International Journal of Food Sciences and Nutrition

ISSN: 0963-7486 (Print) 1465-3478 (Online) Journal homepage: http://www.tandfonline.com/loi/iijf20

Effect of drying methods on the phenolic content

and antioxidant capacity of Brazilian winemaking
byproducts and their stability over storage

Milene Teixeira Barcia, Paula Becker Pertuzatti, Daniele Rodrigues, Vivian

Caetano Bochi, Isidro Hermosín-Gutiérrez & Helena Teixeira Godoy

To cite this article: Milene Teixeira Barcia, Paula Becker Pertuzatti, Daniele Rodrigues, Vivian
Caetano Bochi, Isidro Hermosín-Gutiérrez & Helena Teixeira Godoy (2015) Effect of drying
methods on the phenolic content and antioxidant capacity of Brazilian winemaking byproducts
and their stability over storage, International Journal of Food Sciences and Nutrition, 66:8,
895-903, DOI: 10.3109/09637486.2015.1110688

To link to this article: http://dx.doi.org/10.3109/09637486.2015.1110688

Published online: 11 Nov 2015.

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ISSN: 0963-7486 (print), 1465-3478 (electronic)

Int J Food Sci Nutr, 2015; 66(8): 895–903

! 2015 Taylor & Francis. DOI: 10.3109/09637486.2015.1110688

FOOD COMPOSITION AND ANALYSIS

Effect of drying methods on the phenolic content and antioxidant

capacity of Brazilian winemaking byproducts and their stability over
storage
Milene Teixeira Barcia1, Paula Becker Pertuzatti1,2, Daniele Rodrigues1, Vivian Caetano Bochi1,
Isidro Hermosı́n-Gutiérrez3, and Helena Teixeira Godoy1
1
Faculdade De Engenharia De Alimentos, Universidade Estadual De Campinas (UNICAMP), Cidade Universitária ‘‘Zeferino Vaz’’, São Paulo, Brazil,
2
Engenharia De Alimentos, Instituto De Ciências Exatas E Da Terra, Universidade Federal De Mato Grosso, Mato Grosso, Brazil, and 3Instituto
Downloaded by [Penn State University] at 03:47 03 December 2015

Regional De Investigación Cientı́fica Aplicada, Universidad De Castilla-La Mancha (UCLM), Ciudad Real, Spain

Abstract Keywords
This work aimed to study the antioxidant capacity by different methods, the total content of Anthocyanins, antioxidant capacity,
polyphenols and the stability over time of dried byproducts from Brazilian hybrids and Vitis grape marc, lees, phenolics
vinifera varieties. Oven-dried at 50  C and spray-dried samples were monitored for 90 days of
storage. Under testing conditions, BRS Violeta grapes showed the greatest stability and initial History
high levels of total phenolics and anthocyanins remained almost unchanged until the end of
storage period. The same behavior was observed in BRS Violeta freeze-dried skins, seeds and Received 15 April 2015
lees (8557, 9520 and 4261 mg GAE/100 g DM, respectively, and 829 and 257 mg mv-3-glc/100 g Revised 24 September 2015
DM in skin and lees, respectively). In all methodologies tested, BRS Violeta also showed higher Accepted 17 October 2015
values for antioxidant capacity. These results suggest that dried winemaking byproducts can be Published online 12 November 2015
used as rich sources of polyphenol compounds for industrial extractions with high stability and
antioxidant capacity.

