Journal of Food Engineering 117 (2013) 437442

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Journal of Food Engineering

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The effect of microwave assisted extraction on the isolation of anthocyanins

and phenolic acids from sour cherry Marasca (Prunus cerasus var. Marasca)
Ivona Elez Garofulic , Verica Dragovic-Uzelac, Anet Rezek Jambrak, Marijana Jukic
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia

a r t i c l e

i n f o

Article history:
Available online 5 January 2013
Keywords:
Microwave assisted extraction
Anthocyanins
Phenolic acids
Sour cherry

a b s t r a c t
The aim of this study was to evaluate the inuence of microwave assisted extraction on the isolation of
anthocyanins and phenolic acids from sour cherry Marasca. A general factorial design was used to study
the effect of temperature (from 50 to 70 C), irradiation time (512 min) and microwave power (350
500 W) on individual, total anthocyanins and individual and total phenolic acids. Optimal microwave
assisted extraction conditions differed for anthocyanins and phenolic acids, especially in terms of temperature and irradiation time, so lower temperature (60 C) and shorter time (69 min) was more convenient for anthocyanins extraction, while phenolic acids gave higher extraction yield at higher
temperatures (70 C) and longer irradiation time (10 min). The optimal microwave power did not differ
signicantly for studied compounds, ranging about 400 W. Compared to conventional extraction, microwave assisted one showed higher efciency for all studied compounds.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Marasca sour cherry (Prunus cerasus, var. Marasca) is an important, autochthonous Croatian cultivar. It has signicant role among
other cherry cultivars, due to its characteristics, namely small fruit,
high content of dry matter and sugars and pleasantly sweetbitter
aroma. It is a rich source of polyphenols, among which avonoids,
especially anthocyanins responsible for its dark red color, and several classes of nonavonoids are usually distinguished. The major
anthocyanins in Marasca cherry are derivates of cyanidin, with
cyanidin-3-glucosylrutinoside present in the highest concentration
(Pedisic et al., 2010). Compared to other sour cherry cultivars, Marasca cherry contains higher amount of total anthocyanins, mostly
due to their distribution in both fruit skin and esh (Chaovanalikit
and Wrolstad, 2004; Pedisic et al., 2010). Apart of anthocyanins,
sour cherries are also rich source of colorless polyphenols, with
hydrocinnamates being the major class presented with neochlorogenic, chlorogenic and 3-coumaroylquinic acid (Bonerz et al., 2007;
Kirakosyan et al., 2009).
Because of a great diversity of structures between this two classes of polyphenols in sour cherry Marasca, their different properties and the necessity to isolate them from other constituents
present in fruit, it is of a great importance to optimize the extraction procedure. The polyphenols may occur in plant tissues combined with sugars, proteins or they may build polymerized

Corresponding author. Tel.: +385 1 4605 036; fax: +385 1 4605 072.
E-mail address: ielez@pbf.hr (I. Elez Garofulic).
0260-8774/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2012.12.043

derivatives, making the extraction process difcult. Furthermore,

extraction is also affected by many other factors, such as solvent
composition, time of extraction, temperature, pH, solid-to-liquid
ratio and particle size (Escribano-Bailn and Santos-Buelga,
2003). Generally, polyphenols extraction is a time and solvent consuming process with low efciency. Long time of the process is
especially critical when anthocyanins are present in sample, because long exposure to heat can cause their degradation and decrease the antioxidant activity of the extracts (Lapornik et al.,
2005). In recent years, microwave assisted extraction (MAE), faster
and more automatic technique, has been replacing conventional
methods, such as Soxhlet (Liazid et al., 2007). MAE is a process that
uses microwave energy to heat solvents rapidly and efciently
(Jain et al., 2009). It enables homogeneous heating of solvent and
plant matrix. As plant matrix contains signicant amount of water
which strongly absorbs microwave energy, internal superheating
causes cell disruption, facilitating the extraction process. Furthermore, the migration of dissolved ions, increases solvent penetration into the matrix and subsequently increases the extraction
yield (Wang and Weller, 2006). The main advantages of MAE over
the conventional extraction techniques are reduced solvent consumption, shortened extraction time, moderately high recoveries,
good reproducibility and minimal sample manipulation for extraction process (Armenta et al., 2008). The application of MAE for
extraction of polyphenols present in plant material demands process optimization regarding its operational parameters such as
temperature, time and microwave power. Generally, higher temperatures enhance diffusivity of the solvent into the plant matrix
increasing the extraction yield, but can also reduce extraction

