J. of Supercritical Fluids 46 (2008) 33–39

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journa l homepage: www.e lsev ier .com/ locate /supf lu

Supercritical CO2 extraction of carotenoids from pitangafruits (Eugenia uniflora L.)

Genival L. Filhoa, Veridiana V. De Rossob, M. Angela A. Meirelesa, Paulo T.V. Rosac,d b a,∗

Alessandra L. Oliveira , Adriana Z. Mercadante , Fernando A. Cabral

a Department of Food Engineering, Faculty of Food Engineering, State University of Campinas, 13083-862 Campinas, SP, Brazilb Department of Food Science, Faculty of Food Engineering, State University of Campinas, 13083-862 Campinas, SP, Brazilc Department of Physical-Chemistry, Institute of Chemistry, State University of Campinas, 13084-862 Campinas, SP, Brazild Department of Food Engineering, Faculty of Animal Husbandry and Food Engineering, University of Sao Paulo,

de (Ss (Eugextra

300,nnectin weation

cover4% ofenoidexperd conin th

13635-900 Pirassununga, SP, Brazil

a r t i c l e i n f o

Article history:Received 8 October 2007Received in revised form 21 February 2008Accepted 27 February 2008

Keywords:CarotenoidsEugenia unifloraLycopeneRubixanthinSupercritical fluid extraction

a b s t r a c t

Supercritical carbon dioxidried pulp of pitanga fruitcommercially. The SC-CO2

sures, 100, 150, 200, 250,liquid chromatography coanthin and �-cryptoxanth�-cryptoxanthin concentrditions. The maximum retotal carotenoid content, 7conditions, the total carotand 32% rubixanthin. Theto the extraction yield anbon dioxide was selective

pressure.

1. Introduction

Pitanga (Eugenia uniflora L.) is a tree widely distributed inSouth American countries, mainly in Brazil, Argentina, Uruguay andParaguay [1]. Its fruits are gaining worldwide popularity due to theirnutritional value and exotic flavours that appeal to the consumer.Studies have shown that the pitanga fruit and leaves may be usefulin preventing human diseases [2–4].

In the Brazilian food industry, the pitanga fruit has mostly beenused to produce juice, which shows good economic potential due tothe consumer appeal arising from its high concentration of antiox-idant compounds, such as anthocyanins, flavonols and carotenoids[5]. Some studies have reported on the composition of the essentialoils obtained from its leaves and fruits [2,6].

During the ripening process, the fruits change from green to yel-low, to orange, to red and then to dark red, becoming almost blackin some cases [7] when lycopene is the main carotenoid found. The

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

0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.supflu.2008.02.014

C-CO2) extraction was employed to extract carotenoids from the freeze-enia uniflora L.), an exotic fruit, rich in carotenoids and still little exploredction was carried out at two temperatures, 40 and 60 ◦C, and seven pres-

350 and 400 bar. The carotenoids were determined by high-performanceed to photodiode array and mass spectrometry detectors. Lycopene, rubix-re the main carotenoids present in the freeze-dried pitanga pulp, whereaswas negligible in the SC-CO2 extracts, for all the investigated state con-

y of carotenoids was obtained at 60 ◦C and 250 bar, extracting 55% of thethe rubixanthin and 78% of the lycopene from the pulp. Under these stateconcentration in the extract was 5474 �g/g, represented by 66% lycopeneimental state conditions produced different SC-CO2 extracts with respectcentration of different carotenoids, indicating that the supercritical car-e extraction of the pitanga carotenoids as a function of temperature and

© 2008 Elsevier B.V. All rights reserved.

following carotenoids have been found in pitanga fruits in decreas-ing quantitative order: lycopene, rubixanthin, cis-rubixanthin,�-cryptoxanthin, cis-lycopene, �-carotene, �-carotene, zeaxanthin,lutein, violaxanthin and �-carotene-5,6-epoxide [8].

Carotenoids are the most widespread pigments in nature, themost important being �-carotene, lycopene, lutein and zeaxanthin.The main roles of carotenoids in the human diet are as precursors ofvitamin A and as antioxidants. Lycopene is not a vitamin A precursorbecause it has no �-ionone group, but its capacity to quench singletoxygen is about three times that of �-carotene [9] and it also has agreater colour intensity compared to �-carotene.

Some studies about the SC-CO2 extraction of carotenoids(Table 1) can be found in literature [10–23]. However, there isrelatively little knowledge about the selectivity of SC-CO2 to thedifferent carotenoids.

The search for natural dyes has demanded an increasing searchfor new natural sources and an increase in the amount of researchon their extraction. The carotenoids are important components ofthe human diet, both in disease prevention and for use as natu-ral dyes. In addition, extraction techniques that are non-aggressiveto the environment and that produce products and sub-products

rcritica

athin,

inthin,opene

ents

�-carotene, all-trans-�-carotene and 13-cis-�-carotene were pro-vided by Dr. Werner Simon from DSM Nutritional Products (Basel,Switzerland), showing purity ranges from 95 to 99% by HPLC.

