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Antioxidant properties of blackberry and

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of Turkey
Article in Scientia Horticulturae August 2009
Impact Factor: 1.37 DOI: 10.1016/j.scienta.2009.03.015

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Scientia Horticulturae 121 (2009) 447450

Contents lists available at ScienceDirect

Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Antioxidant properties of blackberry and blueberry fruits grown in the

Black Sea Region of Turkey
Ilkay Koca *, Bulent Karadeniz
Ondokuz Mayis University, Faculty of Engineering, Food Engineering Department, Samsun 55139, Turkey

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 7 October 2008
Received in revised form 4 March 2009
Accepted 6 March 2009

Seven wild and ten cultivated blackberries (Arapaho, Bartin, Black Satin, Bursa 1, Bursa 2, Cherokee,
Chester, Jumbo, Navaho, and Ness), and six lowbush (Vaccinium arctostaphylos) and four highbush
(Vaccinium corymbosum) blueberries fruits (Ivanhoe, Jersey, Northland, and Rekord) were analyzed for
total anthocyanins, total phenolics, and antioxidant activity as ferric reducing antioxidant power (FRAP)
in this study. The respective ranges of total anthocyanin and total phenolic contents of the tested
samples were: blackberries, 0.951.97 and 1.733.79 mg g1 and blueberries, 0.182.94 and 0.77
5.42 mg g1. FRAP values varied from 35.05 to 70.41 mmol g1 for blackberries, 7.41 to 57.92 mmol g1
for blueberries. Wild blackberries had the highest FRAP values while wild blueberries had the highest
total phenolic and total anthocyanin contents. A linear relationship was observed between FRAP values
and total phenolics for blueberries (r = 0.981). The anthocyanin pigments in samples were isolated and
characterized by high-performance liquid chromatography (HPLC) with UVvisible detection. Cyn-3-glu
was the predominant anthocyanin in all blackberry fruits.
2009 Elsevier B.V. All rights reserved.

Keywords:
Anthocyanin
Blackberry
Blueberry
FRAP
HPLC
Phenols

1. Introduction
Blackberries and blueberries are known to contain appreciable
levels of phenolic compounds, including anthocyanins, avonols,
chlorogenic acid and procyanidins, that have high biological
activity and may provide health benets as dietary antioxidants
(Cho et al., 2005). Anthocyanins are probably the largest group of
phenolic compounds in human diet. Anthocyanins have been used
for several therapeutic purposes including the treatment of
diabetic retinopathy, brocystic disease, and vision disorders.
Anthocyanins also have the potential to serve as radiationprotective agents, vasotonic agents and chemoprotective agents
and can act against carbon tetrachloride induced lipoperoxidation
(Veberic et al., 2009; Cacace and Mazza, 2003; Wang et al., 1997).
Phenolic extracts of berries inhibited human low-density lipoprotein (LDL) and liposome oxidation (Heinonen et al., 1998). Berries
have also shown a remarkably high scavenging activity toward

* Corresponding author. Tel.: +90 362 3121919; fax: +90 362 4576035.
E-mail address: itosun@omu.edu.tr (I. Koca).
Abbreviations: Cyn, cyanidin; plg, pelargonidin; peo, peonidin; malv, malvidin;
delp, delphinidin; pet, petunidin; rut, rutinose; glu, glucoside; gal, galactoside; ara,
arabinoside; HPLC, high-performance liquid chromatography; FRAP, ferric reducing
antioxidant power; TACN, total anthocyanin; ORAC, oxygen radical-absorbing
capacity; std dev, standard deviation.
0304-4238/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2009.03.015

chemically generated active oxygen species (Wang and Lin, 2000;

Wang et al., 1997; Heinonen et al., 1998).
Turkey is one of the leading countries in berry cultivation.
Consumption of wild berries used to be popular especially in the
Black Sea Region, however in recent years, cultivated berries are
grown in large areas as a result of breeding studies. Antioxidant
properties of fruits are strongly affected by the type of fruit (species
and variety within species), and cultivation conditions of the plant
(environmental and cultivation techniques) (Scalzo et al., 2005).
Although there are many studies on the phenolic content and
antioxidant activity in blackberris and blueberries (Wang et al.,
1997; Kalt et al., 1999; Deighton et al., 2000; Kalt et al., 2000, 2001;
Ehlenfeldt and Prior, 2001; Prior et al., 2001; Moyer et al., 2002;
Wada and Ou, 2002; Zheng and Wang, 2003; Siriwoharn et al.,
2004; Cho et al., 2005), studies on those fruits grown in Turkey is
limited (Celik et al., 2008). The objective of the present work was to
study the anthocyanin composition and antioxidant activity of
blackberry and blueberry fruits grown in Turkey.
2. Materials and methods
2.1. Chemicals
Cyd-3-glu (kuromanin chloride, lot number 01111218), Cyd3,5-di glu (Cyanin chloride, lot number 01111202), Cyd-3-rut

