Antioxidant activity of vine fruits depending on their colouring

Abstract

Chemical analyses carried out in 2011-2013 aimed at evaluating the contents of flavonoids, free phenolic acids, tannins, anthocyanins, and antioxidant activity (%) by means of DPPH radical neutralization ability in fresh and air-dried fruits of three wild strawberry cultivars. Examinations revealed differences in contents of biologically active substances determined in raw vs. dried material depending on the cultivar. Mean concentrations of flavonoids and tannins were highest in raw fruits of ΄Baron von Solemacher` cv., which amounted to 1.244 mg ∙ g-1 and 6.09%, respectively. Fresh fruits of ‘Regina’ cv. were characterized by the highest average content of phenolic acids and anthocyanins: 4.987 mg ∙ g-1 and 0.636 mg ∙ 100 g-1. The pattern of mean contents of biologically active substances analyzed in air-dried fruits was similar. Significant differences in abilities to neutralize the DPPH radical to diphenylpicrylhydrazine by extracts made of examined wild strawberry fruits were also indicated.

Full text

CHEMIJA. 2014. Vol. 25. No. 1. P. 51–55
© Lietuvos mokslų akademija, 2014
Antioxidant activity of vine fruits depending on their
colouring
MagdalenaKapłan1,
AgnieszkaNajda2
1Chair of Seed Production
and Nurseries,
University of Life Science,
58 Leszczyński Street,
20-068 Lublin, Poland
2Department of Vegetable
Crops and Medicinal Plants,
University of Life Science,
58 Leszczyński Street,
20-068 Lublin, Poland
Fruits of the vine and their products, including wine, are the raw material, which is a valuable
source of biologically active substances for the human organism. e most important
group of health-promoting compounds present in fruits are polyphenols, flavonoids,
phenolic acids, flavones, flavonols, flavanones, flavanonols, catechins, and anthocyanin
pigments. e aim of the present study was to determine the level of selected secondary
metabolites and the antioxidant capacity of vine fruits depending on their colouring. e
experimental material consisted of fruits derived from three vine cultivars characterized
by different skin colouring: ‘Regent’–dark blue fruits, ‘Einset Seedless’–pink fruits, and
‘Jutrzenka’–white fruits. A field experiment was conducted in 2010 and 2011 in Vineyard
Faliszowice in Sandomierz Upland (50°39’N; 21°34’E). Shrubs of three cultivars were
planted in spring 2003. e harvest was dependent on the results of sugar content mea-
surements in grape extract using an Abbe refractometer. Fruits of the studied cultivars
were subject to a comparative analysis of the content of secondary metabolites such as:
total polyphenols, gallic acid, phenolic acids, flavonoids, tannins and anthocyanins. e
results showed that the antioxidant activity of the studied grapes depended significantly
on the colour of berries; fruits of ‘Regent’ and ‘Einset Seedless’ cv. had a significantly higher
antioxidant capacity than the ‘Jutrzenka’ cv. It has been shown that contents of anthocyanins
and flavonoids increased along with increasing colour intensity of grapes. Fruits of ‘Einset
Seedless’ cv. having pink-coloured skin contained significantly more phenolic acids and
tannins than the others.
Keywords: vine, extract, polyphenols, anthocyanins, flavonoids, tannins
*Corresponding author. E-mail:magdalena.kaplan@up.lublin.pl
INTRODUCTION
Due to the taste and nutritional properties, vine fruits are
valuable raw materials for the human organism and an abun-
dant source of biologically active substances such as vitamins
(A, B1, B2, C, PP), minerals (potassium, phosphorus, calcium,
iron, boron, magnesium), pectins, pigments, tannins, oils,
easily digestible carbohydrates, amino acids, fruit acids, and
fiber. e most important group of health-promoting com-
pounds present in fruits are polyphenols, flavonoids, phe-
nolic acids, flavones, flavonols, flavanones, flavanonols, ca-
techins, and anthocyanin pigments [1–6].