2.4. Experimental set-up and procedures

The carotenoids were extracted, in triplicate, from the freeze-dried pitanga pulp (E. uniflora) using SC-CO2 at differenttemperatures (40 and 60 ◦C) and pressures (100, 150, 200, 250, 300,350 and 400 bar).

Fig. 1 shows the diagram of the extraction equipment (Speedfrom Applied Separations, model 7071, Allentown, PA, USA), con-sisting of an extractor installed inside an electrically heated oven.Before entering the oven, the CO2 was cooled to −5 ◦C in an ethy-

34 G.L. Filho et al. / J. of Supe

Table 1Some examples of supercritical extractions of carotenoids

Material Botanical name Compounds analyzed

Marine algae Nannochloropsis gaditana Carotenoids, chlorophyllMarine micro-alga Spirulina pacifica Zeaxanthin, �-cryptoxan

�-caroteneMicro-alga Chlorella vulgaris Astaxanthin, cantaxanthPumpkin Curcurbita moschata �-�-Carotene, cryptoxan

cis-�-carotene, lutein, lycBuriti Mauritia flexuosa Lipids, carotenoidsTomato paste waste – �-Carotene, lycopeneTomato – LycopeneTomato LycopeneTomato Pear type LycopeneChilli peppers Var. Byedige Capsaicinoid, other pigmCarrot n.m. �-Carotene, �-caroteneLeaf protein concentrates – Carotene, luteinSweet potato tissue – �-CarotenePressed palm oil fibers Elaes guineensis Carotene, lipids

free of undesirable solvent residues have been suggested as alter-natives to conventional processes. Thus the present work proposedto study the SC-CO2 extraction of carotenoids from pitanga fruits,a potential source of important carotenoids still not studied bysupercritical extraction, aiming to selectively extract the differ-ent carotenoids present in the fruit, based on the change inselectivity of the carbon dioxide as a function of the operationalvariables.

2. Material and methods

2.1. Raw material

The in natura fruits were obtained from the Sao Paulo Horticul-tural Supply Centre (CEASA) during the harvesting months, fromOctober to January. The fruits were manually pulped, frozen in anultra-freezer (Ecoclima, SC, Brazil) and freeze-dried by the companyTerroni Equipamentos Cientıficos Ltda. (Sao Carlos, SP, Brazil). Thefreeze-dried fruit was ground using a knife mill (Marconi modelMA-340, Piracicaba, Brazil) and stored in sealed bags at −10 ◦C in adomestic freezer.

2.2. Characterization of the freeze-dried ground pitanga fruit

The moisture content was determined by oven drying at 105 ◦Cuntil constant weight was achieved. The particle size distributionwas determined using a vibratory sieve system (Model 1868, Bertel,Caieiras SP, Brazil); using sieves from 16 to 80 mesh (Tyler series,Tyler, Mentor, OH, USA). Particles of 24–80 mesh were selected forthe SFE assays. The ASAE S319.3 method [24] was used to calculatethe mean diameter (dmg) from the following equation:

dmg = log−1

[∑ni=1wi log di∑n

i=1wi

](1)

where di = (didi+1)0.5 is the geometric mean between the diameterof sieve i and of the i + 1, and wi is the mass of solid retained by sievei.

The particle density (dP) was measured by helium gas pyc-nometry (Model 1305 Multivolume, Micromeritics InstrumentCorporation, Norcross, GA, USA). The bulk density (db) was calcu-lated using the extractor volume and the feed mass. The porosity ofthe bed of particles was calculated using the bulk density and theparticle density, as ε = 1 − (db/dP).

l Fluids 46 (2008) 33–39

T (◦C) P (bar) t (min) Ethanol (%) Reference

40–50–60 100 and 500 180 – [10]40–60–80 150–250–350 40–70–100 5–10–15 [11]

40–55 200–350 n.m. – [12]40–80 310–350–500 40 0–10–30 [13]

40–55 200–300 95–210 – [14]35–45–55–65 200–250–300 60 and 180 5–10–15 [15]32 and 86 137.8 and 482.6 – – [16]

[17]40 77–280 30 – [18]40–80 100–400 330–1182 – [19]30–40–50 300–400–500 60 5–10 [20]40 100–700 – – [21]38 and 48 138–414 – – [22]45–55 250–300 – – [23]

2.3. Materials

Carbon dioxide, 99.9% pure, was acquired from Gama GasesEspeciais Ltda. (Campinas, Brazil). Acetronitrile and ethyl acetatehigh-performance liquid chromatography (HPLC) grade wereobtained from Merck (Darmstadt, Germany). The other reagentswere all of analytical grade from Labsynth (Diadema, SP, Brazil).The solvents were filtered through Millipore membranes (0.45 �m)prior to the HPLC analysis.