Introduction in literature, the major phenolic compounds class in grape marcs

and red skins are anthocyanins (Rodrigues et al., 2011), flavonols,
Brazil has agriculture as the major economic activity and,
stilbenes and phenolic acids in seeds and white skins (Karvela
consequently produces a great amount of agro-industrial residues
et al., 2009).
due to industrialization. Grape production in Brazil is mainly used
Phenolic compounds are recognized as potent antioxidants.
for winemaking in which 60% of total grape harvested will
The multiple mechanisms of this activity are expressed in terms of
originate and 5% a first residue, the grape marcs. Moreover, at a
its ability to eliminate free radicals (free radical scavengers),
least 4% of total wine production will be lost by settling as lees, a
metal chelation and synergism with other antioxidants. In general,
second winemaking residue (Rockenbach et al., 2011).
methods used for the determination of the total antioxidant
Grape marc (a mixture of skins and seeds) is an abundant
capacity are divided into two main groups: those based on single
winemaking byproducts being responsible by any economic and
electron transfer (SET), monitored spectrophotometrically by a
environmental problems during storage, processing and disposal
color change due free radical reduction; and those based on
(Shojaee-Aliabadi et al., 2013). After fermentation, fine residual
hydrogen atom transfer (HAT) (Huang et al., 2005), measured by
particles from grape and death yeasts will precipitate as lees
elimination of peroxyl radicals. Oxygen radical absorbance
during a natural decantation process (Cortés et al., 2011).
capacity (ORAC), ferric radical antioxidant power (FRAP) and
Phenolic compounds from grapes are transferred to wine upon
scavenger capacity of 2,2-azinobis (3-ethyl-6-benzothiazoline
characteristics of the winemaking until a solid/liquid partition
sulfonic acid) (ABTS) cation radicals are the most commonly
equilibrium. Consequently, a large proportion of phenolic com-
used method for antioxidant capacity study. The discoloration of
pounds still remains in byprodutcs (Alonso et al., 2002). Since
b-carotene is also widely used to measure the antioxidant capacity
these compounds have importance in human nutrition and health,
of bioactive by scavenger of free radicals formed during oxidation
there is a great interest in exploring extraction processes of
of linoleic acid.
phenolic compounds from wine byproducts to future industrial
The aim of this work was to study total polyphenol and
applications (Arvanitoyannis et al., 2006). As previously reported
antioxidant capacity of winemaking byproducts from Brazilian
hybrid (BRS Violeta and BRS Lorena) and Vitis vinifera
(Cabernet sauvignon and Cabernet franc) grape varieties by
Correspondence: Milene Teixeira Barcia , Faculdade De Engenharia De
Alimentos, Universidade Estadual De Campinas (UNICAMP), Cidade different methods and its correlations. Moreover, polyphenol
Universitária ‘‘Zeferino Vaz’’, S/N, CEP 13083862 Campinas, São Paulo, content stability over time in dry-products was determined as
Brazil. Tel: +55 1935214023. E-mail: milenebarcia@ig.com.br well.
 896 M. T. Barcia et al.
Material and methods
Materials
Chemicals and equipment
The chemicals: 2,20-Azinobis (3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS), 2,4,6-tripyridyltriazine (TPTZ),
b-carotene, acid gallic, 2,20 -azobis (2-amidino-propane) dihy-
drochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox) were purchased from Sigma-Aldrich
(St. Louis, MO), linoleic acid from Fluka (St. Gallen,
Switzerland). Buffer salts and all other chemicals were of
analytical grade.
Equipments: a spectrophotometer UV 1600, Pró-Análise
(São Paulo, Brazil), an automated plate reader BMG Labtech,
Novo Star (Ortenberg, Germany, S/N 700-0120), a microplate
reader (BMG Labtech), an Ultrasound bath SX-20, Arruda, Ultra-
Sons LTDA (Santa Catarina, Brazil), a freeze-dryer Terroni
LS-3000 (São Paulo, Brazil), an oven, Nova Ética (São Paulo,
Brazil), a spray-dryer LAB PLANT SD-05, L.P. Technology
LTDA (Leeds, England) and a centrifuge Harrier 18/80-SANYO-
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MSE (London, UK).
Samples
Entire grapes, fermented skins and seeds and lees of hybrid
varieties (BRS Violeta and BRS Lorena) and Vitis vinifera
(Cabernet sauvignon and Cabernet franc) from 2011 and 2012
vintages were used in this work. For fermented skin of Cabernet
franc and for lees of BRS Lorena, only samples from 2012 vintage
were analyzed. Samples were kindly supplied by a winery from
São Roque region in São Paulo/Brazil (coordinates 23 310 4400
South and 47 080 0600 West, and 771 m above sea level as referred
in datum WGS84, World Geodetic System 1984) characterized by
a subtropical climate with maximum temperatures of 23.1  C and
minimum of 15.5  C). After harvested, samples were stored
at 20  C.
Sample preparation
Grapes, lees, skins and seeds samples were lyophilized in order to
avoid degradation and used as control groups to compare with
proposed drying processes. Thus, skins and seeds were dried in an
oven (50  C forced air circulation until constant weight), whereas
lees were spray-dried (180  C, flow rate of 9 ml/min, outlet
diameter 1.0 mm and exhaust temperature 100  C). Then, samples
were crushed, homogenized and stored in vacuum-sealed
laminated bags.
Dried samples were stored at controlled temperature (25  C in
a standard laboratory incubator, BOD MA 403, Marconi
Equipments) for developing the stability study (time 0, 30 and
90 days of storage). At day 0, samples from drying treatments
(oven-dried and spray-dried) were compared to freeze-dried
samples to determine the amount of phytochemicals that were
lost by the dehydration process. After 30 and 90 days, only
samples from proposed drying treatments were analyzed to
measure the stability over time.
Evaluation of the efficiency of the polyphenol extraction
protocol using the validated Folin–ciocalteu method
The efficiency of the polyphenol extraction protocol was studied
in five complex samples (skins, seeds and lees BRS Violeta, skins
and seeds BRS Lorena). Solid to liquid ratio was determined
testing increasingly amounts of powered sample (tested range:
25–200 mg) to the solvent media. Thus, 25 and 50, 75, 200 mg
were used for phenolic extraction of BRS Violeta’s skins, BRS
Violeta’s lees and seeds, BRS Lorena’s seeds and BRS Lorena’s
Int J Food Sci Nutr, 2015; 66(8): 895–903
skins samples, respectively. Samples were immersed in 25 ml of a
solvent mixture of methanol, water and formic acid (50:48.5:1.5
v/v/v), homogenized for 1 min and centrifuged at 2500 g at 5  C
for 15 min. Supernatants were collected and a second extraction
was performed in pellets at the same conditions. Supernatants
were combined, completed to a known volume (50 ml) and
immediately analyzed (Castillo-Muñoz et al., 2009).
Total Folin–Ciocalteu method for quantitation of total poly-
phenol content was validated as reported by the International
Union of Pure and Applied Chemistry (IUPAC, 2002). The
following validation characteristics were addressed: linearity,
precision and tendency. System linearity was verified with
calibration curves made up of seven points (0.01–0.07 mg/ml).
A lack of fit test for calibration curve was performed to the linear
model. In order to study the efficiency, recovery tests were
performed by spiking samples with gallic acid standard at two
levels: 0.04 and 0.07 mg/ml. The repeatability was performed with
10 repetitions of the extraction protocol in a same day.
Intermediate precision was determined repeating extraction
(n ¼ 3) over three consecutive days.
Total phenolics and anthocyanins
Total phenolic content (TPH) was determined using the Folin–
Ciocalteu colorimetric method as described by Scherer & Godoy
(2014). The absorbance was measured at 740 nm after 120 min at
room temperature. The absorbance values were then compared
with those of standards with known gallic acid concentrations. All
values are the mean value of three replications ± SD (deviation
standard). Quantification was performed using a calibration curve
constructed with gallic acid (0.01–0.07 mg/ml) as standard and
results were expressed as milligrams of gallic acid equivalents per
100 g of dry water sample (DW), and basis of fresh weight (FW)
for the comparison of others results.
Total monomeric anthocyanin content (ACY) was measured
using the pH differential method (Giusti & Wrolstad, 2001).
Extracts were mixed thoroughly with 0.025 M potassium chloride
buffer pH 1 in a ratio of 1:4 extract to buffer ratio. The extracts
were then mixed similarly with a sodium acetate buffer pH 4.5.
The absorbance at 520 and 700 nm was measured against a buffer
blank at pH 1.0 and 4.5.
Absorbance readings were converted to total milligrams of
malvidin 3-glucoside (M3G). The ACY was calculated on the
basis of molecular weight for M3G (494) and its molar
absorptivity (36 400). The ACY was expressed as milligrams of
M3G equivalents per 100 g of dry water sample (DW) and basis of
FW for the comparison of others results. All values are the mean
value of three replications ± SD (deviation standard).
Antioxidant capacity
Ferric-reducing antioxidant power (FRAP) assay
The FRAP assay was carried out as described previously (Benzie
& Strain, 1996). Briefly, the FRAP reagent was daily prepared
and warmed up to 37  C in a water bath immediately before use.
Diluted sample (320 ml) were mixed to 2400 ml of FRAP reagent.
After 15 min at 37  C, the absorbance was recorded at 593 nm.
For quantification purposes, a standard curve with Trolox was
used (160–720 mM).
Capture of free radical ABTS
The ABTS assay was carried out according to a method
previously established (Re et al., 1999). For this measurement,
all samples were diluted approximately to provide 20–80%
inhibition of the blank absorbance. Thus, in the selected dilution
factor, 30 ml of the diluted extract or extraction solution (blank)
 DOI: 10.3109/09637486.2015.1110688 Effect of drying methods on the phenolic content 897
Table 1. Validation of the analysis of phenolic compounds in samples of winemaking byproducts.

