Food Research International 51 (2013) 303–309

Contents lists available at SciVerse ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Red cabbage anthocyanins: Profile, isolation, identification, and antioxidant activity

Wieslaw Wiczkowski ⁎, Dorota Szawara-Nowak, Joanna Topolska
Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn, Tuwima 10, 10-748 Olsztyn, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Red cabbage is rich in a number of bioactive substances, including anthocyanins. The anthocyanin profile of red
Received 31 July 2012 cabbage consisting of twenty derivatives of cyanidin glucosides was described by means of HPLC-DAD-MS/MS.
Accepted 15 December 2012 The base structure of anthocyanins identified was cyanidin-3-diglucoside-5-glucoside. Their glucoside residues
were nonacylated, monoacylated and diacylated. Sinapic, ferulic, caffeic and p-coumaric acids were recognized
Keywords:
as main phenolic acids in this structure. The predominant anthocyanin in red cabbage was nonacylated form
Red cabbage
Acylated anthocyanins
of cyanidin-3-diglucoside-5-glucoside, followed by cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside
HPLC-MS/MS and cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside. Anthocyanins were isolated and purified by Amberlite
Isolation XAD-16 column chromatography and by HPLC on C18 semi-preparative column. Among the seven isolated
Antioxidant activity and purified red cabbage anthocyanins, cyanidin-3-diglucoside-5-glucoside diacylated with sinapic acid showed
the highest radical-scavenging activities toward both 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical
cation and superoxide anion radical. The relative contribution of anthocyanins to the antioxidant capacity of red
cabbage extract was low. The results obtained indicated that red cabbage has its own characteristic anthocyanin
pattern as well as a kind of acylation that affects the antioxidant activity of acylated anthocyanins.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Red cabbage is of high nutritional value as it is rich in minerals, vi-
tamins, oligosaccharides, and a number of bioactive substances, such
as anthocyanins, flavonols, and glucosinolates (Jagdish Singh et al.,
2006; Podsedek, 2007; Volden et al., 2008), having a positive impact
on human health. Apart from a number of nutritional benefits, red
cabbage is also valued by consumers for its taste and for being a source
of an intensive red color which increases esthetic value of the food. For
these reasons, red cabbage is a widely and frequently consumed vegeta-
ble in the form of fresh-cut salads. In addition, red cabbage is character-
ized by high shelf-life, therefore it can be easily stored and available in a
fresh form all year round.
Anthocyanins are red, orange, blue or purple water soluble pigments
occurring in fruit and vegetables. As components of plant food products
anthocyanins are consumed by humans in the amounts which may be
of a significant from the physiological point of view (Clifford, 2000;
Wu & Prior, 2005; Wu et al., 2006). This observation and a fact that
anthocyanins have pro-health functions (anticancer, cardioprotective,
vision improvement, antidiabetic, antineurodegenerative) together
with being non-toxic even after consumption at high doses, contribute
to an increase in the interest in these polyphenols. What more, a
consumer's rejection of artificial pigments stimulates the search for nat-
ural food colorants, and that may be anthocyanins (Azeredo, 2009;
Galvano et al., 2004; Nielsen et al., 2005; Nizamutdinova et al., 2009;
⁎ Corresponding author. Tel.: +48 89 5234604; fax: +48 89 5240124.
E-mail address: w.wiczkowski@pan.olsztyn.pl (W. Wiczkowski).
Rahman, Ichiyanagi, Komiyama, Sato, & Konishi, 2008; Wang & Stoner,
2008; Yanamala, Tirupula, Balem, & Klein-Seetharaman, 2009).
Anthocyanins are substances characterized by complex patterns of
hydroxylation, methoxylation, glycosylation, and acylation (Clifford,
2000; Wu & Prior, 2005). These factors are linked to plant species to
form a characteristic pattern of anthocyanins. Red cabbage has also
its own characteristic anthocyanin pattern. It contains a significant
amount of anthocyanins, main structure of which, in most cases, is
cyanidin glycosides (Wu & Prior, 2005). Anthocyanins with other core
units such as pelargonidin glucoside and peonidin glucosides were also
identified (Charron et al., 2009; McDougall, Fyffe, Dobson, & Stewart,
2007). Furthermore, the dominant derivatives of anthocyanins in red
cabbage occur in acylated forms (Clifford, 2000; Wu & Prior, 2005). Un-
fortunately, there is little information available regarding the relation-
ship between chemical structure of individual acylated anthocyanins of
red cabbage and the antioxidant activity of these natural red colorants.
In the study of Choi, Chang, Ha, and Choi (1997) only one anthocyanin
isolated from red cabbage was tested. In other report (Stintzing,
Stintzing, Carle, Frei, & Wrolstad, 2002) two derivatives of cyanidin
present in red cabbage (cyanidin-3-digucoside-5-glucoside and its
acylated with sinapic acid forms) were used in the Oxygen Radical
Absorbing Capacity (ORAC) assay. In addition, two anthocyanins iso-
lated from red cabbage (monoacylated and diacylated with sinapic
acid derivatives of cyanidin-3-digucoside-5-glucoside) were ana-
lyzed by Degenhardt, Knapp, and Winterhalter (2000). Taking into
account the fact that red cabbage can contain 9–24 different anthocya-
nins (Arapitsas, Sjoberg, & Turner, 2008; Pliszka, Huszcza-Ciolkowska,
Mieleszko, & Czaplicki, 2009), there is a lack of studies comparing the

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2012.12.015
304 W. Wiczkowski et al. / Food Research International 51 (2013) 303–309
antioxidant activity of a wide range of red cabbage anthocyanins in the
same experiment.
In this study, description of the profile of red cabbage anthocyanin
by means of HPLC-DAD-MS/MS as well as isolation and purification of
main anthocyanins from this vegetable were performed. In addition,
the influence of acylation pattern on the antioxidant activity of seven
individual red cabbage anthocyanins measures against both 2,2′-
azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•+)
and superoxide anion radical O2−• was investigated.
2. Material and methods
2.1. Reagents
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox) were purchased from Sigma Chemical Co. (Sigma Chemical
Co., St. Louis, MO). ACW (hydrophilic condition) and ACL (lipophilic
condition) kits (model no. 400.801) for the photochemiluminescence
(PCL) assay were from Analytik Jena AG (Jena, Germany). Cyanidin
aglycone was obtained from Extrasynthese (Genay, France). All other
reagents including acetonitrile, methanol, trifluoroacetic and formic
acid, ethyl acetate, and sodium sulfate were purchased from Merck
KGaA (Dramstad, Germany). Water was purified with a Mili-Q system
(Millipore, Bedford, MA).