Standards of all-trans-�-cryptoxanthin, all-trans-rubixanthin,all-trans-lycopene, 13-cis-lycopene, all-trans-�-carotene, all-trans-

lene glycol bath. For control, the temperature was measured by athermocouple in the outer wall of the extractor, and the extract wascollected in a 50-mL glass flask.

About 5.6 g of ground freeze-dried pitanga with known parti-cle size distribution was manually packed into an extractor with0.78-in. internal diameters and 1.5 in. length (5 mL vessel model,

Fig. 1. Experimental apparatus: (1) CO2 cylinder; (2) CO2cooling bath; (3) boosterpump; (4) heating oven; (5) extractor; (6) sample collection flask; (7) rotometer;(8) digital pressure indicator; (9) (a and b) thermocouples; (10) digital temperatureindicator. Valves: (a) of the CO2 cylinder; (b) entrance of pressurized CO2; (c) vent;(d) of CO2 and product exit; (e) micrometer.

rcritica

G.L. Filho et al. / J. of Supe

Thar Designs, Pittsburgh, PA, USA). The bulk density of the bed wasmaintained constant at 477 kg/m3. Before each extraction, a 10-min static period was adopted to promote greater contact betweenthe particles and the SC-CO2. The global extraction yield (X0) wasmeasured, representing the percent amount of extract that couldbe obtained in an extraction, and the concentration of differentcarotenoids in the extracts were measured for the two isotherms(40 and 60 ◦C) with pressures from 100 to 400 bar (with 50 barincrements).

All the extracts were collected after 120 min of extraction usinga constant CO2 flow rate of 6.8 × 10−5 kg/s, and the line imme-diately following the extractor was then washed internally with10–20 mL ethanol (Ecibra, 99.5%) to recover any rich-pigmentextract deposited in the line. The ethanol used for washing wasthen evaporated from the extract using a rotary evaporator (Hei-dolph Instruments, model Laborota 4001, Schwabach, Germany).All extracts were completely dried.

2.5. Carotenoid analysis

The carotenoids from SC-CO2 extracts were dissolved inpetroleum ether/diethyl ether, saponified overnight with 10%methanolic KOH, washed until alkali free, and concentrated untildryness [25,26]. The carotenoids were exhaustively extracted in a

mortar from 4.0 g of freeze-dried pitanga pulp at room tempera-ture with acetone (five times with 40 mL acetone each) transferredto petroleum ether/diethyl ether, and followed by the same stepsused for the SC-CO2 extracts.

The carotenoid quantitative analysis was carried out in a HPLCfrom Waters (Milford, MA, USA) equipped with a quaternary solventdelivery system (Waters, model 600), on line degasser and a Rheo-dyne injection valve (Rheodyne LCC, Rohnert Park, CA, USA) witha 20-�L loop. The equipment included, connected in series, pho-todiode array detector (PDA) (Waters, model 996). The MillenniumWaters software was used for the data acquisition and process-ing, the chromatograms being processed at 450 nm and the spectraobtained between 250 and 600 nm. The carotenoids were separatedon a C18 Nova-Pak ODS, 300 mm × 3.9 mm (4 �m particle size) col-umn, using a linear gradient of acetonitrile/H2O/ethyl acetate asthe mobile phase, from 88:10:2 to 85:0:15 in 15 min, maintainingthis proportion for further 30 min, with a flow rate of 1 mL/minand column temperature set at 29 ◦C. The carotenoids were quanti-fied using external calibration curves for all-trans-�-cryptoxanthin,all-trans-rubixanthin, all-trans-lycopene, all-trans-�-carotene, all-trans-�-carotene, all-trans-�-carotene, with a minimum of seven

Table 2Characteristics and concentration (�g/g) of carotenoids, extracted with acetone, from free

Peaka Carotenoid tR (min) �max (nm) III/I

1 All-trans-lutein 14.6 423, 447, 476 602 All-trans-zeaxanthin 16.2 430, 453, 481 303 All-trans-rubixanthin 22.1 439, 463, 492 364 cis-Rubixanthin I 22.8 350, 437, 461, 489 305 cis-Rubixanthin II 23.3 350, 437, 460, 488 306 All-trans-�-cryptoxanthin 25.6 432, 454, 481 257 All-trans-lycopene 27.3 447, 474, 505 758 13-cis-Lycopene 28.0 360, 441, 466, 498 549 All-trans-�-carotene 30.9 437, 464, 494 35

10 All-trans-�-carotene 34.5 425, 448, 476 6011 (all-trans + 9-cis)-�-Carotene 35.3 433, 454, 481 2012 (13-cis + 15-cis)-�-Carotene 37.6 343, 420, 449, 475 15

Total carotenoids

(spectral fine estructure): Ratio of the height of the longest wavelength absorption peakheight of the cis peak (AB) and that of the middle main absorption peak (AII).

a Numbered according to the chromatogram shown in Fig. 3.b Average and standard deviation.

l Fluids 46 (2008) 33–39 35

concentration levels. The cis-isomers of lycopene, rubixanthin, �-carotene and �-carotene were quantified using the curve of thecorresponding all-trans-isomer.

