09 e 4150 e 8911736 F 8 D 000000

Journal of Berry Research 1 (2010) 312
DOI:10.3233/BR-2010-001
IOS Press
Antioxidant and antiproliferative properties

of strawberry tree tissues
L. Tavaresa , S. Fortalezasa , C. Carrilhoa , G.J. McDougallb , D. Stewartb , R.B. Ferreiraa,c
and C.N. Santosa,
a Disease
& Stress Biology Laboratory, Instituto de Tecnologia Qumica e Biolgica, Universidade Nova
de Lisboa, Oeiras, Portugal
b Plant Products and Food Quality Programme, Scottish Crop Research Institute, Dundee, Scotland, UK
c Departamento de Botnica e Engenharia Biolgica Instituto Superior de Agronomia, Lisboa, Portugal
Abstract. Strawberry tree (Arbutus unedo) belongs to the Ericaceae family and is endemic to the Mediterranean area. Its fruits
are edible and its fruits and leaves are used in folk medicine for diverse purposes. Previous studies have shown that the fruits are
rich in flavonoids, responsible for their antioxidant properties and compounds isolated from the entire plant were promising in
cancer chemopreventive therapy.
Strawberry tree fruits and leaves extracts enriched in polyphenols, but devoid of organic acids, carotenoids and sugars, were
prepared by solid phase extraction (SPE) and tested for their antioxidant activities and their ability to inhibit metalloproteinases:
attributes that could be related with initiation and proliferation of cancer cells.
After fractionation by SPE, the apparent polyphenol yield was reduced for both leaf and fruit samples by the elimination of
vitamins and organic acids, but the antioxidant and metalloproteinases inhibitory activities were potentiated.
The antioxidant activity and the MMP-9 inhibitory activity of the polyphenol-enriched fractions of A. unedo tissues were similar
or higher than those of blackberry and green tea, which have been recognized in the literature as highly effective.
The phenolic profile of the fruit was dominated by gallic acid and quercetin derivatives with smaller amounts of proanthocyanidins and anthocyanins. The phenolic profile of the leaves was also dominated by gallic acid derivatives, flavonol derivatives and
some tannins but lacked anthocyanins.
The fractions obtained from both strawberry tree tissues seem to be quite promising as antioxidants and antiproliferative agents.
Further cell-based assays are underway to study these possible outcomes.
Keywords: Arbutus unedo, polyphenols, antioxidant activity, MMP-9, HPLC-MS
1. Introduction
There is considerable epidemiological evidence that insufficient intake of fruits and vegetables may predispose
the human body to a range of chronic health disorders, including cancer and cardiovascular disease [9]. Phytochemicals, such as polyphenols, present in many fruits and vegetables may participate in disease prevention and this has
contributed to the growing interest in identifying components in edible plants responsible for anticancer effects [19].
Polyphenols demonstrate diverse biological activities attributed to their general free radical trapping capacity, or
antioxidant activity per se, iron chelation, activation of survival genes, cell signalling pathways and regulation of
mitochondrial function [9, 23]. In cancer, several studies point to the important role of the oxidative stress on the
Corresponding author: C.N. Santos, Disease & Stress Biology Laboratory, Instituto de Tecnologia Qumica e Biolgica, Universidade Nova de
Lisboa, Av. da Repblica, Apartado 127, 2781-901 Oeiras, Portugal. Tel.: +351 214469651; Fax: +351 214433644; E-mail: csantos@itqb.unl.pt.
1878-5093/10/$27.50 2010 IOS Press and the authors. All rights reserved
L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues
oncogenic stimulation. Various cancer cells have a cellular redox imbalance, i.e, a disruption between endogenous
antioxidants and reactive oxygen and nitrogen species (ROS and RNS) balance when compared with normal cells,
which could modify permanently the cell biology. This redox imbalance could lead to damage in cellular biomolecules
that contribute to induction of mutagenesis and carcinogenesis [3739]. Free radical damage could influence DNA
mutations and altered gene expression, lipid peroxidation due to malondialdehyde (MDA) and 4-hydroxynonenal
(HNE) formation [16, 25, 38], and protein oxidation [39].
Cancer cell invasion is a critical point for cancer metastasis. It is generally accepted that remodelling of the
extracellular matrix (ECM) is required for cancer cell invasion [41]. The primary response for the degradation of
ECM components was demonstrated to be the activation of zinc-dependent matrix metalloproteinases (MMPs) [27].
Although MMPs are expressed in normal tissue in remodelling conditions, such as during embryonic development,
wound healing, uterine and mammary involution, cartilage-to-bone transition in ossification, and placenta development, the aberrant expression of various MMPs has been correlated with pathological conditions, such as rheumatoid
arthritis, tumour cell invasion and metastasis [12]. Matrix metalloproteinase inhibitors (MMPIs) have been shown
to inhibit angiogenesis in various models. However the synthetic inhibitors have demonstrated some disadvantages,
such as the lack of selectivity and the secondary effects [24].
Strawberry tree (Arbutus unedo L.; Ericaceae family) is an evergreen shrub, a native Mediterranean species which
is also cultivated in other regions of Eastern Europe [3]. Its fruits are spherical, about 2 cm in diameter, dark red, and
tasty only when fully ripen in autumn. A. unedo berries are rarely eaten as fresh fruits but have some importance in
local agricultural communities which use them for the production of alcoholic beverages, jams, jellies and marmalades
[2, 28]. The fruits are also used in folk medicine as antiseptics, diuretics and laxatives, while the leaves have long
been employed as an astringent, diuretic, urinary anti-septic agent and, more recently, in the therapy of hypertension
and diabetes [4]. Recently, Carcache-Blanco and co-workers isolated ten compounds from A. unedo leaves exhibiting
chemopreventive action through cyclooxygenase-2 inhibitory activity [6]. This work reinforces A. unedo as promising
source of antiproliferative agents. Strawberry tree fruit contains several classes of naturally occurring antioxidants
such as phenolic compounds (e.g. anthocyanins, gallic acid derivatives, tannins and flavonoids), vitamin C, vitamin E
and carotenoids [2, 3, 22, 28, 29], and a high antioxidant capacity [28].
The aim of this work was to assess an in vitro potential of polyphenols from A. unedo fruits and leaves to provide an
antiproliferative effect. The extracts prepared were enriched in polyphenols, but devoid of organic acids (vitamin C),
carotenoids and sugars. Using this approach, we expect to maximize the activities of the compounds through positive
synergisms between the different polyphenols or by eliminating antagonisms, in contrast to the Carcache-Blanco
approach that tested pure compounds [6]. Synergistic positive effects are well described between polyphenols [1,
33, 35]. For example, Seeram et al. detected for cranberry extracts [33], after removing sugars and organic acids,
a higher antiproliferative activity of polyphenols enriched fraction, when compared against the individual purified
phytochemicals.
2. Material and methodology

2.1. Biological material
Arbutus unedo L. fruits and leaves were collected in November 2007, in Arrbida Natural Park (southern region
of Portugal) and frozen. For comparison purposes, the blackberry cv. Apache produced in Fataca experimental field
(Odemira, Portugal) and commercial tea leaves (Lipton Green Tea Pure, Lipton) were used as control samples for
fruits and leaves, respectively. The samples were freeze-dried, ground and stored at 80 C prior to extraction.
2.2. Extract preparation
To each 1 g of lyophilized powder, 12 mL of hydroethanolic solvent (50% (v/v) ethanol/water) was added and
the mixture was shaken for 30 min at room temperature in the dark. The mixture was then centrifuged at 12400 g
for 10 min at room temperature. The supernatant was filtered through paper filter and then through 0.2 m cellulose
acetate membrane filters. The resulting extracts were stored frozen at 80 C.
2.3. Fractionation by solid phase extraction

Hydroethanolic extracts were fractionated by Solid Phase Extraction (SPE) using a Giga tubes 2 g/12 mL, C18-E
units (Phenomenex ). The columns were pre-washed in 0.5% (v/v) glacial acetic acid in acetonitrile and then preequilibrated in 0.5% (v/v) glacial acetic acid in water. The extracts were dried under vaccum and ressuspended in 0.5%
(v/v) glacial acetic acid in water, then they were applied to the columns and unbound material, which contained the
free sugars, organic acids or vitamin C, was discarded. The columns were washed with 0.5% (v/v) aqueous acetic acid
and then polyphenol-enriched bound fractions were eluted with 0.5% (v/v) glacial acetic acid in acetonitrile [30, 32].
2.4. Total phenolic measurement
Determination of total phenolic compounds was performed by the Folin-Ciocalteau method [34]. Briefly, to each
well of a microplate, 235 L water, 5 L sample (or solvent, in the blank), 15 L Folin-Ciocalteaus reagent (Fluka )
and 45 L saturated Na2 CO3 were added. The microplate was incubated for 30 min at 40 C and the absorbance at
765 nm measured. Gallic acid was used as the standard and the results were expressed as mg of gallic acid equivalents
(mg GAE).
2.5. Peroxyl radical scavenging capacity assay
Peroxyl radical scavenging capacity was determined by the ORAC (Oxygen Radical Absorbance Capacity) method
[5, 40]. Briefly, the reaction mixture contained 150 L of sodium fluorescein (0.2 nM) (Uranine, Fluorescein Sodium
Salt TCI Europe), 25 L sample and 25 L of 2,2 -azobis(2-amidopropane) dihydrochloride (41.4 g.L1 ). All solutions are prepared in 75 mM phosphate buffer (pH 7.4). The blank contained 25 L 75 mM phosphate buffer (pH 7.4)
instead of sample, whereas the standards contained 25 L of 10 to 50 M 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox ) in place of the sample. The fluorescent emission at 515 nm was then monitored kinetically
during 30 min at 37 C, after excitation at 493 nm using a FLx800 Fluorescence Microplate Reader (Biotek). The
final results were calculated using the area differences under the fluorescence decay curves between the blank and
the sample, and were expressed as mM Trolox equivalents (mM TE).
2.6. MMP-9 inhibitory activity assay
MMP-9 pro-enzyme was activated using 4-aminophenylmercuric acetate (APMA). The enzyme was incubated
with 1 mM APMA in 100 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2 and 0.05% (v/v)
Brij 35 and 2% (v/v) DMSO for 22 h at 37 C in the dark. Then the reaction was stopped with ice and the active
MMP-9 was kept at 4 C. MMP-9 activity was measured by the direct hydrolysis of MCA-Pro-Leu-Gly-Leu-(Dnpa)-Ala-Ala-Arg-amida substrate [18], which has a fluorescent group 7-metoxicumarin-4-acetil (MCA) and a
quenching group 2,4-dinitrofenilamine (Dnpa). Therefore, an increase in fluorescence intensity occurs as a result
of substrate hydrolysis, namely the cleavage of peptide Gly-Leu. Before inhibition tests, enzymatic activity using
different concentrations of MMP-9 was measured, which revealed a linear relationship between enzyme activity and
enzyme concentration (data not shown). The highest concentration was chosen for inhibition assays. The assays were
performed on 96 well plates measuring a residual enzymatic activity. The enzyme (3.2 nM) was pre-incubated with
100 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 10 mM CaCl2 and 0.05% (v/v) Brij 35 and different
concentrations (025 g GAE. mL1 ) of test fractions at 37 C for 30 min. The reaction was initiated by adding the
substrate (12 M in the same buffer as the enzyme) in a final volume of 100 L. Fluorescence (emission = 393 nm,
excitation = 325 nm) was measured for 10 min at room temperature. Results were transformed using a non-linear
regression with Origin Pro 6.1 software (OriginLab , USA) and the IC50 values were determined.
2.7. HPLC-MS phenolic prole determination
Phenolic extracts were dried by rotary evaporation, ressuspended in 5% (v/v) acetonitrile in water and were analyzed
on a LCQ-DECA system controlled by the XCALIBUR software (2.0, ThermoFinnigan). The LCQ-Deca system
comprised a Surveyor autosampler, pump and photo diode array detector (PDAD) and a Thermo Finnigan mass
spectrometer iontrap. The PDA collected spectral data from 200600 nm and scanned three discrete channels (at 280,
365 and 510 nm). The samples were applied to a C-18 column (Synergi Hydro C18 column with polar end capping,
4.6 mm 150 mm, Phenomonex Ltd) and eluted over a gradient of 95:5 solvent A:B at time = 0 min to 60:40 A:B
at time = 60 min at a flow rate of 400 L/min. Solvent A was 0.1% (v/v) formic acid in ultra pure water and solvent
B 0.1% (v/v) formic acid in acetonitrile. The LCQ-Deca LC-MS was fitted with an ESI (electrospray ionization)
interface and analyzed the samples in positive and negative-ion mode. Two scan events, full scan analysis in mass range
802000 m/z followed by data dependent MS/MS of the most intense ions, were used for compounds detection and
identification. The data-dependent MS/MS used collision energies (source voltage) of 45%. The capillary temperature
was set at 275 C with sheath gas at 60 psi and auxiliary gas at 10 psi. Before the analysis, the system was tuned by
using known concentrations of cyanidin-3-glucoside (positive mode) and quercetin-3-glucoside (negative mode) in
ultrapure water.
2.8. Statistical analysis
The results reported in this work are the averages of at least three independent experiments and are represented as
the mean SD. Differences among treatments were detected by analysis of variance [31] with Tukey HSD (Honestly
Significant Difference) multiple comparison test ( = 0.05) using SigmaStat 3.10 (Systat).
3. Results and discussion

3.1. Assessment of bioactivities recovery in polyphenols enriched fractions
After the hydroethanolic extraction, crude extracts were fractionated by SPE column, to obtain polyphenol-enriched
fractions. The crude extracts and enriched fractions were assessed for total phenolic content, antioxidant activity for
peroxyl radical and the MMP-9 inhibitory activity (Fig. 1).
After fractionation by the SPE column, strawberry tree fruit and leaf extracts showed a decrease in phenolic
content (18% and 62% respectively). The decreases observed are probably due to molecules detected by FolinCiocalteu method, such as vitamins or organic acids that are eliminated by the SPE [13]. Indeed, components such
4
3.5
50
3
40
2.5
30
2
1.5
20
1
10
0.5
MMP-9 inhibitory activity (IC50)
Total phenol content/Anti oxidant activity
60
Extract
Enriched fraction
Fruits
Extract
Enriched fraction
Leaves
Fig. 1. Comparison of total phenol content, antioxidant capacity and metalloproteinases inhibitory activity between crude extracts and polyphenolenriched fractions obtained from A. unedo fruits and leaves. -total phenol content (mg GAE), - IC50 of MMP-9 inhibitory capacity (mg GAE.
mL1 ) and antioxidant activity (10 mmol TE) for peroxyl radical were determined for crude extracts and polyphenols enriched fractions
obtained from A. unedo fruits and leaves. Vertical bars represent SD.
as niacin, ascorbic acid, -carotene, L-malic and quinic acids (detected and quantified in strawberry tree fruits by
Alarco-E-Silva et al. [2] or Ayaz et al. [3]) could account for the apparent loss in total phenol content after SPE.
The greater decrease in the fruit fraction could be due to the higher contents in vitamins and organic acids in fruits.
The strawberry tree fruit polyphenol-enriched fraction presented an increase of 100% of antioxidant activity,
suggesting that the SPE fractionation eliminated some compounds with antagonistic effects. Strawberry tree leaves
had an antioxidant capacity recovery of approximately 100%.
Concerning the MMP-9 inhibitory capacity, the IC50 values for both fractions diminished 50%, which means that
50% of the amount of polyphenols is required to produce the same effect as the crude extract.
Therefore, fractionation of strawberry tree tissues using SPE simultaneously eliminated molecules that could exert
some antagonistic effects (e.g. in the antioxidant activity) and enriched the phenolic compounds (which diminished the
IC50 value for MMP inhibition). However, further fractioning could not be so beneficial, since could eliminate positive
synergistic effects, already detected by others [33]. In fact Carcache-Blanco have tested A. unedo isolated compounds
and detected lower efficacies (high IC50 ) than their positive controls (13-cis-Retinoic acid and trans-Resveratrol) in
chemopreventive assays [6].
3.2. Antioxidant and MMP-9 inhibitory activities
The strawberry tree fruit and leaf extracts gave significantly higher antioxidant capacities (211.66 22.82 mM TE
and 308.56 26.74 mM TE, respectively) than blackberry and green tea, respectively (Table 1). The IC50 values of
MMP-9 inhibitory activity for both strawberry tree tissues were not significantly different of the amount required for
the respective control samples (1.68 0.38 g GAE. mL1 for strawberry tree fruits and 1.31 0.17 g GAE. mL1
for leaves) (Table 1).
Control tissues used in this work, blackberry and green tea leaves, are recognized in the literature as highly
antioxidants and anti-cancer agents [7, 8, 15, 36, 4244], Blackberries are a rich source of polyphenols and present a
recognized in vitro antioxidant activity (6221 mol TE. 100 g1 determined by ORAC method by Wolfe et al.) [42] as
well as a good cellular antioxidant activity detected by a decrease in ROS in cells [42]. These fruits also have shown
to inhibit MMP-2 and MMP-9 activities [36]. Moreover, they also could inhibit HT-29 colon tumor cell growth in a
concentration-dependent manner [8]. Green tea leaves have been studied for many years and it has been shown their
capacity to inhibit carcinogen-induced DNA damage in a number of cell line studies as reviewed by Yang et al. due
to their direct radical scavenging and metal chelation [43, 44]. Tea and tea constituents have been shown to inhibit
the development of several cancer in animal models like oral, esophageal, forestomach, stomach, intestinal, colon,
skin, liver, bladder, prostate, and breast cancer [7, 15, 44].
In this work results for the A. unedo tissues, in comparison with the positive controls, revealed to be promising
sources of effective antiproliferative extracts. To confirm these in vitro results, cell based assays should be performed
as antiproliferative and intracellular antioxidant tests.
Table 1
Comparison of antioxidant capacity and MMP-9 inhibitory capacity (IC50 ) values for
polyphenol-enriched fractions from strawberry tree fruit and leaves with the respective positive
controls (blackberry and green tea). Antioxidant capacity was determined by ORAC method
and expressed as mM TE. IC50 values (g GAE. mL1 ) were obtained by OriginPro 6.1 software. The values presented are the mean value of triplicates SD. The values of each tissue
were statistically compared with the value of respective control. nd: not significantly different
for p < 0.05; *: significantly different for p < 0.05; ***: significantly different for p < 0.001
Tissue
Sample
Fruits
Blackberry
Strawberry tree
Green tea
Strawberry tree
Leaves
Antioxidant capacity
(mM TE)
MMP-9 inhibitory capacity

