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Food Research International 51 (2013) 303309

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Food Research International


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Red cabbage anthocyanins: Prole, isolation, identication, 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

Article history:
Received 31 July 2012
Accepted 15 December 2012
Keywords:
Red cabbage
Acylated anthocyanins
HPLC-MS/MS
Isolation
Antioxidant activity

a b s t r a c t
Red cabbage is rich in a number of bioactive substances, including anthocyanins. The anthocyanin prole of red
cabbage consisting of twenty derivatives of cyanidin glucosides was described by means of HPLC-DAD-MS/MS.
The base structure of anthocyanins identied was cyanidin-3-diglucoside-5-glucoside. Their glucoside residues
were nonacylated, monoacylated and diacylated. Sinapic, ferulic, caffeic and p-coumaric acids were recognized
as main phenolic acids in this structure. The predominant anthocyanin in red cabbage was nonacylated form
of cyanidin-3-diglucoside-5-glucoside, followed by cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside
and cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside. Anthocyanins were isolated and puried by Amberlite
XAD-16 column chromatography and by HPLC on C18 semi-preparative column. Among the seven isolated
and puried 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, vitamins, oligosaccharides, and a number of bioactive substances, such
as anthocyanins, avonols, 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 benets, 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 vegetable in the form of fresh-cut salads. In addition, red cabbage is characterized 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 signicant 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 articial pigments stimulates the search for natural 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).
0963-9969/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2012.12.015

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 signicant
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
identied (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). Unfortunately, there is little information available regarding the relationship 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 isolated from red cabbage (monoacylated and diacylated with sinapic
acid derivatives of cyanidin-3-digucoside-5-glucoside) were analyzed by Degenhardt, Knapp, and Winterhalter (2000). Taking into
account the fact that red cabbage can contain 924 different anthocyanins (Arapitsas, Sjoberg, & Turner, 2008; Pliszka, Huszcza-Ciolkowska,
Mieleszko, & Czaplicki, 2009), there is a lack of studies comparing the

304

W. Wiczkowski et al. / Food Research International 51 (2013) 303309

antioxidant activity of a wide range of red cabbage anthocyanins in the


same experiment.
In this study, description of the prole of red cabbage anthocyanin
by means of HPLC-DAD-MS/MS as well as isolation and purication of
main anthocyanins from this vegetable were performed. In addition,
the inuence of acylation pattern on the antioxidant activity of seven
individual red cabbage anthocyanins measures against both 2,2azinobis(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, triuoroacetic and formic
acid, ethyl acetate, and sodium sulfate were purchased from Merck
KGaA (Dramstad, Germany). Water was puried 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 elds of the
Plantico Zielonki Sp. z o.o. (Zielonki, Poland). The plants harvested
were planted in 2008. The bulbs obtained (7 bulbs) were puried 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 cabbage was obtained using a mixture consisting of methanol/water/
triuoroacetic 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 sonicated and vortexed, and centrifuged (Centrifuge 5415R, Eppendorf,
Niemcy) for 10 min (13,200 g at 4 C). Supernatant was collected
in 5 mL ask. This step was repeated 5 times. Finally, before the analysis, the extract was centrifuged (20 min, 13,000 g).
2.4. Chromatographic analysis
Extracts and solution of isolated red cabbage standards were analyzed by HPLC-DAD method. Aliquots (5 L) 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 m 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 chromatographic determinations were performed at 45 C with the ow 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: 317% B
(077 min), 1780% B (7780 min), 803% B (8084 min), and 3% B
(84105 min). Anthocyanins were identied 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 0.340 M)
was linear with a correlation coefcient of 0.998.
Identication of anthocyanins was carried out using a mass spectrometer QTRAP 5500 (AB SCIEX, USA) equipped with a triple quadrupole, 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 fragmentation ions derived from the selected parent ion for observation of all
the ions formed by the disintegration of the parent ion as well as scanning precursor ion and neutral particles will also be conducted. Optimal
identication of anthocyanins was achieved under the following conditions: 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 50120 V, entrance potential 10 V, collision energy 3070 eV, and collision
cell exit potential 1045 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),
purication and initial separation of anthocyanins on ion-exchange
gel, and isolation of anthocyanins on C18 semipreparative column. Anthocyanins 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 ask. The extract
obtained (about 450 mL) was concentrated with a rotary evaporator
(Rotavapor R-200, Bchi, Switzerland) at 37 C to around 90 mL. Subsequently, the sample obtained was frozen at 80 C and lyophilized.
After lyophilization the sample was dissolved in water containing sodium sulfate (1 g Na2SO3 7H2O/L). Purication 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 eluted 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 purication
was repeated triplicate. The fractions collected were analyzed by
HPLC method presented in Section 2.4.
The fractions from subsequent purication were connected, evaporated to dry and nally dissolved in 10 mL of 5% methanol connecting
0.4% of TFA. Next, anthocyanins were isolated and puried by HPLC on
semipreparative column C18 250 10 mm, 5 m (Luna, Phenomenex,
USA) at 45 C with the ow 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: 813% B (070 min),
13% B (7075 min), 1320% B (75161 min), 2080% B (161163 min),
80% B (163166 min), 808% B (166168 min), and 8% B (168
180 min). The fractions containing puried compounds were collected
and evaporated to dryness. As a result of the applied method of isolation
and purication fteen anthocyanins were collected.

W. Wiczkowski et al. / Food Research International 51 (2013) 303309

305

2.6. Preparation of solutions of isolated anthocyanins

3. Results and discussion

Appropriate amounts of the isolated and puried standards were


dissolved in acidied 80% methanol and their concentration was conrmed by spectroscopic method (Giusti & Wrolstad, 2001). Next, for
the measurements of the antioxidants activity (TEAC and PCL assays),
and HPLC analysis, exactly 100 M concentration of each compound
was prepared.

Analysis of anthocyanins in red cabbage by means of both HPLC-DAD


and HPLC-MS/MS methods were performed. Fig. 1 showed anthocyanin
prole 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 anthocyanin aglycones such as pelargonidin (McDougall et al., 2007), and
peonidin (Charron et al., 2009) were also recognized in the previous
investigation. Twenty anthocyanins were identied (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 comparison, Wu and Prior (2005) and Arapitsas et al. (2008) identied
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 identied may result from varietal diversity, inuence 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-3diglucoside-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 structure. 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 prole obtained. UVVis
analysis of this peak showed maximum at 513 nm and no characteristic

2.7. Antioxidant capacity


2.7.1. Trolox equivalent antioxidant capacity (TEAC) assay
The method presented by Re et al. (1999) with a minor modication
was used to determine the antioxidant activity of isolated and puried
red cabbage anthocyanins and extract of red cabbage. The analysis was
conducted using a spectrophotometer UV-160 1PC (Shimadzu, Japan).
Briey, the ABTS+ solution was diluted with 80% methanol to an absorbance of 0.700.02 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 measured 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 absorbance of the ABTS+ solution at 734 nm. The 80% methanol solution of
Trolox within the concentration range of 0.12.5 mM was used for building 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 puried red cabbage anthocyanins and extract of red cabbage with a Photochem apparatus (Analytik Jena, Leipzig, Germany)
against superoxide anion radicals generated from luminol (a photosensitizer) when exposed to UV light. The antioxidant activity of anthocyanins
and red cabbage extract was analyzed using both ACW (hydrophilic condition) and ACL (lipophilic condition) kits and the protocol of measurement 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 16,000g, and at 4 C prior to the analysis. The antioxidant
assay was carried out in triplicate for each sample. The antioxidant capacity was calculated by means of the comparison with a Trolox standard
curve (0.253.00 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 extract 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 divided by the antioxidant capacity of red cabbage extract and expressed
as % of contribution.
2.8. Statistical analysis
The data are presented as meanSD for triplicate analysis. The results
were subjected to one-way analysis of variation ANOVA with Fisher's
Least Signicant Difference test. Pb 0.05 was considered signicant. The
Pearson Correlation test for correlation analysis was used. The statistical
analysis was performed using Statistica (Stat Soft, USA).

1
20

12

19

15
18

14
3
10
9

5
6 7

2
4

17
8

11
13

16

Fig. 1. HPLC chromatogram of anthocyanin prole of red cabbage detected at 520 nm:
cyanidin-3-diglucoside-5-glucoside (1), cyanidin-3-glucoside-5-glucoside (2), cyanidin3-(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), cyanidin3-(feruloyl)(feruloyl)-triglucoside-5-glucoside (8), cyanidin-3-(feruloyl)-diglucoside-5glucoside (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)-glucoside5-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).

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W. Wiczkowski et al. / Food Research International 51 (2013) 303309

Table 1
The UVVis and MS data of red cabbage anthocyanins.
Peak
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
a
b

Compounds
Cyanidin-3-diglucoside-5-glucoside
Cyanidin-3-glucoside-5-glucoside
Cyanidin-3-(sinapoyl)-diglucoside-5-glucoside
Cyanidin-3-(sinapoyl)-triglucoside-5-glucoside
Cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucosides-5-glucoside
Cyanidin-3-(feruloyl)-triglucosides-5-glucoside
Cyanidin-3-(sinapoyl)-triglucoside-5-glucoside
Cyanidin-3-(feruloyl)(feruloyl)-triglucoside-5-glucoside
Cyanidin-3-(feruloyl)-diglucoside-5-glucoside
Cyanidin-3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside
Cyanidin-3-(caffeoyl)-diglucoside-5-glucoside
Cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside
Cyanidin-3-(caffeoyl)(p-coumaroyl)-diglucoside-5-glucoside
Cyanidin-3-(feruloyl)-diglucoside-5-glucoside
Cyanidin-3-(sinapoyl)-diglucoside-5-glucoside
Cyanidin-3-(feruloyl)-glucoside-5-glucoside
Cyanidin-3-(sinapoyl)-glucoside-5-glucoside
Cyanidin-3-(feruloyl)(feruloyl)-diglucoside-5-glucoside
Cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside
Cyanidin-3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside
Total

Abbreviation
Cy3diG5G
Cy3G5G
Cy3(sin)diG5G
Cy3(sin)triG5G
Cy3(caf)(p-cum)diG5G
Cy3(fer)triG5G
Cy3(sin)triG5G
Cy(fer)(fer)triG5G
Cy3(fer)diG5G
Cy(fer)(sin)triG5G
Cy3(caf)diG5G
Cy3(p-cum)diG5G
Cy3(caf)(p-cum)diG5G
Cy3(fer)diG5G
Cy3(sin)diG5G
Cy3(fer)G5G
Cy3(sin)G5G
Cy3(fer)(fer)diG5G
Cy3(fer)(sin)diG5G
Cy3(sin)(sin)diG5G

vis
(nm)

acyl
(nm)

Eacyl/
Evis (%)

513
512
527
525
521
522
525
536
522
536
522
521
521
523
526
522
527
535
535
535

x
x
330
321
314
320
321
321
328
324
315
312
314
329
328
328
330
328
330
332

x
x
56
53
95
60
54
93
60
95
68
69
97
58
59
62
70
101
109
119

[M]+
(m/z)

MS/MS
(m/z)

mg Cy/g dm

773
611
979
1141
1081
1111
1141
1287
949
1317
935
919
1081
949
979
787
817
1125
1155
1185

611/449/287
449/287
817/449/287
979/449/287
919/449/287
949/449/287
979/449/287
1125/449/287
787/449/287
1155/449/287
773/449/287
757/449/287
919/449/287
787/449/287
817/449/287
449/287
449/287
963/449/287
993/449/287
1023/449/287

0.58
0.06
0.12
0.03
0.06
0.04
0.04
0.03
0.06
0.08
0.02
0.19
0.01
0.14
0.18
0.03
0.04
0.17
0.18
0.26
2.32

The ratio of the absorptivities at the corresponding maxima.


All values were expressed as milligrams of cyanidin (Cy) per gram dry matter of red cabbage.

absorption of acylation acyl at 290340 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 identied as cyanidin triglucosides. Finally, taking into
account these results and the previous data (Wu & Prior, 2005), peak
1 was identied 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 acyl at 290340 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 identied as cyanidin-3-glucoside-5-glucoside.
In contrast to peaks 1 and 2, other peaks have different UVVis
spectrum, with the shoulder absorption acyl at 290340 nm indicating
acylation with aromatic acids (Fossen & Anderson, 1998). The acyl in
the range of 320333 nm showing acylation with sinapic or ferulic acid
gave fourteen peaks (34, 610, 1420) while the acyl in the range of
310315 nm indicating acylation with p-coumaric and caffeic acid gave
four peaks (5, 1113). Moreover, the Eacyl/Evis ratio was used to evaluate
whether the identied anthocyanins are mono- or di-acylated. In this
aspect, the Eacyl/Evis ratio of 5070% indicates monoacylation and the
ratio of 90120% indicates diacylation (Hong & Wrolstad, 1990).
Eleven peaks (34, 67, 9, 1112, 1417) were monoacylated
(Table 1). Peaks 3 and 15 have very similar absorption maximum at
527 nm and 526 nm, the acyl at 330 nm and 328 nm as well as the
Eacyl/Evis ratio of 56% and 59%, respectively. This prole is characteristic of monoacylation of the sugar moiety with synaptic or ferulic acid.
These peaks have also very similar MS/MS spectrum. They both present 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 max at 522 nm
and 523 nm, the acyl 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 identied 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 max at 525 nm and 525 nm, the acyl 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 identied as cyanidin3-(sinapoyl)-triglucoside-5-glucoside. Similar MS and spectra prole
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]+, max at 522 nm, the acyl
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 identied as cyanidin-3-(caffeoyl)-diglucoside-5-glucoside
on the basis of its MS and UVVis spectrum (m/z 935 [M]+, m/z 773
[M-glu]+, m/z 449 [M-2glu-162]+, and m/z 287 [M-3glu-162]+, max
at 522 nm, the acyl 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 identied 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]+,
max at 521 nm, the acyl at 312, the Eacyl/Evis ratio of 69%). The loss
of 146 mu fragment in this MS prole 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 max at 522 nm, the
acyl 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 prole 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) 303309

[M-2glu-206] +, max at 527 nm, the acyl at 330, the Eacyl/Evis ratio of
70%). In this case peak was identied as cyanidin-3-(sinapoyl)glucoside-5-glucoside.
The other seven peaks (5, 8, 10, 13, 1820) 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-3glu162-146]+; the max at 521 nm and the acyl 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 cyanidin3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside, respectively, published
previously (McDougall et al., 2007). Peak 18 was characterized by a molecular 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 max at 535 nm, the acyl at 328. The Eacyl/
Evis ratio of this peak was 101%. Therefore, this anthocyanin was identied 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, max at
535 nm, and the acyl at 330 and the Eacyl/Evis ratio of 109%. These data
are characteristic of cyanidin-3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside. 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, max at
535 nm, the acyl at 332, and the Eacyl/Evis ratio of 119%. By calculating the possible combination it was identied 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
sugarsugar linkage ((1-2), (1-3), (1-6)) occur in vegetables (Wu
& Prior, 2005) which also can inuence on the retention time of compounds. Therefore, taking into account these elucidations, several diand 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-5glucoside, respectively.
Total content of anthocyanins in red cabbage var. Langedijker determined by HPLC-DAD was 2.32 mg of cyanidin/g dm. Dominant forms of
anthocyanins in this red cabbage (calculated based on cyanidin equivalents) 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 anthocyanins covered 38.4% and 34.1%, respectively (Table 1).
According to Charron et al. (2009) the dominant form of anthocyanins 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-3diglucoside-5-glucoside and cyanidin-3-(sinapoyl)-diglucoside-5glucoside.
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 mol Trolox/g dm, respectively.
Results of many experiments on the antioxidant activity of

307

anthocyanins have been published (Castaneda-Ovando, PachecoHernandez, Paez-Hernandez, Rodriguez, & Galan-Vidal, 2009; Kong,
Chia, Goh, Chia, & Brouillard, 2003), but only in a few studies the antioxidant 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 antioxidant activity of seven isolated and puried 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 rst time. Two analytical strategies were used: assay of TEAC and the PCL method using the
Photochem system with the commercial ACW and ACL kits. The results 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 radical than mixture of Cy3(fer)diG5G/Cy3(syn)diG5G. The antioxidant
activity of these natural red colorants analyzed by PCL ACL was highly 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 anthocyanins, 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 capacity 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 rst study, acylated delphinidin-3-rutinoside-5-glucoside was characterized 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 antioxidant activity against DPPH radical than pelargonidin-3-glucoside
and pelargonidin aglycone (Matsufuji et al., 2007). The results indicating 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 derivatives conjugated with both ferulic and p-coumaric acids, whereas
Cy3diG5G binding with p-coumaric acid was characterized by lower activity 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

1
3
12
14/15
18
19
20

Compounds

Cy3diG5G
Cy3(sin)diG5G
Cy3(p-cum)diG5G
Cy3(fer)diG5G/ Cy3(sin)diG5G
Cy3(fer)(fer)diG5G
Cy3(fer)(sin)diG5G
Cy3(sin)(sin)diG5G

Antioxidant activity
ACL PCL
(M Trolox)

ACW PCL
(M Trolox)

TEAC
(mM Trolox)

1.07 0.04a
2.23 0.02b
2.11 0.01c
2.21 0.01b
2.42 0.03d
2.82 0.03e
2.94 0.02f

0.12 0.03a
0.23 0.02b
0.15 0.01a
0.20 0.02b
0.34 0.01c
0.37 0.01d
0.45 0.01e

0.32 0.01a
0.50 0.04b
0.43 0.01c
0.52 0.01b
0.61 0.01b
0.65 0.01d
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 signicantly different (P b 0.05).

308

W. Wiczkowski et al. / Food Research International 51 (2013) 303309

Our results are consistent with the report Fiol et al. (2012) which demonstrated 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 glycosides 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 radicals was in the order of sinapic acid>ferulic acid > p-coumaric acid.
Conrmation 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 antioxidant capacity being higher than in the case of monoacylated anthocyanins. 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 nding was
in agreement with that noted for radical scavenge of monoacylated
anthocyanins. In addition, it is important to remember that conjugation 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 capacity of red cabbage determined by TEAC, PCL ACL, and PCL ACW methods
were demonstrated in Table 3. The lowest contribution to the antioxidant capacity of red cabbage was supplied by Cy3(syn)diG5G, while
the highest contribution was provided by Cy3(syn)(syn)diG5G. Generally, despite the fact that isolated anthocyanins possess a strong antioxidant activity, their contribution to the antioxidant capacity of studied
red cabbage was low. It may indicate that also a number of other compounds present in red cabbage (phenolic acids, avonols, 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

Cy3diG5G
Cy3(sin)diG5G
Cy3(p-cum)diG5G
Cy3(fer)diG5G/ Cy3(sin)diG5G
Cy3(fer)(fer)diG5G
Cy3(fer)(sin)diG5G
Cy3(sin)(sin)diG5G
total contribution

Antioxidant activity
(%)
ACL PCL

ACW PCL

TEAC

1.04
0.45
0.67
0.59
0.69
0.85
1.28
5.57

0.69
0.27
0.28
0.32
0.57
0.66
1.16
3.95

0.75
0.24
0.33
0.33
0.42
0.47
0.77
3.31

Therefore, a further investigation on biological properties and bioavailability of red cabbage anthocyanins is needed.

Acknowledgment
The research was supported by the Polish State Committee for
Scientic Research (project 1902/B/P01/2008/35).

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