For identification purposes, the acetone and SC-CO2 extractswere also analyzed in a Shimadzu HPLC (Kyoto, Japan) equippedwith quaternary pumps (model LC-20AD), on line degasser and aRheodyne injection valve (Rheodyne LCC, Rohnert Park, CA) witha 20-�L loop. The equipment included, connected in series, a PDAdetector (Shimadzu, model SPD-M20A) and a mass spectrometerwith an ion-trap analyser and atmospheric pressure chemical ion-ization (APCI) source from Bruker Daltonics, model Esquire 4000(Bremen, Germany). The UV–vis spectra were obtained between250 and 600 nm and the chromatograms were processed at 450 nm.The MS parameters were as follows—positive mode, current corona:4000 nA, source temperature: 450 ◦C, dry gas N2-temperature:350 ◦C, flow: 60 L/h, nebulizer: 5 psi, MS/MS fragmentation energy:1.4 V. The mass spectra were acquired with scan range of m/z from100 to 700 [26]. The carotenoids were identified according to thefollowing combined information: chromatographic behaviour on aC18 HPLC column [25,27], co-chromatography with authentic stan-dards, UV/visible spectrum (�max, spectral fine structure, peak cisintensity) and mass spectrum compared with data available in theliterature [25–30].

3. Results and discussion

3.1. Carotenoid identification

Twelve carotenoids were identified based on the combinedinformation obtained from chromatographic elution, UV–vis andmass spectra characteristics (Table 2) and the structures of the all-trans-isomers are presented in Fig. 2. The fragments obtained fromMS/MS experiments confirmed the assignment of the protonatedmolecule ([M+H]+) of all identified peaks, as can be seen in Table 2.A detailed description for identification of carotenoids from fruitsusing the above information was already reported by De Rosso andMercadante [26,30].

3.2. Raw material characterization

The sample of ground freeze-dried pitanga showed a mean par-ticle diameter of 0.376 mm, formed from the mean of the materialretained on the following sieves: meshes 16 (0.8%), 24 (6.3%), 32(16.8%), 48 (36.6%) and 80 (31.1%), moisture content of 9.7%, bulkand particle densities of 477 and 1457 kg/m3, respectively, and afixed bed porosity of 67%.

ze-dried pitanga pulp

I (%) AB/AII (%) [M+H]+ (m/z) MS/MS (m/z) Conc (�g/g)b

0 569 551, 533, 477, 459 3.12 ± 0.150 569 551, 533, 463 3.96 ± 0.110 553 535, 497, 461 15.67 ± 0.15

28 553 535, 497, 461 3.62 ± 0.2130 553 n.d. 1.31 ± 0.35

0 553 535, 495, 461 15.24 ± 0.390 537 467, 444 33.22 ± 0.23

62 537 467, 444 6.99 ± 0.130 537 467, 444 1.12 ± 0.160 537 481, 444 0.54 ± 0.320 537 444 2.35 ± 0.15

60 537 444 0.74 ± 0.17

87.88 ± 2.52

(III) and that of the middle absorption peak (II). (cis-peak intensity): ratio of the

rcritica

ll-tran

36 G.L. Filho et al. / J. of Supe

Fig. 2. Structures of the major a

Fig. 3 shows the chromatogram of the carotenoid extract

obtained with acetone from the freeze-dried fruit pulp, andTable 2 the corresponding concentrations. The total carotenoidconcentration in freeze-dried pitanga, was 87.88 �g/g, showingthe following composition in decreasing order of the majorcarotenoids: 33.22 �g/g (37.8%) of all-trans-lycopene, 15.67 �g/g(17.83%) of all-trans-rubixanthin, 15.24 �g/g (17.34%) of all-trans-�-cryptoxanthin, 6.99 �g/g (7.95%) of 13-cis-lycopene and smalleramounts of zeaxanthin, cis-rubixanthin, lutein and �- , �- and�-carotene.