(IC50 ) (g GAE. mL1 )
118.75 17.15
211.66 22.82***
135.95 2.99
308.56 26.74*
1.52 0.16
1.68 0.38nd
1.94 0.39
1.31 0.17nd
3.3. HPLC-MS phenolic prole determination

To identify the main compounds present in fruits and leaves of strawberry tree, phenolic extracts were analysed
by HPLC-ESI-PDA-MS. The compounds were tentatively identified in the bibliography by their PDA and MS
fragmentation patterns [28, 29] (Fig. 2; Table 2). The fruits composition is relatively well-known compared to leaves
[28, 29]. The fruits yielded mainly gallic acid derivatives (as glucosides, galloylquinic acid, galloylshikimic acid and
gallotannins) but also some proanthocyanidins, quercetin derivatives and ellagic acid derivatives. The anthocyanins
are not abundant in these fruits, but were identified in accordance with the bibliography as delphinidin-3-galactoside,
100
NL:
2.35E6
A
90
10
80
70
60
14
12
11
15
50
40
17
18
16 19 22
20 27
9
5 7
30
2 4
20
10
0
100
20
90
NL:
6.98E4
B
21
23 24
16
25
80
70
30
26
29
60
50
40
13
28
30
20
10
0
100
NL:
7.26E4
C
A2
90
80
70
60
50
40
A3
30
A1
20
10
0
0
10
20
30
40
50
60
Time (min)
Fig. 2. HPLC phenolic profile assessed for A. unedo fruit extract. The chromatogram in (A) shows the absorbance of peaks at 280 nm, (B) peaks
at 365 nm and (C) peaks at 560 nm. The peaks are numbered and assignments are given in Table 2 for phenols and Table 3 for anthocyanins.
Table 2
Peaks assignments, retention times and mass spectral data of phenols present in A. unedo fruits extract
Peak no.
RT
PDA
M/Z [M-H]
MS2
Putative identity
1
2
3
4
7.37
8.70
10.64
14.04
280
265
270
255300
331.1
331.1
343.0
331.1
271.0, 211.1, 169.0

169.0, 125.0
191.2, 169.0
169.1
16.29
20.93
21.36
21.95
22.66
23.86
24.31
24.6
25.50
26.10
26.84
28.68
29.44
255300
280
270290
295
280
280
320
285
280, 525
275
270
360
275
169.0, 125.1
289.2
343.0, 191.0
343.0, 191.0
425.0, 407.2, 289.2
261.0, 175.0
577.1
453.1, 301.4, 169.2
325.0, 169.0
325.0, 169.0
463.0, 301.1
301.2
783.1, 492.8
18
19
20
21
22
23
24
25
26
27
31.09
33.08
33.36
34.67
35.21
36.30
36.69
36.96
37.40
38.09
280
275
360
260355
260355
260355
255, 370
345
275355
280
204.1, 186.1, 142.0

633.0, 463.2, 301.2
301.0
463.0, 301.1
463.0, 301.1
463.0, 301.1
301.2, 257.2
301.2
301.2
769.0, 617.2
Unknown
Tannin
Quercetin-3-xyloside
Quercetin hexose galloyl derivative
Ellagic acid
Quercetin 3-galactoside
Quercetin 3-glucoside
Gallotannin
28
29
30
39.81
40.75
41.49
355
355
355
325.0
577.1
495.0, 465.0, 343.0, 191.2
495.0, 465.0, 343.0, 191.2
577.0, 423.2, 407.2, 289.2
289.1
865.1, 453.2, 325.1
541.1, 483.1, 467.3, 321.0, 301.2
477.0, 325.1
477.0, 325.1
633.1, 463.1, 301.2, 275.2
463.2, 301.3
1109.0, 972.9, 647.0, 635.1, 588.1,
441.0, 301.3
366.2, 186.0
953.0
433.1, 301.2
615.2, 463.2, 433.1, 301.1
615.2, 463.2, 433.1, 301.1
615.2, 463.2, 433.1, 301.1
301.2
463.1, 301.2
463.1, 301.2
939.1, 769.1, 729.0, 617.1, 544.2,
480.2, 469.2
599.0, 301.0
433.0, 301.1
447.0, 301.1
Gallic acid glucoside

Galloyl glucoside
3-O- or 5-O-galloylquinic acid [29]
Gallic acid
4-O-B-D-glucopyranoside or
B-D-Glucogalline [29]
Galloyl shikimic acid
Proanthocyanidin dimer [29]
Digalloylquinic acid
Isomer of digalloylquinic acid
Procyanidin dimer B2 [28]
Catechin
Procyanidin trimer
Gallic acid derivative
Digalloyl shikimic acid
Digalloyl shikimic acid
Strictinin ellagitannin
Quercetin-3-glucoside
Gallotannin derivative
5
6
7
8
9
10
11
12
13
14
15
16
17
463.1, 301.1
301.1
301.1
Ellagic acid-hexose derivative

Ellagic acid arabinoside/xyloside
Ellagic acid rhamnoside
The most abundant ions are shown in bold. Numbers in brackets are references.
cyanidin-3-galactoside, cyanidin-3-glucoside and cyanidin-3-arabinoside [28, 29]. The leaf extracts also yielded gallic
acid derivatives common to fruits, (epi)catechin, tannins, myricetin derivatives and kaempferol derivatives. Some of
these phytocompounds, such as tannins, quercetin derivatives and catechin gallate have already been described as
responsible for anti-hypertensive and anti-aggregant effects using strawberry tree leaf extracts [20, 26].
4. Conclusion
In this work the polyphenol-enriched fractions of Arbutus unedo fruits and leaves were characterized for antioxidant
and MMP-9 inhibitory activity, and the results obtained suggest these tissues as promising sources of agents with
pharmacological activities. Many naturally occurring agents have shown chemoprotective potential in a variety of
10

Table 3
Peaks assignments, retention times and mass spectral data of anthocyanins present in A. unedo fruits extract
Peak no.
A1
A2
A3
RT
PDA
20.60
22.94
25.28
280, 525
280, 515
280, 515
M/Z [M+H]
MS2
Pututative identity
579.1, 465.1, 303.2

449.0, 287.2
419.1, 287.2
303.2
287.2
287.2
Delphinidin-3-galactoside [28]
Cyanidin 3-O-glucoside or cyanidin-3-galactoside [28]
Cyanidin 3-O-arabinoside [28]
The most abundant ions are shown in bold. Numbers in brackets are references.
Table 4
Peaks assignments, retention times and mass spectral data of phenols present in A. unedo leaves extract
Peak No.
RT
PDA
M/Z [M-H]
MS2
Putative identity
9.57
280
10.98
12.80
280
260
839.5, 729.2, 676.1, 567.2,

270.9, 160.9, 109.2
271.0, 211.1, 169.1
271.1, 169.1, 125.1
Unknown
2
3
1111.2, 899.3, 839.3, 817.3,

567.3, 317.0
331.2, 271.1, 169.1
331.2, 271.2, 169.1
13.54
270
363.2, 169.2
169.1
5
6
7
8
9
10
11
14.80
20.57
26.48
26.94
27.92
30.82
33.26
275
275
275
275
280
270
275
12
36.91
355
13
14
15
38.57
41.26
43.04
280
360
280, 355
191.2, 169.1
169.1, 125.2
593.0, 575.1, 425.0, 407.2
423.2, 313.1, 169.1
245.2, 231.2, 205.2, 179.1
325.1
914.9, 896.9, 765.0, 613.1,
461.1, 445.1, 301.3
909.1, 801.0, 633.1, 463.2,
301.3
325.1
316.2
787.1, 769.1, 617.2, 601.2
16
17
46.51
50.25
255, 350
350
343.1, 191.2
325.2, 169.1, 125.2
745.1, 577.2, 495.1, 343.1, 289.1
423.3, 313.2
289.2
477.1, 325.2, 169.2
951.1, 933.1, 729.2, 477.1, 301.3,
273.2
1105.0, 1085.0, 953.2, 935.2, 785.1,
633.2, 479.2, 316.2, 301.3
629.0, 477.1, 325.2
463.2, 449.2, 316.2
939.2, 769.2, 617.2, 469.3, 301.3,
169.1
447.2, 300.2
583.2, 417.2, 300.2, 284.2
18
19
20
51.17
38.03
42.56
265, 340
355
350
476.8, 431.2, 285.2, 227.3

479.2, 317.2
463.0, 300.1
285.2
317.1
301.1
301.1
417.2, 327.1, 284.1, 255.3
Gallic acid glucose derivative

B-D-Glucogalline or gallic acid
4-O-B-D-glucopyranoside
B-D-Glucogalline or gallic acid
4-O-B-D-glucopyranoside
derivative
Galloylquinic acid derivative
Galloylshikimic acid
Proanthocyanidin derivative
Galloyl derivative
Catechin
Digalloylshikimic acid derivative
Tannin
Tannin
Galloylshikimic acid derivative
Myricetin-3-rhamnose
Gallotannin
Quercetin 3-rhamnoside
Kaempferol derivative
(Arabinoside/Xyloside)
Kaempferol derivative (Rhamnoside)
Myricetin-Hexoside
Quercetin-Hexoside
The most abundant ions are shown in bold.
bioassay systems and animal models. Phenolic compounds are reported to have antioxidant, antimutagenic and
anticarcinogenic activity and are expected to reduce the risk of disease and to bring health benefits with daily intake
[11].
Gallic acid derivatives dominate the profile of both fruits and leaves of Arbutus unedo. Gallic acid is an intermediate
component of plant metabolism and, together with its analogs, has been associated with a wide variety of biological
actions, including: antioxidant, antifungal, antimalarial, and antiherpetic action [10, 14, 17]. However, the main
interest in gallic acid and its derivatives is related to its antitumoral activity, showing selective cytotoxicity toward
a variety of tumor cells, more cytotoxic for tumor cells than for non-tumor cells [21]. Nevertheless, in studies using
11
pure coumpounds, synergistic effects may be lost in determining anti-proliferative effects. In this study, the use of
polyphenols-enriched fraction, devoid of ascorbic acid and carotenoids, allows interactions between polyphenols and
the detected bioactivity could be directly associated to them and their interactions. Fruits and leaves polyphenolsenriched fractions presented IC50 values similar to the ones obtained for positive control tissues with recognized
activity as anti-cancer and antioxidant agents. Therefore, it is important to confirm the potential of these enriched
fractions as multitarget medicines with antiproliferative efficacy, using cell based assays such as proliferation and
intracellular antioxidant tests.
Acknowledgements
To FCT for financial support of Claudia Santos (SRFH/BPD/26562/2006) and Luclia Tavares (SFRH/BD/37382/
2007) and to Cost Office 863.
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DOI:10.3233/BR-2010-007
IOS Press
Editorial
I would like to begin by first giving a warm welcome to all the readers and contributors to the Journal of Berry
Research.
In the vast current scientific-editorial panorama it is quite understandable to ask oneself why, how and what will
this new scientific journal be like.
Why have a new journal? Because in spite of the many thousands of articles indexed in PubMed under berries
(over 12,000 in 2008 and nearly 15,000 in 2009) there is no specialized journal which can be used for reference,
collection, discussion and promotion of the many excellent studies carried out worldwide. In fact, most articles are
scattered among a large variety of journals, ranging from chemistry of the soil to anti-tumour therapy, with obvious
difficulties both on the part of reviewers who have to evaluate the contents in a rigorously scientific way and on
the part of the readers. Moreover, many excellent articles are not given the visibility they deserve while others are
sacrificed to a concept of necessary priority for journal management, but at the same time penalized if applied to
productive studies and sectors in which the pharmaceutical or food industry still do not have a decisive position.
How will the Journal of Berry Research function? The journal will be multidisciplinary, aimed at improving
knowledge about the quality and production of berries to benefit consumers health; it will cover classical berries
(strawberry, raspberry, blackberry, blueberry, cranberry, etc.) as well as grapes and small fruit in general (e.g., kiwifruit,
tomato) as currently occurs, for example, for the Berry Group in the framework of the International Horticultural
Society. JBR will publish research results covering all areas of plant breeding, including plant genetics, genomics,
functional genomics, proteomics and metabolomics, plant physiology, plant pathology and plant development, as
well as results dealing with the chemistry and biochemistry of bioactive compounds contained in such fruits and
their possible role in human health. Contributions detailing possible pharmacological, medical or therapeutic use or
dietary significance will be welcomed in addition to studies regarding biosafety issues of genetically modified plants.
What will the Journal of Berry Research be like? This depends only partially on us (the Editorial Board) but
above all on you: Our readers and authors. We on the Editorial Board will be committed to following the goals which
we set and which are clearly described in this letter and in the Aims of the journal, and to running the journal with
scientific rigour yet without any kind of prejudice. What JBR becomes depends above all on who reads and, we hope,
appreciates it: It will depend on the quantity but above the quality of your scientific contributions and on how much
these will be read, discussed and cited.
The invitation and wish is therefore the following: Help us to make the Journal of Berry Research an important
journal, your journal.
Editor-in-Chief
Dr. Maurizio Battino, PhD, MD (Hon)
Department of Biochemistry, Biology & Genetics
Faculty of Medicine
Universit Politecnica delle Marche
Via Ranieri
60100 Ancona
Italy
Tel.: +39 071 2204646
Fax: +39 071 2204398
E-mail: m.a.battino@univpm.it

DOI:10.3233/BR-2010-006
IOS Press
53
Nutritional quality of berries and bioactive

compounds in the leaves of black currant
(Ribes nigrum L.) cultivars evaluated
in Estonia
Piret Raudseppa, , Hedi Kaldmeb , Ave Kikasb , Asta-Virve Libekb and Tnu Pssaa
a Department
b Polli
of Food Hygiene, Estonian University of Life Sciences, Tartu, Estonia

Horticultural Research Centre, Viljandimaa, Estonia
Abstract. Polli Horticultural Research Centre (58 7 N; 25 32 E) in Estonia has focused on selecting cultivars with high productivity
and suitable to use in local climatic conditions since 1945. Besides important agronomic characteristics, more attention has been
recently paid to fruit quality and content of various bioactive compounds.
The results of a biochemical analysis of 4 prospective black currant selections (10B, 2-96-51, 1-96-16, 4-96-1), 4 new cultivars
(Karri, Almo, Ats, Elo) from our own breeding program and 7 introduced cultivars (jebyn, Zagadka, Ben Sarek,
Intercontinental, Pamyati Vavilova, Titania and Pilenai) are presented.
In addition to the analysis of main biochemical characteristics, the anthocyanin content of the berries was determined using
high performance liquid chromatography (HPLC). The total anthocyanin content of the berries varied in a wide range. The highest
anthocyanin content was found in the cultivar Almo (212 9 mg/100 g) and the lowest in Ben Sarek (83 24 mg/100 g). The
ascorbic acid content varied from 98 mg/100 g with Ats to 209 mg/100 g with elite selection 4-96-1.
The polyphenol composition of the black currant leaves was determined by HPLC, the compounds were identified using
polyphenol commercial standards and/or compounds mass spectrometric (MS) characteristics.
Keywords: Black currant, seedlings, fruit quality, leaves, poly phenol composition
1. Introduction
Ascorbic acid and anthocyanins are the most highly valued health beneficial compounds in black currants contributing to the antioxidant capacity of the fruit [13]. Anthocyanins have also been associated with fruit colour and
are, therefore, important contributors to the quality of the product, they appear to be very stable and remain active
after a prolonged frozen storage, processing into juice, wine and jam [9, 13]. The change of focus to quality and
nutritional factors in breeding programs in Europe stems from the retail price reduction due to the overproduction of
black currants in Europe in 20032005 [1]. The focus on the nutritional aspects of the berries is an important sales
argument for promoting black currants for fresh consumption. The processors main requirements are high levels
of ascorbic acid and antioxidant properties together with a low acidity and improved sensory characteristics [2].
The anthocyanin content of 4 prospective black currant selections (10B, 2-96-51, 1-96-16, 4-96-1), 4 new cultivars
(Karri, Almo, Ats, Elo) from our own breeding program and 7 introduced cultivars (jebyn, Zagadka, Ben
Corresponding author: P. Raudsepp, Department of Food Hygiene, Kreutzwaldi 58A, Tartu 51014, Estonia. Tel.: +372 7313432; Fax: +372
7313432; E-mail: raudsepp.piret@gmail.com.
54
P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds
Sarek, Intercontinental, Pamyati Vavilova, Titania and Pilenai) were analysed in this study, additionally to
the main biochemical characteristics.
The leaves of black currant have been used in folk medicine for their diaphoretic and diuretic properties as well as
to relieve rheumatic pain [18]. The pharmacological effect other than anti-inflammatory effect of black currant leaves
has not been scientifically proved [3, 4]. Their anti-inflammatory effect may be due to the polyphenol compounds
that are originally synthesised by plants to protect themselves from pathogens [5]. Surveys have indicated that some
of the polyphenol compounds may be exposed on the cuticle of leaves to give a quick response to the microbial attack
[25]. Although black currant leaves are widely in use as tea material [17], there are not many scientific studies about
the composition of the chemical compounds responsible for the health beneficial effect. We used a mixture of known
polyphenol standard compounds, previously known to be present in the leaves of black currant [17, 18] and some
additional standards that have been found to be present in other leaves of pharmacological value [6] to determine the
composition of polyphenol compounds in the leaves.
2. Materials and methods

