Comparative polyphenolic content and antioxidant activities of some wild and cultivated blueberries from Romania

Abstract

Two wild and three cultivated blueberry varieties ('Elliot', 'Bluecrop' and 'Duke') from Romania were analyzed comparatively in order to determine the total polyphenols, total anthocyanins, total flavonoids content and measuring the antioxidant activity using three different single electron transfer-based assays, Trolox equivalent antioxidant capacity (ABTS), ferric reducing ability (FRAP), 2,2-diphenylpicrylhydrazil radical scavenging capacity (DPPH) and one hydrogen atom transfer-assay, oxygen radical absorbance capacity (ORAC). Total polyphenols content ranged from 424.84 - 819.12 mg GAE/100 g FW, total flavonoids ranged from 84.33-112.5 mg QE /100 g FW and total anthocyanins ranged from 100.58-300.02 C3GE/100g FW. The anthocyanins were separated and quantified using RP-HPLC-DAD. In Vaccinium myrtillus, petunidin-3-glucoside and delphinidin-3-glucoside have the highest contribution to the anthocyanin content while in Vaccinium corymbosum, peonidin-3-galactoside represent the major anthocyanin. Except for ORAC assay (r=0.765), all antioxidant activity values obtained were highly correlated with total polyphenol content (0.923. r .0.986). Wild blueberries had higher total polyphenols content and also antioxidant activity compared with cultivated ones.

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Not Bot Horti Agrobo, 2011, 39(2):70-76
Print ISSN 0255-965X; Electronic 1842-4309
Notulae Botanicae Horti Agrobotanici
Cluj-Napoca
Comparative Polyphenolic Content and Antioxidant Activities
of Some Wild and Cultivated Blueberries from Romania
Andrea BUNEA, Dumitriţa O. RUGINĂ, Adela M. PINTEA, Zoriţa
SCONŢA, Claudiu I. BUNEA, Carmen SOCACIU*
1University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, 3-5 Mănăştur Street, 400372,
Cluj-Napoca, România; carmen.socaciu@usamvcluj.ro (*corresponding author)
Abstract
Two wild and three cultivated blueberry varieties (‘Elliot’, ‘Bluecrop’ and ‘Duke’) from Romania were analyzed comparatively in
order to determine the total polyphenols, total anthocyanins, total avonoids content and measuring the antioxidant activity using
three dierent single electron transfer-based assays, Trolox equivalent antioxidant capacity (ABTS), ferric reducing ability (FRAP), 2,2-
diphenylpicrylhydrazil radical scavenging capacity (DPPH) and one hydrogen atom transfer-assay, oxygen radical absorbance capacity
(ORAC). Total polyphenols content ranged from 424.84 - 819.12 mg GAE/100 g FW, total avonoids ranged from 84.33-112.5 mg
QE /100 g FW and total anthocyanins ranged from 100.58-300.02 C3GE/100g FW. e anthocyanins were separated and quantied
using RP-HPLC-DAD. In Vaccinium myrtillus, petunidin-3-glucoside and delphinidin-3-glucoside have the highest contribution to
the anthocyanin content while in Vaccinium corymbosum, peonidin-3-galactoside represent the major anthocyanin. Except for ORAC
assay (r=0.765), all antioxidant activity values obtained were highly correlated with total polyphenol content (0.923≤ r ≤0.986). Wild
blueberries had higher total polyphenols content and also antioxidant activity compared with cultivated ones.
Keywords: Vaccinium myrtillus, Vaccinium corymbosum, total polyphenols, anthocyanins, antioxidant methods
Abbreviations: TPC, total polyphenols content; TA, total anthocyanins; TF, total avonoids; FRAP, ferric reducing antioxidant
potential; ABTS, trolox equivalent antioxidant capacity; ORAC, oxygen radical absorbing capacity; DPPH, 2,2-diphenyl-1-
picrylhydrazyl method, TE, Trolox equivalents, Trolox, 6-hydroxy-2,5,7,8-tetrametylchroman-2-carboxylic acid
Introduction
e interest for natural antioxidants, especially from
fruits and vegetables, has increased in recent years. Epide-
miological studies indicated that a higher level of natural
antioxidants (ascorbic acids, vitamin E, carotenoids and
phenolics) in human everyday diet can protect against
cardiovascular diseases, cataract, cancer and aging-related
disorders (Steen et al., 2003).
