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58 ı Originalarbeiten Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007
# Corresponding author, E-mail: ljakobek@ptfos.hr,
Tel.: +385-31-224-300; Fax: +385-31-207-115
Summary
Anthocyanin content and antioxidant activity of various red fruit juices
(black currant, red raspberry, blackberry, sour cherry, sweet cherry,
strawberry, chokeberry, and elderberry juice) has been evaluated in this
study by using HPLC, pH-differential, and DPPH method. Anthocyanins
were the predominant phenolic components (66 % in elderberry juice,
56 % in black currant juice) or represented considerable portion in to-
tal polyphenol content of some juices (40 % in blackberry juice, 33 % in
chokeberry juice). Amount of anthocyanins determined by HPLC method
ranged from 202 to 6287 mg l –1 in strawberry and elderberry juice, re-
spectively. Anthocyanins present in investigated red fruit juices were de-
rivatives of cyanidin, delphinidin, pelargonidin and peonidin. Chokeberry,
elderberry, blackberry and sour cherry juice were characterized by cyani-
din derivatives, black currant juice by delphinidin and cyanidin derivatives
and strawberry juice by pelargonidin derivatives. The major anthocyanins
in red raspberry and sweet cherry juice were derivatives of cyanidin al-
though peonidin (in sweet cherry juice) and pelargonidin derivatives
(in red raspberry juice) were found in low amount. Antioxidant activity
varied from 4 to 72 μmol TE/ml in sweet cherry and chokeberry juice,
respectively. High correlation was observed between antioxidant activity
and total anthocyanin content of investigated red fruit juices. Overall re-
sults showed that red fruit juices can serve as a good source of bioactive
phytochemicals in human diet. Chokeberry, elderberry and black currant
juice were the richest in anthocyanin content and showed the strongest
antioxidant activity, as well. Therefore, these three juices can be regarded
as good candidates for raw materials in production of functional foods.
Zusammenfassung
In dieser Studie wurden Anthocyane-Konzentration sowie antioxida-
tive Aktivität von Fruchtsäfte aus verschiedenen Beerenfrüchte-Sorten
und Kirschen (schwarze Johannisbeere, Himbeere, Brombeere, Sauer-
und Süßkirschen, Erdbeere, Aronia und Holunderbeere) mittels HPLC,
pH-Differenzial und DPPH-Methode bestimmt. In einigen Säften bilden
Anthocyane den größten Anteil der Gesamtpolyphenole (z. B. 66 % im
Holunderbeersaft, 56 % im schwarzen Johannisbeersaft), oder stellen
einen bedeutenden Anteil am Gesamtpolyphengehalt mancher Säfte
(40 % im Brombeersaft, 33 % im Aroniasaft) dar. Die Anthocyane-Kon-
zentration, mittels HPLC bestimmt, variierte von 202 mg/l im Erdbeer-
saft bis 6287 mg/l im Holunderbeersaft. Bei den aus Beerenfrüchten und
Kirschen hergestellten Fruchsäften vorhandenen Anthocyanen handelt
es sich um Cyanidin-, Delphinidin-, Pelargonidin-, und Peonidinderivate.
So wurden im Aronia-, Holunderbeere-, Brombeere- und Sauerkirschen-
saft Cyanidinderivate gefunden. Im schwarzen Johannisbeersaft wurden
Delphinidin- und Cyanidin-, im Erdbeersaft Pelargonidinderivate gefun-
den. Die im Himbeer- und Süßkirschensaft enthaltenen Anthocyane sind
größtenteils Cyanidinderivate, obwohl auch darin, aber nur in kleinen
Mengen, Peonidinderivate (im Süßkirschensaft) und Pelargonidinderi-
vate (im Himbeersaft) vorhanden sind. Die antioxidative Aktivität variierte
im Bereich von 4 μmol Troloxäquivalent/ml im Süßkirschensaft bis zu
72 μmol Troloxäquivalent/ml im Aroniasaft. Es wurde eine starke Korrela-
tion zwischen der antioxidativen Aktivität und der Anthocyan-Konzentra-
tion in den geprüften Säften aus Beerenfrühten und Kirschen festgestellt.
Alle Ergebnisse weisen darauf hin, dass Säfte aus Beerenfrüchten und
Kirschen eine geeignete Quelle an bioaktiven Phytochemikalien für die
menschliche Ernährung darstellen. Aronia-, Holunderbeer- und schwar-
zer Johannisbeersaft enthalten die größte Anthocyane-Konzentration und
zeigen die stärkste antioxidative Aktivität. Diese Säfte sind daher zur Her-
stellung funktioneller Lebensmittel geeignet.
Keywords: Anthocyanins, red fruit juices, berry juices, antioxidant activ-
ity / Anthocyane, Beerenfrüchtesäfte, antioxidative Aktivität
Introduction
Considerable interest has been developed over the years in
fruits and vegetables due to their potential biological and
health-promoting effects. Numerous epidemiological stud-
ies indicate that an increase in the consumption of fruits
and vegetables is associated with a decrease in the incidence
of various diseases like cardiovascular disease, stroke, and
cancer1–5). The protective effect of fruits and vegetables has
been attributed to their bioactive antioxidant constituents,
including vitamins, carotenoids, polyphenols1,6). Among
various antioxidants present in fruits and vegetables, poly-
phenols (including anthocyanins) have received much at-
tention since being reported to have a positive influence on
human health1).
