Original article

Extraction and identification of anthocyanin from purple corn

(Zea mays L.)
Zhendong Yang,
1
Zhijie Chen,
1
Shulin Yuan,
1
Weiwei Zhai,
1
* Xiangshu Piao
2
& Xianglan Piao
3
1 Food Engineering Department of Jiangsu Food Science College, Huaian, Jiangsu, China, 223003
2 State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China, 100094
3 Institute of Chinese Minority Traditional Medicine, Minzu University of China, Beijing, China, 100081
(Received: 13 November 2008; Accepted in revised from 7 July 2009)
Summary An ecient extraction of anthocyanin from purple corn (Zea mays L.) was investigated in this paper.
Tristimulus colourimetry was used to evaluate the process quantitatively and qualitatively. Purple corn
anthocyanin was extracted with 1 n HCl95% ethanol (15:85, v v) at dierent extraction temperatures (30
70 C), times (60120 min) and solidliquid ratio (1:201:40). The combined eects of extraction conditions
on anthocyanin yield and colour attributes were studied using a three-level three-factor BoxBehnken design.
The results showed that the highest yield of anthocyanin from purple corn (6.02 mg g
)1
) were obtained at
70 C, extraction time 73 min, and solidliquid ratio 1:25. Three kinds of non-acylated anthocyanins were
detected and characterised as cyanidin-3-glucoside, pelargonidin-3-glucoside and peonidin-3-glucoside by
HPLC-MS.
Keywords Anthocyanin, extraction, HPLC-MS, optimisation, purple corn.
Introduction
There are various breeds of corn in the world showing
various colours such as white, yellow, red, purple,
brown, green and blue. Purple corn is a pigmented
variety of Zea mays L., originally cultivated in Andes of
South America and was introduced to China long time
ago. This corn variety is mainly grown in Shanxi and
Anhui Province, China.
Purple corn was rich in anthocyanin. Purple corn
anthocyanin was characterised as cyanidin-3-glucoside,
pelargonidin-3-glucoside, peonidin-3-glucoside and their
respective malonated counterparts (Aoki et al., 2001;
Pascual-Teresa et al., 2002). Currently, it is interesting
to note the relevance of some under utilised cereals as a
potential source of anthocyanin for food-colourant
purposes. Several patens exist that describe various
preparations and application processes for their use as
colourants (Mazza & Miniati, 1993). Recently, antho-
cyanin has been reported to have various biological
activities, such as antioxidant, anti-mutagenic and
anticancer activities (Yoshimoto et al., 2001; Ka hko nen
& Heinoner, 2003; Katsube et al., 2003). Especially,
most of the properties attributed to purple corn extracts
were related to anthocyanin, including colouring
attributes (Duhard et al., 1997; Cevallos-Casals &
Cisneros-Zevallos, 2004), antioxidant (Cevallos-Casals
& Cisneros-Zevallos, 2003a), antimicrobial (Cevallos-
Casals & Cisneros-Zevallos, 200b), anti-obesity activity
and amelioration of hyperglycaemia (Tsuda et al., 2003)
and anticarcinogenic properties (Hagiwara et al., 2001).
Thus, there is a need to develop an ecient extraction
technique of anthoycanins from purple corn.
Anthocyanin extraction is commonly carried out with
ethanol containing a small amount of hydrochloric acid
or formic acid with the purpose of obtaining the
avylium cation, which is red and stable in the acid
medium and recommended to prevent the degradation
of the non-acylated compounds (Gonnet & Fenet, 2000;
Lapornik et al., 2005; Longo & Vasapollo, 2006).
Current studies on anthocyanin from purple corn are
focused on their stability, structure and physiological
functionality (Aoki et al., 2001; Hagiwara et al., 2001;
Pascual-Teresa et al., 2002; Tsuda et al., 2003; Cevallos-
Casals & Cisneros-Zevallos, 2004). However, little
information on the detailed extraction process param-
eters of purple corn anthocyanin is available in open
literatures.