According to Yang et al. [5], 80% of all grapes are used
for wine production, including 13% of dessert fruit, while
the rest of the fruits are used for making raisins. In wine-
producing countries, mainly cultivars derived from Vitis
vinifera (Wine Grape), while in the colder regions, including
Poland, the species belonging to the Vitis labrusca, Vitis ri pa-
ria, Vitis aestivalis, Vitis rupestris, and Vitis rotundifolia are
grown, which are used for wine manufacturing.

MagdalenaKapłan, AgnieszkaNajda
52
In Poland, vine is of little economic importance, but
considerable interest in the cultivation of this species and
wine production has been recently observed [7]. It is es ti-
mated that about 200 thousand vines are planted each year
[8]. e grapevine has been present in home gardens be cause
of its many decorative, taste, and health benefits.
e aim of the present study was to determine the level of
selected secondary metabolites and the antioxidant capacity
of vine fruits depending on their colouring.
EXPERIMENTAL
e experimental material consisted of fruits derived from
three vine cultivars characterized by different skin co lou r-
ing:
1. ‘Regent’– (‘Diana’ (‘Silvaner’× ‘Mueller urgau’) ×
‘Chambourcin’), origin: Germany, dark blue fruits;
2. ‘Einset Seedless’–(‘Fredonia’×‘Canner Seedless’ (‘Hu-
nisia’×‘Sultania’)), origin: USA, pink fruits;
3. ‘Jutrzenka’ – (‘Seyve Villard’ 12-375 × ‘Pinot Blanc’),
origin: Jasło, Poland, white fruits.
A field experiment was conducted in 2010 and 2011
in Vineyard Faliszowice in Sandomierz Upland (50°39’N;
21°34’E). Shrubs of three cultivars were planted in spring
2003. e harvest was dependent on the results of sugar
content measurements in grape extract using an Abbe ref-
ractometer. Fruits of the studied cultivars were subject to a
comparative analysis of the content of secondary metabolites
presented below.
Determination of total phenolic content
e amount of total phenolic was determined using Folin–
Ciocalteu reagent, as described by Singleton and Rossi [9].
About 1g of raw homogenised samples was extracted with
80% aqueous methanol (4.5 ml) on a mechanical shaker
for 2 h. e mixture was centrifuged at 10,000 rpm for
15 min and the supernatant decanted into polypropylene
tubes. e pellets were extracted under identical conditions.
Supernatants were combined and filtered through Whatman
No.1 filter paper. e clear extracts were analysed both for
determination of phenolic content and antioxidant activity.
Results were expressed as milligram gallic acid equivalents
(GAE)/100 g fresh mass.
Total flavonoids estimation
e studied material was investigated for the total content
of flavonoids, using the modified Christ and Müller method,
calculated for quercetin QE [10]. Absorbance was measured
at 425nm on a HITACHI U-2900 spectrophotometer.
e content of flavonoids was calculated from the equa-
tion:
,
where m (g) was the amount of fresh and dry material.
Total phenolic acids estimation
It was carried out according to the Arnov method [11]. One
milliliter of the sample was mixed with 5ml of distilled water,
1ml 0.5M HCl, 1ml of the Arnov reagent and 1ml 1M NaOH,
and subsequently adjusted to 10ml with distilled water. e
absorbance was measured at 490vnm. e total phenolic acid
content was expressed as the caffeic acid equivalent (CAE).
Tannin estimation
e amount of tannin estimation was determined using the
Pharmacopoeia procedure [10]. e content of tannins was
expressed as fresh and dry weight percentage.