Anthocyanins belong to the class of phenolic compounds.
They are water-soluble glycosides and acylglycosides of
anthocyanidins. The most common naturally occurring an-
thocyanins are the 3-O-glycosides or 3,5-di-O-glycosides
of cyanidin, delphinidin, peonidin, petunidin, pelargonidin
and malvidin7). Anthocyanins are important polyphenolic
components of fruits, especially berries8–13). They are potent
antioxidants in vitro14) and may be protective against many
degenerative diseases7). Numerous studies have shown that
anthocyanins are absorbed in their original glycosylated
forms in humans15–18). They appear in urine after supple-
mentation with berries or berry extracts but in very low
Anthocyanin content and antioxidant activity of various red fruit juices
Lidija Jakobek#, Marijan Šeruga, Martina Medvidovi
ć
-Kosanovi
ć
and
Ivana Novak
Department of Applied Chemistry and Ecology, Faculty of Food
Technology, J. J. Strossmayer University of Osijek, Kuha
č
eva 18,
HR-31000 Osijek/Croatia
Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007 Originalarbeiten ı 59
concentrations15,16,18). Anthocyanins have also been found
in human plasma in very low concentrations15–18). Recent
research emphasized the importance of anthocyanins in
development of cancer therapy, because it was found that
the anthocyanins could decrease the in vitro invasiveness of
cancer cells19).
Besides fruits and vegetables, a relevant part of intake of
polyphenolic phytochemicals (including anthocyanins) is
supplied by fruit juices. Juices are suitable food products
in terms of ingestion of health protective phytochemicals.
Bioactive components may even be better absorbed from
juices than from plant tissues, as it was demonstrated for
ascorbic acid20). Some studies have shown that the intake of
antioxidant berry juice (containing white grape, black cur-
rant, elderberry, sour cherry, blackberry and aronia juice)
can increase plasma antioxidant capacity, which suggests
an improvement in antioxidant status and indicates that
antioxidant constituents of the juice might decrease lipid
oxidation within plasma compartment20). Consumption of
polyphenol rich juice (containing apple, orange and mango
extracts) enhanced antioxidant status, reduced oxidative
DNA damage and stimulated immune cell functions21).
There is still not enough knowledge about health effects of
fruits, vegetables, juices or antioxidant concentrates. Much
more research is therefore needed on the composition of
antioxidants in natural antioxidant mixtures, juices or fruits
and on antioxidant effects of these products22). Many reports
have been written about the antioxidant activity and antho-
cyanin profiles of various fruits or fruit extracts8–14,23–27), but
relatively few have been based on red fruit juices28,29). A need
for such data still exists because of increasing popularity of
red fruit juice consumption during the last time, and because
of increasing consumer awareness concerning the nutritional
value of all foods (including these juices).
Therefore, the objective of this work was to evaluate various
natural red fruit juices (black currant, red raspberry, black-
berry, sour cherry, sweet cherry, strawberry, chokeberry,
and elderberry juice) regarding the amount of anthocyanins
and antioxidant activity and to determine individual antho-
cyanic compounds present in these juices. Furthermore, a
concentration of total polyphenolics was measured in order
to determine the portion of anthocyanins in total polyphe-
nol content of red fruit juices.
Materials and methods
Chemicals
For this work, anthocyanin standards cyanidin-3-O-gluco-
side chloride (kuromanin chloride), cyanidin-3-O-rutinoside
chloride (keracyanin chloride), delphinidin-3-O-glucoside
chloride (myrtillin chloride), pelargonidin-3-O-glucoside
chloride (callistephin chloride) and peonidine-3-O-gluco-
side chloride) were purchased from Extrasynthèse (Genay/
France). Methanol (HPLC grade) was obtained from Merck
(Darmstadt/Germany) and ortho-phosphoric acid (85 %,
HPLC grade) was purchased from Fluka (Buchs/Switzer-
land). Hydrochloric acid (36.5 %), potassium chloride,
sodium acetate trihydrate, Folin-Ciocalteau reagent were
obtained from Kemika (Zagreb/Croatia). Trolox ((±)-6-hy-
droxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) and
2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) were pur-
chased from Sigma-Aldrich (St. Louis, MO/USA).
Samples
Fruits [black currant (Ribes nigrum), red raspberry (Rubus
idaeus), blackberry (Rubus fruticosus), sour cherry (Prunus
cerasus), sweet cherry (Prunus avium), strawberry (Fragaria
anannassa), chokeberry (Aronia melanocarpa), and elder-
berry (Sambucus nigra)] were harvested in Slavonia region
(Croatia) at the commercial maturity stage. Immediately af-
ter harvesting, fruits were frozen and stored at –20 º C until
analysis.
Sample preparation
For total anthocyanin, total polyphenols and antioxidant ac-
tivity determination, fruits (~500 g) were thawed at room
temperature for 30 min and then processed in juice extrac-
tor (Juicer, Philips) in order to obtain natural fruit juice.
Three replicates of juice (20 ml each) were centrifuged at
4000 rpm for a 1 h. All juices were analysed the same day
when they were prepared.
Determination of total polyphenols
Total polyphenols were determined by Folin-Ciocalteau mi-
cro method30). An aliquot (20 μl) of appropriately diluted
fruit juice (red raspberry, blackberry, sour cherry, sweet
cherry, strawberry, 1:1 (v/v); black currant 1:2 (v/v); el-
derberry, chokeberry 1:6 (v/v) with H2O) was mixed with
1580 µl of distilled water and 100 μl of Folin-Ciocalteau
reagent. 300 μl of sodium carbonate solution (200 g/l) was
added to the mixture which was then shaken. After incuba-
tion at 40 °C for 30 min in water bath, absorbance of the
mixture was read against the prepared blank at 765 nm.
