Food Research International 54 (2013) 354–366

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Food Research International

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Phenolic composition of the berry parts of hybrid grape cultivar BRS

Violeta (BRS Rubea × IAC 1398-21) using HPLC–DAD–ESI-MS/MS
Ligia Portugal Gomes Rebello a, Ellen Silva Lago-Vanzela b, Milene Teixeira Barcia c, Afonso Mota Ramos d,
Paulo César Stringheta d, Roberto Da-Silva b, Noelia Castillo-Muñoz e,
Sergio Gómez-Alonso f,g, Isidro Hermosín-Gutiérrez g,⁎
a
Instituto Federal Fluminense, Dário Vieira Borges, 235, Parque Trevo, 28360-000 Bom Jesus do Itabapoana, Rio de Janeiro, Brazil
b
Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, Cristovão Colombo, 2265, Jardim Nazareth, 15054-000 São José do Rio Preto, São Paulo, Brazil
c
Universidade Estadual de Campinas, Monteiro Lobato, 80, Cidade Universitária, 13081-970 Campinas, São Paulo, Brazil
d
Universidade Federal de Viçosa, Avenida Peter Henry Rolfs, s/n, Campus Universitário, 36570-000 Viçosa, Minas Gerais, Brazil
e
Instituto de la Vid y el Vino de Castilla-La Mancha, Carretera Toledo-Albacete s/n, 13700 Tomelloso, Spain
f
Fundación Parque Científico y Tecnológico de Albacete, Paseo de la Innovación, 1, 02006 Albacete, Spain
g
Instituto Regional de Investigación Científica Aplicada, Universidad de Castilla-La Mancha, Campus Universitario s/n, 13071 Ciudad Real, Spain

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

Article history: The grape is considered a major source of phenolic compounds when compared to other fruits and vegetables,
Received 22 April 2013 however, there are many cultivars with distinct characteristics directly linked to phenolic profile. Thus, the
Accepted 3 July 2013 present study aimed to identify and quantify, for the first time and in detail, the phenolic compounds present
in the skin, flesh and seeds of BRS Violeta grape berry using combination of SPE methodologies and analytical
Keywords:
HPLC–DAD–ESI-MS/MS. The study was extended to the different berry parts and the most important grape
BRS Violeta
Hybrid grape
and wine phenolic families, and has revealed interesting features. Violeta grape has a very thick skin (46%
Anthocyanins of grape weight) that accumulated the most of grape phenolic compounds: great amount of anthocyanins
Flavonols (3930 mg/kg, as malvidin 3,5-diglucoside), together with also important amounts of flavonols (150 mg/kg,
Proanthocyanidins as quercetin 3-glucoside), hydroxycinnamic acid derivatives (HCAD; 120 mg/kg, as caftaric acid), and
Hydroxycinnamic acid proanthocyanidins (670 mg/kg, as (+)-catechin); in contrast, it seems to be a low resveratrol producer.
Phenolic compounds Violeta grape seeds accounted for similar proportions of low molecular weight flavan-3-ols (mainly mono-
mers; 345 mg/kg, as (+)-catechin) and proanthocyanidins (480 mg/kg, as (+)-catechin). Violeta grape is a
teinturier cultivar, but it only contained traces of anthocyanins and low amounts of all the other phenolic
types in its red-colored flesh. The anthocyanin composition of Violeta grape was dominated by anthocyanidin
3,5-diglucosides (90%). Within flavonols, myricetin-type predominated and kaempferol-type was missing. In
addition to expected hydroxycinnamoyl-tartaric acids, several isomeric esters of caffeic and p-coumaric acids
with hexoses were tentatively identified, accounting for relevant proportions within the pool of HCAD. Al-
though pending of further confirmation over successive vintages, the aforementioned results suggest that
BRS Violeta grape cultivar could be considered an interesting candidate for the elaboration of highly colored
and antioxidant-rich grape juices and wines.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction playing important physiological activities, and their concentration is

The grape is one of the most widely consumed fruits in the world,
both in natural and in processed forms (FAOSTAT, 2012). It is con-
sidered one of the greatest sources of phenolic compounds when com-
pared to other fruits and vegetables, but there is a great diversity among
cultivars resulting in grapes with different characteristics, which is di-
rectly linked to polyphenol profile (Abe, Mota, Lajolo, & Genovese,
2007). The phenolic compounds are secondary metabolites of plants,
⁎ Corresponding author. Tel.: +34 926 295253; fax: +34 926 295351.
E-mail address: isidro.hermosin@uclm.es (I. Hermosín-Gutiérrez).
subjected to great variation. They can be classified into two groups:
non-flavonoids and flavonoid. In the grape, non-flavonoids are mainly
phenolic acids and stilbenes and flavonoids are anthocyanins, flavonols
and flavan-3-ols (monomers, dimers, oligomers and polymers, the latter
also known as tannins) (Garrido & Borges, 2011). In fruits and their
products, phenolic compounds are directly related to sensory character-
istics such as color and flavor. In addition, many beneficial health effects
have been attributed to these compounds present in fruits, vegetables,
teas and wines. Epidemiologic studies, clinical and in vitro studies
have demonstrated some of the biological effects associated with dietary
phenolic compounds such as antioxidant, anti-inflammatory, antimicro-
bial and anti-carcinogenic activities (Dávalos & Lasunción, 2009).

