Separation and Purication Technology 100 (2012) 5158

Contents lists available at SciVerse ScienceDirect

Separation and Purication Technology

journal homepage: www.elsevier.com/locate/seppur

Sweet cherries anthocyanins: An environmental friendly extraction

and purication method
Cristina G. Grigoras a,b, Emilie Destandau a,, Sandrine Zubrzycki a, Claire Elfakir a
a
b

Institut de Chimie Organique et Analytique, Universit dOrlans, CNRS UMR 7311, rue de Chartres BP 67059, 45067 Orlans Cedex 2, France
Vasile Alecsandri University of Bacau, Engineering Faculty, 157 Calea Marasesti, 600115 Bacau, Romania

a r t i c l e

i n f o

Article history:
Received 30 March 2012
Received in revised form 26 July 2012
Accepted 29 August 2012
Available online 7 September 2012
Keywords:
Sweet cherries
Anthocyanins
Solvent free microwave assisted extraction
Green chromatography

a b s t r a c t
Anthocyanins contained in sweet cherries are water soluble compounds responsible for their red colors.
They possess interesting biological activities such as antioxidant or anti-inammatory ones. The aim of
this study was to assess the feasibility of developing a human health and environmental friendly process
to isolate anthocyanins from sweet cherries. Following some green chemistry principles, the use of solvent was reduced, safe solvent and additives were used and waste production and energy consumption
were limited as possible. In this purpose a solvent free microwave assisted extraction method was developed. With only 4 irradiation cycles of 45 s each at a power of 1000 W, anthocyanins were extracted
without solvent added. As anthocyanins are degradable molecules the extract was safely stocked by a
lyophilization step. Then, anthocyanins were puried by semi-preparative liquid chromatography using
a safe and biodegradable isocratic mobile phase consisting in a water/ethanol/formic acid mixture circulating in a closed loop system. From 200 mg of crude cherries extract 1.0 0.3 mg of cyanidin-3-O-glucoside and 2.0 0.5 mg of cyanidin-3-O-rutinoside could be recovered. Their purity was controlled by
HPLC analysis and estimated at around 98% for cyanidin-3-O-glucoside and 97% for cyanidin-3-O-rutinoside respectively.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Sweet cherries are very widespread and appear on the market
as the rst fresh fruits among all. Their consumption has been reported to alleviate arthritis and gout-related pain [1] and to reduce
the proliferation of human colon cancer cells [2]. These benecial
effects have been related to the presence of natural polyphenolic
compounds [37]. Among those, anthocyanins are widely encountered and are responsible for the cyan and red colors of several
fruits regularly consumed in diet. They have several pH-dependent
resonance forms, and the most stable is the avylium cation form
which is prevailing at pH values below 2. In plants, anthocyanins
are located in the vacuoles where they are stabilized by the low
pH and a stacked supramolecular structure involving inter- or intra-co pigmentation, self-association or chelation with metal ions
[8]. Anthocyanins were rst used in food industry as natural colorants. In the last years, the researches started to focus on their possible health applications as nutritional supplements, functional
food formulations, medicines, and cosmetics. Their health benets
have been linked to their antioxidant properties and to their notable effects against chronic inammation, cardiovascular hyperten-

Corresponding author.
E-mail address: emilie.destandau@univ-orleans.fr (E. Destandau).
1383-5866/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2012.08.032

sion, cancer prevention or metabolic syndrome regulation [9].

Actually, their low extraction percentages and their relative instability, when extracted from natural medium, limit their valorization [10]. However, due to the great potential of application for
food, pharmaceutical and cosmetic industries and to their interesting biological activity different kinds of methodologies have been
developed to isolate anthocyanins from fruits [9,10]. Anthocyanins
are polar compounds, thus solvents used for their extraction are
acidied aqueous mixtures of ethanol, methanol or acetone [11
16].
Counter Current Chromatography (CCC), a versatile liquidliquid preparative chromatography based on the partition of solutes
between two immiscible solvents, appears today as a powerful tool
for compounds purication. Indeed, CCC benets of many advantages due to the absence of solid stationary phase as wide injection
capacity, no irreversible adsorption, no solute deactivation and
no solid waste [17,18]. CCC was used for the isolation of anthocyanins from fruits with polar biphasic solvent systems as MTBE/
BuOH/MeCN/H2O, 0.1% TFA or EtOAc/BuOH/H2O, 0.1% TFA [19
21]. These processes allowed obtaining pure anthocyanins from
fruits but they were no friendly for human health and environment. Even if CCC is less solvent consumer than preparative HPLC,
solvent used are often toxic.
The aim of this work was to evaluate the possibility to develop a
friendly method to isolate anthocyanins from sweet cheery using

52

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158

no solvent for extraction, safe solvent and additives for purication, limiting solvent and energy consumption.

set at 1 mL min 1. The elution was achieved at room temperature

using the following linear gradient: 025 min from 5% to 65% solvent B.

