioxidant activity
For the antioxidant effects, grape juice obtained after 7 days
of drying was used. PEDG was extracted from juice by using
C18 cartridges (Waters, Milford, MA, USA). In brief, after acti-
vation of cartridge with methanol and equilibrated with water
a pH 2, juice at pH 2 was washed with water at pH 2 to remove
sugars; phenolics were eluted with methanol and concen-
trated by vacuum. Concentration of phenolics in juice was
determined by absorbance at 280 nm and enzymatically
(Stevanato, Fabris, & Momo, 2004).
The determination of the in vivo antioxidant activity of the
PEDG was performed using S. cerevisiae MML44 (Rodriguez-
Manzaneque, Ros, Cabiscol, Sorribas, & Herrero, 1999). Cells
were grown in liquid YPD medium, using an orbital shaker
at 30 C and 180 rpm. Yeast cells at the exponential phase
(Abs600: 1.5) were transferred to fresh medium (Abs600: 0.2)
and experiments were initiated when Abs600 reached 0.75
units.
2.6. Intracellular oxidation
The oxidant-sensitive probe 2 0 7 0 -dichlorofluorescein diace-
tate (DCFDA) (Invitrogen, Paisley, UK) was used to measure
intracellular oxidation (Dani et al., 2008a). A fresh 5 mM stock
solution of DCFDA dissolved in ethanol was added to the cul-
ture (final concentration 10 lM) and incubated at 30 C for
20 min to allow uptake of the probe. The culture was divided
according to treatment (with or without polyphenols) and
incubated for 45 min. H2O2 was added as indicated and after
45 min cells were harvested by centrifugation and washed
twice with water. The pellets were resuspended in 2 mL of
4 JOURNAL OF FUNCTIONAL FOODS
water, and 200 lL of each sample (containing 10 mg of cells)
and in triplicate were analyzed on a 96 well plate and the fluo-
rescence measured using a Synergy HT (Bio-Tek, Winooski,
VT, USA), excitation 485 nm and emission 520 nm. The fluo-
rescence in cells that had not been exposed to dye or any
treatment was used as a control. All assays were carried out
in quadruplicate.
2.7. Determination of carbonylated proteins
Sixty mililitres of yeast cells at the exponential phase was
pre-incubated or not with 10 lg of phenolics/mL from dried
grapes. Cells were then treated with 4 mM H2O2 for 1 h and
harvested by centrifugation at 7500·g, the pellet was
washed twice with H2O and redissolved in lysis buffer (8 M
urea, 50 mM Tris–HCl, 2 mM (ethylenediaminetetraacetic
acid) EDTA, 0.1% Triton X-100 and 2 mM (phen-
ylmethanesulfonyl fluoride) PMSF, pH 8.0) containing
washed sea sand. Cell extracts were vortexed continually
at 4 C for 30 min and samples were centrifuged at
15,000·g for 10 min at 4 C to obtain protein extract (McDon-
agh et al., 2009). Protein concentrations were measured
using Bradford reagent (BioRad, Hercules, CA, USA) with
BSA used as a standard.
Proteins were derivatised for carbonylation assay as de-
scribed previously (McDonagh & Sheehan, 2006), with 20 lg
of protein loaded in each lane. Following 1DE proteins were
transferred to nitrocellulose membranes and stained with
Ponceau S (0.2%) in 5% acetic acid to check for equivalent pro-
tein loading and transfer efficiency. Membranes were blocked
and probed with anti-DNP (Invitrogen, Paisley, UK) overnight
(dilution 1/1500), washed, incubated with horseradish perox-
idase conjugated goat anti-rabbit (dilution 1/3000) (Dako, Den-
mark) and visualized by enhanced chemiluminescence.
Chemiluminescent signal was detected using a LAS-3000
camera (Fujifilm, Tokyo, Japan) and images analyzed using
Multi Gauge V3.0 (Fujifilm, Tokyo, Japan). All assays were car-
ried out in duplicate.
2.8. Spotting analysis
Exponentially grown cells were incubated for 1 h or 4 h incu-
bated with either 10 or 30 lg/mL of PEDG. For spot analysis,
cells were 10-fold diluted serially and spotted onto solid
YPD medium containing 4 mM H2O2. Plates were incubated
at 30 C for 72 h. Assays were carried out in triplicate.
2.9. Statisitical analysis
