Anthocyanins Contents, Profiles, and Color Characteristics of Red Cabbage Extracts from Different Cultivars and Maturity Stages

Abstract

Red cabbage (Brassica oleracea L.) is an excellent source of food colorant. This study aimed to evaluate the anthocyanin pigment contents and profiles from 7 red cabbage cultivars at two maturity stages (8 weeks apart) and evaluate their color characteristics and behavior under acidic and neutral pH. Anthocyanin concentrations ranged from 1,111 to 1,780 mg Cy3G/100gr DM and did not increase with time. Cultivar and maturation affected pigment profile. Some varieties accumulated ≥30% of di-acylated pigments, and proportions of mono-acylated pigments decreased with time. Extracts from selected varieties at first harvesting time produced colors similar (λmax 520nm and ∆E6.1-8.8) to FD&C Red No.3 at pH 3.5. At pH 7, extracts from the second harvest with higher proportion of di-acylation produced λmax ≃610nm, similar to FD&C Blue No.2. Cultivar selection and maturation affected color and stability of red cabbage extracts at different pH values.

Full text

Anthocyanins Contents, Profiles, and Color Characteristics of Red
Cabbage Extracts from Different Cultivars and Maturity Stages
Neda Ahmadiani,
†
Rebecca J. Robbins,
§
Thomas M. Collins,
§
and M. Monica Giusti*
,†
†
Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Road, Columbus, Ohio 43210, United States
§
Analytical and Applied Sciences Group, Mars Inc., 800 High Street, Hackettstown, New Jersey 07840, United States
ABSTRACT: Red cabbage (Brassica oleracea L.) is an excellent source of food colorant. This study aimed to evaluate the
anthocyanin pigment contents and profiles from seven red cabbage cultivars at two maturity stages (8 weeks apart) and evaluate
their color characteristics and behavior under acidic and neutral pH. Anthocyanin concentrations ranged from 1111 to 1780 mg
Cy3G/100 g DM and did not increase with time. Cultivar and maturation affected pigment profile. Some varieties accumulated
≥30% of diacylated pigments, and proportions of monoacylated pigments decreased with time. Extracts from selected varieties at
first harvesting time produced colors similar (λmax = 520 nm and ΔE= 6.1−8.8) to FD&C Red No. 3 at pH 3.5. At pH 7, extracts
from the second harvest with s higher proportion of diacylation produced λmax ≃610 nm, similar to FD&C Blue No. 2. Cultivar
selection and maturation affected color and stability of red cabbage extracts at different pH values.
KEYWORDS: anthocyanins, red cabbage (Brassica oleracea L.), cultivar, harvesting time, color
■INTRODUCTION
Anthocyanins are water-soluble pigments with potential
application in coloring of different food products.
1,2
Colorants
made of these pigments are currently manufactured for food
use from horticultural crops and processing wastes.
3
Fruit and
vegetable juices containing anthocyanins such as concentrated
red cabbage, black carrot, purple sweet potato, radish, bilberry,
and elderberry are being used as approved food color additives
in most countries.
2,4
In addition, anthocyanins are proven to be
good antioxidant compounds due to their effective free radical
scavenging properties and have shown numerous potential
health benefits in in vitro and vivo studies.
5−9
Red cabbage (Brassica oleracea L.) is an edible source with
high content and high potential yield per unit area of
anthocyanins.
10
Red cabbage anthocyanin extract is known to
have considerable amounts of mono- or diacylated cyanidin
anthocyanins.
11,12
Type and acylation of anthocyanins are two
important factors that determine their color characteristics at
certain pH values.
13−17
Due to its anthocyanin compositions,
red cabbage anthocyanin extracts can exhibit a wide spectrum
of color, ranging from orange through red to purple and blue
based upon the pH of the environment.
18
Acylation of
anthocyanins also influences their antioxidant properties and
stability in the food matrix.
15,19,20
Diacylated anthocyanins are
linked to higher antioxidant activity compared to the other non-
and monoacylated ones.
12
Anthocyanin pigments with higher
number of acylation have also shown good stabilities to light
and processing temperature.
20
There are several known intrinsic and extrinsic factors that
affect anthocyanin content and composition in plants.
21−23
Plants cultivar and maturation time are among the essential
factors that can influence the phytochemical content, including
anthocyanins.
24−26
The objectives of this study were to evaluate
the anthocyanins content and profile from different red cabbage
cultivars at two maturity stages and evaluate their color
characteristics and behavior under acidic and neutral pH.
Knowing the anthocyanin composition of red cabbage cultivar
and maturation time would help us to select the cultivar and
maturation that could provide the desired characteristics for a
specific application.
■MATERIALS AND METHODS
Plant Materials. Seven red cabbage cultivars, Cairo, Kosaro,
Integro, Buscaro, Azurro, Primero, and Bandolero (three heads from
each cultivar), at two maturity stages (harvested 13 and 21 weeks after
transplanting) were donated by Bejo Seeds Inc. (Geneva, NY, USA).
Cabbages were grown side-by-side during the summer season. Samples
were shipped immediately after harvest and refrigerated until analyzed
(within a week). The water content in each sample was determined by
placing 4−5 g of sample in a mechanical convection incubator
(Precision Scientific, Buffalo, NY, USA) at 37 °C for 2 days to dry.
Extraction and Purification. Cabbage heads were sliced, and ≃30
g was frozen with liquid nitrogen and kept frozen until analyzed the
following day. The frozen materials were ground using a stainless steel
Waring Commercial Blender (New Hartford, CT, USA) coupled with
a 0.95 L container.
27
The acetone/chloroform extraction procedure
was adopted from that of Giusti and Wrolstad.