Parameter Skin BRS Violeta Seed BRS Violeta Lees BRS Violeta Skin BRS Lorena Seed BRS Lorena
Coef. angular (sensitivity) 12.216 12.216 12.216 12.817 12.817
Coef. linear 0.0274 0.0274 0.0274 0.0129 0.0129
Coef. correlation (linearity) 0.9984 0.9984 0.9984 0.9983 0.9983
p value (lack of fit test)a 0.500 0.500 0.500 0.447 0.447
Repeatability (n ¼ 10)b 7.11 0.58 4.59 8.42 6.33
Intermediate precision (n ¼ 3)b 7.10 0.55 4.63 8.48 6.29
a
The probability value of the lack of fit test should be greater than 0.05.
b
The repeatability and intermediate precision parameters were evaluated by calculating the relative standard deviation, RSD (%).

was mixed to 3.0 ml ABTS.+ working solution. After 25 min in a
30  C water bath, absorbance at 734 nm was recorded. The percent
of inhibition was assumed as the absorbance difference between
the blank reading and test tube readings (extract or standard
solution) at 734 nm. The calibration curve was constructed using
Trolox (2000–20 mM) as standard.
Oxygen radical absorbance capacity
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shown as the average plus standard deviation of an experiment
performed in triplicate. Correlations among data obtained were
calculated using Pearson’s correlation coefficient (r).
In addition, principal component analysis (PCA; Pirouette
3.11.) was applied to the data matrix composed by the studied
samples (rows) with their corresponding values for each of the
analytical variables (columns), in order to highlight similarities
and/or differences among samples. The graphical representation

