Optimization of microwave-assisted extraction of anthocyanins from purple corn

(Zea mays L.) cob and identication with HPLCMS

Zhendong Yang, Weiwei Zhai
Food engineering department of Jiangsu food science college, Huaian, Jiangsu 223003, China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 13 August 2009
Accepted 9 March 2010
Editor Proof Receive Date 15 April 2010
Keywords:
Purple corn cob
Anthocyanins
Optimization
Extraction
Microwave
HPLCMS
A BoxBehnken design was used to obtain the optimal conditions of microwave-assisted extraction (MAE). The
effects of operating conditions, such as extraction time, solidliquid ratio, and microwave irradiation power, on
the extraction yield of anthocyanins were studied through a response surface methodology (RSM). The highest
total anthocyainin content (TAC) from purple corn cob (185.1 mg/100 g) was obtained at an extraction time of
19 min, a solidliquidratio of 1:20, and a microwave irradiationpower of 555 W. Six major kinds of anthocyanins
were detected and identied as cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside, and their
respective malonated counterparts. In comparison with the conventional solvent extraction, MAE was highly
efcient and rapid in extracting anthocyanins from Chinese purple corn cob.
Industrial relevance: In the last decades, the interest in anthocyanin pigments has increased because of their
possible utilization as natural food colorants and especially as antioxidant and anti-inammatory agents. Purple
corncobwas the byproduct duringthecornprocessing. Purplecorncobis darkpurple toalmost black color due to
its high content of anthocyanins, which makes this byproduct a good source of anthocyanins.
2010 Published by Elsevier Ltd
1. Introduction
Anthocyanins, widely found in the roots, caudexes, leaves, as well
as owers and fruits of tall plants, are used as substitutes for synthetic
pigments because of their attractive color and physiological function-
ality (Mazza & Miniati, 1993). Anthocyanins also possess known
pharmacological properties and are used by humans for therapeutic
purposes (Francis, 1989). An increasing number of studies have
demonstrated that anthocyanins have the ability to protect against a
myriad of human diseases, such as liver dysfunction, hypertension,
vision disorders, microbial infections, and diarrhea (Mazza & Kay,
2008; Smith, Marley, Seigler, Singletary, & Meline, 2000).
Purple cornis a pigmentedvariety of Zea mays L., originally cultivated
in the Andes region of South America. It was introduced to China a long
time ago. This corn variety is now mainly grown in China, especially in
the provinces of Shanxi and Anhui. Purple corn is animportant source of
anthocyanins, which have potential applications as natural food color-
ants and antioxidants. Such applications are widely used in Asia, South
America, and Europe (Aoki, Kuze, & Kato, 2002; Jing &Giusti, 2005). The
anthocyanins present in Andean purple corn, owers, leaves, cobs, and
seeds have previously been characterized, and the major anthocyanins
found were cyanidin-3-dimalonylglucoside, cyanidin-3-glucoside,
pelargonidin-3-glucoside, peonidin-3-glucoside, and their respective
malonated counterparts (Aoki et al., 2002; Fossen, Slimestad, &
Andersen, 2001; Harborne & Self, 1997; Pascual-Teresa, Santos-Buelga,
&Rivas-Gonzalo, 2002). Althoughpurple corn cob canobviously be used
as a natural colorant, existing literature and recent reports have focused
only on analyzing the stability and structure of anthocyanins found in
Peruvian purple corn varieties or their derivatives.
Usually, conventional solvent extraction of anthocyanins is time
and solvent-consuming and has lowefciency (Sun, Liao, Wang, Hu, &
Chen, 2007). Moreover, thermal extraction over a long time can cause
the degradation of anthocyanins and decrease the antioxidant activity
of the extracts (Camel, 2000; Lapornik, Proek, & Wondra, 2005).
Anthocyanins also may also undergo denaturalization when they are
extracted from a natural source. The extraction process involves a loss
of color followed by the formation of brownish degradation products
and insoluble compounds (Castillo-Sanchez, Mejuto, Garrido, &
Garca-Falcn, 2006). Microwave-assisted extraction (MAE) utilizes
the energy of microwaves to cause molecular movement and rotation
of liquids with a permanent dipole, leading to rapid heating of the
solvent and the sample. It has advantages over conventional
