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J. of Supercritical Fluids 86 (2014) 100– 107

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

jou rn al hom epage: www.elsev ier .com/ locate /supf lu

xtraction of anthocyanins and luteolin from Arrabidaea chica byequential extraction in fixed bed using supercritical CO2, ethanol andater as solvents

ulia T. Paulaa, Losiane C. Paviania, Mary A. Fogliob, Ilza M.O. Sousab,ustavo H.B. Duartec, Michelle P. Jorgeb, Marcos N. Eberlinc, Fernando A. Cabrala,∗

Department of Food Engineering, State University of Campinas – UNICAMP, 13083-862 Campinas, SP, BrazilChemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA), State University of Campinas – UNICAMP, 13083-970 Campinas, SP, BrazilThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, State University of Campinas – UNICAMP, 13083-970 Campinas, SP, Brazil

r t i c l e i n f o

rticle history:eceived 25 September 2013eceived in revised form 9 December 2013ccepted 14 December 2013

eywords:rrabidaea chica Verlotnthocyaninsarajurin

a b s t r a c t

Phenolic compounds from Arrabidaea chica Verlot leaves, besides conferring staining properties to theirextracts, also have various biological activities including anti-inflammatory and wound-healing prop-erties. To evaluate new possibilities for obtaining extracts with differentiated yield and composition,sequential extractions in fixed bed were performed at 40 and 50 ◦C, and 300 and 400 bar, using as extract-ing solvents pure supercritical carbon dioxide (scCO2) in a first step, acidified ethanol in a second stepand acidified water in a third extraction step. Four flavonoids of interest were investigated in the extracts,one of them being flavone (luteolin), and three anthocyanin compounds of type 3-desoxyanthocyanidinswhich were quantified by high performance liquid chromatography (HPLC). The extraction curves, the

uteolinupercritical extractionequential extraction

global yields and the concentration and yield of the compounds under study were evaluated. The resultsindicated that the cumulative total yields in the three steps ranged from 22% to 27% in all conditionsof temperature and pressure, with the highest global yield at 50 ◦C and 300 bar. Although the lowestextraction yield was obtained using pure scCO2, this step was highly selective, since only carajurin in itsaglycone form was extracted among the compounds of interest and this was confirmed by analysis of

MS/MS.

. Introduction

Arrabidaea chica Verlot known as “Crajiru”, “Pariri” or “chica”elongs to the family Bignoniaceae, is found in various parts ofrazil and is very common in the Amazon Rainforest [1]. Accordingo literature, acidified hydroalcoholic extracts of this plant promoteecovery and healing of Achilles tendon [2,3]. Dichloromethane andethanolic extracts showed good antimicrobial activity against

andida spp [4,5]. In vitro and in vivo studies demonstrated theealing ability of the crude hydroalcoholic extracts by increasinghe production of fibroblasts [6].

The most common anthocyanins found in A. chica are of type 3-esoxyanthocyanidins (Fig. 1); 6,7,3′-trihydroxy-5-dimetoxifl-vilium (Fig. 1a); 6,7,3′,4′-tetrahidoxi-5-methoxy-flavyliumFig. 1b) known as carajurone; and 6,7-dihydroxy-5,4′-

imetoxiflavilium (Fig. 1c) known as carajurin. Luteolin′,4′,5,7-tetrahydroxyflavone (Fig. 1d) belongs to the flavoneroup of flavonoids, and is usually present in the extracts at low

∗ Corresponding author. Tel.: +55 19 3521 4030; fax: +55 19 3521 4027.E-mail address: cabral@fea.unicamp.br (F.A. Cabral).

896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.supflu.2013.12.008

© 2013 Elsevier B.V. All rights reserved.

concentrations as compared with other flavonoids such asquercetin [7]; however, flavone has strong antioxidant, anti-inflammatory, anti-allergic, and anti-cancer properties [8–11].

The sequential extraction procedures explore the differentproperties of the solvents. CO2 is a nonpolar molecule, thus itsnonpolarity restricts the extraction of either apolar substances orwith low polarity. For that reason, the ethanolic (polarity = 5.2) andaqueous (polarity = 9.0) extraction is important for extracting polarsubstances. In this context, a sequential extraction can be used,in which supercritical CO2 extracts the nonpolar and low polaritycompounds, followed by an ethanolic and/or aqueous extractionthat allow to obtain more concentrated extracts in polar com-pounds [12–14].

Martinez-Correa et al. [12,13] have obtained extracts of rose-mary leaves (Baccharis dracucunfolia L.) and pitanga leaves (Eugeniauniflora) in two stages, using scCO2 at 60 ◦C and 400 bar as asolvent in fixed bed extractor in a first step, followed by a conven-tional extraction using ethanol or water at 60 ◦C and atmospheric

pressure in the second step. The prior extraction with scCO2produced an aqueous extract with the highest yield of phenoliccompounds, or an ethanolic extract more concentrated in phenolicsand flavonoids.

J.T. Paula et al. / J. of Supercritical Fluids 86 (2014) 100– 107 101

F -flavil( eolin

(fatclcetPcsTmw

letla

2

2

p–poo5ee

2

tErsbs(T(wu(d

ig. 1. Structure of aglycones of: (a) pigment (1), 6,7,3′ ,4′-tetrahydroxy-5-metoxyc) pigment (3), 6,7-dihydroxy-5,4′-dimetoxy-flavilium, known as carajurin. (d) Lut

Seabra et al. [14] studied the effect of the solvent mixtureCO2/ethanol/water) in the extraction of anthocyanins derivedrom eldeberry pomace. The experiments were performed at 40 ◦Cnd 210 bar using scCO2 in a first step, and CO2/ethanol/H2O mix-ure at different ratios in a second extraction step in order to obtainoncentrated anthocyanin fractions. In the first step extraction, aipophilic fraction was obtained, and in the second step, a mixtureontaining CO2/ethanol/H2O at different ratios had a great influ-nce on the extraction yield and composition of total phenolics,otal flavonoids, anthocyanins and rutin in the extracts. Likewise,aula et al. [15] obtained extracts of A. chica Verlot using super-ritical carbon dioxide in a first step, followed by a mixture ofcCO2/ethanol/water in a second step, both at 40 ◦C and at 300 bar.he authors concluded that the addition of water in the solventixture was fundamental for obtaining a high extraction yield asell as a high content of anthocyanin compounds.

The aim of this study was to obtain extracts of A. chica Ver-ot leaves using pure supercritical carbon dioxide in a first step,thanol in the second step and water in a third step, and evaluatehe composition of the extracts in terms of phenolic compounds,uteolin and three anthocyanin compounds (pigment 1, carajurone,nd carajurin).

. Materials and methods

.1. Raw material, sampling and fixed bed characterization

The sample of A. chica leaves provided by CPQBA (Multidisci-linary Center for Chemical, Biological and Agricultural Researches

Unicamp, Campinas, Brazil) was the same sample used in arevious study [15], characterized with mean particle diameterf 0.536 mm, calculated by ASAE procedure [16], real densityf 1.32 ± 0.01 g/cm3 obtained by helium gas picnometry, and.30 ± 0.32% moisture content by Karl Fisher method. The appar-nt density of the bed containing milled leaves in the fixed bedxtractor was 0.268 ± 0.019 g/cm2 and porosity of 79.7%.

.2. Extraction procedures

The experiments for obtaining the extracts in fixed bed extrac-or were carried out in the experimental extraction unit LaboratoryxTrAE, UNICAMP, Brazil, whose detailed description of the appa-atus can be found in the previous work [15]. A. chica leaves wereubjected to sequential extraction processes, in three steps, in fixeded extractor using three different solvents. In the first step, pureupercritical CO2 was used with an average flow rate of 1.65 g/min1 L/min) at the system outlet, at 0.93 bar and 25 ◦C (� = 1.65 g/L).he second and third extraction steps were performed with ethanol� = 785 g/L, 25 ◦C) acidified with 0.3% citric acid (pH = 3.1), and

ater (� = 1000 g/L, 25 ◦C) acidified with 0.3% citric acid (pH = 2.4),sing an average flow rate of 0.39 g/min (0.5 mL/min) and 0.5 g/min0.5 mL/min), respectively. Fig. 2 represents the experimental con-itions.

ium; (b) pigment (2), 6,7,4′-trihydroxy-5-metoxy-flavilium, known as carajurone;(3′ ,4′ ,5,7-tetrahydroxyflavone).

During sampling of the extract fractions to construct the extrac-tion curve, browning of the samples was observed in the third stepusing water as extracting solvent. For this reason, the process waschanged and the third step was made by conventional aqueousextraction of the residue 2, at the same operating temperature andambient pressure, thus obtaining the third extract (EA/PESC). Toobtain the third aqueous extract (EW), the residue 2 was dried in avacuum oven (Marconi, MA 030-12, Brazil) at 40 ◦C for an hour anda half [15]. All experiments were performed in triplicate.

2.3. Determination of total phenolics

The determination of total phenolics was performed using theFolin–Ciocalteu reagent, according to the procedure of SINGLETONet al. [17], and expressed as gallic acid equivalents (GAE)/g.

2.4. High performance liquid chromatography (HPLC)

The chromatographic analysis was performed by a Shi-matzu gas chromatograph system SCL-10A; LC-10AT; FCV-10AL;and CTO-10AS, using Shimatzu UV and diode array detec-tor (model SPD-M10A) and C-18 column (Phenomenex Gemine(4.6 mm × 250 mm i.d. × 3 �m)) at a flow rate of 1 mL/min at350 nm, and 0.5 mL/min at 470 nm for the analysis of luteolin andpigments 1, 2, and 3, respectively, present in the extracts of A. chicaVerlot.

The qualitative analysis of pigments 1 and 2 and quantita-tive analysis of pigment 3 carajurin was performed accordingto the method described by Devia et al. [18]. Acetonitrile (HPLCgrade, Mallinckrodt), trifluoroacetic acid (TFA) (Merck), and ultra-pure water (Milli-Q, Millipore) with a conductivity of 18 m� wasused to prepare the mobile phase used to develop the chro-matograms. Since there are no standards for the other anthocyaninpigments (1) 6,7,3′,4′-tetrahydroxy-5-methoxy flavylium and (2)6,7,4′-methoxy-5-trihydroxy flavylium, the concentrations werecalculated using a standard curve of carajurin and the concentrationwas expressed as equivalent of carajurin. The quantitative analysisof luteolin was carried out according to the method described byWang and Li [19]. Methanol (HPLC grade) and phosphoric acid (1 M,adjusted to pH 2 with ultra pure water) were used to prepare themobile phase.

2.5. Mass spectrometry

2.5.1. Sample preparationA 7% formic acid (Merck, Darmstadt, Germany) in deionized

water and methanol (Tedia, Fairfield, OH, USA) (1:1) stock solu-tion was prepared. Sample solutions were prepared by dilutingan aliquot of 5 �L of each sample in polypropylene microtubes

(Eppendorf®) in 1 mL of the formic acid/methanol/water solu-tion. Each tube was agitated in a Vortex mixer for 1 min. Samplesolutions were analyzed by direct infusion into the mass spectrom-eter. The extraction, dilution and analysis sequence were repeated

102 J.T. Paula et al. / J. of Supercritical Fluids 86 (2014) 100– 107

n thre

(ilma

2

a

Fig. 2. Sequential extraction i

duplicate) for each sample. An aliquot of 20 �L of samples werenjected at flow rate of 0.5 mL/min. The concentrations of the ana-yzed samples were of 3.95, 9.01, 4.20 and 3.44 mg of extract per

L of solution, respectively for aqueous, ethanolic, supercriticalnd, hydroalcoholic.

.5.2. Mass spectrometryA Q-TOF mass spectrometer (Micromass, Manchester, UK) with

n electrospray interface and running in the positive ion mode was

Fig. 3. Overall extraction curves in

e steps in fixed bed extractor.

used to perform ESI-MS analyses. The ion source unit was operatedat a desolvation temperature of 100 ◦C, capillary voltage of 3.0 kVand cone voltage of 30 eV. Samples were directly infused into theion source at a rate of 10 �L/min using a Harvard syringe pump.The spectra were acquired in the interval of 50–1000 m/z (mass-

to-charge ratio) and accumulated for 60 s. The data acquisition wasunder the control of Mass Lynx software.

A LTQ-FT Ultra (Thermo Scientific – Germany) with an Elec-trospray interface and running in the positive mode was used to

fixed bed from three steps.

ritical Fluids 86 (2014) 100– 107 103

pipaia

3

3

eeorepw4nt

15svtpde((qe

TE

C

J.T. Paula et al. / J. of Superc

erform collision-induced dissociation (CID) experiments for theon m/z 299. The ion source unit was operated at a desolvation tem-erature of 280 ◦C, capillary voltage of 3.44 kV, and collision energyt 20 eV. Helium was used as CID gas. The spectra were acquiredn the interval of 80–1000 m/z and accumulated for 60 s. The datacquisition was under the control of Excalibur software.

. Results and discussion

.1. Global extraction curves

Initially, once the extraction time was not pre-established, fourxtraction curves were constructed, as shown in Fig. 3, one forach experimental condition. The curves were constructed in termsf cumulative yield of dry extract (discounting the mass of cit-ic acid added) as a function of S/F (mass of solvent used in thextraction/initial mass of raw material). Three different solvents,ure scCO2, ethanol and water both acidified with 0.3% citric acid,ere used at the following operating conditions: 40 ◦C/300 bar,

0 ◦C/400 bar, 50 ◦C/300 bar, and 50 ◦C/400 bar. Overall, there waso significant difference between the total cumulative yields of thehree steps, which ranged from 37% to 39%.

In the first step of the scCO2 extraction, global yields ranged from.2% to 1.9% (mass of extract per 100 g raw material) when using0–200 g CO2 per gram of raw material. Fig. 4 shows the compari-on of the respective extraction curves in Fig. 3, in which for initialalues of S/F = 30 (30 g CO2 per gram of raw material) about 80% ofhe total extract are extracted. Fig. 4 shows than the effect of tem-erature and pressure on vapor pressure of solute and on solventensity, promotes the increase of solubility and increase of yield ofxtraction. Densities of 910.34 kg/m3 (40 ◦C, 300 bar), 956.16 kg/m3

40 ◦C, 400 bar), 872.6 kg/m3 (50 ◦C, 300 bar), and 923.41 kg/m3

50 ◦C, 400 bar) were used in this case [20]. Then, for the subse-uent experiments, S/F = 30 was adopted for the first step (90 minxtraction or 148.5 g CO2 for approximately 5 g raw material).

able 1xtraction yield and total phenolic compounds.

Process Extract Xo

40 ◦C – 300 bar scCO2 040 ◦C – 300 bar Ethanolic

40 ◦C – P atm. Aqueousc

Total

40 ◦C – 400 bar scCO2 040 ◦C – 400 bar Ethanolic

40 ◦C – P atm. Aqueousc

Total

50 ◦C – 300 bar scCO2 050 ◦C – 300 bar Ethanolic

50 ◦C – P atm. Aqueousc 15

Total

50 ◦C – 400 bar scCO2 150 ◦C – 400 bar Ethanolic

50 ◦C – P atm. Aqueousc

Total

Hydroalcoholic 70:30d Ethanol/water ambient conditions

, concentration (mg GAE/g extract); R, yield (mg GAE/g raw material); P atm, atmosphera Xo, global extraction yield (%, d.b.) discounting citric acid added.b Yo, total extraction yield, without discounting the mass of citric acid added.c Conventional aqueous extract from the residue 2 (residue from ethanolic extraction).d Paula et al. [15].

Fig. 4. Extraction kinetics with scCO2 for the four operating conditions using scCO2

as solvent.

In the subsequent ethanolic extraction, the extraction yields var-ied from 11.7% to 13% using a ratio of S/F from 42 to 86 (42–86 gethanol per gram of raw material). However, it can be seen that atvalues of S/F = 23.5 (corresponding to 300 min extraction) approx-imately 70% of the total extract are extracted. Thus, the periodof 300 min was adopted for the ethanolic extraction. The aque-ous extraction was carried out with a ratio of S/F from 38 to 47,with yields ranging from 23% to 26%. Fig. 3 shows that this extrac-tion was very fast, since more than 70% of the total extract wereextracted using initial values of S/F = 10. However, in this latter case,

browning of the extracts was observed, so this last step (aqueousextraction) was carried out by conventional extraction process atthe same temperature, but at atmospheric pressure.

(%a Yo (%)b Total phenolics

C R

.7 ± 0.1 0.7 ± 0.1 69 ± 2 0.50 ± 0.088 ± 1 17 ± 2 107 ± 1 18 ± 2

14 ± 2 20 ± 3 60 ± 1 12 ± 2

23 ± 3 38 ± 5 – 31 ± 4

.8 ± 0.3 0.8 ± 0.3 65 ± 1 0.5 ± 0.210 ± 2 19 ± 4 76 ± 1 14 ± 313 ± 3 19 ± 4 41 ± 1 8 ± 2

23 ± 5 38 ± 8 – 22 ± 5

.8 ± 0.2 0.8 ± 0.2 40 ± 2 0.30 ± 0.0911 ± 2 20 ± 3 115 ± 1 23 ± 3.2 ± 0.8 21 ± 1 43 ± 1 9.0 ± 0.6

27 ± 3 42 ± 4 – 32 ± 4

.5 ± 0.3 1.5 ± 0.3 33 ± 2 0.5 ± 0.111 ± 2 20 ± 3 127 ± 1 25 ± 414 ± 1 20 ± 1 27.8 ± 0.2 5.4 ± 0.3

26 ± 3 41 ± 2 – 31 ± 4

29 ± 2 34 ± 3 122 ± 1 41 ± 3

ic pressure.

104 J.T. Paula et al. / J. of Supercritical Fluids 86 (2014) 100– 107

Fig. 5. Chromatogram of the extract from the sequential extraction at 40 ◦C and 400 bar. Gemini C18 column at 35 ◦C � = 470 nm, flow rate 0.5 mL/min. (a) scCO2 (SC),(b) ethanolic extraction (E), (C) aqueous extraction after (E). Peak 1: 6,7,3′ ,4′-tetrahydroxy-5-metoxiflavilium; Peak 2: 6,7-trihydroxy-5-metoxiflavilium; Peak 3: carajurin(6,7-dihydroxy-5,4′-dimetoxiflavilium).

Table 2Concentration and extraction yield of 3-deoxy antocianidins and luteolin present in the extracts of A. chica Verlot.

Process Yo (%) Carajurin (3) (1)* (2)*∑

(1* + 2* + 3) carajurinequivalent

Luteolin

C (%) R CE (%) CE (%) CE (%)∑

R C (%) R

40 ◦C – 300 bar scCO2 0.7 ± 0.1 3.28 0.21 0 0 3.28 0.21 0 040 ◦C – 300 bar Ethanolic 17 ± 2 8.13 14.0 2.05 6.40 16.6 28.5 0.23 0.1840 ◦C – P atm. Aqueousa 20 ± 3 0.49 0.96 0.28 0.77 1.54 3.0 0 0

Total 38 ± 5 – 15.2 – – – 31.7 – 0.18

40 ◦C – 400 bar scCO2 0.8 ± 0.3 3.68 0.3 0 0 3.68 0.3 0 040 ◦C – 400 bar Ethanolic 19 ± 4 8.15 15.1 1.78 6.04 116.0 29.6 0.19 0.1840 ◦C – P atm. Aqueousa 19 ± 4 0.36 0.68 0.2 0.58 1.14 2.15 0 0

Total 38 ± 8 – 16.08 – – – 32.05 – 0.18

50 ◦C – 300 bar scCO2 0.8 ± 0.2 3.82 0.32 0 0 3.82 0.32 0 050 ◦C – 300 bar Ethanolic 20 ± 3 7.38 14.6 1.59 5.4 14.4 28.5 0.22 0.2450 ◦C – P atm. Aqueousa 21 ± 1 0.38 0.8 0.25 0.71 1.34 2.83 0 0

Total 42 ± 4 – 15.72 – – – 31.7 – 0.24

50 ◦C – 400 bar scCO2 1.5 ± 0.3 0.34 0.05 – – 0.34 0.34 – –50 ◦C – 400 bar Ethanolic 20 ± 3 9.27 18.2 1.72 5.84 16.8 32.9 0.15 0.1650 ◦C – P atm. Aqueousa 20 ± 1 0.5 0.98 0.29 0.85 1.64 3.20 0 0

Total 41 ± 2 – 19.23 – – – 36.5 – 0.16

Hydroalcoholic 70:30b Ethanol/water 34 ± 3 5.78 19.4 1.90 5.00 12.7 42.5 0.901 2.61

Yo, yield (%, d.b.): extracts containing citric acid; C, concentration (wt %); R, yield (mg/g leaf); (1)* and (2)*, compounds (1) and (2) from Arrabidaea chica. CE, carajurinequivalent concentration.

a Conventional aqueous extract from the residue 2 (residue from ethanolic extraction).b Paula et al. [15].

