Separation and Purification Technology 100 (2012) 51–58

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Separation and Purification Technology

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

Sweet cherries anthocyanins: An environmental friendly extractionand purification method

Cristina G. Grigoras a,b, Emilie Destandau a,⇑, Sandrine Zubrzycki a, Claire Elfakir a

a Institut de Chimie Organique et Analytique, Université d’Orléans, CNRS UMR 7311, rue de Chartres BP 67059, 45067 Orléans Cedex 2, Franceb ‘‘Vasile Alecsandri’’ University of Bacau, Engineering Faculty, 157 Calea Marasesti, 600115 Bacau, Romania

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 March 2012Received in revised form 26 July 2012Accepted 29 August 2012Available online 7 September 2012

Keywords:Sweet cherriesAnthocyaninsSolvent free microwave assisted extractionGreen chromatography

1383-5866/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.seppur.2012.08.032

⇑ Corresponding author.E-mail address: emilie.destandau@univ-orleans.fr

Anthocyanins contained in sweet cherries are water soluble compounds responsible for their red colors.They possess interesting biological activities such as antioxidant or anti-inflammatory ones. The aim ofthis study was to assess the feasibility of developing a human health and environmental friendly processto isolate anthocyanins from sweet cherries. Following some green chemistry principles, the use of sol-vent was reduced, safe solvent and additives were used and waste production and energy consumptionwere limited as possible. In this purpose a solvent free microwave assisted extraction method was devel-oped. With only 4 irradiation cycles of 45 s each at a power of 1000 W, anthocyanins were extractedwithout solvent added. As anthocyanins are degradable molecules the extract was safely stocked by alyophilization step. Then, anthocyanins were purified by semi-preparative liquid chromatography usinga safe and biodegradable isocratic mobile phase consisting in a water/ethanol/formic acid mixture circu-lating in a ‘‘closed loop’’ system. From 200 mg of crude cherries extract 1.0 ± 0.3 mg of cyanidin-3-O-glu-coside and 2.0 ± 0.5 mg of cyanidin-3-O-rutinoside could be recovered. Their purity was controlled byHPLC analysis and estimated at around 98% for cyanidin-3-O-glucoside and 97% for cyanidin-3-O-rutino-side respectively.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Sweet cherries are very widespread and appear on the marketas the first fresh fruits among all. Their consumption has been re-ported to alleviate arthritis and gout-related pain [1] and to reducethe proliferation of human colon cancer cells [2]. These beneficialeffects have been related to the presence of natural polyphenoliccompounds [3–7]. Among those, anthocyanins are widely encoun-tered and are responsible for the cyan and red colors of severalfruits regularly consumed in diet. They have several pH-dependentresonance forms, and the most stable is the flavylium cation formwhich is prevailing at pH values below 2. In plants, anthocyaninsare located in the vacuoles where they are stabilized by the lowpH and a stacked supramolecular structure involving inter- or in-tra-co pigmentation, self-association or chelation with metal ions[8]. Anthocyanins were first used in food industry as natural colo-rants. In the last years, the researches started to focus on their pos-sible health applications as nutritional supplements, functionalfood formulations, medicines, and cosmetics. Their health benefitshave been linked to their antioxidant properties and to their nota-ble effects against chronic inflammation, cardiovascular hyperten-

ll rights reserved.

(E. Destandau).

sion, cancer prevention or metabolic syndrome regulation [9].Actually, their low extraction percentages and their relative insta-bility, when extracted from natural medium, limit their valoriza-tion [10]. However, due to the great potential of application forfood, pharmaceutical and cosmetic industries and to their interest-ing biological activity different kinds of methodologies have beendeveloped to isolate anthocyanins from fruits [9,10]. Anthocyaninsare polar compounds, thus solvents used for their extraction areacidified aqueous mixtures of ethanol, methanol or acetone [11–16].

