a

ISSN 0101-2061 Food Science and Technology

DDOI: http://dx.doi.org/10.1590/1678-457X.6731

Microwave-assisted extraction of anthocyanin from Chinese bayberry

and its effects on anthocyanin stability
Wenkai DUAN1,2, Shiping JIN2, Guofu ZHAO2, Peilong SUN1*

Abstract
Anthocyanins are present in high concentrations in Chinese bayberry, Myrica rubra Sieb. & Zucc. Herein, a microwave-assisted
extraction was used to extract the anthocyanins from Chinese bayberry. The HPLC chromatogram of the extracts showed that
the anthocyanin components were slightly hydrolysed during the extraction process. Further experiments confirmed that
microwave irradiation slightly hydrolysed cyanidin-3-O-glucoside to cyanidin, but did not significantly influence the antioxidant
activity of the extracts. Optimized extraction conditions for total anthocyanin content were a solid-to-liquid ratio, extraction
temperature, and extraction time of 1:50, 80 °C, and 15 min, respectively. Under these conditions, the anthocyanin content
was 2.95 ± 0.08 mg·g−1, and the antioxidant activity yield was 279.96 ± 0.1 μmol.·g−1 Trolox equivalent on a dry weight basis.
These results indicated that microwave-assisted extraction was a highly efficient extraction method with reduced processing
time. However, under some extraction conditions it could damage the anthocyanins. These results provide an important guide
for the application of microwave extraction.
Keywords: Chinese bayberry; anthocyanin extraction; microwave-assisted extraction; cyanidin-3-O-glucoside; anthocyanin
stability.
Practical Application: This study showed that microwave irradiation would slightly hydrolysed cyanidin-3-O-glucoside to
cyanidin, it provide an important guide for the application of microwave extraction.

1 Introduction
Chinese bayberry, Myrica rubra Sieb. & Zucc., is one of the supercritical fluid, pressurized fluid, and microwave-assisted
six Myrica species native to China (Chen et al., 2004), and is noted extraction (Schantz et al., 1997; Lopez‑Avila et al., 1994).
for the attractive red colour of its fruits. Many pigments were
Microwave-assisted extraction (MAE) has been demonstrated
extracted, identified, and characterized from different natural to be a fast and efficient unconventional extraction method
sources, especially red fruit extracts, for their safe use as colorants. that was developed for extracting analytes from solid matrixes,
Anthocyanins are among the most broadly distributed pigment in particular, secondary metabolites from plant material
groups in plants, and are present at high concentration in Chinese (Kaufmann & Christen, 2002), such as saponins from ginseng
bayberry. The major anthocyanin present in bayberry fruits was (Kwon et al., 2003; Vongsangnak et al., 2004), glycyrrhizic
identified as cyanidin-3-O-glucoside (C-3-G), which represents acid from licorice root (Pan et al., 2000), anthocyanins in red
more than 95% of the total pigment content (Bao et al., 2005). raspberries (Sun et al., 2007), and alkaloids from the seeds of
Anthocyanins from Chinese bayberry can protect β-cells Lupinus mutabilis (Ganzler et al., 1990). Microwave energy is a
from oxidative-stress mediated injury (Zhang et al., 2011) and non-ionizing radiation that results in molecular movement by
contribute to promoting good health and reducing the risk of migration of ions and rotation of molecules with permanent
chronic disease (Philpott et al., 2004). dipoles in liquids, without altering their molecular structures
Extraction is the first step in the commercial isolation of unless the temperature is too high (Kaufmann & Christen, 2002).
anthocyanins. Conventional extraction techniques for solid Microwave extraction greatly reduces solvent consumption and
matrices include the well-known Soxhlet extraction, sonication, extraction times and improves extraction efficiency (Eskilsson
and blending. Although efficient extractions can be achieved & Björklund, 2000).
using these simple techniques, they present major drawbacks. The stability of anthocyanin is very important during the
In particular, they feature long extraction times (especially for extraction, and is influenced by numerous factors, including
Soxhlet extraction), high solvent consumption, and low temperatures, temperature, pH, water activity, and light (Amr & Tamini, 2007).
and they are not yet readily automated (Heemken et al., 1997; Recently, a study suggested that vitamin C was unstable under
Egizabal et al., 1998). Over the last decade, new techniques have microwave conditions (Yuan et al., 2009), whereas some
emerged that will supersede traditional techniques. These include confirmed that microwaving preserved bioflavonoid content

Received 18 May, 2015

Accepted 03 August, 2015
1
Laboratory of Food Energy, Zhejiang University of Technology, Hangzhou, PR, China
2
Taizhou Vocational College of Science & Technology, Taizhou, PR, China
*Corresponding author: sun_peilong@163.com

524 Food Sci. Technol, Campinas, 35(3): 524-530, Jul.-Set. 2015

 Duan et al.

