Food Chemistry 146 (2014) 7177

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Radical-scavenging-linked antioxidant activities of extracts from black

chokeberry and blueberry cultivated in Korea
Seok Joon Hwang a, Won Byong Yoon a, Ok-Hwan Lee a, Seung Ju Cha b, Jong Dai Kim a,
a
b

Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, South Korea
Legal Personality Icheon Beks & Cornus Co. Ltd, Icheon 467-822, South Korea

a r t i c l e

i n f o

Article history:
Received 8 March 2013
Received in revised form 28 June 2013
Accepted 5 September 2013
Available online 14 September 2013
Keywords:
Black chokeberry
Blueberry
Cultivars
Antioxidant activities
Cyanidin-3-galactoside

a b s t r a c t
The objective of this study was to investigate the radical-scavenging-linked antioxidant properties of the
extracts from black chokeberry and blueberry cultivated in Korea. The 70% ethanol extracts were prepared from black chokeberry and blueberry, and evaluated for total phenolic content, total avonoid content, total proanthocyanidin content, and antioxidative activities, using various in vitro assays, such as
DPPH(2,2-diphenyl-1-picrylhydrazyl), ABTS(2,2-azino-bis-(3-ethylenebenzothiozoline-6-sulphonic acid))
radical-scavenging activity, FRAP(ferric-reducing antioxidant power) and reducing power. The major
phenolic compounds, including cyanidin-3-galactoside, cyanidin-3-arabinoside, neochlorogenic acid,
procyanidin B1, were analysed by HPLC with a photodiode array detector. Results showed that total
phenol, avonoid and proanthocyanidin contents of black chokeberry extract were higher than those
of blueberry extract. In addition, black chokeberry extract exhibited higher free radical-scavenging
activity and reducing power than did blueberry extract. Cyanidin-3-galactoside was identied as a major
phenolic compound, with considerable content in black chokeberry, that correlated with its higher
antioxidant and radical-scavenging effects. These results suggest that black chokeberry extracts could
be considered as a good source of natural antioxidants and functional food ingredients.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Reactive oxygen species (ROS), including superoxide anion
(O2), hydroxyl radical (HO), hydrogen peroxide (H2O2), peroxyl
radical (ROO), singlet oxygen (1O2), and peroxynitrite (ONOO),
can be generated from unbalanced pro-oxidant and antioxidant enzyme response systems (Bursal & Glin, 2011). Furthermore, different environmental stress factors, such as pollution, drought,
temperature, excessive light intensities and nutritional limitation,
increase the production of ROS (Ehling-Schulz & Scherer, 1999).
To inhibit the physiological damage caused by excess ROS, several
enzymatic and non-enzymatic endogenous antioxidant defence
systems have been evolved to compensate the generation of ROS
(Fridovich, 1997; Sies, 2005).
Typical natural antioxidants include tocopherols, carotenoids,
avonoids, and polyphenolic compounds (Amro, Aburjai, & Al-Khalil, 2002) that can potentially provide protection against the development of certain oxidation-linked chronic diseases (kerget et al.,
2005). Regular consumption of bioactive compounds from plant
and fruit may be associated with protecting against oxidative damage and lowering the risk of chronic diseases, such as cancer, heart

Corresponding author. Tel.: +82 33 250 6456; fax: +82 82 33 241 0508.
E-mail address: jongdai@kangwon.ac.kr (J.D. Kim).
0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.035