2.2. Plant material
Red cabbage (Brassica oleracea L. var. capitata L. f. rubra) plants of
the cultivar Langedijker were grown in the experimental fields of the
Plantico Zielonki Sp. z o.o. (Zielonki, Poland). The plants harvested
were planted in 2008. The bulbs obtained (7 bulbs) were purified from
the dried outer leaves and subsequently chopped into eight equal
parts. Two opposite pieces from each bulb (a total of 14 pieces) were
frozen together with liquid nitrogen. After lyophilization, the samples
obtained were pulverized and stored at −80 °C until chromatographic
analysis.
2.3. Extraction
Optimal performance of anthocyanins extraction from red cab-
bage was obtained using a mixture consisting of methanol/water/
trifluoroacetic acid (0.58/0.38/0.04, v/v/v). About 0.05 g of dried
and pulverized red cabbage tissues was extracted by 30 s sonication
(VC 750, Sonics & Materials, USA) with 1 mL of the above mentioned
mixture. Subsequently, the mixture was vortexed for 30 s, again son-
icated and vortexed, and centrifuged (Centrifuge 5415R, Eppendorf,
Niemcy) for 10 min (13,200× g at 4 °C). Supernatant was collected
in 5 mL flask. This step was repeated 5 times. Finally, before the anal-
ysis, the extract was centrifuged (20 min, 13,000× g).
2.4. Chromatographic analysis
Extracts and solution of isolated red cabbage standards were ana-
lyzed by HPLC-DAD method. Aliquots (5 μL) of sample solutions were
injected into a HPLC system (Shimadzu, Kyoto, Japan) equipped with
a 150 × 2.1 mm i.d. XBridge C18 3.5 μm column (Waters, USA). The
HPLC system consisted of two pumps (LC-10 ADVP), DAD detector
(SPD-M10 AVP) set at 520 nm, autosampler (SIL-10 ADVP), column
oven (CTO-10 ASVP) and system controller (SCL-10 AVP). All chromato-
graphic determinations were performed at 45 °C with the flow rate of
0.2 ml/min. The elution was conducted using a solvent gradient system
consisting of solvent A (6% formic acid aqueous solution) and solvent B
(6% formic acid acetonitrile solution). Gradient was as follows: 3–17% B
(0–77 min), 17–80% B (77–80 min), 80–3% B (80–84 min), and 3% B
(84–105 min). Anthocyanins were identified basing on the comparison
of their retention time, UV-visible spectrum and MS/MS fragmentation
spectrum (m/z values) with the previously published data (Charron et al.,
2009; McDougall et al., 2007; Wu & Prior, 2005). Anthocyanin quantity
was calculated from HPLC-DAD peak area at 520 nm against cyanidin as
the external standard. The calibration curve (the range of 0.3–40 μM)
was linear with a correlation coefficient of 0.998.
Identification of anthocyanins was carried out using a mass spec-
trometer QTRAP 5500 (AB SCIEX, USA) equipped with a triple quadru-
pole, ion trap, and ion source of electrospray ionization. Qualitative
analysis will be performed, among other, basing on scanning in positive
ion mode in the quadrupoles and in the ion trap. Scanning of fragmen-
tation ions derived from the selected parent ion for observation of all
the ions formed by the disintegration of the parent ion as well as scan-
ning precursor ion and neutral particles will also be conducted. Optimal
identification of anthocyanins was achieved under the following condi-
tions: curtain gas — 20 L/min, collision gaz — 9 L/min, ionspray
voltage — 5300 V, temperature — 550 °C, 1 ion source gas — 55 L/min,
2 ion source gas — 70 L/min, declustering potential — 50–120 V, en-
trance potential — 10 V, collision energy — 30–70 eV, and collision
cell exit potential — 10–45 V.
2.5. Isolation
The study of anthocyanins isolation from red cabbage was carried
out according to the following procedure: extraction of anthocyanins
with 80% methanol containing sodium sulfate (1 g Na2SO3 ∗ 7H2O/L),
purification and initial separation of anthocyanins on ion-exchange
gel, and isolation of anthocyanins on C18 semipreparative column. An-
thocyanins were extracted from 20 g of lyophilized red cabbage tissues.
The process was carried out three times by homogenization on
Ultra-Turrax T25 homogeniser (Janke & Kunkel, Germany) at room
temperature with 150 mL of above mixture. After each extraction, the
mixture was centrifuged (5000 ×g, 20 min, MPW-360, Poland) and
the supernatant was collected in a 500 mL volumetric flask. The extract
obtained (about 450 mL) was concentrated with a rotary evaporator
(Rotavapor R-200, Büchi, Switzerland) at 37 °C to around 90 mL. Subse-
quently, the sample obtained was frozen at −80 °C and lyophilized.
After lyophilization the sample was dissolved in water containing sodi-
um sulfate (1 g Na2SO3 ∗ 7H2O/L). Purification of anthocyanins was
performed with column chromatography method on Amberlite
XAD-16 ion exchange gel (Rohm & Hass, UK). The extract (about
5 mL) was loaded in column stabilized with 1000 mL 0.02 M HCL and
2000 mL H2O + 1 g Na2SO3 ∗ 7H2O/L. After administering the sample,
the column was washed with 2000 mL of 5% methanol + 1 g
Na2SO3 ∗ 7H2O/L, 1000 mL of ethyl acetate, and then fractions were elut-
ed with 5% formic acid in methanol. Next, chromatography column was
regenerated by washing with 1000 mL of 100% methanol and
2000 mL of H2O + 1 g Na2SO3 ∗ 7H2O/L. The procedure of purification
was repeated triplicate. The fractions collected were analyzed by
HPLC method presented in Section 2.4.
The fractions from subsequent purification were connected, evapo-
rated to dry and finally dissolved in 10 mL of 5% methanol connecting
0.4% of TFA. Next, anthocyanins were isolated and purified by HPLC on
semipreparative column C18 250 × 10 mm, 5 μm (Luna, Phenomenex,
USA) at 45 °C with the flow rate of 0.9 mL/min. The chromatography
system (Shimadzu, Japan) was equipped with two pumps (LC-10 AD),
UV detector (SPD-10AV) set at 520 nm, 1 mL injection loop and system
controller (SIL-6B). A gradient elution was carried out with solvent A
(water with 1% of formic) and solvent B (methanol with 1% of
formic acid). The gradient was as follows: 8–13% B (0–70 min),
13% B (70–75 min), 13–20% B (75–161 min), 20–80% B (161–163 min),
80% B (163–166 min), 80–8% B (166–168 min), and 8% B (168–
180 min). The fractions containing purified compounds were collected
and evaporated to dryness. As a result of the applied method of isolation
and purification fifteen anthocyanins were collected.