Colour is one of the most important attributes of
natural colourants. The application of colourimetric
systems, based on uniform colour spaces (CIELUV and
*Correspondent: Fax: +86 0517 87088039,
e-mail: zaiww6810@126.com
International Journal of Food Science and Technology 2009, 44, 24852492 2485
doi:10.1111/j.1365-2621.2009.02045.x
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
CIELAB) and non-uniform colour spaces (CIEXYZ), is
of great value in the quantication and characterisation
of the colour properties of pigments and foods. The
correlation between some colour parameters and pig-
ments content in food has been evaluated in many
studies (Arias et al., 2000; Lee, 2001; Sinnecher et al.,
2002; Mele ndez-Mart nez et al., 2003; Kammerer et al.,
2004; Montes et al., 2005), although the relationships
between the total anthocyanin content and colour
parameters of purple corn extracts have not been
studied during extraction process.
In this study, the main processing conditions (extrac-
tion temperature, time and solidliquid ratio) on the
extraction of purple corn anthocyanin, was investigated.
The colour properties of anthocyanin extracts were
estimated by Tritimulus Colourimetry. The aim was to
determine the optimal condition for ecient quantitative
and qualitative (related tocolour properties) extraction of
anthocyanin from purple corn in order to be used as food
colourant. In addition, we identied the anthocyanin
constituents of extracts from purple corn by HPLC-MS.
Materials and methods
Sample preparation
Purple corn (Zea mays L. cv. ZiRuo1) was generously
supplied from National Sweet Potato Research Insti-
tute, Xuzhou, China. These corns were pulverised with
the grinder (FSD-100A, Taizhou city, Zhejiang prov-
ince, China) and sifted through a 100 mesh sieve.
Samples were kept at 4 C.
Extraction of purple corn anthocyanin
Purple corn anthocyanins were extracted as described by
Fan et al. (2008a). One gram of sample was used for
determination. The powder of purple corn was put into
a 50 mL conical tube and then 1.5 n HCl95% ethanol
(15:85, v v) with dierent solidliquid ratio (1:151:40)
was added. The mixture was put in thermostatic water
bath at selected temperatures of 2070 C for time of
30180 min, and centrifuged at 4000 g for 15 min.
The supernatant was collected and transferred into
50 mL volumetric ask for the determination of antho-
cyanin yield.
Experimental design
Firstly, the single factor experiment for extraction was
performed with the analysis of the eect of three factors
(temperature, solvent-solid ratio and time) on extraction
of anthocyanin from purple corn. Secondly, the optimi-
sation of anthocyanin extraction parameters through
three parameters was performed using BoxBehnken
design (Table 3) and a model was developed. Thirdly,
the model validation and the eect of extraction times
on anthocyanin yield were carried out (Table 6). Finally,
the anthocyanin constituents of extracts were identied
by HPLC-MS.
Response surface methodology (RSM) is an aective
statistical technique for optimising complex processes. It
is widely used in nding optimal condition of process
variables. The basic theoretical and fundamental aspects
of RSM have been reviewed (Farooq et al., 1997;
Chandrika &Fereidoom, 2005). The experimental design
and statistical analysis were performed using Stat-Ease
software (Design-Expert 6.0.10 Trial, Delaware, USA
Echip, 1993). A three-level three-factor BoxBehnken
design was chosen to evaluate the combined eect of three
independent variables, extraction temperature, time and
solidliquid ratio, coded as A, B and C, respectively. The
minimum and maximum values for extraction tempera-
ture were set at 30 and 70 C, extraction time between
60 and 120 min and solidliquid ratio 1:20 and 1:40
(Table 1). The response values were anthocyanin pigment
yield, L*, C* and h. The complete design consisted of
seventeen combinations including ve replicates of the
centre point (Table 2) (Myers &Montgomery, 2002). The
responses function (Y) was partitioned into linear, qua-
dratic and interactive components,
Y b
0

X
k
i1
B
i
X
i

X
k
i1
B
ii
X
2
i

X
k
i>j
B
ij
X
i
X
j
1
where Y stands for total anthocyanin yield. b
0
denotes
the model intercept, B
i
, B
ii
and B
ij
represent the
coecients of the linear, quadratic and interactive eect,
respectively, and X
i
, and X
j
are the coded independent
variables; k equals to the number of the tested factors
(k = 3). The analysis of variance (anova) tables were
generated and the eect and regression coecients of
individual linear, quadratic and interaction terms were
determined. The signicances of all terms in the poly-
nomial were judged statistically by computing the
F-value at a probability (P) of 0.001, 0.01 or 0.05. The
regression coecients were then used to make statistical
calculations to generate contour maps from the regres-
sion models.