2. Materials and methods

2.5. Anthocyanin purication
2.1. Reagents
Methanol, ethanol, triuoroacetic acid and formic acid used for
HPLC were of analytical grade and were provided by SDS Carlo Erba
(Val-de-Reuil, France).
Water was puried (resistance <18 MX) from distilled water
using an Elgastat UHQ II system (Elga, Antony, France).
Standards of cyanidin-3-O-glucoside chloride, cyanidin-3-Orutinoside chloride and cyanidin-chloride were bought from Phytolab (Vestenbergsgreuth, Germany).

Purication of anthocyanins was realized at room temperature

on a semi-preparative Hypersil H5 C18.25F (L  U = 250  10 mm,
5 lm) column from Interchim (Montluon France). An isocratic
mobile phase consisting of H2O/EtOH both acidied with 1% formic
acid (86:14 v/v) with a ow rate of 4 mL min 1 was used for compound purication. The separation was followed by UV detection
at 280 nm. Injection of 100 lL of 500 mg mL 1 crude extract solution in mobile phase was performed. Puried anthocyanins were
immediately frozen at 80 C and lyophilized.

2.2. Plant material

2.6. Mass spectrometry

Early rivers cherries (Prunus avium L.) were created in the 1900s
in Olivet (France). It is now a specic cultivar from the central region of France. Cherries were harvested in the south of Orleans in
May 2010. The dark red fruits were at commercial ripeness stage
(stage 13 reported by Serrano et al.) [22]. One part was kept at
20 C until the analyses were carried out and the other part
was immediately extracted and analyzed.

Identication of puried anthocyanins was performed after

their dissolution in H2O/MeOH (5:95 v/v) acidied with 0.1% formic acid. Flow injection analysis was realized in positive ionization
mode on an API 300 PE-SCIEX triple quadrupole mass spectrometer
equipped with a TurboIonSpray source (Forster City, CA, USA) and
controlled by Analyst 1.4.2 software (Sciex Applied Biosystems).
The eluent composition was also H2O/MeOH (5:95 v/v) acidied
with 0.1% formic acid and ow rate was set at 0.5 mL min 1. Nitrogen was used as curtain gas and air as nebulizer gas. Operating
conditions were as follows: nebulizer gas ow rate, NEB = 8
(1.2 L min 1); curtain gas ow rate, CUR = 8 (1.2 L min 1); ionspray
voltage, IS = 5800 V; declustering potential, DP = 20 V; focusing potential, FP = 200 V; entrance potential, EP = 10 V. Full scan data
acquisition was performed between 100 and 1000 amu with a step
size of 0.5 amu.

2.3. Solvent Free Microwave Assisted Extraction (SFMAE) procedure

A Milestone MicroSYNTH microwave oven from Milestone
(Sorisole, Italy) was used for extraction. Process parameters (time
and microwave power) were controlled by EasyControl software.
Temperature was followed by an ATC-FO optic ber inserted directly into the vessel and by an infrared external sensor, controlling temperature inside and outside the reactor respectively.
An amount of approximately 50 g of fresh sweet cherries was
introduced in a 250 mL glass vessel without any solvent and submitted to microwave irradiation at 1000 W for 4 cycles of 45 s
each. Extracted juice recovered in the vessel after each irradiation
cycle was removed and collected in a vial. The gathering of the 4
extracts collected after the 4 cycles constitutes the crude extract
which is centrifuged (7000 rpm) for 5 min at 10 C.
The supernatant was immediately frozen at 80 C in order to
be lyophilized. A red thin layer of dry extract was obtained.
2.4. HPLC analysis
Extract HPLC analyses were performed on a Hitachi system
from VWR (Fontenay-sous-Bois, France) equipped with a quaternary pump, an automatic injector (injected volume 20 lL), a Diode
Array Detector (DAD) and a Sedex 55 Evaporative Light Scattering
Detector (ELSD) (Sedere, Alfortville, France) and controlled by EZ
Chrom Elite software. The column used was a Lichrospher 100 RP
18 (L  U = 125  4 mm, 5 lm) from VWR. Ultrapure water and
methanol both acidied with 0.1% triuoroacetic acid were used
as solvent A and B respectively. The mobile phase ow rate was