Berries contain high concentration in bioactive com-
pounds such as polyphenols, including anthocyanins, phe-
nolic acids, tannins, carotenoids, vitamin A, C, E, folic acid
and minerals such as calcium, selenium and zinc (Kresty
et al., 2001; Pineli et al., 2011). Among them, blueber-
ries became well known and oen consumed due to their
uses for treating biliary disorders, coughs, tuberculosis,
diabetes (Martineau et al., 2006; Valentová et al., 2007)
and visual disorders (Canter and Ernst, 2004). Blueber-
ries contain high level of anthocyanin and phenolic com-
pounds with high in vitro antioxidant capacities compared
with other fruits (Wang and Jiao, 2000). e blueberries
phenolic content are aected by genetic dierences, the
cultivar type, growing location and the degree of maturity
at harvest (Zadernowski et al., 2005). e total amount
and the proportion between dierent classes of phenolic
compounds in berries cultivar may vary (Beekwilder et al.,
2005).
e methods used to determine the total antioxidant
capacity can be divided in two major groups: methods
based on single electron transfer (SET) and hydrogen
atom transfer reaction (HAT). e SET methods include
ABTS/TEAC assay (Trolox equivalent antioxidant ca-
pacity), FRAP (ferric reducing ability), CUPRAC assay
(copper reduction) and DPPH assay (2,2-diphenylpic-
rylhydrazil radical scavenging capacity). Hydrogen atom
transfer reaction (HAT) assay include ORAC (oxygen
radical absorbance capacity) and TRAP (total radical-
trapping antioxidant parameter) assay. ese assays are fre-
quently used to measure the total antioxidant capacity of
food extracts. Because their characteristics and because of
the dierences in the mechanisms of the reaction, a single
assay will not reect all the antioxidants present in the sys-
tem (Li et al., 2009). Hence, only by combining dierent
assays, information about the response of the compounds
present in samples in dierent experimental conditions
can be achieved (Sariburun et al., 2010).

Bunea A. et al. / Not Bot Horti Agrobo, 2011, 39(2):70-76
71
was carried out under stirring. e ltrates were combined
in a total extract, which was dried by vacuum rotary evapo-
rator at 40°C. Prior to each analysis, the dry residues were
redisolved in 10 ml of methanol, the samples were centri-
fuged at 5000 rpm and ltered through 0.45 μm nylon l-
ter (Millipore).
Total phenolics
e amount of total polyphenol in the blueberry ex-
tracts was determined using modied Folin-Ciocalteu
colorimetric method (Singleton et al., 1999). Stock so-
lution of sample extracts (25 μl each) were dissolved in
methanol and further dilution were performed to obtain
readings within the standard curve made with gallic acid
(R=0.997). e extracts were oxidized by the Folin-Cio-
calteu reagent (120 μl) and the neutralization was made
with Na2CO3 (340 μl), aer 5 minutes. e absorbance
was measured at 750 nm aer 90 minute in the dark, at
room temperature. e results were expressed as milligram
of gallic acid per 100 grams.
Total avonoids
e total avonoids content was determined using a
colorimetric method (Kim et al., 2003). e alcoholic ex-
tract was diluted to a nal volume of 5 ml with distilled
water. Aer adding 300 μl 5% NaNO2 the mixture was al-
lowed to stay 5 min. en 300 μl AlCl3 10% was added
and, aer 6 minutes, 2 ml NaOH 1N. e solution was
mixed well and the absorbance was measured against pre-
pared water blank at 510 nm. Total avonoid content was
expressed as mg quercetin equivalents/100 g fresh weight
basis.
Quantication of the anthocyanin content
e monomeric anthocyanin content of the blueberry
extract was measured using the pH-dierential method
(Giusti and Wrolstad, 2001). e blueberry extracts dis-
solved in methanol were diluted with 0.025 mol/l potassi-
um chloride (adjusted with HCl to pH 1.0) and 0.4 mol/l
sodium acetate (pH 4.5). Each sample and the standard
(cyanidin-3-glucoside) were diluted with the buer solu-
tion pH 1 and the absorbance was measured at 520 nm
and 700 nm using a UV spectrophotometer (Jasco V-630,
International Co. Ltd, Japan). A second aliquot of each
sample was diluted to the same value with the buer solu-
tion pH 4.5 and measured at 520 and 700 nm. e absor-
bance values were calculated as follows:
A = (Aλ520 - Aλ700)pH1.0 -(Aλ520 - Aλ700)pH4.5
e total anthocyanin content was calculated accord-
ing to the formula:
1 x ε
1000 x DF x MWA x
TA
e results were expressed as mg cyanidin-3-glucoside
chloride per 100 g fruit using the molar absorbtivity (ε)
e literature data about blueberries antioxidant activ-
ity and total polyphenol content are diverse, more on culti-
vated than wild varieties (Castrejón et al., 2008; Dragović-
Uzelac et al., 2010; Garzón et al., 2010; Giovanelli and
Buratti, 2009; Howard et al., 2003; Koca and Karadeniz,
2009; Prior et al., 1998; You et al., 2011).
e objectives of this study were (1) to investigate to-
tal polyphenols, total avonoids, total anthocyanins con-
tent of blueberry extracts, (2) to separate and character-
ize the anthocyanin content using RP-HPLC-DAD, (3)
to measure the antioxidant activity using three dierent
SET-based assays (ABTS, FRAP, DPPH) and one HAT-
based method (ORAC), (4) to correlate the antioxidant
methods applied with total polyphenol, total avonoid
and total anthocyanin content.
Materials and methods
Chemicals
e standard compounds, including cyanidin-3-O-
galactoside (purity 90%), cyanidin-3-O-glucoside (pu-
rity 95%), cyanidin (purity 95%), gallic acid (GAE)
(purity 97.5%), quercetin (purity 98%) and 2,2’-azobis
(2-amidinopropane) dihydrochloride (AAPH) 97% pu-
rity ,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox) 98% purity, uorescein 97% purity, 2,9-di-
metyl-1,10-phenanthroline (Neocuproine) 99% purity,
2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid),
diammonium salt (ABTS) 98% purity, 2,2-diphenyl-1-
picrylhydrazyl (DPPH) 95% purity, 2,4,6-tripyrydyl-S-
triazine (TPTZ) 98% purity, potassium persulfate were
obtained from Sigma-Aldrich (Darmstadt, Germany).
Folin-Ciocalteu’s phenol reagent, HCl, Na2CO3, AlCl3,
NaNO2, H2O2, CuCl2 were purchased from Merck
(Darmstadt, Germany).
Samples extraction
ree varieties of cultivated highbush blueberries (Vac-
cinium corymbosum) ‘Elliot’, ‘Bluecrop’ and ‘Duke’ were
purchased directly from the producers, a farm situated
in North-West of Romania. e two types of wild blue-
berries (Vaccinium myrtillus, Wild 1 and Wild 2) were
harvested from two dierent mountainous geographical
zones: 45°24´44˝N and 46°44´37˝E of Romania. All ber-
ries were picked at the commercially ripe stage. Samples
were stored in a freezer at -20°C until analyzed.
