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Extraction and identification of anthocyanin from purple corn (Zea mays L.)

Article  in  International Journal of Food Science & Technology · October 2009

DOI: 10.1111/j.1365-2621.2009.02045.x

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International Journal of Food Science and Technology 2009, 44, 2485–2492 2485

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 Piao2 & Xianglan Piao3
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 efficient 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 HCl–95% ethanol (15:85, v ⁄ v) at different extraction temperatures (30–
70 C), times (60–120 min) and solid–liquid ratio (1:20–1:40). The combined effects of extraction conditions
on anthocyanin yield and colour attributes were studied using a three-level three-factor Box–Behnken 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 solid–liquid 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.

were related to anthocyanin, including colouring

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; Kähkönen
& Heinoner, 2003; Katsube et al., 2003). Especially,
most of the properties attributed to purple corn extracts
*Correspondent: Fax: +86 0517 87088039,
e-mail: zaiww6810@126.com
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 efficient 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
flavylium 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

doi:10.1111/j.1365-2621.2009.02045.x
 2009 The Authors. Journal compilation  2009 Institute of Food Science and Technology
2486 Extraction of anthocyanin from purple corn Z. Yang et al.
CIELAB) and non-uniform colour spaces (CIEXYZ), is
of great value in the quantification 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; Meléndez-Martı́nez 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 solid–liquid 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 efficient quantitative
and qualitative (related to colour properties) extraction of
anthocyanin from purple corn in order to be used as food
colourant. In addition, we identified 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 HCl–95% ethanol
(15:85, v ⁄ v) with different solid–liquid ratio (1:15–1:40)
was added. The mixture was put in thermostatic water
bath at selected temperatures of 20–70 C for time of
30–180 min, and centrifuged at 4000 · g for 15 min.
The supernatant was collected and transferred into
50 mL volumetric flask for the determination of antho-
cyanin yield.
Experimental design
Firstly, the single factor experiment for extraction was
performed with the analysis of the effect 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 Box–Behnken
design (Table 3) and a model was developed. Thirdly,
International Journal of Food Science and Technology 2009
the model validation and the effect of extraction times
on anthocyanin yield were carried out (Table 6). Finally,
the anthocyanin constituents of extracts were identified
by HPLC-MS.
Response surface methodology (RSM) is an affective
statistical technique for optimising complex processes. It
is widely used in finding 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 Box–Behnken
design was chosen to evaluate the combined effect of three
independent variables, extraction temperature, time and
solid–liquid 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 solid–liquid 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 five replicates of the
centre point (Table 2) (Myers & Montgomery, 2002). The
responses function (Y) was partitioned into linear, qua-
dratic and interactive components,
X
k X
k X
k
Y ¼ b0 þ Bi Xi þ Bii X2i þ Bij Xi Xj ð1Þ
i¼1 i¼1 i>j
where Y stands for total anthocyanin yield. b0 denotes
the model intercept, Bi, Bii and Bij represent the
coefficients of the linear, quadratic and interactive effect,
respectively, and Xi, and Xj 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 effect and regression coefficients of
individual linear, quadratic and interaction terms were
determined. The significances 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 coefficients 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
Code levels
Independents
variables Units Symbol )1 0 1
Temperature C A 30 50 70
Time min B 60 90 120
Solid–liquid ratio 1:X C 20 30 40
 2009 The Authors. Journal compilation  2009 Institute of Food Science and Technology
 Extraction of anthocyanin from purple corn Z. Yang et al. 2487

Table 2 Box–Behnken design and experiment data for anthocyanin extraction from purple corn

Independent variables Dependent variables

Temperature Time Solid–solvent Anthocyanin

No (°C) (min) ratio 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

Determination of total anthocyanin content of purple corn
extract
Purple corn anthocyanins were quantified following the
spectrophotometric method proposed by Francis (1989).
The concentration of anthocyanin was determined using
the Lambert–Beer 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 acid–ethanol,
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) ¼ A530  dilution factor=98:2
ð2Þ
Purification of anthocyanin from purple corn extracts
Anthocyanin extracts of purple corn were purified
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 fine 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 off
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.