3. Results and discussion

3.1. Solvent Free Microwave Assisted Extraction (SFMAE)
Anthocyanins are soluble in polar solvents, and they are extracted from various plant materials by using solidliquid extraction with solvents such as methanol [23], ethanol [24] or water
[25].
Fresh sweet cherries contain a large amount of water, so in order to develop an environmental friendly extraction method it
could be interesting to use only this in situ water as extraction solvent. Moreover, as no solvent was added, compounds were extracted in a low volume corresponding to this in situ water, thus
the extract was already concentrated. To minimize extraction time
and to improve release of water, compounds extraction was assisted by microwave irradiation. Thereby a Solvent-Free Microwave Assisted Extraction (SFMAE) was developed to extract
anthocyanins from sweet cherries. Microwave irradiation heated
water inside cells, leading to temperature and pressure increasing
and thus to vegetal cell destroying and compounds releasing out
off the matrix.

Fig. 1. Picture showing the sweet cherries evolution during the extraction process (A) fresh cherries before extraction; after (B) 1 extraction cycle; (C) 2 extraction cycles; (D)
3 extraction cycles; (E) 4 extraction cycles.

53

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158

400000

ELSD

350000

Intensity (uA)

300000
250000
200000
150000
100000
50000
0
0

10

15

20

25

Time (minutes)

900000

UV 280 nm

Intensity (uA)

750000
600000
450000
300000
150000
0
0

10

15

20

25

Time (minutes)

1050000

UV 520 nm

900000

Intensity (uA)

750000
600000
450000
300000
150000
0
0

10

15

20

25

Time (minutes)
Fig. 2. Chromatographic prole of sweet cherries extract by solvent free microwave assisted extraction. Column: Lichrospher 100 RP 18 (L  U = 125  4 mm, 5 lm) at room
temperature; DAD at 280, 520 nm; ELSD: drift tube temperature: 52 C; nebulizer gas pressure: 2.2 bars; gain: 7; Mobile phase: (A) H2O, (B) MeOH both acidied with 0.1%
TFA; Flow rate: 1 mL min 1; Elution gradient: 025 min from 5 to 65% of B, Injection 20 lL of 1,00,000 ppm of crude extract solution.

In order to dene the appropriate extraction parameters and to

optimize extraction yield assays were done at different extraction
times, irradiation powers and extraction cycles number. The recovered juice volume was measured and HPLC analysis of this juice
was performed to estimate the extraction efciency. Microwave
irradiation power was increased between 200 W and 1000 W, with

a xed irradiation time of 30 s. The intensity of the chromatographic signal and the amount of the extracted compounds increased with power irradiation. Thus 1000 W was found to be
the best irradiation power. Then irradiation time was study from
30 s to 60 s and the best compromise was found to be at 45 s. Below 45 s, lower extracted juice amount and lower chromatographic

54

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158

Table 1
Retention time and UV maximum absorption of standards and main peaks of cherries
extract.
Chromatographic peak

tr (min)

Chlorogenic acid

10.1

k max (nm)
324

1
2
3
4

6.7
7.4
8.1
10.1

324
312
310
323

Cy-3-O-glu chloride
Cy-3-O-rut chloride

15.5
16.3

518
520

5
6

15.5
16.2

518
520

vent-free microwave assisted extraction appears to be more rapid

and efcient. Indeed, amount of extracted juice is higher with
SFMAE than with pressing. Moreover, the chromatographic proles
of the two extracts show similar compounds with higher peak
intensity for the SFMAE extract. In case of pressing, juice is mixed
with small piece of pulp and peel and need centrifugation and ltration before HPLC analysis. On the contrary juice obtained after
microwave irradiation is more limpid. Microwaves facilitate juice
release out off the vegetal matrix. Cherries observed on Fig. 1E
are not completely destroyed just dehydrated. Thus SFMAE extract
does not require other treatments than centrifugation for further
analysis.