of the scores assigned by PCA to each sample in the space

The ORAC procedure used an automated plate reader with 96-
well plates (Huang et al., 2002). Analyses were conducted in
system composed of an indicator, fluorescein; a peroxyl radical
generator, 2,2’-azobis(2-amidinopropane) dihydrochloride
(AAPH); and Trolox as a control standard. All reagents were
prepared in 75 mM potassium phosphate buffer (pH 7.4) just
before analysis. After the addition of 20 ml of diluted samples, it
was added 120 ml of fluorescein (0.378 mg/ml) and 60 ml of AAPH
(41.4 mg/ml) in each well, 80 min for reaction completion at
37  C. Fluorescence conditions were as follows: excitation at
485 nm and emission at 520 nm. The standard curve was
constructed using Trolox (80–1500 mM). The area under de
curve (AUC), relative fluorescence versus incubation time, was
calculated as shown in Equation 1. The AUC differences between
the extract and blank were taken and used for calculations.
f 2 f 3 f 4 fn
AUC ¼ 1 þ þ þ þ ,
f1 f1 f1 f1
were f is the fluorescence reading.
determined by PCA allowed the grouping of the different samples
that can be correlated to their respective analytical results
regarding the content of phenolic compounds and anthocyanins,
as well as their antioxidant capacity.
Results
Extraction of total phenolic compounds and validation of
the Folin–ciocalteu method
The validation results are summarized in Table 1. The results
demonstrated that the standard curve was linear, with high values
for the correlation coefficient (R ¼ 0.99835). Intermediate preci-
sion was evaluated over 3 days using the same sample extraction
under the same conditions. The RSD values,510% for sample,
indicate that the intermediate precision and repeatability were
ð1Þ
acceptable for the range of concentration studied, as described
previously by Horwitz & Albert (1995).
The efficiency of extraction was evaluated by calculating
the percentage of recovery. The results found for recovery
Antioxidant assay using the -carotene bleaching method
The prevention of b-carotene bleaching was determined by the
method of Miller (1970) as described by Pertuzatti et al. (2014). In
brief, 300 ml (1 mg b-carotene in 1 ml chloroform) was mixed with
22 ml of linoleic acid and 200 ml of Tween-40. The chloroform was
evaporated under nitrogen, then 50 ml distilled water (super-
saturated oxygenic) was added and the resulting mixture was
vigorously stirred. The emulsion obtained was freshly prepared
before each experiment. An aliquot (250 ml) of the b-carotene-
linoleic acid emulsion was transferred to microplate wells already
containing 10 ml of extracts at a concentration of 0.5 g/l. Then, after
incubation at 45  C for 2 h, absorbance readings were performed at
470 nm. The percentage of inhibition was determined by the
difference between readings of control wells with extraction
solution (without antioxidants) and the test wells with extract.
Statistical data analysis
Analysis of variance (ANOVA) was performed to evaluate main
effects of a two-factor experiment (year and sample type) and
Tukey test for mean comparison at 95% of confidence level.
Pearson’s correlation coefficient (r) was determined among data
of different variables (total polyphenol content, total monomeric
ACY and antioxidant capacity assays). Statistica 7.0 software
(StatisoftÕ ) was used to perform statistical analysis. All data are
ranged from 79.34 to 114.05%, the higher recovery being
found for the seed samples, very likely due to a combination of
several factors: the presence of interfering substances, once this
sample has a complex matrix regarding its chemical composition
and physical structure; the level of granulometry; and the low
level of gallic acid used to do the recovery. However, these values
were consistent with the range of concentration studied by
Meinhart et al. (2010) and Ballus et al. (2012), whose works
support that recoveries higher than 100% can be achieved in solid/
liquid extraction of complex matrices, because the extraction
procedure is subjected to several intrinsic factors difficult to
totally control. These results are also according to ANVISA
(Agência Nacional de Vigilância Sanitária, 2003) guidelines,
which establish the trueness of an extraction method showing
recovery rates between 80 and 120% of the initial analytical
concentration are satisfactory.
Total phenolic compounds and anthocyanins
Results of total phenolic compound and ACYs are shown in
Table 2. Total phenolic compounds in grape samples ranged from
477 to 4509 mg GAE/100 g DW. Significant differences (p50.05)
were found for the BRS Lorena and Cabernet sauvignon grapes
between vintages with higher amounts of phenolic compounds at
2011 than in 2012.
 898 M. T. Barcia et al. Int J Food Sci Nutr, 2015; 66(8): 895–903

Table 2. Results of total phenolics (TPH) and total anthocyanins (ACY) in grape samples, skin, seed and lees BRS Violeta, BRS Lorena, Cabernet
franc and Cabernet sauvignon in the years 2011 and 2012.

TPH mg GAE/100g DW ACY mg mv-3-glc/100g DW

Samples/years
2011 2012 2011 2012
cA cA bA
BRS Violeta Grape 4509 ± 114 4152 ± 320 444 ± 14 459 ± 17bA
Skin 7832 ± 164aB 9282 ± 207bA 677 ± 33aB 981 ± 26aA
Seed 8062 ± 114aB 10979 ± 922aA NA NA
Lees 4911 ± 157bA 3612 ± 73cB 289 ± 9cA 225 ± 4cB
BRS Lorena Grape 585 ± 15bA 477 ± 33cB NA NA
Skin 491 ± 19cB 1131 ± 31bA NA NA
Seed 974 ± 55aB 2056 ± 78aA NA NA
Lees NA 1149 ± 102b NA NA
Cabernet franc Grape 1672 ± 125bA 1607 ± 67cA 55 ± 4bA 52 ± 5cA
Skin NA 2907 ± 18b NA 85 ± 1a
Seed NA 5026 ± 169a NA NA
Lees 3086 ± 71aA 2697 ± 24bB 90 ± 3aA 64 ± 1bB
Cabernet sauvignon Grape 2462 ± 219cA 1335 ± 94bB 53 ± 2bA 37 ± 2bB
Skin 3203 ± 71aA 1124 ± 60bB 78 ± 8aA 19 ± 1cB
Seed 6312 ± 152aA 3245 ± 274aB NA NA
Lees 2909 ± 47bB 3224 ± 60aA 62 ± 1aA 52 ± 1aB
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Results are mean ± standard deviation. For each grape cultivar, different lower case letters in a same column shows significant differences (p50.05)
among samples. Different capital letters in a same line shows significant differences between years (p50.05) the second test of Tukey.
DW, dry weight; GAE, equivalent acid gallic; mv-3-glc, malvidin-3-glucoside; NA, not analyzed.