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438

selectivity as non-targeted compounds and matrix materials can

also be extracted (Camel, 2000). Additionally, thermal degradation
can occur at higher extraction temperatures as well as with longer
extraction times (Mandal et al., 2007). Microwave power is strongly
dependant on both extraction temperature and time. Increasing the
microwave power together with extraction temperature causes the
rapid cell rupture and therefore, increases the amount of impurities
in extracts. The power must be chosen correctly to avoid excessive
temperatures, which could lead to polyphenols degradation and
overpressure (Camel, 2000). Furthermore, low or moderate power
with longer exposure is considered to be a wiser choice since it results in better purity of the extracts (Mandal et al., 2007). Therefore,
it is essential to optimize these parameters according to the characteristics of the sample matrix and of the target compounds to be
extracted.
Response surface methodology (RSM) is effective technique for
process development and optimization, as it provides more information out of smaller number of experiments, also identifying
the interactions between variables (Dubrovic et al., 2011; Rezek
Jambrak, 2011).
Response surface methodology includes four major steps, which
are the experimental design, model tting, model validation, and
condition optimization (Montgomery, 2001). Experimental designs
such as Central Composite Designs (CCDs) are useful for RSM because they do not require an excessive number of experimental
runs. Response surface methodology (RSM), a statistical method,
uses quantitative data from appropriate experiments to determine
and simultaneously solve multivariate equations (Myers and
Montgomery, 2002). It is a collection of statistical techniques for
designing experiments, building models, evaluating the effects of
factors, and analyzing optimum conditions of factors for desirable
responses.
In recent years, MAE has found its application for fast extraction
of plant polyphenols (Hayat et al., 2009; Inoue et al., 2010; Liazid
et al., 2007; Yang and Zhai, 2010). However, there has been no report about application of MAE for isolation of sour cherry polyphenols. Therefore, the aim of this research was study the inuence of
temperature, extraction time and microwave power in MAE on the
extraction yield of anthocyanins and phenolic acids in sour cherry
Marasca. MAE conditions were optimized using the RSM in order to
determine the extraction parameters for optimal extraction of
anthocyanins and phenolic acids.

2. Materials and methods

2.1. Chemicals and standards
Methanol and hydrochloric acid used for extraction were reagent grade, purchased from Gram-mol (Zagreb, Croatia). Methanol and formic acid used for high performance liquid
chromatography were HPLC grade and purchased from Panreac
(Barcelona, Spain).
Phenolic acid standards (caffeic, chlorogenic and p-coumaric
acid) were purchased from Sigma (Steinheim, Germany), while
cyanidin-3-glucoside chloride was obtained from Extrasynthese,
Lyon, France.
2.2. Plant material
Sour cherry Marasca grown in Zadar area in Croatia was obtained
in June 2011 from local growers. Ripe Marasca cherry samples were
hand picked, placed in an icebox, and immediately frozen upon arrival at the laboratory (within 2 h of picking) packed in polyethylene
bags and kept at 18 C till analysis (approximately 7 days). Before
analysis, sour cherries were defrosted, pitted and homogenized in
house blender (Mixy, Zepter International).
2.3. Microwave assisted extraction of sour cherry Marasca polyphenols
Polyphenols from sour cherry Marasca were extracted using a
single mode focused microwave reactor (Milestone, Start S Microwave Labstation for Synthesis, Italy) operating at 2450 MHz with
adjustable microwave power. General extraction parameters were
kept constant: time required to achieve extraction temperature
2 min; stirring 50%; ventilation after extraction 1 min.
A mass of 5 g 0.001 of homogenized sour cherries was mixed
with 40 mL of 0.1% HCl in 80% methanol and placed into a double
neck, round bottom ask with cooling system. Extraction was performed in triplicate at set temperature, microwave power and irradiation time according to the experimental design given in Table 1.
Afterwards, extracts were cooled at the room temperature, ltered
through Whatman No. 40 lter paper (Whatman International Ltd.,
Kent, UK), transferred in 50 mL volumetric asks, made up with
0.1% HCl in 80% methanol and stored at 18 C in inert gas atmosphere until analysis.