extraction techniques, such as improved efciency, reduced extrac-
tion time, low solvent consumption, and high level of automation
(Buldini et al., 2002; Eskilsson & Bjorklund, 2000). Aside from these
advantages, a wider range of solvents can be used in MAE, as the
technique is less dependent on high solvent afnity (Eskilsson &
Bjorklund, 2000). Recently, many reports have been made on the
application of MAE on the extraction of natural products, such as
artemisinin (Hao, Han, Huang, Xue, & Deng, 2002), ginseng saponins
(Kwon, Blanger, & Par, 2003), essential oils (Gomez & Witte, 2001),
and anthocyanins (Sun et al., 2007). However, no information has yet
Innovative Food Science and Emerging Technologies 11 (2010) 470476
Corresponding author. Tel./fax: +86 517 87088869.
E-mail address: zaiww6810@126.com (W. Zhai).
1466-8564/$ see front matter 2010 Published by Elsevier Ltd
doi:10.1016/j.ifset.2010.03.003
Contents lists available at ScienceDirect
Innovative Food Science and Emerging Technologies
j our nal homepage: www. el sevi er. com/ l ocat e/ i f set
been found on the application of MAE for the extraction of
anthocyanin in purple corn cob.
Response surface methodology (RSM) is an affective statistical
technique for optimizing complex processes. It is widely used in
optimizing the process variables. Upon reviewing the basic theoretical
and fundamental aspects of RSM, color was found to be one of the most
important attribute of natural colorants (Chandrika & Fereidoon, 2005;
Farooq, Imran, &Khaled, 1997). The application of colorimetric systems,
based on uniform color spaces (CIELUV and CIELAB) and non-uniform
color spaces (CIEXYZ), is of great value in the quantication and
characterization of the color properties of pigments and foods. The
correlation between some color parameters and pigment content in
food has been evaluated in many studies (Lee, 2001; Melndez-
Martnez, Vicario, & Heredia, 2003; Montes, Vicario, Raymundo, Fett, &
Heredia, 2005).
In this paper, MAE parameters, such as the ratio of solvents to
materials, microwave irradiation power, and extraction time, were
optimized by RSM in order to obtain the optimal extraction yield of
anthocyanin extracts from purple corn cob. In addition, the anthocya-
nins composition of the extracts was determined by HPLCMS, and the
effect of MAE on the extraction efciency was evaluated in comparison
to the conventional solvent extraction method.
2. Materials and methods
2.1. Material
Purple corn (Z. mays L. cv Heizhenzhu) cob was generously
supplied by Prof. Zhong Zhang of the Anhui Technical Teachers
College (Fengyang City, Anhui Province, China). The cobs were dried
in a heated air dryer (50 C) (ZT-3, Jiangdu City, Jiangsu Province,
China), pulverized by a disintegrator (FSD-100A, Taizhou City,
Zhejiang Province, China), and sifted through a 100-mesh sieve. The
purple corn cob powder (water content b12%) was sealed in a brown
bottle and kept at 4 C.
2.2. Extraction of purple corn cob anthocyanin
The MAE procedure used in the experiment was a modied version
of the method developed by Sun et al. (2007). Briey, the powders of
purple corn cob were put into a 50 mL double-necked ask with a
cooling system. After the ask was lled with a known volume of
solvent, which was selected according to the designed solvent-to-
sample ratio [1.5 M HCl95% ethanol (15:85)], it was exposed in a
microwave extractor (Model NJL07-3, Jiequan microwave equipment
Co., Ltd., Nanjing, China) for a certain period of time under a given
microwave power. The ask was taken out. The solution was cooled
with water under room temperature, ltered through Whatman No. 1
paper under vacuum, and then collected in a volumetric ask. The
residue was taken back and extracted again under the same conditions.
The anthocyaninextracts fromthe two extractionsteps were mixed and
used for the determination of the total anthocyanin content (TAC).
About 5 g of the samples was used for each treatment.
2.3. Experimental design
RSMwas used to determine the optimumconditions for extraction
of anthocyanin in purple corn cob. The experimental design and
statistical analysis were done using Stat-Ease software (Design-Expert
6.0.10 Trial, Delaware, USAEchip, 1993). Athree-level three-factor Box
Behnken design was chosen to evaluate the combined effect of three
independent variables, extraction time, solidliquid ratio, and micro-