The white grape hybrid cultivar BRS Lorena had the lowest
total phenolic content in both sampling dates (585 and 477 mg
GAE/100 g DW for the years 2011 and 2012, respectively). This is
in agreement with a previous work in which lower levels of
phenolic were found for all studied white grape varieties
(Borbalán et al., 2003).
However, according to Yang et al. (2009), the total phenolic
content in different grapes is mainly related to differences among
varieties and not to skin color, the reported average value being
425 mg FW GAE/100 g for Cabernet franc. For comparison with
the latter data, we made a recalculation of the phenolic content on
the basis of the FW used for obtaining the lyophilized grape
sample. Thus, our sample of Cabernet franc grapes accounted for
a total phenolic content of 427 and 440 mg GAE/100 g FW for
vintages of 2011 and 2012, respectively, which are consistent with
the aforementioned study.
In all analyzed varieties, seed showed higher content of
phenolic compounds than skins and lees. This result agrees with
previous literature data (Babbar et al., 2011; Makris et al., 2007;
Rockenbach et al., 2011).
This finding was also observed for the BRS Lorena white
grapes since higher amounts of phenolic compounds were found
in seeds (Table 2) than in skin samples (491 and 1131 mg/100 g
DW, for the years 2011 and 2012, respectively). Moreover, BRS
Lorena seeds for the year 2012 showed a phenolic content similar
to that reported by Rockenbach et al. (2011) for Isabel grape
(2128 mg GAE/100 g DW), that is also a nonvinifera grape
cultivar.
The same work also showed lower concentration of phenolic
compounds in the skin (1065 mg GAE/100 g DW) than in seed
(8249 mg GAE/100 g DW) for Cabernet sauvignon. However, we
observed that there was a considerable variation between vintages
for the same winemaking byproduct.
Our results showed that Cabernet franc had total ACY between
13.9 (year 2011) and 14.1 mg mv-3-glc/100 g (year 2012), with
no differences among the years (p ¼ 0.44). These values were
presented in FW for comparison with Hogan et al. (2009), which
reported a concentration of 17 mg cyanidin-3-glc/100 g of FW for
the same grape. For the other grapes analyzed in this study, no
data were found in the literature. BRS Violeta, a grape hybrid
cultivar, was the variety with higher amounts of anthocyanins, its
content in FW basis from 89.4 mg malvidin-3-glc/100 g FW (year
2011) to 98.6 mg malvidin-3-glc/100 g FW (year 2012). The latter
results are in agreement with other studies where 14 different V.
vinifera grapes and hybrids were analyzed and the content of
anthocyanins were higher in hybrid grapes than in V. vinifera
grapes (Yang et al., 2009).
Antioxidant capacity
For all samples, the ORAC values of antioxidant capacity were
higher than their corresponding FRAP, ABTS and b-carotene/
linoleic acid bleaching test (Table 3).
BRS Violeta grape variety has the greatest antioxidant capacity,
which is in agreement with the high content of phenolic compounds
observed in these samples (Table 2). This hypothesis was reinforced
by the high positive correlation coefficients shown in Table 4 that
were significant values between total polyphenol content (p50.05)
and FRAP, ABTS and ORAC values (0.99, 0.94 and 0.97,
respectively). Similar positive correlations were found between
total ACY and antioxidant capacity measures (0.96, 0.92 and 0.93 for
FRAP, ABTS and ORAC values, respectively), an expectable result
given the also good correlation between TPH and ACY (0.95).
However, there was no significant correlation between
bleaching of b-carotene/linoleic acid emulsions and any of the
phenolic compound concentrations or with any other antioxidant
methods tested (Table 4). Indeed, all grape varieties showed lower
antioxidant capacity to inhibit bleaching in b-carotene/linoleic
acid emulsion. Values ranged from 6.34% for BRS Lorena grapes
of the year 2012 to 20.79% for BRS Violeta grapes of the same
year (Table 3).
Principal component analysis
PCA provides a multivariate study of experimental data, which
facilitates the visualization of the correlation between samples
(scores) and variables (loadings). However, to facilitate this view
was necessary pre-processing, specifically self-scaling. For this,
the data focused on the middle and each one was divided by the
standard deviation, so that all variables associated to each vector
(phenolics, anthocyanins, ORAC, ABTS, FRAP and b-carotene/
linoleic acid assays) were given the same importance, that also
contributes to the dataset variance.
 DOI: 10.3109/09637486.2015.1110688 Effect of drying methods on the phenolic content 899
Table 3. Results of antioxidant capacity by FRAP, ABTS, ORAC and b-carotene/linoleic acid in the samples of grape skin, seed and lees BRS Violeta,
BRS Lorena, Cabernet sauvignon and Cabernet franc in the years 2011 and 2012.

FRAP mM TE/g DW ABTS mM TE/g DW ORAC mM TE/g DW b carotene % inhibition

Samples/years
2011 2012 2011 2012 2011 2012 2011 2012
bA bA bA cB cA bA dB
BRSVioleta Grape 374 ± 10 401 ± 34 344 ± 18 292 ± 10 1408 ± 71 1336 ± 36 14 ± 1 21 ± 2dA
Skin 639 ± 16aB 880 ± 32aA 554 ± 65aA 618 ± 20bA 3132 ± 75aA 2901 ± 113aB 29 ± 6cB 51 ± 8cA
Seed 593 ± 54aB 958 ± 102aA 636 ± 49aB 833 ± 76aA 2487 ± 75bB 3370 ± 330aA 91 ± 6aA 111 ± 15bA
Lees 446 ± 45bA 374 ± 11bB 355 ± 4bA 73 ± 1bB 1568 ± 114cA 781 ± 80cB 58 ± 3bB 133 ± 1Aa
BRSLorena Grape 62 ± 4bA 34 ± 3dB 60 ± 3A 27 ± 1cB 186 ± 14bA 117 ± 12dB 7.2 ± 0bA 6.3 ± 1cA
Skin 54 ± 3bB 127 ± 3bA 47 ± 6cA 21 ± 1cB 203 ± 16abB 257 ± 18cA 4.9 ± 0cA 1.1 ± 0dB
Seed 80 ± 4aB 174 ± 3aA 93 ± 6aA 44 ± 2bB 233 ± 10aB 473 ± 26abA 19 ± 1aA 18 ± 1aA
Lees NA 63 ± 6c NA 65 ± 6a NA 372 ± 17 NA 8.7 ± 0b
Cabernetfranc Grape 116 ± 2bB 131 ± 5dA 64 ± 7bB 121 ± 4cA 339 ± 58bA 400 ± 37cA 18 ± 1bA 19 ± 3bA
Skin NA 257 ± 2b NA 60 ± 1d NA 661 ± 14b NA 24 ± 1b
Seed NA 509 ± 30a NA 449 ± 16a NA 477 ± 49c NA 21 ± 3b
Lees 265 ± 4aA 215 ± 5cB 140 ± 3aB 182 ± 9bA 832 ± 37aA 815 ± 21aA 57 ± 9aB 73 ± 2aA
Cabernetsauvignon Grape 176 ± 18cA 115 ± 9bB 155 ± 13bA 96 ± 4cB 460 ± 68cA 331 ± 18cB 17 ± 1cA 19 ± 2cA
Skin 253 ± 4bA 95 ± 11bB 163 ± 7bA 37 ± 3dB 976 ± 94aA 482 ± 7bB 23 ± 2cA 18 ± 0cB
Seed 551 ± 11A 241 ± 41aB 457 ± 20aA 188 ± 11bB 997 ± 59aA 446 ± 6bB 90 ± 8bA 104 ± 1aA
Lees 202 ± 6cB 246 ± 10aA 137 ± 3bB 217 ± 13aA 716 ± 8bA 794 ± 71aA 65 ± 4aA 71 ± 9A
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For each grape cultivar, different lower case letters in a same column shows significant differences (p50.05) among samples. Different capital letters in
a same line shows significant differences between years (p50.05), the second test of Tukey.
DW, dry weight; NA, not analyzed; TE, equivalent Trolox.

Table 4. Pearson’s correlation coefficients of antioxidant activities, total phenolics and total anthocyanins content.

Trait TPH ACY FRAP ABTS ORAC

ACY 0.95
FRAP 0.99 0.96
ABTS 0.94 0.92 0.91
ORAC 0.97 0.93 0.94 0.95
CAROTENE 0.15 0.01 0.20 0.03 0.04

ABTS, antioxidant capacity based on ABTS assay; ACY, total anthocyanins; CAROTENE, antioxidant capacity based on b-carotene/linoleic acid;
FRAP, antioxidant capacity based on FRAP assay; ORAC, antioxidant capacity based on ORAC assay; TPH, total phenolics.

PC1 explained 74.1% of the total variance, PC2 16.7%, PC3
6.9%, while PC4 explained only 1.6% of the total variance.
However, PC4 versus PC1 were important for the separation of
the samples (Figure 1a and b).
The samples of the also hybrid variety BRS Lorena, were
separated by PC1 and appeared grouped in the most negative part
along the PC1 ax, that corresponded to the lowest values of
phenolic compounds, ABTS, FRAP and ORAC. Cabernet
sauvignon and Cabernet franc were not well separated as they
showed similar phenolic composition and antioxidant behavior.
The PC2 was mainly related to the content of total anthocyanins
(Figure 1d) and made possible the separation of samples of BRS
Violeta grape and lees, and also samples of BRS Violeta skin and
seeds.
Stability of winemaking byproducts
The content of phenolics and anthocyanins, as well as the four
assays of antioxidant capacity were monitored over the storage of
the samples dried after processes oven-drying (skins and seeds
samples) and spray-drying (only lees samples) (Figure 2). With
the exception of BRS Violeta byproducts the other significant
difference (p50.05) was the content of bioactive compounds and
the antioxidant capacity during the interval of 90 days of storage.
Anthocyanins of BRS Violeta remained stable during the 90-
day storage, both in the skin and the lees of red grape cultivars. In
contrast, the ACY in the lees of Cabernet franc already decreased
by 39% after 30 days and remained almost unchanged after 90
days of storage. Similar result was found for lees of Cabernet
sauvignon (reduction of 40% after 30 days) but significant
reduction (p50.05), was also observed after 90 days of storage up
to reach 61% of the initial value. Finally, the ACY of skins of
Cabernet sauvignon only decreased after 90 days (56% of the
initial value).
Regarding the stability of the antioxidant, the observed
behavior differed from that observed for total contents of phenolic
compounds and anthocyanins but also differed according to the
type of determination (Figure 2c–f). FRAP values did not change
so much during storage for all samples (Figure 2c). In the case of
ORAC determinations, little variations (increasing or decreasing
trends) and even maintenance of antioxidant capacity were
observed (Figure 2e).
The BRS Violeta byproducts increased their ABTS (Figure 2d)
and b-carotene/linoleic acid (Figure 2d) values after 30 days of
storage and, after 90 days, the ABTS values decreased for skin and
seed samples (but maintained for lees) and the b-carotene/linoleic
acid values increased for seeds and lees (but maintained for skins).
The behavior for Cabernet sauvignon byproducts was different:
after 30 days, all ABTS values and b-carotene/linoleic acid values
for skins and lees increased, but b-carotene/linoleic acid value for
seed maintained; after 90 days, ABTS of skins and b-carotene/
linoleic acid values for seeds and lees decreased, ABTS values for
seeds and lees maintained and b-carotene/linoleic acid value for
skins increased. The behavior of Cabernet franc lees was similar to
that of lees form Cabernet sauvignon. The singular behavior shown
by BRS Violeta winemaking byproducts could be very likely linked
 900 M. T. Barcia et al. Int J Food Sci Nutr, 2015; 66(8): 895–903

(a) 1.2 (a) 0.6

FRAP
1.0 0.4
ABTS
0.8
0.2 ACY
TPH
0.6
0.0 Carotene/Linoleic Acid
0.4
PC4

PC4
PC4
0.2 -0.2

0.0 -0.4

-0.2
-0.6
-0.4
BRS Violeta ORAC
-0.8
-0.6 BRS Lorena
C. franc
-0.8 -1.0
C. sauvignon 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48
-3 -2 -1 0 1 2 3 4 5 6 7
PC1 PC1
(c) 3 (d) 0.8 Carotene/Linoleic acid

0.6
2

0.4
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1
0.2
TPH
ABTSFRAP
PC2

PC2
0 0.0
ORAC

-0.2
-1

-0.4

-2 BRS Violeta
-0.6 ACY
BRS Lorena
C. franc
-3 C. sauvignon -0.8
-3 -2 -1 0 1 2 3 4 5 6 7 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48
PC1 PC1

Figure 1. Principal component analysis (PCA) on grape, skin, seed and lees of four varieties BRS Violeta, BRS Lorena, Cabernet sauvignon and
Cabernet franc belonging harvests of 2011 and 2012 (a) Graph of scores for PC1 (factor 1) and PC4 (factor 4), differences between classes (grape
varieties), (b) Graph of loadings for PC1 (factor 1) and PC4 (factor 4), (c) Graph of scores for PC1 (factor 1) and PC2 (factor 2), (d) Graphics loadings
for PC1 (factor 1) and PC2 (factor 2). *For abbreviations see Table 4.

with their high contents in anthocyanins that dominated the pool of
total phenolic compounds, as it has been reported for the detailed
phenolic composition of the grapes of this hybrid cultivar (Rebello
et al., 2013).
Discussion
Total phenolics and anthocyanins, antioxidant capacity
and PCA
Very likely, higher levels of phenolic compounds were found in
grape berries samples (BRS Lorena and Cabernet sauvignon) of
year 2011 due to the difference in climate conditions between
years. Thus, considering the climate information about the
cropping area that is available in CEPAGRI (2013), lower average
temperatures and precipitation volumes were observed in the year
2012 (15.3  C and rainfall of 133.4 mm) than in the year 2011
(temperature 20.8–29.8  C and rainfall of 451.6 mm).
Therefore, the significant increase in total phenolic content
between years could be a result of the plant defense mechanism at
high temperatures, since these compounds were reported as able
to act against UV radiation (Barcia et al., 2010).
The grape cultivar showing the highest content of total
phenolics compounds in the present study was BRS Violeta, in
both vintages, which can be related to the fact that this grape
variety showed an intense red-purple color attributed to the high
content of anthocyanins, usually associated to the high content of
phenolics compounds when compared with white grapes, as
described Rebello et al. (2013).
According to Makris et al. (2007), seeds have the highest
contribution to the phenolic content in the grape marcs, mainly in
white grapes, in which the skin shows a lower phenolic content
than in purple cultivars.
Additionally, there is a scarce literature data about phenolic
compounds in winemaking byproducts. For BRS Violeta and BRS
Lorena varieties, e.g. no literature data were found, thus making
difficult the comparison of the results obtained.
For all grape varieties, lees still showed high levels of total
phenolic compounds when compared to the whole grape berry.
Thus, the highest levels was observed again in the case of BRS
Violeta, followed by Cabernet sauvignon and Cabernet franc, with
similar contents, and finally BRS Lorena showed the lowest
amount of phenolics. However, all these values were higher in
lees than those found in the whole grape berry, on a basis of dry
matter.
If compared to other anthocyanin-rich sources ACY in
fermented skin BRS Violeta (146.68 mg malvidin-3-glc/100 g
FW) is lower than values reported for blackberries, 140 mg
cyanidin-3-glc/100 g FW (Jacques et al., 2010) and Jambolão
fruits (Syzygium cumini), 7–17 mg cyanidin-3-glc/100 g FW
(Barcia et al., 2012). And fermented skin BRS Violeta in dried
weight (829 mg malvidin-3-glc/100 g DW) higher than values
found for Ceylon gooseberry pulp, 367 mg cyanidin-3-glc/100 g
 DOI: 10.3109/09637486.2015.1110688 Effect of drying methods on the phenolic content 901
(a)
Lees C. sauvignon