Colour coordinates Identification of anthocyanin from purple corn extracts

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
field and a standard illuminator D65 at Dk of 5 nm (Fan
et al., 2008b).
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 filtered through a

 2009 The Authors. Journal compilation  2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
2488 Extraction of anthocyanin from purple corn Z. Yang et al.
0.45 lm filter. Separations were carried out on a
Lichrospher C18 column (5 lm, 2.1 · 250 mm i.d.).
Simultaneous monitoring was performed at 520 nm at a
flow 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) trifluoroacetic
acid (TFA)] and B (acetonitrile) was used [5% solvent B
(0 min); 20% solvent B (20 min); 4% solvent B
(50 min)]. The flow 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 significance was evaluated using Student’s
t-test and P < 0.05 was taken as significant.
Results and discussion
Preliminary experiments for setting up Box–Behnken
design
The effect of temperature on anthocyain yield was
shown in Table 3. The anthocyanin yield increased
significantly (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. Türker & Erdoğdu (2006)
also suggested that as the temperature raised (25–50 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 significantly (P < 0.05)
when the extraction time was extended from 30 to
90 min. The yield of anthocyanin slight decreased but
not significantly in statistical analysis (P > 0.05) when
Table 3 Effect of different extraction conditions on anthocyanin yield
Temperature (C) (60 min, 1:30)
Index 20 30
Anthocyanin 2.22 ± 0.09d 2.87 ± 0.12c
yield (mg g)1) Time (min) (50 C, 1:30)
30 60
3.95 ± 0.04d 4.37 ± 0.08c
Solid–liquid ratio (50 C, 90 min)
1:15 1:20
2.81 ± 0.05c 2.98 ± 0.14c
Values with different superscripts (a, b, c, etc.) differ significantly (P < 0.05).
International Journal of Food Science and Technology 2009
the time was extended from 90 to 120 min. It decreased
significantly (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 effect of different solid–liquid
ratios on anthocyanin yield. The anthocyanin yield
increased (P < 0.05) when the solid–liquid ratio was
from fifteen to thirty, but it did not increased when the
ratio was higher than 30. This suggested that the
solvent–solid ratio of thirty was the optimal ratio for
anthocyanin extraction.
Analysis of Box–Behnken experiment
The results of each dependent variable with their
coefficients of determination (R2) were summarised in
Table 4. The statistical analysis indicates that the
proposed model was adequate, possessing no significant
lack of fit and with very satisfactory values of the R2 for
all the responses. The R2 values for anthocyanin yield,
L*, C* and h were 0.981, 0.905, 0.854 and 0.852,
respectively. Coefficient of variances (Table 4) for
anthocyanin yield, L*, C* and h were within the
acceptable range. In general, a high coefficient 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 effects of extraction temperature, time and solid–
liquid ratio on anthocyanin yields, L*, C* and h are
reported (Table 4) by the coefficient of the second order
polynomials. Response surface and contour plots were
used to illustrate the effect of extraction temperature,
extraction time and solid–liquid ratio on the responses.
Response surfaces and contour plots for anthocyanin
yield were shown in Figs S1–S3.
Figure S1 showed the contour map for the effect 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
40 50 60 70
3.28 ± 0.04b 3.95 ± 0.02a 3.99 ± 0.04a 4.05 ± 0.11a
90 120 150 180
4.88 ± 0.10a 4.72 ± 0.12ab 4.60 ± 0.07bc 4.48 ± 0.11cd
1:25 1:30 1:35 1:40
3.57 ± 0.10b 3.88 ± 0.05a 3.95 ± 0.08a 4.01 ± 0.05a
 2009 The Authors. Journal compilation  2009 Institute of Food Science and Technology
 Extraction of anthocyanin from purple corn Z. Yang et al. 2489