Total polyphenolics were expressed as mg of gallic acid
equivalents (GAE) per l of fruit juice. Data presented are
mean ± standard deviation (SD).
Determination of total anthocyanins
Total anthocyanins were estimated by a pH-differential
method31). Two dilutions of natural fruit juices were pre-
pared, one with potassium chloride buffer (pH 1.0) (1.86 g
KCl in 1 l of distilled water, pH value adjusted to 1.0 with
concentrated HCl), and the other with sodium acetate buf-
fer (pH 4.5) (54.43 g CH3CO2Na ∙ 3H2O in 1 l of distilled
water, pH value adjusted to 4.5 with concentrated HCl),
diluting each by the previously determined dilution factor
(sweet cherry 1:5 (v/v); strawberry 1:10 (v/v); red raspberry
1:20 (v/v), sour cherry and blackberry 1:30 (v/v); black cur-
rant 1:25 (v/v); elderberry and chokeberry 1:200 (v/v)). Ab-
60 ı Originalarbeiten Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007
sorbance was measured simultaneously at 510 and 700 nm
after 15 min of incubation at room temperature. The con-
tent of total anthocyanins was expressed in mg of cyani-
din-3-O-glucoside equivalents (CGE) per l of fruit juice
using a molar extinction coefficient (ε) of cyanidin-3-O-
glucoside of 26 900 l mol–1 cm–1 and molar weight (MW)
(449.2 g mol–1). Data presented are mean ± standard de-
viation (SD).
Determination of antioxidant activity
Antioxidant activity was determined using the 2,2-diphenyl-
1-picrylhydrazyl (DPPH) method. In the DPPH method9),
five dilutions of each fruit juice with two replicates were
analyzed. Reaction solution was prepared by mixing 50 µl
of diluted fruit juice with 300 μl of methanolic DPPH· solu-
tion (1mM) and brought to 3 ml with methanol. The solu-
tion was kept in dark at room temperature for 15 minutes.
The absorbance (Ajuice) was read against the prepared blank
(50 μl diluted fruit juice, 2950 μl methanol) at 517 nm. A
DPPH· blank solution was prepared (300 μl of 1mM DPPH·
solution, 2.7 ml of methanol) and measured daily. Percent
inhibition of DPPH radical was calculated for each dilution
of juice according to formula:
% inhibition = [(ADPPH–Ajuice)/ADPPH)] × 100
where ADPPH is the absorbance value of the DPPH· blank so-
lution, Ajuice is absorbance value of the sample solution.
Calibration curve of Trolox was constructed by linear re-
gression of the absorbance value versus concentration (0–
2500 μmol l–1). For each dilution of fruit juice, antioxidant
activity was calculated on the basis of the Trolox calibration
curve and expressed in µmol of Trolox equivalent (TE) per
ml of fruit juice. Percent of inhibition of DPPH radical of
each dilution of fruit juice was plotted against antioxidant
activity (μmol TE/ml). Using the curve obtained, antioxidant
activity was calculated for 50 % of DPPH· inhibition. Final
results are expressed as μmol of TE per ml of juice needed to
reduce DPPH radical by 50 %.
HPLC analysis of anthocyanins
For HPLC analysis, fruits (~500 g) (random selection) were
thawed at room temperature for 30 min and then processed
in juice extractor (Juicer, Philips) in order to obtain natural
fruit juice. Three replicates of juice (20 ml each) were cen-
trifuged at 4000 rpm for a 1 hour and diluted 1:3 (v/v) with
0.1 % solution of HCl prepared in methanol. The samples
were filtered through a 0.45 μm syringe filter (VariSep PTFE,
0.45 μm, 25 mm-Varian) before they were injected into
HPLC apparatus. All juices were analysed on HPLC system
immediately, the same day when they were prepared.
The analytical HPLC system employed consisted of a Var-
ian LC system (USA) equipped with a ProStar 230 solvent
delivery module, ProStar 310 UV-Vis Detector, and ProStar
330 PDA Detector. Anthocyanin compounds separation
was done in an OmniSpher C18 column (250 x 4.6 mm
inner diameter, 5 μm, Varian, USA) protected with guard
column (ChromSep 1 cm x 3 mm, Varian, USA). The data
were collected and analysed on IBM computing system
equipped with Star Chromatography Workstation software
(version 5.52).
Elution was employed with a mobile phase A consisting of
0.5 % phosphoric acid in water and mobile phase B consist-
ing of 100 % HPLC grade methanol as follows: 0–38 min
from 3 % B to 65 % B; from 38–45 min, 65 % B; with flow
rate = 1 ml min–1. Operating conditions were as follows:
column temperature 20 ºC; injection volumes, 10 μl of the
standards and samples. A 10-minute re-equilibration period
was used between individual runs. UV-Vis spectra were re-
corded in wavelength range from 190–600 nm (detection
wavelength was 520 nm).