3.3. Global extraction yield

Fig. 4 shows the values obtained (average of the three measure-ments) for the global extraction yield (X0, %) as a function of thedifferent pressure used. At 40 ◦C, the lowest yield of 0.47% wasobtained at 100 bar and the highest overall yield of 0.78% at 400 bar,an increase of 66%. Under these same conditions the solvent den-sity [31] varied from 632.5 to 956.6 kg/m3, an increase in density of51%. Above 200 bar, both the extraction yield and the solvent den-sity were practically constant. At 60 ◦C the variation in density wasmore accentuated, the density at 400 bar (890.7 kg/m3) being three

Fig. 3. HPLC–PDA chromatogram of the freeze-dried pitanga extract extracted byacetone: (1) all-trans-lutein, (2) all-trans-zeaxanthin, (3) all-trans-rubixanthin, (4)cis-rubixanthin I, (5) cis-rubixanthin II, (6) all-trans-�-cryptoxanthin, (7) all-trans-lycopene, (8) 13-cis-lycopene, (9) all-trans-�-carotene, (10) all-trans-�-carotene,(11) all-trans-�-carotene + 9-cis-�-carotene and (12) 15-cis-�-carotene + 13-cis-�-carotene.

l Fluids 46 (2008) 33–39

s-carotenoids found in pitanga.

times higher than that at 100 bar (291.6 kg/m3), resulting in a fivetimes increase in the yield.

At higher pressures, the extraction yield increased with increasein temperature, but at lower pressures, the inverse behaviour wasfound, with the extraction yield decreasing with increasing temper-ature at constant pressure. This behavior can be seen in literaturefor solubility data, which is explained by the effect of the tempera-ture and pressure on the solvent density and of the temperatureon the solute vapour pressures. The crossing point of the yieldisotherms as a function of pressure occurred between 150 and200 bar, with no influence of the temperature on the overall extrac-tion yield at this point. At pressures below 150 bar the behaviourwas similar to that of retrograde condensation as observed in thesolubility of supercritical fluids, where the increase in temperaturedecreases solute solubility.

3.4. Yield and selective extraction of carotenoids by SC-CO2extraction

In order to evaluate the performance and selectivity of SC-CO2as a solvent to selectively extract carotenoids, the concentrationsof the major carotenoids in the SC-CO2 extracts were measured,

Fig. 4. Global yield of the SC-CO2 extracts.

G.L. Filho et al. / J. of Supercritica

Tab

le3

Glo

baly

ield

a,c

once

ntr

atio

ns

ofca

rote

noi

ds

(�g/

g)b

from

extr

acts

obta

ined

bysu

per

crit

ical

extr

acti

onan

dex

trac

tion

yiel

dof

tota

lcar

oten

oidc

T(◦ C

)P

(bar

)G

loba

lyie

ld,

X0

(%)

Ru

bixa

nth

inci

s-R

ubi

xan

thin

�-

Cry

ptox

anth

inLy

cop

ene

cis-

Lyco

pen

e�

-Car

oten

e�

-Car

oten

e(9

-cis

+al

l-tr

ans)

-�-

Car

oten

e

(13

+15

-cis

)-�

-Car

oten

eTo

tal

caro

ten

oidb

Extr

acti

onyi

eld

ofto

tal

caro

ten

oid

c

4010

00.

471

±0.

0526

1014

1814

812

268

138

0.6

815

00.

656

±0.

025

134

68

159

266

123

811

258

802

5.09

200

0.77

2±

0.03

94

4724

615

540

237

29

1542

1316

9911

.75

250

0.73

2±

0.07

74

4731

918

252

640

110

2343

1519

6614

.35

300

0.75

1±

0.01

814

856

44

5911

4747

826

2476

2239

6230

.09

350

0.73

9±

0.04

813

6261

657

528

459

3824

7726

3187

24.9

940

00.

786

±0.

002

1338

550

5338

136

142

2577

2728

5622

.99

6010

00.

193

±0.

019

686

192

126

388

151

4556

171

6618

763.

7215

00.

64

8±

0.0

0712

8662

834

1160

614

3229

9934

3916

23.2

620

00.

771

±0.

066

1182

563

–16

5788

025

2381

264

438

34.3

825

00.

850

±0.

066

1180

547

–26

7591

923

2581

2454

7447

.97

300

0.96

5±

0.07

311

1954

2–

1446

811

2223

7922

406

438

.55

350

1.03

3±

0.05

411

8053

620

182

970

622

2378

2235

9736

.16

400

0.98

2±

0.02

511

4754

324

570

038

522

2574

2131

6333

.30

aA

vera

gean

dst

and

ard

dev

iati

onof

mea

sure

din

trip

lica

te,e

xpre

ssed

asg

ofex

trac

tp

er10

0g

offr

eeze

-dri

edp

itan

ga.

bA

vera

geof

anal

ysis

ind

up

lica

te.C

arot

enoi

dex

pre

ssed

as�

gca

rote

noi

d/g

ofSC

-CO

2ex

trac

tfr

omp

itan

ga.

cTo

talc

arot

enoi

dex

pre

ssed

as�

gca

rote

noi

d/g

offr

eeze

-dri

edp

itan

ga.

l Fluids 46 (2008) 33–39 37

which together with the global extraction yield allowed the calcu-lation of the extraction yield of each carotenoid. Table 3 shows thevalues obtained for global yield in g of extract per 100 g of freeze-

dried pitanga, total carotenoids yield in �g of total carotenoids/gof freeze-dried pitanga and for the concentrations of the differentcarotenoids in the SC-CO2 extract in �g of carotenoids/g of SC-CO2extracts. The concentration was at random measured in two of thethree samples of SC-CO2 extracts. The values of concentration ofminority carotenoids differed between 5 and 12% and of majoritycarotenoids, between 3 and 7%. Table 4 relates the values for thepercentage of recovery and relative concentration. To calculate therecovery, it was considered that the acetone extracted 100% of allthe carotenoids from the freeze-dried pulp.