Counter Current Chromatography (CCC), a versatile liquid–li-quid preparative chromatography based on the partition of solutesbetween two immiscible solvents, appears today as a powerful toolfor compounds purification. Indeed, CCC benefits of many advanta-ges due to the absence of solid stationary phase as wide injectioncapacity, no irreversible adsorption, no solute deactivation andno solid waste [17,18]. CCC was used for the isolation of anthocy-anins 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 fromfruits but they were no friendly for human health and environ-ment. 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 afriendly method to isolate anthocyanins from sweet cheery using

52 C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58

no solvent for extraction, safe solvent and additives for purifica-tion, limiting solvent and energy consumption.

2. Materials and methods

2.1. Reagents

Methanol, ethanol, trifluoroacetic acid and formic acid used forHPLC were of analytical grade and were provided by SDS Carlo Erba(Val-de-Reuil, France).

Water was purified (resistance <18 MX) from distilled waterusing an Elgastat UHQ II system (Elga, Antony, France).

Standards of cyanidin-3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride and cyanidin-chloride were bought from Phy-tolab (Vestenbergsgreuth, Germany).

2.2. Plant material

Early rivers cherries (Prunus avium L.) were created in the 1900sin Olivet (France). It is now a specific cultivar from the central re-gion of France. Cherries were harvested in the south of Orleans inMay 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 partwas immediately extracted and analyzed.

2.3. Solvent Free Microwave Assisted Extraction (SFMAE) procedure

A Milestone MicroSYNTH microwave oven from Milestone(Sorisole, Italy) was used for extraction. Process parameters (timeand microwave power) were controlled by EasyControl software.Temperature was followed by an ATC-FO optic fiber inserted di-rectly into the vessel and by an infrared external sensor, control-ling temperature inside and outside the reactor respectively.

An amount of approximately 50 g of fresh sweet cherries wasintroduced in a 250 mL glass vessel without any solvent and sub-mitted to microwave irradiation at 1000 W for 4 cycles of 45 seach. Extracted juice recovered in the vessel after each irradiationcycle was removed and collected in a vial. The gathering of the 4extracts collected after the 4 cycles constitutes the crude extractwhich is centrifuged (7000 rpm) for 5 min at 10 �C.

The supernatant was immediately frozen at �80 �C in order tobe lyophilized. A red thin layer of dry extract was obtained.

2.4. HPLC analysis

Extract HPLC analyses were performed on a Hitachi systemfrom VWR (Fontenay-sous-Bois, France) equipped with a quater-nary pump, an automatic injector (injected volume 20 lL), a DiodeArray Detector (DAD) and a Sedex 55 Evaporative Light ScatteringDetector (ELSD) (Sedere, Alfortville, France) and controlled by EZChrom Elite software. The column used was a Lichrospher 100 RP18 (L �U = 125 � 4 mm, 5 lm) from VWR. Ultrapure water andmethanol both acidified with 0.1% trifluoroacetic acid were usedas solvent A and B respectively. The mobile phase flow rate was

A B C

Fig. 1. Picture showing the sweet cherries evolution during the extraction process (A) fre3 extraction cycles; (E) 4 extraction cycles.

set at 1 mL min�1. The elution was achieved at room temperatureusing the following linear gradient: 0–25 min from 5% to 65% sol-vent B.

2.5. Anthocyanin purification

Purification of anthocyanins was realized at room temperatureon a semi-preparative Hypersil H5 C18.25F (L �U = 250 � 10 mm,5 lm) column from Interchim (Montluçon France). An isocraticmobile phase consisting of H2O/EtOH both acidified with 1% formicacid (86:14 v/v) with a flow rate of 4 mL min�1 was used for com-pound purification. The separation was followed by UV detectionat 280 nm. Injection of 100 lL of 500 mg mL�1 crude extract solu-tion in mobile phase was performed. Purified anthocyanins wereimmediately frozen at �80 �C and lyophilized.

2.6. Mass spectrometry

Identification of purified anthocyanins was performed aftertheir dissolution in H2O/MeOH (5:95 v/v) acidified with 0.1% for-mic acid. Flow injection analysis was realized in positive ionizationmode on an API 300 PE-SCIEX triple quadrupole mass spectrometerequipped with a TurboIonSpray source (Forster City, CA, USA) andcontrolled by Analyst 1.4.2 software (Sciex Applied Biosystems).The eluent composition was also H2O/MeOH (5:95 v/v) acidifiedwith 0.1% formic acid and flow rate was set at 0.5 mL min�1. Nitro-gen was used as curtain gas and air as nebulizer gas. Operatingconditions were as follows: nebulizer gas flow rate, NEB = 8(1.2 L min�1); curtain gas flow rate, CUR = 8 (1.2 L min�1); ionsprayvoltage, IS = 5800 V; declustering potential, DP = 20 V; focusing po-tential, FP = 200 V; entrance potential, EP = 10 V. Full scan dataacquisition was performed between 100 and 1000 amu with a stepsize of 0.5 amu.

3. Results and discussion

3.1. Solvent Free Microwave Assisted Extraction (SFMAE)

Anthocyanins are soluble in polar solvents, and they are ex-tracted from various plant materials by using solid–liquid extrac-tion with solvents such as methanol [23], ethanol [24] or water[25].

Fresh sweet cherries contain a large amount of water, so in or-der to develop an environmental friendly extraction method itcould be interesting to use only this in situ water as extraction sol-vent. Moreover, as no solvent was added, compounds were ex-tracted in a low volume corresponding to this in situ water, thusthe extract was already concentrated. To minimize extraction timeand to improve release of water, compounds extraction was as-sisted by microwave irradiation. Thereby a Solvent-Free Micro-wave Assisted Extraction (SFMAE) was developed to extractanthocyanins from sweet cherries. Microwave irradiation heatedwater inside cells, leading to temperature and pressure increasingand thus to vegetal cell destroying and compounds releasing outoff the matrix.

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sh cherries before extraction; after (B) 1 extraction cycle; (C) 2 extraction cycles; (D)

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Fig. 2. Chromatographic profile of sweet cherries extract by solvent free microwave assisted extraction. Column: Lichrospher 100 RP 18 (L �U = 125 � 4 mm, 5 lm) at roomtemperature; 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 acidified with 0.1%TFA; Flow rate: 1 mL min�1; Elution gradient: 0–25 min from 5 to 65% of B, Injection 20 lL of 1,00,000 ppm of crude extract solution.

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In order to define the appropriate extraction parameters and tooptimize extraction yield assays were done at different extractiontimes, irradiation powers and extraction cycles number. The recov-ered juice volume was measured and HPLC analysis of this juicewas performed to estimate the extraction efficiency. Microwaveirradiation power was increased between 200 W and 1000 W, with

a fixed irradiation time of 30 s. The intensity of the chromato-graphic signal and the amount of the extracted compounds in-creased with power irradiation. Thus 1000 W was found to bethe best irradiation power. Then irradiation time was study from30 s to 60 s and the best compromise was found to be at 45 s. Be-low 45 s, lower extracted juice amount and lower chromatographic

Table 1Retention time and UV maximum absorption of standards and main peaks of cherriesextract.

Chromatographic peak tr (min) k max (nm)

Chlorogenic acid 10.1 324

1 6.7 3242 7.4 3123 8.1 3104 10.1 323

Cy-3-O-glu chloride 15.5 518Cy-3-O-rut chloride 16.3 520

5 15.5 5186 16.2 520

54 C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58

peak intensity were observed and above 45 s, the increase of tem-perature led to juice evaporation and to sugar degradation makingthe extract brown and smelling caramel. So, cherries were submit-ted to 1000 W irradiation during 45 s. Under these conditions, tem-perature of the extract reached 115 �C. To avoid extractdegradation, glass vessel was frozen immediately in ice to roomtemperature and the red juice produced by microwave irradiationwas removed and stocked in a collection vial. To achieve extractionprocess and to improve extraction yield, cherries were submittedagain to another extraction cycle until no juice was produced. Fi-nally 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 processand the dehydration that occurs following the number of extrac-tion cycles. At the end of the extraction process (4 cycles) cherrieswere exhausted and any juice was no longer available.

Studies show that acidic pH value prevents the degradation ofthe 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 soflavylium ion should be the predominant form of anthocyaninsand their stabilization should be favored.

Compared to the extraction by pressing (data not shown) thatdo not involve solvent or energy consumption, the developed sol-

Fig. 3. Schematic representation of ‘‘closed loop’’ (green arrows) system used for anthocyphase; 2 – HPLC pump; 3 – injection valve; 4 – chromatographic column; 5 – UV detectorof the references to colour in this figure legend, the reader is referred to the web versio

vent-free microwave assisted extraction appears to be more rapidand efficient. Indeed, amount of extracted juice is higher withSFMAE than with pressing. Moreover, the chromatographic profilesof the two extracts show similar compounds with higher peakintensity for the SFMAE extract. In case of pressing, juice is mixedwith small piece of pulp and peel and need centrifugation and fil-tration before HPLC analysis. On the contrary juice obtained aftermicrowave irradiation is more limpid. Microwaves facilitate juicerelease out off the vegetal matrix. Cherries observed on Fig. 1Eare not completely destroyed just dehydrated. Thus SFMAE extractdoes not require other treatments than centrifugation for furtheranalysis.

3.2. Drying of extract

In order to ensure a long time use of extract for further analysesand purification, suitable storing conditions avoiding anthocyaninsdegradation were looked for. Freezing the extracted juice at �20 �Cwas a good way to store it for few days but a degradation of antho-cyanins could be observed after several freezing and thawing steps.Indeed on HPLC extract chromatograms, anthocyanin peaks inten-sity decreased and new peaks at lower retention time appeared.

Drying the fresh extracted juice under a nitrogen flow for onenight at room temperature led also to anthocyanin degradation.

Therefore extract lyophilization was performed. This processwas quite long since the extract was first frozen at �80 �C duringa night and lyophilized during 8 h the following day. A sticky fruitpaste was obtained probably due to the high sugar amount in cher-ries. The extract was then frozen again for a night and submitted tolyophilization. These two steps were repeated three times to ob-tain a dried extract. The HPLC dried extract analysis did not showany degradation of anthocyanins that were preserved thanks tothe low temperature.

The extraction yield, defined as the mass ratio of the dry extractafter lyophilization on the mass of fresh sweet cherries submittedto microwave extraction, was estimated at 5 ± 1%. Dried extractwas stocked at �20 �C and just the amount necessary for analysisor purification has been sampled. Even if lyophilization and freez-

anins from sweet cherries extract purification by semi-preparative HPLC 1 – mobile; 6 – data acquisition system; 7 – collected fraction (blue arrows). (For interpretationn of this article.)

C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58 55

ing are consuming energy process, they ensure a good preservationof anthocyanins for long time (at least 6 months tested time).

3.3. Extracts characterization

Immediately after extraction, the crude fresh extract was ana-lyzed by reversed phase liquid chromatography on Lichrospher100 RP 18 column using a mobile phase consisting of water as sol-vent A and MeOH as solvent B both acidified with 0.1% of TFA in agradient elution program. Chromatograms were monitored by aDAD at k = 280 nm (absorption wavelength of phenolic acids) andat 520 nm (absorption wavelength of anthocyanins) and by ELSD(enable to detect all non volatile compounds with or without chro-mophore group). The extract chromatographic profile shown onFig. 2 revealed the presence of different kind of compounds. Polarcompounds eluted near the void volume detected only with ELSDand presented in high concentration should be sugars as fructose,glucose or saccharose always present in fruits. A second compoundfamily with peak retention times between 3 and 15 min could be de-tected by both ELSD and UV at 280 nm. To identify this family, chlor-ogenic acid standard was injected in the same chromatographicconditions and was eluted at 10.1 min. Its absorption spectrumwas similar to those of extract peaks eluted between 3 and 15 min(Table 1). So these compounds could belong to the phenolic acidsfamily as described in literature [3,6,23,28–30]. Main peaks elutedaround 15–17 min were detected by ELSD and by UV at 280 and520 nm. The intensity of these peaks with ELSD detection indicatedthat these compounds are among the most abundant polyphenolspresent in the cherries extract. but in lower concentration comparedto the sugar amount. UV spectra of these compounds showed anabsorption maximum at 520 nm, specific wavelength of cyanidincompounds (Table 1). In order to confirm this hypothesis, cyani-din-3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride andcyanidin-chloride standards were injected. For the first two theretention times were also around 15–17 min and their UV spectrumwere similar to those observed for the extract. The cyanidin-chloridestandard was more retained on the stationary phase (retention timearound 20 min) and its presence in the cherries extract was not con-firmed. These results are consistent with those reported in the liter-ature in that the sweet cherries contain 3-O-glucoside and 3-O-rutinoside of cyanidin as major anthocyanins [6,31]. Consequentlythe presence of the anthocyanin compounds could be confirmedand the purification process could be focused on these two peaks.

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Fig. 4. Chromatographic profile of sweet cherries extract purification FC – fractio(L �U = 250 � 10 mm; 5 lm) at room temperature; Detection: UV at 280 nm; Mobileelution: 86% A/14% B, Injection 100 lL of 500 mg mL�1 crude extract solution.

3.4. Anthocyanins isolation and purification

Open column chromatography (generally with silica gel) orcounter-current chromatography are commonly used to fraction-ate or isolate molecules from plant extracts. Sometimes thesemethods are time consuming and could require toxic solvents.

Semi-preparative reversed-phase liquid chromatography repre-sents a good compromise since it uses less important volumes ofsolvent compared to the preparative chromatography and it doesnot imply the use of immiscible solvent mixtures (often pollutants)like the counter-current chromatography.

In order to purify the anthocyanin from cherries extract the sep-aration developed at analytical scale was improved enhancing res-olution between the two compounds and developing at semi-preparative scale a more environmental friendly process. Accord-ing to the green chemistry principles applied to chromatographicsystem [32], methanol previously used in the mobile phase was re-placed by ethanol which is considered as a biodegradable solventand which can increase the stability of the mobile phase due toits lower volatility. The higher dimension of the semi-preparativecolumn allowed the use of viscous ethanol without backpressuredrawbacks. In order to ensure a low pH, as a requirement for main-taining the stability of anthocyanins in solution under the flavyli-um cation form, formic acid was used in a relatively smallamount (1%) instead of trifluoroacetic acid known as more corro-sive 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 ofthree solutions with different concentrations (10, 100 and500 mg mL�1) prepared by diluting the crude extract in the mobilephase were injected. For a concentration higher than 500 mg mL�1

the solubility of the extract was limited perhaps due to the highamount of sugar existing in the extract.

Separation was developed under isocratic conditions to mini-mize 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 sol-vent bottle between each fraction when no compound was de-tected (Fig. 3).

Fig. 4 shows the semi preparative HPLC chromatogram obtainedby injecting 100 lL of a 500 mg mL�1 crude extract solution underoptimized conditions a mixture of water/ethanol both acidifiedwith 1% formic acid (86:14 v/v) as mobile phase. The chromato-

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+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) .spc5e2.6.xaM

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

56 C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58

graphic separation, recorded at 280 nm in order to detect all com-pounds, allows an easy recovery of three different fractions con-taining different compounds. The fraction 1 eluted quickly nearthe void volume includes all sugars and phenolic compounds con-tained in the cherries extract, whereas fractions 2 and 3 are consti-tuted by only one purified anthocyanin. The three fractions werecollected at the semi-preparative column outlet and immediatelyfrozen and lyophilized in order to dry them limiting their potentialdegradation.

With 650 mL of mobile phase circulating in ‘‘closed loop’’, up to4 successive injections could be performed. During the last run amore important chromatographic background noise was observedshowing a beginning of mobile phase contamination, but the sep-aration and the purity of the targeted compounds was not affected.Thus, from 200 mg (4 injections of 50 mg each) of dried crude ex-tract, 1.0 ± 0.3 mg of fraction 2 and 2.0 ± 0.5 mg of fraction 3 wererecovered. These measurements correspond to around 30 mg and60 mg of each anthocyanin in 100 g of fresh fruit. Gao et al. re-

C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58 57

ported amounts ranging from 44.10 to 6.32 mg of cyanidin-3-O-glucoside and from 211.40 to 72.16 mg of cyanidin-3-O-rutinosidefor 100 g of flesh (which represents 90% of the whole fruit) in dif-ferent sweet cherry varieties [5]. Seeram et al. purified 21 mg ofcyanidin-3-O-rutinoside from 100 g of fresh sweet cherries [33].Consequently our results were consistent with those previouslypublished on sweet cherries.

3.5. Fractions analysis

After lyophilization, fractions were analyzed by HPLC and massspectrometry in order to control the purity and to identify the col-lected 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 di-vided by the whole chromatogram area, was estimated at98 ± 0.5% for cyanidin-3-O-glucoside and 97 ± 1% for cyanidin-3-O-rutinoside respectively.

MS analyses of these two fractions shown in Fig. 5 were per-formed in positive ionization mode by flow injection analysis inH2O/MeOH (5:95 v/v) acidified with 0.1% formic acid at0.5 mL min�1. The MS spectra generated for fractions 2 and 3 al-lowed identification of cyanidin-3-O-glucoside in fraction 2 andof cyanidin-3-O-rutinoside in fraction 3. Spectra exhibited themolecular ion [M]+ at 449 m/z and at 595.5 m/z corresponding tothe molecular mass of the solute, and the main fragment ion dueto the loss of the sugar moiety corresponding to the loss of glucosefor cyanidin-3-O-glucoside and to the loss of rutinose (glucose andrhamnose) for cyanidin-3-O-rutinoside. These fragment ions wereobserved as a sodium adduct [M–sugar + Na]+ of the cyanidinegroup at m/z 309.5 for the two compounds.

4. Conclusion

Anthocyanins from sweet cherries were extracted by solvent-free microwave assisted extraction. This technique does not re-quire any extraction solvent since it uses the high amount ofin situ water existing in fresh cherries. Thanks to the microwaveirradiation vegetal cells were break down releasing out off the ma-trix juice containing targeted compounds. This technique emergesas an efficient, economic and environmental friendly one savingenergy solvent and waste.

To ensure extract or purified compounds drying and storage,avoiding anthocyanin degradation, lyophilization and freezingwere used.

The purification of the extracted anthocyanins was achieved bysemi-preparative liquid chromatography in accordance with someof the most important green chromatography principles and leadto the recovery of 1.0 ± 0.3 mg of cyanidin-3-O-glucoside and of2.0 ± 0.5 mg of cyanidin-3-O-rutinoside from 200 mg of dried cher-ries crude extract, which represents 30 mg and 60 mg respectivelyfor 100 g of fresh fruit. These sweet cherries major anthocyaninswere successfully identified by mass spectrometry.

These results promote this environmental friendly process touse sweet cherries as natural anthocyanins source. These mole-cules represent a well known alternative for synthetic food colo-rants and their powerful antioxidant activity makes thempotentially useful in different areas such as cosmetics, pharmaceu-tical or food industry. To go further in application of this process itcould be necessary to follow the anthocyanin content in cherrieson several years of harvest and to apply the method to the purifi-cation of anthocyanins from other cherry varieties.

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