448

I. Koca, B. Karadeniz / Scientia Horticulturae 121 (2009) 447450

(keracyanin chloride, lot number 01111213), plg-3-glu (callistephin

chloride, lot number 01111209) and peo-3-glu (peonidin-3-glucoside, lot number 01111204) were purchased from Extrasynthese
(Lyon-Genay-France). Folin-Ciocalteau reagent, formic acid and
hydrochloric acid were purchased from Merck (Darmstadt, Germany). 2,4,6-Tripyridyl-s-triazine (TPTZ) used for the FRAP assay
was purchased from Acros Organics (NJ, USA). HPLC grade methanol
was obtained from JT Baker (Deventer, Holland). Acetone, glacial
acetic acid and phosphoric acid were purchased from Carlo Erba
(Milano, Italy), and acetonitrile of HPLC grade was purchased from JT
Baker (Deventer, Holland).

UV 1000 UVvis detector. Seperation was carried out using a

4.6 mm  250 mm Luna 5u C18 column. The solvents were (A)
0.5% aqueous phosphoric acid (v/v), and (B) water/acetonitrile/
glacial acetic acid/phosphoric acid, 50:48.5:1:0.5 (v/v/v/v). The
solvent gradient was %B, initial, 20%; 26 min, 60%; 30 min, 20%;
35 min, 20% (run time 35 min). Detection wavelengths used were
515 nm for anthocyanins. Flow rate was 1 mL min1, sample
temperature was ambient, and injection volume was 10 mL. The
IBM (300 GL) computer was used to evaulate the peak area.
Chromatographic peaks were characterized by comparison with
UVvisible spectra and retention times of pure standards.

2.2. Samples

2.4. Statistical analysis

Mature berries assessed by full colour development were

harvested in 2002. Seven wild and ten cultivated blackberries
(Arapaho, Bartin, Black Satin, Bursa 1, Bursa 2, Cheroke, Chester,
Jumbo, Navaho, and Ness), and six wild lowbush (Vaccinium
arctostaphylos) and four cultivated highbush blueberry (Vaccinium
corymbosum) fruits (Ivanhoe, Jersey, Northland, and Rekord) were
evaulated in this study. Wild blackberries were collected from
Kavak (418310 N, 358350 E, 600 m) Samsun, while cultivated blackberries were collected from Samsun (418220 N, 368120 E, 200 m),
Turkey. Blueberries were collected from Guneysu (408580 N,
408370 E, 330 m) Rize, Turkey. Samples were packed immediately
into a vacuum and stored in a freezer at 20 8C (for 2 months) until
analyzed.

Correlation analyses were carried out with the SPSS 11.0 for
Windows software package.

2.3. Methods
2.3.1. Determination of antioxidant activity
Antioxidant activity was determined by FRAP assay. To
determine the antioxidant activity, an aliquot of the acetone/
methanol/water/formic acid (40:40:20:0.1) extracts of berry
samples was dried at 30 8C vacuum using Buchi rotary evaporator
(Newcastle, Del, USA), and redissolved in same volume of water.
Aqueous samples were mixed with 0.95 mL of ferric-TPTZ reagent
and measured at 593 nm. FeSO4 was used as a standard and
antioxidant power was expressed as mmol g1 FRAP (Tural and
Koca, 2008).
2.3.2. Determination of total phenolics
Total phenolic content was measured using the Folin-Ciocalteau method (Singleton and Rossi, 1965). Results were expressed
as milligrams of gallic acid equivalents per gram of fresh weight.
2.3.3. Determination of total anthocyanins
Total anthocyanin content was determined with the pH
differential method (Wrolstad, 1976), and expressed as milligrams
of cyanidin-3-glucoside equivalents per gram of fresh weight, on
the basis of the molar absorptivity (29,600) of cyn-3-glu for
blackberry. Calculations were based on malvidin-3-glucoside with
molar absorptivity 28,000 for blueberry. All spectrophotometric
analyses were performed using a UVvisible spectrophotometre
(Jasco V-530, Japan).
2.3.4. HPLC anthocyanin analysis
Sample preparation was carried out according to the procedure
described by Tural and Koca (2008). A berry sample was
homogenized with 1% HCl in methanol, then centrifuged at
3000 rpm for 10 min. After appropriate dilution with 1% HCl in
methanol, the sample was ltered through a 0.45 mm lter
(Biocrom MN 718020, Phonex nylon lter 25 mm), seperated and
characterized by HPLC. Samples were analyzed using a Thermoquest (USA) HPLC system equipped with a model P100 pump, a SN
4000 controller unite, a model AS 2000 autosampler and a model

