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Optimization of Microwave-Assisted Extractionof Anthocyanins from Mulberry and Identificationof Anthocyanins in Extract Using HPLC-ESI-MSTangbin Zou, Dongliang Wang, Honghui Guo, Yanna Zhu, Xiaoqin Luo, Fengqiong Liu, and Wenhua Ling

Abstract: Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants. This studyaims to optimize the microwave-assisted extraction (MAE) conditions of anthocyanins from mulberry (M. atropurpureaRoxb.) using response surface methodology (RSM). A Box–Behnken experiment was employed in this regard. Methanolconcentration, microwave power, and extraction time were chosen as independent variables. The optimized conditionsof MAE were as follows: 59.6% acidified methanol, 425 W power, 25 (v/w) liquid-to-solid ratio, and 132 s time. Underthese conditions, 54.72 mg anthocyanins were obtained from 1.0 g mulberry powder. Furthermore, 8 anthocyanins wereidentified by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) inmulberry extract. The results showed that cyanidin-3-glucoside and cyanidin-3-rutinoside are the major anthocyaninsin mulberry. In addition, in comparison with conventional extraction, MAE is more rapid and efficient for extractinganthocyanins from mulberry.

Keywords: anthocyanins, HPLC-ESI-MS, microwave-assisted extraction, mulberry, response surface methodology

IntroductionMulberry trees are widely distributed worldwide. Mulberry

fruit is a commonly consumed fruit in daily life, since it is de-licious, nutritious, and rich in bioactive compounds (Song andothers 2009). Recent investigations revealed that mulberry fruithas multiple biological and physiological effects, including hypo-glycemic, hypotensive, and diuretic effects due to the abundance ofanthocyanins and phenolic acids (Peng and others 2010; Shih andothers 2010). Anthocyanins are the largest group of water-solublepigments widespread in the plant kingdom (Ghosh and Konishi2007), which exert a wide range of biological activities includingneuroprotective, cardiovascular protective, and anticancer proper-ties (Xia and others 2003; Kang and others 2006; Qin and others2009; Hui and others 2010).

Extraction is a very important stage in the identification andpurification of anthocyanins (Ghafoor and others 2009). Usually,conventional solvent extraction is time consuming and ineffective(Chen and others 2007). In recent years, various novel extrac-tion techniques have been developed for the extraction of activecomponents from plants, such as microwave-assisted extraction(MAE), supercritical fluid extraction, enzymatic extraction, andsoxhlet extraction (Pedersen and Olsson 2003; Hardlei and others2007; Yang and others 2009; Bai and others 2010 ). Among these,MAE is a rapid and efficient extraction technique (Teo and oth-ers 2009). It utilizes the energy of microwaves to cause molecularmovement with a permanent dipole, leads to a fast heating of the

MS 20110679 Submitted 5/30/2011, Accepted 8/3/2011. Authors Zou, Wang,Zhu, Luo, Liu, and Ling are with Guangdong Provincial Key Laboratory of Food,Nutrition and Health, Dept. of Nutrition, School of Public Health, Sun Yat-senUniv. (Northern Campus), Guangzhou 510080, China. Author Guo is with Dept.of Food Science, Yingdong College of Bioengineering, Shaoguan Univ., Shaoguan512000, China. Direct inquiries to author Ling (E-mail: lingwh@mail.sysu.edu.cn).

solvent and the sample, and offers advantages such as improved ef-ficiency and reduced extraction time compared with other extrac-tion techniques (Eskilsson and Bjorklund 2000). Recently, MAEhas been widely applied to the extraction of many natural products(Kaufmann and Christen 2002).

Response surface methodology (RSM) was originally describedby Box and Wilson (1951) as being effective for responses that areinfluenced by many factors and their interactions. The objectivesof this study were to use RSM to determine optimal MAE con-ditions of anthocyanins from mulberry (M. atropurpurea Roxb.).Parameters subject to optimization were methanol concentration,microwave power, and extraction time. In addition, we identifiedthe profiles of anthocyanins in mulberry using high-performanceliquid chromatography-electrospray ionization-mass spectrometry(HPLC-ESI-MS). The results of this study will provide valuableinformation for the exploitation of mulberry resources.