 W. Wiczkowski et al. / Food Research International 51 (2013) 303–309
2.6. Preparation of solutions of isolated anthocyanins
Appropriate amounts of the isolated and purified standards were
dissolved in acidified 80% methanol and their concentration was con-
firmed by spectroscopic method (Giusti & Wrolstad, 2001). Next, for
the measurements of the antioxidants activity (TEAC and PCL assays),
and HPLC analysis, exactly 100 μM concentration of each compound
was prepared.
2.7. Antioxidant capacity
2.7.1. Trolox equivalent antioxidant capacity (TEAC) assay
The method presented by Re et al. (1999) with a minor modification
was used to determine the antioxidant activity of isolated and purified
red cabbage anthocyanins and extract of red cabbage. The analysis was
conducted using a spectrophotometer UV-160 1PC (Shimadzu, Japan).
Briefly, the ABTS•+ solution was diluted with 80% methanol to an absor-
bance of 0.70±0.02 at 734 nm. Next, 1.48 mL of the ABTS•+ ·solution
and 0.02 mL of the isolated red cabbage standard solution or red cabbage
extract or Trolox solution were mixed, and then the absorbance was mea-
sured immediately after 6 min at 734 nm at 30 °C. Appropriate solvent
blanks were analyzed in each assay. The antioxidant assay was carried
out in triplicate for each sample. The TEAC of 80% methanol solution of
isolated anthocyanins and red cabbage extract was calculated, using
Trolox standard curve, on the basis of percent inhibition of the absor-
bance of the ABTS•+ solution at 734 nm. The 80% methanol solution of
Trolox within the concentration range of 0.1–2.5 mM was used for build-
ing the calibration curve.
2.7.2. Determination of ACW and ACL by the photochemiluminescence
(PCL) method
The PCL method was used to measure the antioxidant activity of
isolated and purified red cabbage anthocyanins and extract of red cab-
bage with a Photochem apparatus (Analytik Jena, Leipzig, Germany)
against superoxide anion radicals generated from luminol (a photosensi-
tizer) when exposed to UV light. The antioxidant activity of anthocyanins
and red cabbage extract was analyzed using both ACW (hydrophilic con-
dition) and ACL (lipophilic condition) kits and the protocol of measure-
ment provided by the manufacturer. The assay was carried out as
previously described by Zielinska, Wiczkowski, and Piskula (2008). The
80% methanol solution of standards and extract was centrifuged by
10 min at 16,000×g, and at 4 °C prior to the analysis. The antioxidant
assay was carried out in triplicate for each sample. The antioxidant capac-
ity was calculated by means of the comparison with a Trolox standard
curve (0.25–3.00 nM).
2.7.3. Determination of the relative contribution of isolated anthocyanins
to the antioxidant capacity of red cabbage
The contribution of isolated anthocyanins based on antioxidant
activities of each compound provided by TEAC, PCL ACL and PLC ACW
methods in comparison to the antioxidant capacity of red cabbage ex-
tract measured by the same methods was determined. Therefore, the
mean content of isolated anthocyanins in red cabbage was multiplied
by their antioxidant activities, and then each individual result was di-
vided by the antioxidant capacity of red cabbage extract and expressed
as % of contribution.
2.8. Statistical analysis
The data are presented as mean±SD for triplicate analysis. The results
were subjected to one-way analysis of variation ANOVA with Fisher's
Least Significant Difference test. Pb 0.05 was considered significant. The
Pearson Correlation test for correlation analysis was used. The statistical
analysis was performed using Statistica (Stat Soft, USA).
305
3. Results and discussion
Analysis of anthocyanins in red cabbage by means of both HPLC-DAD
and HPLC-MS/MS methods were performed. Fig. 1 showed anthocyanin
profile of red cabbage using HPLC-DAD chromatogram at 520 nm. The
UV-Vis and MS data of red cabbage anthocyanins were presented in
Table 1. According to the data published (Arapitsas et al., 2008; Wu &
Prior, 2005), the only derivatives of cyanidin were found in red cabbage.
To prove this thesis the presence of a fragment signal at m/z 287 in
MS/MS spectra was only found. However, derivatives of other antho-
cyanin aglycones such as pelargonidin (McDougall et al., 2007), and
peonidin (Charron et al., 2009) were also recognized in the previous
investigation. Twenty anthocyanins were identified (Fig. 1, Table 1)
basing on the comparison of their retention time, UV-visible spectrum
and MS/MS fragmentation spectrum with the data previously published
(Charron et al., 2009; McDougall et al., 2007; Wu & Prior, 2005). In com-
parison, Wu and Prior (2005) and Arapitsas et al. (2008) identified
more than twenty anthocyanins in red cabbage, 23 and 24 compounds,
respectively, whereas McDougall et al. (2007) (18 anthocyanins) and
Dyrby, Westergaard, and Stapelfeldt (2001) (15 anthocyanins) detected
less than twenty derivatives of anthocyanins in the extract of fresh red
cabbage. Only nine derivatives of cyanidin in three cultivars of red
cabbage were found by Pliszka et al. (2009). The different numbers of
anthocyanins identified may result from varietal diversity, influence of
vegetation season (light, level of precipitation), climatic conditions
and cultivation conditions (Wiczkowski & Piskula, 2004).
The data obtained supported a previous observation (Wu & Prior,
2005) that the base structure of anthocyanins found was cyanidin-3-
diglucoside-5-glucosides and the glucoside residues of which can be
nonacylated, monoacylated and diacylated. Sinapic, ferulic, caffeic and
p-coumaric acids were recognized as main phenolic acids in this struc-
ture. The HPLC-DAD chromatograms of the red cabbage extracts were
characterized by eight major peaks and twelve minor peaks (Fig. 1).
Peak 1 was predominant in chromatographic profile obtained. UV–Vis
analysis of this peak showed maximum at 513 nm and no characteristic
1
20
12
15 19
18
14
3
10
5 9
6 7
2
17
4 11 16
8
13
Fig. 1. HPLC chromatogram of anthocyanin profile of red cabbage detected at 520 nm:
cyanidin-3-diglucoside-5-glucoside (1), cyanidin-3-glucoside-5-glucoside (2), cyanidin-
3-(sinapoyl)-triglucoside-5-glucoside (3), cyanidin-3-(sinapoyl)-diglucoside-5-glucoside
(4), cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucoside-5-glucoside (5), cyanidin-3-(feruloyl)-
triglucoside-5-glucoside (6), cyanidin-3-(sinapoyl)-triglucoside-5-glucoside (7), cyanidin-
3-(feruloyl)(feruloyl)-triglucoside-5-glucoside (8), cyanidin-3-(feruloyl)-diglucoside-5-
glucoside (9), cyanidin-3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside (10), cyanidin-3-
(caffeoyl)-diglucoside-5-glucoside (11), cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside
(12), cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucoside-5-glucoside (13), cyanidin-3-
(feruloyl)-diglucoside-5-glucoside (14), cyanidin-3-(sinapoyl)-diglucoside-5-glucoside
(15), cyanidin-3-(feruloyl)-glucoside-5-glucoside (16), cyanidin-3-(sinapoyl)-glucoside-
5-glucoside (17), cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside (18),
cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside (19), and cyanidin-3-
(sinapoyl)(sinapoyl)-diglucoside-5-glucoside (20).