(Gorinstein et al., 2008; El-Adawy, 2002). Thus, the effect of and absorbance of A = [(A520 − A700)pH 1.0 − (A520 − A700)pH 4.5].
microwave irradiation on the stability of anthocyanin needs to The data were reported as mean ± standard deviation for three
be further studied. replications.
Herein, MAE was used to extract antioxidant compounds
from Chinese bayberry. The aim of this work was to elucidate 2.5 HPLC-DAD-ESI-MS analysis
the optimal extraction conditions and evaluate whether it affects HPLC-DAD-ESI-MS analysis was performed using a Waters
the structure or antioxidant activity of the extracted compounds. platform system, which was composed of a Micromass QUATTRO
MICROTM API mass spectrometer, a Waters 600 pump system,
2 Materials and methods and a Waters 2996 photodiode array detector (Waters Corp.,
2.1 Chemicals and reagents Milford, MA, USA). Data were collected and processed on a
personal computer running MassLynx software (Micromass, a
Cyanidin-3-O-glucoside standard was obtained from division of Waters Corp., Beverly, MA, USA). Aliquots of bayberry
Extrasynthèse (Genay, France). 6-Hydroxy-2,5,7,8-tetramethyl- extracts (20 μL) were resolved using a SUNFIRE C-18 column
chroman-2- carboxylic acid (Trolox) and 2,2-Diphenyl-1- (250×4.6 mm, 5μm, Waters Corp., Milford, MA, USA), where
picrylhydrazyl (DPPH) were purchased from Sigma Chemical solvent A was 0.05% (v/v) formic acid in water, and solvent B
Co. (St. Louis, MO, USA). Acetonitrile (HPLC grade), formic was 0.05% (v/v) formic acid in acetonitrile, and the flow rate
acid, ethanol, and hydrochloric acid (analytical grade) were was 0.7 mL·min−1. The injection volume was 20 μL, and the
purchased from Shanghai Chemical Reagent Company (China). detection wavelengths was 520 nm. The elution system was:
All solutions were prepared using distilled-deionized water. 0–5 min = 5% solvent B, 5–10 min = linear gradient from 5–10%
of solvent B, 10–25 min = linear gradient from 10–90% of solvent
2.2 Plant material B, and 25–50 min = 90% solvent B. Cyanidin-3-O-glucoside was
Myrica rubra Sieb. & Zucc. cv. Dongkui belongs to the genus used as a standard for quantitation of cyanidin-3-O-glucoside
Myrica in the family Myricaceae. Mature Chinese bayberry content, the ratio of cyanidin-3-O-glucoside to total anthocyanin
fruits were obtained from a fruit market in Taizhou, Zhejiang was expressed to total anthocyanin content divided by the content
Province, China in June 2013. The samples were immediately of cyanidin-3-O-glucoside.
dried in a lyophilizer (Labconco, Kansas City, USA), and then Mass spectra were obtained using electrospray ionization in
ground and sifted for homogenization before storing at −80 °C positive ion mode. The following ion optics were used: capillary
to avoid the degradation of the compounds. = 3.88 kV and cone = 60 V. The source block temperature was
90 °C and the desolvation temperature was 150 °C. Continuous
2.3 Microwave-assisted extraction mass spectra were recorded over m/z 50–800 with a scan time
of 1 min and interscan delay of 0.15 s.
The microwave-assisted extraction was performed using
a microwave accelerated reaction system (MARSXpress, CEM
2.6 Evaluation of antioxidant activity using the DPPH
Cooperation, Matthews, NC, USA) with a microwave power of
method
800 W, equipped with a digital timer and temperature controller.
The scavenging effects of the samples on the 2,2-diphenyl-1-
The dried bayberry powder (0.50 g) was accurately weighed,
picrylhydrazyl (DPPH) radical were monitored using the previously
placed in a tube, and mixed with an appropriate amount of
reported method (Bao et al., 2005; Zhou et al., 2009). In summary,
extracting solvent (1% HCl in 95%ethanol). After thorough
0.1 mL of the diluted sample was added to 3.9 mL of 0.1 mmol·L−1
mixing, the tube containing the suspension was irradiated,
DPPH solution. It was vortexed and then allowed to react at
using a predetermined extraction time and temperature, in the
ambient temperature for 30 min in the dark. The absorbance at
microwave device. After the microwave extraction, the sample
517 nm was measured using a spectrophotometer. A calibration
was centrifuged at 8000 rpm for 10 min, and then the supernatant
curve was constructed for the decrease in absorbance based on
was collected and all samples were diluted to the same volume.
trolox concentration, and 0.1 mL of 95% ethanol was used as a
All of the samples were filtered through a 0.45-μm syringe filter
control. The inhibition ratio was calculated as follows:
(Pall Life Sciences, Ann Arbor, MI, USA).
Inhibition ratio (%) = 100×(Ao−As)/Ao, where Ao and As
2.4 Determination of anthocyanin content represent the absorbance of the control and sample solutions,
respectively. The antioxidant activity was expressed as μmol
The total anthocyanin content of extracts was determined Trolox equivalent g−1 dry weight.
using a modified pH differential method described previously
(Kim et al., 2003; Zhou et al., 2009). A general spectrophotometer
2.7 Experimental design and statistical analysis
(T6 New Century, Purkinje General Instrument Co., Beijing, China)
was used to measure absorbance at 520 and 700 nm in buffers at pH A three-level three-factor Box–Behnken design was
1.0 and 4.5. The absorbance measurements were converted into total selected to evaluate the combined effect of three independent
milligrams of cyanidin-3-glucoside per gram dry weight of bayberry variables: solid to liquid ratio, extraction temperature, and
using the molar extinction coefficient, ε = 26 900 L·mol−1·cm−1, time, which were coded as X1, X2, and X3, respectively (Table 1).