Table 1 Independent variables and their coded and actual values used
for optimisation
Independents
variables Units Symbol
Code levels
)1 0 1
Temperature C A 30 50 70
Time min B 60 90 120
Solidliquid ratio 1:X C 20 30 40
Extraction of anthocyanin from purple corn Z. Yang et al. 2486
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
Determination of total anthocyanin content of purple corn
extract
Purple corn anthocyanins were quantied following the
spectrophotometric method proposed by Francis (1989).
The concentration of anthocyanin was determined using
the LambertBeer law. The factor 98.2 is the molar
absorption value of cyanidin-3-glucoside for the acid
ethanol solvent and it refers to the absorption of a
mixture of cranberry anthocyanin in acidethanol,
measured in a 1-cm cell at 535 nm, at a concentration
of 1% (w v).
The spectra recorded in a diode array spectropho-
tometer (UV-2802, UNICO, FL, USA) were measured
at 25 C and 530 nm, against the solvent. For that
purpose 1-cm quartz cells were used. The anthocyanin
yield (mg g
)1
) was calculated using the following
equation:
Total anthocyanin (TA) A
530
dilution factor=98:2
2
Colour coordinates
Tristimulus parameters (L*, C* and h) were calculated
using CR-S4w software (CR400, Konica Minolta,
Japan), based on CIELAB colour space, and with
reference to standard observation angle of ten visual
eld and a standard illuminator D65 at Dk of 5 nm (Fan
et al., 2008b).
Purication of anthocyanin from purple corn extracts
Anthocyanin extracts of purple corn were puried
according to the procedure described by Fuleki &
Francis (1968). The Amberlite CG-50 resins (50 g each
time) were hydrated by placing in a beaker with repeated
decantation and removed the ne particles with distilled
water. Water slurry of the hydrated resin was poured
into a 26 400 mm column, and the excess water was
allowed to drain out without letting the column dry.
Approximately 15 mL of aqueous extract was poured
on the top of the column until the entire resin bed
became red due to the absorbed anthocyanin. Anthocy-
anin were absorbed onto the resin column while sugars,
acids and other water-soluble compounds were removed
by washing the column with 100 mL of distilled water as
judged by the refractive index of the liquid coming o
the column. The pigments were eluted by adding ethanol
containing 0.01% HCl (approximately 50 mL) until the
resin returned to its original colour. The eluate was
concentrated with a rotary evaporator at 45 C under
vacuum until the ethanol was evaporated, and the
residue was dissolved in 0.5% HCl solvent. The solution
was stored at )20 C until further analysis.
Identication of anthocyanin from purple corn extracts
by HPLC-MS
High-performance liquid chromatography (HPLC) sys-
tem consisted of a Waters 2690 pump coupled with a
Waters 2996 photodiode array detector. Data analysis
was performed with Waters HPLC chem-station soft-
ware. Solvents and samples were ltered through a
Table 2 BoxBehnken design and experiment data for anthocyanin extraction from purple corn
No
Independent variables Dependent variables
Temperature
(C)
Time
(min)
Solidsolvent
ratio
Anthocyanin
yield (mg g
)1
) L* C* h
1 30()1) 60()1) 30(0) 4.13 0.05 20.55 0.08 4.28 0.05 24.31 0.12
2 70(1) 60()1) 30(0) 5.99 0.05 19.52 0.10 5.46 0.07 23.15 0.07