Homogeneous group analyses (HGA) were made to identify
differences among the phenolic concentration and intra-
cellular oxidation of yeast exposed to different phenolic
concentration and/or to hydrogen peroxide. Differences in
the intensity of bands of carbonylated proteins were also
analyzed by HGA. All analysis were carried out by
triplicate.
All these analyses were made by means of the statistical
package Statgraphics Centurion XV.II, StatPoint Technologies,
Inc. (Warrenton, VA, USA).
Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
x x x ( 2 0 1 3 ) x x x –x x x
3. Results and discussion
3.1. Phenolics content in sun dried grapes
UV light induces the synthesis of phenolics, included resvera-
trol, in skins of postharvested grapes exposed to UV-C for
2 min and these grapes have been proposed as a nutraceutical
food (Cantos et al., 2002). Additionally, dehydration of post-
harvested shadded grapes also induces the synthesis of phen-
olics (Bonghi et al., 2012; Mencarelli et al., 2010), and in both
cases optimal temperature is between 20 and 30 C. Pedro
Ximenez grapes are dehydrated by exposure to sunlight and
the diurnal temperature can reach 40 C, with variations be-
tween day and night about 20 C. In this work, we have used
similar HPLC separation conditions to the method employed
to detect phenolics from skins, including resveratrol, in
grapes exposed to UV pulses (Selma et al., 2008). The phenolic
composition of phenolics in skins from sundried grapes was
evaluated by HPLC coupled with a diode array detector and
an ion trap MS spectrometer. The identification of the com-
pounds was revealed by comparison with standards as caftar-
ic acid (1) and catechin (2), and by MS/MS and assigned as
quercetin-3-O-glucoside (3), engeletin (4) and astilbin (5)
(Fig. 1 and Table 1).
Compounds 3, 4 and 5 have been identified in the pomace
(Lu & Foo, 1999) and skins of white grapes, and also in wines
(Trousdale & Singleton, 1983). Compared to the control (day 0)
the concentration of compounds 3–5 displayed a maximum at
2 days and declined until 7 days (Table 1). A similar pattern for
quercetin has been described in skins of dehydrated grapes,
detecting a high increase in quercetin when the weight loss
of grapes was 10% and then, to decrease when reached a
30% (Bonghi et al., 2012). Additionally, the effect of decompo-
sition of phenolics by UV light cannot be discarded (Galeano-
Dı́az, Durán-Merás, & Airado-Rodrı́guez, 2007). The absence of
resveratrol was notable, although temperature can be a nega-
tive factor for its induction; in fact, Antelmi et al., 2010
showed that resveratrol appeared in dehydrated grapes at
least two days later than quercetin when the temperature
was 30 C. A similar result has been recently described in
skins of red grapes exposed to sunlight (López de Lerma, Pei-
nado, & Peinado, in press), as the concentration of phenolics
in skins of red grapes exposed to sunlight clearly decreased
two days after the maximum. The HPLC separation also de-
tected a decrease in the levels of catechin and t-caftaric acid
levels (Fig. 1).
Coincident with these changes in grape skins, the concen-
tration of phenolics also increased in the flesh of dried
grapes. The phenolic composition of juice of dried grapes is
shown in Table 2, with catechin as the most abundant phe-
nolic determined. In addition, compounds 3–5 detected in
skins were also present in the juice. This may occur by diffu-
sion from the skin to the flesh of grape, since dehydration of
grapes causes a destruction of the cells (Margaris & Ghiaus,
2007). Very similar levels of phenolics in juice were described
by Serratosa, Lopez-Toledano, Medina, and Merida (2008a)
when Pedro Ximenez grapes were dried in the absence of
sunlight by using a chamber at 40 C, and the authors sug-
gested that the increase in the concentration of most of the
 JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x 5