28
Frozen plant powder
was mixed with 30 mL of acetone. The mixture was filtered through a
Whatman no. 1 filter (Whatman Inc., Florham, NJ, USA), and the
residual cake was washed with 70% aqueous acetone acidified with
0.1% formic acid (≃250 mL) until the powder was white and the
filtrate was clear. The filtrates were combined, transferred to a
separatory funnel, and mixed with 1 volume of chloroform. The phases
were allowed to separate for 4−5 h. The aqueous phase was collected,
and the residual acetone was evaporated using a Büchi rotavapor
(Brinkmann Instruments, Inc., Westbury, NY, USA). The aqueous
extract was purified using a Sep-Pak C18 cartridge (Waters Corp.,
Milford, MA, USA). The cartridge was activated with methanol and
washed with acidified water before the sample was loaded. The
cartridge was further washed with acidified water (0.1% formic acid),
Received: January 15, 2014
Revised: July 2, 2014
Accepted: July 3, 2014
Published: July 3, 2014
Article
pubs.acs.org/JAFC
© 2014 American Chemical Society 7524 dx.doi.org/10.1021/jf501991q |J. Agric. Food Chem. 2014, 62, 7524−7531

and the anthocyanins were recovered with acidified methanol (0.1%
formic acid). The methanol was removed using the rotavapor, and the
volume was taken to 25 mL with acidified water (0.1% formic acid).
Anthocyanins Content. The total monomeric anthocyanin was
determined by using the pH differential method according to Giusti
and Wrolstad.
29
The extract was diluted using pH 1 (0.025 M
potassium chloride) and pH 4.5 (0.4 M sodium acetated) buffers with
a dilution factor of 100. The solutions were allowed to equilibrate for
15 min in the dark. Absorbance was read on 1 cm path length cuvettes
at 520 and 700 nm using a Shimadzu UV−visible spectrophotometer
(Shimadzu, Columbia, MD, USA). The total monomeric anthocyanin
was calculated on the basis of the dry matter (DM) and fresh matter
(FM) and reported as milligrams of cyanidin-3-glucoside (Cy3G) per
100 g of sample using the equation
ε
=− −− ×
×× ×
AA AA
P
total monomeric anthocyanin (mg/L)
[(()())DF
1000 MW]/( )
520 700 pH1 520 700 pH4.5
where DF is the dilution factor, MW is the molecular weight (449.2 for
Cy3G), εis the molar absorptivity coefficient (26900 cm−1mg−1for
Cy3G), and Pis the cuvette path length.
Alkaline Hydrolysis of Anthocyanins. Alkaline hydrolysis
(saponification) was adopted from the method of Giusti and
Wrolstad.
28
Purified red cabbage anthocyanin extract (10 mL) was
mixed with 10 mL of 10% KOH in a capped test tube and set aside for
15 min at room temperature in the dark. The solution was neutralized
using 2 N HCl until the color turned pink. The neutralized sample was
then purified using a Sep-Pak C18 cartridge (Waters Corp., Milford,
MA, USA) and prepared for HPLC analysis.
Chromatographic Analysis. A Shimadzu Prominence reverse
phase high-pressure LC-MS was coupled to an SPD-M20-A photo-
diode array and a single-quadrupole electrospray ionization (ESI) mass
spectrophotometer (Shimadzu Scientific, Inc.). For data analysis, LC-
MS Solution software was used (Shimadzu Scientific, Inc.). The
column was a 100 ×4.5 mm Kinetex PFP 2.6 μm (Phenomenex Inc.,
Torrance, CA, USA). The solvents were phase A, 4.5% formic acid in
LC-MS grade water, and phase B, LC-MS acetonitrile (Fisher
Scientific Inc., Fair Lawn, NJ, USA), and the gradient was 0−50
min, 0−30% B. Injection volume was 20 μL. Spectral data were
obtained from 250 to 700 nm, and elution of anthocyanins was
monitored at 510−540 nm. Peak areas at this region were then
integrated and normalized. The proportion of the total peak area of
each individual anthocyanin was calculated and reported as percentage
of total peak area at 510−540 nm.
For MS analyses, a 0.2 mL/min volume was diverted into the MS
and ionized under positive ion condition using an electrospray probe.
Data were monitored using total ion scan (SCAN) (m/z200−1200)
and selected ion monitoring at m/z271 (pelargonidin), m/z287
(cyanidin), m/z301 (peonidin), m/z303 (delphinidin), m/z317
(petunidin), and m/z331 (malvidin).
Color and Spectrophotometric Analyses. A ColorQuest XE
colorimeter (HunterLab, Hunter Associates Laboratories Inc., Reston,
VA, USA) was used to measure the color characteristics (Hunter CIE
LCh) of the samples. The equipment was set for transmittance with
specular included, and D65/10°was used for the measurements.
Samples were placed in a 1 cm path length plastic cuvette and CIE L*,
a*,b*, chroma (c*), and hue angle (h°) were measured.
To measure the color in acidic condition, the extract was mixed (in
triplicate) with distilled (DI) water (1:20 v/v). The pH of the
solutions were measured after 30 min of equilibration and was 3.5. To
measure the color in neutral conditions, the extract was diluted with
water (1:2 v/v, in triplicate). The diluted solutions were then mixed
with 0.1 M potassium phosphate buffer (pH 7) with a dilution factor
(DF) of 20. The maximum absorbances (λmax) in the visible range of
the neutral solutions were recorded using a Shimadzu UV−visible
spectrophotometer 2450.
FD&C Red No. 3, FD&C No. 40, and FD&C Blue No. 2 (Noveon
Hilton Davis, Inc., Cincinnati, OH, USA) were also dissolved in DI
water to concentrations that most closely matched the lightness (L*)
and chroma (c*) of the samples.
Statistical Analysis. Seven different cultivars at two maturity
stages (three heads each) for a total of 42 samples were analyzed using
principal component analysis (PCA). PCAs for total monomeric
anthocyanin, nonacylated, monoacylated, and diacylated pigments
(based on the proportional peak areas shown in Table 2) were
performed. Autoscaling was used to normalize each variable before the
analysis.