306 W. Wiczkowski et al. / Food Research International 51 (2013) 303–309

Table 1
The UV–Vis and MS data of red cabbage anthocyanins.

Peak Compounds Abbreviation λvis λacyl Eacyl/ [M]+ MS/MS mg Cy/g dm b

a
(nm) (nm) Evis (%) (m/z) (m/z)

1 Cyanidin-3-diglucoside-5-glucoside Cy3diG5G 513 x x 773 611/449/287 0.58

2 Cyanidin-3-glucoside-5-glucoside Cy3G5G 512 x x 611 449/287 0.06
3 Cyanidin-3-(sinapoyl)-diglucoside-5-glucoside Cy3(sin)diG5G 527 330 56 979 817/449/287 0.12
4 Cyanidin-3-(sinapoyl)-triglucoside-5-glucoside Cy3(sin)triG5G 525 321 53 1141 979/449/287 0.03
5 Cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucosides-5-glucoside Cy3(caf)(p-cum)diG5G 521 314 95 1081 919/449/287 0.06
6 Cyanidin-3-(feruloyl)-triglucosides-5-glucoside Cy3(fer)triG5G 522 320 60 1111 949/449/287 0.04
7 Cyanidin-3-(sinapoyl)-triglucoside-5-glucoside Cy3(sin)triG5G 525 321 54 1141 979/449/287 0.04
8 Cyanidin-3-(feruloyl)(feruloyl)-triglucoside-5-glucoside Cy(fer)(fer)triG5G 536 321 93 1287 1125/449/287 0.03
9 Cyanidin-3-(feruloyl)-diglucoside-5-glucoside Cy3(fer)diG5G 522 328 60 949 787/449/287 0.06
10 Cyanidin-3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside Cy(fer)(sin)triG5G 536 324 95 1317 1155/449/287 0.08
11 Cyanidin-3-(caffeoyl)-diglucoside-5-glucoside Cy3(caf)diG5G 522 315 68 935 773/449/287 0.02
12 Cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside Cy3(p-cum)diG5G 521 312 69 919 757/449/287 0.19
13 Cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucoside-5-glucoside Cy3(caf)(p-cum)diG5G 521 314 97 1081 919/449/287 0.01
14 Cyanidin-3-(feruloyl)-diglucoside-5-glucoside Cy3(fer)diG5G 523 329 58 949 787/449/287 0.14
15 Cyanidin-3-(sinapoyl)-diglucoside-5-glucoside Cy3(sin)diG5G 526 328 59 979 817/449/287 0.18
16 Cyanidin-3-(feruloyl)-glucoside-5-glucoside Cy3(fer)G5G 522 328 62 787 449/287 0.03
17 Cyanidin-3-(sinapoyl)-glucoside-5-glucoside Cy3(sin)G5G 527 330 70 817 449/287 0.04
18 Cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside Cy3(fer)(fer)diG5G 535 328 101 1125 963/449/287 0.17
19 Cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside Cy3(fer)(sin)diG5G 535 330 109 1155 993/449/287 0.18
20 Cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside Cy3(sin)(sin)diG5G 535 332 119 1185 1023/449/287 0.26
Total 2.32
a
The ratio of the absorptivities at the corresponding maxima.
b
All values were expressed as milligrams of cyanidin (Cy) per gram dry matter of red cabbage.

absorption of acylation λacyl at 290–340 nm. MS/MS analysis of peak 1
revealed a molecular ion at m/z 773 [M] + and fragment ions at m/z
611 [M-glu]+, m/z 449 [M-2glu]+ and m/z 287 [M-3glu] +. The m/z
287 [M]+ corresponds to cyanidin and was produced after a loss of
486 mass unite (mu) from the molecular ion. The neutral loss of 162
mu corresponded to one molecule of dehydrohexose [-glu]+. Three
neutral losses of 162 were found in the structure therefore the peak
was tentatively identified as cyanidin triglucosides. Finally, taking into
account these results and the previous data (Wu & Prior, 2005), peak
1 was identified as cyanidin-3-diglucoside-5-glucoside. Peak 2 has UV–
Vis spectra shaped very similarly to peak 1, with maximum at 512 nm,
and also no characteristic absorption of acylation λacyl at 290–340 nm.
This peak gave molecular ion at m/z 611 [M]+ with fragment ions at
m/z 449 [M-glu]+ and m/z 287 [M-2glu]+. Through comparison with
the data published (Arapitsas et al., 2008; Wu & Prior, 2005), peak 2
was identified as cyanidin-3-glucoside-5-glucoside.
In contrast to peaks 1 and 2, other peaks have different UV–Vis
spectrum, with the shoulder absorption λacyl at 290–340 nm indicating
acylation with aromatic acids (Fossen & Anderson, 1998). The λacyl in
the range of 320–333 nm showing acylation with sinapic or ferulic acid
gave fourteen peaks (3–4, 6–10, 14–20) while the λacyl in the range of
310–315 nm indicating acylation with p-coumaric and caffeic acid gave
four peaks (5, 11–13). Moreover, the Eacyl/Evis ratio was used to evaluate
whether the identified anthocyanins are mono- or di-acylated. In this
aspect, the Eacyl/Evis ratio of 50–70% indicates monoacylation and the
ratio of 90–120% indicates diacylation (Hong & Wrolstad, 1990).
Eleven peaks (3–4, 6–7, 9, 11–12, 14–17) were monoacylated
(Table 1). Peaks 3 and 15 have very similar absorption maximum at
527 nm and 526 nm, the λacyl at 330 nm and 328 nm as well as the
Eacyl/Evis ratio of 56% and 59%, respectively. This profile is characteris-
tic of monoacylation of the sugar moiety with synaptic or ferulic acid.