Seed C. sauvignon

Skin C. sauvignon

90 days
Lees C. franc
30 days
Samples Seed BRS Lorena
0 days

Skin BRS Lorena

Lees BRS Violeta

Seed BRS Violeta

Skin BRS Violeta

0 1000 2000 3000 4000 5000 6000

mg GAE/100g DM

(b)
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Lees C. sauvignon

Skin C. sauvignon
90 days

30 days
Sample

Lees C. franc
0 days

Lees BRS Violeta

Skin BRS Violeta

0 25 50 75 100 125 150 175 200 225 250 275 300

mg malv-3-glc/100g DM

(c) (d)
Lees C. sauvignon Lees C. sauvignon

Seed C. sauvignon Seed C. sauvignon

Skin C. sauvignon Skin C. sauvignon

Lees C. franc 90 days Lees C. franc 90 days

Sample
Sample

30 days Seed BRS Lorena 30 days

Seed BRS Lorena
0 days 0 days
Skin BRS Lorena Skin BRS Lorena

Lees BRS Violeta Lees BRS Violeta

Seed BRS Violeta Seed BRS Violeta

Skin BRS Violeta Skin BRS Violeta

0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 500

µM TE/g DM µM TE/g DM

(e) (f)
Lees C. sauvignon Lees C. sauvignon

Seed C. sauvignon Seed C. sauvignon

Skin C. sauvignon Skin C. sauvignon

90 days
Lees C. franc Lees C. franc 90 days
30 days
Sample

Sample

30 days
Seed BRS Lorena 0 days Seed BRS Lorena
0 days
Skin BRS Lorena Skin BRS Lorena

Lees BRS Violeta Lees BRS Violeta

Seed BRS Violeta Seed BRS Violeta

Skin BRS Violeta Skin BRS Violeta

0 250 500 750 1000 1250 1500 1750 0 15 30 45 60 75 90 105 120 135 150
µM TE/g DM %Inibition

Figure 2. Phenolic compounds (a), anthocyanins (b), antioxidant capacity by FRAP (c), ABTS (d) and ORAC (e) b-carotene/linoleic acid (f) in skin,
seed (drying oven-dried at 50  C) and lees (spray-drying) at 0, 30 and 90 days of storage of four varieties BRS Violeta, BRS Lorena, Cabernet
sauvignon and Cabernet franc (year 2011).
 902 M. T. Barcia et al.
DW (Bochi et al., 2014), blueberries, 140–318 mg cyanidin-3-glc/
100 g DW (Pertuzatti et al., 2014).
Regarding winemaking byproducts, skins presented higher
content of anthocyanins than lees. An exception was observed for
Cabernet sauvignon, in which there was no significant difference
between skin and lees for samples of the year 2011 (Table 2), and
skin samples of the year 2012 showed lower ACY than in lees.
Another important finding was reported by Rebello et al.
(2013), showing that in the fresh skin of BRS Violeta, anthocya-
nins accounted for 393 mg mv-3,5-diglc/100 g FW, a value higher
than that found in our study (108 and 184 mg/100 g FW, in 2011
and 2012, respectively). However, we believe that anthocyanins
were transferred from the skin to the wine.
The results very likely suggest that grape and winemaking
byproduct phenolic compounds have an intense activity as
scavenger of peroxyl radicals, and in a less extension by
mechanisms of ABTS cation radical neutralization, electron
donation to ferric ions, or prevention of carotenoid bleaching by
lipid peroxyl radicals.
Results were in agreement with previous reported values.
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Recently, Rodrigues et al. (2011) published values of 19.5% for a
blueberries cultivar variety with the highest antioxidant capacity
by other methods. The antioxidant capacity of winemaking
byproducts (Table 3) reached the highest values for seeds
(p50.05) for all studied varieties. This can be explained by
the high level of phenolic compounds found in this grape part
(Table 2). However, this behavior has not been always reported for
fruits in general, since there are some previous works that showed
high levels of phenolic compounds in fruit seed but the highest
antioxidant capacity was found in other fruit part, using ABTS
method (Babbar et al., 2011). It was hypothesized that it could be
due to the presence of other non-phenolic antioxidants like
ascorbate, carotenoids or terpenes.
In the PCA it can be noted that all samples of BRS Violeta
(grape, skins, seeds and lees) had highly positive scores (mainly
related to ORAC, FRAP, ABTS and total phenolic content) and
separation of these samples was possible along the positive ax of
principal component. So, facilitates the visualization of the
correlation between samples and variables.
Stability of winemaking byproducts
The phenolic compounds are highly unstable and can be lost
during processing, particularly when thermal treatment is
involved (Srivastava et al., 2007), as was observed in this
study when data from Figure 2(a) were compared with those in
Table 2. A reduction in the total phenolic content compared to
initial time was measured, could be attributed to oxidative
conditions during storage, as reported by other researchers
(Srivastava et al., 2007).
Some studies claim that prolonged storage at high tempera-
tures can affect glycosylation and hydroxylation of phenolic
compounds (Srivastava et al., 2007), besides an increase in
glycosidic substitution, acylation and methoxylation tends to
improve the stability of anthocyanins, which may have influenced
so that there was an increase in antioxidant capacity in most of the
samples analyzed in this study.
Conclusion
The grape cultivar BRS Violeta and their corresponding wine-
making residues had the highest content of total phenolic
compounds and anthocyanins, thus contributing to an important
enhancement of their antioxidant capacity measured by four
methods.
The winemaking byproducts of all the studied grape varieties
can be suggested as interesting sources for the extraction of
Int J Food Sci Nutr, 2015; 66(8): 895–903
bioactive compounds, with the seed, regardless of the variety, the
largest source of phenolic compounds.
With regard to stability of phenolic compounds present in
winemaking byproducts, the behavior shown by BRS Violeta was
the most interesting. The total phenolic and ACYs were almost
maintained over 90 days of storage, in parallel with maintenance
and even increase of antioxidant capacity.
Acknowledgements
The authors are grateful to Mr. Fernando José de Góes, the owner of Góes
winery (São Roque, Brazil) for supplying the grape, grape marc and lees
samples. The authors would like to thank Brazilian agency, CAPES, for
the productivity scholarship.
Declaration of interest
The authors declare that there is no conflict of interests.
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