To compare the anthocyanin contents and profile, normality of the
variables were first checked using the Kolmogorov−Smirnov test (α=
0.05). Analysis of variance (ANOVA) was then used to analyze the
total monomeric anthocyanin and the proportion of each group of
pigments separately using the following model: Yijk =μ+vi+tj+vtij +
Figure 1. HPLC chromatograms of two representative red cabbage anthocyanin extracts: second-harvested Cairo (top) and first-harvested Integro
(bottom) red cabbage at 510−540 nm. Refer to Table 1 for major peak identifications. HPLC conditions: solvent A, 4.5% formic acid in LC-MS
grade water; solvent B, LC-MS acetonitrile; gradient, 0−50 min, 0−30% B. The major peaks (1−8) were found in all other red cabbage samples.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf501991q |J. Agric. Food Chem. 2014, 62, 7524−75317525

αk+εijk, where Yis the individual variable, μis the grand mean, viis
the cultivar effect, tjis the harvesting time effect, vtij is the interaction
between the main factors, αkis the heads effect defined as random
factor, and εijk is the random error of the model. When a significant
difference was obtained (Pvalue < 0.05), the Tukey means-
comparison test was used to compare each pair of means.
All statistical analyses were done on the basis of at least three
independent replicate samples from each individual head. Results were
analyzed by using Minitab 16 statistical software (Minitab Inc.).
■RESULTS AND DISCUSSION
HPLC-PDA-MS and Identification of Major Pigments.
According to the HPLC-PDA data obtained at 510−540 nm,
up to 23 peaks were observed. In previous studies, up to 36
different anthocyanins have been detected in red cabbage.
29−31
Figure 1 shows examples of anthocyanin profiles (Cairo and
Integro extracts) obtained by HPLC-PDA. The eight major
peaks, representing ≃90% of the total anthocyanins and
common to all seven cultivars at both maturity stages, were
selected for further identification and analyses. The λmax at the
UV and vis ranges, molecular ions, and fragments along with
tentative identification of each peak are presented in Table 1.
The pigments were identified using HPLC-PDA and HPLC-
MS and compared with data reported in the literature.
11,12
As shown in Table 1, m/z287 was the fragment in all eight
anthocyanins indicating that cyanidin derivatives were the
major aglycon as reported in previous studies.
12,30,32−34
All of
the pigments were nonacylated, monoacylated, and diacylated
derivatives of cyanidin-3-diglucoside-5-glucoside (Cy-3diG-
5G), which was also confirmed by saponification (results are
not shown). The acylating groups were aromatic acids: sinapic,
ferulic, and p-coumaric acids (Table 1).
Anthocyanin Content and Proportion of the Pig-
ments. Principal Component Analysis of Red Cabbage
Anthocyanin Extracts. The analysis of the objects (i.e., red
cabbage cultivars at two maturity stages) is performed visually
using the scores plot (Figure 2), where the objects are
represented as a function of the principal components (PCs).
As shown in Figure 2, PC1 correlated positively with
monoacylated pigments as opposed to the diacylated pigments.
PC2, on the other hand, was more affected by the nonacylated
pigments and the total monomeric anthocyanin. PC1 and PC2
extracted 83.7 and 12.8% of the total variances, respectively.
According to this analysis, the samples were clearly separated
diagonally on the basis of their maturation time. With
maturation the percentage of diacylation increased for most
samples, so for most of the second-harvested samples PC1 is
negative. Also, because the early mature samples (week 13) had
slightly higher amounts of total monomeric anthocyanin, PC2
tend to be more positive (Figure 2).
PCA is a proper way to obtain relevant information from the
original variables into fewer new latent variables (PCs).
According to our results, this analysis was helpful in
classification of the samples at two maturity stages. Most
samples with different maturity times were separated on the
basis of the first two principal components. Azurro and Primero
cultivars, however, were exceptions because their pigment
profiles were not significantly changed with maturation.
Anthocyanin Content. The average anthocyanin contents
for the 13- and 21-week-harvested plants were ≃1442 and 1269
mg Cy3G/100 g DM respectively, and these values for the fresh
matter were ≃150 and 145 mg Cy3G/100 g FM, respectively.
Piccaglia et al. also reported the anthocyanin content of three
red cabbage cultivars in Italy to be >1000 mg/100 g DM.
10
The
anthocyanin content for the fresh weight red cabbage reported
by Oregon State University database and Wu et al. were,
however, 25 and 322 ±40.8 mg/100 g FW, respectively.
35,36
According to our findings, cultivar made a difference in
anthocyanin contents at both maturity stages.
As shown in Table 2, Buscaro and Integro harvested after 13
weeks had the highest anthocyanin contents. Anthocyanin
contents, however, did not change significantly from the first to
the second harvest except for the Buscaro (DM) cultivar, which
showed a lower anthocyanin content when plants were left
longer on the ground (Table 2). Accumulation of anthocyanins
can be explained by developmental factors, which could be
different in different varieties.
23
Proportion of Major Pigments. Although red cabbage is
known to have more than 20 different anthocyanins, relative
proportions of 8 of them (Figure 1 and Table 1) represented
≃90% of the total anthocyanins and were measured and
Table 1. PDA Absorbance and MS Data for Red Cabbage
Anthocyanins
a
peak RT
b
(min)
λvis
c
(nm)
λacyl
d
(nm) M+
e
identification
1 12.13 513 773 (287) Cy-3diG-5G
2 16.95 528 334 979 (287) Cy-3diG-5G + sinapic
f
3 27.41 523 313 919 (287) Cy-3diG-5G + p-
coumaric
4 28.33 523 326 949 (287) Cy-3diG-5G + ferulic
5 28.85 524 329 979 (287) Cy-3diG-5G + sinapic
6 30.53 536 319 1125 (287) Cy-3diG-5G + ferulic
and ferulic
7 31.55 536 330 1155 (287) Cy-3diG-5G + sinapic
and ferulic
8 32.31 536 331 1185 (287) Cy-3diG-5G + sinapic
and sinapic
a
m/z287 was the major fragment in all eight peaks. Cy-3diG-5G,
cyanidin-3-diglucoside-5-glucoside.
b
Retention time.
c
λvis−max.
d
λof
acylation.
e
Mass ion.
f
Tentative identification.