Materials and Methods

MaterialsThe ripe fruits of mulberry (M. atropurpurea Roxb.) were

bought from a local market. The samples were freeze-dried with alyophilizer (Labconco, Kansas City, Mo., U.S.A.), ground powderwas stored at −80 ◦C to avoid the degradation of active compo-nents. Standards of cyanidin-3-glucoside, cyanidin-3-rutinoside,and pelargonidin-3-glucoside were kindly provided by Polyphe-nol AS (Sandnes, Norway). Analytical grade methanol and triflu-oroacetic acid (TFA) were obtained from Guangzhou ChemicalIndustry (Guangzhou, China). HPLC grade acetonitrile andformic acid were purchased from Merck (Darmstadt, Germany).

Microwave-assisted extractionMicrowave irradiation was performed by using a Galanz (800 W,

2450 MHz) microwave oven (Guangzhou, China). Four gram

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of mulberry powder was suspended in 100 mL extraction sol-vent and mixed with a stirrer bar for 15 min to give sufficienttime for penetration (Yoshida and others 2010). Then sampleswere treated under predetermined conditions. Preliminary extrac-tion trials were carried out with 10%, 30%, 50%, 70% acidifiedmethanol (1% TFA) (Barnes and others 2009); 240, 320, 400,480 W microwave power; and 40, 80, 120, 160 s extraction time.After the MAE, samples were centrifuged at 700 g for 10 min atroom temperature, and then the supernatant was collected. Theprecipitation was taken back and extracted again under the sameconditions. The extracts of the twice-extraction were mixed andfiltered using a 0.22-μm syringe filter (Pall Life Sciences, AnnArbor, Mich., U.S.A.) for anthocyanin analysis.

Experimental designRSM was employed to optimize the MAE of anthocyanins

from mulberry. A Box–Behnken experiment was employed inthis regard. Methanol concentration (X1), microwave power (X2),and extraction time (X3) were chosen for independent variables.The range and center point values of the 3 independent vari-ables presented in Table 1 are selected on the basis of preliminaryexperiments. The experimental design consists of 12 factorialexperiments and 3 replicates of the central point. Anthocyaninyield was selected as the responses for the combination of theindependent variables given in Table 2. Experimental runs wererandomized, to minimize the effects of unexpected variability onthe observed responses. The variables were coded according to thefollowing equation:

x = (Xi − X0)/�X,

where x is the coded value, Xi is the corresponding actual value,X0 is the actual value in the center of the domain, and �X is theincrement of Xi corresponding to a variation of 1 unit of x. The

Table 1–Coded and actual levels of 3 variables.

Coded levels

Independent variables −1 0 1

Methanol concentration (X1, %) 30 50 70Microwave power (X2, W) 320 400 480Time (X3, second) 80 120 160

Table 2–Experimental designs using Box–Behnken and results.

Coded levels

Treatment nr X1 X2 X3 Anthocyanin yield (mg/g)

1 1 0 1 52.122 0 1 −1 44.623 1 −1 0 46.394 1 0 −1 46.255 −1 0 1 45.646 0 −1 −1 43.087 0 −1 1 45.038 −1 0 −1 42.879 −1 1 0 46.21

10 0 1 1 49.8311 −1 −1 0 44.1612 1 1 0 51.7813 0 0 0 53.4214 0 0 0 53.8615 0 0 0 54.14

mathematical model corresponding to the Box–Behnken design is

Y = b0 +3∑

i=1

b i Xi +3∑

i=1

b i i X2i +

2∑

i=1

3∑

j=i+1

b i j Xi X j ,

where Y is the predicted response, b0 is a constant, and b i , b i i , andb i j are the model coefficients. They represent the linear, quadratic,and interaction effects of the variables. The adequacy of the modelwas determined by evaluating the lack of fit, coefficient of deter-mination (R2), and the Fisher test value (F-value) obtained from theanalysis of variance (ANOVA) that was generated by the DesignExpert software.