2
Table 4 Regression coefficients, R , and CV values for four dependents increased rapidly at the beginning but with a decreasing
variables for the extraction of purple corn towards the end. It can be seen that the positive linear
and negative quadratic effects of solid–liquid ratio and
Anthocyanin
Coefficient yield L* C* h°
temperature explained the observed nature of the curve
shown in Fig. S3.
b0 (intercept) 5.270 19.960 4.760 24.540 The contour plots showed the optimum conditions of
Liner the extraction process to anthocyanin yield. There are a
B1 0.810*** )0.420*** 0.360 )0.380** number of combinations of variables that could give
B2 )0.200** 0.120 )0.130 )0.081 maximum levels of anthocyanin yield. Since the opti-
B3 0.220** )0.110 0.044 0.130
mum response for each dependent variable did not fall
Quadratic
exactly in the same region, the superimposition of all the
B11 )0.050 )0.034 )0.024 )0.480**
B22 )0.420*** 0.250* )0.120 )0.250
contour plots obtained was done. During the anthocy-
B33 )0.400*** 0.210* )0.061 0.430** anin extraction, the extraction temperature, time and
Cross product solid–liquid ratio are important. Therefore, the best
B12 )0.100 0.075 )0.170 0.110 combination of process variables for response functions
B13 )0.300** 0.130 )0.093 )0.200 was found. The process variables for the best combina-
B23 0.012 0.067 0.020 )0.013 tion of response function are extraction temperature
R2 0.981 0.905 0.854 0.852 70 C, time 73 min and solid–liquid ratio 1:25. The
CV 3.040 0.900 3.960 1.210 response functions were calculated from the final poly-
Probability (P) <0.0001*** 0.007** 0.029* 0.030**
nomial, and the response were 6.02 mg g)1 for antho-
Lack of fit 0.356 0.989 0.191 0.340
cyanin yield, 19.52 for L*, 5.23 for C*and h for 23.69,
*Significant at 0.05 level; **significant at 0.01 level; ***significant at respectively. Cevallos-Casals & Cisneros-Zevallos
0.001 level. (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
its negative linear effect (P < 0.01) was significant, fruits, such as grape (0.25–2.60 mg g)1, fresh weight)
whereas it’s quadratic effect were not significant (Arozarena et al., 2002), blackberry (0.67–2.30 mg g)1,
(P > 0.05), which resulted in a curvilinear increase in fresh weight) (Wang & Xu, 2007) and Jaboticaba
anthocyanin yield for all the extraction times. It can be (0.044–0.163 mg g)1, fresh weight) (Montes et al.,
seen that the extraction temperature was the main effect 2005), the yield of anthocyanin was relatively high in
on the anthocyanin yield. In this case, the extraction purple corn, which made this cereal a good source for
time had only a slight influence, especially when the anthocyanin.
extraction temperature is low. When the extraction time The effects of temperature, time and solid–liquid on
was longer than 90 min, the anthocyanin yield decreased the yield of anthocyanins from purple corn were
significantly (P < 0.01). investigated in this paper. However, the other important
The contour map for effect of temperature and solid– factor, such as diffusion coefficients, particle size,
liquid ratio on anthocyanin yield was shown in Fig. S2. characteristics of biological material, co-pigmentation
Anthocyanin yield depends on the solid–liquid ratio as reactions and complexation etc., were not taken into
its linear (P < 0.01) and quadratic effects (P < 0.001) account and could play a crucial role in the extraction
were significant (Table 4). The linear effect (P < 0.01) yield. So, these factors need thus further research.
was positive, whereas the quadratic effect (P < 0.001)
was negative, which resulted in an overall curvilinear
Correlations between anthocyanin yield and the colour
increase in anthocyanin yield for all extraction temper-
parameters
ature. When the solid–liquid ratio increased up to about
thirty, temperature became the critical factor for Correlations between the yield of anthocyanin and the
improving anthocyanin yield. colour parameters were explored in this study (Fig. S4).
Figure S3 showed the contour map for the effect of A negative correlation (r = )0.9263) was found with
the time and solid–liquid ratio on anthocyanin yield. L*, indicating that higher L* values were related to
The solid–liquid ratio where it’s linear (P < 0.01) and lower yield of anthocyanin. The positive correlation
quadratic effects (P < 0.001) were significantly influ- were found between the yield of anthocyanins and C*
enced the anthocyanin yield (Table 4). The anthocyanin (r = 0.7808). This positive correlation indicated that
yield increased when the solvent–solid ratio increased high C* values were related to high yield of anthocy-
from twenty to thirty but it did not continue to increase anins. These results were in agreement with Montes
significantly when the ratio was higher than thirty. At et al. (2005) who evaluated the correlations between the
the lowest level of time, anthocyanin yield of purple corn anthocyanin yield and C* and L* in Juboticaba fruit.