For sample extraction, 5 g of blueberries, in three rep-
licated each, was extracted by grinding the sample 1 min
at 20,000 rpm in a blender (Ultra-Turrax Miccra D-9 KT
Digitronic, Germany) with 10 ml of acidied methanol
(85:15 v/v, MeOH:HCl) (El-Sayed and Hucl, 2003).
e homogenate was centrifuged at 3500 rpm for 10 min.
e extract was separated and the residual tissue was re-
extracted until the extraction solvents became colorless
(the total solvent volume was between 100-250 ml). Aer
adding 10 ml of the same solvent mixture, the extraction

Bunea A. et al. / Not Bot Horti Agrobo, 2011, 39(2):70-76
72
in methanol/HCl of 34300 (M-1 cm-1), molecular weight
(MW) of 484.8 and dilution factor (DF).
Anthocyanin determination by RP-HPLC-DAD
Analyses were performed on a Shimadzu HPLC sys-
tem equipped with a binary pump delivery system LC-20
AT (Prominence), a degasser DGU-20 A3 (Prominence),
diode-array SPDM20 A UV-VIS detector (DAD) and a
Luna Phenomenex C-18 column (5μm, 25 cm x 4.6 mm).
e mobile phase consisted in: solvent A - formic acid
(4.5%) in bidistilled water and solvent B - acetonitrile. e
gradient elution system was: 10% B, 0-9 min; 12% B, 9-17
min; 25% B 17-30 min; 90% B, 30-50 min; 10% B, 50-55
min. e ow rate was 0.8 ml/min and the analyses were
performed at 35°C. e chromatograms were monitored
at 520 nm. e anthocyanins identication and peak as-
signments are based on their retention times, UV-VIS
spectra comparing with standards and published data. e
anthocyanin quantication was performed using cyanidin
3-O-galactoside.
Oxygen radical absorbance activity (ORAC)
e oxygen radical absorbance capacity (ORAC) mea-
sure the peroxyl radical scavenging activity using as stan-
dard 6-hydroxy-2,5,7,8-tetrametylchroman-2-carboxylic
acid (Trolox) (Huang et al., 2002). A uorescein stock
solution (4x10-3 μM) was made in phosphate buer (75
mM, pH 7.4) and kept in the dark at 4°C. Before utiliza-
tion, the uorescein stock solution was diluted with the
phosphate buer. e uorescein solution was added to
each Trolox standard and blueberry sample (25 μl) made
in phosphate buer and incubated for 30 min, at 37°C.
e reaction was initiated by adding 25 μl 2,2’-azobis-2-
amidinopropane (AAPH) and the uorescence was mea-
sured kinetically at excitation wavelength 485 nm and
emission wavelength 535 nm, every minute using a uo-
rescence microplate reader BioTek (Synergy HT, BioTek
Instruments, Winooski, VT). e ORAC values for each
blueberry extract were calculated using the net area under
the decay curves and were expressed as micromoles Trolox
equivalents per gram sample (μmol TE/g).
ABTS radical cation decolorization assay (ABTS)
e ABTS assay is based on the capacity of a sample
to scavenge the ABTS radical cation (ABTS.+) compared
to a standard antioxidant (Trolox), adapted to 96 wells
microplate, described by Arnao et al. (2001). e ABTS.+
solution was produced by reacting 7mM ABTS stock solu-
tion with 2.45 mM potassium persulfate (nal concentra-
tion) for 12-16 h, in the dark, at room temperature. Prior
to use, the ABTS.+ working solution was prepared by di-
luting the stock solution with EtOH to an absorbance of
0.70 ±0.02 at 734 nm. e samples and Trolox standards
(20 μl) were combined with the ABTS.+ working solution
(170μl, absorbance 0.70 ±0.02) in 96-well microplate.
Aer 6 min of incubation at 30°C, the absorbance at 734
nm was read with a microplate reader. If the absorbance
of ABTS value was higher than the standard, the samples
were diluted and re-evaluated. e results were expressed
as micromoles Trolox equivalents per gram sample (TE
μmol /g).