Conventional extractionConventional extraction procedure used in this experiment has

been described by Fuleki and Ricardo-Da-Silva (2003) and a littlemodification was made. A total of 4.0 g mulberry powder wassuspended in 100 mL of 60% methanol (1% TFA, v/v) and mixedwith a stirrer bar for 15 min to give sufficient time for penetration.Extraction temperature was set at 60 ◦C, which was equivalent tothe mean temperature under optimized conditions in the MAEprocess. Extractions were carried out for 15, 30, 45, 60 min,respectively. After the extraction, the anthocyanin extracts weretreated the same as MAE.

HPLC-ESI-MS analysisAn Agilent 1200 series high-performance liquid chromatogra-

phy (Agilent Technologies, Palo Alto, Calif., U.S.A.) coupled toan Agilent 6410 triple quadrupole mass spectrometer and an Ag-ilent Zorbax SB-C18 column (2.1 mm × 50 mm, 1.8 μm) wereused to identify anthocyanins in mulberry. The mobile phase wascomposed of eluant A (5% formic acid in water, v/v) and eluantB (acetonitrile), the gradient elution program was performed asfollows: 0 to 1 min, 2% B; 1 to 3 min, 2% to 8% B; 3 to 10 min,8% to 10% B; 10 to 15 min, 10% to 2% B. Column tempera-ture was 30 ◦C, flow rate was 0.2 mL/min, and injection volumewas 5 μL. For the analysis of anthocyanins, electrospray ioniza-tion (ESI) was operated in the positive ion mode in the range200 to 800 (m/z) and under the following conditions: capillaryvoltage, 5000 V; nebulizer pressure, 40 psi; drying gas temperatureat 350 ◦C was used at a flow rate of 8.0 L/min. Anthocyanin yieldwas quantified by using calibration curves and expressed as mg/gcyanidin-3-glucoside.

Statistical analysisAll determinations were carried out in triplicate, and the re-

sults obtained were expressed as means ± SD (standard deviation).Analysis of the experimental design data and calculation of pre-dicted responses were carried out using the Design Expert software(Version 7.1.6, Stat-Ease, Inc., Minneapolis, Minn., U.S.A.). Dif-ferences were considered significant if P < 0.05.

Results and Discussion

Optimization of the anthocyanin yieldThe anthocyanin yield of mulberry was optimized using the

RSM approach. A fixed liquid-to-solid ratio (25:1) was chosen.The coded and actual levels of the 3 variables in Table 1 wereselected to maximize the yield. Table 2 shows the treatments withcoded levels and the experimental results of anthocyanin yieldin mulberry. The yield ranged from 42.87 to 54.14 mg/g. The

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MAE and identification of anthocyanins from mulberry . . .

maximum yield was recorded under the experimental conditionsof X1 = 50%, X2 = 400 W, and X3 = 120 s. By applying multipleregression analysis to the experimental data, the response variableand the test variables are related by the following second-orderpolynomial equation:

Y = 53.81 + 2.21X1 + 1.72X2 + 1.98X3 + 0.84X1 X2

+ 0.77X1 X3 + 0.81X2 X3 − 2.80X21 − 3.88X2

2 − 4.29X23 .

In the preceding equation, X1 is the methanol concentration,X2 is the microwave power, and X3 is the extraction time. Table 3shows the analysis of variance (ANOVA) for the regression equa-tion. The linear term and quadratic term were very significant(P < 0.01). The lack of fit was used to verify the adequacy ofthe model and was not significant (P > 0.05), indicating that themodel fit the experiment data adequately.

The adequate precision measures the signal-to-noise ratio. Aratio greater than 4 is desirable. In this study, the ratio was found

Table 3–Analysis of variance (ANOVA) for the regressionequation.