Figure 2. Correlation between the first two principal components and
the variables as well as score plot with respect to cultivars and
harvesting time for seven different red cabbage cultivars: a, total
monomeric anthocyanin; b, diacylated pigments; c, monoacylated
pigments; d, nonacylated pigments. AZ, Azurro; Ba, Bandolero; Bu,
Buscaro; Ca, Cairo; In, Integro; Ko, Kosaro; Pr, Primero. Symbols in
black and gray represent harvest times after 13 or 21 weeks,
respectively.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf501991q |J. Agric. Food Chem. 2014, 62, 7524−75317526

compared among cultivars and maturation stages as they are
more likely to affect the color and stability of the extract. The
pigments were also grouped into nonacylated, monoacylated,
and diacylated.
According to our results, nonacylated pigments represented
an average of ≃19.6% (±4.5) of the major pigments (Table 2).
Charron et al. also found the percentage of nonacylated
pigment in red cabbage to be 21.3%.
31
Among the major
pigments identified, the amounts of mono- and diacylated
Table 2. Anthocyanin Contents (Total Monomeric Anthocyanin) and Percentage of Major Pigments (Percent Total Peak Area
at 510−540 nm) in Seven Red Cabbage Cultivars at Two Different Harvesting Times
a
total monomeric
anthocyanin
(mg Cy3G/100 g)
cultivar harvest time (week) DM
b
FM
c
nonacylated pigments
d
(%) monoacylated pigments
e
(%) diacylated pigments
f
(%)
Primero 13 1111 e 109 gh 21.24 bc 67.63 a 4.56 g
21 1026 e 104 h 26.84 a 63.71 ab 4.09 g
Integro 13 1660 ab 185 a 18.17 cde 65.22 ab 7.23 fg
21 1637 ab 188 a 25.59 ab 53.32 cde 12.28 ef
Azurro 13 1392 bcd 144 bcdef 15.85 def 69.33 a 9.19 fg
21 1217 de 137 cdef 19.33 cde 65.01 ab 9.21 fg
Kosaro 13 1247 cde 128 efgh 15.51 ef 55.43 cd 11.42 f
21 1001 e 115 fgh 20.25 cd 46.74 ef 19.41 cd
Cairo 13 1389 bcd 153 bcde 20.08 cde 58.23 bc 12.87 ef
21 1256 cde 168 ab 28.5 a 43.96 fg 17.74 de
Bandolero 13 1517 abc 165 abcd 13.1 f 48.67 def 24.54 bc
21 1512 abc 165 abcd 19.07 cde 36.75 gh 29.1 b
Buscaro 13 1780 a 170 abcd 13.36 f 52.1 cde 23.59 bcd
21 1236 de 137 defg 17.04 cdef 35.48 h 35.45 a
a
Different letters in the same column indicate significant differences (p< 0.05).
b
Dry matter.
c
Fresh matter.
d
Peak 1 (Table 1).
e
Peaks 2, 3, 4, and 5
(Table 1).
f
Peak 6, 7, and 8 (Table 1).
Table 3. CIE L*a*b*, Chroma (c*), Hue (h°), and λmax of Seven Red Cabbage Cultivar Extracts at Two Different Harvesting
Times at pH 3.5 and Their Comparison with Two FD&C Synthetic Red Dyes
cultivar HT
a
(week) L*a*b*c*h°λmax (nm) ΔE1
b
ΔE2
c
Primero 13 76.2 ±0.8 45.3 ±0.6 −3.5 ±0.7 47.4 ±3.5 359.2 ±4.4 520 ±1.2 15.9 ±218±0.7
21 77.1 ±2 42.1 ±4−2.7 ±0.7 42.2 ±4 356.3 ±0.9 520 ±1.4 18.5 ±4.2 17.9 ±1
Integro 13 67.4 ±2.6 59.5 ±1.7 −0.5 ±0.3 59.6 ±4.6 357.5 ±2.6 520 ±0.8 8.8 ±3.1 23.4 ±1.3
21 66.2 ±1.8 61 ±2.5 −1.9 ±1.2 61 ±2.5 358.2 ±1.2 520 ±1.1 9.3 ±2.2 24.8 ±1.4
Azurro 13 69.6 ±3 56.1 ±1−2.8 ±2 56.6 ±5.3 357.7 ±2.7 520 ±0.6 6.8 ±2.9 20.7 ±2
21 71.2 ±2.9 53.2 ±4.7 −2.9 ±1.4 53.3 ±4.6 356.7 ±1.9 520 ±0.5 9.1 ±2.2 20.2 ±1.4
Kosaro 13 69 ±3.2 57 ±3.5 −5.9 ±1.2 57.3 ±5 354.1 ±2.6 520 ±0.3 6.1 ±2.7 23.7 ±3.7
21 69.5 ±1.5 55.5 ±2.4 −7.1 ±0.9 56 ±2.4 352.7 ±1 520 ±0.8 7 ±0.8 24.8 ±1.6
Cairo 13 68.9 ±3.5 57.1 ±1.6 −4.5 ±0.6 55.9 ±3.6 357 ±1.4 520 ±1.4 6.6 ±3.2 23 ±1.3
21 64.9 ±1.5 63 ±2−4.3 ±0.4 63.2 ±2 356.1 ±0.5 520 ±0.6 10.1 ±2 28.1 ±1.6
Bandolero 13 62.5 ±1.6 67.3 ±4.3 −4.7 ±1.3 68.4 ±1.5 356.8 ±4.4 520 ±0.8 14.8 ±332±1.5
21 60.7 ±1.8 68.2 ±2.1 −4.8 ±1.5 68.4 ±2 356 ±1.4 530 ±1.2 15.9 ±2.7 33.6 ±1.3
Buscaro 13 62.9 ±4.1 66.9 ±2.3 −3.7 ±1 64.5 ±1.2 357.9 ±1.4 520 ±0.9 12.8 ±3 29.9 ±4.4
21 61.7 ±4.2 66 ±4.5 −6.4 ±2.9 66.4 ±4.2 354.4 ±2.8 530 ±1.5 14.2 ±5.7 33 ±3
FD&C Red No. 3 74.2 59.99 −6.1 60.3 354.19 520
FD&C Red No. 40 75.64 45.11 14.48 47.38 17.79 500
a
Harvesting time.