2.1. Materials
The anthocyanidin standards and other chemicals, used in sample preparation or chromatographic separation were
all with HPLC purity grade: catechin, kaempherol, quercetin-3-glucoside, myricetin, quercetin-3-galactoside from
Sigma-Aldrich Inc.; cyanidin-chloride from Carl Roth 4545.1, Germany; delphinidin-chloride from Carl Roth 4537.1,
Germany; formic acid from Fluka, USA; quercitrin, Alfa-Aesar; acetonitril, ethanol 96%, hydrochloric acid 37%,
NaOH and methanol were with analytical grade; water, ultra pure made prior to analysis using Milli Q by Millipore
Corporation.
The chemicals used for biochemical analysis: potassium ferricyanide (III), sulfuric acid, lead subacetate, potassium
iodate, NaOH, 2,6-dichloroindophenol, methylene blue and oxalic acid were of analytical grade.
2.2. Collecting of samples
The research was carried out in 20052007 and 2009. 500 g of berry samples per cultivar were collected in the middle
of the harvesting season from the cultivar evaluation test plots established in 2000. Four prospective selections (10B,
2-96-51, 1-96-16, 4-96-1) and 4 new cultivars (Karri, Almo, Ats, Elo) [11] from our own breeding program
and 7 introduced cultivars (jebyn, Zagadka, Ben Sarek, Intercontinental, Pamyati Vavilova, Titania and
Pilenai) were analysed in three consecutive years 2005, 2006 and 2007.
Fully developed leaves for the qualitative analysis of polyphenols were collected in August 2009 randomly from
6 different bushes of the same cultivar (Karri) including equal proportions of leaves from different sides and inner
and outer range of the bush.
2.3. Methods
The representative samples were prepared from the field samples, 200 g of berries were homogenized using a
kitchen blender and analysed on the same day for sugar, soluble solids and ascorbic acid content. The soluble solids
content in the homogenized samples were recorded in a refractometer (Abbe WYA-1S, Optic Ivymen System) at 20 C,
organic acids were determined by titration with 0.1 NaOH. Ascorbic acid was determined using the modified Tillmans
method. Ascorbic acid was titrated with 2,6-dichloroindophenol under acid conditions [16]. The ferricyanide method
was used for the sugar content analysis [23]. The results are expressed in mg per 100 g of fresh berries. The average
weight of berries was determined by weighing 20 berries per sample.
The samples for the anthocyanin content determination were collected at the same time and frozen at 40 C for
further preservation.
The leaves were dried at 50 C for 24 h until air-dry and were ground prior to analysis.
55
2.4. Sample preparation for HPLC analysis of anthocyanins and leaf polyphenols
For the anthocyanidin analysis, 200 g of fresh frozen berries were homogenized with a Heidolph DIAX 900, 2 g of
homogenizate was weighed with an OHAUS analytical standard scale into a 50 ml centrifuge tube, 3 parallel samples
of each. The samples were extracted for 30 min at room temperature with 20 ml ethanol/water/HCl (70:30:1), after
which the samples were shaken on a minishaker IKA MS 1 by IKA-WORKS for 30 s twice after 5 min. The tubes were
then centrifuged with an Eppendorf centrifuge 5810 R at 3220 g at 20 C for 10 min, the supernatant was removed and
the samples were extracted once more with 20 ml ethanol/water/HCl (70:30:1) using the same procedure, and for the
third time with 5 ml ethanol/water/HCl (70:30:1). The supernatants were combined and 1 ml of the final solution was
filtered through a Spartan 13 mm regenerated cellulose membrane filter with a pore size of 0.2 m (modified [26]
and [24]). For the acid hydrolysis of the samples, the final filtrate was taken to 1 M HCl concentration and heated in
the Binder oven at 90 C for 60 min [25].
For the polyphenol analysis of the leaves: 0.1 g of dried leaves were weighed into a 15 ml centrifuge tube, 10 ml of
40% ethanol was added, and 3 parallel samples were made. The samples were extracted at room temperature for 24 h.
The extracts were centrifuged at 3220 g at 20 C for 10 min. The supernatant was filtered through Spartan 13 mm
regenerated cellulose membrane filter with a pore size 0.2 m
2.5. Chromatographic conditions
For detection and quantification of anthocyanins, Agilent 1100 Series LC/MSD Trap-XCT with an ESI interface
(m/z interval 501000 in positive ion mode) and UV-Vis diode array detector was used. HPLC reversed phase column
Zorbax 300SB-C18 with dimensions of 2.1 150 mm with a particle size of 5 m was used at 35 C, and the speed of
the mobile phase was 0.3 ml min1 . The elution was carried out with 1% formic acid in water (mobile phase A) and
acetonitril (solvent B), a multistep mobile phase gradient was used as follows: 0 min 95:5 (A:B), linear gradient until
30 min 70:30 (A:B), by 40 min the ratio of A:B was 10:90 and maintained for 50 min, after what the concentration
of mobile phase A was raised again to 95% while mobile phase B was dropped to 5% and the system was reequilibrated 10 min. For the quantification of the anthocyanins, the peak area at 510 nm was used. The detection limit
for anthocyanins was 0.1 g/ml and the quantification limit 0.2 g/ml. The calibration curve was constructed using
a standard mixture of delphinidin and cyanidin.
The analysis of the polyphenol composition of the leaves was conducted on the same chromatographic system
but in the negative ionisation mode. The elution gradient was set as follows: 0 min 90:10 [0.1% formic acid in water
(A):acetonitril (B)], the linear gradient until 30 min 70:30 (A:B), by 40 min the ratio of A:B was 10:90 and maintained
until 50 min and from 50.1 min the concentration of solvent A was raised again to 90% and the concentration of solvent
B dropped to 10% and maintained until 60 min to re-equilibrate the system. The calibration curves were constructed
using a standard mixture of catechin, chlorogenic acid, quercetin galactoside, quercetin glucoside, myricetin, quercetin
rhamnoside also known as quercitrin, and kaempherol.

3.1. Biochemical and morphological characteristics of fruit
The average fruit weight was 1.25 g. The cultivar jebyn had the smallest fruits (0.9 g) and Karri the largest
ones 1.7 g.
The ascorbic acid content varied from 98 mg/100 g with Ats to a very high level of 209 mg/100 g with elite selection
4-96-1 with average ascorbic acid content of 132.5 mg/100 g (Table 1). 130 mg has been set as the requirement by
some producers to ensure the healthiness of the processed product [12].
The sugar content and sugar acid ratio, that are major factors affecting the sweetness of the taste of the berries,
were highest with the cultivar Pilenai (12.6% and 4.7, respectively) and lowest with Zagadka (8.4% and 2.7
respectively). Above-the-average sugar acid ratio (3.7) was recorded for the cultivars Pamyati Vavilova Titania
Intercontinentaland elite selections 4-96-1 and 2-96-51. These selections both have the cultivar Pamyati Vavilovaas
56

Table 1
The average biochemical and morphological characteristics of berries, measured in 2005 and 2007
Cultivar
10 B
1-96-16
2-96-51
4-96-1
Almo
Ats
Ben Sarek
Elo
Intercontinental
Karri
Pamyati Vavilova
Pilenai
Zagadka
Titania
jebyn
Soluble solids
in juice (%)
Water
(%)
Titratable
acids (%)
Sugars
(%)
Sugar acid
ratio
Ascorbic acid
(mg/100g)
Average fruit
weight (g)
17.3
17.2
15.6
16.3
17.8
16.9
16.3
17
18.5
16.9
16.4
16
16
16
17.9
79
76
83
79
79
78
80
79
79
77
79
81
78
77
2.6
2.7
2.7
2.6
2.8
2.4
3.6
2.6
2.8
2.4
2.7
2.7
3.1
3.1
2.7
9.8
11
10.4
10.7
9.5
8.8
8.7
9.2
10.4
8.9
11.9
12.6
8.4
12.6
8.7
3.5
4.1
3.9
4.2
3.4
3.7
2.4
3.6
3.7
3.7
4.4
4.7
2.7
4.1
3.2
138
134
141
209
112
98
115
117
132
134
164
110
107
141
133
1.2
1
1.2
1.2
1.2
1.4
1.5
1.2
1.5
1.7
1.2
1.3
1.2
1.1
0.9
one of the crossing parents. Both selections have also high acorbic acid contents (209 mg and 141 mg/100 g) similar
to their parental genotype Pamyati Vavilova(ascorbic acid content 164 mg/100 g). Comparison with our earlier
research [11] including the 4 new cultivars from our breeding program and cultivar jebynused as the standard
cultivar, showed that the sugar content in the current study years was slightly higher than average and might have
been influenced by the weather conditions and relatively modest yields (data not shown) in these years. The ascorbic
acid levels obtained with the above listed cultivars are consistent with the average results obtained previously and
show a similar variation between genotypes.
3.2. Analysis of the anthocyanin content of the berries
Our analysis revealed that anthocyanins are present in form of cyanidin and delphinidin glycosides in black currant
berries. The prevailing glycosides are delphinidin glucoside (1), delphinidin 3-O-rutinoside (2), cyanidin-glucoside
(3), cyaniding 3-O-rutinoside (4) (Fig. 1). Previous studies have revealed similar results [10, 14, 19]. Some authors
have discovered a total of 15 different anthocyanin glycosides in black currant berries [21, 22]. After the acid
hydrolysis of the samples, it was revealed that cyanidin was slightly dominant over delphinidin (Table 2) in most
of the studied cultivars. It is interesting to remark that at the beginning of the berry ripening in the example of
Pilenai, the cyanidin was largely prevailing over delphinidin 73:23, but in the fully ripened berries, the ratio 57:43
was closer to equal (Table 2). The highest content of anthocyanins was found in cultivars Titania, Almo, jebyn,
Ats, Intercontinental and Elo from which Almo, Elo and Ats are from our own breeding programme.
Seedling 1-96-16 from our breeding programme showed also a high content of anthocyanins in three consecutive
years (Table 2).
It has to be mentioned that in the second year of the study (2006), the weather conditions were not favourable for
the berry production, and the content of anthocyanins in the berries gathered approximately at the same time as in the
two other years was lower than in the first (2005) and the third (2007) year. It may be the result of incomplete ripening
by that time. Previous studies have revealed that anthocyanin content depends on the ripening stage of the berries,
increasing during ripening and reaching the maximum when berries are fully ripened [7, 8, 15]. The anthocyanin
content may also be influenced by weather conditions [12]. The overall crop yield of berries was likewise lower in
the year 2006.
Intens. mAU
40
57
30
2
20
3
1
10
0
5
10
20
15
25
30
Time (min)
Fig. 1. UV-chromatogram of fruit anthocyanin analysis at 510 nm. 1) delphinidin glucoside, 2) delphinidin 3-O-rutinoside, 3) cyanidin-glucoside,
4) cyaniding 3-O-rutinoside.
Table 2
The average anthocyanin content of black currant berries in 3 consecutive
years and the percentage of the main aglycones after acid hydrolysis
10B
1-96-16
1-96-51
4-96-1
Almo
Ats
Ben Sarek
Elo
Intercontinental
Karri
Pamjati Vavilova
*Pilenai I
*Pilenai II
*Pilenai III
*Pilenai IV
Zagadka
Titania
jebyn
Anthocyanin content
of berries (ug/ml)
Delphinidin
(%)
Cyanidin
(%)
158 21
221 29
150 66
148 33
213 9
185 65
84 24
172 21
177 6.5
154 89
143 22
0
14 4
111 32
177 93
179 43
216 29
194 52
44
55
41
42
38
39
37
40
44
41
40
27
40
43
52
49
42
56
45
59
58
63
61
63
60
56
59
60
73
60
57
48
51
58
*The cultivar Pilenai was measured in four different phases of ripening;

stage I being green berries and IV stage the fully ripened stage, collected at
the same time as the berries of the other cultivars and seedlings.
3.3. Polyphenol composition of leaves

The results of the HPLC analysis of the leaves are presented in Fig. 2. The analysis was conducted using polyphenol
standards (2-catechin, 3-chlorogenic acid, a-quercetin galactoside, 6-quercetin glucoside, b-myricetin, 8-quercitrin,
58
BPC of blackcurrant leaves 0.1g diluted in 10 ml of 40% ethanol (1/100 m/v)

8
6
7
1
10
3
2
Intens.
10 6
11
BPC of standard mixture with concentration 12.5 ug/ml

6
5
8
4
a
d
b
2
2
1
0
10
20
30
40
50
Time (min)
Fig. 2. The base peak chromatogram (BPC) of analysis of the leaves and standard mixture of the known leaf specific polyphenols. The
polyphenols found in black currant leaves are marked with numbers repeating the peak numbers on leaf BPC and the standard compounds
not found in black currant leaves are marked with letters. 1. gallocatechin; 2. epigallocatechin; 3. chlorogenic acid; 4. unidentified compound
with [M-H] = 383; 5. myricetin glucoside; 6. quercetin glucoside; 7. quercetin malonylhexoside; 8. mixture of kaempferol glucoside, quercetin
rutinoside and quercetin acetylglucoside; 9. mixture of isorhamnetin rutinoside and an unidentified glucoside with [M-H] = 373; 10. kaempferol
malonylhexoside; 11. isorhamnetin glucoside.
c-quercetin, d-kaempherol) previously known as present in the black currant leaves [17, 18] or in the other leaves of
pharmacological value [6]. Some of the standard compounds were present in the black currant leaves as well (peaks
2, 3, 6 and 8). The compounds that were not overlapping with any of the used standards were identified using their
molecular weight and MS2 collision fragments.
The quantitative analysis was performed using the peak area under MS extracted ion chromatograms. The analysis
revealed that the concentration of catechin in the dried leaves was 786 mg/100 g; chlorogenic acid 1493 mg/100 g;
quercetin glucoside 1947 mg/100 g and quercetin rutinoside 399 mg/100 g. The content of catechin was lower than
found in the green tea by Rusak et al. (3330 mg/100 g), but the content of quercetin was significantly higher than
found in green tea by Rusak et al. (28 mg/100 g) [20]. The health beneficial effect of black currant leaves compared
to the green tea needs to be investigated further.
59
Acknowledgements
We thank the Estonian Science Foundation (research grants no. 6801 and 7703) and Ministry of Education and
Research of Estonia (target financing project no. 1092711s06) for financial support.
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DOI:10.3233/BR-2010-005
IOS Press
45
The inhibitory effects of berry-derived

flavonoids against neurodegenerative
processes
David Vauzour, Katerina Vafeiadou, Catarina Rendeiro, Giulia Corona and Jeremy P.E. Spencer
Molecular Nutrition Group, Department of Food and Nutritional Sciences, School of Chemistry,
Food and Pharmacy, University of Reading, Reading, UK
Abstract. Evidence suggests that a combination of oxidative stress, neuroinflammation and the formation of endogenous neurotoxins contribute to the underlying neuronal death associated with neurodegenerative diseases. In this study we have investigated
the ability of the berry-derived flavonoids to protect against neuronal damage induced by neuroinflammation and the neurotoxin
5-S-cysteinyl-dopamine. The flavanols (+)-catechin and ()-epicatechin, but not the anthocyanidin pelargonidin, were observed
to attenuate LPS/IFN--induced TNF- production in glial cells and associated neuronal injury. In contrast, pre-treatment of primary cortical neurons with pelargonidin, (+)-catechin and ()-epicatechin (0.1 and 0.3 M) resulted in concentration-dependant
protection against 5-S-cysteinyl-dopamine-induced neurotoxicity. Together these data suggest that berry-derived flavonoids may
offer some protection against the neuronal injury relevant to the aetiology of the Parkinsons and Alzheimers diseases.
Keywords: Brain, neuroinflammation, flavonoids, Parkinson disease, neurotoxins
1. Introduction
Abundant evidence exists to suggest that increased oxidative stress may contribute to the neuropathology of agerelated brain disorders such as Alzheimers and Parkinsons disease [11, 21]. Although the precise mechanisms of
neuronal death in these disorders remains uncertain, it is likely that a combination of elevated oxidative stress, the
formation endogenous neurotoxic cysteinyl-catecholamine conjugates [26] and neuroinflammation [9] are believed
to play a role in the underlying pathogenesis. For example, 5-S-cysteinyl-dopamine (CysDA) has been shown to
possess strong neurotoxicity and may initiate a sustained increase in intracellular reactive oxygen species (ROS) in
neurons leading to DNA oxidation, caspase-3 activation and delayed neuronal death [28]. Furthermore, glial cells
may become activated by various inflammatory stimuli to produce cytokines such as TNF- which contribute to
neuronal injury [7]. Deleterious effects of TNF- have been reported [19], and appear to be mediated by its binding
to neuronal tumour necrosis factor receptor-1 (TNFR1) and the subsequent activation of downstream caspase-8 and
caspase-3-mediated apoptotic pathways [29].
Recent studies have focused on the ability of dietary-derived phenolic compounds to protect against neuronal
damage resulting from aging and other neurodegenerative processes [18, 32, 38]. Much attention has centred on the
potential neuroprotective effects of flavonoids, which have been shown to protect against age-related cognitive decline
[12, 39], against 6-hydroxydopamine neurotoxicity [8] and against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Corresponding author: J.P.E. Spencer, School of Chemistry, Food and Pharmacy, The University of Reading, PO Box 226, Whiteknights,
Reading, RG2 6AP, UK. Tel.: +44 118 378 8724; Fax: +44 118 931 0080; E-mail: j.p.e.spencer@reading.ac.uk.
46
D. Vauzour et al. / The inhibitory effects of berry-derived avonoids against neurodegenerative processes
(MPTP) lesioning of the nigrostriatal tract [16]. For example, the consumption of flavonoid-rich blueberries and
strawberries has been shown to reverse age-related cognitive and motor behavioural deficits [12, 27, 35, 39], whilst
green tea and isolated EGCG protect against MPTP induced striatal dopamine depletion and neuronal loss [16] in
animal models. Furthermore, flavonoids have also been shown to inhibit inflammatory processes mediated by glial
cells [32], with the flavones wogonin and baicalein [5, 14], the flavonol quercetin [6], the isoflavone genistein [37]
and the flavanols catechin and epigallocatechin gallate [10, 17] all expressing anti-inflammatory activity.
In the present study, we provide evidence that both 5-S-cysteinyl-dopamine and LPS/IFN--induced TNF- release
resulted in concentration-dependent neurotoxicity. However, pre-treating the neuronal cells with the berry-derived
flavonoids pelargonidin, (+)-catechin and ()-epicatechin significantly decreased the injury elicited by these endogenous neurotoxins at low micromolar concentrations. The same results were observed for LPS/IFN--induced TNF-
release treated glial cells for the flavanols (+)-catechin and ()-epicatechin, but not the anthocyanidin pelargonidin.