These peaks have also very similar MS/MS spectrum. They both pres-
ent a molecular ion at m/z 979 [M] +, and fragment ion at m/z 817
[M-glu] +, m/z 449 [M-2glu-206] +, and m/z 287 [M-3glu-206] +. The
interpretation concerning neutral loss of 162 mu corresponding to
one molecule of dehydrohexose has already been presented above. The
fragment of 206 mu is commonly characterized as sinapic acid residue
in anthocyanin analysis. The data collected indicate that these peaks can
be assigned to cyanidin-3-(sinapoyl)-diglucoside-5-glucoside. What is
more, peaks 3 and 15 may be isomers with a different position of the
sinapoyl group connection to the sugar moiety, which gives different
retention. Identical phenomenon associated with the appearance of
two peaks with a similar pattern of MS/MS fragmentation (m/z 949
[M] +, m/z 787 [M-glu] +, m/z 449 [M-2glu-176] +, and m/z 287
[M-3glu-176] +), and spectral characteristics (the λmax at 522 nm
and 523 nm, the λacyl at 328 nm and 329 nm) was found in case of
peaks 9 and 14. Moreover, these peaks have the Eacyl/Evis ratio of
60% and 58%, respectively, indicating monoacylated structure. The
fragment of 176 mu is commonly characterized as ferulic acid residue.
On the basis of these data and previous information (McDougall et al.,
2007; Wu & Prior, 2005) both peaks were identified as cyanidin-3-
(feruloyl)-diglucoside-5-glucoside but with a different position of
acylation causing different retention. Another two peaks with similar
pattern of MS/MS fragmentation (m/z 1141 [M] +, m/z 979 [M-glu]+,
m/z 449 [M-3glu-206]+, and m/z 287 [M-4glu-206] +), and spectral
characteristics (the λmax at 525 nm and 525 nm, the λacyl at 321 nm
and 321 nm, the Eacyl/Evis ratio of 53% and 54%) were found in case of
peaks 4 and 7. These peaks were putatively identified as cyanidin-
3-(sinapoyl)-triglucoside-5-glucoside. Similar MS and spectra profile
was observed in peak 6 (m/z 1111 [M]+, m/z 949 [M-glu]+, m/z 449
[M-3glu-176]+, and m/z 287 [M-4glu-176]+, λmax at 522 nm, the λacyl
at 320, the Eacyl/Evis ratio of 60%). Considering these data peak 6 was
putatively described as cyanidin-3-(feruloyl)-triglucoside-5-glucoside.
Peak 11 was identified as cyanidin-3-(caffeoyl)-diglucoside-5-glucoside
on the basis of its MS and UV–Vis spectrum (m/z 935 [M]+, m/z 773
[M-glu]+, m/z 449 [M-2glu-162]+, and m/z 287 [M-3glu-162]+, λmax
at 522 nm, the λacyl at 315, the Eacyl/Evis ratio of 68%). The molecular
weight of the acylated group was 162 which corresponds to caffeic acid
residue. Peak 12 was one of the dominant ones and was identified as
cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside (m/z 919 [M]+, m/z
757 [M-glu]+, m/z 449 [M-2glu-146]+, and m/z 287 [M-3glu-146]+,
λmax at 521 nm, the λacyl at 312, the Eacyl/Evis ratio of 69%). The loss
of 146 mu fragment in this MS profile corresponds to acylated
group of p-cummaric residue. Peak 16 had a molecular weight of
787 ([M] + m/z), and two ions m/z 449 [M-glu-176] +, and m/z 287
[M-2glu-176] + as well as UV-Vis spectrum of λmax at 522 nm, the
λacyl at 328. Moreover, the Eacyl/Evis ratio (62%) of this peak indicates
monoacylation. Thus, the structure could be cyanidin-3-(feruloyl)-
glucoside-5-glucoside. Similar MS and spectra profile was observed
in peak 17 (m/z 817 [M] +, m/z 449 [M-glu-206] +, and m/z 287
 W. Wiczkowski et al. / Food Research International 51 (2013) 303–309
[M-2glu-206] +, λmax at 527 nm, the λacyl at 330, the Eacyl/Evis ratio of
70%). In this case peak was identified as cyanidin-3-(sinapoyl)-
glucoside-5-glucoside.
The other seven peaks (5, 8, 10, 13, 18–20) were diacylated (Table 1).
There were two isomers of cyanidin-3-(caffeoyl)(p-coumaroyl)-
diglucoside-5-glucoside (peaks 5 and 13; m/z 1081 [M]+, m/z 919
[M-glu]+, m/z 449 [M-2glu-162-146]+, and m/z 287 [M-3glu-
162-146]+; the λmax at 521 nm and the λacyl at 314 nm; the Eacyl/Evis
ratio of 95% and 97%, respectively). Peaks 8 and 10 had similar MS data as
cyanidin-3-(feruloyl)(feruloyl)-triglucoside-5-glucoside and cyanidin-
3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside, respectively, published
previously (McDougall et al., 2007). Peak 18 was characterized by a mo-
lecular weight of 1125 ([M]+ m/z), and three fragment ions m/z 963
[M-glu]+, m/z 449 [M-2glu-2x176]+, and m/z 287 [M-3glu-2x176]+ as
well as UV-Vis spectrum of λmax at 535 nm, the λacyl at 328. The Eacyl/
Evis ratio of this peak was 101%. Therefore, this anthocyanin was iden-
tified as cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside. Peak
19, in of this group, yielded [M]+ m/z 1155, [M-glu]+ m/z 993,
[M-2glu-176-206]+ m/z 449, [M-3glu-176-206]+ m/z 287, λmax at
535 nm, and the λacyl at 330 and the Eacyl/Evis ratio of 109%. These data
are characteristic of cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glu-
coside. Peak 20 gave [M] + m/z 1185, [M-glu] + m/z 1023,
[M-2glu-2 × 206] + m/z 449, [M-3glu-2 × 206] + m/z 287, λmax at
535 nm, the λacyl at 332, and the Eacyl/Evis ratio of 119%. By calcu-
lating the possible combination it was identified as cyanidin-3-
(sinapoyl)(sinapoyl)-diglucoside-5-glucoside.
Peaks 3 and 15; 4 and 7; 9 and 14 have very similar maximum of
absorption as well as MS/MS spectrum. As indicated, in the above
studies (McDougall et al., 2007; Wu & Prior, 2005), different position
of acylation in cyanidin glucoside molecule is possible, resulting in
different retention time. On the other hand, the different types of
sugar–sugar linkage (β(1-2), β(1-3), β(1-6)) occur in vegetables (Wu
& Prior, 2005) which also can influence on the retention time of com-
pounds. Therefore, taking into account these elucidations, several di-
and tri-glucoside structures with different position of acyl group are
possible. Since the analytical equipment used in our investigation had
not permitted for determination of the acylation position and type of
sugar-sugar linkage, both compounds from each couples were designated
as cyanidin-3-(sinapoyl)-diglucoside-5-glucoside, cyanidin-3-(sinapoyl)-
triglucoside-5-glucoside, and cyanidin-3-(feruloyl)-diglucoside-5-
glucoside, respectively.