Table 1
Observed concentrations of individual and total anthocyanins and phenolic acids at different combinations of temperature, irradiation time and microwave power used in the
experimental design. The results are expressed as mg/g of fresh sample.
Experiment
no.

X1:
temperature
(C)

X2:
irradiation
time (min)

X3:
microwave
power (W)

Cy-3-Sa

Cy-3-GRa

Cy-3-Ga

Cy-3-Ra

TAa

CAa

ChAa

p-CAa

TPAa

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

70
70
50
60
70
50
43
70
60
60
77
60
60
50
60
50

5
12
5
3
5
12
8.5
12
8.5
8.5
8.5
8.5
8.5
12
14
5

350
350
500
425
500
350
425
500
425
425
425
300
550
500
425
350

0.11 0.01
0.16 0.01
0.10 0.01
0.10 0.01
0.09 0.00
0.09 0.00
0.09 0.00
0.10 0.01
0.10 0.01
0.12 0.01
0.18 0.01
0.11 0.01
0.11 0.01
0.10 0.01
0.14 0.01
0.14 0.01

0.95 0.04
1.14 0.06
0.87 0.02
0.87 0.03
0.79 0.03
0.77 0.02
0.62 0.03
0.88 0.04
0.87 0.04
0.96 0.05
1.29 0.06
0.96 0.04
0.76 0.03
0.82 0.04
0.94 0.05
0.91 0.04

0.08 0.00
0.11 0.01
0.06 0.00
0.07 0.00
0.06 0.00
0.05 0.00
0.05 0.00
0.06 0.00
0.06 0.00
0.08 0.00
0.11 0.01
0.08 0.00
0.05 0.00
0.06 0.00
0.08 0.00
0.06 0.00

0.28 0.02
0.51 0.03
0.24 0.02
0.26 0.02
0.23 0.01
0.22 0.01
0.18 0.01
0.25 0.02
0.26 0.02
0.29 0.02
0.52 0.03
0.29 0.02
0.21 0.01
0.25 0.01
0.28 0.02
0.25 0.02

1.43 0.07
1.91 0.08
1.27 0.05
1.29 0.06
1.17 0.07
1.13 0.05
0.93 0.03
1.30 0.05
1.29 0.06
1.45 0.07
2.10 0.09
1.44 0.07
1.13 0.05
1.22 0.05
1.44 0.08
1.36 0.06

0.07 0.00
0.08 0.00
0.07 0.00
0.08 0.00
0.05 0.00
0.07 0.00
0.04 0.00
0.06 0.00
0.06 0.00
0.08 0.00
0.08 0.00
0.08 0.00
0.05 0.00
0.07 0.00
0.07 0.00
0.07 0.00

0.99 0.06
1.14 0.05
0.91 0.04
1.00 0.05
0.79 0.03
0.91 0.04
0.61 0.02
0.89 0.04
0.88 0.04
1.06 0.05
1.20 0.05
1.31 0.07
0.77 0.03
0.96 0.04
1.02 0.06
0.97 0.05

0.04 0.00
0.05 0.00
0.04 0.00
0.05 0.00
0.04 0.00
0.04 0.00
0.02 0.00
0.04 0.00
0.04 0.00
0.05 0.00
0.06 0.00
0.05 0.00
0.03 0.00
0.04 0.00
0.04 0.00
0.04 0.00

1.10 0.05
1.28 0.06
1.01 0.05
1.12 0.04
0.89 0.03
1.02 0.04
0.67 0.03
0.99 0.04
0.98 0.04
1.19 0.06
1.34 0.05
1.44 0.07
0.85 0.03
1.07 0.04
1.13 0.05
1.08 0.04

a
Cyanidin-3-sophoroside (Cy-3-S), cyanidin-3-glucosylrutinoside (Cy-3-GR), cyanidin-3-glucoside (Cy-3-G), cyanidin-3-rutinoside (Cy-3-R), total anthocyanins (TA),
caffeic acid (CA), chlorogenic acid (ChA), p-coumaric acid (p-CA), total phenolic acids (TPA).

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439

Table 2
ANOVA for the effect of temperature (X1), irradiation time (X2) and microwave power (X3) on the concentration of cyanidin-3-glucosylrutinoside, total anthocyanins, chlorogenic
acid and total phenolic acids, using a quadratic response surface model, at 95% condence level.
Source

X1
X2
X3
X1X1
X1X2
X1X3
X2X2
X2X3
X3X3

Cy-3-GR

TA

ChA

TPA

F-ratio

p-Value

F-ratio

p-Value

F-ratio

p-Value

F-ratio

p-Value

0.23
0.03
0.59
1.33
1.56
0.03
0.44
0.80
1.29

0.65
0.87
0.47
0.29
0.26
0.86
0.53
0.41
0.30

0.34
0.00
0.65
1.37
1.50
0.17
0.60
0.40
1.43

0.58
0.98
0.45
0.29
0.27
0.69
0.47
0.55
0.28

2.70
0.10
0.03
0.73
0.18
0.74
0.48
0.09
1.94

0.15
0.77
0.87
0.43
0.68
0.42
0.51
0.77
0.21

2.62
0.08
0.02
0.84
0.20
0.70
0.45
0.07
1.92

0.16
0.78
0.88
0.39
0.67
0.43
0.53
0.80
0.22

2.4. Conventional extraction of sour cherry Marasca polyphenols

Sour cherry Marasca polyphenols were extracted from
5 g 0.001 of homogenized samples using 20 mL of 0.1% HCl in
80% methanol. The mixture was extracted for 20 min in Erlenmeyer ask with reux, ltered through Whatman No. 40 lter paper (Whatman International Ltd., Kent, UK), using a Buchner
funnel. Extraction of the residue was repeated at the same conditions. The ltrates were combined and made up with extraction
solvent to 50 mL in a volumetric ask. Extracts were stored at
18 C in inert gas atmosphere before analysis. Extracts were prepared in triplicate.
2.5. HPLC analysis
Separation of polyphenols was performed by HPLC analysis,
using a Varian ProStar System equipped with a ProStar Solvent
Delivery Module 230, Injector Rheodyne 7125, ProStar 330 UV/
Vis-Photo Diode Array Detector. Chromatographic separation was
performed on a Zorbax ODS C18 column (250  4.6 mm i.d.,
5 lm) including Zorbax ODS C18 guard column (10  4.6 mm i.d.,
5 lm) (Agilent, USA). The solvent composition and used gradient
conditions were described previously by Toms-Barbern et al.
(2001) with some modications. Instead of four, three mobile
phases (A, B, and C) were used. Furthermore, instead of 5% of
formic acid, solvents contained 2.5% formic acid. The elution was
carried using 2.5% formic acid solutions in methanol and in
water, mixed in different proportions: A water/methanol, 95/5,

B water/methanol, 88/12 and C water/methanol, 20/80. The following gradient was used: 015 min, 100% A; 1535 min, 100% B;
3550 min, 75% B and 25% C; 5052 min, 50% B and 50% C; 52
60 min, 100% C. Operating conditions were: constant ow rate
1 mL/min, column temperature 20 C, injection volume 20 lL,
UV/Vis-Photo Diode Array detection at 278 and 510 nm.
Identication of phenolic compounds was carried out by comparing retention times and spectral data of separated peaks with
those of authentic standards. Phenolic acids were identied at
278 nm and anthocyanins at 510 nm using UV/Vis-Photo Diode Array detection.
The quantications of anthocyanins and phenolic acids were
performed by the external standard method. All standards were
prepared as stock solutions in methanol at following concentrations: caffeic and p-coumaric acid 500 mg/L, chlorogenic acid and
cyanidin-3-glucoside chloride 200 mg/L. Working standards solutions were prepared by diluting the stock solution to yield ve concentrations in range from 50 to 250 mg/L for caffeic and pcoumaric acid, and from 25 to 200 mg/L for chlorogenic acid and
cyanidin-3-glucoside chloride. Quantitative determinations
were carried out using calibration curves of standards (caffeic
acid: y = 406506x, R2 = 0.9898; chlorogenic acid: y = 188160x,
R2 = 0.9919; p-coumaric acid: y = 661409x, R2 = 0.9902; cyanidin3-glucoside: y = 255287x, R2 = 0.9978). All identied anthocyanins
are cyanidin derivates differing in sugar moiety bonded to the
aglyconic part of molecule. For anthocyanins lacking reference
standards (cyanidin-3-sophoroside, cyanidin-3-glucosylrutinoside
and cyanidin-3-rutinoside) identication was done according to

Fig. 1. Response surface plots for concentration of cyanidin-3-glucosylrutinoside, total anthocyanins, chlorogenic acid and total phenolic acids in sour cherry Marasca
extracts obtained at constant microwave power of 425 W. Concentrations are expressed in mg/g of fresh sample.

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440

Table 3
The equations of regression models for concentration (mg/g of fresh fruit) of cyanidin-3-glucosylrutinoside, total anthocyanins, chlorogenic acid and total phenolic acids in sour
cherry Marasca extracts.
Response

Models

Cy-3-GR

2.81801 + 0.0876278  X1  0.215674  X2 + 0.0106965  X3  0.000835391  X 21 + 0.00278214  X1  X2  0.0000191667X1X3  0.00393334  X 22

TA

7.16692 + 0.174417  X1  0.287  X2 + 0.0233197  X3  0.00147227  X 21 + 0.00473214  X1  X2  0.0000751667X1X3  0.00796343 X 22

ChA

5.59449 + 0.0980154  X1  0.0171726  X2 + 0.0169836  X3  0.000531881  X 21 + 0.000821429  X1  X2  0.000077  X1  X3

TPA

6.38098 + 0.114258  X1  0.0204186  X2 + 0.0189416  X3  0.000643357  X 21 + 0.000957143  X1  X2  0.0000843333  X1  X3

+0.000265238  X2  X3  0.0000146  X 23
+0.000327143X2X3  0.000026708  X 23
 0.00354818  X 22 + 0.0000771429  X2  X3  0.0000154583  X 23
 0.00385209  X 22 + 0.0000780952  X2  X3  0.0000172515  X 23

their retention times, polarity, characteristic spectra and previous

literature reports (Kirakosyan et al., 2009; imunic et al., 2005;
Toms-Barbern et al., 2001) and quantication was done according to the cyanidin-3-glucoside calibration curve. All identied
anthocyanins show characteristic UV/Vis anthocyanin spectra,
especially as all of them are cyanidin derivates. The spectra of
the anthocyanins were recorded between 200 and 600 nm. As they
are differing in sugar moiety, they can be discriminated according
to their polarity, as they elute in order of falling polarity, as it has
also been previously reported by other authors (Mitic et al., 2012;
Kirakosyan et al., 2009; Blando et al., 2005). Furthermore, HPLC results of anthocyanin identication were similar to those obtained
in another study on sour cherry Marasca products done with LC
MSMS (results not shown).
All HPLC determinations were performed in triplicate and results were expressed as mean values standard deviation.
2.6. Experimental design and statistical analysis
The experimental design and statistical analysis were done
using Statgraphics Centurion software (StatPoint Technologies,
Inc, VA, USA). A general factorial design comprising 16 experimental trials with one replication of the central point was chosen to
evaluate the combined effect of three independent variables, temperature, irradiation time and microwave power, termed as X1, X2
and X3, respectively (Table 1). In order to determine the inuence
of each factor on the individual and total anthocyanins, individual
and total phenolic acids, central composite design (CCD) model
was chosen. Experiments were performed in triplicate, in randomized order according to the trial number as arranged by the software. The operating variables were considered at three levels,
namely low (1), central (0), and high (1). Accordingly, 16 experiments were conducted with experiments organized in a central
composite design (including factorial points, axial points, and center point) and the remaining involving the replication of the central
point to get a good estimate of experimental error. The minimum
and maximum values for temperature were set at 50 C (1) and
70 C (1), irradiation time at 5 (1) and 12 min (1) and microwave
power between 350 (1) and 500 W (1). Repetition experiments
were carried out after other experiments followed by the order
of runs designed by the program. The responses obtained from
the experimental design were concentrations of individual and total anthocyanins, individual and total phenolic acids expressed in
mg/g of fresh sample.
The design matrix for the experiment and the regression model
for each response were calculated as follows (Khuri and Cornell,
1996):

Y b0

bi X i

bii X 2i

bij X i X j

where Y is predicted response, b0 is the xed response at central

point, bi, bii and bij are the linear, quadratic and interaction coefcients, respectively.

Analysis of variance (ANOVA) was carried out to determine any

signicant differences (p < 0.5) among the applied treatments.
Three dimensional response surface plots for cyanidin-3-glucosylrutinoside, total anthocyanins, chlorogenic acid and total phenolic acids concentration were generated by keeping one response
variable at its optimal level and plotting it against the two other
independent variables. While demonstrating the signicant effects,
3-dimensional tted surfaces were drawn (Lu et al., 2008). The
model was tted by multiple linear regressions (MLRs). The validity of the quadratic empirical model was tested using the analysis
of variance (ANOVA). The condence level used was 95%.
3. Results and discussion
Microwave assisted extraction is a useful process for fast and
economical extraction of plant polyphenols, which demands optimization of process parameters in order to experience its benets.
Sour cherry Marasca is fruit cultivar especially rich in polyphenols,
mainly anthocyanins which are responsible for its intensive dark
red color. Because they are unstable and easily degradable, new,
fast extraction techniques, such as MAE are suitable for their isolation. Therefore, the aim of this study was to optimize MAE of sour
cherry Marasca anthocyanins and phenolic acids in terms of selecting the optimal temperature, irradiation time and microwave
power.
3.1. Microwave assisted extraction of sour cherry Marasca polyphenols
Sour cherry Marasca polyphenols were extracted according to
the experimental design in order to select temperature, irradiation
time and microwave power, which are optimal for extraction of
anthocyanins and phenolic acids. The parameter span taken into
account was selected according to the previous reports. Sun et al.
(2007) reported the optimal conditions for MAE of red raspberry
anthocyanins, temperature 55 C, irradiation time 12 min and
microwave power 366 W. Liazid et al. (2007) extracted the anthocyanins from grape skins and reported the optimized parameters
to be temperature 100 C, 5 min irradiation time and microwave
power 500 W. Unfortunately, there have been no reports of MAE
of phenolic acids from red colored fruit, so we have considered
our previous ndings when extracting phenolic compounds from
plant material, showing the decrease in total phenolic content
when using microwave power higher than 500 W and irradiation
time longer than 10 min (Dragovic-Uzelac et al., 2012). Therefore,
the selected temperature range was from 50 to 70 C, in order
not to reach the boiling point of the solvent, irradiation time from
5 to 12 min and microwave power from 350 to 500 W.
In all obtained extracts, individual anthocyanins and phenolic
acids were determined using HPLC with UV/Vis PDA. Concentrations of all individual compounds, as well as sums of identied
anthocyanins and sums of phenolic acids are shown in Table 1.
In all 16 experimental trials four major anthocyanins and three

I. Elez Garofulic et al. / Journal of Food Engineering 117 (2013) 437442

Table 4
Optimal values of temperature, irradiation time and microwave power for microwave
assisted extraction of cyanidin-3-glucosylrutinoside, total anthocyanins, chlorogenic
acid and total phenolic acids from sour cherry Marasca and their predicted
concentrations at optimized extraction conditions.

X1 temperature C
X2 irradiation time min
X3 microwave power W
Predicted concentration (mg/g)

Cy-3-GR

TA

ChA

TPA

58.20
6.09
383.49
1.13

63.71
9.19
403.22
1.77

71.21
10.14
397.28
1.18

70.07
10.11
400.67
1.31

Fig. 2. Comparison of concentrations of cyanidin-3-glucosylrutinoside, total anthocyanins, chlorogenic acid and total phenolic acids extracted from sour cherry
Marasca using conventional (CE) and microwave-assisted extraction (MAE). Results
are expressed in mg/g of fresh sample.

phenolic acids were identied, anthocyanins in order of elution,

cyanidin-3-sophoroside (Cy-3-S), cyanidin-3-glucosylrutinoside
(Cy-3-GR), cyanidin-3-glucoside (Cy-3-G) and cyanidin-3-rutinoside (Cy-3-R), and phenolic acids, caffeic acid (CA), chlorogenic acid
(ChA), and p-coumaric acid (p-CA). The results for total anthocyanins and phenolic acids and dominant compounds from each
group, namely Cy-3-GR and ChA, were analyzed by RSM using Statgraphics Centurion software. Calculations were done at 95% condence level. Analysis of variance for MAE experimental trials at
specic temperature, irradiation time and microwave power is
shown in Table 2. From the ANOVA results, there is no signicant
inuence (p < 0.05) of MAE parameters or their combination on
the concentration of dominant anthocyanin, Cy-3-GR, total anthocyanins and dominant acid, ChA and total phenolic acids. The lack
of the signicance may be due to investigated parameters span
which was selected upon previous research showing it to be suitable for MAE of plant polyphenols. Nevertheless, there are big differences in obtained concentrations in different trials, so it is
important to set MAE parameters carefully as concentration of
TA may vary from 1.13 to 2.10 mg/g or for TPA from 0.67 to
1.44 mg/g. Response surface plots for TA and TPA content at microwave power of 425 W are shown in Fig. 1. Temperature and irradiation time had similar effect on Cy-3-GR and ChA as on TA and TPA,
respectively, as those compounds are dominant in concentration in
each group. It can be seen that the effect of temperature and irradiation time differs from TA to TPA. Moderate temperature increase to 60 C and irradiation time to 9 min, enhanced the
anthocyanins extraction yield. On the other hand, concentration
of phenolic acids was higher when applying higher temperatures
(70 C) and moderate irradiation time of 10 min. Zhang et al.
(2008) have reported that the optimal temperature for MAE of
ChA from ower buds of Lonicera Japonica Thunb. is 60 C as there
was a rapid increase in concentration at 60 and 80 C after 5 min,
but without signicant difference in concentration at 60 and at
80 C. Liazid et al. (2011) have reported similar results for MAE
of anthocyanins from grape skins, showing no signicant differ-

441

ences between 5 and 15 min extraction time, but with highest

yield at 100 C.
In our study, exposure to higher temperatures, during short
time did not cause the increase of extraction yield for both TA
and TPA, so that both groups show the highest yield during moderate irradiation time from 9 to 10 min.
Table 3 shows the equations of regression model for Cy-3-GR, TA,
ChA and TPA in sour cherry Marasca extracts. In these models, studied extraction parameters are combined in linear, quadratic and
interaction coefcients so that concentrations of Cy-3-GR, TA, ChA
and TPA in mg/g can be calculated for desired value of temperature,
irradiation time and microwave power used in MAE process. The
optimal MAE conditions were obtained from response surface analysis for each response value (Table 4). It can be observed that optimal
temperature for MAE of anthocyanins is lower (58.2 C for Cy-3-GR,
63.71 C for TA) than the one optimal for the extraction of phenolic
acids (71.21 C for ChA, 70.07 C for TPA). Anthocyanins are highly
unstable and susceptible for degradation (Giusti and Wrolstad,
2003) and are affected by many factors, among other, the temperature. Liazid et al. (2007) have found that the fewer are the substituents present in the aromatic ring, the higher is the stability of
phenolic compounds during the MAE. Therefore, Cy-3-GR shows
lower stability than ChA because of its structure with three aromatic
rings, hydroxylated groups and two bounded sugars. Furthermore,
optimal irradiation time for ChA and TPA is 10.14 and 10.11 min,
respectively, while for anthocyanins it is shorter (6.09 min for Cy3-GR, 9.19 min for TA). It can be also related to different structure
and stability of these compounds during exposure to microwaves.
Also, Cy-3-GR is the only anthocyanin identied in sour cherry Marasca with two bounded sugars in its structure which may be the reason why its optimal irradiation time is shorter than for other
anthocyanins. The optimal microwave power did not differ signicantly for studied compounds, ranging around 400 W for both
anthocyanins and phenolic acids. It was determined that at optimal
temperature, irradiation time and microwave power for each compound, their predicted optimal concentrations in mg/g are as follows: Cy-3-GR 1.13, TA 1.77, ChA 1.18 and TPA 1.31. Further
extractions have been carried out at these optimized conditions
for each studied compound. Under these conditions the experimental concentrations were 1.10 mg/g for Cy-3-GR, 1.73 mg/g for TA,
1.16 mg/g for ChA and 1.29 mg/g for TPA. The experimental concentrations were about 97% of predicted values for anthocyanins and
about 98% of those predicted for phenolic acids, showing the model
to be successful to predict the MAE efciency for extraction of polyphenols from sour cherry Marasca.
3.2. Comparison of MAE and conventional extraction method
Sour cherry polyphenols were extracted by conventional
extraction method and by the optimized MAE procedure for Cy3-GR, TA, ChA and TPA in order to evaluate the effect of MAE on
extraction efciency. MAE did not affect the composition of sour
cherry polyphenols, as the same compounds were identied in extracts obtained by conventional extraction and MAE. Results show
that MAE was more efcient than conventional method (Fig. 2). All
studied compounds were present in higher concentrations in MAE
extracts. Sun et al. (2007) also reported the MAE to be more efcient for extraction of anthocyanins from red raspberry, even when
the conventional extraction was prolonged from 12 to 60 min.
Yang and Zhai (2010) reported the similar results, also showing
MAE to be more efcient than conventional method regardless
the extraction time. The reason of MAEs high extraction yields is
generally the microwave irradiation mechanism (Sun et al., 2007;
Zhang et al., 2008). Microwaves cause localized temperature rise
in plant tissue which leads to their disruption and consequently
to the migration of phenolic compounds to surrounding solvent.

442

I. Elez Garofulic et al. / Journal of Food Engineering 117 (2013) 437442

Therefore, MAE presents an efcient and rapid method for the

extraction of polyphenols.

4. Conclusions
In this study, MAE was optimized for the extraction of anthocyanins and phenolic acids from sour cherry Marasca. Optimized conditions are different for those two large groups of polyphenols,
especially in terms of temperature and irradiation time, so lower
temperature and shorter time of exposure is more convenient for
anthocyanins extraction, while phenolic acids give higher extraction yield at higher temperatures and longer irradiation time.
When compared to conventional extraction, MAE showed higher
efciency for all studied compounds. The main advantage of microwaves has been showed in this application, higher concentration of
sour cherry polyphenols extracted in shorter time then with conventional extraction.

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