2.1. Materials
The following chemicals were obtained from Sigma Chemical Company (Poole, UK): (+)-catechin, ()-epicatechin,
bacterial endotoxin LPS (Escherichia Coli, serotype 0127:B8) and mouse interferon- (IFN-)). Pelargonidin was
purchased from Extrasynthese (Genay, France). The 5-S-CysDA was prepared as previously described [36]. All
tissue culture reagents were purchased from Invitrogen (Paisley, UK). Ultrapure water (18.2 M.cm) passed through
a purification system (Purite Ltd, Oxon, UK) was used for all purposes.
2.2. Cell culture
Neuronal cells: Primary culture of neuronal cells were prepared as described previously [34]. Cells (106 /ml)
were plated in a serum-free medium composed of a mixture of DMEM and F-12 nutrient (1:1 v/v) supplemented
with glucose (33 mM), glutamine (2 mM), sodium bicarbonate (6.5 mM), HEPES (pH 7.4, 5 mM), streptomycin
(100 g/ml) and penicillin (100 UI/ml) (all Invitrogen, Paisley, UK). A mixture of hormones and salts composed
of insulin (25 g/ml), transferrin (100 g/ml), putrescine (60 g/ml), progesterone (20 nM) and sodium selenate
(30 nM) (all from Sigma, Poole, Dorset, UK) was also added to the culture medium. Cells were cultured at 37 C
in a humidified atmosphere of 95% air and 5% CO2 and after 56 days the vast majority of cells were neuronal
(>98%) with <2% astrocytes as determined by -tubulin and GFAP immunocytochemistry respectively as reported
previously [4].
Glial cells: Primary culture of glial cells was conducted as described previously [30]. Cells (5 105 /mL) were
cultured in a medium composed of Dulbeccos Modified Eagle Medium and F-12 nutrient (1:1 v/v) supplemented with
glucose (33 mmol/L), glutamine (2 mmol/L), sodium bicarbonate (13 mmol/L), HEPES buffer (pH 7.4, 5 mmol/L),
streptomycin (100 g/mL), penicillin (100 IU/mL), and 10% heat inactivated FBS (37 C; 5% CO2 ). The culture
medium was replaced once per week and glial cells were used following 1416 days in vitro, at which time they
consisted of astrocytes (90%) and microglia (10%). Cell morphology was determined by immunocytochemistry
with antibodies against GFAP (astrocytes) and -tubulin (neurons) as reported previously [4].
2.3. Cell treatments
Medium levels of TNF- were determined using a mouse enzyme-linked immunosorbent assay (R&D Systems,
Abingdon, UK) following the manufacturer instructions. To investigate the ability of flavonoids to inhibit TNF-
release, glial cells were pre-treated with different concentrations of flavonoid (0.1 and 0.3 mol/L) or vehicle
(methanol) for 24 h prior to the addition of LPS (1g/mL) and IFN- (10 ng/mL) for 24 h. To avoid chemical
interaction between the flavanones and LPS or IFN- cells were washed thoroughly with phosphate buffer saline
(PBS) supplemented with glucose (33 mmol/L) prior to their addition. Following exposure to LPS and IFN-, the
medium was retained for TNF- analysis.
47
To investigate the protective effects of flavonoids, neurons were pre-treated with (+)-catechin, ()-epicatechin and
pelargonidin (0.1 and 0.3 M) for 18 h prior the addition of CysDA (100 M) for a further 24 h. Following exposure,
cultures were washed twice with sterile PBS before the addition of Alamar Blue solution (10% v/v) in DMEM:F12.
Plates were then returned to the incubator for 2, 3 hours, before fluorescence was measured (Ex: 540 nm; Em: 612 nm)
on a Tecan GENios multiplate reader (Tecan GENios, Theale, UK). Results were expressed as percentage of neuronal
injury relative to vehicle treated cells.
All results are expressed as means SD of three separate experiments unless otherwise stated. The statistical evaluation of the results was performed by one-way analysis of variance (ANOVA) followed by a post-hoc t-test using
GraphPad InStat version 3.05 (GraphPad Software, San Diego, CA, USA). Significance was defined as p < 0.05.
Significant changes are indicated as follows: ***p < 0.001; **p < 0.01; *p < 0.05 and a = p < 0.001; b = p < 0.01;
c = p < 0.05.
3. Results
3.1. Flavonoids protect neurons challenged with 5-S-cysteinyl-dopamine
Pelargonidin and the flavanols (+)-catechin and ()-epicatechin (0.13.0 M) did not induce neuronal injury
as assessed by the Alamar blue assay 24 h post exposure (>98% compared to vehicle, 100). Exposure of primary cortical neurons to 5-S-cysteinyl-dopamine (10500 M) led to concentration-dependent increases in neuronal
injury (IC50 = 100.2 2.1 M) when assessed 24 h after exposure (Fig. 1). The strongest neuroprotective effect was
observed when neurons were pre-treated with pelargonidin (0.13 M). Indeed, concentrations ranging from 0.1
to 1 M significantly protected neurons from injury induced by 5-S-CysDA (Fig. 2A), with 1 M being the most
potent (90.1 4.2%, p < 0.001). Pelargonidin (3 M) was also observed to be significantly protective, although to
a lesser degree (34.2 2.5%, p < 0.001). Significant protection against 5-S-CysDA-induced neuronal damage was
also observed for ()-epicatechin (0.1 to 3 M), the greatest degree of protection apparent at the 1 M concentration
(46.7 4.0%; p < 0.001) (Fig 2B). Finally, a small but significant protection was also afforded by (+)-catechin, with
the 300 nM concentration being the most protective (19.0 5.0%; p < 0.05) (Fig 2C).
Percentage of viable neurons
100
90
80
***
70
60
***
50
40
*** ***
30
0
10
100
1000
CysDA concentration (M)

Fig. 1. 5-S-cysteinyl-dopamine induces toxicity on primary cortical neurons. 5, 6 days in vitro primary neurons were exposed to 5-S-cysteinyldopamine (10500 M) or vehicle for 24 h. After 24 h, cell viability was determined by Alamar blue reduction. Results are expressed as mean SD
of quadruplicate wells from single experiments repeated three times with similar results (***p < 0.001; compared with control, as analysed by
one way ANOVA followed by post-hoc t-test).
48
***
Percentage viable cells
100
80
60
40
20
0
CysDA (100M)
Pelargonidin (M)
0.1
0.3
***
B
100
a
80
c
60
40
20
0
CysDA (100M)
Epicatechin (M)
0.1
0.3
***
C
100
80
c
60
40
20
0
CysDA (100M)
Catechin (M)
0.1
0.3
Fig. 2. Protective effects of flavonoids against 5-S-cysteinyl-dopamine-induced neuronal injury. Following pre-treatment with (A) pelargonidin,
(B) ()-epicatechin or (C) (+)-catechin (0.13 M) for 18 h, 56 DIV primary neurons were exposed to 5-S-cysteinyl-dopamine (100 M)
for 24 h. Alamar Blue solution (10% v/v) was added to plates and they were returned to the incubator for 23 h, before fluorescence was
measured (Ex: 540 nm; Em: 612 nm). Results are expressed as mean SD of quadruplicate wells from single experiments repeated three times
with similar results. Data were analysed by one way ANOVA followed by post-hoc t-test. ***p < 0.001 respresent a significant decrease of
neuronal viability relative to control; a = p < 0.001, b = p < 0.01, c = p < 0.05 represent a significant increase in neuronal viability relative CysDA
treated cells.
49
3.2. Inhibitory effect of avonoids on LPS/IFN--induced TNF-a release

Pelargonidin and the flavanols (+)-catechin and ()-epicatechin (0.13.0 M) did not induce glial cell injury as
assessed by the Alamar blue assay 24 h post exposure. Treatment of glial cells with LPS (1 g/mL) and IFN-
(10 ng/mL) for 24 h resulted in a significant increase in TNF- production (p < 0.001, n = 6) (Fig. 3). However, preincubation of glial cells with either (+)-catechin or ()-epicatechin (0.13 M; 24 h), prior to LPS/IFN- exposure,
resulted in a significant decrease in medium TNF- levels (Fig. 3A and B). In contrast, pre-treatment with pelargonidin
(0.13 M; 24 h) had no significant effect on LPS/IFN--induced TNF- release (Fig. 3C).
4. Discussion
Abundant evidence exists to suggest that increased oxidative stress may contribute to the neuropathology of
age-related brain disorders such as Alzheimers and Parkinsons diseases [11, 21]. In this study, we show that
5-cysteinyl conjugates of dopamine possess a strong cytotoxicity towards primary cortical neurons. 5-S-CysDA,
have been reported previously to be neurotoxic by inducing both oxidative DNA base modification and the activation
of caspase-3 activity in neurons [20, 26]. Once formed, 5-S-CysDA may be further oxidised, yielding more complex
conjugated species, such as DHBT-1 [24], an event which may be accelerated in the presence of peroxynitrite [33],
a powerful oxidant and cytotoxic molecule formed from the reaction of nitric oxide (NO ) with superoxide (O2 -)
[13]. Presence of such oxidant molecules may be the results of activation of the iNOS enzyme, an event triggered
under neuro-inflammatory processes. Indeed, under inflammatory conditions, excess release of cytokines, such as
TNF-, is a central event in glial-induced neurotoxicity. Such detrimental actions of TNF- induced neurotoxicity
have had been reported in neuronal models [19], and are thought to be the result of the activation iNOS expression
and the subsequent NO release, thus enhancing its neurotoxic potential [15]. As a consequence, there has been
much interest in the development of novel therapeutic agents that can selectively attenuate neurotoxins-induced
toxicity.
Recent evidence from human, animal and cell studies suggest that flavonoids may exert neuroprotective effects [3, 8,
25]. In the brain, the potential actions of flavonoids on neurons will be influenced by interactions with both astrocytes
and microglia, which occupy the majority of the brain volume and play a key role in the maintenance of neuronal
integrity. In this study, we show that specific berry-derived flavonoids are capable of inhibiting glial activation and of
reducing cysteinyl-dopamine neurotoxicity at physiologically achievable plasma concentrations (nanomolar). Whilst
the three flavonoids tested (pelargonidin, (+)-catechin and ()-epicatechin) were able to decrease the neuronal cell
death in primary neurons challenged with CysDA, only the flavanols (+)-catechin and ()-epicatechin were able to
inhibit TNF-, production in LPS/IFN--activated glial cells. Such protective effects may be the results of an inhibition
of the inducible nitric oxide synthase (iNOS) in glial cells as reported previously for the flavanone naringenin [30], or
an activation of the detoxification enzymes such as the glutathione-S-transferase as observed previously in primary
neurons in presence of ()-epicatechin [4]. Action of such enzymes may decrease the neurotoxic potential of some
molecules, as observed for quercetin where its neurotoxicity was significantly reduced by glial cell metabolism,
notably by its conversion to 2 -glutathionyl quercetin [31].
Although flavonoids have been reported to exert strong antioxidant effects in vitro, accumulating evidence suggests
that dietary polyphenols may cross the blood brain barrier [2, 40], accumulate in the brain at nanomolar concentrations
[1, 2] and exert neuromodulatory effects through selective actions on different components of a number of protein
kinase and lipid kinase signalling cascades, such as PI 3-kinase, Akt/PKB, tyrosine kinase, PKC and members of
the MAP kinase family [23]. Modulation of these pathways may underlie the ability of berry-derived flavonoids to
exert their protective effects, as previously reported for the flavan-3-ol, ()-epicatechin, which has been observed
to stimulate the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a
regulator of neuronal viability and synaptic plasticity [22]. Moreover, both hesperetin and 5-nitro-hesperetin were
also effective at preventing neuronal apoptosis via a mechanism involving the activation of both Akt/PKB and ERK1/2
[34]. Further research is required in order to establish the underlying mechanisms involving the neuroprotective effects
of the berry-derived flavonoids.
50
***
350
TNF- (pg/ml)
300
250
200
150
100
50
0
LPS/IFN-
Epicatechin (M)
0.1
0.3
***
350
TNF- (pg/ml)
300
250
200
150
100
50
0
LPS/IFN-
Catechin (M)
0.1
0.3
0.1
0.3
***
450
400
TNF- (pg/ml)
350
300
250
200
150
100
50
LPS/IFN-
Pelargonidin (M)
Fig. 3. Flavonoids inhibit TNF- production in LPS/IFN- activated primary glial cells. Cells were treated with LPS (1 g/mL) and IFN-
(10 ng/mL) for 24 h alone (LPS/IFN- group) or pre-treated with naringenin (A), ()-epicatechin (B), (+)-catechin or (C), pelargonidin
(0.13 mol/L) for 24 h before LPS/IFN- activation for another 24 h. After incubation TNF- concentrations were measured in the cell culture
medium by enzyme-linked immunosorbent assay. Results are presented as means SD of three independent experiments. ***p < 0.001, indicates significant increase in TNF- production relative to control; a = p < 0.001; b = p < 0.01; c = p < 0.05 indicates significant decrease in TNF-
production relative to LPS/IFN- group.
51
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DOI:10.3233/BR-2010-004
IOS Press
35
Evaluating the health benefits of fruits for

physical fitness: A research platform
Birgit Schragea , David Stevensona , Robyn W. Wellsa , Kirsty Lyalla , Selena Holmesa ,
Dawei Denga and Roger D. Hurstb,
a Food
& Wellness Group, The New Zealand Institute for Plant & Food Research Ltd, Hamilton, New Zealand
b Food & Wellness Group, The New Zealand Institute for Plant & Food Research Ltd, Palmerston North, New Zealand
Abstract. To evaluate the health promoting attributes of fruits and their compounds the New Zealand Institute for Plant & Food
Research Ltd (PFR) is using exercise as a model for oxidative stress and immune depression. Regular exercise has health benefits
believed to be derived from adaptive responses to moderate oxidative stress. However, following exhaustive or unaccustomed
exercise, excessive and prolonged oxidative stress and inflammation can be detrimental and the right balance of modulation
from nutritional support via fruit phytochemicals (and vitamins) may prevent damage, aid recovery, and/or enhance muscular
and immune function. We have developed a research platform to evaluate physical health, performance and recovery to position
new fruit varieties in this area. Utilising compositional analysis of fruit extracts, in vitro screening of muscle cells, electrically
stimulated muscle ex vivo, and animal and human intervention and exercise trials, we are evaluating the physical health-promoting
effects of polyphenolic phytochemicals derived from fruit, particularly berry fruits. Our research demonstrates that certain fruits
may complement the benefits of regular exercise through appropriate modulation of excessive oxidative stress and inflammation.
Keywords: Berry fruit, kiwifruit, fruit, polyphenolics, phytochemicals, anthocyanins, oxidative stress, inflammation, exercise,
skeletal muscle
1. Introduction
The New Zealand Institute for Plant & Food Research Ltd (PFR) provides research and innovation for New
Zealands horticultural sector. PFR has developed a research platform to evaluate physical performance and recovery.
Regular exercise has health benefits derived from moderate oxidative stress. However, depending on the mode,
intensity and duration, exercise can inflict high levels of mechanical and metabolic stress on muscle [28]. Dietary
supplementation is an attractive choice for health-conscious individuals. However, the right balance of modulation
from nutritional support needs to be established to prevent damage/injury, maximise recovery for enhanced muscle
function, but also to optimise immune function.
Utilising fruit varieties from breeding programmes at PFR with well-defined compositional data combined with a
range of cell-based screening, ex vivo muscle experiments, and animal/human trials, we are evaluating the physical
health-promoting effects of phytochemicals derived from some new fruit varieties, including berry fruits. The determination of biochemical markers of oxidative stress, inflammation and immune function in cell and tissue assays, and
animal and human trials enables us to evaluate the effects of fruit consumption and to ultimately guide the breeding
of health promoting fruit.
Corresponding author: Roger D. Hurst, Food & Wellness Group, The New Zealand Institute for Plant & Food Research Ltd, Private Bag
11600, Palmerston North 4442, New Zealand. Tel.: +64 6 355 6203; Fax: +64 6 351 7050; E-mail: Roger.Hurst@plantandfood.co.nz.
36
B. Schrage et al. / Evaluating the health benets of fruits for physical tness: A research platform