3.2. Drying of extract

peak intensity were observed and above 45 s, the increase of temperature led to juice evaporation and to sugar degradation making
the extract brown and smelling caramel. So, cherries were submitted to 1000 W irradiation during 45 s. Under these conditions, temperature of the extract reached 115 C. To avoid extract
degradation, glass vessel was frozen immediately in ice to room
temperature and the red juice produced by microwave irradiation
was removed and stocked in a collection vial. To achieve extraction
process and to improve extraction yield, cherries were submitted
again to another extraction cycle until no juice was produced. Finally 4 irradiation cycles were carried out and a volume of 20
30 mL of juice was gathered from a 50 g amount of fresh cherries.
Fig. 1 shows the aspect of cherries during the extraction process
and the dehydration that occurs following the number of extraction cycles. At the end of the extraction process (4 cycles) cherries
were exhausted and any juice was no longer available.
Studies show that acidic pH value prevents the degradation of
the non-acylated anthocyanin pigments. Thus hydrochloric [26],
formic or acetic [27] acids could be added to the extraction solvent.
Without any acid addition, pH of SFMAE extract was around 2 so
avylium ion should be the predominant form of anthocyanins
and their stabilization should be favored.
Compared to the extraction by pressing (data not shown) that
do not involve solvent or energy consumption, the developed sol-

In order to ensure a long time use of extract for further analyses

and purication, suitable storing conditions avoiding anthocyanins
degradation were looked for. Freezing the extracted juice at 20 C
was a good way to store it for few days but a degradation of anthocyanins could be observed after several freezing and thawing steps.
Indeed on HPLC extract chromatograms, anthocyanin peaks intensity decreased and new peaks at lower retention time appeared.
Drying the fresh extracted juice under a nitrogen ow for one
night at room temperature led also to anthocyanin degradation.
Therefore extract lyophilization was performed. This process
was quite long since the extract was rst frozen at 80 C during
a night and lyophilized during 8 h the following day. A sticky fruit
paste was obtained probably due to the high sugar amount in cherries. The extract was then frozen again for a night and submitted to
lyophilization. These two steps were repeated three times to obtain a dried extract. The HPLC dried extract analysis did not show
any degradation of anthocyanins that were preserved thanks to
the low temperature.
The extraction yield, dened as the mass ratio of the dry extract
after lyophilization on the mass of fresh sweet cherries submitted
to microwave extraction, was estimated at 5 1%. Dried extract
was stocked at 20 C and just the amount necessary for analysis
or purication has been sampled. Even if lyophilization and freez-

Fig. 3. Schematic representation of closed loop (green arrows) system used for anthocyanins from sweet cherries extract purication by semi-preparative HPLC 1 mobile
phase; 2 HPLC pump; 3 injection valve; 4 chromatographic column; 5 UV detector; 6 data acquisition system; 7 collected fraction (blue arrows). (For interpretation
of the references to colour in this gure legend, the reader is referred to the web version of this article.)

55

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158

3.4. Anthocyanins isolation and purication

ing are consuming energy process, they ensure a good preservation

of anthocyanins for long time (at least 6 months tested time).

Open column chromatography (generally with silica gel) or

counter-current chromatography are commonly used to fractionate or isolate molecules from plant extracts. Sometimes these
methods are time consuming and could require toxic solvents.
Semi-preparative reversed-phase liquid chromatography represents a good compromise since it uses less important volumes of
solvent compared to the preparative chromatography and it does
not imply the use of immiscible solvent mixtures (often pollutants)
like the counter-current chromatography.
In order to purify the anthocyanin from cherries extract the separation developed at analytical scale was improved enhancing resolution between the two compounds and developing at semipreparative scale a more environmental friendly process. According to the green chemistry principles applied to chromatographic
system [32], methanol previously used in the mobile phase was replaced by ethanol which is considered as a biodegradable solvent
and which can increase the stability of the mobile phase due to
its lower volatility. The higher dimension of the semi-preparative
column allowed the use of viscous ethanol without backpressure
drawbacks. In order to ensure a low pH, as a requirement for maintaining the stability of anthocyanins in solution under the avylium cation form, formic acid was used in a relatively small
amount (1%) instead of triuoroacetic acid known as more corrosive and toxic compound.
Separation was carried out on Hypersil H5 C18 (250  4.6 mm)
column. In order to evaluate the column overloading, 100 lL of
three solutions with different concentrations (10, 100 and
500 mg mL 1) prepared by diluting the crude extract in the mobile
phase were injected. For a concentration higher than 500 mg mL 1
the solubility of the extract was limited perhaps due to the high
amount of sugar existing in the extract.
Separation was developed under isocratic conditions to minimize the equilibration time between injections and to allow a
closed loop mobile phase circulation between fractions collect.
Indeed to use less solvent the mobile phase was directed to the solvent bottle between each fraction when no compound was detected (Fig. 3).
Fig. 4 shows the semi preparative HPLC chromatogram obtained
by injecting 100 lL of a 500 mg mL 1 crude extract solution under
optimized conditions a mixture of water/ethanol both acidied
with 1% formic acid (86:14 v/v) as mobile phase. The chromato-