Reducing power (FRAP)
e reducing capacity of methanolic blueberry ex-
tracts was measured as ferric reducing antioxidant power
(FRAP). Antioxidants are evaluated as reducers of Fe3+
to Fe2+, which is chelated by TPTZ to form Fe2+ - TPTZ
complex, with a maximum absorbance at 593 nm (Benzie
and Strain, 1996). e absorbance of the colored product
was monitored by Biotek Synergy HT spectrophotometer.
All solutions were used in the day of preparation. Briey,
2.5 ml TPTZ (10 mM in 40 nM HCl), 25 ml acetate buf-
fer (300 mM, pH=3.6) and 2.5 ml FeCl3 (20 mM) were
mixed and aer adding 180 μl FRAP reagent the mixture
was incubated for 3 min. en, 20 μl of each sample added
to each well and the absorbance was read immediately at
593 nm with a microplate reader. Samples dilution were
perfomed when the values were over the linear range of the
curve of 0 to 1 μM Fe2+/ml, using FeSO4x7H2O.
DPPH· scavenging activity assay
e DPPH scavenging activity assay was done accord-
ing to a method reported by Brand-Williams et al. (1995).
A DPPH· solution (80 μM) was freshly prepared in 95%
methanol. A volume of 250 μl of this solution was allowed
to react with 35 μl sample and the absorbance was mea-
sured at 515 nm, for 30 minutes. e chemical kinetics of
blueberries extract was recorded. e antioxidant activity
was calculated as follows:
% DPPH· scavenging activity = (1-[Asample/Acontrol t=o]) 100
Statistical analysis
e data’s are expressed as mean ± standard deviation
(SD) from three parallel measurements. In order to deter-
mine the signicant dierences between values, analysis
of variance (ANOVA) and Duncan’s multiple range tests
were performed. Signicance of dierence was dened at
the 5% level (p<0.05). All statistical analysis was carried
out using Graph Pad Version 4.0 (Graph Pad Soware
Inc; San Diego, CA, USA). Pearson’s correlation coe-
cient was calculated using Microso Excel 2003.
Results and discussion
Total polyphenols content
e comparative data about total polyphenols, a-
vonoids and anthocyanins content in wild and cultivated
blueberries are presented in Tab. 1.
e total polyphenols content (TPC) was determined
using the Folin-Ciocalteu method. Gallic acid was used
as calibration standard and the results (expressed as gallic
acid equivalents) were expressed as means ±standard devia-
tion of triplicate analysis. e TPC values in the blueberry
extracts analyzed was in the range of 424.84 - 819.12 mg

Bunea A. et al. / Not Bot Horti Agrobo, 2011, 39(2):70-76
73
GAE/100 g fresh weight. Among all the varieties analyzed,
the Wild 1 blueberry revealed the highest TPC at 819.12
gallic acid equivalents/100 g FW followed by Wild 2 blue-
berries (672.59 mg GAE/100g). Between the cultivated
blueberries, ‘Bluecrop’ has the highest TPC at 652.27 mg
GAE/100 g while the lowest value was found for ‘Duke’
variety (424.84 mg GAE/100g). Signicant dierences
were found in total phenolic content when compared all
the varieties analyzed (p<0.05).
e TPC data’s obtained are comparable to previ-
ous ndings which reported values between 251-310 mg
GAE/100 g for some cultivated blueberries and between
577 and 614 mg GAE/100 g for wild Italian blueberries
(Giovanelli and Buratti, 2009). Lee et al. (2004) obtained
between 367-1286 mg GAE/100 g total polyphenols for
V. membranaceum and for V. ovalifolium species 677-
1054 mg GAE/100 g. Prior et al. (1998) reported values
between 181 and 390 mg/100g for V. corymbosum L spe-
cies. Dragovič-Uzelac et al. (2010) reported for ‘Bluecrop’
variety higher amount compared with ‘Duke’, while Prior
et al. (1998) obtained higher TPC values for ‘Duke’ vari-
ety. It is known that phenolics show quantitative variation
at dierent genetic levels within species.