2.1. Fruit extract preparation and composition
The ZESPRI GOLD kiwifruit (Actinidia chinensis Hort16A) extract was prepared by homogenising freeze-dried
fruit (20 g) with methanol (50 mL), followed by 30 min in an ultrasonic bath. Solvent was recovered by filtration and
the residue extracted twice more in methanol. The extract was rotary evaporated, dissolved in the minimum volume of
water and applied to a 10 g C-18 solid-phase extraction cartridge (Strata, Phenomenex, Torrance, CA). The cartridge
was flushed with deionised water (30 mL) and eluted with methanol (40 mL). Evaporation of the methanol eluate
yielded 0.24 g of extract which was prepared in buffer for the analysis of ex vivo muscle performance.
The polyphenolic extract of blueberry fruit (Vaccinium corymbosum, Reka) was prepared from approximately
800 g of frozen fruit homogenised with acetone and the residue recovered by filtration. Following a further
homogenisation and filtration with acetone/water (70:30), acetone extracts were combined and concentrated by
rotary evaporation. Lipids were removed by partitioning the aqueous extract with heptane and the residual heptane in
the aqueous layer removed by rotary evaporation. The polyphenolics were isolated from this extract by absorption,
recovery, and elution (methanol) of XAD-7 (Sigma, Sydney, Australia) and dried by rotary evaporation to yield
a friable powder. In all experiments, the blueberry fruit extracts were prepared from a stock in DMSO. DMSO
concentrations in the final experimental conditions were below 1% and had no observable effect.
Juices of various cultivars of blackcurrant (Ribes nigrum) were prepared by blending fruit (180 g) with a domestic
hand blender, then treating with Pectinex (0.18 mL; Novozymes, Bagsvaerd, Denmark) at 48 C for 4 h. After centrifugation (4000 rpm, 10 min) the yield of juice was typically 50%. Blackcurrant juice samples were analysed by liquid
chromatography-mass spectrometry (LC-MS) using a Shimadzu (Auckland, New Zealand) 20-Series UFLC system
including an autosampler, column oven and photodiode-array detector, linked to an LCMS 2020 single-quadrupole
mass spectrometer. The LC system was fitted with a 150 2 mm, 4 Synergi Fusion RP column (Phenomenex).
For analysis, blackcurrant samples were diluted 10 into 1% aqueous formic acid and kiwifruit extracts dissolved
in water at 2 mg/mL. Samples of 10 L were injected and eluted by the following solvent programme. Flow rate
0.6 mL/min, Solvent A: 2% formic acid, solvent B: methanol. The initial conditions were 8% B and ramped linearly
to 10% at 2.5 min, 18% at 5 min, 38% at 8.5 min, 63% at 10.5 min, held at 70% between 11.5 and 12.5 min, then back
to the starting conditions between 13 and 15 min. Column oven temperature was 50 C. The mass spectrometer was
operated under standard tuning conditions, as specified by the manufacturer and half the LC flow was diverted to
waste through a split valve. Identification for compounds of interest was achieved by comparison of a combination
of UV-visible and mass spectra with standard compounds.
The commercially available juices GHO Natural Quenchers (Good Health Organisation, Nelson, New Zealand)
contained 65% of either ZESPRI GOLD kiwifruit or blackcurrant juice.
2.2. Determination of muscle cell intracellular free radical levels
The rat skeletal muscle cell line L6 (American Type Culture Collection, Manassas, USA) was cultured and
differentiated as described elsewhere [13]. Muscle cells were plated at 5000 cells/well in 96 well plates and incubated
with 2 ,7 -dichlorohydrofluorescein diacetate (DCFDA) (10 M) for 60 min at room temperature. DCFDA is cleaved
intracellularly into DCF, which produces an increase in fluorescence in the presence of reactive free radicals. Following
a wash in D-PBS (2 ), cells were equilibrated at room temperature for 10 min prior to incubation (simultaneously
[time zero] and at the times indicated) with the blueberry fruit extract (50 g/mL), followed by exposure to H2 O2
(0.5 mM). Fluorescence intensity was determined over time using a fluorescence platereader (BMG FluorStar Optima,
Alphatech Systems Ltd) with excitation and emission wavelengths of 485 and 520 nm respectively.
2.3. Evaluation of muscle cell total glutathione (GSH) levels and glutathione peroxidase (GPx) activity
The total amount of muscle cell GSH and GPx was determined colourimetrically using Bioxytech assay kits
(GSH-420TM and GPx-340TM , Sapphire Bioscience Pty. Ltd, Auckland). Differentiated L6 myotubes were incubated
with the blueberry fruit extract both simultaneously and for 24 h before GSH and GPx determination. For GSH
37
analysis, cells were plated at a density of 1 106 cells/well in 6 well plates and grown until confluent. Following
extract exposure, cells were harvested using TrypleTM , washed in D-PBS, and permeabilised by sonication in the
precipitation reagent provided by the kit. All other analysis steps were then as recommended by the supplier. To
determine muscle cell GPx levels, cells were grown in T75 flasks until confluent. Following extract exposure, cells
were harvested using a cell scraper and prepared into 3 106 cell aliquots before sonication in D-PBS containing
1 mM mercaptoethanol. All other analysis steps were as recommended by the supplier.
2.4. Animals
Male Swiss mice, 812 weeks old, were used for the muscle bath and animal exercise experiments. Animals were
cared for in accordance with the Code of Ethical Conduct, Regulations Act, 1987 (New Zealand) and all procedures
were approved by our local Animal Ethics Committee.
2.5. Ex vivo model of muscle performance
To investigate the effect of fruit extracts on muscle tissue, a model bath system was established in which isolated
fresh muscle (Soleus) was exposed to extracts and subsequently electrically stimulated to examine the effects on
contractile activity (both maximum force produced and fatigue profiles).
Animals were killed by CO2 asphyxiation. Soleus muscles were isolated from both hindlimbs, leaving tendons
attached. Muscles were placed in an organ bath in Krebs-Ringer solution (in mM: 118 NaCl, 4.75 KCl, 1.18 MgSO4 ,
2.54 CaCl2 , 24.8 NaHCO3 , 1.18 KH2 PO4 , 10 glucose), bubbled continuously with 95% O2 5% CO2 to maintain
a pH of 7.5 at 25 C. Muscles were attached to a force transducer (World Precision Instruments, Sarasota, USA) and
positioned between electrodes. Using a micrometer, muscles were adjusted to their optimum length to yield maximal
force at 120 Hz stimulation. The output of the force transducer was monitored using Data-Trax software (World
Precision Instruments, Sarasota, USA).
Baseline tetanic force production (3 millisecond pulses at 20 Hz over 14 s) in each individual muscle was measured
in the absence of fruit extract exposure. After a recovery period of 10 min, the buffer was replaced with fresh KrebsRinger solution for control muscles, or Krebs-Ringer solution containing 5 g/mL ZESPRI GOLD kiwifruit extract
or 2% GHO Natural Quenchers containing 65% of either ZESPRI GOLD kiwifruit or blackcurrant juice. Muscles
were incubated for 15 min before stimulating another tetanus response. Muscles were then removed and length and
weight measured; the cross sectional area was estimated according to Mendez and Keys [19] and force expressed as
mN/mm2 . Force measured during the fatigue protocols was assessed in relation to the peak force generated in each
individual muscles baseline stimulation.
2.6. Animal exercise
Mice were randomly assigned to the following five groups: no-exercise controls, exercising mice sacrificed at
0, 1, 3, or 24 h after fatiguing uphill running. All mice were acclimated to exercise conditions on the motorized
treadmill (Muromachi Kikai, Tokyo, Japan) for five days, during which they walked/ran for 15 min at a speed of
015 metres/min and a maximum incline of +2 . The running adaptation was followed by at least five days of rest.
During the fatiguing exercise, mice ran on the treadmill with a maximum speed of 36 metres/min and a maximum
incline of +8 until the point of fatigue, at which they were no longer able to maintain pace despite gentle prodding and discharges of compressed air. Control mice were kept in the room with the treadmill to expose them to
the same noise and handling as the exercising mice. After completion of the exercise, mice in the control and 0
hour group were sacrificed immediately, all other mice were sacrificed after 1, 3, or 24 h of recovery. Animals were
killed by CO2 asphyxiation and blood was collected by heart puncture. Blood was centrifuged (9000g, 10 min) and
plasma was stored at 80 C until further analysis. Plasma creatine kinase (CK) activity was analysed by Gribbles
Veterinary Laboratory (Hamilton, New Zealand). Results are expressed as international units per litre of plasma
(IU/L). Plasma with a haemolysis index greater than 60 was excluded from the analysis. Interleukin-6 (IL-6) concentration in plasma was analysed using the Quantikine Mouse IL-6 ELISA from R&D Systems (Minneapolis,
38
USA) and was performed according to the manufacturers instructions. Results are expressed as pg IL-6 per mL
plasma (pg/mL).
Results are expressed as means standard errors of the mean (SEM) for at least four observations in each case.
Statistical significance for the comparison of two groups was assessed by the Students t-test. A probability value (P)
of less than 0.05 was considered significant.

3.1. Anthocyanin content of fruit juices and effects of fruit on muscle cell oxidative stress
Many fruits, and especially berries, contain bioactive compounds whose health-promoting properties are widely
recognised [1, 27]. Utilising fruit varieties from breeding programmes at PFR with compositional analysis we are
evaluating the physical health-promoting effects of phytochemicals derived from some new fruit varieties, including
berry fruits. Breeding programmes at PFR have targeted high anthocyanin content as one of the possible desirable
characteristics of new fruit cultivars for health benefits. A compositional survey of juices prepared from commercially
grown non-New Zealand as well as New Zealand blackcurrant cultivars and new selections from the PFR breeding
programmes demonstrates that the anthocyanin content of the New Zealand fruit juice is approximately 1.5 times
that of the non-New Zealand and one new cultivar is particularly high in anthocyanins (Table 1). The fruits for the
juices were grown under New Zealand conditions and the data suggest that New Zealand cultivars perform well
when compared with those grown in North America [20]. The exceptionally high anthocyanin content makes some
New Zealand blackcurrants cultivars unique and might provide potential strategies to counter/modulate the stress
and damage associated with over-exercise, as well as to improve overall body wellness.
Cellular studies can be used to determine the underlying mechanisms of action of active fruit extracts/compounds.
We report here insights into the protective mechanism of action of a blueberry fruit extract. We analysed the antioxidant
ability of skeletal muscle myotubes (differentiated from muscle myoblast cell lines) following exposure to the extract.
Using DCFDA, which accumulates intracellularly and is cleaved to produce the radical sensitive product DCF, enabled
the determination of intracellular free radical levels by monitoring of relative fluorescence intensities after exposure
to H2 O2 . Figure 1 displays the antioxidant ability of muscle cells following different lengths of incubation exposure
Table 1
Anthocyanin content of blackcurrant juice from New Zealand and non-New
Zealand cultivars
Non-New Zealand
Cultivars*
Baldwin
Ben Dorain
Ben Lomond
Ojebyn
Anthocyanins
New Zealand
Anthocyanins
(mg/100 mL)
Cultivars
(mg/100 mL)
310
297
249
179
Ben Ard
Ben Rua
Blackadder
Magnus
New Selection 1
New Selection 2
463
477
446
336
471
850
Juices were made from fruit harvested in the 2009 season from bushes on
research plots in Canterbury, New Zealand.
*A comparable range of anthocyanin content was obtained from the large
study carried out on 32, mostly European, blackcurrant fruit cultivars grown
in North America [20].
Relative Fluorescence (% from time zero)
Time 0 hours
90%
80%
70%
60%
50%
H2O2
BP (64.3%)
40%
30%
20%
10%
0%
Control
90%
Time 4 hours
80%
70%
60%
50%
H2O2
40%
BP (49.6%)
30%
20%
10%
Control
0%
0
500
1000
1500
500
1000
1500
Time (sec)
Time (sec)
90%
39
90%
Time 8 hours
Time 24 hours
80%
80%
70%
70%
60%
60%
50%
50%
H2O2
40%
H2O2
40%
30%
30%
20%
20%
BP (77.3%)
Control
10%
0%
0
500
1000
Time (sec)
1500
10%
BP (91.9%)
0%
Control
0
500
1000
1500
Time (sec)
Fig. 1. Effect of incubation time on the intracellular free radical scavenging ability of muscle cells following exposure to a blueberry polyphenolic
extract. Muscle cells were loaded with the reactive oxygen species indicator DCF as described in Materials and Methods and incubated (simultaneously [time zero] and at the times indicated on the figures) with a blueberry fruit (Vaccinium corymbosum Reka) polyphenolics extract (BP,
50 g/mL) prior to challenge with H2 O2 and fluorescence intensity determination. Values are means SEM from at least four experiments and
are expressed as relative fluorescence intensity (% from time zero). The percentage inhibition of H2 O2 -induced fluorescence by the blueberry
extract is shown in parentheses.
(simultaneously [time zero] to 24 h) with the blueberry extract (50 g/ml) before oxidative challenge. H2 O2 elicited
a sustained, progressive, and large increase in muscle intracellular free radical levels. The magnitude of this response
was reduced from 4 h onwards and was probably due to the leakage of the DCF from the cells over time, but this did not
affect the determination of the intracellular antioxidant ability. With the simultaneous incubation of the muscle cells
with the blueberry fruit extract, a marked increase in cellular antioxidant ability was observed, which was reflected by
a 64.3% inhibition of fluorescence signal elicited by H2 O2 . This immediate antioxidant ability was further enhanced
by longer extract pre-incubations (Fig. 1). After a 24-h extract pre-incubation, the antioxidant scavenging ability of
the muscle cells had increased and was reflected by a 91.9% inhibition of the fluorescence signal elicited by H2 O2 .
We further evaluated whether washing the blueberry extract from the cells prior to challenge with H2 O2 prevented the
immediate and longer-term antioxidant effects. There was no significant difference between washed and non-washed
cells (data not shown).
In vitro models of oxidative stress using skeletal muscle cells have been used successfully in the past to monitor,
under controlled conditions, the responses of muscle to oxidative stress that cannot be easily evaluated in human
trials, and to provide a cost-effective method for the screening of beneficial substances. A useful and simple system is
the use of skeletal muscle myotubes (differentiated from muscle myoblast cell lines) exposed to calcium ionophores
[17].
We have previously demonstrated that the blueberry fruit (Vaccinium corymbosum Reka) polyphenolic extract
mediated protection against muscle cell oxidative stress and damage [13]. The data presented here give insights into
the potential process of action. We compared the functional activity of anthocyanin rich sub-extracts derived from
the total extract with individual anthocyanin components and demonstrated that the most likely actives responsible
40
for the protection against the oxidative stress were malvidin glycosides, particularly malvidin galactoside, and/or
malvidin glucoside [13].
3.2. Effects on muscle cell GSH and GPx activity
The data in Fig. 1 suggest that while there is an immediate antioxidant ability of the fruit extract, the increase in
antioxidant ability over time is not associated with the extract itself. Tissues and cells are equipped with efficient
enzymatic and non-enzymatic antioxidant defence systems, which serve to counter the damaging action of oxidative
stress [10]. Figure 2 shows the determination of muscle cell GSH and GPx activity following blueberry fruit extract
exposure. While there were no changes in GPx activity, GSH levels after a 24-h exposure were significantly enhanced
and suggest the induction of endogenous antioxidant pathways. These data support other reports that highlight that
polyphenolics can modulate the levels of antioxidant defence enzymes such as superoxide dismutase, catalase, GSH
and GPx [29]. Evidence suggests that these adaptive actions to ensure cell and tissue survival following oxidative stress
may be mediated by the induction of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Nrf2 is a transcription factor
that binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes,
where it initiates the transcription of many cytoprotective proteins including endogenous antioxidant enzymes [15,
22]. More research is warranted to elucidate the significance of fruit polyphenolic-mediated induction of endogenous
antioxidant pathways for modulation of unregulated oxidative stress and the potential long-term effects for muscle
performance.
3.3. Effect of fruit on muscle function
Exposure of isolated muscle tissue to some fruit extracts also modulates muscle tissue contractile activity and
performance. Assessing force production and fatigue profile in electrically stimulated murine soleus muscles, we
have previously reported [26] that a ZESPRI GOLD kiwifruit extract mediated an increased maximum tetanic force
in a similar manner to the antioxidant superoxide dismutase (1000 IU/mL).
Here we report (Fig. 3) the contractile performance of soleus muscle following exposure to a ZESPRI GOLD
Kiwifruit extract and two commercially prepared ZESPRI GOLD kiwifruit and blackcurrant juices (2% GHO
140
40
120
Reduced Glutathione (uM)
Glutathione Peroxidase (mUnits/ml)
100
80
60
40
20
30
20
10
0
0 hours
24 hours
0 hours
24 hours
Fig. 2. Muscle cell reduced glutathione levels and glutathione peroxidase activity following incubation with a blueberry fruit polyphenolic extract.
The effect of a blueberry (Vaccinium corymbosum Reka) polyphenolics extract (BP, 5 and 50 g/mL) on muscle cell glutathione peroxidase
(GPx) activity and reduced glutathione (GSH) levels was evaluated. Muscle cell GPx (A) and GSH levels (B) were determined immediately
and 24 h after incubation with the blueberry extract (BP) as described in Materials and Methods. Values are means SEM from at least four
experiments and are expressed as mU/mL and M (GPx and GSH respectively). *Represents p < 0.05 statistical significance (paired Student
t-test) compared with the control.
B
Force (% max)
Force (% max)
41
Time (sec)
Time (sec)
Fig. 3. Effect of fruit extract and juices on fatigue in mouse soleus muscles induced by electrical stimulation. Muscles incubated with (A)
ZESPRI GOLD Kiwifruit (Actinidia chinensis Hort16A) phenolic extract (5 g/ml, control, kiwifruit extract), or (B) GHO ZESPRI
GOLD Kiwifruit and blackcurrant juices (2%, control, kiwifruit, blackcurrant) were compared with buffer-only controls. The recorded force
was divided by the cross sectional area of the muscle and normalized to the maximum force generated during baseline stimulation. A minimum of
five muscles were analysed for each group. Values are means SEM. *Represents p < 0.05 statistical significance (paired Student t-test) between
the area under the curve for muscles incubated with buffer-only and fruit extract.
Natural Quenchers). Mouse soleus muscles were electrically stimulated ex vivo after 15 min of incubation with the
ZESPRI GOLD Kiwifruit extract (5 g/ml), or control buffer and tetanic force production was measured over 14 sec
(Fig. 3A). Mean peak force in fruit extract exposed muscles was 86.1 9.7%, 23.8% higher than the peak force in the
controls (62.3 14.2%). After the 14-sec tetanus stimulation the muscles demonstrated fatigue a decline in muscle
performance due to muscle activity. With kiwifruit extract exposure, muscle fatigue was reduced, with the mean force
retaining 71.4% of the peak baseline force, whereas force in the controls dropped to 34.4%. Comparison of the area
under the curves highlighted that the ZESPRI GOLD Kiwifruit mediated protection against fatigue was statistically
significant (kiwifruit 1108.5 147.3, control 615.2 151, p < 0.05). The commercially prepared ZESPRI GOLD
Kiwifruit and blackcurrant juices mediated a greater enhancement in muscle performance and protection against
fatigue (Fig. 3B). Mean peak force in blackcurrant and kiwifruit juice-exposed muscles was 147.1 20.6% and
148.9 13.8% respectively, 85% higher than the peak force in controls. After the 14 sec tetanus stimulation, muscle
fatigue was reduced by the blackcurrant and kiwifruit juice extracts with the mean force retaining 93.6% (blackcurrant)
and 89.2% (kiwifruit) of the peak baseline force. Protection against fatigue, analysed as area under the curve, was
statistically significant for both blackcurrant (1546.5 225.8) and kiwifruit juices (1498.4 170.1, p < 0.05).
These data demonstrate that ZESPRI GOLD Kiwifruit and blackcurrant fruit extracts and juices have an effect
on muscle performance, which could be mediated by antioxidant scavenging of exercise-mediated reactive oxygen
species (ROS). Active muscles produce excess ROS and it is widely accepted that they play a role in fatigue. During
intensive exercise, muscle oxygen consumption increases up to 100-fold, and in parallel, superoxide anion production
in mitochondria increases to a level where endogenous ROS scavengers are overwhelmed and hydrogen peroxide
diffuses into the muscle cell cytoplasm. Some exogenous antioxidants like N-acetylcysteine (NAC), vitamins C and
E, have been reported to reduce fatigue in isolated muscles, but only NAC has so far been shown to improve fatigue
performance in vivo [23].
The fruit compound responsible for the enhanced force production and reduced fatigue in our isolated mouse
soleus muscles remains to be elucidated, but it is possible that vitamin C and/or a variety of polyphenolics present
in the fruits and extracts play a role. Indeed, Dorchies and colleagues showed that five weeks of feeding green tea
extract and ()-epigallocatechin gallate (EGCG) to muscular dystrophic mice protected the animals from fatigue and
improved muscle force generation [11]. However, while many antioxidants have been shown to provide protection
against oxidative stress and fatigue ex vivo, this has not always translated into the in vivo situation. With this in
mind we have recently initiated the establishment of animal models of exercise at PFR. Figure 4 shows biochemical
data in blood samples taken at various times from mice undertaking an exhaustive uphill running exercise. While
not statistically significant, there was a trend towards plasma CK activity (an indicator of muscle tissue damage),
42
2500
5.0
4.0
Plasma IL-6 (pg/ml)
Plasma Creatinine Kinase (IU/L)
2000
1500
1000
3.0
2.0
1.0
500
0.0
0
Control
Time (hours)
24
Control
24
Time (hours)
Fig. 4. Markers of stress and inflammation in plasma of exercising mice. Mice performed uphill running on a treadmill until exhaustion and were
sacrificed at 0, 1, 3 or 24 h after the exercise. Resting mice functioned as a control group. Blood samples were taken at the time of sacrifice and
plasma was isolated. (A) CK activity was expressed as IU/L and (B) IL-6 concentration was expressed as pg/mL. Each group consisted of 12
mice. Values are means SEM. *Represents p < 0.05 statistical significance (paired Student t-test) between control and exercise group.
slightly higher one hour after the exercise, compared with the activity in the control (non-exercised mice Fig. 4A).
The release of IL-6 from muscle post exercise is known to be dependent upon both exercise intensity and duration
[6, 12]. Figure 4B shows the plasma levels of IL-6 in exercising mice following our exercise regime. Immediately
after the exercise (0 h), the mean plasma IL-6 concentration significantly increased (7-fold) from 0.45 0.37 pg/mL
in resting control mice to 3.11 1.04 pg/mL in exercised mice (p < 0.05). After only 1 hour of recovery, plasma IL-6
concentrations returned to pre-exercise levels. While the exact role of the release of muscle tissue IL-6 is not clear,
it is probably released in response to metabolic changes and to oxidative stress and/or tissue damage. It is suspected
that IL-6 may be involved in activating aspects of the immune system to assist with tissue repair and recovery. These
data also suggests that our uphill running protocol was inducing IL-6 response in exercising mice, without being
strenuous or damaging enough to significantly increase CK levels. This is consistent with the findings of others in
which no CK increase was observed with an uphill run, while downhill running, which mediates more tissue damage
because of its eccentric nature, consistently increased CK in plasma [4, 18]. Others have utilised exercise routines in
animals to evaluate the effects of nutrient supplementation on exercise-induced stress and performance. For example,
curcumin [9], green tea extract [21], and Ginseng extract [3] have all been shown to decrease markers of damage,
oxidative stress, and inflammation in exercising animals. Our preliminary data are therefore encouraging and in the
near future we hope to be able to evaluate the benefits of fruits on stress and inflammation robustly using animal
models of exercise.
In humans, exercise can have benefits on the immune system including an ability to positively modulate the acute
inflammatory response to a simulated bacterial infection [14]. Furthermore, appropriate fruit extract consumption may
assist these health benefits. In a recent study we reported the effect of supplementation with a blackcurrant fruit extract
on exercise-induced health benefits [16]. Consumption of the anthocyanin-rich blackcurrant extract prior to a moderate
rowing exercise mediated an amelioration of plasma markers of oxidative stress and micro muscle damage, but also
complemented the ability of exercise to stimulate an acute inflammatory response to a simulated bacterial infection
in ex vivo experiments. These findings could be significant in the augmented activation (by fruit polyphenolics) of
43
appropriate adaptive immune responses and of associated positive health benefits derived from moderate and regular
exercise. Other studies have evaluated the potential of fruits and/or polyphenolic compound supplementation as
antioxidants and reported potential protective effects and benefits to muscle and exercise performance [2, 7, 25].
In contrast however, some studies show that antioxidant supplementation may in fact neutralize the health benefits
of regular exercise [5, 24] and that in some individuals undergoing long-term strenuous exercise regimes immune
suppression can be observed [8].
Further studies are being undertaken at PFR in this area to elucidate the health potential of fruit polyphenolics as
appropriate modulators of oxidative stress and inflammation.
4. Summary
Our emerging evidence suggests that some fruits (and derived phenolic compounds) offer significant health and
wellness potential. Our aim is to utilise unique New Zealand germplasm to unleash opportunities for the creation
of premium functional foods (backed by science), to prevent muscle damage/injury, aid recovery, and/or enhance
muscular and immune function.
Acknowledgements
We are grateful for the preparation of the blueberry fruit phenolic extract and the blackcurrant juice by Drs Tony
McGhie and Catherine Snelling of PFR. We are grateful for funding support from the New Zealand Foundation
for Research, Science and Technology (contract C06X0807, cell and human work) and internal funding from PFR
(animal studies).
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DOI:10.3233/BR-2010-003
IOS Press
25
Antioxidative efficiency of an anthocyanin