3.3. Extracts characterization

Immediately after extraction, the crude fresh extract was analyzed by reversed phase liquid chromatography on Lichrospher
100 RP 18 column using a mobile phase consisting of water as solvent A and MeOH as solvent B both acidied with 0.1% of TFA in a
gradient elution program. Chromatograms were monitored by a
DAD at k = 280 nm (absorption wavelength of phenolic acids) and
at 520 nm (absorption wavelength of anthocyanins) and by ELSD
(enable to detect all non volatile compounds with or without chromophore group). The extract chromatographic prole shown on
Fig. 2 revealed the presence of different kind of compounds. Polar
compounds eluted near the void volume detected only with ELSD
and presented in high concentration should be sugars as fructose,
glucose or saccharose always present in fruits. A second compound
family with peak retention times between 3 and 15 min could be detected by both ELSD and UV at 280 nm. To identify this family, chlorogenic acid standard was injected in the same chromatographic
conditions and was eluted at 10.1 min. Its absorption spectrum
was similar to those of extract peaks eluted between 3 and 15 min
(Table 1). So these compounds could belong to the phenolic acids
family as described in literature [3,6,23,2830]. Main peaks eluted
around 1517 min were detected by ELSD and by UV at 280 and
520 nm. The intensity of these peaks with ELSD detection indicated
that these compounds are among the most abundant polyphenols
present in the cherries extract. but in lower concentration compared
to the sugar amount. UV spectra of these compounds showed an
absorption maximum at 520 nm, specic wavelength of cyanidin
compounds (Table 1). In order to conrm this hypothesis, cyanidin-3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride and
cyanidin-chloride standards were injected. For the rst two the
retention times were also around 1517 min and their UV spectrum
were similar to those observed for the extract. The cyanidin-chloride
standard was more retained on the stationary phase (retention time
around 20 min) and its presence in the cherries extract was not conrmed. These results are consistent with those reported in the literature in that the sweet cherries contain 3-O-glucoside and 3-Orutinoside of cyanidin as major anthocyanins [6,31]. Consequently
the presence of the anthocyanin compounds could be conrmed
and the purication process could be focused on these two peaks.

30000

Intensity (uA)

25000
20000
15000
10000
5000

CL

FC

0
0

10

20

30

FC
40

50

CL

FC

60

70

CL
80

Time (minutes)
Fig. 4. Chromatographic prole of sweet cherries extract purication FC fraction collect; CL closed loop; 1, 2, 3 fractions. Column: Hypersil H5 C18
(L  U = 250  10 mm; 5 lm) at room temperature; Detection: UV at 280 nm; Mobile phase (4 mL min 1): (A) H2O; (B) EtOH both acidied with 1% HCOOH; Isocratic
elution: 86% A/14% B, Injection 100 lL of 500 mg mL 1 crude extract solution.

56

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158
+Q1: 0.323 to 0.434 min from Sample 1 (sandrine vial 2) of sandrine vial 2 pos.wiff (Turbo Spray), subtracted (0.777 to 1.332min)
6.2e5
6.0e5

Max. 6.2e5 cps.

Cyanidin-3-O-Glucoside

35000

163.0

30000

101.0
25000
Intensity (uA)

5.5e5

5.0e5

20000
15000
10000

4.5e5

5000

195.0
4.0e5

0
0

[M-Glu+Na] +
309.5
309.5

3.5e5

10

15

20

25

Time (minutes)

3.0e5

OH
241.0

OH

2.5e5

2.0e5

HO

149.0

117.0

1.5e5

[M] +
449
449.0

180.5
263.0

O
OH

Glucose

341.0

1.0e5
135.0

5.0e4

166.5

100

295.0

155.0

123.0
150

231.0
189.0

200

249.0

253.0
250

387.0

325.0

318.0 335.0 389.0 420.5

300

350

400

447.0 507.0 565.0 575.5 596.0 653.0 662.5 717.0 753.0 782.5 843.5 853.0 883.0
450

500

550
600
m/z, amu

650

700

750

800

850

935.5 949.5

900

950

+Q1: 0.313 to 0.424 min from Sample 1 (sandrine vial 3) of sandrine vial 3 pos.wiff (Turbo Spray), subtracted (0.656 to 1.473 min)

19800

4.2e5

17600

4.0e5

15400

Intensity (uA)

4.4e5

3.8e5
3.6e5
3.4e5

Max. 4.5e5 cps.