Anthocyanins estimation by means of colorimetry
Samples of raw material (1.0g) were extracted with 50 ml
HCl (1mol∙dm3) and heated in a water bath for 1hour. e
obtained extract was hydrolyzed with 20 ml n-buthanol,
and then two 10 ml n-buthanol portions were added as a
solution. Anthocyanin extracts were rinsed in a 50ml flask
with n-buthanol. e absorbance was measured immediately
at 533nm [12].
e percentage of anthocyanins, as delphynidyn chloride,
was calculated from the expression:
,
where P is total anthocyanins (mg∙100g–1), A is the ab sor-
bance at 533nm, V is the value of buthanol phase (50ml), F
is the coefficient for delphinidyn chloride (2.6), M is the mass
of the sample to be examined (mg).
Determination of antiradical activity (AA)
A 0.1 ml aliquot of the methanol extract prepared above
was mixed with 3.9ml of an 80% ethanolic 0.6mM DPPH
solution. e tubes were vortexed for 15s and allowed to
stand for 180min, as described by Caietal. [13]. Aer this,
the absorbance of the mixture was measured at λ=517nm
wavelength using the HITACHI UV-Vis spectrophotometer
(UV-Vis model U-2900, Shimadzu, Kyoto, Japan). Most test-
ed compounds react completely within 180min in this con-
dition. Reaction time for vitamin C is less than 1 min due
to its fast oxidation. Ethanol (80%) was used as a blank so-
lution, and the DPPH solution without test samples (3.9ml
of DPPH+0.1ml of 80% ethanol) served as the control. All
tests were performed in triplicate. e antiradical activity
of the test samples was expressed as the median effective
concentration for radical scavenging activity (EC50): TP (mg)
of the antioxidant (test sample) required for a 50% decrease
in the absorbance of DPPH radicals, and inhibition (%) of
the DPPH absorbance=(Acontrol–Atest )×100/Acontrol. A plot of
the absorbance of DPPH vs concentration of the antioxidant
was made to establish the standard curves (dose-response
curves) and to calculate that EC50. Acontrol is the absorbance
of the control (DPPH solution without the test sample), and
Atest is the absorbance of the test sample (DPPH solution plus

53
Antioxidant activity of vine fruits depending on their colouring
0.1 ml of 5 μM test compound). Ascorbic acid served as a
standard. e results of the assay were expressed relative to
an ascorbic acid equivalent.
Chemicals
All reagents and solvents were analytical grade chemicals
from Merck (Darmstadt, Germany) or Sigma Chemical Co.
(St. Louis, MO, USA).
Results achieved from laboratory experiments were sta-
tistically processed by means of the variance analysis method
and Tukey confidence intervals at 5% confidence level.
RESULTS AND DISCUSSION
It was shown that extract contents for the studied fruits signi-
ficantly differed between cultivars and amounted to: ‘Einset
Seedless’–17.5%, ‘Jutrzenka’–18.3%, and ‘Regent’–19.3%
(Figure). Studies performed by Krośniaket al. [4] revealed
that extract content in vine fruits ripening under Polish con-
ditions was very divergent depending on the cultivar. In the
case of light-fruit cultivars, values of the parameter were at
the levels: 16.4% for ‘Muscat Odeski’, 21.1% for ‘Jutrzenka’,
and 22.8% for ‘Seyval Blanc’, whereas for dark-berry cultivars:
‘Mareachal Foch’ and ‘Rondo’–19.2%.
e results unveiled that the antioxidant activity of
tested vine fruits significantly depended on their colouration
(Table 1). It has been shown that fruits of ‘Einset Seedless’
cv. and ‘Regent’ cv. contained remarkably more polyphenols
than the ‘Jutrzenka’ cv. It has been observed that pink-colored
fruits of ‘Einset Seedles’ cv. had slightly more polyphenols
Table 1. The total phenolic, phenolic acid and tannins contents in 3 grape varieties (mean for 2010–2011)
Cultivar Colour Total phenolic, mg 100g FM Phenolic acid, mg 100g FM Tannins, %
‘Regent’ Dark purple 337.2±3.71a 5.10 ±0.01c 0.12±0.006b
‘Einset Seedless’ Red, purple 365.8±1.03a 26.7±0.11a 0.30±0.011a
‘Jutrzenka’ White 258.8±1.44b 13.2±0.04b 0.07±0.00c
Explanation: Means followed by the same letter are not signicantly dierent at α=0.05.