disease, and cerebrovascular disease (Block, Patterson, & Subar,

1992; Hung et al., 2004; Liu et al., 2000; Willis, Shukitt-Hale, &
Joseph, 2009). Moreover, several authors have recently reported
that phytochemicals, including phenolic compounds, have gastroprotective effects due to their antioxidant properties (La Casa,
Villegas, Alarcon de La Lastra, Motilva, & Martn Calero, 2000;
Martin et al., 1998).
Berries are well known as good natural antioxidants (Benvenuti,
Pellati, Melegari, & Bertelli, 2004; Garzn, Riedl, & Schwartz, 2009;
Meyers, Watkins, Pritts, & Liu, 2003), which contain phenolic compounds, avonoids, anthocyanidins and antioxidant vitamins
(Kammerer, Schillmller, Maier, Schieber, & Carle, 2007). Among
the berries, high demand for black chokeberry and blueberry has
increased for their use in foodstuffs and as beverage ingredients
in Korea. In addtion, black chokeberries (Aronia melanocarpa) Ellot
have received considerable attention, because they are known to
function as chemopreventive agents against oxidative damage
(Pool-Zobel, Bub, Schrder, & Rechkemmer, 1999). Black chokeberry is very rich in phenolic compounds from the anthocyanin
subclass, namely cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, cyanidin-3-O-xyloside and cyanidin-3-O-glucoside (Oszmianski & Sapis, 1988) that have been reported to possess antioxidative
activities (Noda, Kaneyuki, Mori, & Packer, 2002; Tsuda, Shiga,
Ohshima, Kawakishi, & Osawa, 1996; Wang et al., 1999). Although
the antioxidant activity of black chokeberry cultivated in North

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S.J. Hwang et al. / Food Chemistry 146 (2014) 7177

America and Europe has been extensively investigated

(Oszmianski & Wojdylo, 2005; Skupien & Oszmianski, 2007; Wu,
Gu, Ronald, & McKay, 2004; Zheng and Wang, 2003), there are no
reports documenting radical-scavenging-linked antioxidant activity of black chokeberry cultivated in Korea.
The objective of this study was to investigate the total
phenolics, total avonoid proanthocyanidin contents, DPPH
(2,2-diphenyl-1-picrylhydrazy) radical-scavenging activity, ABTS
(2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay,
ferric reducing ability (FRAP) assay, major phenolic compounds
of black chokeberry and blueberry (Vaccinium corymbosum L.)
cultivated in Korea by HPLC with a photodiode array detector.
2. Materials and methods
2.1. Chemicals
The following compounds were used as standards for analysis by
HPLC-DAD: cyanidin 3-galactoside and cyanidin 3-arabinoside were
obtained from Polyphenols Laboratories AS (Sandnes, Norway);
neochlorogenic acid and procyanidin B1 were also obtained from
Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China).
FolinCiocalteau reagent, gallic acid, rutin and catechin were obtained from SigmaAldrich (St. Louis, MO, USA). The chemicals used
for antioxidant activities, such as DPPH (2,2-diphenyl-1-pic-rylhydrazy), ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid), TPTZ (2,4,6-tripyridyl-s-triazine), trichloroacetic acid,
ascorbic acid, were purchased from SigmaAldrich (St. Louis, MO,
USA). All other chemicals were of reagent grade.

2.5. Determination of proanthocyanidin contents

The proanthocyanidin contents of black chokeberry and blueberry were evaluated by the method of Sun, Ricardo-da-Silva, &
Spranger (1998). The sample solutions (0.5 ml) were mixed with
3 ml of vanillin (4%) and 1.5 ml of conc-HCl. After incubation for
15 min, the absorbance was measured at 490 nm and proanthocyanidin content was calculated as catechin (mg CE/g).
2.6. The antioxidant activity of extracts by DPPH radical
In the DPPH assay, the natural and synthetic antioxidants were
able to reduce the stable radical DPPH to the yellow-coloured
DPPH. This method is based on the reduction of DPPH in alcoholic
solution in the presence of a hydrogen-donating antioxidant due to
the formation of the non-radical form DPPH-H in the reaction. The
use of DPPH provides an easy way to evaluate antioxidant activity
(Oyaizu, 1986). The antioxidant activity of these extracts was measured on basis of their electron-donating ability (EDA) of the stable
DPPH as previously described (Chu, Chang, & Hsu, 2000) with
slight modication. Different concentrations (10500 lg/ml) of
black chokeberry and blueberry were prepared. Then, 1 ml of ethanolic DPPH solution (4  104 M) was added to the samples.
These samples were vortexed and incubated in the dark for
10 min at room temperature. DPPH radical-scavenging activities
were measured by spectrophotometer at 490 nm and were calculated and expressed as a percentage using the following formula:

DPPH radical-scavenging activity % 1  A=B  100

A : absorbance value of testing solution

2.2. Preparation of 70% ethanol extract of black chokeberry and

blueberry

B : absorbance value of control solution

The black chokeberry and blueberry were collected from Legal

personality Icheon beks & cornus of Korea and kept in a deep freezer (70 C). The black chokeberry and blueberry were extracted
with 10 vol (v/w) of 70% ethanol at 70 C for 3 h, and extraction
was repeated three times. The extracts were ltered through
Whatman lter paper (No. 2), concentrated with a vacuum evaporator, and completely dried with a freeze-drier. The freeze-dried
powder was dissolved in 50% DMSO (dimethyl sulfoxide) and ltered through a membrane lter (0.45 lm) and used for antioxidant activity.

2.7. ABTS radical-scavenging activity

2.3. Determination of total phenol contents

The total phenolic contents of extracts from black chokeberry
and blueberry were determined by a modied method of Gutnger
(1981). The sample solution (1 ml) was placed in a test tube with
FolinCiocalteau reagent (1 ml) and sodium carbonate solution
(1 ml). After incubation for 1 h at 25 C, the absorbance was measured at 750 nm and total phenolic content was calculated as gallic
acid equivalents (mg GAE/g).

The antioxidant activity was determined using ABTS free radical, as previously described by Re et al. (1999). Before analysis,
the stock solution was prepared by stirring ABTS+ (7 mM) and
potassium persulfate (2.45 mM) in water at room temperature
for 16 h. The ABTS+ solution was diluted with ethanol to achieve
an absorbance of 0.75 0.025 at 750 nm. Then, 1 ml ABTS+ solution
was added to 10 ll of different concentrations (10500 lg/ml) of
black chokeberry and blueberry extracts. These samples were vortexed and incubated in the dark for 6 min. ABTS radical-scavenging
activities were measured by spectrophotometer at 750 nm, were
calculated and expressed as a percentage using the following
formula:

ABTS radical-scavenging activity % 1  A=B  100

A : absorbance value of testing solution
B : absorbance value of control solution
2.8. FRAP assay

2.4. Determination of total avonoid contents

The total avonoid contents of extracts from black chokeberry
and blueberry were determined according to the method of
Moreno, Isla, Sampietro, and Vattuone (2000). The sample solution
(0.5 ml) was mixed with 1.5 ml of ethanol (95%), followed by
0.1 ml of aluminium chloride (10%), 0.1 ml of potassium acetate
(1 M) and distilled water. After incubation at room temperature
for 30 min, the absorbance was measured at 415 nm and total
avonoids content was calculated as rutin equivalents (mg RE/g).

The FRAP assay is based on the ability of the antioxidant to reduce Fe3+ to Fe2+ in the presence of TPTZ, forming an intense blue
Fe2+TPTZ complex with an absorbance maximum at 593 nm,
which is pH-dependent. The absorbance decrease is proportional
to the antioxidant activity (Benzie & Strain, 1996). The FRAP values
of black chokeberry and blueberry extract were obtained by using
the method of Benzie and Strain, (1996) with modication. FRAP
reagent was prepared from 300 mM acetate buffer (pH 3.6),
10 mM TPTZ in 40 mM HCl and 20 mM iron chloride in proportions

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S.J. Hwang et al. / Food Chemistry 146 (2014) 7177

of 10:1:1(v/v), respectively. Different concentrations (10500 lg/ml)