3 30()1) 120(1) 30(0) 3.82 0.04 20.67 0.09 4.11 0.07 24.22 0.13
4 70(1) 120(1) 30(0) 5.27 0.07 19.94 0.07 4.62 0.03 23.52 0.03
5 30()1) 90(0) 20()1) 3.43 0.10 20.78 0.14 4.28 0.11 24.34 0.09
6 70(1) 90(0) 20()1) 5.60 0.04 19.72 0.04 5.04 0.09 24.16 0.01
7 30()1) 90(0) 40(1) 4.64 0.08 20.29 0.09 4.49 0.07 25.20 0.18
8 70(1) 90(0) 40(1) 5.60 0.07 19.74 0.11 4.88 0.05 24.22 0.07
9 50(0) 60()1) 20()1) 4.47 0.07 20.47 0.06 4.54 0.06 24.90 0.06
10 50(0) 120(1) 20()1) 4.15 0.09 20.54 0.04 4.47 0.18 24.46 0.05
11 50(0) 60()1) 40(1) 4.73 0.04 20.15 0.07 4.65 0.07 24.99 0.09
12 50(0) 120(1) 40(1) 4.46 0.01 20.49 0.09 4.66 0.01 24.50 0.14
13 50(0) 90(0) 30(0) 5.10 0.06 20.34 0.07 4.71 0.09 24.80 0.07
14 50(0) 90(0) 30(0) 5.30 0.08 19.80 0.06 4.87 0.15 24.81 0.08
15 50(0) 90(0) 30(0) 5.26 0.07 19.91 0.15 4.56 0.03 24.49 0.11
16 50(0) 90(0) 30(0) 5.21 0.14 20.00 0.07 4.73 0.04 24.38 0.08
17 50(0) 90(0) 30(0) 5.47 0.07 19.74 0.01 4.92 0.02 24.20 0.02
Extraction of anthocyanin from purple corn Z. Yang et al. 2487
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
0.45 lm lter. Separations were carried out on a
Lichrospher C
18
column (5 lm, 2.1 250 mm i.d.).
Simultaneous monitoring was performed at 520 nm at a
ow rate 0.3 mL min
)1
. The running temperature was at
35 C and the injection volume was 10 lL. A gradient
elution system of solvent A [0.05% (v v) triuoroacetic
acid (TFA)] and B (acetonitrile) was used [5% solvent B
(0 min); 20% solvent B (20 min); 4% solvent B
(50 min)]. The ow rate was 0.8 mL min
)1
. The antho-
cyanin were checked using electrospray MS with a
Waters Platform ZMD 4000 mass spectrometer
equipped with an ion spray interface (ISV = 4400).
Spectra were recorded in positive ion mode between m z
200 and 1300.
Statistics
All trials were carried out in triplicate and all the data
were reported as means standard deviation (SD).
The statistics signicance was evaluated using Students
t-test and P < 0.05 was taken as signicant.
Results and discussion
Preliminary experiments for setting up BoxBehnken
design
The eect of temperature on anthocyain yield was
shown in Table 3. The anthocyanin yield increased
signicantly (P < 0.05) with the temperature from 10 to
50 C, but it did not increase any further above 50 C.
This suggested that the optimal temperature for antho-
cyanin extraction was 50 C. Tu rker & Erdog du (2006)
also suggested that as the temperature raised (2550 C),
the anthocyanin yield increased. However, higher tem-
perature may also result in degradation of anthocyanin
as reported by Cacace & Mazza (2003).
The results showed in Table 3 indicated that the
anthocyanin yield increased signicantly (P < 0.05)
when the extraction time was extended from 30 to
90 min. The yield of anthocyanin slight decreased but
not signicantly in statistical analysis (P > 0.05) when
the time was extended from 90 to 120 min. It decreased
signicantly (P < 0.05) when the extraction time were
between 120 and 180 min. This indicated that the
optimal time for anthocyanin extraction was 60
120 min.
Table 3 showed the eect of dierent solidliquid
ratios on anthocyanin yield. The anthocyanin yield
increased (P < 0.05) when the solidliquid ratio was
from fteen to thirty, but it did not increased when the
ratio was higher than 30. This suggested that the
solventsolid ratio of thirty was the optimal ratio for
anthocyanin extraction.