3. Results and discussion

The total anthocyanin, total phenolic content, and total
antioxidant activity (expressed as FRAP value) in fruits of
blackberry and blueberry are shown in Table 1. The percentage
contribution to the total anthocyanins is presented in Tables 2
and 3.
The antioxidant activities of samples were determined using
the FRAP assay. Antioxidant activities differed among cultivars as
indicated in literature (Scalzo et al., 2005). FRAP values differed
among cultivars and varied from 35.05 to 70.41 mmol g1 for
blackberries. The antioxidant activity of blackberries reported by
Moyer et al. (2002) and Siriwoharn et al. (2004) is generally higher
than the value determined for blackberries in the present study.
Our data were lower than values observed (139.1285.4 mmol g1)
by Reyes-Carmona et al. (2005) while our results were in
agreement with values (4.766.61 mmol 100 g1) reported by
Halvorsen et al. (2002). The differences in results may be due, in
part, to the use of different blackberry genotypes in these studies.
Wild blackberries had the highest antioxidant activity compared to
cultivated blackberries, lowbush and highbush blueberries (Fig. 1).
Although the antioxidant activities obtained in our study were
Table 1
Antioxidant activity, anthocyanin and phenolic contents of blackberry and
blueberry.
FRAP (mmol g1)

Total phenolics
(mg g1)

TACN (mg g1)

Wild blackberries (n = 7)
Ranges
52.8870.41
Means  std dev
59.36  5.84

2.643.79
3.26  0.41

1.301.97
1.59  0.22

Cultivated blackberries
Arapaho
Bartin
Black Satin
Bursa 1
Bursa 2
Cherokee
Chester
Jumbo
Navaho
Ness

2.19
1.73
1.76
2.10
2.34
3.05
2.25
2.39
2.10
1.76

1.15
0.96
1.20
1.27
0.95
1.15
1.41
1.07
0.98
1.58

Wild blueberries (n = 6)
Ranges
34.4557.92
Means  std dev
43.97  8.93

3.085.42
3.95  0.91

0.592.94
1.50  0.87

Cultivated blueberries
Jersey
7.41
Ivanhoe
12.36
Northland
13.69
Rekord
7.56

8.20
1.40
1.84
0.77

0.25
0.25
0.29
0.18

38.08
35.05
39.16
38.67
40.32
43.44
42.16
38.68
36.32
41.36

I. Koca, B. Karadeniz / Scientia Horticulturae 121 (2009) 447450

Table 2
Percent contribution of individual anthocyanins to total anthocyanins in blackberries.
Cyn-3,5-di
glu (%)

Cyn-3-glu
(%)

Cyn-3-rut Plg-3-glu
(%)
(%)

Peo-3-glu
(%)

Wild blackberries (n = 7)
Ranges
0.000.14 80.0682.71
Means  std dev 0.10  0.02 81.47  1.10

0.220.48 0.000.06
0.32  0.10 0.05  0.01

Cultivated blackberries
Arapaho
0.31
Bartin

Black Satin
0.12
Bursa 1
0.12
Bursa 2
0.17
Cherokee
0.24
Chester
0.15
Jumbo
0.14
Navaho
0.15
Ness
0.20

0.10
0.33
0.11
0.11
0.17
0.15
0.14
0.06
0.11
0.11

90.42
78.19
77.47
81.05
85.79
84.60
82.14
82.29
80.58
82.81

9.09

0.38
0.11

0.04
0.09
0.12
0.10
0.13

Individual compounds presented as percentage of total peak area monitored at

515 nm.