than the ‘Regent’ cv. with dark skin of the fruit. ese results
confirm the study by Krośniak et al. [4], which showed that
the polyphenol content significantly depended on the co lour
of the skins and the cultivar of grapes. e authors proved that
red-colored fruits (‘Rondo’ and ‘Marechal Foch’ cv.) con tained
considerably more polyphenols than white cultivars (‘Jutr-
zenka’, ‘Seyval Blanc’, and ‘Muscat Odeski’). e influence of a
cultivar on the studied parameter among cultivars with the
same colour of berries appeared to be sig nificant.
e content of phenolic acids in the studied fruits de-
pended remarkably on the cultivar and ranged from 5.10 up
to 26.7mg 100g FM (Table1). It has been shown that fruits
of ‘Regent’ cv. with dark-coloured skin contained significant-
ly less phenolic acids than ‘Einset Seedless’ cv. (red, purple)
and ‘Jutrzenka’ cv. (white). Similar results were obtained by
Bu neaetal. [15], who studied ferrulic acid, quercetin, and re-
sveratrol in 9 cultivars of vine grapes grown by means of the
conventional and organic method. ey found that fruits of
‘Muscat de Hamburg’ cv. with dark-coloured skin con tain ed
less ferrulic acid as compared to those with white-co loured
skin ‘Timpuriu de Cluj’ cv. Yangetal. [5] also re port ed that
‘Ba co Noir’ contained less total phenolic acids and flavo-
noids than white cultivars such as ‘Riesling’ cv. and ‘Vi dal
Blanc’ cv. According to Yangetal. [5], Anastasiadietal. [16],
and Buneaet al. [15], the quantitative and qualitative com-
po si tion of phenolic acids is significantly affected by genetic
features, environmental conditions, as well as by agro tech-
nical management.
e analysis showed that the level of tannins in vine fruits
substantially depended on their colouring (Table1). Among
Figure. Soluble solids content of investigated grape cultivars (g100g–1)

MagdalenaKapłan, AgnieszkaNajda
54
studied cultivars, significantly higher level of tannins was
found in fruits of ‘Einset Seedless’ cv., while remarkably lower
level was in ‘Jutrzenka’ cv. According to Matthews and Nuzzo
[17], tannins are present in the skin, seeds, and peduncles.
eir content in fruit juice (a must) and wine depends on
the crop technique, shrub loading, and climatic conditions,
methods of maceration and fermentation circumstances.
ese compounds have a spectrum of important properties
that affect the colour, colour stability, astringency, and wine
depth [18].
e content of flavonoids in the studied fruits rang-
ed from 84.8 to 396 mg of cyanidin 3-glucoside equi va-
lents / 100g of grapes, which differed significantly between
the assessed cultivars (Table 2). It was observed that the
fla vonoid content significantly increased with an increase
in the colour intensity of fruit. However, these facts do not
confirm findings of Yangetal. [5], who did not show any
univocal effect of fruit colour on the tested parameter.
Among cultivars from Vitis vinifera species, significantly
high er flavonoid content was found for ‘Pinot Noir’ cv. (dark
pur ple) rather than for others: ‘Cabernet Franc’ cv. (dark pur-
ple), ‘Chardonnay’ cv. (green), and ‘Riesling’ cv. (green), while
for hybrids, significant differences were observed between
‘Catawba’ cv. (pink) and ‘Cayuga White’ cv. (green) vs ‘Baco
Noir’ cv. (dark purple), ‘Chancellor’ cv. (dark purple), ‘De
Chaunac’ cv. (dark blue), ‘Marechal Foch’ cv. (dark purple),
and ‘Vidal Blanc’ cv. (green).