Analysis of BoxBehnken experiment
The results of each dependent variable with their
coecients of determination (R
2
) were summarised in
Table 4. The statistical analysis indicates that the
proposed model was adequate, possessing no signicant
lack of t and with very satisfactory values of the R
2
for
all the responses. The R
2
values for anthocyanin yield,
L*, C* and h were 0.981, 0.905, 0.854 and 0.852,
respectively. Coecient of variances (Table 4) for
anthocyanin yield, L*, C* and h were within the
acceptable range. In general, a high coecient of
variances indicates that variation in the mean value is
high and does not satisfactorily develop an adequate
response model (Chandrika & Shahidi, 2005). The
probability (P) values of all regression models were less
than 0.05.
The eects of extraction temperature, time and solid
liquid ratio on anthocyanin yields, L*, C* and h are
reported (Table 4) by the coecient of the second order
polynomials. Response surface and contour plots were
used to illustrate the eect of extraction temperature,
extraction time and solidliquid ratio on the responses.
Response surfaces and contour plots for anthocyanin
yield were shown in Figs S1S3.
Figure S1 showed the contour map for the eect of
extraction temperature and time on the yield of antho-
cyanin from purple corn. As shown in Table 4, antho-
cyanin yield depends on the extraction temperature as
Table 3 Effect of different extraction conditions on anthocyanin yield
Index
Temperature (C) (60 min, 1:30)
20 30 40 50 60 70
Anthocyanin
yield (mg g
)1
)
2.22 0.09
d
2.87 0.12
c
3.28 0.04
b
3.95 0.02
a
3.99 0.04
a
4.05 0.11
a
Time (min) (50 C, 1:30)
30 60 90 120 150 180
3.95 0.04
d
4.37 0.08
c
4.88 0.10
a
4.72 0.12
ab
4.60 0.07
bc
4.48 0.11
cd
Solidliquid ratio (50 C, 90 min)
1:15 1:20 1:25 1:30 1:35 1:40
2.81 0.05
c
2.98 0.14
c
3.57 0.10
b
3.88 0.05
a
3.95 0.08
a
4.01 0.05
a
Values with different superscripts (a, b, c, etc.) differ signicantly (P < 0.05).
Extraction of anthocyanin from purple corn Z. Yang et al. 2488
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
its negative linear eect (P < 0.01) was signicant,
whereas its quadratic eect were not signicant
(P > 0.05), which resulted in a curvilinear increase in
anthocyanin yield for all the extraction times. It can be
seen that the extraction temperature was the main eect
on the anthocyanin yield. In this case, the extraction
time had only a slight inuence, especially when the
extraction temperature is low. When the extraction time
was longer than 90 min, the anthocyanin yield decreased
signicantly (P < 0.01).
The contour map for eect of temperature and solid
liquid ratio on anthocyanin yield was shown in Fig. S2.
Anthocyanin yield depends on the solidliquid ratio as
its linear (P < 0.01) and quadratic eects (P < 0.001)
were signicant (Table 4). The linear eect (P < 0.01)
was positive, whereas the quadratic eect (P < 0.001)
was negative, which resulted in an overall curvilinear
increase in anthocyanin yield for all extraction temper-
ature. When the solidliquid ratio increased up to about
thirty, temperature became the critical factor for
improving anthocyanin yield.
Figure S3 showed the contour map for the eect of
the time and solidliquid ratio on anthocyanin yield.
The solidliquid ratio where its linear (P < 0.01) and
quadratic eects (P < 0.001) were signicantly inu-
enced the anthocyanin yield (Table 4). The anthocyanin
yield increased when the solventsolid ratio increased
from twenty to thirty but it did not continue to increase
signicantly when the ratio was higher than thirty. At
the lowest level of time, anthocyanin yield of purple corn
increased rapidly at the beginning but with a decreasing
towards the end. It can be seen that the positive linear
and negative quadratic eects of solidliquid ratio and
temperature explained the observed nature of the curve
shown in Fig. S3.