Cyanidin-3-O-Rutinoside

22000

101.0

1000

13200
11000
8800
6600

195.0

3.2e5

4400

3.0e5

2200

2.8e5

149.0 163.0

2.6e5

10

15

20

25

Time (minutes)

2.4e5
2.2e5

OH
180.5

2.0e5
1.8e5
1.6e5

309.5
309.5

1.4e5

OH

[M] +
595.5
595.5

[M-Rut+Na] +

HO

1.2e5 117.0
1.0e5
8.0e4

O
OH

135.0 167.0

Rutinose

121.5

247.0 263.0
215.0
341.0
155.0 211.0 227.5 281.0 294.5
387.0
313.0
2.0e4 139.0 175.0
273.0
331.0
449.5 470.0 509.0
193.0 224.0
364.0 414.5
549.0
6.0e4
4.0e4

100

150

200

250

300

350

400

450

500

635.5

550
600
m/z, amu

649.5 665.0
650

697.0 726.5 807.0 827.0 847.0 910.0 925.5 965.0

700

750

800

850

900

950

1000

Fig. 5. MS spectra (in ESI + mode) and UV (k = 280 nm) chromatograms of puried anthocyanins.

graphic separation, recorded at 280 nm in order to detect all compounds, allows an easy recovery of three different fractions containing different compounds. The fraction 1 eluted quickly near
the void volume includes all sugars and phenolic compounds contained in the cherries extract, whereas fractions 2 and 3 are constituted by only one puried anthocyanin. The three fractions were
collected at the semi-preparative column outlet and immediately
frozen and lyophilized in order to dry them limiting their potential
degradation.

With 650 mL of mobile phase circulating in closed loop, up to

4 successive injections could be performed. During the last run a
more important chromatographic background noise was observed
showing a beginning of mobile phase contamination, but the separation and the purity of the targeted compounds was not affected.
Thus, from 200 mg (4 injections of 50 mg each) of dried crude extract, 1.0 0.3 mg of fraction 2 and 2.0 0.5 mg of fraction 3 were
recovered. These measurements correspond to around 30 mg and
60 mg of each anthocyanin in 100 g of fresh fruit. Gao et al. re-

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158

ported amounts ranging from 44.10 to 6.32 mg of cyanidin-3-Oglucoside and from 211.40 to 72.16 mg of cyanidin-3-O-rutinoside
for 100 g of esh (which represents 90% of the whole fruit) in different sweet cherry varieties [5]. Seeram et al. puried 21 mg of
cyanidin-3-O-rutinoside from 100 g of fresh sweet cherries [33].
Consequently our results were consistent with those previously
published on sweet cherries.

3.5. Fractions analysis

After lyophilization, fractions were analyzed by HPLC and mass
spectrometry in order to control the purity and to identify the collected compounds.
HPLC analysis of each fraction, monitored by UV at 280 nm,
showed no other peaks excepting those of targeted anthocyanins.
The fraction purity, calculated by the anthocyanin peak area divided by the whole chromatogram area, was estimated at
98 0.5% for cyanidin-3-O-glucoside and 97 1% for cyanidin-3O-rutinoside respectively.
MS analyses of these two fractions shown in Fig. 5 were performed in positive ionization mode by ow injection analysis in
H2O/MeOH (5:95 v/v) acidied with 0.1% formic acid at
0.5 mL min 1. The MS spectra generated for fractions 2 and 3 allowed identication of cyanidin-3-O-glucoside in fraction 2 and
of cyanidin-3-O-rutinoside in fraction 3. Spectra exhibited the
molecular ion [M]+ at 449 m/z and at 595.5 m/z corresponding to
the molecular mass of the solute, and the main fragment ion due
to the loss of the sugar moiety corresponding to the loss of glucose
for cyanidin-3-O-glucoside and to the loss of rutinose (glucose and
rhamnose) for cyanidin-3-O-rutinoside. These fragment ions were
observed as a sodium adduct [Msugar + Na]+ of the cyanidine
group at m/z 309.5 for the two compounds.