0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.07.024
 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366
The Brazilian viticulture is booming, according to Food and
Agricultural Organization—FAO (FAOSTAT, 2012), in 2011 about
1.5 million tons of grapes were produced, with approximately 50%
aimed at making wines and juices. Grape production in Brazil is lo-
cated mainly in the South, Southeast and Northeast. It constitutes a
consolidated activity in the production of wines and juice, with
socio-economic importance, particularly in the states of Rio Grande
do Sul and Santa Catarina, with increasing activity in the state of
São Paulo (Lafka, Sinanoglou, & Lazos, 2007; Llobera & Cañellas,
2008; Torres et al., 2002). The grape cultivar BRS Violeta is a hybrid
cultivar, obtained in 2006 from the crossing between BRS Rubea ×
IAC 1398-21, and was launched by the Brazilian Agricultural Research
Corporation Grape and Wine (Embrapa Uva e Vinho) as an alternative
to increase quality and competitiveness of table wine and grape
juice produced in Brazil, because it is characterized by high levels of
sugar concentration and color, and is especially suitable for cultiva-
tion in subtropical climate zones like the state of São Paulo. This
grape is being made available to the Brazilian wine industry, and is in-
dicated for the direct preparation of juices and the production of
wines or for addition in juices and wines made from Concord and
Isabel grapes, thus adding more color (Camargo, Maia, & Nachtigal,
2005). For instance, reported values revealed high anthocyanin con-
tent of BRS Violeta must and grape juice (2340 ± 4 mg/L, as malvidin
3-glucoside equivalents; Silva et al., 2011). As a consequence, the
production of BRS Violeta grape has increased in the few years of its
existence and, in year 2011, reached 2.4 million kg in the Brazilian
State of Rio Grande do Sul (De Mello & Machado, 2011). However,
little is known about BRS Violeta grape composition and only the
global chemical composition of derived products like must and grape
juices as well as the positive effects on the sensory evaluation of grape
juices obtained by blending of different grape cultivars including BRS
Violeta have been reported (Azevedo, Gonçalves, Schneider, Portela, &
Rufato, 2009; Borges, Prudêncio, Roberto, & de Assis, 2011; Silva et al.,
2011).
To the best of our knowledge, BRS Violeta grape has not been yet
well characterized with regard to its phenolic composition. However,
the phenolic composition of grapes represents important information
since, by being raw material in the production of wines and fruit
juices, they can influence the quality of final products. Thus, the
present study aimed to identify and quantify, for the first time and
in detail, the phenolic compounds present in the skin, flesh and
seeds of BRS Violeta grape using methodology HPLC–DAD–ESI-MS/
MS. The pool of compounds considered in this study includes the
most important phenolic types with regard to the main sensory char-
acteristics (color, bitterness, and astringency) and possible biological
effects (e.g., antioxidant capacity) of grapes and their products (grape
juice and wine): anthocyanins, flavonols, hydroxycinnamic acid deriva-
tives, stilbenes, flavan-3-ol monomers and dimers, and proanthocyanins.
2. Material and methods
2.1. Chemicals
All solvents were of HPLC quality, and all chemicals were analytical
grade (N 99%). Water was of Milli-Q quality. The following commercial
standards from Phytolab (Vestenbergsgreuth, Germany) were used:
malvidin 3-glucoside, malvidin 3,5-diglucoside, peonidin 3,5-diglucoside,
caffeic acid, p-coumaric acid, trans-caftaric acid, trans-piceid, (−)-
epigallocatechin, and (−)-gallocatechin. Commercial standards from
Extrasynthese (Genay, France) were used: cyanidin 3-glucoside,
cyanidin 3,5-diglucoside, procyanidins B1 and B2, kaempferol,
quercetin, isorhamnetin, myricetin, syringetin, and the 3-glucosides of
kaempferol, quercetin, isorhamnetin, and syringetin. The following
commercial standards from Sigma (Tres Cantos, Madrid, Spain) were
used: gallic acid, trans-resveratrol, (+)-catechin, (−)-epicatechin, (−)-
355
epicatechin 3-gallate, and (−)-gallocatechin 3-gallate. Some other
non-commercial flavonol standards (myricetin 3-glucoside, quercetin
3-glucuronide, and laricitrin 3-glucoside) were previously isolated
from Petit Verdot grape skins (Castillo-Muñoz, Gómez-Alonso, et al.,
2009). Finally, a sample of procyanidin B4 was kindly supplied by
Prof. Fernando Zamora (Department of Biochemistry and Biotechnolo-
gy, Universitat Rovira i Virgili, Spain). The trans isomers of resveratrol
and its 3-glucoside (piceid) were transformed into their respective cis
isomers by UV-irradiation (366 nm light during 5 min in quartz vials)
of 25% MeOH solutions of the trans isomers.
All standards were used for identification. Quantification (in mg
per kg of grape) was done as equivalents of the most representative
compounds for each family of phenolic compounds: malvidin 3,5-
diglucoside was used for anthocyanidin 3,5-diglucosides; caffeic acid
for hydroxycinnamic acid derivatives; quercetin 3-glucoside for flavo-
nol 3-glycosides and their free aglycones; (+)-catechin for polymeric
flavan-3-ols (total proanthocyanidins); individual flavan-3-ol mono-
mers and dimers by their corresponding standards, but their total
sum as (+)-catechin equivalents.
2.2. Grapes
During the 2011 harvest season, healthy BRS Violeta grapes were
collected at optimum ripeness for harvesting in the municipal district
of São Roque (São Paulo, Brazil), which lies at 23° 31′44″ S and 47° 08′
06″ W, and 771 m above sea level (referred to datum WGS84, World
Geodetic System 1984) and which has a subtropical climate (maximum,
23.1 °C; minimum, 15.5 °C). Three batches of grape clusters (5 kg each)
were sampled following a zigzag path between two marked rows of
vines in three different zones of the vineyard. Once in the lab, grape
berries from each batch were randomly separated by picking berries
from the top, central, and bottom parts of every cluster. The picked
grapes were bulked and separated in two subsamples of ca. 100 g
each. Every grape subsample was used for all the different classes of
analysis, which were made in triplicate for each one. The results were
given as the average of the data obtained for all the grape batches
(n = 3). The sampled grapes showed the following characteristics,
measured following the official analysis methods proposed by OIV
(2011): sugar content, 19.75 ± 0.35 °Brix; total acidity, 11.13 ±
0.19 g/kg, as tartaric acid; pH, 3.75 ± 0.07; berry diameter, 15.7 mm;
berry weight, 1.93 ± 0.4 g.
2.3. Sample preparation
The three samples (ca. 100 g) of grapes were manually and care-
fully peeled and the resulting skins (yield, 46% of fresh grape weight)
were immediately frozen (− 80 °C for 12 h) and then freeze-dried for
24 h and weighed. The dried skins were homogenized in a porcelain
mortar with the aid of a pestle, weighed and used for analysis. The
samples (ca. 0.25 g) were immerged in 25 mL of a solvent mixture
of methanol, water, and formic acid (50:48.5:1.5 v/v) and subjected
to an ultrasonic bar for 10 min. Samples were then centrifuged at
2500 g at 5 °C for 5 min. A second extraction of the resulting pellets
was made using the same volume of the solvent mixture (25 mL). Al-
iquots of joined skin extracts were diluted with HCl 0.1 N (1:10, v/v),
filtered (0.20 μm, polyester membrane, Chromafil PET 20/25, Macherey-
Nagel, Düren, Germany) and directly injected onto the HPLC system for
anthocyanin determination.
The peeled berries were finger-pressed to separate the flesh
(yield, 52% of fresh grape weight) and the seeds (yield, 2% of fresh
grape weight). The separated flesh was immediately homogenized
with 100 mL of a solvent mixture of methanol, water, and formic
acid (70:28.5:1.5, v/v), thus avoiding oxidation. Then maintained
under an ultrasonic bath for 10 min, and the flesh extract was
356 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366
centrifuged at 2500 g at 5 °C for 10 min. The supernatant was sepa-
rately dried in a rotary evaporator (35 °C) and its volume was made
up to 100 mL with water. The separated seeds (ca. 1 g) were crushed
and homogenized (Heidolph DIAX 900) after immersion in 50 mL of
the solvent mixture of methanol, water, and formic acid (50:48.5:1.5
v/v) during 3 min and then centrifuged at 2500 g at 5 °C for 10 min; a
second extraction was repeated with the resulting pellets and both
supernatants were combined and stored at −18 °C until needed. The
necessary extraction steps for quantitative extraction of phenolic com-
pound from the different samples of skins, flesh and seeds were deter-
mined by chromatographic analysis of up to five successive extraction
steps.
ECX SPE cartridges (40 μm, 500 mg, 6 mL; Scharlab, Sentmenat,
Barcelona, Spain) allowed the isolation of non-anthocyanin phenolic
compounds from BRS Violeta grape skin and flesh extracts, and
these anthocyanin-free fractions were used to analyze flavonols and
hydroxycinnamic acid derivatives. To carry out this step, 3 mL of
grape skin and flesh extracts were diluted with 9 mL of HCl 0.1 N,
and the prepared samples were then passed through the SPE car-
tridges that had been previously conditioned with 5 mL of methanol
and 5 mL of water. After the cartridges were washed with 5 mL of
HCl 0.1 N acid and 5 mL of water, the anthocyanin-free fraction was
eluted with 3 × 5 mL of methanol. This eluate was dried in a rotary
evaporator (35 °C) and re-dissolved in 1 mL of 20% methanol in
water and directly injected onto the HPLC equipment.
Finally, the flavan-3-ols (monomers, B-type dimers, and polymeric
proanthocyanidins) and stilbenes were isolated from BRS Violeta
grape skin, flesh and seed extracts using SPE on C18 cartridges
(Sep-Pak Plus C18, Waters Corp., Mildford, MA; cartridges filled
with 820 mg of adsorbent). A mixture of 2 mL of each extract with
12 mL of water was then passed through the C18 cartridge, which
had been previously conditioned with methanol (5 mL) and water
(5 mL); after the cartridge was dried under reduced pressure, methanol
(15 mL) and ethyl acetate (5 mL) were added in order to recover
adsorbed phenolics; after the solvent was evaporated in a rotary evap-
orator (35 °C), the residue was dissolved in methanol (2 mL) and
stored at −18 °C until needed.
2.4. HPLC–DAD–ESI-MSn identification of BRS Violeta grape phenolic
compounds
Anthocyanins and non-anthocyanin phenolic compounds from
grape skin and flesh were separately analyzed after adaptation of
previously described methods (Castillo-Muñoz, Gómez-Alonso, García-
Romero, & Hermosín-Gutiérrez, 2007; Castillo-Muñoz, Gómez-Alonso,
et al., 2009) to the use of narrow bore, smaller particle size, chromatog-
raphy columns. For the analysis of anthocyanins, 10 μL of diluted ex-
tracts was injected, whereas 20 μL of anthocyanin-free extract fractions
was used for the analysis of non-anthocyanin phenolics. The injections
were made after filtration (0.20 μm, polyester membrane, Chromafil
PET 20/25, Machery-Nagel, Düren, Germany) on a reversed-phase col-
umn Zorbax Eclipse XDB-C18 (2.1 × 150 mm; 3.5 μm particle; Agilent,
Germany), thermostated at 40 °C. Flow rate was 0.19 mL/min. The sol-
vents were: solvent A (acetonitrile/water/formic acid, 3:88.5:8.5, v/v/
v), solvent B (acetonitrile/water/formic acid, 50:41.5:8.5, v/v/v), and sol-
vent C (methanol/water/formic acid, 90:1.5:8.5, v/v/v). The linear sol-
vents' gradient for anthocyanin analysis was: zero min, 94% A and 6%
B; 10 min, 70% A and 30% B; 30 min, 50% A and 50% B; 34 min, 100%
B; 36 min, 100% B; 42 min, 96% A and 4% B; 50 min, 96% A and 4% B.
The linear solvents' gradient for non-anthocyanin analysis was: zero
min, 98% A and 2% B; 8 min, 96% A and 4% B; 37 min, 70% A, 17% B and
13% C; 51 min, 50% A, 30% B and 20% C; 51.5 min, 30% A, 40% B and
30% C; 56 min, 50% B and 50% C; 57 min, 50% B and 50% C; 64 min,
96% A and 4% B. For identification, Ion Trap ESI-MS/MS detector was
used in both positive (anthocyanins) and negative (flavonols and
hydroxycinnamic acid derivatives) ion modes, setting the following pa-
rameters: dry gas, N2, 8 L/min; drying temperature, 325 °C; nebulizer,
N2, 50 psi; ionization and fragmentation parameters were optimized
by direct infusion of appropriate standard solutions (malvidin 3,5-
diglucoside in positive ionization mode; quercetin 3-glucoside and
caftaric acid in negative ionization mode); scan range, 50–1200 m/z.
Identification was mainly based on spectroscopic data (UV–vis and
MS/MS) obtained from authentic standards or previously reported
(Castillo-Muñoz, Gómez-Alonso, et al., 2009; Lago-Vanzela, Da-Silva,
Gomes, García-Romero, & Hermosín-Gutiérrez, 2011a, 2011b). For
quantification, DAD-chromatograms were extracted at 520 (anthocya-
nins), 360 (flavonols) and 320 nm (hydroxycinnamic acid derivatives).
In the case of overlapping DAD peaks (peaks A–F in Figs. 1–4), quantifi-
cation was made with the help of extracted ion chromatograms at the
m/z values of each overlapping compound: the EIC integral value was
used for an estimation of contribution of each overlapping compound
to the joint DAD peak. Analyses were performed in triplicate.
2.5. Identification and quantification of BRS Violeta grape skin, flesh and
seeds flavan-3-ols and grape skin stilbenes using multiple reaction
monitoring HPLC–ESI-MS/MS
In the analysis of flavan-3-ol monomers and B-type dimer pro-
cyanidins from grape skin and seeds, and stilbenes from grape skin,
0.25 mL of the SPE-C18 extract was diluted with 4.75 mL of water:
formic acid (98.5:1.5) in a chromatographic vial that was sealed, and
the extract was then injected.
The structural information of proanthocyanidins was obtained
following the method of acid-catalyzed depolymerization induced by
pyrogallol, a recently proposed alternative nucleophile trapping agent
that offers similar results when compared to the classic phloroglucinol
method, but which also functions under milder experimental condi-
tions (Bordiga, Coïsson, Locatelli, Arlorio, & Travaglia, 2013; Bordiga et
al., 2009). In this study, 0.50 mL of pyrogallol reagent solution (100 g/L
of pyrogallol and 20 g/L ascorbic acid in methanolic 0.3 N HCl) was
added to 0.25 mL of SPE-C18 extract, and the mixture was then
maintained at 30 °C for 40 min. After the reaction was finalized with
the addition of 2.25 mL of 67 mM sodium acetate and 2 mL of water,
the reaction mixture was then injected.
The analysis was performed using an Agilent 1200 series system
equipped with DAD (Agilent, Germany), and coupled to an AB Sciex
3200 Q TRAP (Applied Biosystems) electrospray ionization mass spec-
trometry system (ESI-MS/MS). The chromatographic system was man-
aged by the Agilent ChemStation (version B.01.03) data-processing
station. The mass spectra data was processed with the Analyst MSD
software (Applied Biosystems, version 1.5). The samples (before and
after acid-catalyzed depolymerization reaction) were injected (10 μL)
on a reversed-phase column Agilent Eclipse XDB-C18 (2.1 × 150 mm;
3.5 μm particle; Agilent, Germany), thermostated at 16 °C. The solvents
used were water/methanol/formic acid (97:2:1, v/v/v, solvent A) and
methanol (solvent B), and the flow rate was 0.1 mL/min. The linear gra-
dient for solvent B was as follows: 0 min, 5%; 2 min, 5%; 25 min, 30%;
40 min, 55%; 50 min, 65%; 55 min, 95%; 65 min, 95%; 70 min, 5%; and
80 min, 5%. Two MS scan types were used: enhanced MS (EMS) for
compound identification; and multiple reaction monitoring (MRM)
for quantification. MS conditions for both scan types were as follows:
ion spray voltage, −4000 V; ion source temperature, 500 °C; collision
gas, high; curtain gas, 15 psi; ion source gas 1, 50 (arbitrary units);
ion source gas 2, 50 (arbitrary units); declustering potential, −35 V;
entrance potential, −10 V; collision energy, −30 V; and collision cell
exit potential, −3 V. Data of initial concentrations of flavan-3-ol mono-
mers, obtained before depolymerization reaction, was used for correc-
tion of the concentrations of released flavan-3-ol monomers (terminal
subunits of polymeric proanthocyanidins) during the depolymerization
reaction of proanthocyanidins.
 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366 357