rich bilberry extract in the human colon
tumor cell lines Caco-2 and HT-29
Markus Schantz, Christiane Mohn, Matthias Baum and Elke Richling
Food Chemistry and Toxicology, Molecular Nutrition, University of Kaiserslautern, Kaiserslautern, Germany
Abstract. Bilberries (Vaccinium myrtillus L.) and its major polyphenolic constituents, the anthocyanins, are discussed to be
preventive against diseases, such as colon cancer or inflammatory bowel diseases (e.g. Crohns disease, ulcerative colitis), are
associated with oxidative stress. Therefore the gastrointestinal tract (GIT) might be a target for prevention of these diseases. In
this study antioxidative efficiency of a commercially available anthocyanin rich bilberry extract (BE) was investigated in vitro
in the human colon tumor cell lines Caco-2 and HT-29. The cell cytotoxicity of the BE was measured by alamar blue assay.
Modulation of intracellular generated reactive oxygen species (ROS) levels was investigated by dichlorfluorescein assay (DCF).
Oxidative DNA damage was monitored by single-cell gel electrophoresis (comet assay) with additional treatment of the DNA
with formamido-pyrimidinglycosylase (FPG) to enhance sensitivity towards ROS induced DNA lesions. Modulation of the total
glutathione (tGSH) level was assayed in a photometric kinetic assay. In a two step protocol cells were first treated with the
protective extract (5500 g/ml; 1 and 24 h) and then with the redox-cycler menadione (Md) (HT-29: 20 M and Caco-2: 6 M)
or the oxidant TBH (tert-butyl hydroperoxide) (250 M, 40 min). Under all conditions tested BE was not cytotoxic in Caco-2 and
HT-29 cells. The data achieved revealed that BE significantly reduce ROS level in HT-29 (250 g/ml; 24 h, p < 0.05) and Caco-2
(50 g/ml; 1 h, p < 0.05) cells. Significant decrease of induced DNA damage was detected in Caco-2 cells after BE treatment
(5 g/ml; 24 h; FPG, p < 0.05). Trend towards increase of tGSH was observed at concentrations of 50500 g/ml BE in Caco-2
cells after 24 h incubation.
In total, the BE was shown to possess antioxidative activity under the used assay conditions towards prevention of oxidative
DNA damage, reduction of intracellular ROS and cellular tGSH.
Keywords: Anthocyanins, bilberries, antioxidants, DNA damage, glutathione, ROS, cytotoxicity
1. Introduction
Anthocyanins (ACN) and their glycosides are polyphenols that play an important role in fruits, flowers and
vegetables and are responsible for their bright colours like orange, blue and red, which help to attract animals
[11, 27, 48] and prevent plants from oxidative stress due to their photoprotective functions such as light induced
photooxidation or against UVB damage [7]. Polyphenols showed beneficial effects on degenerative diseases and
are regarded to exhibit protective effects against cardiovascular diseases and cancer [33]. The main daily intake
of anthocyanins averages 12.7 mg/person in USA and 82 mg/person in Finland [45]. Among dietary sources of
anthocyanins, bilberry (vaccinium myrtillus L.) is one of the richest [23, 29], with varying anthocyanin patterns [34].
In contrast to other flavonoids, anthocyanidins consist of a flavylium ion whereas the structural variations are due
Corresponding author: Prof. Dr. Elke Richling, Food Chemistry and Toxicology, Molecular Nutrition, University of Kaiserslautern, ErwinSchroedinger-Str. 52, 67663 Kaiserslautern, Germany. Tel.: +49 631 205 4061; Fax: +49 631 205 3085; E-mail: richling@chemie.uni-kl.de.
26
M. Schantz et al. / Antioxidative efciency of anthocyanins in vitro
to differences in the number of hydroxyl and methoxyl groups at the B-ring. To the anthocyanidin aglycons sugar
moieties (like glucose, galactose, arabinose, rhamnose and xylose [8, 12]) are attached to the phenolic molecule at
position 3 and 7; and less frequently to 3 and 5 positions [11, 26]. Structures of the ACN found in bilberries are
shown in Fig. 1.
During the last decades, there has been intense interest in beneficial health effects of anthocyanins because of
increasing evidence demonstrating potential biological effects after consumption of fruits and vegetables [3, 41].
A number of studies in vitro and in vivo have been reported that anthocyanins from bilberries are strong antioxidants
[17, 38, 43], inhibit the growth of a range of tumor cells [16, 24, 31], induce apoptosis [14, 19] and have anticarcinogenic [6, 10] properties.
Diseases of the gastrointestinal tract like inflammatory bowel diseases (crohns disease, ulcerative colitis) or colon
cancer, are associated with an imbalance in the cellular redox system based on an increased level of reactive oxygen
species (ROS) [36]. The antioxidant potential of flavonoids and other polyphenols is their ability to scavenge ROS
and protect the organism against oxidative stress induced damage [41].
The specific object of this study was to investigate the in vitro antioxidant effectiveness of a BE. In vitro human
adenocarcinoma cell lines HT-29 and Caco-2, representing characteristics of adult colon cells. As markers for antioxidant capacity the intracellular ROS level (DCF assay), oxidative DNA damage (comet assay), cellular tGSH level
(kinetic assay) and cytotoxicity (alamar blue assay) was used.

2.1. Chemicals, cells and media
All chemicals and solvents were of analytical grade or complied with the standards needed for cell culture
experiments. The BE was provided from Kaden Biochemicals (Hamburg, Germany) (BE, 600761 Bilberry Extract;
consisted of 25% anthocyanins), formamido-pyrimidinglycosylase (FPG) was a gift of Prof. A.R. Collins (University
Oslo, Norway), HT-29 and Caco-2 cells were obtained from DSMZ (Braunschweig, Germany), resazurin, tertbutyl hydroperoxide (TBH), menadione (Md), reduced/oxidized glutathione, glutathione reductase (GSR), catalase,
potassium persulfate, 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), 5-sulfosalicylic acid (SSA), dimethylsulfoxide
(DMSO) and ethidium bromide were purchased from Sigma Aldrich (Munich, Germany). NADPH and 2 ,7 dichlorfluorescein diacetat (DCFH-DA) were purchased from Fluka (Deisenhofen, Germany), low and normal melting
agarose from Bio-Rad (Munich, Germany), bicinchoninic acid (BCA) protein quantification kit was from Interchim
(Sankt Augustin-Buisdorf, Germany). Cell culture media (DMEM and DMEM/F-12 (1:1)) and reagents (fetal calf
serum (FCS), penicillin/streptomycin, Trypsin 0.05%) were purchased from Invitrogen (Karlsruhe, Germany). Cell
culture material (Petri dishes, well plates, flasks, etc.) were from Greiner Bio-One (Essen, Germany).
2.2. Bilberry extract (BE)
The BE was made from European bilberry pomace (Vaccinium myrtillus L.) by extraction with methanol, filtration,
evaporation and lyophilisation. It was stored dry and cold at 24 C. The anthocyanin content was adjusted at 25%.
Fig. 1. Chemical structure of the most common anthocyanins found in bilberry.
27
The BE also contains other polyphenols, tannins, carbohydrates and roughages. Basic characterisation of the anthocyanin profile was performed by Symrise GmbH & Co.KG. Control measurements of BE were performed by using a
Jasco HPLC-UV system equipped with Jasco PU-2080 intelligent HPLC pump, Jasco LG-2080-02 ternary gradient
unit, Jasco UV-2075 plus intelligent UV/VIS detector, Jasco DG-2080-53 3-line-degaser, AS-2055 plus intelligent
sampler (Jasco, Gross-Umstadt, Germany). Separation of 15 anthocyanins was performed on a phenomenex Luna C18
(2) column (Phenomenex, Aschaffenburg, Germany) equipped with a security guard column; injection volume: 20 l;
flow rate: 0.5 ml/min; solvent A (acetonitrile/water/HCOOH; 87/3/10 v/v/v); solvent B (acetonitrile/water/HCOOH;
50/40/10 v/v/v); gradient elution: 020 min from 214% B, 2040 m hold 14% B, 4050 min to 15% B, 5055 min
to 19% B, 5565 min to 20% B, 65110 min washing and reequilibration, detection wavelength 520 nm.
2.3. Cell culture
HT-29 cells were cultivated in 175 cm2 flasks in DMEM with addition of 10% FCS, Caco-2 cells in DMEM/F12
(1:1) with addition of 20% FCS. Both were supplemented with 100 U/ml penicillin and 100 g/ml streptomycin in
humidified atmosphere with 5% CO2 at 37 C.
2.4. Incubations with BE
HT-29 and Caco-2 cells were seeded in Petri dishes (6 cm for HT-29, 9 cm for Caco-2; tGSH level and comet
assay: for 1 h incubation 1.5 106 cells per dish and for 24 h incubation 106 cells per dish, respectively), cultivated
for 24 h (cultures were semiconfluent and undifferentiated), washed with PBS and incubated in the first step with
the BE in incubation medium containing 10% FCS for Caco-2 and 5% for HT-29 cells (dissolved in DMSO at a
final concentration of 1%). In the second step, cells were treated with Md (HT-29: 20 M and Caco-2: 6 M). After
incubation, cells were washed with PBS and isolated by Trypsin (0.05%) treatment. Cell suspensions were directly
used for tGSH level analysis and comet assay. For DCF assay, cells were seeded in 96 well plates (HT-29: 3.2 104
and Caco-2: 2.0 104 ) and incubated with BE (dissolved in DMSO at a final concentration of 1%). The alamar
blue assay was used to check for cytotoxicity, therefore cells were seeded in 48 well plates (HT-29: 6.0 104 and
Caco-2: 5.5 104 ) and incubated with BE (dissolved in DMSO at a final concentration of 1%). All incubations were
performed in the presence of catalase (100 U/ml) to inhibit formation of extracellular hydrogen peroxide (H2 O2 ),
resulting from pro-oxidative interaction of phenolic compounds with cell media constituents like metal ions [13, 22].
Addition of catalase provides an opportunity to preclude effects resulted from extracellular generated H2 O2 .
2.5. Intracellular ROS level (DCF assay)
DCF assay was performed according to Wang and Joseph [42] with modifications [5]. Briefly, after BE treated
cells were incubated for 30 min with DCFH-DA dissolved in DMSO (50 M in PBS; pH 7.0), washed and incubated
with TBH (250 M in PBS; pH 7.4) for another 40 min. After TBH treatment, formation of DCF was measured as
increase of fluorescence intensity between 0 and 30 min in a microplate reader (MWG, Ebersberg, Germany) and
stated as relative fluorescence intensity (FI) in percent control (without BE treatment).
2.6. Cellular GSH level (kinetic assay)
The cellular GSH level was measured by photometric determination of 5-thio-2-nitrobenzoate (TNB), formed from
DTNB [35]. After BE incubation, cells were treated with Md (HT-29: 20 M and Caco-2: 6 M for 1 h in serum free
medium) suspension. The formation of TNB after 2 min reaction time was measured in 96 well plates by microplate
reader (MWG, Ebersberg, Germany) at 412 nm. The GSH level was specified as nanomoles per milligram of protein
and expressed as percent of Md-treated control.
2.7. Oxidative DNA damage (comet assay)
After extract incubation, cells were incubated with Md (see above) and isolated by trypsin treatment. The detailed
process of the alkine single cell gel electrophoresis was described previously [35]. Briefly, 70,000 cells per preparation
28
were centrifuged (10 min, 2000 rpm, 4 C) and the pellet was resuspended with low melting agarose, applied on a
previously prepared microscope slide (per condition, 2 slides with and without FPG respectively), added to lysis
and treated with FPG enzyme to enhance sensitivity towards induced DNA lesions. After DNA unwinding and gel
electrophoresis, slides were neutralized, stained with ethidium bromide and analyzed by fluorescence microscope
(Zeiss, Germany). Per slide 2 50 cells were scored, averaged and the DNA damage was calculated as tail intensity
(TI%: DNA in the comet tail in percent of total DNA) [9, 37, 39]. Results were expressed as percent of Md-treated
control.
2.8. Cytotoxicity (alamar blue assay)
Metabolic activity results in the chemical reduction of resazurin (blue and nonfluorescent) to resorufin (pink and
highly fluorescent) [30]. After extract incubation, cells were washed with PBS, treated with serum free medium
(500 l) containing 10% resazurin solution and measured by a microplate reader (MWG, Ebersberg, Germany)
(excitation wavelength 530 nm, emission wavelength 590 nm, 37 C). Treatment with 0.1% saponin was used as
positive control for cytotoxicity. Results were calculated as percent of control (without BE treatment).
2.9. Statistics
Results of the performed assays are presented as mean SD of 3 to 6 independent experiments. Differences
(p < 0.05) were determined by independent one-sided t-test by origin analysis 6.0 (MicrocalTM Origin , Northampton,
USA).

3.1. Cytotoxity
Resorufin formation from resazurin (alamar blue assay) was analysed as a measure of cytotoxicity in terms of
mitochondrial integrity and cell growth [30]. Incubation time of 1 h was used to minimize confounding factors like
instability and degradation of the anthocyanins as reported earlier [20, 44]. As a positive control resorufin formation
was completely inhibited (0.1% saponin), whereas under all other treatment conditions resorufin formation reached
median 95% of the negative control. Only in HT-29 cells a significant effect at the highest concentration of 500 g/ml
BE (Fig. 2) was detectable. Our data are in good agreement with a study by Yi et al., who reported that higher doses
from 1 to 3 mg/ml crude blueberry extract inhibited cell growth in HT-29 and Caco-2 cells [47], while other studies
investigating cytotoxicity of corresponding anthocyanidins (aglycons) at longer incubation times, cell growth was
inhibited also at lower concentrations [16, 20, 24].
3.2. Intracellular ROS level
The TBH induced modulation of the intracellular ROS level in HT-29 and Caco-2 cells after BE incubation (1 and
24 h) was measured by DCF assay. Quercetin (30 M), used as a positive control, reduced cellular ROS level in the
range of 20% as compared to the control as also demonstrated earlier [35]. With the BE (1 to 250 g/ml) a significant,
concentration dependent decrease of intracellular ROS was observed at 50 g/ml in Caco-2 and 250 g/ml in HT-29
cells after 1 and 24 h incubation. Maximum reduction of ROS level in Caco-2 cells was detected at concentrations
>100 g/ml. The BE was less efficient in HT-29 cells, a significant reduction of ROS level was observed beginning
at 250 g/ml (24 h), respectively (Fig. 3). Our results are comparable to a study of Milbury et al. [28], demonstrating
significant decreases of intracellular ROS level in ARPE-19 cells after bilberry extract treatment (0.01 g/ml).
With a fermented apple extract a significant decrease of ROS level was observed only at higher concentration
ranges [4].
29
Caco-2: 1 h incubation
HT-29: 1 h incubation
120
rel. viability [% control]
100
80
60
40
20
0
Saponin 0 (control)
10
50
100
250
500
concentration [g/ml BE]

Fig. 2. Alamar blue assay to assess the cytotoxicity of an anthocyanin rich bilberry extract (BE) toward Caco-2 and HT-29 cells. For incubation,
cells were treated with 10 to 500 g/ml BE for 1 and 24 h and incubated with resazurin for another 1 h whereby the fluorescence of resorufin was
measured. Saponin was used as positive control for cytotoxicity. Results were calculated as percent of DMSO treated control; n = 4 (mean SD).
Differences were determined by independent one-sided t-test related to the DMSO treated control: *p < 0.05, ***p < 0.001.
140
120
rel. FI [% control]
100
80
60
40
20
0
ue
tin
rce
Q
M
30
l)
BH
tro
con
0(
+T
10
50
100
250
500
concentration [g/ml BE] + TBH
Fig. 3. Modulation of tert-butyl hydroperoxide (TBH)-induced intracellular ROS level in Caco-2 and HT-29 cells after 1 and 24 h incubation
with an anthocyanin rich bilberry extract (BE) (1 to 500 g/ml). Results were calculated as percent of TBH treated control; n = 35 (mean SD).
Differences were determined by independent one-sided t-test related to the TBH treated control: *p < 0.05, **p < 0.01, ***p < 0.001.
30
3.3. Oxidative DNA damage