Total content of anthocyanins in red cabbage var. Langedijker deter-
mined by HPLC-DAD was 2.32 mg of cyanidin/g dm. Dominant forms of
anthocyanins in this red cabbage (calculated based on cyanidin equiva-
lents) were nonacylated compound, cyanidin-3-diglucoside-5-glucoside
(peak 1, 25.0%). High concentration of anthocyanins was also estimated
in case of cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside
(peka 20, 11.2%), cyanidin-3-(p-coumaroyl)-diglucosides-5-glucoside
(peak 12, 8.2%), cyanidin-3-(sinapoyl)-diglucoside-5-glucoside (peak
15, 7.8%), cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside
(peak 19, 7.8%), cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside
(peak 18, 7.3%), cyanidin-3-(feruloyl)-diglucoside-5-glucoside (peak 14,
6.0%) and cyanidin-3-(sinapoyl)-diglucoside-5-glucoside (peak 3,
5.2%). Totally, nonacylated anthocyanins comprised 27.6% of total
red cabbage anthocyanins while monoacylated and diacylated an-
thocyanins covered 38.4% and 34.1%, respectively (Table 1).
According to Charron et al. (2009) the dominant form of anthocya-
nins in red cabbage was also cyanidin-3-diglucoside-5-glucoside.
In contrast, the study of Wu and Prior (2005) showed that
the major anthocyanins in red cabbage were both cyanidin-3-
diglucoside-5-glucoside and cyanidin-3-(sinapoyl)-diglucoside-5-
glucoside.
The results of antioxidant capacity of red cabbage var. Langedijker
derived from TEAC, PCL ACL and PLC ACW assays were 86.51 ± 2.89,
208.04 ± 41.04, and 35.22 ± 0.64 μmol Trolox/g dm, respectively.
Results of many experiments on the antioxidant activity of
307
anthocyanins have been published (Castaneda-Ovando, Pacheco-
Hernandez, Paez-Hernandez, Rodriguez, & Galan-Vidal, 2009; Kong,
Chia, Goh, Chia, & Brouillard, 2003), but only in a few studies the an-
tioxidant capacity of acylated anthocyanins was compared (Azuma et
al., 2008; Fiol et al., 2012; Kano, Takayanagi, Harada, Makino, &
Ishikawa, 2005; Matsufuji et al., 2007). In this study, a comparison of an-
tioxidant activity of seven isolated and purified red cabbage derivatives of
cyanidin (Cy3dig5G, Cy3(syn)diG5G, Cy3(p-cum)diG5G, Cy3(fer)diG5G/
Cy3(syn)diG5G, Cy3(fer)(fer)diG5G, Cy3(fer)(syn)diG5G, Cy3(syn)(syn)
diG5G) was determined for the very first time. Two analytical strate-
gies were used: assay of TEAC and the PCL method using the
Photochem system with the commercial ACW and ACL kits. The re-
sults obtained varied depending on the method used and the kind
of acyl moiety conjugated (Table 2). In both systems, Cy3(syn)
(syn)diG5G showed the highest antioxidant activity followed by
Cy3(fer)(syn)diG5G and Cy3(fer)(fer)diG5G, while Cy3dig5G showed
the lowest antioxidant activity. The order of scavenging activity
against ABTS+ was as follows: Cy3(syn)(syn)diG5G > Cy3(fer)(syn)
diG5G > Cy3(fer)(fer)diG5G > Cy3(fer)diG5G/Cy3(syn)diG5G>Cy3
(syn)diG5G>Cy3(p-cum)diG5G>Cy3dig5G. In case of the antioxidant
activity provided by the PCL assay the order of analyzed anthocyanins
characterized one change in comparison to TEAC assay; Cy3(syn)
diG5G showed higher ability to scavenge the superoxide anion radi-
cal than mixture of Cy3(fer)diG5G/Cy3(syn)diG5G. The antioxidant
activity of these natural red colorants analyzed by PCL ACL was high-
ly correlated with those provided by TEAC (r = 0.97), while PLC ACW
correlated with PCL ACL (r = 0.84) and TEAC (r = 0.87).
When comparing the antioxidant activity of red cabbage anthocy-
anins, it seems crucial to consider the presence or the lack of acyl unit
in the molecular structure of these natural red colorants. In our study,
all acylated cyanidin glycosides demonstrated higher antioxidant ca-
pacity than the nonacylated form of cyanidin glycosides. Other results
which also show that acylation increases the antioxidant activity have
been presented by Azuma et al. (2008) and Matsufuji et al. (2007). In
the first study, acylated delphinidin-3-rutinoside-5-glucoside was char-
acterized by higher activity toward both 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical and linoleic acid radical than that provided by both
delphinidin-3-rutinoside and delphinidin-3-glucoside (Azuma et
al., 2008). In comparison in the second report, acylated form of
pelargonidin-3-sophoroside-5-glucoside showed stronger antioxi-
dant activity against DPPH radical than pelargonidin-3-glucoside
and pelargonidin aglycone (Matsufuji et al., 2007). The results indicat-
ing a higher antioxidant ability of acylated anthocyanins glycosides
when compared to their corresponding anthocyanins glycosides have
been also shown by Terahara et al. (2001).
In the case of monoacylated derivatives of cyanidin, the compound
acylated with sinapic acid had higher activity than cyanidin deriva-
tives conjugated with both ferulic and p-coumaric acids, whereas
Cy3diG5G binding with p-coumaric acid was characterized by lower ac-
tivity when compared to Cy3diG5G binding with ferulic acid (Table 2).
Table 2
The antioxidant activity of individual red cabbage anthocyanins determined by TEAC,
ACL PLC and ACW PCL assays.
Peak Compounds Antioxidant activity
ACL PCL ACW PCL TEAC
(μM Trolox) (μM Trolox) (mM Trolox)
1 Cy3diG5G 1.07 ± 0.04a 0.12 ± 0.03a 0.32 ± 0.01a
3 Cy3(sin)diG5G 2.23 ± 0.02b 0.23 ± 0.02b 0.50 ± 0.04b
12 Cy3(p-cum)diG5G 2.11 ± 0.01c 0.15 ± 0.01a 0.43 ± 0.01c
14/15 Cy3(fer)diG5G/ Cy3(sin)diG5G 2.21 ± 0.01b 0.20 ± 0.02b 0.52 ± 0.01b
18 Cy3(fer)(fer)diG5G 2.42 ± 0.03d 0.34 ± 0.01c 0.61 ± 0.01b
19 Cy3(fer)(sin)diG5G 2.82 ± 0.03e 0.37 ± 0.01d 0.65 ± 0.01d
20 Cy3(sin)(sin)diG5G 2.94 ± 0.02f 0.45 ± 0.01e 0.74 ± 0.01e
Data are expressed as means ± SD (n = 3). Means in column related to a respective test
followed by the different letters are significantly different (P b 0.05).