Food Sci. Technol, Campinas, 35(3): 524-530, Jul.-Set. 2015 525

 Microwave extract anthocyanin and antioxidant

In total, 15 experiments with three replicates at the centre were 3 Results and discussion
employed. The response surface methodology (RSM) was used 3.1 HPLC analysis of anthocyanins extracted from chinese
to determine the optimum conditions for the extraction of bayberry
anthocyanins from Chinese bayberry. The experimental design
Anthocyanins were extracted from Chinese bayberry using
and statistical analysis were performed using Stat-Ease software
a microwave-assisted method. The HPLC chromatogram of the
(Design-Expert 7.0.10 Trial, Delaware, USA). The generalized anthocyanins is shown in Figure 1. Peak 1 displayed at m/z 287,
second order polynomial model used for the response surface corresponding to the molecular of cyanidin, was assumed to be
analysis was as follows: cyanidin according to the reported by Zhao et al. (2013). Peak 2 has
an m/z 449 corresponding to [M]+, with a characteristic fragmentation
3 3 2
b0 + ∑ bi X i + bii ∑ X i2 + ∑
3
[M-162]+ at 287 corresponding to the cleavage of glucose, hence, it
Y= ∑ bim X i X m (1)
was assigned as cyanidin-3-O-glucoside. By comparing its HPLC
i= 1 i= 1 i = 1 m = i +1
retention time to a cyanidin-3-O-glucoside standard, peak 2 was
where the response function, Y, was partitioned into linear, further confirmed as cyanidin-3-O-glucoside. We suggest that
quadratic, and interactive components, b0, bi, bii, and bim are cyanidin might be the hydrolysis product of cyanidin-3-O-glucose
defined as the constant, linear coefficient, quadratic coefficient, formed during the microwave-assisted extraction. Although it
has been reported that malvidin-3-glucoside and malvidin-3,5-
and cross-product coefficient, respectively, and Xi and Xm are
diglucoside might hydrolyze to the products of anthocyanone,
the levels of the independent variables. Analysis of variance hydroxycoumarins and dihydroxy phenylacetaldehyde under
(ANOVA) tables were generated, and the effect and regression microwave treatment (Ramirez et al., 2015), this finding imply
coefficients of the individual linear, quadratic, and interaction that there may be other anthocyanin degradation pathway
terms were determined. under different conditions, such as cyanidin, delphinidin and

Table 1. Code and actual levels of three variables.

Code levels
Independent variables Units Symbol
–1 0 1
Solid to liquid ratio 1:X X1 30 40 50
T [°C] X2 40 60 80
t [min] X3 5 10 15

Figure 1. HPLC profile of the anthocyanin extracted from Chinese bayberry using a microwave-assisted method. 1 = Cyanidin; 2 = Cyanidin-3-O-glucoside
(C-3-G). UV-Vis detection at 520 nm. (a) under 80 °C extract condition; (b) under 60 °C extract condition.