Total avonoids content
e total avonoids content of the extracts was deter-
mined using aluminium chloride colorimetric method
and the results are shown in Tab. 1. e total avonoids
content ranged from 84.33 mg QE/100 g in ‘Duke’ vari-
ety to 112.5 mg QE/100 g in Wild 2 blueberries. For the
other blueberry varieties the TFC were as follows: Wild 1
(110.36 mg QE/100 g), ‘Bluecrop’ (103.18 mg QE/100
g), ‘Elliot’ (92.82 mg QE/100 g ) and ‘Duke’ (84.33 mg
QE/100 g ). ere were no signicant dierences in total
avonoid content among ‘Elliot’, ‘Bluecrop’, Wild 1 and
Wild 2 (p<0.05). ‘Duke’ variety has the lowest total a-
vonoid content, signicantly dierent than other varieties
analyzed. ere are no data regarding the total content
of avonoids in blueberries. In blackberry varieties, the
TFC content range between 29.07-82.21 mg QE/100 g
(Sariburun et al., 2010).
Total anthocyanins content
e total anthocyanins content (TAC) of the blueber-
ries extract were also determined (Tab. 1). e highest
anthocyanins content was found in wild blueberries Wild
1 (300.02 mg/100g), followed by wild blueberries Wild
2 (252.23 mg/100g ), ‘Elliot’ (163.4 mg/100g ), ‘Blue-
crop’ (160.76 mg/100g) and the lowest TAC was found
in ‘Duke’ variety (69.58 mg/100g). ere were signicant
dierences (p<0.05) in anthocyanins content between
Wild 1, Wild 2 and ‘Duke’. However, signicant dierenc-
es in the total anthocyanin content were not observed be-
tween ‘Elliot’ and ‘Bluecrop’ (p<0.05). e levels of TAC
in TPC are between 23% and 37% and they are in agree-
ment with the data’s reported by other authors (Giovanelli
and Buratti, 2009). e present TAC values obtained are
in agreement with other studies (Dragovič-Uzelac et al.,
2010; Prior et al., 1998).
Tab. 1. Total polyphenols, total avonoids and total
anthocyanins content in wild and cultivated blueberries
Total
polyphenols
GAE mg/100 g
Total
avonoids
mg QE/100 g
Total
anthocyanins
C3GE mg/100 g
Vaccinium corymbosum
‘Elliot’ 526.3 ± 26d92.82 ± 8.4a163.40 ± 16.4c
‘Bluecrop’ 652.27 ± 30c103.18 ±10.2a160.76 ± 13.9c
‘Duke’ 424.84 ± 20e84.33 ± 8b100.58 ± 13.5d
Vaccinium myrtillus
Wild 1 819.12 ± 36a110.36 ±12.3a300.02 ± 27.9a
Wild 2 672.59 ± 30b112.50 ±15a252.23 ± 18b
GAE mg/100g, QE mg/100g, and C3GE mg/100g represent mg of gallic
acid equivalents, mg of quercetin equivalents and mg of cyanidin-3-glucoside
equivalents per 100g of fresh blueberry, respectively. For each measurements,
the data marked by dierent letters indicate signicant dierence (p<0.05)
Tab. 2. Concentration of individual anthocyanins in blueberries (expressed in mg/100 g fruit)
Compound Retention
time
Maximum
absorbance
Vaccinium myrtillus Vaccinium corymbosum
Wild 1 Wild 2 ‘Bluecrop’ ‘Elliot’ ‘Duke’
Delphinidin-3-galactoside 9.2 217; 277; 523 113.67±11 73.43±8.1 53.29±7.2 ND ND
Delphinidin-3-glucoside 10.6 217; 277; 523 119.86±14 87.14±8.2 24.53±2 53.62±7 23.69±2.4
Cyanidin-3-galactoside 12.4 217; 279; 518 91.85±10 37.95±4.3 9.96±1.8 ND ND
Delphinidin-3-arabinoside 13.01 217; 277; 529 66.64±9.2 76.35±8.8 31.78±2.6 41.07±5.1 13.64±1.1
Cyanidin-3-glucoside 14.4 217; 279; 518 96.48±11 43.34±6.1 2.08±0.4 3.09±0.6 0.11±