of sample solution (50 ll) were mixed with distilled water (150 ll)
and FRAP reagent (1.5 ml) was added. The absorbance of the reaction mixture was then measured at 595 nm after 4 min.
2.9. Reducing power
In this reducing power assay, the presence of reducers (namely,
black chokeberry and blueberry extract) converted the Fe3+/ferricyanide complex present in the assay system to the ferrous form. By
measuring the absorbance at 750 nm to determine the Fe2+ concentration, it is possible to estimate the reducing power of the
reducers. The reducing power of black chokeberry and blueberry
extracts were evaluated by the method of Jayaprakasha, Singh,
and Sakariah (2001). Different concentrations (10500 lg/ml) of
black chokeberry and blueberry extracts in distilled water
(0.5 ml) were mixed with 2.5 ml of sodium phosphate buffer (pH
6.6) and 2.5 ml of potassium ferricyanide (1%). The mixtures were
incubated at 50 C for 20 min. 2.5 ml of trichloroacetic acid (10%)
were then added and the mixture was centrifuged 1790g for
10 min. Then, 2.5 ml of the supernatant were withdrawn and
mixed with 2.5 ml of distilled water. After adding 0.5 ml of iron
(III) chloride (0.1%), the absorbance was spectrophotometrically
measured at 750 nm.
2.10. Analysis of phenolic compounds using HPLC-DAD
HPLC analysis of black chokeberry and blueberry extracts was
done as described by Dyrby, Westergaard, and Stapelfeldt (2001).
10 mg quantities of extracts were dissolved in 10 ml of water/formic acid (90/10, v/v,%) and ltered with a Millipore membrane lter (0.45 lm) prior to HPLC analysis. The HPLC equipment was an
Agilent 1260 with photodiode array detector at 280 nm. HPLC
analysis was carried out using a C18 column (Agilent Technologies,
Palo Alto, CA, USA) (4.6  150 mm, 3.5 lm). The mobile phase
consisted of water/formic acid (90/10, v/v,%) (A) and water/
acetonitrile/formic acid (40/50/10, v/v/v,%) (B). The ow rate was
1.0 ml/min with the following gradient programme: 0 min
88%A + 12%B, 26 min: 70%A + 30%B, 29 min: 0%A + 100%B, 30 min:
88%A + 12%B. The standards were cyanidin 3-galactoside, cyanidin
3-arabinoside, neochlorogenic acid, procyanidin B1.
2.11. Statistical analysis
All measurements were repeated three times. The results were
statistically analysed by ANOVA and Duncans multiple range tests.
Statistical signicance was accepted at a level of p < 0.05.
3. Results
3.1. Total phenolic, avonoid and proanthocyanidin contents of
extracts from black chokeberry and blueberry
The extraction yields of black chokeberry and blueberry were
14.2 and 8.7%, respectively. In addition, the total phenolic contents
of chokeberry and blueberry, determined using the regression
equation of the calibration curve (y = 14.826 + 0.0337, R2: 0.9985)
and expressed in gallic acid equivalents, were found to be
110 5.6 mg GAE/g, and 27.4 7.4 mg GAE/g, respectively
(Table 1). Our results are slightly different from another study by
Khknen, Hopia, and Heinonen (2001), in which total phenolic
content of black chokeberry was 4210 100 mg/100 g. Benvenuti
et al. (2004) also showed that total phenolic content of black
chokeberry extract was 690 8.8 mg/100 g. The total avonoid
contents of chokeberry and blueberry extracts, determined using

Table 1
Extraction yields, total phenol, avonoid and proanthocyanidin contents of 70%
ethanol extracts from black chokeberry and blueberry cultivated in Korea.

Extraction yields (%)

Total phenol content(mg GAE/g)
Total avonoids content(mg RE/g)
Proanthocyanidin content(mg CE/g)

Black chokeberry

Blueberry

14.2
110 5.6
5.3 0.8
107 6.6

8.7
27.4 7.4
1.6 0.3
11.8 5.0

Signicantly different at p < 0.05 by student t-test. The values are means standard
deviation from three replications.
GAE: Gallic acid equivalents.
RE: Rutin equivalents.
CE: Catechin equivalents.

the regression equation of the calibration curve (y = 2.0637 +

0.05, R2: 0.9996) and expressed in rutin equivalents, were found
to be 5.3 0.8 mg RE/g, and 1.6 0.3 mg RE/g, respectively
(Table 1).
The proanthocyanidins constitute a complex mixture of monomers, oligomers and polymers which generally consist of (+)-catechin, ()-epicatechin, (+)-gallocatechin, ()-epigallocatechin and
their 3-O-gallic acid esters (Prieur, Rigaud, Cheynier, & Moutounet,
1994). They are also an important sensory component, and are
responsible for the antioxidant efciency of berries products (Kanner, Frankel, Granit, German, & Kinsella, 1994). The proanthocyanidin contents of chokeberry and blueberry extracts, determined
using the regression equation of the calibration curve
(y = 1.5811 + 0.0343, R2: 0.9985) and expressed in catechin equi
valents, were found to be 107 6.6 mg CE/g and 11.8 5.0 mg
CE/g, respectively (Table 1).

3.2. Antioxidant activities of black chokeberry and blueberry extracts,

using in vitro model
The DPPH radical-scavenging activities of black chokeberry,
blueberry extracts and ascorbic acid were in the following order:
ascorbic acid > black chokeberry extract > blueberry extract at the
same concentration of test samples (500 lg/ml) (Fig. 1A). The
DPPH radical-scavenging percentages of various concentrations
(10, 50 and 500 lg/ml) of black chokeberry extract were 31.1%,
37.0% and 72.7%, respectively. In addition, the DPPH radical-scavenging activities of various concentrations (10, 50 and 500 lg/ml)
of blueberry extract were 29.4%, 29.6% and 40.6%, respectively.
The black chokeberry exhibited markedly higher DPPH free radical-scavenging activity than did blueberry extract. Benvenuti
et al. (2004) reported that the EC50 (DPPH radical-scavenging
activity) of chokeberry cultivated in Italy was 1.8 mg. Oszmianski
and Wojdylo, (2005) also showed the DPPH radical-inhibition
of black chokeberry cultivated in Poland to be 38.1%. Moreover,
Bermdez-Soto and Toms-Barbern (2004) determined that the
antioxidant activity of chokeberry juice cultivated in Spain was
60.0 1.2 mg TEAC (trolox equivalent antioxidant capacity)/ml.
Another effective method to measure radical-scavenging activity is the ABTS radical cation decolorisation assay, which showed
results similar to those obtained in the DPPH reaction. The ABTS
radical-scavenging activities of black chokeberry, blueberry and
ascorbic acid were in the following order: ascorbic acid > black
chokeberry extract > blueberry extract at a concentration of
500 lg/ml (Fig. 1B) The ABTS radical-scavenging percentages of
various concentrations (10500 lg/ml) of black chokeberry extract
were 4.6%, 10.3% and 46.3%, respectively. In contract, the ABTS radical-scavenging percentages at concentrations of 10, 50 and
500 lg/ml of blueberry extract were 2.3%, 4.2% and 8.6%, respectively. These results show that black chokeberry extract had significantly higher ABTS radical-scavenging activity than had blueberry

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S.J. Hwang et al. / Food Chemistry 146 (2014) 7177

(b) 120

100

80

Chokeberry
Blueberry
Ascorbic acid

60

40

d
e

20

ABTS radical scavenging activity (%)

DPPH radical scavenging activity (%)

(a)

Chokeberry
Blueberry
Ascorbic acid

100
80
60
40

20

ed

ed

ef

(d)

2.5
Chokebery
Blureberry
Ascorbic acid

0.5

b
d d cd
10

cd d
50

50

10

500

Concentration (g/mL)

Absorbance (750 nm)

Absorbance (595 nm)

0
500

50

Concentration (g/mL)

0.0

0
10

(c)

0.8
Chokeberry
Blueberry
Ascorbic acid

a
b

0.2

c
ed

c
0.0

500

d
e

10

Concentration (g/mL)

eded

ed

50

500

Concentration (g/mL)

Fig. 1. Antioxidant activities of black chokeberry and blueberry extracts. (A) DPPH radical-scavenging activity, (B) ABTS radical-scavenging activity, (C) FRAP assay, (D)
reducing power. aeValues are means standard deviation (n = 3); means in the same column not sharing a common letter are signicantly different (p < 0.05) by Duncans
multiple range test.

extract. Moreover, these results are in good agreement with

Bermdez-Soto and Toms-Barbern (2004) who reported that
chokeberry juice exhibited strong ABTS radical-scavenging activity
(103 2.2 mg TEAC/ml).
In the present study, the trend for FRAP of black chokeberry,
blueberry extracts and ascorbic acid are shown in Fig. 1C. For black
chokeberry and blueberry extract, the absorbance was increased
due to the formation of the Fe2+TPTZ complex with increasing
concentration (Fig. 1C). The black chokeberry extract, at a concentration of 500 lg/ml, had FRAP value similar to 50 lg/ml of ascorbic acid and higher than that of blueberry extract.
The reducing powers of the black chokeberry and blueberry extracts were increased along with the treatment concentrations
(Fig. 1D). The black chokeberry extract, at a concentration of
500 lg/ml, had reducing power (about 0.71 at 750 nm) similar to
a concentration of 50 lg/ml of ascorbic acid and higher than that
of blueberry extract.
3.3. Correlation among the antioxidant characteristics

and proanthocyanidin contents. A recent report (Zheng and Wang,

2001) demonstrated that some bioactive compounds present in
medical plants possessed high total antioxidant activity, which
was due to the presence of phenolics, carotenoids and avonoids.
Therefore, we evaluated the correlation coefcient between

Table 2
Correlations (ra) between different antioxidant capacity parameters (by DPPH, ABTS,
FRAP, reducing power activity) and total phenolic contents, total avonoid contents or
proanthocyanidin contents of extracts from black chokeberry.

TPC
TFC
PC
DPPH
ABTS+
FRAP
RPA
a
b

Total phenolic, total avonoids and proanthocyanidin contents

have been reported to be responsible for the antioxidant activities
of botanical extracts. DPPH, ABTS, FRAP, and reducing power activities have been used to measure antioxidant activity and these results should correlate with those of total phenolic, total avonoids

c
d
e
f
g
h

TPCb

TFCc

PCd

DPPHe

ABTSf

FRAPg

RPAh

0.9999

0.9999
0.9999

0.9953
0.9964
0.9964

0.9960
0.9970
0.9970
0.9999

0.9997
0.9994
0.9994
0.9928
0.9937

0.9995
0.9998
0.9998
0.9979
0.9984
0.9984

r, correlation coefcient.
TPC, total phenolic content.
TFC, total avonoid content.
PC, proanthocyanidin content.
DPPH radical-scavenging activity.
ABTS, ABTS radical-scavenging activity.
FRAP, ferric reducing ability of plasma.
RPA, reducing power activity.

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S.J. Hwang et al. / Food Chemistry 146 (2014) 7177

Table 3
Correlations (ra) between different antioxidant capacity parameters (by DPPH, ABTS+,
FRAP, reducing power activity) and total phenolic contents, total avonoid contents or
proanthocyanidin contents of extracts from blueberry.

TPC
TFC
PC
DPPH
ABTS+
FRAP
RPA
a
b
c
d
e
f
g
h

TPCb

TFCc

PCd

DPPHe

ABTSf

FRAPg

RPAh

0.9999

0.9999
0.9999

0.9977
0.9968
0.9968

0.9454
0.9492
0.9492
0.9214

0.9771
0.9796
0.9796
0.9605
0.9930

0.7895
0.7965
0.7965
0.7489
0.9431
0.8981

r, correlation coefcient.
TPC, total phenolic content.
TFC, total avonoid content.
PC, proanthocyanidin content.
DPPH radical-scavenging activity.
ABTS, ABTS radical-scavenging activity.
FRAP, ferric reducing ability of plasma.
RPA, reducing power activity.

in vitro antioxidant activities and antioxidant compounds in black

chokeberry and blueberry, respectively (Tables 2 and 3). Our results show that high correlation coefcients were found between
the in vitro antioxidant activities (DPPH, ABTS, FRAP, and reducing
power activity) and antioxidant compounds (total phenolics, total
avonoids and proanthocyanidin contents). These results suggest
that strong antioxidant and radical-scavenging activities of black
chokeberry and blueberry extracts can be attributable to high levels of the antioxidant compounds (total phenolics, total avonoids
and proanthocyanidin contents) present in these extracts, protecting against damage from reactive oxygen radicals.
3.4. HPLC analysis of phenolic compounds
We further investigated the contents of cyanidin 3-galactoside,
cyanidin 3-arabinoside, neochlorogenic acid, procyanidin B1, using
HPLC (Fig. 2). The levels of cyanidin 3-galactoside, cyanidin 3-arabinoside, neochlorogenic acid and procyanidin B1 in black chokeberry extract were 93.3 0.1 mg/g, 37.1 0.6 mg/g, 35.7 0.1 mg/
g, 25.4 0.3 mg/g, respectively (Table 4). Among the four standard
contents of black chokeberry and blueberry extracts, the cyanidin
3-galactoside was present in the largest amounts, followed by
cyanidin 3-arabinoside, neochlorogenic acid and procyanidin B1.
Our results show that total content of the four standards in black
chokeberry extract was higher than that of blueberry extract.
These results are in good agreement with Pool-Zobel et al.
(1999), who reported that the contents of both cyanidin-3-galactoside and cyandidin-3-arabinoside of black chokeberry were 166
and 39 mg/g, respectively. Bijak et al. (2011) reported that cyanidin
3-galactoside, cyanidin 3-arabinoside, neochlorogenic acid, procyanidin B1 contents of black chokeberry were 64.0 3.53,