526 Food Sci. Technol, Campinas, 35(3): 524-530, Jul.-Set. 2015

 Duan et al.

pelargonidin can be extracted from fresh floral and vegetative yield of anthocyanins extracted from Chinese bayberry using a
tissue by using 2M HCl leaching (Des Marais & Rausher, 2010). microwave-assisted method. The C-3-G and total anthocyanin
contents in the microwave-assisted extract were determined,
3.2 Effects of microwave irradiation on and the ratio of C-3-G to total anthocyanin was calculated.
Cyanidin-3-O-glucose hydrolysis The antioxidant activity of the microwave-assisted extracts was
also determined. These results are listed in Table 3.
Cyanidin is an anthocyanin and is bound to glucose to form
C-3-G, which is the main component of anthocyanins in the A positive correlation between the total anthocyanins and
fruit of Chinese bayberry (Tanaka et al., 2008). Although it is the antioxidant activity (R2 = 0.8431, Figure 2) was observed,
well documented that there is no significant difference between which agrees with previous findings (Kähkönen & Heinonen,
the antioxidant activity of cyanidin and C-3-G (Kähkönen & 2003). Because C-3-G is hydrolysed into cyanidin in human
Heinonen, 2003), we used a C-3-G standard to evaluate the effect bodies (Min et al., 2010), it is better to optimize the extraction
of microwaves on the hydrolysis and release of cyanidin and its
antioxidant activity. A two-factor and two-level experimental
design was conducted to evaluate the influence of temperature
and extraction time on the hydrolysis of C-3-G. The results
are shown in Table 2. Two-way ANOVA results showed that
increasing the temperature increased the total anthocyanin
content, but decreased the ratio of C-3-G to total anthocyanin,
whereas it had no significant effect on the antioxidant activity
of the extracts. Increasing the extraction time did not increase
the antioxidant activity of the extracts or the total anthocyanin
content, but significantly decreased the ratio of C-3-G to total
anthocyanin, especially when extracting at 80 °C.

3.3 Optimization of the microwave-assisted extraction

conditions
A three-level three-factor Box–Behnken design was selected
to evaluate the combined effect of three independent variables: Figure 2. The correlation between total anthocyanin content and
solid to liquid ratio, extraction temperature, and time on the antioxidant activity (R2 = 0.8431).

Table 2. Effect of microwave irradiation on the hydrolysis of C-3-G.

40 °C 80 °C
5 min 15 min 5 min 15 min
DPPH [%] 60.73 ± 1.36 63.43 ± 3.20 64.17 ± 2.21 64.60 ± 0.96
Total anthocyanin [mg·L–1] 26.33 ± 1.15 26.67 ± 1.15 28.67 ± 0.58 29.67 ± 1.15
C-3-G/total anthocyanin [%] 98.00 ± 0.61 97.00 ± 0.56 76.00 ± 0.92 48.00 ± 1.15

Table 3. Response surface design and experimental dataa.

Total anthocyanin yield Total antioxidant
Run X1 X2 X3 C-3-G yield [mg·g–1] C-3-G/total anthocyanin
[mg·g–1] activityb [μmol·g–1]
1 1 0 1 1.97 2.85 0.69 271.07
2 0 1 –1 1.83 2.51 0.73 230.56
3 1 –1 0 0.73 1.10 0.66 120.78
4 1 0 –1 1.30 1.86 0.70 161.26
5 –1 0 1 1.70 2.20 0.77 226.38
6 0 –1 –1 1.38 1.87 0.74 162.8
7 0 –1 1 1.39 2.07 0.67 153.56
8 –1 0 –1 1.60 2.29 0.70 173.14
9 –1 1 0 1.77 2.37 0.75 240.24
10 0 1 1 1.89 2.69 0.70 261.8
11 –1 –1 0 0.90 1.41 0.64 119.68
12 1 1 0 1.93 2.57 0.75 250.80
13 0 0 0 1.43 1.97 0.73 185.24
14 0 0 0 1.48 2.02 0.73 195.58
15 0 0 0 1.37 1.96 0.70 194.92
a
Yields and activities are based on dry weight. X1, X2 and X3 are defined in Table 1. bAntioxidant activity was expressed as μmol of Trolox equivalent per g of dry weight.

Food Sci. Technol, Campinas, 35(3): 524-530, Jul.-Set. 2015 527

 Microwave extract anthocyanin and antioxidant

to target the total anthocyanin content if we are only concerned

with their health benefits. However, the extraction conditions
should be optimized to target the C-3-G content if purified
C-3-G from Chinese bayberry is required.
The total anthocyanin content was optimized using an RSM
analysis. The polynomial equation of the quadratic model was
as follows:
Y=1.98+0.014X1+0.46X2+0.16X3+0.13X1X2+0.27X1X3−2.5
×10-3X2X3−0.054X12 −0.067X22+0.37X32 (Fmodel =7.32; p < 0.05;
R2 =0.9295), where Y, X1, X2, and X3 are the total anthocyanin
yield, solid to liquid ratio, extraction temperature, and extraction
time, respectively.
The maximum total anthocyanin yield of 2.95 mg·g–1 on a
dry weight basis was obtained when using the optimal extraction
conditions: solid to liquid ratio, extraction temperature, and time
of 1:50, 80 °C, and 15 min, respectively. The ANOVA statistics
showed that the experimental data had correlation coefficient
R2 = 0.9295 with the calculated model.
To confirm these results, Chinese bayberry powder was
extracted in triplicate using the above-mentioned conditions.
The anthocyanin yield was 2.95 ± 0.08 mg·g−1 with an antioxidant
activity yield of 279.96 ± 0.1 μmol·g−1 equivalent of Trolox on
a dry weight basis.
The C-3-G content was also optimized using an RSM analysis.
The polynomial equation of the quadratic model was as follows:
Figure 3. The response surfaces for the effects of temperature
Y=1.43+0.005X1+0.43X2+0.16X3+0.083X1X2+0.14X1X3+0.11X2X3 and solid to liquid ratio on the yield of total anthocyanin (a) and
−0.088X12 −0.006X22+0.30X32 (Fmodel =15.47; p < 0.05; R2 =0.965), cyanidin-3-O-glucoside (b).
where Y, X1, X2, and X3 are the yield of C-3-G, solid to liquid
ratio, extraction temperature, and extraction time, respectively.
a high solid to liquid ratio because low extract concentrations
The maximum C-3-G yield of 2.01 mg·g-1 on a dry weight would reduce the solubility.
basis was obtained when using the optimal extraction conditions:
solid to liquid ratio, extraction temperature, and time of 1:50,
4 Conclusion
75.8 °C, and 14.7 min, respectively. The ANOVA statistics
showed that the experimental data had correlation coefficient We first investigated the effect of microwave irradiation
R2 = 0.956 with the calculated model. on the extracted components. HPLC-DAD-ESI-MS analysis
of the extract implied that cyanidin-3-O-glucoside have been
The total anthocyanin and C-3-G contents extracted were hydrolysed to cyanidin during the extraction process. To confirm
affected by the three variables studied, but they were most whether microwave irradiation damaged the anthocyanins,
greatly influenced by the extraction temperature. Increased a cyanidin-3-O-glucoside standard was used to evaluate the
temperature significantly increased the extraction of total effects of microwave irradiation on the hydrolysis and release
anthocyanins and C-3-G (Figure 3), and was able to modify of cyanidin and its antioxidant activity. Our results showed that
the equilibrium and mass transfer conditions of the solid-liquid cyanidin-3-O-glucoside hydrolysed to cyanidin, but the antioxidant
extraction. Although higher temperatures had a positive effect activity was not significantly changed during the extraction
on the extraction yields, the temperature cannot be increased process featuring high temperature and long extraction times.
indefinitely because of the thermal instability of anthocyanin
compounds (Liazid et al., 2011). Finally, we used the RSM method to optimize the extraction
conditions and a positive correlation was found between the
The solid to liquid ratio also had an effect on the extraction total anthocyanin content and antioxidant activity, R2=0.8431.
of total anthocyanins and C-3-G. An increase in the solid to The maximum total anthocyanin yield of 2.95 mg·g−1, and an
liquid ratio resulted in increased total anthocyanin and C-3-G antioxidant activity yield of 279.96 ± 0.1 μmol·g−1 equivalent of
extraction (Figure 3). At high extract concentrations, the Trolox on a dry weight basis were obtained under the optimal
solubility would have increased, and then an increase in the conditions where the solid to liquid ratio, extraction temperature,
solid to liquid ratio resulted in a greater extraction of the total and extraction time were 1:50, 80 °C, and 15 min, respectively,
anthocyanin and C-3-G; however, values increased slowly at whereas the maximum cyanidin-3-O-glucoside yield of 2.01 mg·g−1

528 Food Sci. Technol, Campinas, 35(3): 524-530, Jul.-Set. 2015

 Duan et al.

on a dry weight basis was obtained under conditions there the Kaufmann, B., & Christen, P. (2002). Recent extraction techniques for
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were 1:50, 75.8 °C, and 14.7 min, respectively. solvent extraction. Phytochemical Analysis: PCA, 13(2), 105-113.
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These results provide an important guide for the application of
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