The contour plots showed the optimum conditions of
the extraction process to anthocyanin yield. There are a
number of combinations of variables that could give
maximum levels of anthocyanin yield. Since the opti-
mum response for each dependent variable did not fall
exactly in the same region, the superimposition of all the
contour plots obtained was done. During the anthocy-
anin extraction, the extraction temperature, time and
solidliquid ratio are important. Therefore, the best
combination of process variables for response functions
was found. The process variables for the best combina-
tion of response function are extraction temperature
70 C, time 73 min and solidliquid ratio 1:25. The
response functions were calculated from the nal poly-
nomial, and the response were 6.02 mg g
)1
for antho-
cyanin yield, 19.52 for L*, 5.23 for C*and h for 23.69,
respectively. Cevallos-Casals & Cisneros-Zevallos
(2003a) reported that the anthocyanin content is
16.42 mg g
)1
(fresh weight) in whole purle corn. For
purple corn cob, the anthocyanin content is 5.90 mg g
)1
(dry weight) (Yang et al., 2008). Compared with other
fruits, such as grape (0.252.60 mg g
)1
, fresh weight)
(Arozarena et al., 2002), blackberry (0.672.30 mg g
)1
,
fresh weight) (Wang & Xu, 2007) and Jaboticaba
(0.0440.163 mg g
)1
, fresh weight) (Montes et al.,
2005), the yield of anthocyanin was relatively high in
purple corn, which made this cereal a good source for
anthocyanin.
The eects of temperature, time and solidliquid on
the yield of anthocyanins from purple corn were
investigated in this paper. However, the other important
factor, such as diusion coecients, particle size,
characteristics of biological material, co-pigmentation
reactions and complexation etc., were not taken into
account and could play a crucial role in the extraction
yield. So, these factors need thus further research.
Correlations between anthocyanin yield and the colour
parameters
Correlations between the yield of anthocyanin and the
colour parameters were explored in this study (Fig. S4).
A negative correlation (r = )0.9263) was found with
L*, indicating that higher L* values were related to
lower yield of anthocyanin. The positive correlation
were found between the yield of anthocyanins and C*
(r = 0.7808). This positive correlation indicated that
high C* values were related to high yield of anthocy-
anins. These results were in agreement with Montes
et al. (2005) who evaluated the correlations between the
anthocyanin yield and C* and L* in Juboticaba fruit.
Table 4 Regression coefcients, R
2
, and CV values for four dependents
variables for the extraction of purple corn
Coefcient
Anthocyanin
yield L* C* h
b
0
(intercept) 5.270 19.960 4.760 24.540
Liner
B
1
0.810*** )0.420*** 0.360 )0.380**
B
2
)0.200** 0.120 )0.130 )0.081
B
3
0.220** )0.110 0.044 0.130
Quadratic
B
11
)0.050 )0.034 )0.024 )0.480**
B
22
)0.420*** 0.250* )0.120 )0.250
B
33
)0.400*** 0.210* )0.061 0.430**
Cross product
B
12
)0.100 0.075 )0.170 0.110
B
13
)0.300** 0.130 )0.093 )0.200
B
23
0.012 0.067 0.020 )0.013
R
2
0.981 0.905 0.854 0.852
CV 3.040 0.900 3.960 1.210
Probability (P) <0.0001*** 0.007** 0.029* 0.030**
Lack of t 0.356 0.989 0.191 0.340
*Signicant at 0.05 level; **signicant at 0.01 level; ***signicant at
0.001 level.
Extraction of anthocyanin from purple corn Z. Yang et al. 2489
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
However, the correlations between yield of anthocya-
nins and h was too low (r = )0.367).
Verication of model
Within the scopes of the variables investigated in
BoxBehnken design, additional experiments with
dierent conditions for anthocyanin extraction were
carried out in order to assess the validity of the model
(eqn 1). The arrangement and result of the conrma-
tory trials were shown in Table 5. It was demon-
strated that there was a high t degree between the
values observed in experiment and the values pre-
dicted by eqn 1.
Effect of repeated extraction on the anthocyanin
yield
Four times of repeated extraction of anthocyanin from
purple corn under the optimal conditions (70 C,
73 min, 1:25) were performed in order to obtain the
maximum anthocyanin yield (Table 6). In fact, the rst
two extractions (6.63 mg g
)1
) produced as much as
94.31% of the entire anthocyanin content (7.03 mg g
)1
)
in purple corn, implying that two times of extraction
would be enough to get a desired anthocyanin yield
from purple corn.
Identications of anthocyanin from purple corn extracts by
HPLC-MS analysis
Reverse phase HPLC and MS analysis were used to
rapidly identify the anthocyanin from purple corn
extracts. The identication was carried out by compar-
ison of their retention time and by conrming the
molecular weight with ESI-MS (Aoki et al., 2001;
Pascual-Teresa et al., 2002).