Table 3
Percent contribution of individual anthocyanins to total anthocyanins in blueberries.
Cyn-3-glu (%)
Wild blueberries (n = 6)
Ranges
9.2012.52
Means  std dev 10.44  1.53
Cultivated blueberries
Jersey
3.11
Ivanhoe
3.51
Northland
3.30
Rekord
1.79

Cyn-3-rut (%)

Plg-3-glu (%)

Peo-3-glu (%)

8.3118.72
12.31  4.13

1.293.74
2.50  0.89

34.0550.26
44.23  6.68

1.50
4.70
0.90
4.35

36.21
40.00
26.04
35.22

3.87
5.64
2.29
5.45

Individual compounds presented as percentage of total peak area monitored at

515 nm.

Fig. 1. Antioxidant activities of blackberries and blueberries.

generally lower than the values obtained in other studies, the

results are still considerable to say that the blackberries and
blueberries grown in the Black Sea Region of Turkey are good
sources of antioxidants.
Anthocyanin contents of wild blackberries were higher than
those of cultivated blackberry fruits. Total anthocyanin contents of
blackberries ranged from 0.12 to 3.26 mg g1 according to several
researchers (Sapers et al., 1986; Wang et al., 1997; Moyer et al.,
2002; Sellappan et al., 2002; Wada and Ou, 2002; Siriwoharn et al.,
2004; Fan-Chiang and Wrolstad, 2005; Pantelidis et al., 2007).
These results are in agreement with our results. Also, phenolic
contents of wild blackberries were higher than those of cultivated
fruits. Total phenolic values were somewhat lower than the range
of 2.6110.56 mg g1 reported by several researchers (Moyer et al.,

449

2002; Sellappan et al., 2002; Wada and Ou, 2002; Siriwoharn et al.,
2004).
Cho et al. (2004) determined the total anthocyanins in arapaho
and navaho genotypes as 1.798 and 1.823, respectively. Dai et al.
(2009) determined the total anthocyanins and total phenolics in
Chester purees as 7.95 mg g1 vs. and 25.31 mg g1, respectively
and in Black Satin purees as 7.16 mg g1 vs. and 22.91 mg g1,
respectively. These ndings are generally higher than our results.
Ehlenfeldt and Prior (2001) evaulated antioxidant activity, total
phenolic and total anthocyanin contents in highbush blueberry,
including Ivanhoe, Jersey and Northland varieties. They found that
the respective average values of antioxidant activity, total
anthocyanin and total phenolic of samples were: Ivanhoe, 16.1
ORAC (oxygen radical-absorbing capacity) units, 2.01 and
0.95 mg g1; Jersey, 19.3 ORAC units, 2.05 and 1.06 mg g1 and
Northland, 17.2 ORAC units, 1.29 and 1.37 mg g1. Kalt et al. (1999)
reported that phenolics and anthocyanins values were 27.7 and
4.35 mmol g1 for lowbush blueberry, respectively. Also, they
found that phenolics and anthocyanins values 22.7 and
2.67 mmol g1 for highbush blueberry, respectively. In addition,
Zheng and Wang (2003) found that antioxidant activity (assessed
by ORAC), total anthocyanin and total phenolic contents in
blueberry were 28.9 mmol g1, 1.20 and 4.12 mg g1, respectively.
Total phenolic content of blueberries reported by Ehlenfeldt
and Prior (2001) is generally lower than the value determined for
highbush blueberries in the present study, whereas anthocyanin
values reported by them is higher. The total anthocyanin content of
blueberries was lower than the values (1.438.22 mg g1) reported
by Cho et al. (2004), which may be attributed to differences in
environmental growing conditions and genotypes. Kalt et al.
(2001) also indicated that synthesis of anthocyanins and other
phenolic compounds can be inuenced by various abiotic and
biotic factors, including temperature, irradiation, herbivory, and
pathogenic infection.
Antioxidant activity, total anthocyanin and total phenolic
values of lowbush blueberries were higher than highbush blueberries. On average, lowbush blueberries had higher levels of
anthocyanins, total phenolics, and antioxidant activity than
highbush blueberries; which is consistent with the earlier reports
(Kalt et al., 1999, 2000).
FRAP values in blueberries were highly correlated with phenol
content (r = 0.981), whereas a less linear correlation between total
antioxidant activity and anthocyanin content was recorded
(r = 0.817), implying that the antioxidant activity of blueberries
is largely due to presence of phenolic compounds. Similar results
have been reported by other researchers (Wang and Lin, 2000),
who found a linear correlation between total antioxidant activity
and phenol content in blackberries (r = 0.961). In addition,
Deighton et al. (2000) reported that there were apparent linear
relationships between antioxidant activity (assessed by FRAP) and
total phenols (r = 0.965), whereas anthocyanin content had a
minor inuence on antioxidant activity (r = 0.588) of Rubus juices.
Also, the correlation coefcient for antioxidant activity and total
phenols content (r = 0.798) in blackberries is better than that for
antioxidant activity and anthocyanin content (r = 0.530). Similar
results have been reported by other researchers (Wada and Ou,
2002) for blackberries.
In this study, both wild and cultivated blackberries generally
contained 12 kinds of anthocyanins. Six peaks of anthocyanins
were identied. The amounts of anthocyanins in the blackberries
varied markedly. Cyn-3-glu was the major anthocyanin in blackberries. Cyn-3-glu ranged from 77.47% to 90.42% of peak area
(Table 2). The data were consistent with previous reports (Sapers
et al., 1986; Wada and Ou, 2002; Cho et al., 2004; Fan-Chiang and
Wrolstad, 2005). The other minor anthocyanins identied included
cyn-3,5-di glu, peo-3-glu, plg-3-glu, cyn-3-rut (only in Nessy