Table 4
Phenolic composition of extracts from black chokeberry and blueberry.
Phenolic compounds

Black chokeberry extract

(mg/g)

Blueberry extract
(mg/g)

Cyanidin 3galactoside
Cyanidin 3arabinoside
Neochlorogenic acid
Procyanidin B1
Total

93.3.0.1

6.5 0.0

2.6 0.1

35.7 0.1
25.4 0.3
192 1.1

N.D.
3.0 0.1
12.1 0.2

37.1 0.6

N.D.: Not detected.

Signicantly different at p < 0.05 by student t-test. The values are means standard
deviation from three replications.

23.4 1.12, 24.1 0.94, and 10.3 0.06 mg/g, respectively. Furthermore, Oszmianski and Wojdylo (2005) indicated that cyanidin
3-galactoside, cyanidin 3-arabinoside and neochlorogenic acid of
black chokeberry were 1,282, 582, and 291 mg/100 g, respectively.

4. Discussion
Phenolics have a common structure composed of an aromatic
hydroxyl nucleus and approximately 8000 known in nature (Karaman, Ttem, Szgen Baskan, & Apak, 2010). Furthermore, the phenolic compounds are plant secondary metabolites extensively
spread throughout the plant kingdom (Andreasen, Christensen,
Meyer, & Hansen, 2000). The recent focus of interest on phenolic
compounds stems from their potential protective role, through
ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers) (Othman, Ismail, Abdul Ghani, & Adenan, 2007; Robbins, 2003). The
avonoids are the most common group of polyphenolic compounds in the human diet and are commonly found in fruits and
vegetables. They can prevent coronary heart disease and have antioxidant properties (Pietta, 2000).
Plant polyphenols and avonoids are multifunctional in that
they can act as reducing agents, hydrogen atom donors, and singlet
oxygen scavengers. They are also effective as antioxidants, capable
of chelating transition metal ions, which may induce Fenton-type
oxidation reactions in their free states (Karaman et al., 2010;
Rice-Evans, Miller, & Paganga, 1996). It was reasonable to experiment with their total amount in the selected vegetables or fruits.
In addition, avonoids also possess antioxidant activity (Lotito &
Frei, 2004), and are the most common type of polyphenolic compound in the plant (Bursal & Glin, 2011) and are capable of chelating Fe3+, Fe2+ (Moridani, Pourahmad, Bui, Siraki, & OBrien,
2003).
Recently, many experimental studies have been carried out on
black chokeberries from the United States and Europe, owing to

Fig. 2. HPLC chromatograms of major phenolic compounds in black chokeberry and blueberry extracts. STD 1-cyanidin 3-galactoside; STD 2-cyanidin 3-arabinoside; STD 3neochlorogenic acid; STD 4-procyanidin B1. 0 min 88%A (90/10, v/v) + 12%B (40/50/10, v/v/v), 26 min: 70%A + 30%B, 29 min: 0%A + 100%B, 30 min: 88%A + 12%B.