Petunidin-3-galactoside 15.09 217; 277; 524 38.36±5.2 17.53±0.9 28.54±2.5 ND ND
Petunidin-3-glucoside 16.9 217; 278; 521 146.27±18 85.19±7.8 25.14±2.5 21.35±1.9 8.18±0.9
Peonidin-3-galactoside 19.4 217; 278; 520 8.71±1.3 ND ND 125.79±12 37.114.3
Petunidin-3-arabinoside 20.3 217; 277; 525 12.80±1.9 9.66±1.2 12.70±1.9 ND ND
Peonidin-3-glucoside 22.4 217; 278; 521 108.81±12 28.91±2.2 54.37±7.5 12±0.9 12.14±1.1
Malvidin-3-galactoside 25.3 217; 278; 527 119.53±12 39.00±4.3 37.97±2.6 67.45±9 27.55±2.4
Malvidin-3-glucoside 27.4 217; 277; 528 17.51±3.2 6.04±1.1 34.75±2.5 ND ND
Malvidin-3-arabinoside 29.5 217; 277; 528 tr tr ND ND ND

Bunea A. et al. / Not Bot Horti Agrobo, 2011, 39(2):70-76
74
Total antioxidant activity
For measuring the total antioxidant activity of the
blueberry extracts, four dierent assays were used. Tab.
3 includes the mean values for antioxidant activity mea-
sured.
ORAC assay is probably the most widely used HAT-
based assay and indicate the free-radical scavenging ability
of antioxidant against peroxyl radical. e values obtained
were not signicantly dierent among samples, ranging
from 34.85-38.49 μmol TE/g fresh weight. e present
ORAC results are similar to those obtained by Prior et al.
(1998) (17-37.1 μmol TE/g) and lower than the values re-
ported for rabbiteye blueberries by You et al. (2011) (44-
55 μmol TE/g). For ‘Duke’ variety, it has been obtained
34.85 μmol TE/g comparable with the values reported
by Wang et al. (2009) (40.4 μmol TE/g) and Moyer et
al. (2002) (32.6 μmol TE/g). Regarding the ORAC val-
ues for ‘Bluecrop’ variety it is a great discrepancy in the
literature data. Prior et al. (1998), reported 17 mM TE/
kg, Moyer et al. (2002), 50 mM TE/kg and Howard et al.
(2003), between 21-38 mM TE/kg, the last values being
comparable with the present value. e last mention study
indicates that genotypes inuence more the TPC and
ORAC values than growing season.
ABTS assay is based on the antioxidant ability of the
extracts to react with ABTS·+ radical cation generated in
the system. In contrast, the ferric reducing antioxidant
power assay (FRAP) measures the ability of the extracts
to reduce ferric complex to the ferrous form. e averages
values obtained for ABTS and FRAP assay are given in
Tab. 2. Blueberries Wild 1 extract showed the highest an-
tioxidant activity based on FRAP and ABTS assay (73.71
μM Fe2+/g and 56.65 μmol TE/g). e lowest level in both
assays was obtained for ‘Duke’ variety. e antioxidant ac-
tivity order in both assays, FRAP and ABTS, was: Wild
1>Wild 2>’Bluecrop’>’Elliot’ >’Duke’. In FRAP assay,
there were signicant dierences found between Wild 1,
Wild 2, ‘Bluecrop’ and ‘Duke’ (p<0.05). In ABTS assay,
signicant dierences were found between Wild 1, Wild
2, ‘Elliot’ and ‘Duke’ (p<0.05). ere were no statistical
dierences between ‘Elliot’ and ‘Bluecrop’ varieties.
Identication and quantication of anthocyanins
Using HPLC-DAD, 13 anthocyanins were identied
based on their retention time, UV-VIS spectra compared
with standards and published data. HPLC chromatogram
for anthocyanins separation in ‘Bluecrop’ variety is pre-
sented in Fig. 1.