Sum of Degrees MeanSource squares of freedom square F value P value

Model 234.73 9 26.08 106.98 <0.0001X1 38.98 1 38.98 159.91 <0.0001X2 23.74 1 23.74 97.36 0.0002X3 31.21 1 31.21 128.00 <0.0001X1 X2 2.79 1 2.79 11.44 0.0196X1 X3 2.40 1 2.40 9.85 0.0257X2 X3 2.66 1 2.66 10.90 0.0214X2

1 28.86 1 28.86 118.39 0.0001X2

2 55.74 1 55.47 227.51 <0.0001X2

3 67.98 1 67.98 278.84 <0.0001Residual 1.22 5 0.24Lack of it 0.96 3 0.32 2.42 0.3060

to be 27.41, indicating that this model can be used to navigate thedesign space. The relationship between the experimental valuesand predicted values showed that the plotted points cluster aroundthe diagonal line, indicating good fitness of the model becausethe value of predicted R-squared of 0.9327 is in reasonable agree-ment with the adjusted R-squared of 0.9855. A very low value ofcoefficient of the variance (C.V.%) (1.03) clearly indicated a veryhigh degree of precision and reliability of the experimental values.Figure 1 shows the three-dimensional response surface plots. Anincrease of methanol concentration (X1) results in an increase ofanthocyanin yield to a maximum at a certain level, while an in-crease of microwave power (X2) and extraction time (X3) resultin an initial increase of anthocyanin yield that then decrease as thepower and time continue to increase.

The optimal values of the selected variables were obtained bysolving the regression equation. After calculation by the DesignExpert software, the optimal conditions for extracting mulberryanthocyanins were 59.6% methanol (1% TFA, v/v), 425 W mi-crowave power, 25 liquid-to-solid ratio, and 132 s extraction time,with the corresponding Y = 54.90 mg/g. To confirm the result,tests were performed in triplicate under optimized conditions. Theanthocyanin yield was 54.72 ± 0.41 mg/g, which clearly showedthat the model fitted the experimental data and optimized theextraction procedure of anthocyanins from mulberry.

Comparison of MAE to conventional extraction ofanthocyanins in mulberry

Mulberry samples were extracted by MAE and conventionalextraction, respectively. Compared with conventional extraction,MAE was more efficient. Table 4 shows that when mulberry sam-ples were extracted for 15 min, anthocyanin yield by conventionalmethod was only 67% of that by MAE. Given more time in theconventional extraction, such as 60 min, anthocyanin yield was justabout 82% of that by MAE. Therefore, MAE is a more efficientextraction technique.

-1.00

-0.50

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46.75

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52.25

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A: methanol concentration B: power

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BA

yiel

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yiel

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yiel

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Figure 1–Response surface plots of the anthocyanin yield of mulberryextract as affected by methanol concentration, microwave power, andextraction time in MAE. (A) methanol concentration (X1) and microwavepower (X2); (B) methanol concentration (X1) and extraction time (X3);(C) microwave power (X2) and extraction time (X3).

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Identification of anthocyanins in mulberry extractAnthocyanins in mulberry extract were identified by HPLC-

ESI-MS. For the standards were definite, the identification wascarried out by comparison of retention time and molecularweight. Moreover, previous studies of other researchers weretaken as reference for us (Song and others 2000; Mullen andothers 2002; Chaovanalikit and others 2004; Wu and Prior2005a, 2005b; Montoro and others 2006; Chen and others 2007;Zhao and others 2010). Table 5 shows 8 anthocyanins foundin mulberry: cyanidin-3-(2G-glucosylrutinoside), delphinidin-3-rutinoside-5-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside,pelargonidin-3-rutinoside, and delphinidin-3-rutinoside.