Identification and peak assignment of anthocyanins in all
fruit juices was based on comparison of their retention times
and spectral data (190–600 nm) with those of authentic
standards. Additional identification of anthocyanins pres-
ent in fruit juices was carried out by spiking the juices with
anthocyanin standards. Anthocyanin profiles of fruit juices
were compared with those found in literature8,10–12,14,23–29)
which gave additional information on anthocyanin identi-
fication. Calibration curves of the standards were made by
diluting stock standards in 0.1 % HCl solution prepared in
methanol to yield 1–100 mg l–1 (cyanidin-3-glucoside); 0.5–
200 mg l–1 (cyanidin-3-rutinoside); 5–100 mg l–1 (peonidin-
3-glucoside); 1–100 mg l–1 (delphinidin-3-glucoside) and
1–100 mg l–1 (pelargonidin-3-glucoside). Identified antho-
cyanins were quantified using calibration curve of cyanidin-
3-glucoside. Data presented are mean ± standard deviation
(SD).
Analytical quality control
All anthocyanic compounds showed a linear response within
range studied (r2 = 0.9958 – 0.9997). Precision of method
was evaluated by determining within-day variation of the
HPLC analysis (within-day precision-repeatability). To gain
the data for studying repeatability, each juice sample was
analysed three times within one day. Coefficients of varia-
tion (CV) of peak areas varied between 0.04 and 5.95 %.
Recoveries were measured by adding known amounts
of standards (10–50 mg l–1) to fruit juices prior to HPLC
analysis. The recoveries ranged from 80 to 92 % for cyani-
din-3-glucoside, from 74 to 84 % for cyanidin-3-rutinoside,
from 90 to 95 % for peonidin-3-glucoside, from 89 to 93 %
for delphinidin-3-glucoside and from 95 to 101 % for pel-
argonidin-3-glucoside. In the calculation of final results, no
correction for recovery was applied to data. The following
limits of detection were estimated using a signal-to-noise
ratio of 3:0.64 mg l–1 by cyanidin-3-glucoside, 1.18 mg l–1
by cyanidin-3-rutinoside, 0.41 mg l–1 by peonidin-3-gluco-
side, 0.35 mg l–1 by delphinidin-3-glucoside and 0.92 mg l–1
by pelargondin-3-glucoside.
Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007 Originalarbeiten ı 61
Statistical analysis
Correlation and regression analyses were
performed using Statistica 7.1 (Statsoft,
Tulsa/USA). Differences at p ≤ 0.05 were
considered significant.
Results and discussion
Natural red fruit juices were analyzed us-
ing a pH-differential and Folin-Ciocalteau
method in order to examine their total an-
thocyanin (TA) and total polyphenol (TP)
content. The portion of anthocyanins in
total polyphenol concentration was evalu-
ated by calculating TA/TP ratio. The con-
centrations of total anthocyanins, total
polyphenols and TA/TP ratio are shown
in Table 1. Anthocyanins were found in
the highest concentrations in elderberry,
chokeberry and black currant juices
(4189 mg l–1, 3042 mg l–1, 1544 mg l–1, re-
spectively) whereas the concentrations of
anthocyanins in blackberry, sour cherry,
sweet cherry, red raspberry and strawberry
juice were significantly lower (740 mg l–1,
369 mg l–1, 257 mg l–1, 217 mg l–1, 206 mg
l–1, respectively). Data presented by other
authors also confirmed that the amount of
total anthocyanins were higher in choke-
berry and black currant than in other red
fruits like blackberry, raspberry or red
currant9).
Polyphenols were found in the highest
concentrations in chokeberry, elderberry
and black currant juices (9154 mg l–1,
6362 mg l–1, 2771 mg l–1, respectively).
High concentrations of polyphenols were
found in sour cherry and blackberry juice
as well (2054 mg l–1, 1831 mg l–1, respec-
tively), while sweet cherry, strawberry and
red raspberry juice had relatively lower
concentrations of polyphenols (1567 mg
l–1, 1272 mg l–1, 1234 mg l–1, respectively).
According to data presented by other au-
thors, the concentrations of polyphenols
were higher in chokeberry and black cur-
rant than in red currant, blackberry and
raspberry9).