Table 3 shows distinct behaviours for the extractions madeby the two isotherms and also for the different carotenoids.In the SC-CO2 extracts obtained at 60 ◦C the concentrations oftotal carotenoids and of lycopene were always higher than thoseobtained at 40 ◦C, the reverse being observed for rubixanthin andcryptoxanthin.

The maximum carotenoid extraction yield occurred at 60 ◦C and250 bar, extracting 47.97 �g total carotenoids/g of pitanga (Table 3).Considering that the acetone extracted all the carotenoids, givinga total of 87.88 �g carotenoids/g of freeze-dried pitanga (Table 1),the SC-CO2 extract obtained under these conditions corresponded

Fig. 5. HPLC–PDA chromatograms of the SC-CO2 extracts (A) obtained at 300 barand 40 ◦C and (B) obtained at 300 bar and 60 ◦C (B): (3) all-trans-rubixanthin, (4)cis-rubixanthin I, (6) all-trans-�-cryptoxanthin, (7) all-trans-lycopene and (8) 13-cis-lycopene.

38 G.L. Filho et al. / J. of Supercritical Fluids 46 (2008) 33–39

Table 4Recovery (a) and relative concentration (b) of carotenoids in the SC-CO2 extracts

T (◦C) P (bar) (all-trans + cis) Rubixanthin (%) All-trans-�-cryptoxanthin (%) (all-trans + cis) Lycopene (%) (�,�,�) Carotene (%) Total (%)

(a) (b) (a) (b) (a) (b) (a) (b) (a)

40 100 0.9 26 0.45 10 0.4 23 5.7 39 0.77150 6.2 25 6.7 20 6.1 49 7.0 6.5 5.8200 23.3 41 7.0 9 13.3 46 11.4 4.6 13.4250 27.1 39 8.7 9 16.8 47 13.9 4.6 16.3300 78.5 54 3.0 1.5 30.7 41 23.8 3.7 34.2350 75.3 62 3.0 2 19.3 31 27.2 5.2 28.4400 73.8 66 2.8 2 14.9 26 29.1 6.0 26.2

60 100 8.4 47 1.6 7 2.7 29 14.1 18 4.2150 55.2 49 1.3 1 26.2 46 24.2 5 26.5200 65.6 40 0 0 48.9 57 25.3 3.5 39.1250 73.5 32 0 0 78.4 66 28.2 2.8 54.6300 76.5 41 0 0 53.2 56 29.3 3.6 43.9350 83.7 48 13.3 6 38.4 43 30.5 4.0 41.1400 86.4 53 16.9 8 28.4 34 31.4 4.5 37.9

Fig. 6. Profiles of the carotenoid concentrations as a function

to the extraction of 55% of the total carotenoids, 74% of rubix-anthin isomers and 78% of the lycopene isomers. Under theseconditions the concentration of total carotenoids in the SC-CO2extract was 5474 �g/g of extract, represented by 66% lycopeneand 32% rubixanthin. At 40 ◦C the maximum extraction occurredat 300 bar, producing a SC-CO2 extract containing 3962 �g totalcarotenoids/g of extract, extracting 30.09 �g total carotenoids/gcrude pitanga (Table 3), corresponding to the extraction of approx-

Fig. 7. Recovery of carotenoids in the SC-CO

of pressure at temperatures of: (a) 40 ◦C and (b) 60 ◦C.

imately 34% of the total carotenoids, 78% of rubixanthin isomersand only 22% of the lycopene isomers. Of this total, 41% werelycopene and 54% rubixanthin isomers (Table 4). These twoconditions clearly showed that the operational conditions oftemperature and pressure strongly influenced the selectivity ofthe CO2 and the extraction yield. In Fig. 5, the chromatogramsobtained under two distinct conditions showed the consider-able difference between the extract compositions, indicating, for

2 extracts at: (a) 40 ◦C and (b) 60 ◦C.

rcritica

[

[

[

[

[

[

[

[

[

[

[

[

[

Fluids 18 (2000) 35–47.

G.L. Filho et al. / J. of Supe

example, that under the conditions of 300 bar and 60 ◦C, the SC-CO2 is strongly selective to preferentially extract rubixanthin andlycopene.