Figure S4 shows the chromatograms of anthocya-
nins from purple corn, which were detected at 520 nm.
Three peaks were identied in the chromatograms,
indicating the existence of three types of anthocya-
nin. Their contents were 73.3%, 9.3% and 17.4%,
respectively.
Three types of anthocyanin were identied shown in
Figs S5 and S6. They were identied as cyanidin-3-
glucoside, pelargonidin-3-glucoside and peonidin-3-glu-
coside, respectively (Fig. S5). Peak 1 showed [M]
+
at
m z 449 and fragment ion of loss of a glucose [M-162]
+
at m z 287. It was identied as cyanidin-3-glucoside.
Peak 2, with [M]
+
at m z 433, fragment ion [M-162]
+
at
m z 271, was identied as pelargonidin-3-glucoside.
Peak 3 appeared a [M]
+
at m z 463 and the fragment
ion of less of glucose [M-162]
+
at m z 301. It was
identied as peonidin-3-glucoside (Fig. S5).
In this study, some anthocyanin components of
purple corn extracts were identied similar with the
previous reports (Aoki et al., 2001; Pascual-Teresa
et al., 2002). However, cyanidin-3-(6-malon-glucoside),
pelargonidin-3-(6-malon-glucoside) and peonidin-3-(6-
malon-glucoside) were not detected, which have been
identied by Aoki et al. (2001).
Conclusions
The dierent conditions for anthocyanin extraction
recealed that extraction temperature, time and solid
liquid ratio markedly aect the anthocyanin yield.
These can be related to the anthocyanin extraction
conditions by second-order polynomials. Using the
contour plots, the optimum set of the operating
variables are obtained graphically in order to obtain
the desired levels of these properties of the purple corn
anthocyanin, which is suitable for the subsequent
analysis based clarication processes. The best combi-
nation of response function are extraction temperature
70 C, time 73 min, and solidliquid ratio 1:25. Three
types of anthocyanin from purple corn extracts were
separated and identied them as cyanidin-3-glucoside,
pelargonidin-3-glucoside and peonidin-3-glucoside
using HPLC-MS analysis.
Table 5 Arrangement and result of conrmatory trials
Trials Temperature (C)
Time
(min)
Solventsolid
ratio
Anthocyanin yield (mg g
)1
)
Observed value Predicted value
Optimum condition 70 73 25 5.98 0.15 6.02
Random condition 1 50 100 40 5.05 0.11 4.98
Random condition 2 70 60 30 5.90 0.18 5.91
Table 6 Effect of extraction times on anthocyanin yield and extraction
rate of purple corn anthocyanins (70 C, 90 min, 1:30)
Index
Extraction stage
First Second Third Fourth
Anthocyanin
yield (mg g
)1
)
4.79 0.24 1.84 0.13 0.29 0.09 0.11 0.02
Extraction
ratio (%)
68.14 26.17 4.12 1.57
Extraction of anthocyanin from purple corn Z. Yang et al. 2490
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Contour plots for the eects of temperature
and time at a constants solid-liquid ratio of 1:30 on the
yield of anthocyanin from purple corn.
Figure S2. Contour plots for the eects of temperature
and solid liquid ratio at a constants time of 90 min on
the yield of anthocyanin from purple corn.
Figure S3. Contour plots for the eects of time and
solid-liquid ratio at a constants of 50 C on the yield of
anthocyanin from purple corn.
Extraction of anthocyanin from purple corn Z. Yang et al. 2491
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
Figure S4. The calibration curve of corrections between
anthocyanin yeild and the colour parameters (L* and
C*).
Figure S5. HPLC chromatogram at 520 nm correspond-
ing to purple corn extract.
Figure S6. Positive ion mass spectra of anthocyanin
constituents in purple corn extract: (1) cyanin-3-gluco-
side; (2) pelargonidin-3-glucoside; (3) peonidin-3-gluco-
side; Glu = glucoside.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
Extraction of anthocyanin from purple corn Z. Yang et al. 2492
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology