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Sweet cherries anthocyanins: An environmental friendly extraction and purification method
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 extraction and purification method Cristina G. Grigoras a,b, Emilie Destandau a,⇑, Sandrine Zubrzycki a, Claire Elfakir a a b
Institut de Chimie Organique et Analytique, Université d’Orléans, CNRS UMR 7311, rue de Chartres BP 67059, 45067 Orléans 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-inflammatory 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 purified 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. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Sweet cherries are very widespread and appear on the market as the first 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 beneficial effects have been related to the presence of natural polyphenolic compounds [3–7]. 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 flavylium 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 first 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 benefits have been linked to their antioxidant properties and to their notable effects against chronic inflammation, cardiovascular hyperten-
⇑ Corresponding author. E-mail address: [email protected] (E. Destandau). 1383-5866/$ - see front matter Ó 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 acidified aqueous mixtures of ethanol, methanol or acetone [11– 16]. Counter Current Chromatography (CCC), a versatile liquid–liquid preparative chromatography based on the partition of solutes between two immiscible solvents, appears today as a powerful tool for compounds purification. Indeed, CCC benefits 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
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no solvent for extraction, safe solvent and additives for purification, limiting solvent and energy consumption.
set at 1 mL min 1. The elution was achieved at room temperature using the following linear gradient: 0–25 min from 5% to 65% solvent B.
2. Materials and methods 2.5. Anthocyanin purification 2.1. Reagents Methanol, ethanol, trifluoroacetic acid and formic acid used for HPLC were of analytical grade and were provided by SDS Carlo Erba (Val-de-Reuil, France). Water was purified (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).
Purification 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 (Montluçon France). An isocratic mobile phase consisting of H2O/EtOH both acidified with 1% formic acid (86:14 v/v) with a flow rate of 4 mL min 1 was used for compound purification. 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. Purified 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 specific 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.
Identification of purified anthocyanins was performed after their dissolution in H2O/MeOH (5:95 v/v) acidified 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) acidified with 0.1% formic acid and flow 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 flow rate, NEB = 8 (1.2 L min 1); curtain gas flow 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 fiber 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 acidified with 0.1% trifluoroacetic acid were used as solvent A and B respectively. The mobile phase flow rate was
A
B
C
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 solid–liquid 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.
D
E
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.
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400000
ELSD
350000
Intensity (uA)
300000 250000 200000 150000 100000 50000 0 0
5
10
15
20
25
Time (minutes)
900000
UV 280 nm
Intensity (uA)
750000 600000 450000 300000 150000 0 0
5
10
15
20
25
Time (minutes)
1050000
UV 520 nm
900000
Intensity (uA)
750000 600000 450000 300000 150000 0 0
5
10
15
20
25
Time (minutes) 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 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 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.
In order to define 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 efficiency. Microwave irradiation power was increased between 200 W and 1000 W, with
a fixed 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
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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 efficient. Indeed, amount of extracted juice is higher with SFMAE than with pressing. Moreover, the chromatographic profiles 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 filtration 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 flavylium 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 purification, 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 flow 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 first 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, defined 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 purification 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 purification 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 figure legend, the reader is referred to the web version of this article.)
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3.4. Anthocyanins isolation and purification
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 flavylium cation form, formic acid was used in a relatively small amount (1%) instead of trifluoroacetic 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 acidified 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 acidified 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 profile 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 [3,6,23,28–30]. Main peaks eluted around 15–17 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, specific wavelength of cyanidin compounds (Table 1). In order to confirm this hypothesis, cyanidin-3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride and cyanidin-chloride standards were injected. For the first two the retention times were also around 15–17 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 confirmed. 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 confirmed and the purification process could be focused on these two peaks.
30000
2
1
3
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 profile of sweet cherries extract purification 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 acidified with 1% HCOOH; Isocratic elution: 86% A/14% B, Injection 100 lL of 500 mg mL 1 crude extract solution.
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C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58 +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
5
10
15
20
25
Time (minutes)
3.0e5
OH 241.0
OH
2.5e5
2.0e5
+
HO
149.0
O
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
0
149.0 163.0
2.6e5
0
5
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
O
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 purified 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 purified 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-
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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 flesh (which represents 90% of the whole fruit) in different sweet cherry varieties [5]. Seeram et al. purified 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 flow injection analysis in H2O/MeOH (5:95 v/v) acidified with 0.1% formic acid at 0.5 mL min 1. The MS spectra generated for fractions 2 and 3 allowed identification 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 [M–sugar + 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 efficient, economic and environmental friendly one saving energy solvent and waste. To ensure extract or purified compounds drying and storage, avoiding anthocyanin degradation, lyophilization and freezing were used. The purification 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 identified 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 purification of anthocyanins from other cherry varieties.
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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, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS, J. Agric. Food Chem. 49 (2001) 3515–3521. [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 flavonols in petals of lotus (Nelumbo) cultivars by high-performance liquid chromatography-photodiode array detection/electrospray ionization mass spectrometry, J. Chromatogr. A 1216 (2009) 106–112. [15] E.N. Bridgers, M.S. Chinn, V.-D. Truong, Extraction of anthocyanins from industrial purple-fleshed sweetpotatoes and enzymatic hydrolysis of residues for fermentable sugars, Indust. Crops. Prod. 32 (2010) 613–620. [16] J. Srivastava, P.S. Vankar, Canna indica flower: new source of anthocyanins, Plant Physiol. Biochem. 48 (2010) 1015–1019. [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) 2195–2208. [18] L. Marchal, J. Legrand, A. Foucault, Centrifugal partition chromatography: a survey of its history, and our recent advances in the field, The Chem. Rec. 3 (2003) 133–143. [19] J.-H. Renault, P. Thépenier, M. Zéches-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) 345–352. [20] F. Qiu, J. Luo, S. Yao, L. Ma, L. Kong, Preparative isolation and purification of anthocyanins from purple sweet potato by high-speed counter-current chromatography, J. Sep. Sci. 32 (2009) 2146–2151. [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) 2741–2745. [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) 9921–9927. [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) 214–222. [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) 719–724. [27] A. Kirakosyan, E.M. Seymour, D.E.U. Llanes, P.B. Kaufman, S.F. Bolling, Chemical profile and antioxidant capacities of tart cherry products, Food Chem. 115 (2009) 20–25.
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[28] D. Bonerz, K. Würth, H. Dietrich, F. Will, Analytical characterization and the impact of ageing on anthocyanin composition and degradation in juices from five 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) 302–309. [30] B. Mozetic, P. Trebse, J. Hribar, Anthocyanins and hydroxycinnamic acids in sweet cherries, Food Technol. Biotechnol. 40 (2002) 207–212.
[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) 185–192. [32] P. Sandra, K. Sandra, A. Pereira, G. Vanhoenacker, F. David, Green chromatography: part 1: introduction and liquid chromatography, LC-GC Eur. 23 (2010) 242–259. [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) 362–369.
Contents lists available at SciVerse ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Sweet cherries anthocyanins: An environmental friendly extraction and purification method Cristina G. Grigoras a,b, Emilie Destandau a,⇑, Sandrine Zubrzycki a, Claire Elfakir a a b
Institut de Chimie Organique et Analytique, Université d’Orléans, CNRS UMR 7311, rue de Chartres BP 67059, 45067 Orléans 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-inflammatory 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 purified 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. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Sweet cherries are very widespread and appear on the market as the first 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 beneficial effects have been related to the presence of natural polyphenolic compounds [3–7]. 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 flavylium 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 first 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 benefits have been linked to their antioxidant properties and to their notable effects against chronic inflammation, cardiovascular hyperten-
⇑ Corresponding author. E-mail address: [email protected] (E. Destandau). 1383-5866/$ - see front matter Ó 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 acidified aqueous mixtures of ethanol, methanol or acetone [11– 16]. Counter Current Chromatography (CCC), a versatile liquid–liquid preparative chromatography based on the partition of solutes between two immiscible solvents, appears today as a powerful tool for compounds purification. Indeed, CCC benefits 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
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no solvent for extraction, safe solvent and additives for purification, limiting solvent and energy consumption.
set at 1 mL min 1. The elution was achieved at room temperature using the following linear gradient: 0–25 min from 5% to 65% solvent B.
2. Materials and methods 2.5. Anthocyanin purification 2.1. Reagents Methanol, ethanol, trifluoroacetic acid and formic acid used for HPLC were of analytical grade and were provided by SDS Carlo Erba (Val-de-Reuil, France). Water was purified (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).
Purification 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 (Montluçon France). An isocratic mobile phase consisting of H2O/EtOH both acidified with 1% formic acid (86:14 v/v) with a flow rate of 4 mL min 1 was used for compound purification. 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. Purified 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 specific 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.
Identification of purified anthocyanins was performed after their dissolution in H2O/MeOH (5:95 v/v) acidified 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) acidified with 0.1% formic acid and flow 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 flow rate, NEB = 8 (1.2 L min 1); curtain gas flow 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 fiber 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 acidified with 0.1% trifluoroacetic acid were used as solvent A and B respectively. The mobile phase flow rate was
A
B
C
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 solid–liquid 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.
D
E
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.
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400000
ELSD
350000
Intensity (uA)
300000 250000 200000 150000 100000 50000 0 0
5
10
15
20
25
Time (minutes)
900000
UV 280 nm
Intensity (uA)
750000 600000 450000 300000 150000 0 0
5
10
15
20
25
Time (minutes)
1050000
UV 520 nm
900000
Intensity (uA)
750000 600000 450000 300000 150000 0 0
5
10
15
20
25
Time (minutes) 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 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 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.
In order to define 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 efficiency. Microwave irradiation power was increased between 200 W and 1000 W, with
a fixed 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
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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 efficient. Indeed, amount of extracted juice is higher with SFMAE than with pressing. Moreover, the chromatographic profiles 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 filtration 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 flavylium 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 purification, 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 flow 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 first 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, defined 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 purification 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 purification 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 figure legend, the reader is referred to the web version of this article.)
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3.4. Anthocyanins isolation and purification
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 flavylium cation form, formic acid was used in a relatively small amount (1%) instead of trifluoroacetic 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 acidified 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 acidified 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 profile 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 [3,6,23,28–30]. Main peaks eluted around 15–17 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, specific wavelength of cyanidin compounds (Table 1). In order to confirm this hypothesis, cyanidin-3-O-glucoside chloride, cyanidin-3-O-rutinoside chloride and cyanidin-chloride standards were injected. For the first two the retention times were also around 15–17 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 confirmed. 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 confirmed and the purification process could be focused on these two peaks.
30000
2
1
3
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 profile of sweet cherries extract purification 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 acidified with 1% HCOOH; Isocratic elution: 86% A/14% B, Injection 100 lL of 500 mg mL 1 crude extract solution.
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C.G. Grigoras et al. / Separation and Purification Technology 100 (2012) 51–58 +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
5
10
15
20
25
Time (minutes)
3.0e5
OH 241.0
OH
2.5e5
2.0e5
+
HO
149.0
O
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
0
149.0 163.0
2.6e5
0
5
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
O
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 purified 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 purified 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 Purification Technology 100 (2012) 51–58
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 flesh (which represents 90% of the whole fruit) in different sweet cherry varieties [5]. Seeram et al. purified 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 flow injection analysis in H2O/MeOH (5:95 v/v) acidified with 0.1% formic acid at 0.5 mL min 1. The MS spectra generated for fractions 2 and 3 allowed identification 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 [M–sugar + 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 efficient, economic and environmental friendly one saving energy solvent and waste. To ensure extract or purified compounds drying and storage, avoiding anthocyanin degradation, lyophilization and freezing were used. The purification 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 identified 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 purification of anthocyanins from other cherry varieties.
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