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S.J. Hwang et al. / Food Chemistry 146 (2014) 7177

their biological activities (Skupien and Oszmianski, 2007;

Valcheva-Kuzmanova et al., 2005; Wu et al., 2004; Zheng and
Wang, 2003). In the present study, we attempted to assess antioxidant activities of black chokeberry and blueberry extracts cultivated in Korea. The present study shows that the total phenol,
avonoids, and proanthocyanidin contents of chokeberry extract
were signicantly higher than those of blueberry extract. Khknen
et al. (2001) reported that total phenolic content of black chokeberry was 4,210 100 mg/100 g. Benvenuti et al. (2004) also
showed that total phenolic content was 690 8.8 mg/100 g,
whereas our results indicated that the total phenolic contents of
black chokeberry and blueberry extract cultivated in Korea were
110 5.6 and 27.4 7.4 mg GAE/g, respectively. These results
suggest that different total phenol contents of black chokeberry extract from diverse countries are inuenced by environmental factors such as soil, climate and temperature. Environmental factors
may account for the differences in phenolic content among previous reports (Benvenuti et al., 2004; Herms & Mattson, 1992).
We also examined antioxidant activity of black chokeberry and
blueberry extracts by various in vitro methods (DPPH, ABTS+,
FRAP, and reducing power). The DPPH and ABTS+ are widely used
radical-scavenging methods because of their ease and convenience
(Brand-Williams, Cuvelier, & Berset, 1995). They offer a rapid
screening for radical-scavenging activity of various natural substances. DPPH is scavenged by bioactive compounds through
donation of hydrogen (Matthus, 2002). ABTS+ assay involves an
electron transfer process. The FRAP and reducing power are also
antioxidant assays. These methods reect the electron donation
capacity of antioxidant compounds and are associated with antioxidant activity. Natural antioxidants can be reductants and inactivate oxidants. Both FRAP and reducing power are designed to
measure overall antioxidant activity, or reducing potential (Glin,
Bursal, Sehitoglu, Bilsel, & Gren, 2010). Our results show that the
antioxidant activity of black chokeberry, blueberry extracts and
ascorbic acid possess effective radical-scavenging ability. At a concentration of 500 lg/ml, black chokeberry extract was markedly
higher in free radical-scavenging activity than was blueberry extract, and indicated values similar to vitamin C at a concentration
of 50 lg/ml. These antioxidant activities may depend on the
amount of total phenolics, avonoids and proanthocyanidin contents. Furthermore, the antioxidant activity of both berries could
be due to the individual phenolic compounds. Therefore, we further investigated cyanidin-3-galactoside, cyanidin-3-arabinoside,
neochlorogenic acid and procyanidin B1, using HPLC with a photodiode array detector. Cyanidin-3-galactoside (93.3 mg/g) in black
chokeberry extract was found to be a major phenolic compound.
The total amount of these phenolic compounds in black chokeberry
extract was higher than that in blueberry extract (P < 0.05). The
phenolic compounds in black chokeberry are known to have strong
antioxidant activities and may also be responsible for the pharmaceutical effects of black chokeberry extract. However, since the
black chokeberry and blueberry were evaluated on a dry basis
and black choke berry has higher antioxidant activity than has
blueberry, further studies on fresh black chokeberry and blueberry
with respect to antioxidant properties are needed.
In conclusion, the black chokeberry cultivated in Korea was
effective in scavenging radicals and containing various phenolic
compounds. These radical-scavenging activities are likely due to
total phenol, avonoids, proanthocyanidin and phenolic compounds present in the black chokeberry extracts. Therefore, our results strongly suggest that black chokeberry extract is a good
source of natural antioxidants and is a protective ingredient for
oxidative stress-related chronic diseases. However, the extent of
in vivo antioxidant and protective effects of black chokeberry depend on their bioavailability. The apparent absorption of several
anthocyanidins after black chokeberry and blueberry consumption

has been reported in animal models and clinical tests (Kaume,

Howard, & Devareddy, 2012; Zafra-Stone et al., 2007). Pedersen
et al. (2000) reported that consumption of cranberry juice resulted
in a signicant increase in the ability of plasma to reduce oxidative
stress of healthy female volunteers, whereas consumption of blueberry juice had no such effect. These studies strongly indicate that
the antioxidant activity of black chokeberry is mainly due to their
rich antioxidant compounds (total phenolics, avonoid and proanthocyanidin contents) and bioavailability.

Acknowledgements
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (NO20110022812) to J-D Kim.

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