wave irradiation power. There were termed as X
1
, X
2
and X
3
,
respectively. The minimum and maximum values for extraction time
were set at 10 and 30 min. The solidliquid ratio was set at 1:10 and
1:30. The microwave irradiationpower was set between400 and600 W
(Table 1). The complete design consisted of 17 combinations, including
ve replicates of the center point (Table 2), and the response function
(Y) was partitioned into linear, quadratic, and interactive components,
Y =
0
+
k
i =1
B
i
X
i
+
k
i =1
B
ii
X
2
i
+
k
i N j
B
ij
X
i
X
j
1
where Y stands for total anthocyanin yield;
0
denotes the model
intercept; B
i
, B
ii
, and B
ij
represent the coefcients of the linear,
quadratic, and interactive effects, respectively; X
i
, and X
j
are the coded
independent variables; and k equals the number of tested factors
(k=3). Tables of the analysis of variance (ANOVA) were generated,
and the effect and regression coefcients of individual linear,
quadratic, and interactive terms were determined. The signicances
of all terms in the polynomial were evaluated statistically by
computing the F-value at the probability (p) of 0.001, 0.01, or 0.05.
The regression coefcients were then used to make statistical
calculations to generate contour maps from the regression models.
2.4. Determination of the total anthocyanin content (TAC)
The TAC was determined according to the pH-differential methods
(Lee, Durst, & Wrosltad, 2005). An aliquot of the sample (1 mg) was
placedintoa 25 mL volumetric askandmadeuptothenal volume with
pH 1.0 buffer (0.025 M potassium chloride). Another 1 mg of the sample
was placed into a 25 mL volumetric ask and made up to a nal volume
withpH4.5buffer (0.4 Msodiumacetate). Absorbancewas measuredbya
spectrophotometer (UV-2802, UNICO) at 510 and 700 nm. Absorbance
was calculated as Abs=[(A
510
A
700
) at pH 1.0][(A
510
A
700
) at pH
4.5] with a molar extinction coefcient of 26,900 for cyanindin-3-
glucoside. The TAC was calculated using Eq. (2) and expressed as
milligrams of cyanindin-3-glucoside equivalents per 100 g dry cods. All
TCA shown was normalized with the amount of cyanindin-3-glucoside.
TAC mg=100g
AB
eL
MW D
V
G
100 2
where Abs is absorbance, e is the cyanindin-3-glucoside molar
absorbance (26,900), L is the cell path length (1 cm), MW is the
molecular weight of anthocyanin (449.2 Da), Dis the dilutionfactor, Vis
the nal volume (mL), and M is the dry cob weight (mg).
2.5. Color coordinates
Tristimulus parameters (L*, C*, and h) were calculated using CR-
S4wsoftware (CR400, Konica Minolta, Japan), basedonthe CIELABcolor
space. A standard illuminator D65 was used at a of 5 nm (Fan, Han,
Gu, & Gu, 2008).
2.6. Conventional extraction of anthocyanins from purple corn cob
The conventional extraction procedure used in this study was a
modied version of the process described by Fuleki and Francis (1968).
Atotal of 1 g of samples was mixedwith30 mL of 1.5 NHCl95%ethanol
(15:85) in a 50 mL round bottomask tted with a cooling system. The
extraction temperature was set at 551 C, equivalent to the mean
temperature under optimized conditions determined through the MAE
Table 1
Independents variables and their coded and actual values used for optimization.
Independents variables Units Symbol Code levels
1 0 1
Extraction time min X
1
10 20 30
Solidliquid ratio 1: X X
2
10 20 30
Microwave irradiation power W X
3
400 500 600
471 Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470476
process. Extractions were carried out for 30, 60, 90, and 120 min. The
anthocyaninextracts werecooledtoroomtemperature, lteredthrough
Whatman No. 1 paper in a vacuum, and collected in a volumetric ask.
The residue was gathered and extracted again under the same
conditions. The anthocyanin extracts of the two extractions were
combined and used to determine the TAC.
2.7. Purication of anthocyanin from purple corn cob extracts
Anthocyanin extracts of purple corn were puried according to the
procedure described by Sun et al. (2007). Amberlite CG-50 resins
(50 g each time) were hydrated by placing in a beaker with water and
performing repeated decantation. The ne particles were removed
with distilled water. An aqueous slurry of the hydrated resin was
poured into a 26400 mmcolumn, and the excess water was allowed
to drain out without letting the column dry. Approximately 15 mL of
the aqueous extract was poured on the top of the column until the
entire resin bed became red due to the absorbed anthocyanin.
Anthocyanins were absorbed onto the resin column, while sugars,
acids, and other water-soluble compounds were removed by washing
the column with 100 mL distilled water. This was conrmed by the
refractive index of the liquid coming out of the column. The pigments
were eluted by adding ethanol containing 0.01% HCl (approximately
50 mL) until the resin returned to its original color. The eluate was
concentrated with a rotary evaporator at 45 C in a vacuum until the
ethanol evaporated, and the residue was dissolved in 0.5% HCl solvent.
The solution was stored at 20 C until further analysis.
2.8. Identication of anthocyanin from purple corn cob extracts by
HPLCMS
Identication was performed by hA High-pPerformance lLiquid
cChromatography (HPLC), Waters 2690) and with a Waters 2996
photodiode array detector (Waters 2996) were used. Data analysis
was performed with Waters HPLC chem-station software. Solvents
and samples were ltered through a 0.45 m lter. Chromatographic
analysis was carried outdone using a Lichrospher C-18 (5 m
2.1 mm250 mm i.d.) prodigy. Simultaneous monitoring was per-
formed at 520 nm at a ow rate of 0.8 mL/min. The running
temperature was at 35 C, and the injection volume was 10 L. The
mobile phase Phase A was a mixture of 0.05% (v/v) triuoroacetic acid
(TFA) in distilled water, whereas Mobile B consisted of 100% HPLC-
grade acetonitrile. The separation of anthocyanins was carried
outdone for 40 min. The elution prole was a linear gradient elution
with solvent A at 9580% solvent A from 0 to 20 min and, at 8060%
from 20 to 50 min. The ow rate was 0.8 mL/min. The identity of
anthocyanins was checked conrmed by using electrospray mass
spectrometry (MS) with a Waters Platform ZMD 4000 mass
spectrometer equipped with an ion spray interface (ISV=4400)
operated in the positive-ion mode. Spectra were recorded in positive
ion mode between m/z 200 and 1200.
2.9. Statistical analysis
All trials were carried out in triplicate and all the data were
reported as meanstandard deviation (n=3). The statistical signif-
icance was established using the Students's t-test at pb0.05.
3. Results and discussion
3.1. Statistical analysis
The results of each dependent variable with their coefcients of
determination (R
2
) are summarized in Table 3. Statistical analysis
indicated that the proposed model was adequate, possessing no
signicant lack of t and with satisfactory values of the R
2
for the TAC
and h. The R
2
values for the TAC, L*, C*, and h were 0.996, 0.958, 0.942,
and 0.936, respectively. The coefcients of variances (Table 3) for the
TAC, L*, C*, andh were 1.55, 1.40, 1.20, and4.93, respectively. Ingeneral,
a high coefcient of variances indicates that variation in the mean value
is high and does not satisfactorily develop an adequate response model
(Ravikumar, Ramalingam, Krishnan, & Balu, 2006). The probability (p)
values of all the regression models were less than 0.05.
According to the model (Table 3), the linear terms of extraction
time (X
1
, pb0.01), solidliquid ratio (X
2
, pb0.01), microwave
irradiation power (X
3
, pb0.001), the quadratic terms of extraction
time (X
1
2
, pb0.01), solidliquid ratio (X
2
2
, pb0.01), microwave
irradiation power (X
3
2
, pb0.001) reached statistical signicance. The
interaction of extraction time and solidliquid ratio (X
1
X
2
, pb0.01),
and the extraction time and microwave irradiation power (X
1
X
3
,
pb0.001) also reached statistical signicance. The results suggested
that the change in the above three factors had a signicant effect on
the TAC in the extracts. In contrast, the interaction between the solid
liquid ratio and the microwave irradiation power (X
2
X
3
) was not
statistically signicant. Meanwhile, the whole model, including the
linear level (pb0.01), quadratic level (pb0.001), and cross-product
level (pb0.01), all reached statistical signicance (pb0.01), but the
Table 2
Experimental data and the observed response value with different combinations of extraction time (A), solidsolvent ration (B) and microwave irradiation power (C) used in the
BoxBehnken design.
No Independent variables Dependent variables
X
1
(min) X
2
(1:X) X
3
(W) TAC (mg/100 g) L* C* h
1 10 (1) 10 (1) 500 (0) 129.95.5 32.110.08 30.240.05 10.930.12
2 30 (1) 10 (1) 500 (0) 129.83.5 32.470.10 30.340.07 12.980.07
3 10 (1) 30 (1) 500 (0) 133.16.4 32.820.09 30.020.07 9.530.13
4 30 (1) 30 (1) 500 (0) 110.87.7 19.940.07 4.620.03 12.020.03
5 10 (1) 20 (0) 400 (1) 143.43.1 31.280.14 31.140.11 10.600.09
6 30 (1) 20 (0) 400 (1) 125.14.4 33.100.04 30.730.09 10.270.01
7 10 (1) 20 (0) 600 (1) 148.45.8 30.360.11 31.770.07 8.870.18
8 30 (1) 20 (0) 600 (1) 155.57.7 30.540.08 31.170.05 12.720.07
9 20 (0) 10 (1) 400 (1) 132.62.7 30.470.06 32.540.16 12.030.06
10 20 (0) 30 (1) 400 (1) 125.68.9 32.310.04 32.350.18 12.920.05
11 20 (0) 10 (1) 600 (1) 153.25.4 30.110.07 32.370.07 12.090.09
12 20 (0) 30 (1) 600 (1) 149.63.1 30.860.09 31.370.01 12.490.14
13 20 (0) 20 (0) 500 (0) 182.35.6 29.320.07 32.850.09 13.920.07
14 20 (0) 20 (0) 500 (0) 183.34.8 29.490.06 32.980.15 13.850.08
15 20 (0) 20 (0) 500 (0) 180.82.7 29.310.15 32.580.03 13.240.11
16 20 (0) 20 (0) 500 (0) 183.55.4 29.350.07 32.990.04 14.030.08
17 20 (0) 20 (0) 500 (0) 184.63.7 29.310.01 32.850.02 13.300.02
472 Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470476
lack of t was not signicant, indicating excellent agreement of the
experiment values with the predicted values.
3.2. Analysis of BoxBehnken experiment
When one factor was xed as the optimal value calculated from
the BoxBehnken experiment, the effects of both factors on the
extraction of anthocyanins were shown by the contour optimizer
plots. Contour plots for the TAC are shown in Figs. 13.
The effects of extraction time (X
1
) and solidliquid ratio (X
2
) on
the TAC of the extracts are reected in Fig. 1. With an increase of X
1
and X
2
, the TAC sharply increased, achieving a saturated value when
the extraction was conducted for 20 min at 1:20. It remained at this
value despite further increases in X
1
and X
2
. The relationship between
the TAC and extraction time (X
1
) as well as the microwave irradiation
power (X
3
) is illustrated in Fig. 2. The factor X
3
is as signicant as X
1
in
relation to the TAC. Therefore, we can conclude that the microwave
irradiation power and extraction time is important for attaining a
highly efcient MAE extraction of anthocyanin. This result is in
agreement with a study done by Eskilsson and Bjorklund (2000) on
analytical-scale MAE. Due to the acceleration of the disruption of
tissues and the immigration of solutes from tissues under microwave
power, extraction became more efcient. The effects of the solid
liquid ratio (X
2
) and microwave irradiation power (X
3
) on the TAC in
the extracts are shown in Fig. 3. When the microwave irradiation
power is about 550 W, X
2
becomes the critical factor for improving the
TAC. The uctuation of X
2
can lead to a large difference in the TAC. The
optimal X
2
and X
3
for anthocyanin extraction were about 1:20 and
550 W. If the X
3
was lower than 550 W, the dissolution of
anthocyanins did not reach an equilibrium and part of the anthocya-
nins remained in purple corn cob. If the X
3
was higher than 550 W, the
TAC in the extracts slightly decreased.
The contour plots show the optimum conditions of the extraction
process for anthocyanins. There are a number of combinations of
variables that can give maximum levels of the TAC. During the
anthocyanin extraction, the extraction time, solidliquid ratio, and
microwave irradiation power were the most important process
variables needed to optimize the response in this study. The best
combination of process variables for the best set of response
properties included an extraction time of 19 min, a solidliquid ratio
of 1:20, and a microwave irradiation power of 555 W. The responses
calculated from the nal polynomial functions were 185.1 mg/100 g
for the TAC, 29.05 for L*, 32.88 for C*, and 13.34 for h, respectively.
In this paper, the purple corn (Z. mays L. cv Heizhenzhu) cob
contained higher amounts of anthocyanins than pigmented corns
from the United States (30.7 mg/100 g), Mexico (72.1 mg/100 g), and
Canada (127.7 mg/100 g) (Dep Pozo-Insfran, Brenes, Serna Saldivar, &
Talcott, 2006). Thus, the variety and growth conditions of purple corn
could inuence their anthocyanin content. To prove this, Moreno and
co-workers (Moreno, Snchez, Hernadez, & Lobato, 2005) found that
the TAC of four Mexican blue corn varieties ranged from54 to 115 mg/
100 g. In the present study, the anthocyanin yield in the purple corn
cob was relatively high, which made this material a good source of
anthocyanin.
Table 3
Regression coefcients, R
2
, and CV values for four dependents variables for the
extraction of purple corn cob.
Coefcient TAC (mg/100 g) L* C* h

0
(intercept) 182.90 29.36 32.85 13.67
Liner
X
1
4.20

0.38

0.22 1.01

X
2
3.30

0.41

0.11 0.13
X
3
10.00

0.68

0.01 0.04
Quadratic
X
11
27.08

1.77

1.72

2.04

X
22
29.93

1.31

0.76

0.27
X
33
12.72

0.25 0.07 1.02

Cross product
X
12
5.55

0.03 0.24 0.11

X
13
6.35

0.46 0.05 1.05

X
23
0.85 0.30 0.20 0.12
R
2
0.996 0.958 0.942 0.936
CV 1.55 1.40 1.20 4.93
Probability (p) b0.0001

0.0005

0.0015

0.0021

Lack of t 0.0811 0.0006

0.0209

0.0833
Signicant at 0.05 level.
Signicant at 0.01 level.
Signicant at 0.001 level.
Fig. 1. Contour plots for the effects of extraction time and solidliquid ratio at a
constants microwave irradiation power of 500 W on the TAC from purple corn cob.
Fig. 2. Contour plots for the effects of extraction time and microwave irradiation power
at a constants solidliquid ratio of 1:20 on the TAC from purple corn cob.
473 Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470476
3.3. Correlations between the TAC and the color parameters
The correlations between the TAC and the color parameters were
explored in this study (Table 4). A negative correlation (r=0.899)
was found with L*, indicating that higher L* values corresponded to
lower TAC. In contrast, a positive correlation was found between the
yield of anthocyanins and C* (r=0.730). This means that high C*
values were correlated to high TAC. These results were in agreement
with a study done by Montes et al. (2005), who evaluated the
correlations between the anthocyanin yield and C* and L* in
Jaboticaba fruit. There are no correlations between the TAC and h.
3.4. Comparison of MAE with conventional extraction of anthocyanins in
purple corn cob
Samples were extracted by MAE and conventional extraction,
respectively, in order to evaluate the effects of MAE on the extraction
efciency. As shown in Table 5, the MAE method was more efcient
than the conventional extraction. When purple corn cob samples
were extracted for 10 min, the TAC in the extracts obtained by
conventional method was only 67.8% that of MAE. Even when the
conventional extraction was done for a longer time like 60 min, the
TAC was just 85.6% that of MAE. The TAC did increase as the extraction
time increased. Microwave energy is an electromagnetic source of
radiation that causes molecular motion through the migration of ions
and rotation of dipoles (Beejmohun et al., 2007). Conventional solvent
extraction without microwave assistance is a time-consuming process
that uses heat to increase the mass transfer rate of the extraction
system (Proestos & Komaitis, 2008). In contrast, microwave-assisted
extraction is a fast extraction process where microwave energy is
delivered efciently delivered to materials through molecular
interaction with in the electromagnetic eld. It offers a rapid transfer
of energy to the extraction solvent and raw plant materials (Criado,
Torre, Pereiro, & Torrijos, 2004; Zhou & Liu, 2006). Furthermore, the
direct interaction of microwaves with solvent also results in the
rupture of the plant cells and a quick release of intracellular products
into the solvent (Lay-Keow & Michel, 2003) quickly.
3.5. Identication of anthocyanin from purple corn cob extracts by
HPLCMS analysis
Gradient reversed-phase HPLC with UV detection and MS analysis
were used to rapidly identify anthocyanins in purple corn cob extracts.
The HPLC prole is shown in Fig. 4, and the mass spectral fragmentation
patterns are shown in Fig. 5. If the standard anthocyanins were
unavailable, identication was performed by comparing their retention
timeandmolecular weight withdatafromliterature(Pascual-Teresaet al.,