A)
mAU 11+A

250 1

200
5+D

150
4
3 13+C
22
100 2
12+B
18 9
50 6 20 21 10 15+E
7 8 16 17 23
19 14 24 25
0

5 10 15 20 25 min

B)
mAU

12 peak16 (dp-3-cfglc-5-glc)
peak17 (cy-3-cfglc-5-glc)
10

300 350 400 450 500 550 nm

Fig. 1. Anthocyanins of BRS Violeta grape skin: A) HPLC–DAD chromatogram (detection at 520 nm); B) on-line DAD–UV–vis spectra of the newly described delphinidin
3-(6″-caffeoyl)-glucoside-5-glucoside (dp-3cfglc-5-glc) and cyanidin 3-(6″-caffeoyl)-glucoside-5-glucoside (cy-3cfglc-5-glc). For peak assignation see Table 1.

For the identification and quantification of diverse flavan-3-ols
and stilbenes we used standards of: the monomers (+)-catechin,
(−)-epicatechin, (−)-epigallocatechin, (−)-gallocatechin, and (−)-
epicatechin 3-gallate; the dimers procyanidins B1, B2 and B4; and
the trans and cis isomers of resveratrol and its 3-glucoside (piceid).
The total polymeric proanthocyanidin content was also quantified
as equivalents of (+)-catechin, and their structural features were
characterized (molar percentage of extension and terminal subunits;
mean degree of polymerization, mDP; molar percentage of galloylation;
and molar percentage of prodelphinidins). We followed a previously
developed method based on the use of the — EMS (enhanced mass
spectrum; MS conditions) scan mode for identification, MRM (multiple
reaction monitoring; MS/MS conditions) scan mode for quantification,
(+-catechin as external standard, and acid-catalyzed depolymerization
induced by pyrogallol for structural characterization of proanthocyanidins
(Lago-Vanzela et al., 2011a, 2011b). The identification and quantification
of stilbenes (trans and cis isomers of resveratrol and piceid) were made
from the extracted ion chromatograms obtained by MRM after selection
of the following characteristic m/z transitions: 389-227 for piceid isomers;
and 227-185 for resveratrol isomers.
3. Results and discussion
3.1. Anthocyanins
The content of anthocyanins of BRS Violeta grapes was high (ca.
3950 mg/kg of grape, as malvidin 3,5-diglucoside equivalents) and
mainly occurred in their skin (99.57%), but they were also found as
minor compounds in their flesh. Total anthocyanin content reported
for other non-vinifera grape cultivars (mg/kg, as malvidin 3,5-
diglucosides) are lower: Bordô (Vitis labrusca), 1360 (Lago-Vanzela
et al., 2011b); Concord (V. labrusca), Marechal Foch (Vitis rupestris ×
Vitis vinifera) and Norton (Vitis aestivalis), 1116–2750 (Muñoz-
Espada, Wood, Bordelon, & Watkins, 2004); Folha de Figo (V. labrusca)
and Niágara Rosada (V. labrusca), 61–1550 (in this case mg/kg,
as cyanidin equivalents, which correspond to 146–3779 mg/kg of
malvidin 3,5-diglucoside equivalents) (Abe et al., 2007). In contrast,
other hybrid teinturier grape varieties such as Colobel and Lomanto
and their offspring are seemingly higher in anthocyanins content
than Violeta grapes (mean values ranging 4210–6030 mg anthocya-
nin/kg berry) (Mazza, 1995).
358 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366

Intens.
x106 789.3 +MS, 15.7min #576
1.5 A)
1.0

0.5
0.0
x105 627.1 +MS2(789.7), 15.7min #577
5
4
3
2 303.0
1 465.1
0
200 400 600 800 1000 m/z
x106
1.5 773.4 +MS, 16.9min #620
B)
1.0

0.5

0.0
x105 611.1 +MS2(773.6), 16.9min #621
6

2 287.0
449.1
0
200 400 600 800 1000 m/z
x107
773.3 +MS, 17.5min #654
1.0 C)
0.8
0.6
0.4
0.2 803.3
0.0
x105 641.1 +MS2(803.7), 17.5min #655
3

2
1 317.1
479.1
0
200 400 600 800 1000 m/z
x107 757.4 +MS, 18.9min #706
0.8 D)
0.6
0.4
0.2
787.3
0.0
x105 625.1 +MS2(787.5), 18.9min #707
2.0
1.5
1.0
0.5 301.1 463.2
0.0
200 400 600 800 1000 m/z

x10 6 787.4 +MS, 19.7min #732

6 E)
4

2 817.3

0
655.1 +MS2(817.6), 19.7min #733
x105
2.5
2.0
1.5
1.0 331.1
0.5 493.1
0.0
200 400 600 800 1000 m/z

Fig. 2. MS and MS/MS spectra of the suggested anthocyanidin 3-(6″-caffeoyl)-glucoside-5-glucosides found in BRS Violeta grape skins, derived from the anthocyanidins:
A), delphinidin; B), cyanidin; C), petunidin; D), peonidin; and E), malvidin.
 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366 359

Intens.
11+A
mAU A) DAD, 520 nm
300

200
16 17 12+B 13+C
100

0
x105
6 16 B) EIC 789 +AllMS
4

0
x107
11
1.00
C) EIC 773 +AllMS
0.75
0.50
17
0.25
0.00
x105
4
D) EIC 803 +AllMS A

3
2
1
0
x106
3 12
E) EIC 757 +AllMS
2

0
x106
13
4 F) EIC 787 +AllMS
3
2
1 B

0
x105
C
G) EIC 817 +AllMS
2

0
15 16 17 18 19 20 Time [min]

Fig. 3. Caffeoylated anthocyanins in BRS Violeta grape skin: A) HPLC–DAD chromatogram (detection at 520 nm); and selected extracted ion chromatograms (EIC) at m/z values
corresponding to the molecular ions of 3-(6″-caffeoyl)-glucoside-5-glucoside derivatives (3-cfglc-5-glc) and some co-eluting 3-(6″-p-coumaroyl)-glucoside-5-glucoside deriva-
tives (3-cmglc-5-glc); B) EIC at m/z 789 (delphinidin 3-cfglc-5-glc); C) EIC at m/z 773 (cyanidin 3-cfglc-5-glc and delphinidin 3-cmglc-5-glc); D) EIC at m/z 803 (petunidin
3-cfglc-5-glc); E) EIC at m/z 757 (cyanidin 3-cmglc-5-glc); F) EIC at m/z 787 (delphinidin 3-cfglc-5-glc and petunidin 3-cmglc-5-glc); G) EIC at m/z 817 (delphinidin 3-cfglc-5-glc).
For peak assignation see Table 1.

Another remarkable difference between skin and flesh anthocya-
nins of BRS Violeta grapes was found in their respective anthocyanin
molar profiles (Table 1) including the lack of many of the minor indi-
vidual anthocyanins in the case of grape flesh. The aforementioned
findings were in agreement with reported data dealing with other
teinturier red grapes, as the V. vinifera L. cultivars Garnacha Tintorera
and BRS Morena (Castillo-Muñoz, Fernández-González, Gómez-Alonso,
García-Romero, & Hermosín-Gutiérrez, 2009; Lago-Vanzela et al.,
2011a). The typical chromatographic profile of BRS Violeta grape
skin anthocyanins showed a predominance of anthocyanidin 3,5-
diglucosides (Fig. 1A), as can be expected for American non-vinifera
grape cultivars and their hybrids (Lago-Vanzela et al., 2011b; Wang,
Race, & Shrikhande, 2003; Zhu, Zhang, & Lu, 2012). Anthocyanidin
3-glucosides are the only kind of anthocyanins present in V. vinifera
grape cultivars and their intraspecific hybrids (Hermosín-Gutiérrez &
García-Romero, 2004; Lago-Vanzela et al., 2011a) and they are also
360 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366

mAU 28

175

150

125

100

75

50 31

27 30
25 F+32
26 29 33
34
0

20 25 30 35 40 min

Fig. 4. HPLC–DAD chromatogram (detection at 360 nm) corresponding to the flavonols found in BRS Violeta grape skins. For peak assignation see Table 2.

found in American non-vinifera grapes and their hybrids (Lago-Vanzela
et al., 2011b; Wang et al., 2003; Zhu et al., 2012). The occurrence of
anthocyanidin 3,5-diglucosides is a quality indicator used in distin-
guishing vinifera from non-vinifera grapes and their products. For
marketing and import purposes, many European countries test for
diglucoside content, with wine testing above a specified limit (e.g.,
15 mg/L) being prohibited for sale (OIV, 2011). In the case of BRS
Violeta grape the proportion of anthocyanidin 3-glucosides was low,
10.25% in the skin and 1.27% in the flesh. The chromatographic and
spectral characteristics (molecular and product ions generated under
ESI-MS/MS, on-line DAD UV–vis maximum absorbance wavelengths,
and retention times) of BRS Violeta grape anthocyanins are presented
in Table 1 together with the molar profiles for both skin and flesh.
The identification of each anthocyanin was tentatively based on the
comparison of spectroscopic data, mainly MS/MS spectra data, with
those obtained from authentic standards or previously reported (Lago-
Vanzela et al., 2011b; Nixdorf & Hermosín-Gutiérrez, 2010). The usual
grape anthocyanidin structures, namely, delphinidin, cyanidin, petunidin,
peonidin, and malvidin were identified (the product ions at m/z values of
303, 287, 317, 301, and 331, respectively, were detected in the MS/MS

Table 1
Chromatographic and spectroscopic (UV–vis and MS/MS spectra) characteristics of the anthocyanins identified in BRS Violeta grape by HPLC–DAD–ESI-MS/MS (positive ionization
mode), molar proportions (mean value ± standard deviation, n = 3), and total concentration (as malvidin 3,5-diglucoside equivalents), in both parts of fruit (skin and flesh). Peak
numbers as in Fig. 1.

Peak Assignationa Rt (min) UV–vis (nm) Molecular ion; product ions (m/z) % molar (skin) % molar (flesh)

1 dp-3,5-diglc 4.73 274, 295 (sh), 342, 400 (sh), 522 627; 465, 303 17.99 ± 0.59 NQ
2 cy-3,5-diglc 6.80 277, 290 (sh), 330 (sh), 375 (sh), 514 611; 449, 287 7.99 ± 0.26 7.44 ± 0.22
3 pt-3,5-diglc 9.21 274, 295 (sh), 342, 400 (sh), 524 641; 479, 317 14.11 ± 0.27 NQ
4 pn-3,5-diglc 11.38 277, 290 (sh), 330 (sh), 375 (sh), 516 625; 463, 301 8.80 ± 0.42 44.60 ± 1.08
5 mv-3,5-diglc 12.46 274, 295 (sh), 342, 400 (sh), 526 655; 493, 331 14.03 ± 0.45 34.79 ± 0.69
6 dp-3-acglc-5-glc 12.20 274, 295 (sh), 342, 400 (sh), 525 669; 507, 465, 303 0.64 ± 0.01 ND
7 cy-3-acglc-5-glc 13.73 ~516 653; 491, 449, 287 0.08 ± 0.00 ND
8 pt-3-acglc-5-glc 14.58 ~529 683; 521, 479, 317 0.25 ± 0.01 ND
9 pn-3-acglc-5-glc 15.50 NR 667; 505, 463, 301 NQ ND
10 mv-3-acglc-5-glc 16.62 274, 295 (sh), 342, 400 (sh), 525 697; 535, 493, 331 0.07 ± 0.00 ND
11 dp-3-cmglc-5-glc 17.49 279, 298, 316 (sh), 529 773; 611, 465, 303 13.86 ± 0.28 2.12 ± 0.19
12 cy-3-cmglc-5-glc 18.91 280, 290 (sh), 312, 521 757; 595, 449, 287 3.48 ± 0.19 2.76 ± 0.38
13 pt-3-cmglc-5-glc 19.67 279, 298, 316 (sh), 531 787; 625, 479, 317 4.29 ± 0.31 1.34 ± 0.17
14 pn-3-cmglc-5-glc 21.61 280, 290 (sh), 312, 521 771; 609, 463, 301 0.98 ± 0.02 2.08 ± 0.05
15 mv-3-cmglc-5-glc 22.27 280, 298, 312 (sh), 534 801; 639, 493, 331 1.83 ± 0.04 3.59 ± 0.29
16 dp-3-cfglc-5-glc 15.70 278, 298 (sh), 328 (sh), 532 789; 627, 465, 303 0.57 ± 0.03 ND
17 cy-3-cfglc-5-glc 16.89 281, 294 (sh), 322, 521 773; 611, 449, 287 0.30 ± 0.00 ND
A pt-3-cfglc-5-glc 17.49 NR 803; 641, 479, 317 0.29 ± 0.01 ND
B pn-3-cfglc-5-glc 18.91 NR 787; 625, 463, 301 NQ ND
C mv-3-cfglc-5-glc 19.67 NR 817; 655, 493, 331 0.19 ± 0.01 ND
18 dp-3-glc 7.81 277, 298 (sh), 346, 440 (sh), 524 465; 303 3.72 ± 0.30 1.19 ± 0.19
19 cy-3-glc 10.58 280, 292 (sh), 325 (sh), 380 (sh), 440 (sh), 517 449; 287 0.62 ± 0.02 0.08 ± 0.01
D pt-3-glc 12.46 NR 479; 317 0.15 ± 0.01 NQ
20 pn-3-glc 14.04 280, 292 (sh), 325 (sh), 380 (sh), 440 (sh), 518 463; 301 0.07 ± 0.01 NQ
21 mv-3-glc 15.14 276, 298 (sh), 398, 440 (sh), 528 493; 331 0.25 ± 0.01 NQ
22 dp-3-cmglc 20.12 282, 298 (sh), 316 (sh), 440 (sh), 530 611; 303 4.00 ± 0.12 ND
E cy-3-cmglc 22.27 NR 595; 287 0.94 ± 0.02 ND
23 pt-3-cmglc 23.46 282, 298 (sh), 316 (sh), 440 (sh), 531 625; 317 0.37 ± 0.02 ND
24 pn-3-cmglc 26.21 283, 313, 440 (sh), 521 609; 301 0.05 ± 0.00 ND
25 mv-3-cmglc 27.22 284, 298 (sh), 316 (sh), 440 (sh), 532 639; 331 0.08 ± 0.01 ND
Total (mg/kg grape) 3932.4 ± 292.4 16.9 ± 2.9
a
Nomenclature abbreviations: dp, delphinidin; cy, cyanidin; pt, petunidin; pn, peonidin; mv, malvidin; glc, glucoside; diglc, diglucoside; acglc, 6″-acetyl-glucoside; cmglc,
6″-p-coumaroyl-glucoside; cfglc, 6″-caffeoyl-glucoside; NR, non-registered; ND, non-detectable; NQ, detectable but non-quantifiable.
 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366
spectra). Pelargonidin-based anthocyanins were not found, as indicated
the absence of product ion signals at m/z 271 (Lago-Vanzela et al.,
2011b). The assignation of 3-glucoside and 3,5-diglucoside substitution
patterns was made on the basis of the fragmentation patterns observed
in the MS/MS spectra: only one product ion signal for 3-glucosides (the
entire glucoside moiety was lost under MS/MS ion trap conditions inde-
pendently of whether the glucose was acylated or not); two or more
product ion signals indicated the occurrence of 3,5-diglucosides (a set of
product ion signals were produced by the independent and consecutive
losses of the acylated or not 3-glucoside moiety, and the non-acylated
5-glucoside moiety). We assumed that glucose was the sugar linked in
both 3 and 5 glycosylation positions and that only acylated derivatives
could occur in the glucose unit linked to the C-3 position of antho-
cyanidins (Lago-Vanzela et al., 2011b; Nixdorf & Hermosín-Gutiérrez,
2010; Wang et al., 2003).
The tentative assignation of occurring anthocyanins in BRS Violeta
grape comprised six complete series for the five identified antho-
cyanidins: the non-acylated 3,5-diglucosides and their corresponding
acetyl, p-coumaroyl, and caffeoyl derivatives; and the non-acylated
3-glucosides and their p-coumaroyl derivatives. In the case of grape
flesh, only the complete series of non-acylated anthocyanidin 3,5-
diglucosides and their p-coumaroyl derivatives, together with the
non-acylated 3-glucosides, were detected. It is remarkable that antho-
cyanidin 3-(6″-caffeoyl)-glucoside-5-glucosides had not been previous-
ly reported in grapes and we have obtained spectroscopic evidence for
suggesting the occurrence of that complete series in Violeta grapes
(Table 1). On one hand, the UV–vis spectra of the most abundant peak
16, tentatively assigned as delphinidin 3-(6″-caffeoyl)-glucoside-5-
glucoside, showed the expected features on the basis of its suggested
structure (Fig. 1B): a visible maximum at 532 nm (attributable to B-ring
tri-substitution pattern), no shoulder around 440 nm (3,5-diglucoside,
instead 3-glucoside that shows this shoulder), and a shoulder at
328 nm attributable to the caffeoyl moiety; similarly, the compound elut-
ing under peak 17 was tentatively assigned as cyanidin 3-(6″-caffeoyl)-
glucoside-5-glucoside, showing a visible maximum at 521 nm (B-ring
di-substitution pattern), absence of shoulder at 440 nm (3,5-diglucoside),
and maximum at 322 nm (caffeoyl residue); for the rest of compounds
of this series, the co-elution with other more abundant anthocyanins
made not possible getting any information about their UV–vis spectra.
On the other hand, the MS spectra showed the signal of the expected
molecular ions (m/z 789, 773, 803, 787, and 817 for the respective de-
rivatives of delphinidin, cyanidin, petunidin, peonidin, and malvidin)
and the MS/MS spectra always showed three ion product signals
(Fig. 2), generated by independent and consecutive neutral fragment
losses of 162 (glucose moiety linked to C-5) and 324 units (caffeoyl-
glucose moiety linked to C-3). As the 3-(6″-caffeoyl)-glucoside-5-
glucoside derivatives of petunidin, peonidin, and malvidin (peaks A, B,
and C, respectively) co-eluted with other more abundant chromato-
graphic peaks (11, 12, and 13, respectively), their quantification was
made from the extracted ion chromatograms (Fig. 3) at the m/z values
of their expected molecular ions (803, 787, and 817, respectively).
Non-acylated derivatives were the main anthocyanin type for
3,5-diglucosides (62.92% in skin and 86.83% in flesh). Within acylated an-
thocyanins, the p-coumaroyl derivatives dominated in 3,5-diglucosides
by 24.44% in skin and 11.89% in flesh (they were the only detected
acylated derivatives in grape flesh). In the case of 3-glucosides, the
p-coumaroyl derivatives slightly dominated over the non-acylated
ones (5.44% vs. 4.81%) in the skin, and only non-acylated derivatives
were found in the flesh. The dominant anthocyanidin structures with-
in 3,5-diglucoside derivatives were the B-ring tri-substituted ones
(delphinidin, petunidin, and malvidin; joint average proportion around
68%) with predominance of delphinidin-based anthocyanidins, especial-
ly in the acylated derivatives. The anthocyanin profile showed by Violeta
grape was similar to previously reported anthocyanin profiles of other
non-vinifera or hybrid grape cultivars and their wines, and were very dif-
ferent to the predominance of malvidin-based anthocyanins usually
361
found in V. vinifera grapes and wines (Hermosín-Gutiérrez & García-
Romero, 2004; Lago-Vanzela et al., 2011a, 2011b; Nixdorf & Hermosín-
Gutiérrez, 2010; Wang et al., 2003). Similarly, the anthocyanin profile
of anthocyanidin 3-glucosides of Violeta wine was dominated by B-ring
tri-substituted anthocyanidin with predominance of delphinidin-based
anthocyanins. In contrast, the profile of anthocyanidin 3,5-diglucosides
showed by grape flesh differed mainly in a decrease of the importance
of delphinidin-based anthocyanins and an increase of the malvidin-
and peonidin-based anthocyanins (Table 1).
In summary, the most remarkable features found in hybrid BRS
Violeta grapes with regard to their composition in anthocyanins
were their high concentration and the prevalence of anthocyanidins
3,5-diglucoside structures that are p-coumaroylated in an important
extent. Making comparison with another interesting grape cultivar
studied by our group and also used in Brazil, the non-tenturier
V. labrusca L. Bordô grape, BRS Violeta grape accounted for a three-fold
anthocyanin content (3950 mg/kg, mainly located in the skins, vs.
1360 mg/kg in Bordô); in addition, Violeta grape skins accounted for
similar proportions of the predominant 3,5-diglucosides (89.8% vs.
89.9% in Bordô), but the proportion of non-acylated anthocyanidin
3,5-diglucosides was higher (62.9% vs. 41.2% in Bordô) and, conse-
quently, the proportion of their p-coumaroylated derivatives was lower
(24.4% vs. 47.7% in Bordô). Concerning the use of Violeta grapes for
elaboration of red wines, it has to bear in mind that anthocyanidin
3,5-diglucosides are less reactive than anthocyanidin 3-glucosides, the
only type of anthocyanins occurring in V. vinifera grape cultivars, espe-
cially with regard to formation of anthocyanin–tannin adducts (the
position C-4 of the anthocyanins is spatially hindered for those reactions)
and pyranoanthocyanins (it is needed that the hydroxyl group attached
to C-5 was free) (Monagas & Bartolomé, 2009). Moreover, the
p-coumaroylation of anthocyanins likely reinforce their stability by
means of intramolecular copigmentation and can also be involved in
processes of intermolecular copigmentation, thus improving the red
color characteristics of the future wine. Therefore, Violeta grapes could
be suggested as an important source of anthocyanins that should give
rise to intensely colored grape juices and young red wines. In this con-
text, the possibility of using Violeta grapes for making wines able for fur-
ther aging (high-quality red wines) seems to be initially less useful and
needs further study. In particular, Violeta anthocyanin 3,5-diglucosides
and their subsequent expression in wine color, could likely follow dif-
ferent reaction pathways during wine aging than those deeply studied
for V. vinifera wines (Monagas & Bartolomé, 2009; Waterhouse &
Kennedy, 2004). Finally, as anthocyanins are transferred from grape
to wine during winemaking in relatively low proportion (average of
40%; Boulton, 2001) it is obvious that grape pomace will still contain a
high amount of anthocyanins and that their recovery should be of great
interest to food industry.
3.2. Flavonols
Flavonols were found mainly in the skins of BRS Violeta grapes
(153.4 ± 30.7 mg/kg of grape, vs. 2.4 ± 2.4 mg/kg in the flesh, as
quercetin 3-glucoside equivalents). The distribution of flavonols be-
tween skin and flesh (Table 2) was similar to that found in the Brazilian
non-vinifera grape cultivar Bordô (Lago-Vanzela et al., 2011b), but their
total content almost doubled that of Bordô grapes (calculated total
content of 335 μmol/kg grape, vs. 154 μmol/kg in Bordô grapes). It
is important to highlight that flavonols are the best cofactors for
copigmentation of anthocyanins in wine and that the higher the content
of grape flavonols the higher the proportion of anthocyanins transferred
to wine during winemaking (Schwarz, Picazo-Bacete, Winterhalter, &
Hermosín-Gutiérrez, 2005).
The flavonol profile of Violeta grape skin (Fig. 4) only showed
3-glycoside derivatives and was dominated by myricetin-based flavo-
nols (average of 74%), being myricetin 3-glucoside the main individual
flavonol found (68.06 ± 0.72%), in a similar way that also reported for
362 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366