Fig. 1 – HPLC chromatograms of skin extracts of sun dried Pedro Ximenez grapes. A and B: day 0; C: 2 days of drying. Peaks
identified by MS/MS: (1) t-caftaric acid; (2) catechin; (3) quercetin-3-O-glucoside; (4) engeletin; (5) astilbin.

phenolics in the juice could be explained by the concentra-
tion effect due to the loss of water in the grapes. For in-
stance, the levels of the two more abundant phenolics
detected, epicatechin or catechin, were 21.4 or 23.9 mg/L in
the absence of sunlight (Serratosa et al., 2008a), while in
our study we have determined 21.3 or 22.6 mg/L, respectively;
in this paper we report the presence of c-caftaric acid
(formed by UV-isomerization from t-caftaric acid) and of
quercetin-3-O-glucoside, engeletin and astilbin. Finally, the
chamber shortens the drying-time and avoids a potential
fungal infection, but the tasters prefer the musts obtained
from grapes dried by the traditional sunlight method in order
to prepare the sweet wines (Serratosa, Marquez, Lopez-Toled-
ano, & Merida, 2012).

Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
6 JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x

Table 1 – Identification of flavonols in extracts from grape skins by LCQqTOF MS/MS analysis and changes in their
concentrations during the drying.

Compound Retention time Mass m/z Concentration of flavonols of skins during the drying1
Day 0 Day 1 Day 2 Day 4 Day 7
a b c bd
Quercetin-3-O-b-glucoside 20,559 464,0975 463,0897 6.40 ± 0.50 12.11 ± 1.29 23.41 ± 1.48 14.97 ± 1.10 9.76 ± 1.28be
Engeletin 22.845 434,1222 433,1141 1.06 ± 0.12a 1.94 ± 0.15b 3.89 ± 0.28c 2.82 ± 0.29d 2.31 ± 0.20b
Astilbin 23,135 450,1159 449,1088 0.26 ± 0.08a 0.53 ± 0.14b 0.91 ± 0.18c 0.78 ± 0.16b 0.63 ± 0.09b
Different letters indicate significant differences at the 95% confidence level.
1
Concentrations are expressed as micrograms of quercetin-3-O-glucoside equivalents/kg of grape.

Table 2 – Mean values for some phenolic compounds (mg/L) in juice of grapes along the drying process.
Phenolic Day 0 Day 2 Day 7 HGA

t-Caftaric acid 1.10 ± 0.09 1.00 ± 0.10 0.41 ± 0.06 aab

c-Caftaric acid 0.58 ± 0.09 0.68 ± 0.09 3.51 ± 0.24 aab
Caffeic acid 0.29 ± 0.03 0.57 ± 0.09 2.13 ± 0.22 abc
Coumaric acid 0.34 ± 0.05 0.40 ± 0.08 0.77 ± 0.08 aab
Ferulic acid 0.38 ± 0.07 0.49 ± 0.10 1.20 ± 0.13 aab
Procyanidin B1 0.27 ± 0.03 0.39 ± 0.09 1.01 ± 0.10 aab
Procyanidin B2 0.41 ± 0.02 0.44 ± 0.08 1.48 ± 0.20 aab
Catechin 4.21 ± 0.32 6.72 ± 0.30 22.6 ± 1.90 abc
Epicatechin 4.17 ± 0.31 6.40 ± 0.43 21.3 ± 1.36 abc
Quercetin-3-O-glucoside 2.82 ± 0.21 3.66 ± 0.31 4.74 ± 0.43 abc
Engeletin 0.45 ± 0.06 0.59 ± 0.10 0.97 ± 0.18 aab
Astilbin 1.1 ± 0.08 1.92 ± 0.18 3.58 ± 0.42 abc
Rutin 0.16 ± 0.03 0.21 ± 0.06 0.59 ± 0.07 aab
Different letters indicate significant differences at the 95% confidence level.