J.T. Paula et al. / J. of Supercritical Fluids 86 (2014) 100– 107 105

F bar (�

eougfa

wvbc

3

lh4cma4

Atpw

ig. 6. Chromatogram of the extracts from (a) ethanolic extraction at 40 ◦C and 400 = 350 nm, flow rate 0.5 mL/min. Peak 1: luteolin.

After establishing the time intervals (or solvent mass S/F), threexperiments were performed in each experimental condition, andnly one accumulated extract was collected. Table 1 shows the val-es of global yield, concentration and yield of total phenolics (inallic acid equivalents). The global yield was calculated by two dif-erent ways: as Xo, where the added citric acid was deducted, ands Yo, where the citric acid was incorporated.

Table 1 shows that the highest yield values were obtained whenater was used as solvent, indicating that the polarity of the sol-

ent influenced the extraction yield. Similar findings were observedy Paula et al. [15], who obtained Crajiru extracts using a mixtureontaining scCO2/ethanol/water as solvent.

.2. Total phenolic compounds

As shown in Table 1, the ethanolic extracts have higher pheno-ics concentration (approximately 100 mg GAE/g extract), and theighest value of 127 mg GAE/g extract was obtained at 50 ◦C and00 bar, similar to the value of 122 mg GAE/g extract obtained byonventional hydroalcoholic extraction. Paula et al. [15] obtained aore concentrated extract containing 178 mg GAE/g extract, using

mixture of scCO2/ethanol/water as solvent at a ratio of 80:20:0 at0 ◦C and 300 bar.

The aqueous extracts presented lower phenolics concentration.

ll extracts obtained with scCO2 were significantly different, once

he phenolics content decreased with increasing temperature andressure, and the highest concentration (69.3 mg GAE/g extract)as obtained at 40 ◦C and 300 bar.

E); (b) conventional hydroalcoholic extraction (HEC). Gemini C18 column at 35 ◦C,

The phenolics concentrations in the ethanolic extracts would bemuch higher if the citric acid was not added to the solvent, becausedilutes the dry extracts. For example, at 300 bar and 50 ◦C, the con-centration would be on the order of 211 mg GAE per gram extract,against 115 mg GAE obtained in this condition.

3.3. Anthocyanins and luteolin quantification by HPLC

Fig. 5 shows the chromatograms at 470 nm of the extractsobtained at 40 ◦C and 400 bar for the three anthocyanin compounds.Fig. 6 shows the chromatograms at 350 nm for both ethanolicextract obtained at 40 ◦C and 400 bar and conventional hydroal-coholic extract, where the peak 1 corresponds to luteolin. Table 2presents the global yields and the respective carajurin and luteolinconcentrations. The carajurin and luteolin concentration was pre-sented as a percentage by weight (gram of solute per 100 g extract),and the extraction yield was calculated based on global yield andconcentration in the extract, obtaining the extraction yield in mgof solute per gram of A. chica Verlot leaves.

In general, the ethanolic extracts had higher carajurin con-tent when compared to the extracts obtained with other solvents.Moreover, the prior extraction with scCO2 increased the cara-jurin content in the ethanolic extracts of three-step sequential

extraction. However, as can be seen in the chromatograms, thesupercritical extracts were more selective, having only cara-jurin, which can be explained by the polarity of this compound[smaller amount of OH groups as compared to the pigments

106 J.T. Paula et al. / J. of Supercritical Fluids 86 (2014) 100– 107

360m/z0

100

%

0

100

%

0

100

%

C_44 ms 44 (0.856) Cm (1:50) TOF MS ES+ 641299.1027

249.0012 277.2276262.9726 291.2378

300.1186

429.3877409.3879391.2804365.2796

301.1158331.1193 7693.8440992.973 463.3847

e44 - ms 2 (0.041) Cm (1:54) TOF MS ES+ 5.99e3299.1027

285.1005

261.0156

291.0374301.0955

385.0821305.0395 463.1320407.0636 423.0382 483.0725

a44 ms 39 (0.733) Cm (1:53) TOF MS ES+ 1.13e3245.0348

279.0875

247.0601 263.0487

423.0623287.0859

301.1158299.1230

311.1174

345.0399329.0080

317.1089377.0620349.0417

370.8874407.0872379.0705

463.1320

437.0842

430.9215443.0188

455.0833

477.1564464.1431 478.1570

(a)