Table 2
Chromatographic and spectroscopic (UV–vis and MS/MS spectra) characteristics of the flavonols identified in BRS Violeta grape by HPLC–DAD–ESI-MS/MS (negative ionization
mode), molar proportions (mean value ± standard deviation, n = 3), and total concentration (as quercetin 3-glucoside equivalents), in both parts of fruit (skin and flesh). Peak
numbers as in Fig. 3.

Peak Assignationa Rt (min) UV–vis (nm) Molecular ion; % molar (skin) % molar (flesh)
product ions (m/z)

26 M-3-glcU 19.45 257 (sh), 260, 305 (sh), 354 493; 317 2.69 ± 0.06 8.12 ± 5.19
27 M-3-gal 20.59 ~354 479; 317 1.87 ± 0.22 2.34 ± 2.33
28 M-3-glc 21.08 255 (sh), 261, 305 (sh), 355 479; 317 68.06 ± 0.72 18.39 ± 28.22
F free M 32.05 ~372 317; 317 1.66 ± 0.16 ND
29 Q-3-gal 27.47 ~353 463; 301 1.04 ± 0.12 3.65 ± 1.74
30 Q-3-glcU 27.77 254, 264 (sh), 300 (sh), 353 477; 301 5.82 ± 0.25 4.49 ± 1.68
31 Q-3-glc 29.13 256, 264 (sh), 300 (sh), 354 463; 301 12.79 ± 0.17 39.14 ± 14.37
32 L-3-glc 32.28 255, 262 (sh), 305 (sh), 357 493; 331 5.10 ± 0.26 5.00 ± 2.45
33 I-3-glc 38.84 ~354 477; 315 NQ 8.92 ± 5.92
34 S-3-glc 40.74 253, 264 (sh), 305 (sh), 357 507; 345 0.98 ± 0.05 9.94 ± 5.34
Total (mg/kg grape) 153.4 ± 30.7 2.4 ± 2.4
a
Nomenclature abbreviations: M, myricetin; Q, quercetin; L, laricitrin; I, isorhamnetin; S, syringetin; glcU, glucuronide; glc, glucoside; gal, galactoside; ND, non-detectable.

Bordô grapes (Lago-Vanzela et al., 2011b). The second most important
type of flavonols was based on quercetin (average of 20%; 3-glucoside
derivative, 12.79 ± 0.17%), followed by lower proportions of the 3-
glucosides of laricitrin, syringetin, and isorhamnetin. The 3-glucoside
of isorhamnetin co-eluted with other compound and was not possible
in its quantification in the skin extracts, whereas in flesh extracts that
handicap did not happen. No flavonols based on kaempferol were de-
tected, in contrast to reported data for Bordô grapes and V. vinifera
grape cultivars (Hermosín-Gutiérrez, Castillo-Muñoz, Gómez-Alonso,
& García-Romero, 2011; Lago-Vanzela et al., 2011b). In the case of
V. vinifera red grape cultivars, the proportions of flavonols based on
myricetin and quercetin are usually more balanced and one or other
type of flavonols slightly predominates (Castillo-Muñoz et al., 2007;
Hermosín-Gutiérrez et al., 2011; Mattivi, Guzzon, Vrhovsek, Stefanini,
& Velasco, 2006). With regard to flavonol profile showed by Violeta
grape flesh, it was very variable (high values for standard deviation)
very likely due to the difficulty in determining the exact separation
between flesh and skin that was performed manually. However, the re-
sults suggested that the proportions of myricetin-based and quercetin-
based flavonols interchanged their predominance (29% vs. 47%, respec-
tively). We have observed a higher abundance of triply substituted
flavonols and anthocyanins in the skin of BRS Violeta grape, compared
to the flesh from within the berry, as is commonly reported for grapes.
Transcriptomics analysis of grape has revealed much higher levels of
flavonoid 3′-5′-hydroxylase (F3′5′H) gene expression in grape skins,
perhaps inducible by light (UV) exposure (Falginella et al., 2010). As
there is no (or little) light penetrating to any appreciable depth within
the berry, we would expect this result.
3.3. Hydroxycinnamic acid derivatives (HCAD) and stilbenes
Usual hydroxycinnamic acid derivatives (HCAD) reported in V. vinifera
grapes, namely, trans-caftaric acid, trans- and cis-coutaric acids, and trans-
and cis-fertaric acids, were detected in BRS Violeta grapes. In addi-
tion, three caffeic-type (peaks 36, 37 and 40 in Fig. 5) and three
p-coumaric-type (peaks 41, 42 and 45 in Fig. 5) HCAD were also
found. The spectral characteristics of compounds eluting under peaks
36 and 41 (Table 3) matched with those previously found in Bordô
grapes and were tentatively assigned as 1″-glucose esters of caffeic
and p-coumaric acids, respectively (Lago-Vanzela et al., 2011b). The
other two compounds of each trio (37 and 40, 42 and 45, respectively)
showed spectral characteristics very similar to the initially assigned
HCAD (36 and 41, respectively), thus they were suggested to be isomers
within each trio. The possibilities of isomerization could be related with
the configuration of the double bound of the hydroxycinnamoyl moiety
(trans and cis isomers), the esterification position on the glucose mole-
cule (positional isomers), or even the nature of the hexose (hexose
isomers). One of these newly found compounds, that eluting under
peak 42, was not detected in Violeta grape flesh. The occurrence of
such esters between hydroxycinnamic acids and hexoses is not usually
found, especially among V. vinifera grape varieties and their wines
(Baderschneider & Winterhalter, 2001), and their detection in BRS
Violeta grapes constitutes a new source of HCAD in this variety. HCAD
are involved in anthocyanin copigmentation processes and help antho-
cyanins to be transferred from grape to wine during winemaking
(Schwarz et al., 2005).
As found for the non-vinifera Bordô grape (Lago-Vanzela et al.,
2011b), the main amount of HCAD in hybrid BRS Violeta grape was lo-
cated in the skin (120.2 ± 26.1 mg/kg grape, vs. 13.7 ± 3.8 mg/kg in
the flesh, as caftaric acid equivalents). The total amount of HCAD in
Violeta grape (calculated total content, ca. 429 μmol/kg grape) was
similar to that found in Bordô grape (483 μmol/kg grape) and was
within the usual range reported for V. vinifera grape cultivars (Lago-
Vanzela et al., 2011b). The HCAD profile was slightly dominated by
p-coumaric-type HCAD in Violeta grape skin whereas the caffeic-type
was predominant in the flesh (average of 50.39% and 74.15%, respective-
ly). It is also remarkable that the newly described hydroxycinnamoyl es-
ters with glucose accounted for the half or more of their respective
hydroxycinnamic-type groups in the skin of Violeta grape (22.34% vs.
43.10% for caffeic-type; 36.43% vs. 50.39% for p-coumaric-type) and
that this pattern will be likely transmitted to the wines made with this
grape cultivar.
Finally, grape stilbenes usually can be analyzed in the same
DAD-chromatograms at 320 nm obtained for DAHC analysis. Howev-
er, in the case of Violeta grape samples, those compounds accounted
for concentrations below their quantification thresholds. Using the
MRM LC–MS chromatographic method described for the analysis of
flavan-3-ols monomers and dimers with little adaptations (the inclu-
sion of the characteristic MRM transitions observed for resveratrol
and its 3-glucoside, piceid), it was possible to analyze them. Only the
two isomers of piceid were detected, accounting for 0.058 ± 0.040
(trans isomer) and 0.080 ± 0.042 mg/kg (cis isomer). These stilbene
concentrations were lower than those described for other non-vinifera
grape cultivars, like the 10.91 mg/kg of total resveratrol reported for
V. labrusca Bordô grapes (Lago-Vanzela et al., 2011b). Moreover,
according to the classification proposed for V. vinifera grape cultivars
(Gatto et al., 2008), the total amounts of resveratrol found in the Violeta
grape (total sum of 0.083 ± 0.027 mg/kg, as resveratrol equivalents)
suggest that it could be considered a grape cultivar that is a low resver-
atrol producer (less than 1.8 mg/kg), pending of additional data for
more samples of different vineyards and successive vintages.
3.4. Flavan-3-ol monomers and dimers
Three flavan-3-ol monomers and three B-type dimers were found
in the different parts of analyzed BRS Violeta grape, showing different
 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366 363

mAU 35
A) Violeta grape skin
100
41
36
80

60
38
39 42
40 43
37 45
40 44
20

4 5 6 7 8 9 10 min

mAU 35

B) Violeta grape flesh

250

200

150 36

100
43
50 39 41
37 38 40 45
44
0
4 5 6 7 8 9 10 min

Fig. 5. HPLC–DAD chromatograms (detection at 320 nm) corresponding to the hydroxycinnamic acid derivatives (HCAD) found in BRS Violeta grape: A) grape skin; B) grape flesh.
For peak assignation see Table 3.

concentrations and distribution within these parts (Table 4). Violeta
grape seeds contained 346.3 mg/kg grape, as (+)-catechin equiva-
lents, an amount of around forty-times higher than the content
found in grape skin (8.6 mg/kg). Traces of these compounds were
found in grape flesh (2.0 mg/kg). Seed flavan-3-ol monomers in
Violeta grape were mainly (+)-catechin, followed by lower amounts
of (−)-epicatechin and (−)-epicatechin 3-gallate. (+)-catechin was
also the major monomer found in Violeta grape skin and flesh, to-
gether with only (−)-epicatechin. With regard to flavan-3-ol dimers,
procyanidin B1 was predominant in both grape seeds and skin, but
was absent in grape flesh; procyanidin B2 was the second most im-
portant dimer found in grape seeds and skin, and the only dimer
found in grape flesh; finally, low concentrations of procyanidin B4
were also found in grape seeds. The aforementioned results suggest
that Violeta grape seeds accounted for a relevant amount of bitter
compounds (flavan-3-ols of low molecular weight) and should be
desirable to avoid their transfer from grape to wine during wine-
making, that is, long macerations should be avoided.
3.5. Proanthocyanidins
As could be expected, the seeds of BRS Violeta grape were an im-
portant source of proanthocyanidins, their content ranging within
23944 ± 2680 mg/kg seed, as (+)-catechin equivalents. However,
since seeds represented only the 2% of Violeta grape weight, this value
was reduced up to 478.9 ± 52.8 mg/kg of grape (Table 5). Moreover,
Violeta grape has a very thick skin (representing the 46% of grape
weight) and contributed with a higher amount of proanthocyanidins,
673.0 ± 228.9 mg/kg grape, in comparison with seeds. Reported data
for V. vinifera grape cultivars usually show higher content of pro-
anthocyanidins in the seeds (e.g., 1560–2100 mg/kg grape) than in

Table 3
Chromatographic and spectroscopic (UV–vis and MS/MS spectra) characteristics of the hydroxycinnamic acid derivatives identified in BRS Violeta grape by HPLC–DAD–ESI-MS/MS
(negative ionization mode), molar proportions (mean value ± standard deviation, n = 3), and total concentration (as caftaric acid equivalents), in both parts of fruit (skin and
flesh). ND, non-detectable. Peak numbers as in Fig. 1.

Peak Assignation Rt (min) UV–vis (nm) Molecular ion; product ions (m/z) % molar (skin) % molar (flesh)

35 trans-Caftaric acid 4.03 300 (sh), 328 311; 179, 149, 135 20.76 ± 0.52 50.64 ± 8.79
36 Caffeoyl-glucose-1 4.38 302 (sh), 329 341; 179, 161, 135 15.16 ± 0.18 21.85 ± 2.39
37 Caffeoyl-glucose-2 5.10 302 (sh), 325 341; 179, 161, 135 4.63 ± 0.27 1.04 ± 0.49
38 trans-Coutaric acid 5.74 300 (sh), 313 295; 163, 149, 119 10.52 ± 0.82 1.22 ± 1.18
39 cis-Coutaric acid 5.87 300 (sh), 311 295; 163, 149, 119 3.44 ± 0.27 0.47 ± 0.39
40 Caffeoyl-glucose-3 6.18 302 (sh), 328 341; 179, 161, 135 2.55 ± 0.34 0.61 ± 0.26
41 p-Coumaroyl-glucose-1 6.71 300 (sh), 315 325; 163, 145 22.48 ± 0.14 4.37 ± 1.46
42 p-Coumaroyl-glucose-2 8.18 300 (sh), 313 325; 163, 145 8.24 ± 0.79 ND
43 trans-Fertaric acid 8.33 300 (sh), 326 325; 193, 149 4.70 ± 0.02 14.14 ± 1.64
44 cis-Fertaric acid 8.92 300 (sh), 322 325; 193, 149 1.81 ± 0.27 1.40 ± 0.28
45 p-Coumaroyl-glucose-3 10.40 300 (sh), 312 325; 163, 145 5.70 ± 0.08 4.25 ± 0.69
Total (mg/kg grape) 120.2 ± 26.1 13.7 ± 3.8
364 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366

Table 4 identification allowed us to detect other minor, usually non-reported,

Monomeric flavan-3-ol and B-type procyanidin dimer molar proportions (mean ± subunits (Lago-Vanzela et al., 2011a, 2011b).
standard deviation) in the skin, flesh and seeds of BRS Violeta grape, and total content
(as (+)-catechin equivalents). ND, non-detectable.
The extension subunits that formed Violeta grape skin PA were the
same as those expected in V. vinifera grape (Souquet et al., 1996): (−)-
Flavan-3-ols Skin Flesh Seeds epicatechin accounted for around 73% of total subunits, followed by
(+)-catechin 49.38 ± 2.20 83.49 ± 1.86 86.73 ± 0.28 the characteristic grape skin (−)-epigallocatechin (a prodelphinidin),
(−)-epicatechin 13.55 ± 1.64 10.99 ± 1.94 6.81 ± 0.21 which accounted for approximately 19%; (+)-catechin and (−)-
(−)-epicatechin 3-gallate ND ND 0.21 ± 0.02
epicatechin 3-gallate were minor extension subunits (less than 3%
Procyanidin B1 27.49 ± 3.59 ND 3.65 ± 0.16
Procyanidin B2 9.57 ± 2.84 5.53 ± 0.98 2.30 ± 0.07 each); however, a newly detected prodelphinidin, (−)-gallocatechin,
Procyanidin B4 ND ND 0.30 ± 0.01 accounted for a relevant proportion of approximately 6%. With regard
Total (mg/kg grape) 8.6 ± 1.5 2.0 ± 0.5 346.3 ± 32.6 to the skin PA terminal subunits, most studies have reported only the oc-
currence of (+)-catechin as the main flavan-3-ol and, to a lesser extent,
(−)-epicatechin (Busse-Valverde et al., 2010; Labarbe et al., 1999;
the skin (e.g., 280–720 mg/kg grape) (Busse-Valverde et al., 2010; Souquet et al., 1996; Travaglia et al., 2011). Our results also showed
Travaglia, Bordiga, Locatelli, Coïsson, & Arlorio, 2011). that (+)-catechin was the main terminal subunit (ca. 57%), but two
The results of the structural analysis of the proanthocyanidins prodelphinidins, (−)-epigallocatechin and (−)-gallocatechin accounted
(PA) found in BRS Violeta grape (Table 5) were generally in agree- for similar relevant percentages (ca. 17% each), followed by lower contri-
ment with previously reported data for V. vinifera grape cultivars butions of (−)-epicatechin and (−)-epicatechin 3-gallate (ca. 8% and
(Busse-Valverde et al., 2010; Cosme, Ricardo-Da-Silva, & Laureano, 3%, respectively). Reported data for prodelphinidins in V. vinifera grape
2009; Labarbe, Cheynier, Brossand, Souquet, & Moutounet, 1999; skin are of similar magnitude, 28.5–41.8% (Cosme et al., 2009).
Souquet, Cheynier, Broussaud, & Moutounet, 1996; Travaglia et al., In the case of seeds PA, most reported data for V. vinifera grape
2011). When PA from skin and seeds were compared, the mean de- cultivars indicate that (+)-catechin and (−)-epicatechin are the
gree of polymerization (mDP) was higher in skin PA (mDP, 12.9 vs. main terminal subunits and that (−)-epicatechin predominated over
4.1), the galloylation percentage (% of 3-gallate esters) was higher (+)-catechin within the extension subunits; in both cases, relevant
in seeds PA (5.86% in terminal subunits and 6.20% in extension sub- proportions of characteristic (−)-epicatechin 3-gallate are also found
units; 2.71% and 0.68%, respectively, in skin PA) but their values (Busse-Valverde et al., 2010; Labarbe et al., 1999; Souquet et al., 1996;
were lower than those reported for V. vinifera grape seeds (12.4– Travaglia et al., 2011). In Violeta grape seeds (Table 5), (+)-catechin
19.7%) (Busse-Valverde et al., 2010; Cosme et al., 2009); and the percent- was the main terminal subunit (ca. 69%) whereas (−)-epicatechin
age of B-ring tri-hydroxylated subunits (also known as prodelphinidins) was the main extension subunit (ca. 88%), and characteristic (−)-
was higher in skin PA (33.24% in terminal subunits, and 24.51% in exten- epicatechin 3-gallate accounted for relevant proportions (ca. 6% for
sion subunits; 0.74% and 1.25%, respectively, in the seeds of PA). The little both terminal and extension subunits); minor proportions of (−)-
amount of PA found in the flesh of Violeta grapes showed structural fea- epigallocatechin contributed to terminal and extension subunits (less
tures simultaneously similar to the PA of both skin and seeds: mDP of than 1%) and (−)-gallocatechin also contributed with only 1% to exten-
5.3, closer to that of seeds PA; high galloylation percentage in terminal sion subunits. As previously described, the flesh PA showed a distribu-
subunits, but low value in extension subunits (6.06% vs. 2.38%); and tion of extension and terminal subunits that remembered both skin
high prodelphinidin percentages (29.76% in terminal subunits, and and seeds PA (Table 5). The most remarkable finding was related to
39.95% in extension subunits). The extension and terminal subunits usu- the prodelphinidin subunits, being (−)-gallocatechin only found as ex-
ally reported in V. vinifera grape PA were detected (Busse-Valverde et al., tension subunit whereas (−)-epigallocatechin was only as terminal
2010; Labarbe et al., 1999; Souquet et al., 1996; Travaglia et al., 2011), subunit.
but the higher sensitivity of the MRM technique used for detection and Proanthocyanidins (PA) have been related to the wine mouth-feel
perception called astringency and it has been proposed that astrin-
gency of PA (also known as condensed tannins) increases with mo-
Table 5 lecular size and degree of galloylation. However, the influence of
Structural characterization of proanthocyanidins (molar percentages; mean ± stan- large PA (mDP N 7) on astringency is a matter of controversy; their
dard deviation) found in the skin, flesh, and seeds of BRS Violeta grape. suggested low solubility seems to avoid astringency, although it has
Proanthocyanidina Skin Flesh Seeds been demonstrated that highly polymerized PA (mDP up to 70)
were soluble in water–alcohol solution and highly astringent (Santos-
Total PA (mg/kg grape) 673.0 ± 228.9 12.7 ± 1.1 478.9 ± 52.8
mDP 12.9 ± 1.2 5.3 ± 0.4 4.1 ± 0.1
Buelga & de Freitas, 2009). Concerning PA of Violeta grape, the obtained
% terminal-galloylation 2.72 ± 0.53 6.06 ± 1.28 5.86 ± 0.36 results suggested that their skin PA accounted for similar amounts and
% terminal-prodelphinidin 33.24 ± 4.45 29.76 ± 2.19 0.74 ± 0.18 show similar structural composition than PA from skins of V. vinifera
% terminal-C 56.52 ± 5.62 50.04 ± 3.88 69.00 ± 0.95 grape cultivars. However, Violeta grape seeds seem to account for re-
% terminal-EC 7.53 ± 0.75 14.14 ± 2.86 24.40 ± 0.64
markable lower content of PA together with lower degree of galloylation,
% terminal-GC 16.53 ± 2.10 ND ND
% terminal-EGC 16.71 ± 3.01 29.76 ± 2.20 0.74 ± 0.19 thus being potentially less astringent.
% terminal-ECG 2.72 ± 0.54 6.06 ± 1.29 5.86 ± 0.37
% extension-galloylation 0.68 ± 0.16 2.38 ± 0.07 6.20 ± 0.19 4. Conclusions
% extension-prodelphinidin 24.51 ± 1.30 39.95 ± 2.32 1.25 ± 0.07
% extension-C 1.74 ± 0.37 0.80 ± 1.39 4.60 ± 0.13
% extension-EC 73.07 ± 1.74 56.87 ± 1.01 87.96 ± 0.34
The development by Embrapa Uva e Vinho of the hybrid grape
% extension-GC 5.92 ± 1.58 39.95 ± 2.32 1.03 ± 0.05 cultivar BRS Violeta has covered the needs of grape-growers and
% extension-EGC 18.60 ± 0.28 ND 0.22 ± 0.03 winemakers of sub-tropical zones of Brazil, which demanded a
% extension-ECG 0.68 ± 0.16 2.38 ± 0.07 6.20 ± 0.19 grape cultivar able to reach higher sugar content and color. The de-
a
Total PA, total concentration of proanthocyanidins, as (+)-catechin equivalents, tailed phenolic composition of BRS Violeta grape has been studied
calculated by total sum of the concentrations of all the extension and terminal sub- by a combination of SPE methodologies and analytical HPLC–DAD–
units; mDP, mean degree of polymerization; % galloylation, % of 3-gallate subunits; ESI-MS/MS. The study was extended to the different berry parts and
% prodelphinidin, % of epigallocatechin subunits; and % of each of the flavan-3-ol mono-
mers found as terminal and extension subunits; C, (+)-catechin; EC, (−)-epicatechin;
the most important grape and wine phenolic families, and has re-
GC, (−)-gallocatechin; EGC, (−)-epigallocatechin; ECG, (−)-epicatechin 3-gallate; CG, vealed interesting features. Violeta grape has a very thick skin (ca.
(−)-catechin 3-gallate; ND, non-detectable. 46% of grape weight) that accumulated the most of grape phenolic
 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366
compounds: great amounts of anthocyanins (3930 mg/kg, as malvidin
3,5-diglucoside), together with also important amounts of flavonols
(150 mg/kg, as quercetin 3-glucoside), hydroxycinnamic acid deriva-
tives (HCAD; 120 mg/kg, as caftaric acid), and proanthocyanidins
(670 mg/kg, as (+)-catechin); in contrast, this grape cultivar seems to
be a low resveratrol producer (only 0.083 mg/kg, as resveratrol).
Violeta grape seeds accounted for similar proportions of low molecular
weight flavan-3-ols (mainly monomers; 345 mg/kg, as (+)-catechin)
and proanthocyanidins (480 mg/kg, as (+)-catechin). Violeta grape is
a teinturier cultivar having a red-colored flesh, but it only contained
traces of anthocyanins and low amounts of all the other phenolic
types. The anthocyanin composition of Violeta grape was dominated
by anthocyanidin 3,5-diglucosides (90%) that were p-coumaroylated
in a relevant degree (24%), being both factors considered to enhance
stability of anthocyanins. Evidence for the tentative assignation of the
complete series of caffeoylated anthocyanidin 3,5-diglucosides has
been found and that likely constitutes the first report of such anthocya-
nin derivatives in grape. Within flavonols, myricetin-type predomi-
nated and kaempferol-type were missing. In addition to expected
hydroxycinnamoyl-tartaric acids, several isomeric esters of caffeic and
p-coumaric acids with hexoses were tentatively identified, accounting
for relevant proportions within the pool of HCAD. Although pending
of further confirmation over successive vintages and different location
vineyards, the aforementioned results suggest that BRS Violeta grape
cultivar could be considered an interesting candidate for the elaboration
of highly colored, antioxidant-rich, grape juice and young red wines.
Moreover, the solid residues generated during the elaboration of Violeta
grape-derived products could be of additional interest, because they
have still contain high amounts of phenolic compounds.
Acknowledgments
The authors would like to thank Brazilian agency, CNPq, for the
productivity scholarship (Da-Silva, R.; Ramos, A. M.; Stringheta, P. C.)