3.2. Antioxidant activity of sundried grapes
Grapes dried after seven days are used to prepare fortified
sweet wines in the South of Spain by addition of ethanol to
the juice obtained by pressing the dried grapes. We have ex-
tracted the polyphenols from grapes along the drying time
and have studied the biological and antioxidant activity of
the phenolic extracts by exposing yeast cells to an oxidant
insult.
H2O2 is rather inert at low concentrations and has been
considered as a signaling molecule (Gough & Cotter, 2011).
H2O2 at high concentrations provokes deleterous effects on
yeast, such as reduced viability (Dani et al., 2008a), inhibition
of antioxidant enzymes and irreversible oxidation of proteins
(Costa, Amorim, Quintanilha, & Moradas-Ferreira, 2002). In
this paper we have studied the protection against induced
oxidative damage by preadaptation of the yeast cells with a
phenolic fraction. The range of phenolic concentrations used
(0.5–30 lg/mL) was wider than that previously used (Dani
et al., 2008a). Firstly, the possible H2O2 scavenging activity of
the PEDG was analyzed. The decrease in the concentration
of H2O2 at 234 nm (Ruch, Cheng, & Klauning, 1989) was mon-
itored over time, and no H2O2 scavenging activity was de-
tected for at least ninety minutes (maximum time of the
experiments).
Polyphenols exhibit a wide range of biological effects (Xia
et al., 2010) many of them have been attributed to their free
radical scavenging activity. We have studied the protection
in yeast cells by phenolics from dried grapes using DCFDA,
which is susceptible to oxidation by a large number of differ-
ent ROS, particularly H2O2 and hydroxyl radical (He et al.,
2012). This lack of specificity, regarding which ROS can oxi-
dize DCFDA, makes this compound a valuable reagent for
use in the early stages of a pathogenesis investigation. This
assay can be used to screen for an oxidative cellular environ-
ment regardless of which oxygen radical or combination of
ROS are responsible for the cellular conditions (Testa et al.,
2011) and has been recently proposed as a rapid mitochon-
dria-based assay for assessing antioxidant properties of pure
compounds and herb extracts (He et al., 2012).
Yeasts were exposed to H2O2 and the effects of catechin
and quercetin-3-O-glucoside (used as a positive controls),
and PEDG along the drying time were evaluated. Fig. 2A shows
that DCFDA is oxidized when H2O2 is added; however we have
observed that a substantial oxidation of the probe occurs in
the absence of peroxide, suggesting a basal level of oxidation
in yeast cells. In these conditions, using 10 lg/mL of catechin,
quercetin-3-O-glucoside or PEDG at 7 days more efficiently
protected DCFDA from oxidation; although, PEDG at 7 days
protect more efficiently than catechin or quercetin-3-O-gluco-
side. When the yeast cells were exposed to H2O2, all the sam-
ples were protected from oxidation but catechin and PEDG at
7 days displayed the most protective effects (Fig. 2A).
We have studied the protective effect of PEDG at 7 days in a
wide concentration range (0.5–30 lg/mL) in the presence or
the absence of H2O2 (Fig. 2B). The curves are quite similar,
although the protective effect of PEDG was more pronounced
in the absence of H2O2, since with 30 lg/mL there was a

Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
 JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x 7

A 2000
d
1800

1600

Fluorescence intensity
1400
f f
f
1200
e e
1000

800 a
c
600 c
b b
400 b

200

0
FA h in 3G ay ay ay 2O
2
2O
2
2O
2
2O
2
2O
2
2O
2
DC tec in - 0d 2d 7d
Ca e rcet DG DG E DG FA/H hin/H -3G/H 0d
/H
2d
/H
7d
/H
E E C c n G G G
Qu P P P D
Ca
te et i D D D
erc PE PE PE
Qu

B 1800 A
1600 A
Fluorescence intensity

1400
B
1200 B
C C
1000
A
800
B
600 C
400 D
D D
200
0
-5 0 5 10 15 20 25 30 35
Concentration polyphenols (µg/ml)

Fig. 2 – Intracellular oxidation, measured as fluorescence intensity of oxidized dichlorofluorescein (DCFDA). (A) Yeast cells
were incubated with catechin, quercetin-3-O-glucoside or phenolic extract from dried grapes (PEDG) at 0, 2, and 7 days of
drying in the presence or absence of response of 4 mM H2O2. Different letters indicate significant differences among the
treatments. (B) Concentration effect of phenolics in the oxidation of of 2 0 7 0 -dichlorofluorescein diacetate (DCFDA). The results
were expressed as the difference in fluorescence intensity of oxidized dichlorofluorescein between yeasts exposed to or not
to 4 mM H2O2 and in the presence of variable concentrations of phenolics from PEDG at 7 days. Different letters indicate
significant differences due to the phenolic concentration.