e anthocyanin content in the studied vine fruits sub-
stantially depended on their colouration and ranged from
2.5mg100 g–1 FM to 396.4 mg 100 g–1 FM (Table2). Fruits
of ‘Regent’ cv. had significantly more anthocyanins than ‘Ein-
set Seedless’ cv. and ‘Jutrzenka’ cv. e content of these com-
pounds in ‘Regent’ cv. was almost 150 times greater than that
in ‘Jutrzenka’ cv. and 62 times higher than in ‘Einset Seedless’
cv. Studies carried out by Yangetal. [5] involving cultivars with
green-coloured skin did not reveal any presence of antho-
cya nins, whereas for pink-coloured fruits of ‘Catawa’ cv., this
parameter reached 8.1mg·100mL of fresh must. In the case
of grapes with dark coloured skin, those authors demonstrat ed
a significant impact of a cultivar on the evaluated parame ter;
the anthocyanin content ranged from 49.8 to 239.6mg 100mL
and it differed significantly. Similarly, in the study performed
by Mazza [1], the total content of anthocyanins in the fresh
weight of ripe vine fruits ranged from 30 to 750mg g–1 de-
pend ing on the cultivar. Numerous studies have demonstrated
that the anthocyanin and tannin contents largely depend on
the cul tivar, species, ripeness degree of the fruits, produc tion
lo calization, and climate [1, 5, 19, 20].
e antioxidant activity of fruit extracts made from the
studied fruits as determined by the DPPH method ranged
from 41.1 to 81.4µM TE g–1 and it significantly depended
on the cultivar (Table 2). It has been shown that fruits of
‘Regent’ cv. and ‘Einset Seedless’ cv. had considerably higher
antioxidant activity than ‘Jutrzenka’ cv. Research by Katalinić
et al. [6], which involved assessing the antioxidant activity
of 14 vine cultivars, showed no significant effect of the vine
fruit colour and cultivar on the tested parameter; considering
white cultivars, the DPPH value ranged from 52.8 to 291.0µM
TEg–1, while for red ones it was from 58.0 to 239.0µM TEg–1.
CONCLUSIONS
e antioxidant activity of the studied vine grapes determined
as the total polyphenols and the DPPH parameter depended
significantly on the colour of berries. Fruits of ‘Regent’ cv.
and ‘Einset Seedless’ cv. had significantly higher antioxidant
capacity than ‘Jutrzenka’ cv. Fruits of ‘Einset Seedless’ cv.
characterized by pink-coloured skin contained more phe-
no lic acids and tannins than others. It has been shown that
the flavonoid content remarkably increased along with the
increase of grape colour intensity. e anthocyanin level in
fruits of the studied vine cultivars significantly depended on
their colouration.
Received 20 June 2013
Accepted 20 November 2013
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Cultivar Total avonoids, mg 100g–1 FM Total anthocyanin, mg 100g–1 FM DPPH, µM TE/g–1 FM
‘Regent’ 396.4±21.3a 55.1±4.8a 81.4±2.5a
‘Einset Seedless’ 104.1±13.1b 6.4±1.2b 68.1±4.2a
‘Jutrzenka’ 84.8±7.3c 2.5±0.7b 41.1±1.7b
Explanation: Means followed by the same letter are not signicantly dierent at α=0.05.

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MagdalenaKapłan, AgnieszkaNajda
VYNUOGIŲ ANTIOKSIDACINIO AKTYVUMO RYŠYS
SU JŲ SPALVA
Santrauka
Trijose skirtingose vynuogių rūšyse buvo nustatyti ir tarpusavyje
palyginti antrinių metabolitų– polifenolių, galo rūgšties, fenolių
rugščių, flavonoidų, taninų ir antocianinų–kiekiai. Nustatyta, kad
tirtųjų vynuogių antioksidacinės savybės yra glaudžiai susiję su jų
spalva.