450

I. Koca, B. Karadeniz / Scientia Horticulturae 121 (2009) 447450

blackberries). Arapaho contained the highest amount of cyn-3-glu

(90.42%), while Black Satin contained the lowest amount of cyn-3glu (77.47%). Cyn-3-glu values agreed well with the values
reported by Cho et al. (2004) for Navaho, but were higher than
range of values reported by Cho et al. (2004) for Arapaho.
Sapers et al. (1986) showed in addition the presence of cyn-3xyloside along with 2 cyanidin derivatives that were vaguely
characterized as being substituted with dicarboxylic acids. FanChiang and Wrolstad (2005) detected ve pigments: cyn-3-glu,
cyn-3-rut, cyn-3-xyloside, cyn-3-glu with malonic acid, and an
unidentied acylated derivative of cyn-3-glu in blackberries.
The blueberry fruits generally contained 15 kinds of anthocyanins. Five peaks of anthocyanins were identied (Table 3).
Identied predominant anthocyanin in blueberries was peo-3glu. The relative distribution of anthocyanins in blueberries was
different from the previous reports (Skrede et al., 2000; Prior et al.,
2001; Cho et al., 2004).
Skrede et al. (2000) identied the anthocyanins in highbush
blueberries as malv-3-gal (20.2%), malv-3-ara (13.5%), delp-3-gal
(12.3%), delp-3-ara + cyn-3-gal (12.0%), malv-3-glu (10.6%), pet-3gal (9.1%), pet-3-glu (7.2%), pet-3-ara (6.3%) and delp-3-glu (5.4%).
Prior et al. (2001) found that the total anthocyanin fraction was
composed of more than 16 individual components in blueberries.
They detected major anthocyanin pigments: malv-3-gal (14.4%),
malv-3-glu (14.1%), pet-3-glu (10.7%), delp-3-glu (7.8%), and delp3-gal (7.7%) in lowbush blueberries. Cho et al. (2004) found 14
anthocyanin monoglycosides, and three acylated anthocyanins in
ve blueberry genotypes. They stated that delphinidin (27.1
40.4%), malvidin (22.132.9%) and petunidin (18.926.2%) were
the predominant monomeric anthocyanins, followed by cyanidin
(5.714.0%) and peonidin (1.44.5%) in blueberries.
4. Conclusion
Blackberries and blueberries are good sources of natural
antioxidants and possess potent antioxidant activity. In the
present study, we showed that antioxidant activity values of wild
blackberries and blueberries were higher than those of cultivated
berries. It was observed that the antioxidant activity values in
blueberries and blackberries were more highly correlated with
total phenolics than total anthocyanins.
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