The comet assay was used to study the effects of the BE on the reduction of Md induced oxidative DNA damage in
Caco-2 and HT-29 cells with and without FPG to enhance sensitivity towards ROS induced DNA lesions. Preventive
effects of quercetin (positive control, 30 M) reducing DNA damage in the range of 60% tail intensity (TI) compared
to the control was demonstrated earlier in the literature [35]. After treatment of cells only with DMSO, a basal level
of 24 to 31% TI was measured in Caco-2 cells. A slight decrease of DNA damage was obtained in a range from 5 to
100 g/ml BE in Caco-2 cells. Concentration curves, resembling a U-shape, were observed reaching a minimum
at 5 g/ml. Significant DNA-protecting effects were shown after 24 h of incubation with 50 g/ml BE and 5 g/ml
BE treated with FPG, respectively (Fig. 4). No reduction of Md induced DNA damages in HT-29 cells was observed
(data not shown). The U-shaped form of concentration curve in the same test system was also observed earlier
with apple juice extract [35]. In low BE (0.01 to 1 g/ml) concentrations no effects and in high BE concentrations
(250 to 500 g/ml) a prooxidative effect was detectable. These data are also in agreement with the results of an
intervention study with hemodialysis patients consuming daily 200 ml anthocyanin rich fruit juice, resulting in a
significant decrease of oxidative DNA damage and a significant increase of GSH level in blood [38]. In another
in vivo study with healthy volunteers who consumed an anthocyanin/polyphenolic-rich fruit juice, a decrease of
oxidative DNA damage during the juice-uptake phase was observed [43].
3.4. Cellular tGSH level
In a photometric kinetic assay, the cellular tGSH level in Caco-2 and HT-29 cells after BE and Md treatment was
measured. DMSO without further treatment as negative control, to quantify the basal level of tGSH in the cells, was
used as a positive control, demonstrated tGSH levels in the range of 160% as compared to negative Md-control as published earlier [35]. After 24 h incubation, Caco-2 cells showed slight but insignificant tGSH increases in the highest
used BE concentrations (50500 g/ml). In contrast, after 1 h incubation 500 g/ml extract significantly reduced the
1 h incubation
1 h incubation + FPG
24 h incubation
24 h incubation +FPG
180
tail intensity [% control]
160
140
120
100
80
60
40
20
0
tin
30
ce
uer
d
l)
+ M ontro
c
0(
0.0
0.1
10
50
100
250
500
concentration [g/ml BE] + Md
Fig. 4. Modulation of Menadione (Md)-induced (HT-29: 20 M; Caco-2: 6 M; 1 h) oxidative DNA damage in Caco-2 cells after 1 and 24 h
incubation with an anthocyanin rich bilberry extract (BE) (0.01 to 500 g/ml); quercetin (30 M) was used as positive control. Results were
calculated as percent of Md treated control; n = 36 (mean SD). Differences were determined by independent one-sided t-test related to the
TBH treated control: *p < 0.05, ***p < 0.001.
31
200
1 h incubation
24 h incubation
180
tGSH level [% control]
160
140
120
100
80
60
40
20
0
d
-M
DM
SO
l)
tro
con
0(
0.0
0.1
10
50
100
250
500
concentration [g/ml BE] + Md

Fig. 5. Modulation of Md-induced total glutathione (tGSH) level in Caco-2 cells after 1 and 24 h incubation with an anthocyanin rich bilberry
extract (BE) (0.01 to 500 g/ml); quercetin (30 M) was used as positive control. Results were calculated as percent of Md treated control; n = 36
(mean SD). Differences were determined by independent one-sided t-test related to the TBH treated control: *p < 0.05, **p < 0.01, ***p < 0.001.
tGSH level down to about 70% of the negative control. BE in a range between 0.01 to 10 g/ml induced no effects after
1 and 24 h of incubation in comparison to the negative control (Fig. 5). This suggests that low anthocyanin concentrations used have no effect on the tGSH level in the used cell systems. In contrast to our in vitro data, in vivo studies, one
with hemodialysis patients and the other with healthy volunteers which consumed an anthocyanin/polyphenolic-rich
fruit juice, an increased level of tGSH in blood after juice uptake was detectable [38, 43].
Taken together, our data indicate that anthocyanins from berries (bilberries) exert antioxidant activity contributing
to the health preventing properties of fruits and vegetables. The anthocyanin profile of the used BE corresponds to
those of native bilberries. Anthocyanin content of 1 g of enriched BE is equivalent to 17 g of native bilberries [23]. In
a study with ileostomy volunteers who consumed 300 g blueberries (with a total anthocyanin amount of 7834 mg/kg)
up to 85% of the anthocyanins reached the colon [18]. He et al. quantified the anthocyanin uptake by small intestine
tissue to be 7.5% of the administrated dose after 120 min in rats [15]. Due to these data concentrations in a range of
0.01 to 500 g/ml BE were investigated in our cellular systems.
As found by us, ROS level increases in Caco-2 cells significantly at 50 g/ml BE. These data are comparable
to a study in which anthocyanins showed significant positive effects on intracellular ROS and lipid peroxidation
in rat pheochromocytoma cells (PC-12) after incubation with sweet potato anthocyanins (50 g/ml) [46]. Further
in vitro studies with anthocyanins demonstrated the high antioxidative capacity due to their ability to scavenge free
radicals [17, 21, 40]. Due to these scavenging effects, anthocyanins (2.5 nm in media) were able to stabilize the
DNA against hydroperoxide radicals, associated with a decrease in DNA damage [25]. Acquaviva et al. monitored
a concentration dependent (100 to 500 mol/l) reduction of plasmid DNA damage after cyanidin and cyanidin 3-Oglucoside treatment [1]. In agreement with these data our comet assay results showed significant DNA-protecting
effects at 5 and 50 g/ml BE in Caco-2 cells. In two in vivo studies with mice under oxidative stress, positive effects of
anthocyanin rich extracts (concentration range 100 to 200 mg/kg KG) like increasing tGSH level and other oxidative
stress markers were reported [2, 32].
In total, we suppose that the preventive properties of the anthocyanin rich bilberry extract to reduce oxidative DNA
damage, lower ROS level and elevate tGSH in human colon tumor cell lines, is associated with protection of the
colon from oxidative stress.
32
Acknowledgements
The authors gratefully acknowledge the gift of FPG enzyme by Prof. A.R. Collins (Institute for Nutrition Research,
University of Oslo, Norway) and the Symrise GmbH & Co.KG for BE characterization. This work was supported by
Bundesministerium fr Wirtschaft und Technologie/AiF via the Forschungskreis der Ernhrungsindustrie e.V. (FEI),
Bonn, project No. 15614 N for financial support.
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DOI:10.3233/BR-2010-002
IOS Press
13
Superoxide anion radical scavenging activity

of bilberry (Vaccinium myrtillus L.)
Vesna Tumbasa, , Jasna Canadanovi

c-Bruneta , Lars Gilleb , Sonja ilasa and Gordana Cetkovi
ca
a Faculty
of Technology, University of Novi Sad, Novi Sad, Serbia

Pharmacology and Toxicology Unit, Department of Biomedical Sciences, University of Veterinary
Medicine Vienna, Vienna, Austria
b Molecular
Abstract. Naturally occurring antioxidants present in bilberry were separated into three groups: vitamin C, phenolic acids and
flavonoids using solid phase extraction (SPE). Chemical composition of bilberry extract fractions was obtained by spectrophotometry and high performance liquid chromatography (HPLC). The results of HPLC analysis point out the high content of vitamin C
(1529 g/g) in fraction Fr1, flavonoids (1328.58 g/g) in fraction Fr2 and phenolic acids (494.31 g/g) in fraction Fr3. The free
radical scavenging activities of these antioxidant fractions on superoxide anion radicals and detection of free radical intermediates
was studied using electron spin resonance (ESR) spectroscopy. Superoxide anion free radical assay demonstrated very potent free
radical scavenging activity of bilberry extract fractions. The results of correlation analysis showed that the separated classes of
antioxidants from bilberry (vitamin C, flavonoids and phenolic acids) are responsible for its antioxidant activity.
Keywords: Bilberry, ESR spectroscopy, superoxide anion radicals, phenolic acids, flavonoids, vitamin C
1. Introduction
Many of the human age-related degenerative diseases are associated with oxidative processes. Many edible plants
are capable of producing natural chemopreventive compounds which have no synthetic counterparts and play a
protective role in human health maintenance. It has been well established that many of the phytochemicals present in
plant derived foods have antioxidant capacity, i.e. are able to remove damaging radical species, as shown by a range
of in vitro assays. Fruits, vegetables and teas contain a wide range of antioxidant compounds, including phenolic
compounds and vitamins [41]. Phenolic secondary metabolites play an important role in plant-derived food quality,
as they affect quality characteristics such as appearance flavour and health-promoting properties.
Edible berries have been a part of mans diet for centuries. Berries are one of the most important dietary sources
of fibre and essential vitamins, minerals and vast number of phytochemicals such as phenolic compounds, including
anthocyanins, phenolic acids, flavonol glycosides and flavan-3-ols [26, 33]. It has been reported that berry extracts
have cardioprotective effects and beneficial effects on platelet aggregation, they are very effective inhibitors of low
density lipoprotein oxidation and inhibitors of the growth of cultured cancer cells [3]. Research at the Scottish Crop
Research Institute and the University of Ulster has shown that berry extracts can inhibit the initiation, progression, and
invasiveness of colon cancer cells [40]. Different berry components are responsible for the inhibition of -glucosidase
and -amylase, which suggests considerable potentiation of effects on blood glucose levels [34]. Similar effects on
lipid digestion have been noted [35].
Corresponding author: V. Tumbas, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia. Tel.: +381
21 485 3652; Fax: +381 21 450 413; E-mail: vesnat@uns.ac.rs.
14
V. Tumbas et al. / Radical scavenging activity of bilberry
Compounds present in the fruits of Vaccinium species are reported to have beneficial influence on human health [38,
44]. Bilberry (Vaccinium myrtillus L.) is a member of the Eriacaceae family and is also known as European blueberry,
huckleberry, whortleberry or blueberry. Fruits of wild bilberry have a well-established role in pharmacognosy and it
has been used as food for centuries, due to its high nutritive value [48]. Bilberry is a rich source of anthocyanins, and
its extracts are extensively used in food/dietary supplements and pharmaceutical products.
Biological properties of the bilberry fruit extract include antioxidant capacity, astringent and antiseptic properties,
ability to decrease the permeability and fragility of capillaries, inhibition of platelet aggregation, inhibition of urinary
tract infection and strengthening of collagen matrices via cross linkages [15]. Bilberry crude extracts are marketed
as pharmaceutical preparations for the treatment of both ophthalmological diseases and blood vessel disorders. More
recently, they have been used for the treatment of diarrhea, dysentery, and mouth and throat inflammations [48].
Bilberries may be eaten fresh or dried. Bilberry tea may also be made from fresh or dried berries, or from the leaves.
Dried bilberries are high in tannin and pectin, which have an astringent action that controls the inflammation that
causes diarrhea. Dried billberies are traditionally used as herbal tea remedy. Distribution and contents of antioxidant
compounds (anthocyanins, flavonoids, phenolic acids) as well as antioxidant activity of fresh bilberries has already
been confirmed in the literature [12, 19, 22, 37]. Laplaud et al. [28] reported that bilberry extract, characterised by
74.2 4.9 mg of polyphenols/g, inhibited copperinduced oxidative modifcation of human LDL. In another study,
Viljanen et al. [49] found that raspberry, bilberry, lingonberry and black currant extracts inhibited copper-induced
protein and lipid oxidation in a lactalbumin-liposome oxidation system, bilberry extract being one of the most
effective.
However, contents of phenolic compounds and vitamin C, as well as antioxidant activity of dried brilberry fruits has
not been evaluated. Yue and Hue [52] examined changes of anthocyanins, anthocyanidins, and antioxidant activity in
fresh bilberry extract during dry heating at 80 C, 100 C and 125 C. Conjugated sugars of anthocyanins were cleaved
from the anthocyanins to produce their corresponding anthocyanidins or aglycones during heating and the heated
extracts had higher free radical scavenging capability than unheated extract.
In this study we used dried bilberries as a potential source of naturally occuring antioxidants. Extract from dried
bilberries was fractionated into three groups of naturally occuring antioxidants: vitamin C, neutral and acidic phenolics using solid phase extraction (SPE). Present study describes behaviour of obtained bilberry extract fractions in
superoxide anion generating system their free radical scavenging activities and free radical intermediates formed
during this reaction. In addition, correlation matrix was conducted in order to evaluate the contribution of separated
phytochemicals from bilberry and relationship with free radical scavenging activity.

2.1. Plant material
Dried bilberry fruits were obtained in a local drugstore. The air dried fruits were ground using a coffee mill and
passed through a 0.36 mm sieve.
2.2. Preparation, purication and fractionation of bilberry extract
20 g of dried and ground bilberry fruits were macerated with 500 ml of 80% acetone during 24 hours. The macerat
was filtered (Whatman No. 4) and the maceration was repeated once more. Two macerates were mixed and evaporeted
to dryness under reduced pressure. In order to separate vitamin C from the phenolic antioxidants and to remove organic
acids, residual sugars, amino acids, proteins and other hydrophilic compounds as well as to exchange solvents, a
cleanup of the bilberry extract by solid phase extraction (SPE) was performed according to Rigo et al. [39], with
Chromabond C-18 (1000 mg, J.T. Baker, Holland). The dry bilberry extract was redissolved in 5 ml of 0.5 M H2 SO4 ,
filtered through 0.45 m (pore size) membrane filters (Millipore, Bedford, MA) and loaded on the Chromabond
C-18 preconditioned with 2 ml of methanol followed by 5 ml of 5 mM H2 SO4 . The polar substances, including
vitamin C, were removed with 2 ml of 5 mM H2 SO4 and this fraction was marked as Fr1. The phenolic compounds
were eluted with 2 ml of methanol followed by 5 ml of distilled water and this solution was considered as purified
15
bilberry extract. Spectrophotometrical determiantion of total phenols, flavonoids and anthocyanins was conducted
with purified bilberry extract. Further, extraction and fractionation of neutral and acidic phenolics was conducted
according to Chen et al. [10]. The purified bilberry extract was adjusted to pH 7.0 with 2.0 M NaOH solution and
loaded onto the Chromabond C-18 preconditioned for neutral phenolics with 8 ml of methanol followed by 4 ml
of distilled-deionized water adjusted to pH 7.0. The column was washed with 10 ml of distilled-deionized water
and eluted with 12 ml of methanol. Fraction eluted in this step contains neutral phenols and is used further as Fr2.
The effluent portion was adjusted to pH 2.0 with 2.0 M HCl, passed through the preconditioned acidic column. For
isolation of acidic phenolics, cartridge was preconditioned by passing 8 ml of methanol and 4 ml of 0.01 M HCl.
Then the column was washed with 5 ml of 0.01 M HCl and the adsorbed fraction was eluted with 12 ml of methanol.
This fraction contains acidic phenols and is labeled as Fr3. The obtained fractions of bilberry extract, Fr1, Fr2 and
Fr3, were evaporated using a rotary evaporator until dryness at 35 C under reduced pressure.
2.3. Spectrophotometrical determination

2.3.1. Total phenol concentration in puried bilberry extract
The amount of total soluble phenolics in extracts was determined spectrophotometrically according to the FolinCiocalteu method [45]. The reaction mixture was prepared by mixing 0.1 ml of water solution (concentration 1 mg/ml)
of purified bilberry extract, 7.9 ml of distilled water, 0.5 ml of Folin-Ciocalteus reagent and 1.5 ml of 20% sodium
carbonate. After 2 h, the absorbance at 750 nm (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan) was obtained
against control that had been prepared in a similar manner, by replacing the extract with distilled water. The total
phenolic content, expressed as mg of chlorogenic acid equivalents per g dry weight of purified bilberry extract, was
determined using calibration curve of chlorogenic acid standard.
2.3.2. Total avonoids in puried bilberry extract

Total flavonoids (expressed as mg rutin per g dry weight) in purified extract were estimated spectrophotometrically according to Markham [30]. Flavonoids from purified bilberry extract (0.5 ml) were extracted with 1 ml of
extraction medium (70% [v/v] methanol, 5% [v/v] acetic acid and 25% [v/v] distilled water) at room temperature for 60 min. The resulting solution was filtered trough Whatman paper No. 4 and filtrate volume was adjusted
to 100 ml. The probes were prepared by mixing: 5 ml of dilluted extract, 1 ml of distilled water and 2.5 ml of
AlCl3 solution (26.6 mg AlCl3 6H2 O and 80 mg CH3 COONa dissolved in 20 ml distilled water). A blank probe was
prepared by replacing AlCl3 solution with distilled water. The absorbance of probes and blank probe were measured
immediately at 430 nm (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan). Total flavonoid content, expressed
as mg rutin per g dry weight of purified bilberry extract, was calculated from a calibration curve using rutin as
standard.
2.3.3. Total and monomeric anthocyanins in puried bilberry extract

Total anthocyanin content in purified extract was estimated spectrophotometrically using the pH single and
differential method [9]. The spectrophotometric single pH method was used to determine total (monomeric
plus polymerized) anthocyanins and differential pH method for determination of monomeric anthocyanins in
the extract. The aqueous extract was diluted with two buffer solutions at pH 1 and 4.5. The absorbance
of each dilution was measured at 510 and 700 nm against a distilled water control using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The total anthocyanin concentration was obtained from equation:
Ctot (mg/l) = (Atot MW DF 1000)/ L while monomeric anthocyanin concentration was calculated from the
equation: Cmon (mg/l) = (Amon MW DF 1000)/ l, where Atot is calculated as Atot = A510 A700 , Amon is
calculated as Amon = (A515 A700 )pH 1 (A515 A700 )pH 4,5 , is cyanidin-3-glucoside molar absorbance coefficient
(26900 l/(mol cm)), MW is cyanidin-3-O-glucoside molecular weight (449.2 g/mol), DF is dilution factor and L is
cell path length (1 cm). Total anthocyanin and monomeric anthocyanin content was expressed as mg cyanidin-3-Oglucoside equivalents per g of purified bilberry extract [1].
16
2.4. HPLC analysis of bilberry extract fractions