Anthocyanins were the predominant
polyphenolic components in elderberry
(66 %) and black currant juice (56 %) or
represented significant portion in total
polyphenol concentration of chokeberry
(33 %) or blackberry juice (40 %). The
portion of anthocyanins in red raspberry
Tab. 1 Concentrations of total anthocyanins (TA) (mg CGE/l)a, total polyphenols (TP) (mg GAE/l)a,
antioxidant activity of red fruit juices (μmol TE/ml)b, and TA/TP ratio
Total anthocyanins
[mg/l]
Total polyphenols
[mg/l]
Antioxidant activity
[μmol TE/ml]
TA/TP
Black currant
Red raspberry
Blackberry
Sour cherry
Sweet cherry
Strawberry
Chokeberry
Elderberry
1543.89 ± 5.5
217.39 ± 5.2
739.93 ± 37.5
369.36 ± 2.4
256.60 ± 2.5
205.98 ± 2.2
3042.20 ± 196.3
4188.63 ± 257.0
2770.94 ± 63.9
1234.27 ± 54.8
1831.21 ± 111.6
2054.43 ± 140.2
1566.84 ± 130.2
1271.85 ± 106.9
9154.47 ± 595.4
6361.89 ± 298.9
30.15
8.20
8.75
12.52
4.07
4.39
72.44
62.14
0.56
0.18
0.40
0.18
0.16
0.16
0.33
0.66
a values are means ± SD (n=3); b antioxidant activity determined after 15 min
0.02
0.04
0.06
0.08
0.10
0.00
6
2
9
10
Black currant
AU
AU
AU
AU
AU
Sour cherry
3
4
9
10
18 20 22 24 26 28 30
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
7
9
11
Chokeberry
14
AU
Tim e / min
0.02
0.04
0.06
0.08
0.10
0.00
3
9
8
Red raspberry
0.04
0.02
0.06
0.08
0.00
9
10
13
16
Sweet cherry
0.00
0.01
0.02
0.03
0.04
0.05
0.06
9
12
15
Strawberry
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1
5+9
10
Elderberry
0.00
0.05
0.10
0.15
0.20
0.25 9
10
Blackberry
AU
14
AU
AB
CD
E F
GH
Tim e / min
Tim e / min
Tim e / min
Tim e / min
Tim e / min
Tim e / min
Tim e / min
0.00
0.05
0.10
0.15
0.20
0.25
18 20 22 24 26 28 30
18 20 22 24 26 28 30
18 20 22 24 26 28 30
18 20 22 24 26 28 30
18 20 22 24 26 28 30
18 20 22 24 26 28 30
18 20 22 24 26 28 30
Fig. 1 HPLC chromatograms of the anthocyanin compounds of red fruit juices. Peak identification:
(1) cyanidin-3-sambubioside-5-glucoside; (2) delphinidin-3-glucoside; (3) cyanidin-3-sophoroside;
(4) cyanidin-3-glucosyl-rutinoside, (5) cyanidin-3-sambubioside; (6) delphinidin-3-rutinoside;
(7) cyanidin-3-galactoside; (8) pelargonidin-3-sophoroside; (9) cyanidin-3-glucoside; (10) cyanidin-
3-rutinoside; (11) cyanidin-3-arabinoside, (12) pelargonidin-3-glucoside; (13) peonidin-3-glucoside;
(14) cyanidin-3-xyloside; (15) pelargonidin-3-rutinoside; (16) peonidin-3-rutinoside
62 ı Originalarbeiten Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007
(18 %), sour cherry (18 %), sweet cherry (16 %) and straw-
berry juice (16 %) was considerably lower (Tab. 1).
In order to separate and determine individual anthocyanic
compounds present in red fruit juices, HPLC method was
applied. The HPLC chromatograms of red fruit juices re-
corded at 520 nm are presented in Figure 1. The amounts
of anthocyanins in red fruit juices are shown in Table 2.
Chokeberry, elderberry, blackberry and sour cherry juices
contained only cyanidin based pigments. Chokeberry juice
contained a mixture of four different cyanidin-glycosides:
3-galactoside, 3-arabinoside, 3-glucoside and 3-xyloside
of cyanidin. Cyanidin-3-galactoside and cyanidin-3-ara-
binoside were found in high concentrations (1817 mg l–1,
647 mg l–1, respectively, >93 % of total anthocyanin con-
tent) whereas the concentrations of cyanidin-3-xyloside
(100 mg l–1) and cyanidin-3-glucoside (74 mg l–1) were rela-
tively low. The data presented by other authors are showing
that the main anthocyanins in chokeberry are cyanidin-3-
galactoside and cyanidin-3-arabinoside; the following are
cyanidin-3-xyloside and cyanidin-3-glucoside8,12,28), which is
consistent with our results.
The major anthocyanins in elderberry juice were cyanidin
based [cyanidin-3-sambubioside and cyanidin-3-glucoside
eluted together (5227 mg l–1), cyanidin-3-sambubioside-5-
glucoside (949 mg l–1) and cyanidin-3-rutinoside (112 mg
l–1)], with cyanidin-3-sambubioside and cyanidin-3-glucoside
predominating (83 % of total anthocyanin amount). Data
presented by other authors also confirmed that the major
anthocyanins in elderberry are cyanidin-based, with cyani-
din-3-sambubioside and cyanidin-3-glucoside predominat-
ing8,12,28,29) like in our study.
Cyanidin-3-glucoside was the major anthocyanin in black-
berry juice (743 mg l–1, 90 % of total anthocyanin content)
whereas the concentrations of cyanidin-3-xyloside (68 mg
l–1) and cyanidin-3-rutinoside (11 mg l–1) were considerably
lower. Previous study confirmed that the main anthocyanin
in blackberry is cyanidin-3-glucoside (from 44 to 95 % of
total peak area in various blackberry samples)23). Cyanidin-
3-rutinoside ranged from trace amount to 53 %, whereas
cyanidin-3-xyloside, cyanidin-3-malonylglucoside and cy-
anidin-3-dioxalylglucoside were detected but at much lower
concentrations23).
Sour cherry juice contained -3-glucosylrutinoside (183 mg
l–1, 61.4 %), -3-rutinoside (93 mg l–1, 31.2 %), -3-glucoside
(11 mg l–1, 3.5 %) and -3-sophoroside (12 mg l–1, 3.9 %) of
cyanidin. These data are in accordance with those found in
literature24,25,29).
Black currant juice was characterized by the presence of
rutinosides and glucosides of delphinidin and cyanidin
with the rutinosides being the most abundant (delphinidin-
3-rutinoside 706 mg l–1, cyanidin-3-rutinoside 580 mg l–1;
79.9 % of total anthocyanin content). The data reported
in the literature are showing that the main anthocyanins in
black currant are rutinosides and glucosides of delphinidin
and cyanidin8,12,14,28,29) which agrees with our results.