4. Conclusion
Anthocyanins from sweet cherries were extracted by solventfree microwave assisted extraction. This technique does not require any extraction solvent since it uses the high amount of
in situ water existing in fresh cherries. Thanks to the microwave
irradiation vegetal cells were break down releasing out off the matrix juice containing targeted compounds. This technique emerges
as an efcient, economic and environmental friendly one saving
energy solvent and waste.
To ensure extract or puried compounds drying and storage,
avoiding anthocyanin degradation, lyophilization and freezing
were used.
The purication of the extracted anthocyanins was achieved by
semi-preparative liquid chromatography in accordance with some
of the most important green chromatography principles and lead
to the recovery of 1.0 0.3 mg of cyanidin-3-O-glucoside and of
2.0 0.5 mg of cyanidin-3-O-rutinoside from 200 mg of dried cherries crude extract, which represents 30 mg and 60 mg respectively
for 100 g of fresh fruit. These sweet cherries major anthocyanins
were successfully identied by mass spectrometry.
These results promote this environmental friendly process to
use sweet cherries as natural anthocyanins source. These molecules represent a well known alternative for synthetic food colorants and their powerful antioxidant activity makes them
potentially useful in different areas such as cosmetics, pharmaceutical or food industry. To go further in application of this process it
could be necessary to follow the anthocyanin content in cherries
on several years of harvest and to apply the method to the purication of anthocyanins from other cherry varieties.

57

References
[1] H. Wang, M.G. Nair, G.M. Strasburg, Y.-C. Chang, A.M. Booren, J.I. Gray, D.L.
DeWitt, Antioxidant and antiinammatory activities of anthocyanins and their
aglycon, cyanidin, from tart cherries, J. Nat. Prod. 62 (1999) 294296.
[2] S.-Y. Kang, N.P. Seeram, M.G. Nair, L.D. Bourquin, Tart cherry anthocyanins
inhibit tumor development in ApcMin mice and reduce proliferation of human
colon cancer cells, Cancer Lett. 194 (2003) 1319.
[3] K. Robards, P.D. Prenzler, G. Tucker, P. Swatsitang, W. Glover, Phenolic
compounds and their role in oxidative processes in fruits, Food Chem. 66
(1999) 401436.
[4] C. Kaur, H.C. Kapoor, Antioxidants in fruits and vegetables the millenniums
health, Int. J. Food Sci. Technol. 36 (2001) 703725.
[5] L. Gao, G. Mazza, Characterization, quantitation, and distribution of
anthocyanins and colorless phenolics in sweet cherries, J. Agric. Food Chem.
43 (1995) 343346.
[6] H. Wang, M.G. Nair, A.F. Iezzoni, G.M. Strasburg, A.M. Booren, J.I. Gray,
Quantication and characterization of anthocyanins in balaton tart cherries, J.
Agric. Food Chem. 45 (1997) 25562560.
[7] H. Wang, M.G. Nair, G.M. Strasburg, A.M. Booren, J.I. Gray, Antioxidant
polyphenols from tart cherries (Prunus cerasus), J. Agric. Food Chem. 47
(1999) 840844.
[8] P. Bridle, C.F. Timberlake, Anthocyanins as natural food colours-selected
aspects, Food Chem. 58 (1997) 103109.
[9] J. Valls, S. Milln, M.P. Mart, E. Borrs, L. Arola, Advanced separation methods
of food anthocyanins, isoavones and avanols, J. Chromatogr. A 1216 (2009)
71437172.
[10] A. Castaeda-Ovando, M.d.L. Pacheco-Hernndez, M.E. Pez-Hernndez, J.A.
Rodrguez, C.A. Galn-Vidal, Chemical studies of anthocyanins: a review, Food
Chem. 113 (2009) 859871.
[11] A. Chandra, M.G. Nair, A. Iezzoni, Evaluation and characterization of the
anthocyanin pigments in tart cherries (Prunus cerasus L.), J. Agric. Food Chem.
40 (1992) 967969.
[12] A. Chandra, M.G. Nair, A.F. Iezzoni, Isolation and stabilization of anthocyanins
from tart cherries (Prunus cerasus L.), J. Agric. Food Chem. 41 (1993) 1062
1065.
[13] A. Chandra, J. Rana, Y. Li, Separation, identication, quantication, and method
validation of anthocyanins in botanical supplement raw materials by HPLC
and HPLC-MS, J. Agric. Food Chem. 49 (2001) 35153521.
[14] R.-Z. Yang, X.-L. Wei, F.-F. Gao, L.-S. Wang, H.-J. Zhang, Y.-J. Xu, C.-H. Li, Y.-X.
Ge, J.-J. Zhang, J. Zhang, Simultaneous analysis of anthocyanins and avonols
in petals of lotus (Nelumbo) cultivars by high-performance liquid
chromatography-photodiode array detection/electrospray ionization mass
spectrometry, J. Chromatogr. A 1216 (2009) 106112.
[15] E.N. Bridgers, M.S. Chinn, V.-D. Truong, Extraction of anthocyanins from
industrial purple-eshed sweetpotatoes and enzymatic hydrolysis of residues
for fermentable sugars, Indust. Crops. Prod. 32 (2010) 613620.
[16] J. Srivastava, P.S. Vankar, Canna indica ower: new source of anthocyanins,
Plant Physiol. Biochem. 48 (2010) 10151019.
[17] K. Ingkaninan, A. Hazekamp, A.C. Hoek, S. Balconi, R. Verpoorte, Application of
centrifugal partition chromatography in a general separation and
dereplication procedure for plant extracts, J. Liquid Chromatogr. Related
Technol. 23 (2000) 21952208.
[18] L. Marchal, J. Legrand, A. Foucault, Centrifugal partition chromatography: a
survey of its history, and our recent advances in the eld, The Chem. Rec. 3
(2003) 133143.
[19] J.-H. Renault, P. Thpenier, M. Zches-Hanrot, L. Le Men-Olivier, A. Durand, A.
Foucault, R. Margraff, Preparative separation of anthocyanins by gradient
elution centrifugal partition chromatography, J. Chromatogr. A 763 (1997)
345352.
[20] F. Qiu, J. Luo, S. Yao, L. Ma, L. Kong, Preparative isolation and purication of
anthocyanins from purple sweet potato by high-speed counter-current
chromatography, J. Sep. Sci. 32 (2009) 21462151.
[21] E. Salas, M. Duenas, M. Schwarz, P. Winterhalter, V. Cheynier, H. Fulcrand,
Characterization of pigments from different high speed countercurrent
chromatography wine fractions, J. Agric. Food Chem. 53 (2005) 4536
4546.
[22] M. Serrano, F. Guillen, D. Martinez-Romero, S. Castillo, D. Valero, Chemical
constituents and antioxidant activity of sweet cherry at different ripening
stages, J. Agric. Food Chem. 53 (2005) 27412745.
[23] D.-O. Kim, H.J. Heo, Y.J. Kim, H.S. Yang, C.Y. Lee, Sweet and sour cherry
phenolics and their protective effects on neuronal cells, J. Agric. Food Chem. 53
(2005) 99219927.
[24] B. Lapornik, M. Prosek, A. Golc Wondra, Comparison of extracts prepared from
plant by-products using different solvents and extraction time, J. Food Eng. 71
(2005) 214222.
[25] J.E. Cacace, G. Mazza, Extraction of anthocyanins and other phenolics from
black currants with sulfured water, J. Agric. Food Chem. 50 (2002) 5939
5946.
[26] C.A. Nayak, P. Srinivas, N.K. Rastogi, Characterisation of anthocyanins from
Garcinia indica Choisy, Food Chem. 118 (2010) 719724.
[27] A. Kirakosyan, E.M. Seymour, D.E.U. Llanes, P.B. Kaufman, S.F. Bolling, Chemical
prole and antioxidant capacities of tart cherry products, Food Chem. 115
(2009) 2025.

58

C.G. Grigoras et al. / Separation and Purication Technology 100 (2012) 5158

[28] D. Bonerz, K. Wrth, H. Dietrich, F. Will, Analytical characterization and

the impact of ageing on anthocyanin composition and degradation in juices
from ve sour cherry cultivars, Eur. Food Res. Technol. 224 (2007) 355
364.
[29] B. Mozetic, M. Simcic, P. Trebse, Anthocyanins and hydroxycinnamic acids of
Lambert Compact cherries (Prunus avium L.) after cold storage and 1methylcyclopropene treatment, Food Chem. 97 (2006) 302309.
[30] B. Mozetic, P. Trebse, J. Hribar, Anthocyanins and hydroxycinnamic acids in
sweet cherries, Food Technol. Biotechnol. 40 (2002) 207212.

[31] V. Usenik, J. Fabcic, F. Stampar, Sugars, organic acids, phenolic composition and
antioxidant activity of sweet cherry (Prunus avium L.), Food Chem. 107 (2008)
185192.
[32] P. Sandra, K. Sandra, A. Pereira, G. Vanhoenacker, F. David, Green
chromatography: part 1: introduction and liquid chromatography, LC-GC
Eur. 23 (2010) 242259.
[33] N.P. Seeram, R.A. Momin, M.G. Nair, L.D. Bourquin, Cyclooxygenase inhibitory
and antioxidant cyanidin glycosides in cherries and berries, Phytomedicine 8
(2001) 362369.