decrease of 74% of the initial control value while a 40% de- protective effects of grape juice (Dani et al., 2008b) and cloudy
crease was observed in the presence of H2O2. apple juice in rats (Kujawska, Ignatowicz, Ewertowska, Mar-
Protein carbonylation is an early marker during protein kowski, & Jodynis-Liebert, 2011). In our case, the oxidation
oxidation, as a result of oxidative modifications on arginine, of proteins was studied by exposure of yeast to 4 mM H2O2,
proline, lysine, and histidine residues, and by oxidative cleav- and protection against protein oxidation was assayed by add-
age of the peptide chain (Berlett & Stadtman, 1997). Protein ing PEDG at 7 days one hour prior to the oxidative insult.
oxidation elicited by free radicals may cause protein function Fig. 3A is the Ponceau S staining of the membrane to ensure
disruptions, thereby implicated in diseases, such as Alzhei- equivalent protein loading, and immunodetection of carbony-
mers (Butterfield et al., 2012) or used as a tool for environmen- lated proteins of the corresponding membrane is shown in
tal monitoring (McDonagh & Sheehan, 2006). Fig. 3B. It is normal that in the absence of treatment there is
High concentrations of H2O2 induce specific protein oxida- a basal level of carbonylation (lane 1), which is clearly in-
tion in yeast cells, resulting in inactivation of specific protein creased in the presence of H2O2 (lane 2). Catechin was used
targets, such as glycolytic enzyme glyceraldehyde-3-phos- at a concentration of 10 lg/mL as a positive control of protec-
phate dehydrogenase (Gough & Cotter, 2011). Although, there tion against oxidation and a clear decrease in the oxidation of
are large numbers of potential oxidation sites, oxidation proteins was observed even in the absence or presence of
seemed to be restricted to a small area in most of the proteins H2O2 (lanes 3 and 4, respectively). When PEDG was added at
identified (Mirzaei & Regnier, 2007). There are only a few re- an identical concentration, it was even more effective at pro-
ports about the protection against protein oxidation by tection against oxidation than catechin (lanes 5–6). The inten-
phenolic compounds from grapes and they include, the sity of bands of carbonylated proteins were analyzed and

Please cite this article in press as: Peinado, J. et al., Sunlight exposure increases the phenolic content in postharvested white grapes. An evaluation
of their antioxidant activity in Saccharomyces cerevisiae, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.06.007
8 JOURNAL OF FUNCTIONAL FOODS x x x ( 2 0 1 3 ) x x x –x x x

A Lan
anes 1 2 3 4 5 6
confirmed that PEDG protected the yeast cells more efficiently
against protein carbonylation (Fig. 3C).
Cat
atechi
chin - - + + - - The survival of exponential growing cells to H2O2 was also
examined (Fig. 4). One hour prior to spotting on plates con-
PED
EDG - - - - + +
taining H2O2, cells were preincubated with PEDG at 7 days.
H 2O 2 - + - + - + 10 lg/mL of phenolics was enough to increase the survival
of yeast to H2O2. At 30 lg/mL the survival was clearly en-
hanced and when preincubation time of yeasts with pheno-
lics continued for 4 h, a supplementary increase in the
survival of yeast was observed. Similar results were obtained
when the survival experiment was carried out with grape
juice containing similar concentrations of phenolics (not
shown).
B
4. Conclusion

The results described here support the hypothesis that grape

juice has an immediate and significant antioxidant effect
(Andrade et al., 2011; Falchi et al., 2006; Ludke et al., 2010;
Mullen, Marks, & Crozier, 2007; Park, Park, Kim, & Kang,
2003). In this work we identify the phenolics induced in the
skins of sunlight dried grapes, with a maximum concentra-
tion at 2 days in the skin, while after 7 days there is signifi-
C
Fluorescence intensity

cantly higher concentration in grape flesh. In this paper, we

report the in vivo protective effects on yeast cells are concen-
tration dependent. These results provide further evidence of
the potential beneficial of natural concentrated sources of
polyphenols and antioxidants such as sundried Pedro Xime-
nez grapes.

Acknowledgement
2

2
G
n
ol

2O

2O

2O
hi

D
tr

ec

PE
on

/H

/H
at
C

in

G
C

Authors thank to Dr. R. Peinado his valuable comments and

D
h
ec

PE
at

assistance in the statistical analysis, to J.L. Trapero for his

C

technical assistance, to winery Cooperativa San Acacio

Fig. 3 – Protection from irreversible of protein carbonylation
(Montemayor, Spain) and grape grower A. Requena for supply-
in yeast. (A) Ponceau staining and samples of yeast crude
ing grape samples. This research was supported by an FPU
extract. (B) Immunodetection of protein carbonylation by
Scholarship (2008 official call) from Spain’s Ministry of Educa-
chemiluminescence. (C) Chemiluminescence analysis of the
tion, grants from the Spanish Ministry of Science and
protein carbonylation. Intensity of bands of carbonylated
Education (BFU2006-02990-BMC) and the Andalusian Govern-
proteins were analyzed and different letters indicate
ment (CVI-1611), and group BIO-261 from the Andalusian
significant differences at 95% of confidence level.
Government.

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