All analyte solutions and solvents were filtered prior to analysis through 0.45 m (pore size) membrane filters
(Millipore, Bedford, MA). Quantification of phenolics in Fr2 and Fr3 was done by HPLC analysis by a Waters
Breeze chromatographic system (Waters, Milford, MA), which consisted of 1525 binary pumps, thermostat and
717+ autosampler connected to the Waters 2996 diode array detector (Waters, Milford, USA). Chromatograms were
recorded in 3D mode. Separation was performed on a Symmetry C-18 RP column 125 4 mm with 5 m particle
size (Waters, Milford, USA) with an appropriate guard column. Two mobile phases, A (0.1% phosphoric acid) and
B (acetonitrile), were used at flow rates of 1 ml/min with the following gradient profile: the first 20 m from 10 to
22% B; next 20 m of linear rise up to 40% B, and for last 10 m 55% of B, followed by 10 m reverse to initial 10%
B with additional 5 m of equilibration time. The data acquisition and spectral evaluation for peak confirmation were
carried out by the Waters Empower 2 Software (Waters, Milford, USA). Quantification of total phenolic compounds
in bilberry fractions Fr2 and Fr3 were performed using quercetin and p-coumaric acid as secondary standards for
flavonoids and phenolic acids, respectively.
HPLC analysis of vitamin C content in Fr1 was performed on a liquid chromatograph Agilent 1100, USA,
equiped with a ultraviolet diode array detector (UV-DAD). For separation a C-18 column with a 5 m particle size
was used at a flow rate of 0.4 ml/min and temperature 37 C. Reference substance (vitamin C) and samples were
dissolved/extracted in solution of meta-phosphoric acid (3% w/w) in 8% acetic acid. Ammoniumacetate (0.1 mol/l,
pH 5.1) was used as a mobile phase. The injected volume was 20 l and the total running time was 6 min.
2.5. ESR measurements

2.5.1. Superoxide anion radical scavenging activity of bilberry extract fractions
A solution containing superoxide anion radicals was prepared by dissolving KO2 /crown ether (10 mM/20 mM)
in dry dimethylsulfoxide (DMSO) and 0.005 ml of this solution were added to 0.5 ml of dry DMSO and 0.005 ml
of an DMSO spin trap solution (DMPO, 5,5-dimethyl-1-pyrroline-N-oxide, 2 M). The influence of extracts on the
formation of DMPO/ OOH adducts was studied by adding the N,N-dimethylformamide (DMF) solution of bilberry
extract fraction to the superoxide anion reaction system at a final concentration range of 0.0011 mg/ml. Synthetic
antioxidant, butylated hydroxyanisole (BHA), was used for comparison. Then the solution was transferred to a
quartz flat cell ER-160FT and 2 m after mixing ESR spectra were recorded on an EMX spectrometer from Bruker
(Rheinstetten, Germany) using a TE102 cavity. The following instrument settings were used: field modulation 100 kHz,
modulation amplitude 4.00 G, receiver gain 1 104 , time constant 327.68 ms, conversion time 40.96 ms, center field
3440.00 G, sweep width 100.00 G, x-band frequency 9.64 GHz, power 20 mW, temperature 23 C.
The extent of scavenging by antioxidant fractions was expressed as scavenfing activity (SA) values. The SAo2
value of the extract was defined as: SAo2 (%) = 100 (h0 hx )/h0 , where h0 and hx are the hight of the second
peak in the ESR spectrum of DMPO/ OOH spin adduct of the sample without and with antioxidant fractions,
respectively.
2.5.2. Detection of antioxidant-derived radicals

The antioxidant-derived radicals were determined in the reaction system containing 0.5 ml of DMSO solution
of KO2 /crown ether (10 mM/20 mM) and 0.5 ml of DMF solution of bilberry extract fraction (5 mg/ml). These
solutions were aspirated from autosampler vial into the quartz flat cell, which was located in the TE102 -resonator of
the EMX spectrometer (Bruker, Rheinstetten, Germany). The mixing of both solutions was performed in the lower
part of the cell prior to reaching the active zone of the flat cell. ESR measurements were started 20 s after mixing.
The following instrument settings were used: microwave frequency, 9.73 GHz; modulation frequency, 100 kHz;
microwave power, 20 mW; center field, 3491.1 G; sweep, 25 G; modulation amplitude, 0.48 G; receiver gain, 8 105 ;
scan rate, 35.77 G/min; time constant, 0.163 s; scans 5. ESR spectral files were imported into the WINSIM program
[16] for the analysis of the hyperfine splitting constants.
17

All analysis were run in triplicate and were expressed as means standard deviation (SD). Statistical analysis was
done by using Statistica 8.0 software package (StatSoft Inc., 19842007). Significant differences were calculated by
ANOVA test and then least significant difference (LSD) test (p < 0.05, unless noted otherwise).
3. Results
3.1. Chemical composition of bilberry
The contents of total phenols, flavonoids, anthocyanins and monomers of anthocyanins in purified bilberry extract
are given in Table 1. According to these results, flavonoids are the most abundant class of phenolic compounds present
in bilberry extract (82.24%). Anthocyanins are one of the main flavonoid subgroups in fruits and berries and they
are responsible for their red, violet, purple and blue colours. The anthocyanins are considered the most important
of the pharmacologically active constituents. Anthocyanin concentration in the fresh bilberry fruit is approximately
0.10.5%, while concentrated bilberry extracts are usually standardized to 25% anthocyanins [24, 53]. Valentov
et al. [48] detected cyanidin, delphinidin, malvidin, paeonidin and petunidin and its glycosides in bilberry extract,
summary 25% of extract. Presented results show that 35% of flavonoids and less than 30% of total phenols in bilberry
extract represent anthocyanins.
The compounds of interest in this study were vitamin C, flavonoids and phenolic acids. Total phenolic content
measured by the Folin-Ciocalteu method may give false and overestimated results because possible interference from
other chemical components present in the extract (sugars, aromatic amines, sulfur dioxide, ascorbic acid, organic acids,
Fe(II), and other nonphenolic organic substances that react with Folin-Ciocalteu reagent) [42, 45]. High performance
liquid chromatography (HPLC) method was used for further quantification of total phenolic compounds, flavonoids
in Fr2 and phenolic acids in Fr3, as well as vitamin C in Fr1.
As presented in Table 2. bilberry extract contains two times higher levels of flavonoids than phenolic acids.
Herrmann [23] and Wildanger and Herrmann [51] reported that the far predominant phenolic compound in bilberry
is quercetin. Beside quercetin, the detectable levels of myricetin and kaempferol were found in several European
bilberry varieties [46]. High contents of quercetin were confirmed also by Hkkinen et al. [20] where quercetin
was abundant in 21.4% of total phenolic compounds detected in bilberry. In that study the main phenolic acid in
bilberry was p-coumaric acid followed by ferulic and caffeic acids. Also, total contents of phenolic compounds
obtained in this study were lower comparing to the values obtained in the study of Prior et al. [37] probably due to
the variability of plant material used for extraction. Their data are based on fresh fruit flesh and we used dried plant
material. According to the study of Hkkinen [21] quercetin content in bilberry decreased markedly during 9 months
of storage, the lower values we observed seem to be reasonable. Also, the contents of vitamin C, as expected, were
much lower than reported [2] due to the the sensitivity and instability of this vitamin during storage and processing
Table 1
Total contents of phenols, flavonoids and anthocyanins in purified bilberry extract
Compounds
phenolsa
Total
Total flavonoidsb
Total anthocyaninsc
Monomeric anthocyaninsc
a mg
b mg
c mg
chlorogenic acid/g.
rutin/g.
cyanidin-3-glycoside/g.
Total content
mg/g dry weight
mg/g plant material
273.25 10.69
136.29 4.28
224.71 7.34
112.08 3.87
78.50 3.34
58.45 2.28
39.15 1.34
29.15 0.98
18

Table 2
Contents of phenolic compounds and vitamin C in bilberry extract fractions
Compounds
Vitamin C (Fr1)
Flavonoids (Fr2)
Phenolic acids (Fr3)
Total content
Expressed as mg per
100 g of bilberry
extract fraction
Expressed as mg per
100 g of bilberry
extract
Expressed as mg per
100 g of bilberry
sample extracted
152.90 14.60
132.86 0.05
49.43 0.02
2.02 0.19
0.77 0.01
0.36 0.00
1.01 0.09
0.38 0.01
0.18 0.00
[17]. In general, vitamin C content of berries declines during storage by up to 56% depending on duration and
temperature [4]. Finally, actual levels of antioxidants in plants depend greatly on the variety of plant analysed, the
way in which it was cultivated (since synthesis of antioxidants can be a response to stress), and how it is harvested
and stored [11].
3.2. Superoxide anion radical scavenging activity of bilberry extract fractions
Although the superoxide anion radical is a weak oxidant, it gives rise to the generation of more powerful and
dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to the oxidative stress [29]. Superoxide
anion radical can be produced enzymatically, in xanthine/xanthine oxidase system or chemically, as in this study.
The advantage of the latter system for determination of superoxide anion radical scavenging activity is the simplicity
in interpretation of results, since possible inhibition of the xanthine oxidase enzyme by antioxidant fractions is not a
problem.
The superoxide adduct of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) has been detected by ESR spectroscopy
using KO2 solubilized in crown ether as a source of superoxide (Fig. 1). The ESR hyperfine splitting constants
of the DMPO/ OOH adduct were: aN = 12.65 G; aH = 10.4 G; aH = 1.3G, which is in accordance with literature
data [8].
Fig. 1. ESR spectrum of DMPO/ OOH spin adduct (blank) recorded 2 min in after mixing of 0.005 ml of KO2 /crown ether (10 mM/20 mM)
dissolved in dry DMSO, 0.5 ml of dry DMSO and 0.005 ml of an DMSO spin trap solution (DMPO, 2 M).
Fig. 2. Superoxide anion radical scavenging activity of bilberry extract fractions:

of three determinations.
Fr1;
Fr2;
19
Fr3. Each value is presented as mean SD
All three fractions of bilberry extract have efficiently scavenged superoxide anion radicals (Fig. 2). These fractions showed similar, not significantly different (p < 0.05) scavenging activity, when applied at lower concentrations
(0.0010.01 mg/ml). Complete elimination of superoxide anion radicals (SAo2 = 100%) was achieved with the
same concentration, 0.75 mg/ml, of Fr2 and Fr3, and with 1 mg/ml of Fr1. The SAo2 of bilberry extract fractions
decreased in the following order: Fr3 > Fr2 > Fr1.
In the present study bilberry fractions scavenged superoxide anion radicals with an efficiancy higher than BHA.
Calculated IC50 values for Fr1 (49 g/ml), Fr2 (21 g/ml) and Fr3 (15 g/ml) were much lower than IC50 value
for BHA (2680 g/ml). IC50 values of bilberry extract fractions were comparable to the superoxide anion radical
scavenging activity values of bilberry extract obtained in the study of Valentov et al. [48] (final concentration
47.6 g/ml scavenged 25.7% of superoxide anion radicals formed) and Martin-Aragon et al. [32] (final concentration
50 g/ml scavenged 63% of superoxide anion radicals formed). Difference in reactivity is due to the difference in the
contents of active compounds, resulting from different extraction methods, and different experimental conditions,
especially because in these studies superoxide anion radical was produced enzymatically.
The scavenging properties of antioxidant compounds are often associated with their ability to form stable radicals. It
is well known that aromatic compounds containing hydroxyl groups, especially those having ortho di- or trihydroxyfunction, can give rise to radicals stable enough to be directly detected by ESR spectroscopy [14]. Seyoum et al.
[43] proposed a mechanism of antioxidant reaction of flavonoids, via hydrogen donation leading to formation of a
flavonoid radical and termination of flavonoid radicals by further loss of a hydrogen atom via disproportionation or
other subsequent reactions. An ESR signal assignable to a flavonoid semiquinone radical was found in the superoxide
generating system in the presence of Fr2 and without adding the spin trap (Fig. 3). After simulation of the obtained

ESR spectrum (Fig. 3) and comparing the obtained hyperfine constants (aH 2 = 1.7 G; aH 5 = 0.8 G and aH 6 = 2.8 G)

2
5
6
with the reports of Canadanovic-Brunet et al. [6] (aH = 1.5 G; aH = 0.7 G and aH = 2.7 G), it was determined that
the observed radical was quercetin semiquinone radical. This is consistent with the findings of Herrmann [51] and
Wildanger and Herrmann [46] who determined that the dominant bilberry flavonoid is quercetin. The stability of
flavonoid, i.e. quercetin is proposed to be due to the large conjugated system providing delocalization of the unpaired
electron. However, the reaction of superoxide anion radical with Fr3 without presence of a spin trap did not result in
an ESR visible product, probably due to the low stability of the radicals of phenolic acids. This could be caused by the
lack of stabilization by resonance due to the smaller conjugated system comparing to the flavonoid molecule. On the
other hand, the free-radical product obtained by oxidation of Fr1 with superoxide anion radicals was characterized
by ESR spectroscopy as a simple doublet showing coupling constants of aH = 1.84 G (Fig. 4). According to literature
data and due to the high content of vitamin C confirmed by HPLC analysis (Table 2), this can be assigned to an
ascorbyl radical [18, 36]. It has been reported that the reaction of ascorbic acid with more aggressive radicals results
in the production of an intermediate radical, ascorbyl, of low reactivity. The lower activity comes from the ability of
ascorbate to delocalize the radical electron through its -system.
20
Experiment
Simulation
Fig. 3. ESR spectrum of antioxidant-derived radical obtained in the reaction system containing 0.5 ml of DMSO solution of KO2 /crown ether
(10 mM/20 mM) and 0.5 ml of DMF solution of bilberry extract fraction Fr2 (5 mg/ml) 20 s after mixing. Simulated spectrum was obtained using
WINSIM program.
Experiment
Simulation
Fig. 4. ESR spectrum of antioxidant-derived radical obtained in the reaction system containing 0.5 ml of DMSO solution of KO2 /crown ether
(10 mM/20 mM) and 0.5 ml of DMF solution of bilberry extract fraction Fr1 (5 mg/ml) 20 s after mixing. Simulated spectrum was obtained using
WINSIM program.
Berries of the genus Vaccinium have been reported to express antioxidative and anticarcinogenic effects in vitro,
which are partly proposed to be due to phenolic compounds in these berries [2, 13, 20, 27]. In these studies berry
extracts, including bilberry, had high total phenolic contents and high antioxidant activity when compared to other
plant materials. Kay et al. [25] reported that ingestion of 100 g of freeze dried blueberries (100 g) and berry juices
increases the antioxidant capacity of blood plasma by 1430%. A commercial extract of bilberry scavenged superoxide
anion and hydroxyl radicals, inhibited lipid peroxidation in rat liver microsomes and liver lipid peroxidation in vivo
in mice [31].
21
Understanding the link between the antioxidant capacity of individual components and the bioactivities of different
berries may direct the biotechnological improvement of new berry varieties [3]. Many researchers have found a strong
correlation between antioxidant activity and total phenolics [7, 20, 50]. Beekwilder et al. [5] reported that about half
of antioxidant activity of raspberries was due to ellagitannins, 20% due to vitamin C and 25% to anthocyanins, while
Tulipani et al. [47] found greater contribution of vitamin C (3035%) in some strawberry varieties. Positive linear
correlation between SAo2 and total conents of vitamin C (r2 = 0.810), flavonoids (r2 = 0.707) and phenolic acids
(r2 = 0.608) obtained in this study indicate that these phytochemicals are one of the main components responsible
for antioxidant behaviour of bilberry.
4. Conclusion
Analysis of bilberry extract fractions containing vitamin C (Fr1), neutral phenols (Fr2) and acidic phenols (Fr3)
showed high superoxide anion radical scavenging activities, better than synthetic antioxidant BHA. The highest
activity was achieved with Fr3. HPLC analysis confirmed significant levels of vitamin C, flavonoids and phenolic
acids in investigated bilberry extract fractions. Correlation analysis proved the involvement of these compounds
in antioxidative activity. Further research is focused in the investigation of exact and comlete polyphenol profile of
bilberry extract, especially anthocyanin contents, and other biological activities contributing the well known beneficial
health effects of bilberry.
Acknowledgements
This research is part of the Project No. 142036 which is financially supported by the Ministry of Science and
Technological Development of the Republic of Serbia. A part of this research was supported by a Grant from AD
Austria.
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Journal of Berry Research 1 (2010) 312

Antioxidant and antiproliferative properties

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

2. Material and methodology

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

2.3. Fractionation by solid phase extraction

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

3. Results and discussion

MMP-9 inhibitory activity (IC50)

Total phenol content/Anti oxidant activity

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

MMP-9 inhibitory capacity

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

3.3. HPLC-MS phenolic prole determination

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

271.0, 211.1, 169.0

204.1, 186.1, 142.0

Gallic acid glucoside

Ellagic acid-hexose derivative

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

579.1, 465.1, 303.2

839.5, 729.2, 676.1, 567.2,

1111.2, 899.3, 839.3, 817.3,

476.8, 431.2, 285.2, 227.3

Gallic acid glucose derivative

The most abundant ions are shown in bold.

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

L. Tavares et al. / Antioxidant and antiproliferative properties of strawberry tree tissues

Journal of Berry Research 1 (2010) 1

Journal of Berry Research 1 (2010) 5359

Nutritional quality of berries and bioactive

of Food Hygiene, Estonian University of Life Sciences, Tartu, Estonia

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

2. Materials and methods

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

3. Results and discussion

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

*The cultivar Pilenai was measured in four different phases of ripening;

3.3. Polyphenol composition of leaves

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

BPC of blackcurrant leaves 0.1g diluted in 10 ml of 40% ethanol (1/100 m/v)

BPC of standard mixture with concentration 12.5 ug/ml

P. Raudsepp et al. / Nutritional quality of berries and bioactive compounds

Journal of Berry Research 1 (2010) 4552

The inhibitory effects of berry-derived

2. Materials and methods

Percentage of viable neurons

CysDA concentration (M)

Percentage viable cells

3.2. Inhibitory effect of avonoids on LPS/IFN--induced TNF-a release

Journal of Berry Research 1 (2010) 3544

Evaluating the health benefits of fruits for

2. Materials and methods

3. Results and discussion

Relative Fluorescence (% from time zero)

Reduced Glutathione (uM)

Glutathione Peroxidase (mUnits/ml)

Plasma IL-6 (pg/ml)

Plasma Creatinine Kinase (IU/L)

Journal of Berry Research 1 (2010) 2533

Antioxidative efficiency of an anthocyanin

M. Schantz et al. / Antioxidative efciency of anthocyanins in vitro