(b)

(c)

tial ex

(tedttac

gd

3350340330320310300290280270260250240

Fig. 7. ESI(+)-MS of the extracts from the sequen

1) 6,7,3′,4′-tetrahydroxy-5-methoxy flavylium and (2) 6,7,4′-rihydroxy flavylium-methoxy-5]. It is observed in the supercriticalxtracts obtained at 50 ◦C that increasing pressure considerablyecreased the carajurin content. The highest carajurin content inhe supercritical CO2 extracts was obtained at 50 ◦C and 300 bar. Inhe ethanolic extracts, the highest carajurin content was obtainedt 50 ◦C and 400 bar. The aqueous extracts showed low carajurin

ontent.

The molecular structure of luteolin (Fig. 1) has four hydroxylroups, which makes luteolin a molecule insoluble in carbonioxide. This fact explains the absence of luteolin in the scCO2

C44msms299 #1-98 RT: 0.00-0.98 AV: 98 NL: 4.40E5T: ITMS + p ESI Full ms2 299.00@cid20.00 [80.00-1000.00]

80 10 0 12 0 14 0 16 0 180 200 220 240 260 m

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

281253.0871.93280.531 191.08163.08 211.17121.0895.08

OHO

HO

OCH3

O

Fig. 8. ESI(+)-MS/MS of the

48047046045044043042041040039038070

traction at 40 ◦C and 400 bar (a) SC, (b) E e (c) A.

extracts. In general, the extracts showed low luteolin concentration(Table 2). The highest luteolin concentration was obtained both inthe ethanolic extract (2nd step) at 40 ◦C and 300 bar, and in theconventional hydroalcoholic extract.

3.4. Mass spectrometry

Once the extract obtained with pure CO2 presented only cara-jurin, mass spectrometry was performed to confirm whetherthe carajurin was in the aglycone form. Fig. 8 shows themass spectrum of the extracts obtained at 40 ◦C and 400 bar.

280 300 320 340 360 380 400 420 44 0 460/z

284.17

299.08.17 395.75 434.25341.08 355.00 456.9233.01429.213

OHO

HOOCH3

OCH3

cationic 3 (m/z 299).

ritical

I6tpr

tmp1efs

ftrF2

4

dpeta(mttp

A

f

R

[

[

[

[

[

[

[

[

[

[

[

J.T. Paula et al. / J. of Superc

ons of m/z 301, 285 and 299 correspond to the pigment (1),7,3′,4′-tetrahydroxy-5-metoxy-flavilium; pigment (2) 6,7,4′-rihydroxy-5-metoxy-flavilium known as carajurone; andigment (3) carajurin, 6,7-dihydroxy-5,4′-dimetoxy-flavilium,espectively.

In the supercritical extract (Fig. 7a), it is possible to visualizehe presence of carajurin as aglycone (m/z 299) and the absence of/z 285 and 301. In contrast, in the ethanolic extract (Fig. 7b) isossible to visualize not only the carajurin but also the pigments

and 2. In the aqueous extract (Fig. 7c), it is observed the pres-nce of three anthocyanin majority pigments in their glycosylatedorm (m/z 423, 437 and 463). The information provided by the masspectra confirms the data obtained by HPLC-UV analysis (Table 2).

In order to confirm the identity of carajurin, MS/MS was per-ormed for the ion of m/z 299. The fragmentation spectrum showshe ions of m/z 299 and m/z 284. The difference of 15 Da in massepresents the loss of a methyl group from the carajurin structure.ig. 8 provides suggestions for the fragmentation of the ion of m/z99 [21].

. Conclusions

The three step sequential extraction using three solvents withifferent polarities allowed obtaining different extracts in terms ofhenolics composition. In the first step, the scCO2 was selective toxtract only carajurin among the four target components. In addi-ion, the ESI MS confirmed that carajurin was present in the extracts aglycone. The ethanolic extracts after supercritical extractionsecond step), and the conventional hydroalcoholic extract were

ore concentrated in phenolic compounds. This technique is viableo obtain high yields of extraction and, the three steps can enablehe production of differentiated extracts containing the main com-onents of interest..

cknowledgment

The authors thank FAPESP (Process 2012/50182-5) and CAPESor their financial support.

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