2002; Pedreschi & Cisneros-Zevallos, 2007).
Six anthocyanins were identied fromthe purple corn cob extracts
(Fig. 5): Peak 1 was cyanidin-3-glucoside with a molecular ion peak
[M+H]
+
at m/z 449 and a fragment ion peak [M+H-162]
+
at m/z
287. This correspondedto the loss of a glucoside residue fromcyanidin-3-
glucoside, yielding cyanidin. Peak 2, with [M]
+
at m/z 433, was identied
as pelargonidin-3-glucoside. The fragment ion peak [M-162]
+
at m/z 271
corresponded to a loss of glucoside from pelargonidin-3-glucoside,
yielding pelargonidin. Peak 3, with [M]
+
at m/z 463, is identied as
peonidin-3-glucoside. The fragment ion peak at m/z 301, indicating a loss
of 162 from peonidin-3-glucoside, was identied as peonidin. Peak 4,
with [M]
+
at m/z 535, was identied as cyanidin-3-(6-malon)-glucoside.
The fragment ion peak [M-16286]
+
at m/z 287 was referred to as
cyanidin, from a loss of glucoside (162) and a malonic acid (86) from
cyanidin-3(6-malon)-glucoside. Peak 5, with [M]
+
at m/z 519, was
identied as pelargonidin-3-(6-malon)-glucoside. The fragment ion peak
at m/z 433, indicating a loss of 162 from pelargonidin-3-glucoside, was
referred to as pelarginidin. Peak 6, with [M]
+
at m/z 549, was identied as
peonidin-3-(6-malon)-glucoside. The fragment ion peaks at m/z 463 and
301 referred to peonidin-3-glucoside and peonidin, respectively.
In this study, the anthocyanins in the purple corn (Z. mays L. cv.
Heizhenzhu) cob extracts showed little differences with those reported
by Pascual-Teresa et al. (2002), such as cyanidin-3-glucoside, pelargo-
nidin-3-glucoside, peonidin-3-glucoside, and their respective
Fig. 3. Contour plots for the effects of microwave irradiation power and solidliquid
ratio at a constants extraction time of 20 min on the TAC from purple corn cob.
Table 4
Correlation coefcients for relations between TAC and color parameters.
Index L* C* h
TAC 0.899 0.730 0.489
Table 5
The comparison of the extraction efciency by MAE method to conventional solvent
extraction.
MAE
a
Conventional solvent extraction
b
19 30 40 60
TAC (mg/100 g) 184.84.2 125.32.7 132.74.4 148.83.1 158.23.8
a
The MAE was carried out at optimized conditions (19 min, 1:20, 555 W).
b
The conventional solvent (1.5 N HC95% ethanol, 15:85) extraction was carried out
55 C, solidsolvent ration 1:30.
Fig. 4. HPLC chromatogram at 520 nm corresponding to purple corn cob extract.
474 Z. Yang, W. Zhai / Innovative Food Science and Emerging Technologies 11 (2010) 470476
malonatedderivatives inthe purple corn(Z. mays L. cv. Heizhenzhu) cob
extract. Cyaniding-3-6-ethylmalonylglucoside, cyaniding-3-6-ethylma-
lonylglucoside, and cyaniding-3-6-ethylmalonylglucoside were not
found. The difference may be attributed to different variants and
growing conditions (Ferreyra, Vina, Mygridge, & Chaves, 2007; Fossen
et al., 2001).
4. Conclusions
In this study, process variables, such as extraction time, solid
liquid ratio, and microwave irradiation power, signicantly affected
the anthocyanin yield. When MAE was conducted at an extraction
time of 19 min, a solidliquid ratio of 1:20, and a microwave
irradiation power of 555 W, the TAC in the extract reached
185.1 mg/100 g (98.85%). Six kinds of anthocyanins were identied
using HPLCMS analysis: cyanidin-3-glucoside, pelargonidin-3-glu-
coside, peonidin-3-glucoside, and their respective malonated coun-
terparts. In this study, the anthocyanins identied in the purple corn
cob extract showed a small difference with those reported by Pascual-
Teresa et al. (2002).
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