b
Color difference with FD&C Red No. 3.
c
Color difference with FD&C Red No. 40.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf501991q |J. Agric. Food Chem. 2014, 62, 7524−75317527

anthocyanins were on average 54.4 (±12.6) and 15.8% (±8.7),
respectively (Table 2). Red cabbage has been identified as a
highly acylated anthocyanin source according to previous
research.
30,31
Before the percentages of each group of pigments were
compared using ANOVA, the normality test was done to verify
the validity of the test, and none of the variables had a pvalue
smaller than 0.05.
Cultivar had a significant (pvalue < 0.05) impact on pigment
profile. The proportions of monoacylated and diacylated
pigments were the major differences found among the cultivars.
Primero and Azurro had the highest proportions of
monoacylated pigments and the lowest proportions of
diacylated pigments, whereas Buscaro and Bandolero varieties
had the lowest proportions of monoacyltaed pigments and the
highest proportion of diacylated pigments. The effect of
maturity on pigment profile was largely dependent on the
cultivar for monoacylated and diacylated pigments (pvalue <
0.05); this interaction was not observed for the nonacylated
pigments.
As shown in Table 2, the proportion of nonacylated
pigments increased significantly in the 21-week-mature plants
except for Azurro and Buscaro cultivars, which did not show a
significant increase. More mature Primero, Cairo, and Integro
had highest amounts of nonacylated pigments.
The proportions of monoacylated anthocyanins were,
however, decreased with longer maturation time, except for
Primero and Azurro cultivars, which did not show a significant
decrease on monoacylated pigments. The proportion of
monoacylated pigments was highest (≃67%) for cultivar
Primero, Integro, and Azurro first harvests as compared to
the other cultivars (≃54%) harvested at the same time (Table
2).
The proportion of diacylated pigments varied remarkably
among cultivars. They increased significantly in Kosaro and
Buscaro due to maturation. Buscaro and Bandolero, harvested
after 21 weeks, had the highest (≃30%) proportion of
diacylated pigments, whereas Primero (≃4%) had the lowest
at both maturity stages (Table 2).
Because the plants were grown side by side, the main reason
for the profile differences could arise from intrinsic factors such
as plant genetics and enzymes and their activities throughout
the maturation, which influence the anthocyanin synthesis
within the plant. Variation in anthocyanin structures can be
correlated with alteration of single genes, which influence the
enzymatic step of anthocyanin synthesis pathways.
37
The
reason for accumulation of certain anthocyanins in certain
cultivars could be due to the difference in the activities of
different genes. Also, during the maturation, the activity of the
genes controlling the synthesis of monoacylated pigments
could have slowed, whereas those responsible for the synthesis
of nonacylated anthocyanins or for the addition of a second
acyl group may have remained active.
Color Characteristics and Stability. Color in Acidic pH.
Table 3 shows the color characteristics of solutions colored
with red cabbage anthocyanin extracts from the different
cultivars at pH 3.5 and their color comparisons to synthetic
colorants FD&C Red No. 3 and No. 40. The lightness of the
samples was approximately between 60 and 77 (Table 3). The
synthetic colorant solutions were also adjusted to have a similar
lightness. Extracts from all seven cultivars produced a deep pink
color at pH 3.5 with a hue angle close to 350°. Chroma values
of the samples were higher for solutions prepared with
Bandolero and Buscaro extracts as opposed to Primero. The
wavelengths of maximum absorbances (λmax) of the samples
were similar to that of Red No. 3 (≃520 nm) except for late-
harvested Bandolero and Buscaro cultivars (λmax ≃530 nm).
Generally, samples showed more similar color characteristics to
FD&C Red No. 3 than to FD&C Red No. 40 synthetic dyes
(ΔE1<ΔE2). Under acidic pH, maturation of the plants did
not seem to significantly affect the color of the solutions. Early-
harvested Azurro, Kosaro, Cairo, and Integro at the tested
concentrations were the samples that produced colors most
similar to FD&C Red No. 3 with similar λmax and ΔEbetween
6.1 and 8.8 (Table 3).
Stability in Neutral pH. The spectral characteristics of the
samples at pH 7 were monitored over 72 h of refrigerated
storage. Figure 3 shows the changes in λmax for three
representative samples. Solutions colored with Buscaro and
Bandolero red cabbage extracts showed the highest variations in
the λmax, whereas solutions colored with Primero extract
showed the least changes over the 72 h. Most of the changes
observed happened during the first 5−6 h (Figure 3). Table 4
shows the λmax of all seven varieties 30 min after the extracts
were mixed with buffer pH 7 and 6 h afterward (refrigerated
storage). The λmax for samples containing significantly higher
amounts of diacylated anthocyanins (Buscaro and Bandolero)
at this pH seemed to be the highest (≃600 nm) compared to
the other samples. Torskangerpoll and Andersen also
investigated the absorbance and color change in cyanidin 3-
(2″-(2‴-sinapoylglucosyl)-6″-sinapoylglucoside)-5-glucoside (a
diacylated pigment isolated from red cabbage) at different pH
values. They also found out that at pH 7.2 the pigment had λmax
of ≃605 nm.
38
A bathochromic effect of up to 7 nm was
observed for most samples after 6 h of refrigerated storage.
More mature Buscaro and Bandolero varieties produced a λmax
(≃610 nm) similar to that of FD&C Blue No. 2. For other
samples such as Primero and Integro, however, minute
bathochromic effects were observed (Table 4).
Anthocyanins tend to have lower stabilities at neutral to
alkaline pH values.
14,39
After mixing with buffer pH 7 followed
by 6 h of refrigeration, the color degradation was highest
(≃53%) and lowest (≃19%) for the second-harvested Primero
and Buscaro, respectively (Table 4). Higher stabilities of the
Buscaro and Bandolero cultivars at the tested pH values could
be explained by the larger number of diacylated anthocyanins,
which have higher stabilities due to the pigments intramolecular
and/or intermolecular copigmentation and self-association
reactions.
15
Torskangerpoll and Andersen also demonstrated
Figure 3. Change in λmax for the second-harvested Buscaro, Bandolero,
and Primero at pH 7 over 72 h of refrigeration. Most color changes
happened during the first 6 h of storage.
Journal of Agricultural and Food Chemistry Article
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that diacylation of anthocyainins increases their color
stabilities.
38
The color characteristics of pH 7 buffer solutions colored
with the different red cabbage extracts after 6 h of refrigeration
compared to FD&C Blue No. 2 are shown in Table 5. The
lightness of the samples ranged between ≃60 and 85. Chroma
values were higher for Bandolero and Buscaro cultivars at the
tested pH value (Table 5). The synthetic FD&C Blue No. 2
was diluted until the lightness was similar to that of the
evaluated samples. ΔEvalues of the samples when compared to
this synthetic dye were between 17.6 and 26.4 (Table 5). The
color difference with FD&C Blue No. 2 at the tested
concentration for the late-harvested Kosaro sample was the
smallest; however, the λmax values for the second-harvested
Buscaro and Bandolero cultivars were closer to this value for
FD&C Blue No. 2 (Tables 4 and 5). Acylation of anthocyanins
with aromatic acids (e.g., cinnamic acid) has proven to increase
the λmax and shift the hue angle to purple color under acidic
conditions.
16,17
As shown in Table 1, late-harvested Buscaro
and Bandolero cultivars, with the highest percentages of
diacylated pigments, also exhibited the highest λmax under
neutral conditions.
In conclusion, anthocyanin content varied among red
cabbage cultivars, and leaving the cabbages in the ground for
additional time did not increase the pigment content. The
pigment profiles changed among the cultivars and maturation
stages. For most cultivars, the amount of nonacylated and
diacylated pigments increased as opposed to the monoacylated
pigments. Cultivars with lower proportions of diacylated
pigments better reproduced the color of FD&C Red No. 3
under acidic conditions, whereas cultivars with higher
proportions of diacylated pigments better matched the colors
of FD&C Blue No. 2 at neutral pH. Pigment profile also
affected the color stability at neutral pH. For future studies,
Table 4. Maximum Absorbance (λmax) of Red Cabbage
Extracts at Two Different Harvesting Times Measured after
30 min and 6 h of Refrigeration Storage in Buffer pH 7
Compared to FD&C Synthetic Blue No. 2 and Stabilities
(Percent Degradation) during This Time
a
λmax (nm)
cultivar HT
b
(weeks) 30 min 6 h % degradation
Primero 13 591.4 ±0.6 591.6 ±0.8 50.1 ±5.5
21 591.3 ±0.4 591.9 ±1 53.3 ±8.9
Integro 13 591.4 ±1.1 592.1 ±0.1 38.9 ±5.4
21 598.6 ±2.3 599.7 ±244±9.9
Azurro 13 590.8 ±0.8 593.5 ±0.5 41.5 ±4.9
21 595.9 ±0.7 597.8 ±2.1 49.3 ±7.9
Kosaro 13 596.3 ±1 599.9 ±1.4 31 ±3.3
21 602 ±0.3 604.8 ±0.8 39.5 ±5.2
Cairo 13 593.6 ±0.2 595.3 ±4.2 42.1 ±8.4
21 599.7 ±2.4 602.9 ±0.5 36.7 ±8.2
Bandolero 13 601.8 ±1.2 609.4 ±1.2 27.1 ±4
21 599.7 ±2.3 610.1 ±1.6 22.5 ±3.8
Buscaro 13 600.1 ±0.7 606.7 ±1.9 28.6 ±2.3
21 601.8 ±0.4 610.1 ±1.8 19.1 ±3.8
FD&C Blue No. 2 610 610
a
Percent degradation was calculated by dividing the absorbance at λmax
after 6 h by the absorbance at λmax after 30 min in buffer pH 7 ×100.
b
Harvesting time.
Table 5. CIE L*a*b*, Chroma (c*), and Hue (h°) of Seven Red Cabbage Extracts at Two Different Harvesting Times after 6 h
of Refrigeration Storage at pH 7 Compared to FD&C Blue No. 2
cultivar HT
a
(weeks) L*a*b*c*h°ΔE
b
Primero 13 83.6 ±0.6 2.6 ±0.2 −9.9 ±0.6 10.2 ±2.4 284.6 ±1.5 25.7 ±0.5
21 84.8 ±2 2.1 ±0.6 −8.7 ±2.2 9 ±2.2 284.5 ±5.1 26.4 ±2
Integro 13 70.5 ±2.9 1.7 ±2.2 −22.4 ±2.8 22.5 ±2.7 274.7 ±5.8 21.4 ±1
21 69.9 ±3.2 −0.2 ±0.6 −23.5 ±2.6 23.5 ±2.6 269.5 ±1.3 19.8 ±0.7
Azurro 13 77.7 ±1.5 1.4 ±0.2 −15.8 ±1.5 15.9 ±1.4 275.3 ±1.3 21.2 ±1.3
21 78.5 ±3.7 1.2 ±0.4 −15.2 ±3.1 15.3 ±3.1 274.7 ±1.9 21.5 ±1.3
Kosaro 13 72.1 ±1.5 −1.3 ±0.7 −22.1 ±1.9 22.1 ±2.4 266.7 ±1.3 17.9 ±2.3
21 71.9 ±2.5 −1.8 ±1.8 −22.5 ±2 22.6 ±2.2 265.6 ±4.2 17.6 ±1.1
Cairo 13 69.3 ±4.4 −0.5 ±1.7 −24.7 ±4.2 24.7 ±4.2 269.2 ±3.5 20.2 ±1.1
21 66.2 ±3.4 −2.3 ±0.6 −27.9 ±328±3 265.3 ±0.8 20.2 ±1.8
Bandolero 13 60.8 ±1.8 −4.9 ±0.7 −31.9 ±2.7 32.3 ±4.3 261.2 ±1.4 22.9 ±1.2
21 59.6 ±3.1 −5.7 ±0.6 −33.2 ±2.6 33.7 ±2.5 260.2 ±1.6 23.9 ±3.6
Buscaro 13 63.4 ±3−3.8 ±1.5 −29.9 ±2.9 30.2 ±2.1 262.9 ±2.9 21.2 ±1.6
21 63.4 ±1.4 −5.4 ±0.2 −30.6 ±0.8 31 ±0.8 260 ±0.7 20.3 ±1.4
FD&C Blue No. 2 78.06 −17.95 −24.28 30.19 233.52
a
Harvesting time.
b
Color difference with FD&C Blue No. 2.
Journal of Agricultural and Food Chemistry Article
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however, year-to-year variability of these cultivars should be
investigated.
■AUTHOR INFORMATION
Corresponding Author
*(M.M.G.) Phone: (614) 247-8016. Fax: (614) 292-0218. E-
mail: giusti.6@osu.edu.
Funding
We are thankful to MARS Global Chocolate, Hackettstown, NJ,
USA, for providing funding for the project.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
We thank Ken McCammon and Bejo Seeds, Inc., for providing
the plant materials. Also, we especially thank Marçal Plans
Pujolras from Universitat Politècnica de Catalunya for his help
with data statistical analysis.
■ABBREVIATIONS USED
DM, dry matter; FM, fresh matter; AZ, Azurro; Ba, Bandolero;
Bu, Buscaro; Ca, Cairo; In, Integro; Ko, Kosaro; Pr, Primero
■REFERENCES
(1) Giusti, M. M.; Schwartz, S.; Elbe, H. V. Colorants. In Fennema’s
Food Chemistry, 4th ed.; Damodaran, S., Parkin, K., Fennema, O. R.,
Eds.; CRC Press/Taylor & Francis: Boca Raton, FL, USA, 2008; pp
571−638.
(2) Henry, B. S. Natural food colours. In Natural Food Colorants, 2nd
ed.; George, A. F., Hendry, J. D., Eds.; Blackie Chapman & Hall:
London, UK, 1996; pp 40−79.
(3) Wrolstad, R. E.; Smith, D. E. Color analysis. In Food Analysis, 4th
ed.; Nielsen, S. S., Ed.; Springer Science+Business Media: Berlin,
Germany, 2010; pp 573−586.
(4) Socaciu, C. Food Colorants: Chemical and Functional Properties;
Taylor & Francis: Boca Raton, FL, USA, 2008.
(5) Wrolstad, R. E. Symposium 12: Interaction of natural colors with
other ingredients −anthocyanin pigments −bioactivity and coloring
properties. J. Food Sci. 2004,69, C419−C421.
(6) Kong, J.-M.; Chia, L.-S.; Goh, N.-K.; Chia, T.-F.; Brouillard, R.
Analysis and biological activities of anthocyanins. Phytochemistry 2003,
64, 923−933.
(7) Heins, A.; Stockmann, H.; Schwarz, K. Designing “anthocyanin-
tailored”food composition. In Biologically-Active Phytochemicals in
Food; Pfannhauser, W., Fenwick, G. R., Khokhar, S., Eds.; Springer:
Berlin, Germany, 2001; pp 378−381.
(8) He, J. A.; Giusti, M. M. Anthocyanins: natural colorants with
health-promoting properties. Doyle, M. P., Klaenhammer, T. R., Eds.
Annu. Rev. Food Sci. Technol. 2010,1, 163−186.
(9) Ghosh, D.; Konishi, T. Anthocyanins and anthocyanin-rich
extracts: role in diabetes and eye function. Asia Pac. J. Clin. Nutr. 2007,
16, 200−208.
(10) Piccaglia, R.; Marotti, M.; Baldoni, G. Factors influencing
anthocyanin content in red cabbage (Brassica oleracea var capitata Lf
rubra (L) Thell). J. Sci. Food Agric. 2002,82, 1504−1509.
(11) McDougall, G. J.; Fyffe, S.; Dobson, P.; Stewart, D.
Anthocyanins from red cabbage −stability to simulated gastro-
intestinal digestion. Phytochemistry 2007,68, 1285−1294.
(12) Wiczkowski, W.; Szawara-Nowak, D.; Topolska, J. Red cabbage
anthocyanins: profile, isolation, identification, and antioxidant activity.
Food Res. Int. 2013,51, 303−309.
(13) Mazza, G.; Miniati, E. Anthocyanins in fruits, vegetables, and
grains; CRC Press: Boca Raton, FL, USA, 1993.
(14) Cabrita, L.; Fossen, T.; Andersen, O. M. Colour and stability of
the six common anthocyanidin 3-glucosides in aqueous solutions. Food
Chem. 2000,68, 101−107.
(15) Giusti, M. M.; Wrolstad, R. E. Acylated anthocyanins from
edible sources and their applications in food systems. Biochem. Eng. J.
2003,14, 217−225.
(16) Stintzing, F. C.; Stintzing, A. S.; Carle, R.; Frei, B.; Wrolstad, R.
E. Color and antioxidant properties of cyanidin-based anthocyanin
pigments. J. Agric. Food Chem. 2002,50, 6172−6181.
(17) Giusti, M. M.; Rodriguez-Saona, L. E.; Wrolstad, R. E. Molar
absorptivity and color characteristics of acylated and non-acylated
pelargonidin-based anthocyanins. J. Agric. Food Chem. 1999,47, 4631−
4637.
(18) Walkowiak-Tomczak, D.; Czapski, J. Colour changes of a
preparation from red cabbage during storage in a model system. Food
Chem. 2007,104, 709−714.
(19) Tamura, H.; Yamagami, A. Antioxidative activity of mono-
acylated anthocyanins isolated from Muscat Bailey A grape. J. Agric.
Food Chem. 1994,42, 1612.
(20) Dyrby, M.; Westergaard, N.; Stapelfeldt, H. Light and heat
sensitivity of red cabbage extract in soft drink model systems. Food
Chem. 2001,72, 431−437.
(21) Chalker-Scott, L. Environmental significance of anthocyanins in
plant stress responses. Photochem. Photobiol. 1999,70,1−9.
(22) Connor, A. M.; Luby, J. J.; Tong, C. B. S.; Finn, C. E.; Hancock,
J. F. Genotypic and environmental variation in antioxidant activity,
total phenolic content, and anthocyanin content among blueberry
cultivars. J. Am. Soc. Hortic. Sci. 2002,127,89−97.
(23) Awad, M. A.; de Jager, A.; van der Plas, L. H. W.; van der Krol,
A. R. Flavonoid and chlorogenic acid changes in skin of ‘Elstar’and
‘Jonagold’apples during development and ripening. Sci. Hortic.−
Amsterdam 2001,90,69−83.
(24) Solomon, A.; Golubowicz, S.; Yablowicz, Z.; Grossman, S.;
Bergman, M.; Gottlieb, H. E.; Altman, A.; Kerem, Z.; Flaishman, M. A.
Antioxidant activities and anthocyanin content of fresh fruits of
common fig (Ficus carica L.). J. Agric. Food Chem. 2006,54, 7717−
7723.
(25) Fawole, O. A.; Opara, U. L. Effects of maturity status on
biochemical content, polyphenol composition and antioxidant capacity
of pomegranate fruit arils (cv. ‘Bhagwa’). S. Afr. J. Bot. 2013,85,23−
31.
(26) Josuttis, M.; Verrall, S.; Stewart, D.; Krueger, E.; McDougall, G.
J. Genetic and environmental effects on tannin composition in
strawberry (Fragaria ×ananassa)cultivarsgrownindifferent
European locations. J. Agric. Food Chem. 2013,61, 790−800.
(27) Wrolstad, R. E.; Durst, R. W.; Giusti, M. M.; Rodriguez-Saona,
L. E. Analysis of anthocyanins in nutraceuticals. In Quality Management
of Nutraceuticals; American Chemical Society: Washington, DC, USA,
2001; Vol. 803, pp 42−62.
(28) Giusti, M. M.; Wrolstad, R. E. Characterization of red radish
anthocyanins. J. Food Sci. 1996,61, 322−326.
(29) Arapitsas, P.; Sjoberg, P. J. R.; Turner, C. Characterisation of
anthocyanins in red cabbage using high resolution liquid chromatog-
raphy coupled with photodiode array detection and electrospray
ionization-linear ion trap mass spectrometry. Food Chem. 2008,109,
219−226.
(30) Wu, X. L.; Prior, R. L. 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. J. Agric. Food Chem. 2005,53,
3101−3113.
(31) Charron, C. S.; Clevidence, B. A.; Britz, S. J.; Novotny, J. A.
Effect of dose size on bioavailability of acylated and nonacylated
anthocyanins from red cabbage (Brassica oleracea L. var. capitata). J.
Agric. Food Chem. 2007,55, 5354−5362.
(32) Park, S.; Arasu, M. V.; Lee, M. K.; Chun, J. H.; Seo, J. M.; Lee, S.
W.; Al-Dhabi, N. A.; Kim, S. J. Quantification of glucosinolates,
anthocyanins, free amino acids, and vitamin C in inbred lines of
cabbage (Brassica oleracea L.). Food Chem. 2014,145,77−85.
(33) Scalzo, R. L.; Genna, A.; Branca, F.; Chedin, M.; Chassaigne, H.
Anthocyanin composition of cauliflower (Brassica oleracea L. var.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf501991q |J. Agric. Food Chem. 2014, 62, 7524−75317530

botrytis) and cabbage (B. oleracea L. var. capitata) and its stability in
relation to thermal treatments. Food Chem. 2008,107, 136−144.
(34) Sun, J. H.; Xiao, Z. L.; Lin, L. Z.; Lester, G. E.; Wang, Q.;
Harnly, J. M.; Chen, P. Profiling polyphenols in five brassica species
microgreens by UHPLC-PDA-ESI/HRMSn. J. Agric. Food Chem.
2013,61, 10960−10970.
(35) Wu, X. L.; Beecher, G. R.; Holden, J. M.; Haytowitz, D. B.;
Gebhardt, S. E.; Prior, R. L. Concentrations of anthocyanins in
common foods in the United States and estimation of normal
consumption. J. Agric. Food Chem. 2006,54, 4069−4075.
(36) Linus Pauling Institute: Micronutrient Research for Optimum
Health, Oregon State University; http://lpi.oregonstate.edu/
infocenter/phytochemicals/flavonoids/flavtab2.html (accessed July
15, 2013).
(37) Holton, T. A.; Cornish, E. C. Genetics and biochemistry of
anthocyanin biosynthesis. Plant Cell 1995,7, 1071−1083.
(38) Torskangerpoll, K.; Andersen, Ø. M. Colour stability of
anthocyanins in aqueous solutions at various pH values. Food Chem.
2005,89, 427−440.
(39) Fossen, T.; Cabrita, L.; Andersen, O. M. Colour and stability of
pure anthocyanins influenced by pH including the alkaline region.
Food Chem. 1998,63, 435−440.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf501991q |J. Agric. Food Chem. 2014, 62, 7524−75317531