Fig. 6 compares the concentrations of total carotenoids, lycopeneand rubixanthin in the SC-CO2 extracts. At 40 ◦C, both the lycopeneand rubixanthin had their maximum concentrations under thesame conditions that presented the maximum recovery of totalcarotenoids, indicating that these components were determinantin the extraction yield. At 60 ◦C, the extraction of rubixanthinremained high (Table 4) under practically all the conditions, withlittle variation in its concentration in the SC-CO2 extracts, butthe lycopene concentration was predominant and determined therecovery of total carotenoids. Fig. 7 compares the SC-CO2 recoveryof carotenoids based on the extraction with acetone. The recoveryof rubixanthin was important at the two temperatures, reachingapproximately 80% at higher pressures, whereas the recovery oflycopene presented maximums at 300 and 250 bar at 40 and 60 ◦C,respectively. The recovery of the carotenes showed a maximum of30%, always increasing with increasing pressure.

The oxygen-containing molecules, lutein and zeaxanthin, werenot extracted by the SC-CO2 and the extraction of �-cryptoxanthinwas negligible, but the recovery of rubixanthin was excellent. Interms of the chemical structure of these carotenoids (Fig. 2), luteinand zeaxanthin contain two OH groups and two cyclic end-groups,cryptoxanthin contains one OH group and two cyclic �-iononegroups, whereas rubixanthin contains one OH group, one cyclic �-ionone end-group and an acyclic �-end-group on the other side ofthe structure. Differences can be observed between the moleculesdue to the presence of OH and different end-groups, promotingdifferences in polarity and molecule shape, and consequently influ-encing selectivity by the SC-CO2. Although rubixanthin has an OHgroup in its molecule, this was the component showing the highestextraction yield, most probably due to the presence of end-groupswith different shapes. In general, the extraction yield of lycopene,with acyclic �-end-group, was lower than those observed for rubix-anthin, probably due to its ease crystal formation and furtherorganization in multilayers or aggregates in the cell [32,33].

4. Conclusions

Varying the operational conditions of temperature and pressureindicated that the supercritical CO2 selectively extracted differentcarotenoids. Molecules presenting OH groups were not extracted

by the SC-CO2 with the exception of rubixanthin, for which a highextraction yield was obtained. Lycopene and rubixanthin (all-transand cis isomers) were the most important carotenoids in the SC-CO2 extracts. The maximum recovery of carotenoids was obtainedat 60 ◦C and 250 bar, extracting 74% of the rubixanthin and 78% ofthe lycopene isomers.

Acknowledgement

The authors would like to thank the Brazilian Funding AgencyFAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo).

References

[1] A.E. Consolini, M.G. Sarubbio, Pharmacological effects of Eugenia uniflora (Myr-taceae) aqueous crude extract on rat’s heart, J. Ethnopharmacol. 81 (2002)57–63.

[2] I.A. Ogunwande, N.O. Olawore, O. Ekundayo, T.M. Walker, J.M. Schmidt, W.N.Setzer, Studies on the essential oil composition, antibacterial and cytotoxicityof Eugenia uniflora L., Int. J. Aromather. 15 (2005) 147–152.

[3] A.E. Consolini, O.A.N. Bandini, A.G. Amat, Pharmacological basis for empiricaluse of Eugenia uniflora L. (Myrtaceae) as antihypertensive, J. Ethnopharmacol.66 (1999) 33–39.

[

[

[

[

[

[

[

[

[

[

l Fluids 46 (2008) 33–39 39

[4] Y. Momose, Pentahydoryndolizidine and �-glucosidaseinhibitors containingproducts of Eugenia uniflora, Jpn. Kokai Tokkyo Koho 72 (2000) 770.

[5] V.L.A.G. Lima, E.A. Melo, D.E.S. Lima, Fenolicos e carotenoides totais em pitanga,Sci. Agric. 59 (2002) 447–450.

[6] A.L. Oliveira, R.B. Lopes, F.A. Cabral, M.N. Eberlin, Volatile compounds frompitanga fruit (Eugenia uniflora L.), Food Chem. 99 (2006) 1–5.

[7] J.E.F. Bezerra, J.F.S. Junior, I.C. Lederman, Botanical and ecology, in: L.C. Donadio(Ed.), Pitanga (Eugenia uniflora L.), vol. 1, 1st ed., Funep, Jaboticabal, Brazil, 2000,p. 30.

[8] C.H. Azevedo-Meleiro, D.B. Rodrigues-Amaya, Confirmation of the identity ofthe carotenoids of tropical fruits by HPLC-DAD and HPLC–MS, J. Food Comp.Anal. 17 (2004) 385–396.

[9] P. Di Mascio, S. Kaiser, H. Sies, Lycopene as the most efficient biologi-cal carotenoid singlet oxygen quencher, Arch. Biochem. Biophys. 274 (1989)532.

10] M.D. Macıas-Sanchez, C. Mantell, M. Rodrıguez, E. Martınez de la Ossa, L.M.Lubian, O. Montero, Supercrıtical fluid extraction of carotenoids and chlorophylla from Nannochloropsis gaditana, J. Food Eng. 66 (2005) 245–251.