In Vaccinium myrtillus, petunidin-3-glucoside and del-
phinidin-3-glucoside have the highest contribution to the
anthocyanin content (15.6% and 16.9%; 17.3 and 12.7%
respectively). In Vaccinium corymbosum, peonidin-3-galac-
toside represent the major anthocyanin content in ‘Elliot’
and ‘Duke’ variety (40.3% and 30.3%) but in ‘Bluecrop’
peonidin-3-glucoside is absent, peonidin-3-glucoside be-
ing the major contributors (17.3%).
Fig. 1. Anthocyanin HPLC proles for ‘Bluecrop’ variety. Peak
identication: 1- Delphinidin-3-galactoside; 2- Delphinidin-
3-glucoside; 3-Cyanidin-3-galactoside; 4- Delphinidin-3-ara-
binoside; 5- Cyanidin-3-glucoside; 6-Petunidin-3-galactoside;
7-Petunidin-3-glucoside; 8-Peonidin-3-galactoside; 9-Petuni-
din-3-arabinoside; 10-Peonidin-3-glucoside; 11- Malvidin-3-ga-
lactoside; 12-Malvidin-3-glucoside; 13-Malvidin-3-arabinoside
Tab. 3. Antioxidant activity for blueberry fruits, using four
dierent complementary assays (FRAP, ABTS, ORAC, DPPH)
FRAP
μM Fe2+/g
ABTS
TE μmol /g
ORAC
TE μmol/g
DPPH·
scavenging
activity (%)
Vaccinium corymbosum
‘Elliot’ 50.74 ± 1.9c36.46 ± 4.26c38.05±1.56a43.48± 1.9d
‘Bluecrop’ 60.39 ± 1.6b37.96 ± 2.98bc 37.04 ± 1.7 a46.64± 1.65c
‘Duke’ 33.03 ± 2.54d24.33 ± 3.76d34.85 ± 1.3b29.96± 1.38e
Vaccinium myrtillus
Wild 1 73.71 ± 3.2a56.65 ± 3.79a38.49± 1.01a59.79± 1.24a
Wild 2 64.87 ± 2.9b43.08 ± 2.3b37.78± 0.89a49.93± 1.6b
Fig. 2. Kinetics of antioxidant activity of blueberry extracts us-
ing DPPH method. e inhibition percentage represents the
antioxidant activity

Bunea A. et al. / Not Bot Horti Agrobo, 2011, 39(2):70-76
75
standards. e highest Pearson’s coecient between was
obtained when it has been compared ABTS and DPPH
antioxidant methods (0.986). As mentioned before, these
two assays are also based on similar mechanisms.
Conclusions
ere were analyzed, for the rst time, the antioxidant
activity in relation to the polyphenol content in wild and
some cultivated blueberry fruits from Romania. Total
polyphenol, total anthocyanin, total avonoid content
and antioxidant activity measured with six dierent as-
says were higher in wild blueberry varieties, as compared
with cultivated ones. e FRAP, ABTS and DPPH assays
showed higher correlation, statistically signicant, with
total polyphenol content compared with total anthocya-
nin content. To measure adequately the antioxidant po-
tential ABTS and DPPH methods are considered to be
most appropriate, in good agreement with the concentra-
tions of phenolic derivatives (polyphenols, anthocyanins,
avonoids).
Acknowledgment
is research was supported by CNCSIS-UEFISCSU,
project number PNII-TE_168, code 109/2010. e sec-
ond author’s work was supported by European Postdoc-
toral Program POSDRU/89/1.5/S/60746. e authors
thank Natural Invest for providing the blueberry culti-
vars.
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TF - 0.95* 0.853ns 0.739ns 0.896*
FRAP - 0.951* 0.858ns 0.985**
ABTS - 0.853ns 0.986**
ORAC - 0.891*
DPPH -
*,** signicant at p<0.05 and 0.01, respectively

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