Pelargonidin derivatives predominated in strawberry juice
(pelargonidin-3-glucoside, 159 mg l–1, pelargonidin-3-rutin-
oside, 36 mg l–1). These anthocyanins represented together
Tab. 2 Concentrations of anthocyanins in natural red fruit juices (mg/l)a de-
termined by HPLC method and percentage distribution of anthocyanins
Juice Concentration as Cy-3-
glu equivalents [mg/l]
Total anthocyanins
[%]
Chokeberry
cy-3-gal
cy-3-glu
cy-3-ara
cy-3-xyl
1816.6 ± 4.9
74.3 ± 0.1
647.1 ± 1.0
99.8 ± 0.1
68.9
2.8
24.5
3.8
Total 2637.8 ± 6.1 100.0
Elderberry
cy-3-sam-5-glu
cy-3-sam + cy-3-glu
cy-3-rut
949.1 ± 9.9
5226.6 ± 18.9
111.7 ± 5.9
15.1
83.1
1.8
Total 6287.4 ± 34.7 100.0
Blackberry
cy-3-glu
cy-3-rut
cy-3-xyl
743.3 ± 2.7
10.8 ± 0.0
68.1 ± 1.2
90.4
1.3
8.3
Total 822.2 ± 3.9 100.0
Sour Cherry
cy-3-sopho
cy-3-glu-rut
cy-3-glu
cy-3-rut
11.7 ± 0.8
183.3 ± 0.7
10.6 ± 0.3
93.0 ± 1.6
3.9
61.4
3.5
31.2
Total 298.6 ± 3.4 100.0
Black Currant
dp-3-glu
dp-3-rut
cy-3-glu
cy-3-rut
222.3 ± 10.9
706.4 ± 38.9
99.9 ± 1.7
579.5 ± 12.3
13.8
43.9
6.2
36.0
Total 1608.1 ± 63.8 99.9
Strawberry
cy-3-glu
pg-3-glu
pg-3-rut
7.3 ± 0.7
159.2 ± 2.0
35.9 ± 0.0
3.6
78.7
17.7
Total 202.4 ± 2.7 100.0
Red Raspberry
cy-3-sopho
pg-3-sopho
cy-3-glu
239.4 ± 2.8
18.0 ± 0.5
42.5 ± 0.7
79.8
6.0
14.2
Total 299.9 ± 4.0 100.0
Sweet Cherry
cy-3-glu
cy-3-rut
pn-3-glu
pn-3-rut
71.6 ± 1.5
252.9 ± 8.0
16.0 ± 1.1
14.7 ± 0.2
20.2
71.2
4.5
4.1
Total 355.2 ± 10.8 100.0
Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007 Originalarbeiten ı 63
> 96 % of total anthocyanin amount. The concentration
of cyanidin-3-glucoside was low (7 mg l–1, 4 %). Previous
studies confirmed that strawberry is characterized by the
same pelargonidin and cyanidin glycosides as in our study
and that pelargonidin glycosides predominate in straw-
berry11,26).
The main anthocyanin found in red raspberry juice, which
represented 79.8 % of total anthocyanin content, was cyani-
din-3-sophoroside (239 mg l–1). Cyanidin-3-glucoside and
pelargonidin-3-sophoroside were found in relatively lower
amount (43 mg l–1, 18 mg l–1, respectively). Previous study
confirmed that anthocyanins present in red raspberry (early
cultivar Heritage) are cyanidin-3-sophoroside, cyanidin-3-
glucoside and pelargonidin-3-sophoroside which is in ac-
cordance with our results27). However, anthocyanin profiles
of various raspberry cultivars are very heterogenous; the
main red raspberry anthocyanins are cyanidin and pelargo-
nidin derivatives 11,27,29) but in some late cultivars malvidin
and delphinidin derivatives were found as well27).
The dominant anthocyanin in sweet cherry juice was cyani-
din-3-rutinoside (253 mg l–1), with cyanidin-3-glucoside be-
ing second (72 mg l–1). These two anthocyanins represented
together 91.4 % of total anthocyanin amount, while the
concentrations of peonidin-3-rutinoside and peonidin-
3-glucoside were low (15 mg l–1, 16 mg l–1, respectively).
The data reported in the literature are showing that sweet
cherry contains cyanidin, peonidin and pelargonidin deriva-
tives10,11). The major anthocyanins are cyanidin-3-rutinoside
and cyanidin-3-glucoside like in our study, while 3-rutino-
side and 3-glucoside of pelargonidin and peonidin are pres-
ent but at much lower concentrations10,11).
In order to evaluate antioxidant activity of chosen red fruit
juices, DPPH· assay was applied and the results are pre-
sented in Table 1. All investigated juices exhibited potent
radical scavenging activity. The strongest radical scavenging
activity showed chokeberry, elderberry, and black currant
juice (72 µmol TE/ml, 62 μmol TE/ml μmol, 30 μmol TE/ml
respectively) while activities of other juices were consider-
ably lower (sour cherry 13 μmol TE/ml; blackberry 9 μmol
TE/ml; red raspberry 8 μmol TE/ml; strawberry 4 μmol TE/
ml; sweet cherry 4 μmol TE/ml). There are already a num-
ber of reports on the antioxidant activity of berry extracts
by several methods such as oxygen radical absorbance capa-
city12) or DPPH radical scavenging capacity8,9) indicating
that chokeberry, elderberry and black currant possess strong
antiradical activities. Antioxidant activity of chokeberry
juice concentrate against DPPH radical was stronger than
antioxidant activity of black currant, elderberry, red cur-
rant, strawberry, red raspberry and cherry concentrate28).
The correlation between antioxidant activity measured by
DPPH methods and total polyphenols and total anthocya-
nins are presented in Figure 2. The concentration of total
polyphenols were found to correlate with the antioxidant
activities of juices (r = 0.97***). The concentration of to-
tal anthocyanins correlate with antioxidant activity as well
(r = 0.95***) but the correlation coefficient was lower for
total anthocyanins versus antioxidant activity than for the
total polyphenols versus antioxidant activity. According to
the data presented by others, the best linear relationship
was observed between antioxidant capacity and total poly-
phenols of various red fruits (r2 = 0.96) than between anti-
oxidant capacity and total anthocyanins (r2 = 0.95)12) which
agrees with our results.
Red fruit juices evaluated in this study are showing signifi-
cant variations in anthocyanin content and profile. Some
juices have low amount of anthocyanins, like strawberry
and red raspberry juice, whereas the amount of these phy-
tochemicals is very high in chokeberry, elderberry and black
currant juice. Anthocyanins are the predominant phenolic
components in elderberry and black currant juice or re-
present significant part in total polyphenol concentration
of chokeberry and blackberry juice. Every juice evaluated
Fig. 2 Correlation plots of DPPH values versus total polyphenol and total
anthocyanin contents of red fruit juices. The correlation coefficients (r)
are marked in each plot; significant correlations are marked with asterisks
(*** = significance at P ≤ 0.001)
64 ı Originalarbeiten Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007
possess unique anthocyanin pattern. Chokeberry, elder-
berry, blackberry and sour cherry juice contain only cyani-
din based pigments, black currant juice is characterized by
delphinidin and cyanidin, and strawberry juice by pelargo-
nidin derivatives. The major anthocyanins in red raspberry
and sweet cherry juice are derivatives of cyanidin although
peonidin (in sweet cherry juice) and pelargonidin deriva-
tives (in red raspberry juice) are present in low amount.
Although all juices can serve as a good source of bioactive
phytochemicals in the human diet, chokeberry, elderberry
and black currant juice stand out in high anthocyanin con-
tent and high antioxidant activity. From the view of the
anthocyanin content and antioxidant activity these juices
can be regarded as good candidates for raw materials in
production of health beneficial functional foods. Moreover,
delphinidin-3-glucoside, delphinidin-3-rutinoside and cy-
anidin-3-glucoside were reported to have strong antiradical
activity among various anthocyanins32). Therefore, black
currant (which is abundant in delphinidin derivatives) and
blackberry (abundant in cyanidin-3-glucoside) juice can
serve as good source of these individual anthocyanins.
Acknowledgements
This work was supported by the Croatian Ministry of Sci-
ence, Education, and Sports (Project number 0113006).
References
1) Kris-Etherton, P. M., K. D. Hecker, A. Bonanome, S. M. Coval, A. E. Binko-
ski, K. F. Hilpert, A. E. Griel, and T. D. Etherton: Bioactive compounds in
foods: their role in the prevention of cardiovascular disease and cancer.
Am. J. Med. 113, 71–88 (2002).
2) Joshipura, K. J., F. B. Hu, J. E. Manson, M. J. Stampfer, E. B. Rimm, F.
E. Speizer, G. Colditz, A. Ascheiro, B. Rosner, D. Spiegelman, and W. C.
Willett: The effect of fruit and vegetable intake on risk for coronary heart
disease. Ann. Intern. Med. 134, 1106–1114 (2001).
3) Knekt, P., J. Kumpulainen, R. Järvinen., H. Rissanen, M. Heliövaara, A.
Reunanen, and T. Hakulinen: Flavonoid intake and risk of chronic di-
seases. Am. J. Clin. Nut. 76, 560–568 (2002).
4) Le Marchand, L., S. P. Murphy, J. H. Hankin, L. R. Wilkens, and L. N.
Kolonel: Intake of flavonoids and lung cancer. J. Natl. Cancer Inst. 92,
154–160 (2000).
5) Garcia-Closas, R., C. A. Gonzalez, A. Agudo, and E. Riboli: Intake of spe-
cific carotenoids and flavonoids and the risk of gastric cancer in Spain.
Cancer Causes Control 10, 71–75 (1999).
6) Shi, H., N. Noguchi, and E. Niki: Introducing natural antioxidants. In Po-
korny, J., N. Yanishlieva, and M. Gordon (eds.): Antioxidants in food, pp.
147–158, Woodhead Publishing Limited, Cambridge (2001).
7) Clifford, M. N.: Anthocyanins-nature occurence and dietary burden. J.
Sci. Food Agr. 80, 1063–1072 (2000).
8) Nakajima, J., I. Tanaka, S. Seo, M. Yamazaki, and K. Saito: LC/PDA/ESI-
MS profiling and radical scavenging activity of anthocyanins in various
berries. J. Biomed. Biotechnol. 5, 241–247 (2004).
9) Benvenuti, S., F. Pellati, M. Melegari, and D. Bertelli: Polyphenols, antho-
cyanins, ascorbic acid, and radical scavenging activity of Rubus, Ribes,
and Aronia. J. Food Sci. 69, 164–169 (2004).
10) Mozeti
č
, B., and P. Trebše: Identification of sweet cherry anthocyanins
and hydroxycinnamic acids using HPLC coupled with DAD and MS
detector. Acta Chim. Slov. 51, 151–158 (2004).
11) Wu, X., and R. L. Prior: Systematic identification and characterization
of anthocyanins by HPLC-ESI-MS/MS in common foods in the United
States: fruits and berries. J. Agr. Food Chem. 53, 2589–2599 (2005).
12) Wu, X., L. Gu, R. L. Prior, and S. McKay: Characterization of anthocyanins
and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus
and their antioxidant capacity. J. Agr. Food Chem. 52, 7846–7856 (2004).
13) Kim, D. O., H. J. Heo, Y. J. Kim, H. S. Yang, and C. Y. Lee: Sweet and sour
cherry phenolics and their protective effects on neuronal cells. J. Agr.
Food Chem. 53, 9921–9927 (2005).
14) Kähkönen, M. P., J. Heinämäki, V. Ollilainen, and M. Heinonen: Berry an-
thocyanins: isolation, identification and antioxidant activities. J. Sci. Food
Agr. 83, 1403–1411 (2003).
15) Netzel, M., G. Strass, M. Herbst, H. Dietrich, R. Bitsch, and T. Frank: The
excretion and biological antioxidant activity of elderberry antioxidants in
healthy humans. Food Res. Int. 38, 905–910 (2005).
16) Mülleder, U., M. Murkovic, and W. Pfannhauser: Urinary excretion of cya-
nidin glycosides. J. Biochem. Biophys. Methods 53, 61–66 (2002).
17) Miyazawa, T., K. Nakagawa, M. Kudo, K. Muraishi, and K. Someya: Direct
intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and
cyanidin-3,5-diglucoside, into rats and humans. J. Agr. Food Chem. 47,
1093–1091 (1999).
18) Milbury, P. E., G. Cao, R. L. Prior, and J. Blumberg: Bioavailability of el-
derberry anthocyanins. Mech. Ageing Dev. 123, 997–1006, (2002).
19) Chen,P. N., S. C. Chu, H. L. Chiou, W. H. Kuo, C. L. Chiang, and Y. S.
Hsieh: Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glu-
coside, exhibited an inhibitory effect on the migration and invasion of a
human lung cancer cell line. Cancer Lett. 235, 248–259 (2006).
20) Netzel, M., G. Strass, C. Kaul. I. Bitsch, H. Dietrich, and R. Bitsch: In
vivo antioxidative capacity of a composite berry juice. Food Res. Int. 35,
213–216 (2002).
21) Bub, A., B. Watzl, M. Blockhaus, K. Briviba, U. Liegibel, H. Müller, B. L.
Pool-Zobel, and G. Rechkemmer: Fruit juice consumption modulates an-
tioxidant status, immune status and DNA damage. J. Nutr. Biochem. 14,
90–98. (2003).
22) Heinonen, I. M., and A. S. Meyer: Antioxidants in fruits, berries and ve-
getables. In Jongen, W. (ed.): Fruit and vegetable processing, pp.23–51,
Woodhead Publishing Limited, Cambridge (2002).
23) Fan-Chiang, H. J., and R. E. Wrolstad: Anthocyanin pigment composition
of blackberries. J. Food Sci. 70, C198–C202 (2005).
24) Blando, F., C. Gerardi, and I. Nicoletti: Sour cherry (Prunus cerasus L.)
anthocyanins as ingredients for functional foods. J. Biomed. Biotechnol.
5, 253–258 (2004).
25) Blando, F., A. P. Scardino, L. De Bellis, I. Nicoletti, and G. Giovinazzo:
Characterization of in vitro anthocyanin-producing sour chery (Prunus
cerasus L.) callus cultures. Food Res. Int. 38, 937–942 (2005).
26) Gill, M. I., D. M. Holcroft, and A. A. Kader: Changes in strawberry antho-
cyanins and other polyphenols in response to carbon dioxide treatments.
J. Agr. Food Chem. 45, 1662–1667 (1997).
27) De Ancos, B., E. Gonzalez, and M. P. Cano: Differentiation of raspberry
varieties according to anthocyanin composition. Z. Lebensm. Unters.
Forsch. A 208, 33–38 (1999).
28) Bermúdez-Soto, M., and F. A. Tomás-Barberán: Evaluation of commercial
red fruit juice concentrates as ingredients for antioxidant functional jui-
ces. Eur. Food Res. Technol. 219, 133–141 (2004).
29) Goiffon, J., P. P. Mouly, and E. M. Gaydou: Anthocyanic pigment deter-
mination in red fruit juices, concentrated juices and syrups using liquid
chromatography. Anal. Chim. Acta 382, 39–50 (1999).
30) Waterhouse, A.: (May, 10th 2006). http://waterhouse.ucdavis.edu/phenol/
folinmicro.htm
31) Giusti, M. M., and R. E. Wrolstad: Anthocyanins. Characterization and
measurement with UV-visible spectroscopy. In Wrolstad, R. E. (ed.): Cur-
rent protocols in food analytical chemistry, pp. F1.2.1.–F1.2.13. Wiley,
New York (2001).
32) Kähkönen, M. P., and M. Heinonen: Antioxidant activity of anthocyanins
and their aglycons. J. Agr. Food Chem. 51, 628–633 (2003).
VI ı Impressum Deutsche Lebensmittel-Rundschau ı 103. Jahrgang, Heft 2, 2007
Impressum
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