and Project 486967/21012-3-CNPq. Authors Rebello, L. P. G. and Barcia,
M. T., thanks the National Council for the Improvement of Higher
Education (CAPES) for their scholarship in the Program of Doctoral
Sandwich Abroad (PDSE). The authors are also grateful to Mr. Fernando
José de Góes, the owner of Góes winery (São Roque, Brazil) for supply-
ing the grape samples. Author Gómez-Alonso, S., thanks the Fondo So-
cial Europeo and the Junta de Comunidades de Castilla-La Mancha for
co-funding his contract through the INCRECYT program. Author
Castillo-Muñoz, N., thanks the Fondo Social Europeo and the Junta de
Comunidades de Castilla-La Mancha for co-funding his contract (project
POII10-0061-4432). Authors Gómez-Alonso, S. and Hermosín-Gutiérrez,
I., thank Spanish Ministerio de Economía y Competitividad for financial
support (project AGL2011-29708-C02-02).
References
Abe, L. T., Mota, R. V., Lajolo, F. M., & Genovese, M. I. (2007). Compostos fenólicos e
capacidade antioxidante de cultivares de uvas Vitis labrusca L. e Vitis vinifera L.
Ciência e Tecnologia de Alimentos, 27(2), 394–400.
Azevedo, F. Q., Gonçalves, M. A., Schneider, E. P., Portela, M. N., & Rufato, A. R. (2009).
Caracterizaçao de mosto e suco da uva “BRS Violeta” producida en Pelotas-RS.
XVIII Congresso de Iniciação Científica; XI Encontro de Pós-Graduação - I Mostra
Científica Outubro, 2009, Pelotas (Available in: http://www.ufpel.edu.br/cic/2009/
cd/pdf/CA/CA_02009.pdf).
Baderschneider, B., & Winterhalter, P. (2001). Isolation and characterization of novel
benzoates, cinnamates, flavonoids, and lignans from Riesling wine and screening
for antioxidant activity. Journal of Agricultural and Food Chemistry, 49, 2788–2798.
Bordiga, M., Coïsson, J. D., Locatelli, M., Arlorio, M., & Travaglia, F. (2013). Pyrogallol: An
alternative trapping agent in proanthocyanidins analysis. Food Analytical Methods,
6, 148–156.
Bordiga, M., Travaglia, F., Coïsson, J. D., Locatelli, M., Arlorio, M., & Martelli, A. (2009).
Pyrogallol: A new trapping nucleophile in proanthocyanidins analysis. XXXII
World Congress of Vine and Wine. OIV. Zagreb (Croatia) 28 June–3 July, 2009.
Borges, R. S., Prudêncio, S. H., Roberto, S. R., & de Assis, A. M. (Outubro). Avaliação sen-
sorial de suco de uva cv. Isabel em cortes com diferentes cultivares. Revista
Brasileira de Fruticultura, Jaboticabal-SP, Volume Especial, E. (pp. 584–591).
365
Boulton, R. (2001). The copigmentation of anthocyanins and its role in the color
of red wine: A critical review. American Journal of Enology and Viticulture, 52,
67–87.
Busse-Valverde, N., Gómez-Plaza, E., López-Roca, J. M., Gil-Muñoz, R., Fernández-
Fernández, J. I., & Bautista-Ortín, A. B. (2010). Effect of different enological practices
on skin and seed proanthocyanidins in three varietal wines. Journal of Agricultural
and Food Chemistry, 58, 11333–11339.
Camargo, U. A., Maia, J. D. G., & Nachtigal, J. C. (2005). BRS Violeta: Nova cultivar de uva
para suco e vinho de mesa. Comunicado Técnico, v. 63, (Available in: http://www.
cnpuv.embrapa.br/publica/comunicado/cot063.pdf).
Castillo-Muñoz, N., Fernández-González, M., Gómez-Alonso, S., García-Romero, E., &
Hermosín-Gutiérrez, I. (2009a). Red-color related phenolic composition of Garnacha
Tintorera (Vitis vinifera L.) grapes and red wines. Journal of Agricultural and Food
Chemistry, 57, 7883–7891.
Castillo-Muñoz, N., Gómez-Alonso, S., García-Romero, E., Gómez, M. V., Velders, A. H., &
Hermosín-Gutiérrez, I. (2009b). Flavonol 3-O-glycosides series of Vitis vinifera
cv. Petit Verdot red wine grapes. Journal of Agricultural and Food Chemistry, 57,
209–219.
Castillo-Muñoz, N., Gómez-Alonso, S., García-Romero, E., & Hermosín-Gutiérrez, I.
(2007). Flavonol profiles of Vitis vinifera red grapes and their single-cultivar
wines. Journal of Agricultural and Food Chemistry, 55, 992–1002.
Cosme, F., Ricardo-Da-Silva, J. M., & Laureano, O. (2009). Tannin profiles of Vitis vinifera
L. cv. red grapes growing in Lisbon and from their monovarietal wines. Food Chem-
istry, 112, 197–204.
Dávalos, A., & Lasunción, M. A. (2009). Health-promoting effects of wine phenolics. In
M. V. Moreno-Arribas, & M. C. Polo (Eds.), Wine chemistry and biochemistry
(pp. 571–593). New York: Springer Science + Business Media LLC.
De Mello, L. M. R., & Machado, C. A. E. (2011). Banco de dados de uva, vinho e derivados.
Quantidade de uvas processadas no Rio Grande do Sul. Available in: http://vitibrasil.
cnpuv.embrapa.br/index.php?sopcao=sopt_07&opcao=opt_03
Falginella, L., Castellarin, S. D., Testolin, R., Gambetta, G. A., Morgante, M., & Di Gaspero,
G. (2010). Expansion and subfunctionalisation of flavonoid 3′,5′-hydroxylases in
the grapevine lineage. BMC Genomics, 11, 562.
FAOSTAT (2012). FAO Statistical Database. Agriculture Data. Available in: http://
faostat.fao.org/site/339/default.aspx (Accessed on: 20 march 2013).
Garrido, J., & Borges, F. (2011). Wine and grape polyphenols. A chemical perspective.
Food Research International, 44, 3134–3148.
Gatto, P., Vrhovsek, U., Muth, J., Segala, C., Romualdi, C., Fontana, P., et al. (2008).
Ripening and genotype control stilbene accumulation in healthy grapes. Journal
of Agricultural and Food Chemistry, 56, 11773–11785.
Hermosín-Gutiérrez, I., Castillo-Muñoz, N., Gómez-Alonso, S., & García-Romero, E. (2011).
Flavonol profiles for grape and wine authentication. Chapter 8. In S. E. Ebeler, G. R.
Takeoka, & P. Winterhalter (Eds.), Progress in authentication of food and wine. ACS
Symposium Series. (pp. 113–129). Washington, DC: American Chemical Society.
Hermosín-Gutiérrez, I., & García-Romero, E. (2004). Antocianos de variedades tintas
cultivadas en La Mancha: perfiles varietales característicos de la uva y de los vinos
monovarietales, y evolución durante la maduración de la baya. Alimentaria, 41,
127–139.
Labarbe, B., Cheynier, V., Brossand, F., Souquet, J., & Moutounet, M. (1999). Quantitative
fractionation of grape proanthocyanidins according to their degree of polymeriza-
tion. Journal of Agricultural and Food Chemistry, 47, 2719–2723.
Lafka, T., Sinanoglou, V., & Lazos, E. S. (2007). On the extraction and antioxidant activity
of phenolic compounds from winery wastes. Food Chemistry, 104, 1206–1214.
Lago-Vanzela, E. S., Da-Silva, R., Gomes, E., García-Romero, E., & Hermosín-Gutiérrez, I.
(2011a). Phenolic composition of the Brazilian seedless table grape varieties BRS
Clara and BRS Morena. Journal of Agricultural and Food Chemistry, 59, 8314–8323.
Lago-Vanzela, E. S., Da-Silva, R., Gomes, E., García-Romero, E., & Hermosín-Gutiérrez, I.
(2011b). Phenolic composition of the edible parts (flesh and skin) of Bordô grape
(Vitis labrusca) using HPLC–DAD–ESI-MS/MS. Journal of Agricultural and Food
Chemistry, 59, 13136–13146.
Llobera, A., & Cañellas, J. (2008). Antioxidant activity and dietary fibre of Prensal Blanc
white grape (Vitis vinifera) by-products. International Journal of Food Science and
Technology, 43, 1953–1959.
Mattivi, F., Guzzon, R., Vrhovsek, U., Stefanini, M., & Velasco, R. (2006). Metabolite
profiling of grape: Flavonols and anthocyanins. Journal of Agricultural and Food
Chemistry, 54, 7692–7702.
Mazza, G. (1995). Anthocyanins in grape and grape products. Critical Reviews in Food
Science and Nutrition, 35, 341–371.
Monagas, M., & Bartolomé, B. (2009). Anthocyanins and anthocyanin-derived com-
pounds. In M. V. Moreno-Arribas, & M. C. Polo (Eds.), Wine chemistry and biochemistry
(pp. 439–462). New York: Springer Science + Business Media LLC.
Muñoz-Espada, A. C., Wood, K. V., Bordelon, B., & Watkins, B. A. (2004). Anthocyanin
quantification and radical scavening capacity of Concord, Norton, and Marechal
Foch grapes and wines. Journal of Agricultural and Food Chemistry, 52, 6779–6786.
Nixdorf, S. L., & Hermosín-Gutiérrez, I. (2010). Brazilian red wines made from the hy-
brid grape cultivar Isabel: Phenolic composition and antioxidant capacity. Analytica
Chimica Acta, 659, 208–215.
Organisation Internationale de la Vigne et du Vin (2011). Recueil des methods interna-
tionals d'analyse des vins et des mouts, edition 2011. 8th Assemblée Générale, 21
June 2010, Paris.
Santos-Buelga, C., & de Freitas, V. (2009). Influence of phenolics on wine organoleptic
properties. In M. V. Moreno-Arribas, & M. C. Polo (Eds.), Wine Chemistry and Bio-
chemistry (pp. 529–570). New York: Springer Science+Business Media LLC.
Schwarz, M., Picazo-Bacete, J. J., Winterhalter, P., & Hermosín-Gutiérrez, I. (2005). Effect
of copigments and grape cultivar on the color of red wines fermented after the addi-
tion of copigments. Journal of Agricultural and Food Chemistry, 53, 8372–8381.
366 L.P.G. Rebello et al. / Food Research International 54 (2013) 354–366
Silva, G. G., Nascimento, R. L., Oliveira, V. S., Araújo, A. J. B., Oliveira, J. B., & Pereira, G. E.
(2011). Características físico-químicas de sucos de uvas “Isabel Precoce” e “BRS Violeta”
elaborados no Nordeste do Brasil. Jornada de Iniciação Científica da Embrapa Semiárido.
Embrapa Semiárido. Documentos, 238. (pp. 353–359). Petrolina: Embrapa Semiárido
(2011, Available in: http://www.alice.cnptia.embrapa.br/handle/doc/916687).
Souquet, J., Cheynier, V., Broussaud, F., & Moutounet, M. (1996). Polymeric proantho-
cyanidins from grape skins. Phytochemistry, 43, 509–512.
Torres, J. L., Varela, B., Garcia, M. T., Carilla, J., Matito, C., Centelles, J. J., et al. (2002). Val-
orization of grape (Vitis vinifera) byproducts. Antioxidant and biological properties
of polyphenolic fractions differing in procyanidin composition and flavonol con-
tent. Journal of Agricultural and Food Chemistry, 50, 7548–7555.
Travaglia, F., Bordiga, M., Locatelli, M., Coïsson, J. D., & Arlorio, M. (2011). Polymeric
proanthocyanidins in skins and seeds of 37 Vitis vinifera L. cultivars: A methodolog-
ical comparative study. Journal of Food Science, 76, 742–749.
Wang, H., Race, E. J., & Shrikhande, A. J. (2003). Characterization of anthocyanins
in grape juices by ion trap liquid chromatography–mass spectrometry. Journal of
Agricultural and Food Chemistry, 51, 1839–1844.
Waterhouse, A., & Kennedy, J. A. (2004). Red wine color. Revealing the mysteries. ACS
Symposium Series, 886, Washington, DC: American Chemical Society.
Zhu, L., Zhang, Y., & Lu, J. (2012). Phenolic contents and compositions in skins of red
wine grape cultivars among various genetic backgrounds and originations. Interna-
tional Journal of Molecular Sciences, 13, 3492–3510.