Peak 1 (retention time, Rt = 9.37 min) was cyanidin-3-(2G-glucosylrutinoside) with molecular ion at m/z 757. The fragmention was at m/z 287, which corresponds to cyanidin, and m/z 611,with −146 for lose of rhamnose and −324 for lose of 2 moleculesof indicans. Peak 2 (Rt = 9.96 min) was delphinidin-3-rutinoside-5-glucoside with molecular ion at m/z 773. The fragment ion wasat m/z 303 for delphinidin, −308 and −162 was accorded withrutinoside and glucoside, respectively. Peak 3 (Rt = 10.64 min)was cyanidin-3,5-diglucoside with molecular ion at m/z 611. Thefragment ion was at 287, which corresponds to cyanidin, and −324for loss of diglucoside. Peak 4 (Rt = 11.38 min) was cyanidin-3-glucoside with molecular ion at m/z 449. The fragment ionwas at m/z 287 for cyanidin, and −162 for loss of glucoside. Peak5 (Rt = 12.39 min) was cyanidin-3-rutinoside with molecularion at m/z 595. The fragment ion was at m/z 287 for cyanidin,and −308 for loss of rutinoside. Peak 6 (Rt = 13.45 min) waspelargonidin-3-glucoside with molecular ion at m/z 433. Thefragment ion was at m/z 271 for pelargonidin, and −162 forloss of glucoside. Peak 7 (Rt = 14.74 min) was pelargonidin-3-rutinoside with molecular ion at m/z 579. The fragment ion wasat m/z 271 for pelargonidin, and −308 for loss of rutinoside. Peak8 (Rt = 17.98 min) was delphinidin-3-rutinoside with molecularion at m/z 611. The fragment ion was at m/z 303 for delphinidin,and −308 for loss of rutinoside.

The results of this study showed that the species of an-thocyanins in mulberry extract include those previously re-ported by Dugo and others (2001). Moreover, we foundother 4 anthocyanins, such as cyanidin-3-(2G-glucosylrutinoside),cyanidin-3,5-diglucoside, delphinidin-3-rutinoside-5-glucoside,

Table 4–The comparison of MAE and conventional extraction.

Conventional extraction

MAE 15 min 30 min 45 min 60 min

Yield (mg/g) 54.72 ± 0.41 36.71 ± 0.46 41.20 ± 0.37 43.56 ± 0.52 44.83 ± 0.48

Table 5–Identification of anthocyanins from mulberry extract byHPLC-ESI-MS.

Retention Parent Producttime ion ion

Peak (min) (m/z) (m/z) Compound

1 9.37 757 611/287 Cyanidin-3-(2G-glucosylrutinoside)2 9.96 773 611/303 Delphinidin-3-rutinoside-5-glucoside3 10.64 611 449/287 Cyanidin-3,5-diglucoside4 11.38 449 287 Cyanidin-3-glucoside5 12.39 595 287 Cyanidin-3-rutinoside6 13.45 433 271 Pelargonidin-3-glucoside7 14.74 579 271 Pelargonidin-3-rutinoside8 17.98 611 303 Delphinidin-3-rutinoside

and delphinidin-3-rutinoside. The difference in anthocyanin com-position is probably linked to different mulberry varieties.

ConclusionsIn the present study, 2 extraction methods have been developed

for extracting anthocyanins from mulberry. The main factors af-fecting the extraction process, including methanol concentration,microwave power, and extraction time were optimized. Underthe optimum conditions, anthocyanin yield reached 54.72 mg/gpowder in MAE, significantly more than using conventional ex-traction. Moreover, we identified 8 anthocyanins in mulberry ex-tract, which can be used either in some mulberry-related healthcare products or for further isolation and purification of specificanthocyanin. Thus, microwave is a powerful tool and has the po-tential to be used in natural anthocyanin industry.

AcknowledgmentsThis research was supported by the Key Projects in the Na-

tional Science & Technology Pillar Program during the EleventhFive-year Plan Period (Nr 2008BAI58B06) and grants from theNational Natural Science Foundation of China (Nr 30730079).

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