308 W. Wiczkowski et al. / Food Research International 51 (2013) 303–309
Our results are consistent with the report Fiol et al. (2012) which dem-
onstrated that the conjugation with the sinapic acid leads to a higher
antioxidant capacity than with the ferulic acid. Similarly, the data
obtained by Matsufuji et al. (2007) indicated that pelargonidin glyco-
sides acylated with ferulic acid had a higher ability to scavenge radicals
than that acylated with p-coumaric acid. All data mentioned indicated
that the kind of monoacylation promoting the ability to scavenge radi-
cals was in the order of sinapic acid>ferulic acid > p-coumaric acid.
Confirmation of these results may be sought in the previous study
which showed that antiradical activity of selected phenolic acid against
DPPH• was in the order of caffeic> protocatechuic >sinapic > ferulic >
vanillic> p-coumaric > o-coumaric (Karamac, Kosinska, & Pegg, 2005).
In this study, diacylated anthocyanins were characterized by anti-
oxidant capacity being higher than in the case of monoacylated an-
thocyanins. What is more, Cy3diG5G acylated with two sinapic acids
showed the strongest ability to scavenge radicals (Table 2). Higher
antioxidant activity of anthocyanins diacylated with sinapic acid in
comparison to anthocyanins monoacylated with sinapic acid was
consistent with that reported by Degenhardt et al. (2000). The results
obtained for diacylated anthocyanins indicate also that acylation with
sinapic acid increased the ability to radical scavenge. This finding was
in agreement with that noted for radical scavenge of monoacylated
anthocyanins. In addition, it is important to remember that conjuga-
tion site of acyl residue can affect antioxidant activity of anthocyanins
(Matsufuji et al., 2007).
The relative contributions of Cy3dig5G, Cy3(syn)diG5G,
Cy3(p-cum)diG5G, Cy3(fer)diG5G/Cy3(syn)diG5G, Cy3(fer)(fer)diG5G,
Cy3(fer)(syn)diG5G, and Cy3(syn)(syn)diG5G to the antioxidant capac-
ity of red cabbage determined by TEAC, PCL ACL, and PCL ACW methods
were demonstrated in Table 3. The lowest contribution to the antioxi-
dant capacity of red cabbage was supplied by Cy3(syn)diG5G, while
the highest contribution was provided by Cy3(syn)(syn)diG5G. Gener-
ally, despite the fact that isolated anthocyanins possess a strong antiox-
idant activity, their contribution to the antioxidant capacity of studied
red cabbage was low. It may indicate that also a number of other com-
pounds present in red cabbage (phenolic acids, flavonols, ascorbic acid)
and/or their interactions are responsible for the antioxidant activity of
red cabbage extract.
4. Conclusion
In order to understand the functions of plant anthocyanins in the
human organism, it is important to discover their chemical structures
which determine their biological properties including antioxidant
activity. These results indicate that red cabbage is a rich source of
acylated anthocyanins possessing strong antioxidant activity. It is
especially crucial to note that acylated anthocyanins display marked
stability, and can be used as natural food colorants as well as dietary
antioxidants, what is important when taking into consideration their
role in the prevention of diseases associated with oxidative damage.
Table 3
Relative contribution of Cy3dig5G, Cy3(syn)diG5G, Cy3(p-cum)diG5G, Cy3(fer)diG5G/
Cy3(syn)diG5G, Cy3(fer)(fer)diG5G, Cy3(fer)(syn)diG5G, Cy3(syn)(syn)diG5G to the
antioxidant capacity of red cabbage.
Compounds Antioxidant activity
(%)
ACL PCL ACW PCL TEAC
Cy3diG5G 1.04 0.69 0.75
Cy3(sin)diG5G 0.45 0.27 0.24
Cy3(p-cum)diG5G 0.67 0.28 0.33
Cy3(fer)diG5G/ Cy3(sin)diG5G 0.59 0.32 0.33
Cy3(fer)(fer)diG5G 0.69 0.57 0.42
Cy3(fer)(sin)diG5G 0.85 0.66 0.47
Cy3(sin)(sin)diG5G 1.28 1.16 0.77
total contribution 5.57 3.95 3.31
Therefore, a further investigation on biological properties and bio-
availability of red cabbage anthocyanins is needed.
Acknowledgment
The research was supported by the Polish State Committee for
Scientific Research (project 1902/B/P01/2008/35).
References
Arapitsas, P., Sjoberg, P. J. R., & Turner, Ch. (2008). Characterization of anthocyanins in
red cabbage using high resolution liquid chromatography coupled with photodiode
array detection and electrospray ionization-linear ion trap mass spectrometry. Food
Chemistry, 109, 219–226.
Azeredo, H. M. C. (2009). Betalains: properties, sources, applications, and stability — A
review. International Journal of Food Science and Technology, 44, 2365–2376.
Azuma, K., Ohyama, A., Ippoushi, K., Ichiyanagi, T., Takeuchi, A., Saito, T., et al. (2008).
Structures and antioxidant activity of anthocyanins in many accessions of eggplant
and its related species. Journal of Agricultural and Food Chemistry, 56, 10154–10159.
Castaneda-Ovando, A., Pacheco-Hernandez, Ma. L., Paez-Hernandez, Ma, Rodriguez, J. A., &
Galan-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review. Food Chemistry,
113, 859–871.
Charron, C. S., Kurilich, A. C., Clevidence, B. A., Simon, P. W., Harrison, D. J., Britz, S. J.,
et al. (2009). Bioavailability of anthocyanins from purple carrot juice: effects of ac-
ylation and plant matrix. Journal of Agricultural and Food Chemistry, 57, 1226–1230.
Choi, S. W., Chang, E. J., Ha, T. Y., & Choi, K. H. (1997). Antioxidative activity of acylated
anthocyanin isolated from fruit and vegetables. Journal of Food Science and Nutrition,
2(3), 191–196.
Clifford, M. N. (2000). Anthocyanins — Nature, occurrence and dietary burden. Journal
of the Science of Food and Agriculture, 80, 1063–1072.
Degenhardt, A., Knapp, H., & Winterhalter, P. (2000). Separation and purification of
anthocyanins by high-speed countercurrent chromatography and screening for
antioxidant activity. Journal of Agricultural and Food Chemistry, 48, 338–343.
Dyrby, M., Westergaard, N., & Stapelfeldt, H. (2001). Light and heat sensitivity of red
cabbage extract in soft drink model systems. Food Chemistry, 72, 431–437.
Fiol, M., Adermann, S., Neugart, S., Rohn, S., Mugge, C., Schreiner, M., et al. (2012). High-
ly glycosylated and acylated flavonols isolated from kale (Brassica oleracea var.
sabellica) — Structure–antioxidant activity relationship. Food Research International,
47, 80–89.