 2009 The Authors. Journal compilation  2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
2490 Extraction of anthocyanin from purple corn Z. Yang et al.

Table 5 Arrangement and result of confirmatory trials

Anthocyanin yield (mg g)1)

Time Solvent–solid
Trials Temperature (°C) (min) ratio 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

However, the correlations between yield of anthocya-
nins and h was too low (r = )0.367).
Verification of model
Within the scopes of the variables investigated in
Box–Behnken design, additional experiments with
different conditions for anthocyanin extraction were
carried out in order to assess the validity of the model
(eqn 1). The arrangement and result of the confirma-
tory trials were shown in Table 5. It was demon-
strated that there was a high fit 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 first
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.
ison of their retention time and by confirming 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 identified 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 identified shown in
Figs S5 and S6. They were identified 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 identified as cyanidin-3-glucoside.
Peak 2, with [M]+ at m ⁄ z 433, fragment ion [M-162]+ at
m ⁄ z 271, was identified 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
identified as peonidin-3-glucoside (Fig. S5).
In this study, some anthocyanin components of
purple corn extracts were identified 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
identified by Aoki et al. (2001).

Identifications of anthocyanin from purple corn extracts by Conclusions

HPLC-MS analysis
The different conditions for anthocyanin extraction
Reverse phase HPLC and MS analysis were used to recealed that extraction temperature, time and solid–
rapidly identify the anthocyanin from purple corn liquid ratio markedly affect the anthocyanin yield.
extracts. The identification was carried out by compar- These can be related to the anthocyanin extraction
conditions by second-order polynomials. Using the
contour plots, the optimum set of the operating
Table 6 Effect of extraction times on anthocyanin yield and extraction variables are obtained graphically in order to obtain
rate of purple corn anthocyanins (70 C, 90 min, 1:30) the desired levels of these properties of the purple corn
anthocyanin, which is suitable for the subsequent
Extraction stage
analysis based clarification processes. The best combi-
Index First Second Third Fourth nation of response function are extraction temperature
70 C, time 73 min, and solid–liquid ratio 1:25. Three
Anthocyanin 4.79 ± 0.24 1.84 ± 0.13 0.29 ± 0.09 0.11 ± 0.02 types of anthocyanin from purple corn extracts were
yield (mg g)1)
separated and identified them as cyanidin-3-glucoside,
Extraction 68.14 26.17 4.12 1.57
ratio (%)
pelargonidin-3-glucoside and peonidin-3-glucoside
using HPLC-MS analysis.

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 effects 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 effects 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 effects of time and
solid-liquid ratio at a constants of 50 C on the yield of
anthocyanin from purple corn.
International Journal of Food Science and Technology 2009
2492 Extraction of anthocyanin from purple corn Z. Yang et al.
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-
International Journal of Food Science and Technology 2009
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side; (2) pelargonidin-3-glucoside; (3) peonidin-3-gluco-
side; Glu = glucoside.
Please note: Wiley-Blackwell are not responsible for the
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 2009 The Authors. Journal compilation  2009 Institute of Food Science and Technology