Fossen, T., & Anderson, M. (1998). Cyanidin 3-O-(6′-succinyl-glucopyranoside) and
other anthocyanins from Phragmites australis. Phytochemistry, 49, 1065–1068.
Galvano, F., La Fauci, L., Lazzarino, G., Fogliano, V., Ritieni, A., Cappellano, S., et al.
(2004). Cyanidins: Metabolism and biological properties. The Journal of Nutritional
Biochemistry, 15, 2–11.
Giusti, M. M., & Wrolstad, R. E. (2001). Characterization and measurements of anthocy-
anins by UV–visible spectroscopy. In R. E. Wrolstad (Ed.), Pigments and colorants.
Current protocols in food analytical chemistry (pp. 1–13). New York: Wiley.
Hong, V., & Wrolstad, R. E. (1990). Characterization of anthocyanin-containing color-
ants and fruits juices by HPLC/Photodiode Array Detection. Journal of Agricultural
and Food Chemistry, 38(3), 698–708.
Jagdish Singh, A. K., Upadhyay, A., Bahadur, B., Singh, B., Singh, K. P., & Mathura Rai, A. K.
(2006). Antioxidant phytochemicals in cabbage (Brassica oleracea L. var. capitata).
Scientia Horticulture, 108, 233–237.
Kano, M., Takayanagi, T., Harada, K., Makino, K., & Ishikawa, F. (2005). Antioxidative activ-
ity of anthocyanins from purple sweet potato, Ipomoera batatas cultivar Ayamurasaki.
Bioscience, Biotechnology, and Biochemistry, 69(5), 979–988.
Karamac, M., Kosinska, A., & Pegg, R. B. (2005). Comparison of radical-scavenging activities
for selected phenolic acids. Polish Journal of Food and Nutrition Sciences, 14/55, 165–170.
Kong, J. M., Chia, L. S., Goh, N. K., Chia, T. F., & Brouillard, R. (2003). Analysis and biolog-
ical activities of anthocyanins. Phytochemistry, 64, 923–933.
Matsufuji, H., Kido, H., Misawa, H., Yaguchi, J., Otsuki, T., Chino, M., et al. (2007). Stability to
light, and hydrogen peroxide at different pH values and DPPH radical scavenging ac-
tivity of acylated anthocyanins from red radish extract. Journal of Agricultural and Food
Chemistry, 55, 3692–3701.
McDougall, G. J., Fyffe, S., Dobson, P., & Stewart, D. (2007). Anthocyanins from red
cabbage — Stability to simulated gastrointestinal digestion. Phytochemistry, 68,
1285–1294.
Nielsen, I. L. F., Rasmussen, S. E., Mortensen, A., Ravn-Haren, G., Ma, H. P., Knuthsen, P.,
et al. (2005). Anthocyanins increase low-density lipoprotein and plasma cholester-
ol and do not reduce atherosclerosis in Watanabe heritable hyperlipidemic rabbits.
Molecular Nutrition & Food Research, 49, 301–308.
Nizamutdinova, I. T., Jin, Y. C., Chung, J. I., Shin, S. C., Lee, S. J., Seo, H. G., et al. (2009).
The anti-diabetic effect of anthocyanins in streptozotocin-induced diabetic rats
through glucose transporter 4 regulation and prevention of insulin resistance
and pancreatic apoptosis. Molecular Nutrition & Food Research, 53, 1419–1429.
Pliszka, B., Huszcza-Ciolkowska, G., Mieleszko, E., & Czaplicki, S. (2009). Stability and
antioxidative properties of acylated anthocyanins in three cultivars of red cabbage
(Brassica oleracea L. var. capitata L. f. rubra). Journal of the Science of Food and Agri-
culture, 89, 1154–1158.
Podsedek, A. (2007). Natural antioxidants and antioxidant capacity of Brassica vegetables:
A review. LWT- Food Science and Technology, 40, 1–11.
 W. Wiczkowski et al. / Food Research International 51 (2013) 303–309
Rahman, M. M., Ichiyanagi, T., Komiyama, T., Sato, S., & Konishi, T. (2008). Effects of an-
thocyanins on psychological stress-induced oxidative stress and neurotransmitter
status. Journal of Agricultural and Food Chemistry, 56, 7545–7550.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. A. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology & Medicine, 26, 1231–1237.
Stintzing, F. C., Stintzing, A. S., Carle, R., Frei, B., & Wrolstad, R. E. (2002). Color and
antioxidant properties of cyaniding-based anthocyanin pigments. Journal of Agri-
cultural and Food Chemistry, 50, 6172–6181.
Terahara, N., Callebaut, A., Ohba, R., Nagata, T., Ohnishi-Kameyama, M., & Suzuki, M.
(2001). Acylated anthocyanin 3-sophoroside-5-glucosides from Ajuga reptans
flowers and the corresponding cell cultures. Phytochemistry, 58, 493–500.
Volden, J., Borge, G. I. A., Bengtsson, G. B., Hansen, M., Thygesen, I. E., & Wicklund, T.
(2008). Effect of thermal treatment on glucosinolates and antioxidant-related pa-
rameters in red cabbage (Brassica oleracea L. ssp. capitata f. rubra). Food Chemistry,
109, 595–605.
Wang, L. S., & Stoner, G. D. (2008). Anthocyanins and their role in cancer prevention.
Cancer Letters, 269, 281–290.
309
Wiczkowski, W., & Piskula, M. K. (2004). Food flavonoids. Polish Journal of Food and
Nutrition Sciences, 13/54, 101–114.
Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. F., & Prior, R. L.
(2006). Concentrations of anthocyanins in common foods in the United States
and estimation of normal consumption. Journal of Agricultural and Food Chemistry,
54, 4069–4075.
Wu, X., & Prior, R. L. (2005). Identification and characterization of anthocyanins by
high-performance liquid chromatography-electrospray ionization-tandem mass
spectrometry in common foods in the United States: Vegetables, nuts, and grains.
Journal of Agricultural and Food Chemistry, 53, 3101–3113.
Yanamala, N., Tirupula, K. C., Balem, F., & Klein-Seetharaman, J. (2009). pH-dependent
interaction of Rhodopsin with cyanidin-3-glucoside. 1. Structural aspects. Photo-
chemistry and Photobiology, 85, 454–462.
Zielinska, D., Wiczkowski, W., & Piskula, M. K. (2008). Determination of the relative
contribution of quercetin and its glucosides to the antioxidant capacity of onion
by cyclic voltammetry and spectrophotometric methods. Journal of Agricultural
and Food Chemistry, 56, 3524–3531.