[11] M. Careri, L. Furlattini, A. Mangia, M. Musci, E. Anklam, A. Theobald, C. vonHolst, Supercritical fluid extraction for liquid chromatographic determinationof carotenoids in Spirulina Pacifica algae: a chemometric approach, J. Chro-matogr. A 912 (2001) 61–71.

12] R.L. Mendes, H.L. Fernandes, J.P. Coelho, E.C. Reis, J.M.S. Cabral, J.M. Novais,A.F. Palavra, Supercritical CO2 extraction of carotenoids and other lipids fromChlorella vulgaris, Food Chem. 53 (1995) 99–103.

13] J.S. Seo, B.J. Burri, Z. Quan, T.R. Neidlinger, Extraction and chromatography ofcarotenoids from pumpkin, J. Chromatogr. A 1073 (2005) 371–375.

14] L.F. de Franca, G. Reber, M.A.A. Meireles, N.T. Machado, G. Brunner, Supercriticalextraction of carotenoids and lipids from buriti (Mauritia flexuosa), a fruit fromthe Amazon region, J. Supercrit. Fluids 14 (1999) 247–256.

15] T. Baysal, S. Ersus, D.A. Starmands, Supercritical CO2 extraction of �-caroteneand lycopene from tomato paste waste, J. Agric. Food Chem. 48 (2000)5507–5511.

16] N.L. Rozzi, R.K. Singh, R.A. Vierling, B.A. Watkins, Supercritical fluid extractionof lycopene from tomato processing byproducts, J. Agric. Food Chem. 50 (2002)2638–2643.

17] G. Vasapollo, L. Longo, L. Rescio, L. Ciurlia, Innovative supercritical CO2 extrac-tion of lycopene from tomato in the presence of vegetable oil as co-solvent, J.Supercrit. Fluids 29 (2004) 87–96.

18] M.S. Gomez-Prieto, M.M. Caja, M. Herraiz, G. Santa-Marıa, Supercritical fluidextraction of all-trans-lycopene from tomato, J. Agric. Food Chem. 51 (2003)3–7.

19] A. Perva-Uzunalic, M. Skerget, B. Weinreich, Z. Knez, Extraction of chillipepper (var. Byedige) with supercritical CO2: effect of pressure and temper-ature on capsaicinoid and colour extraction efficiency, Food Chem. 87 (2004)51–58.

20] M. Margaret Barth, C. Zhou, K.M. Kute, G.A. Roenthal, Determination of optimumconditions for supercritical fluid extraction of carotenoids from carrot (Daucuscarota L.) tissue, J. Agric. Food Chem. 43 (1995) 2876–2878.

21] F. Favati, J.W. King, J.P. Friedrich, K. Eskins, Supercritical CO2 extraction ofcarotene and lutein from leaf protein concentrates, J. Food Sci. 53 (1988) 1532–1536.

22] G.A. Spanos, H. Chen, S.J. Schwartz, Supercritical CO2 extraction of �-carotenefrom sweet potatoes, J. Food Sci. 58 (1993) 817–820.

23] L.F. Franca, M.A.A. Meireles, Modeling the extraction of carotene and lipids frompressed palm oil (Elaes guineensis) fibers using supercritical CO2, J. Supercrit.

24] ASAE Standards Method of Determining and Expressing Fineness of Feed Mate-rials by Sieving. ASAE, 1997, S319.3, 547.

25] V.V. De Rosso, A.Z. Mercadante, Carotenoid composition of two Brazilian geno-types of Acerola (Malpighia punicifolia L.) from two harvests, Food Res. Int. 38(2005) 1073–1077.

26] V.V. De Rosso, A.Z. Mercadante, Identification and quantification of carotenoids,by HPLC–PDA–MS/MS, from Amazonian fruits, J. Agric. Food Chem. 55 (2007)5062–5072.

27] C.F. Zanatta, A.Z. Mercadante, Carotenoid composition from the Brazilian trop-ical fruit camu–camu (Myrciaria dubia), Food Chem. 101 (2007) 1526–1532.

28] G. Britton, S. Liaaen-Jensen, H. Pfander, Carotenoids Handbook, Birkhauser,Basel, Switzerland, 2004.

29] G. Britton, UV/Visible Spectroscopy, in: G. Britton, S. Liaaen-Jensen, H.Pfander (Eds.), Carotenoids, vol 1B: Spectroscopy, Birkhauser, Basel, 1995,pp. 13–62.

30] V.V. De Rosso, A.Z. Mercadante, HPLC–PDA–MS/MS of anthocyanins andcarotenoids from dovyalis and tamarillo fruits, J. Agric. Food Chem. 55 (2007)9135–9141.

31] S. Angus, B. Armstrong, K.M. Reuck, Carbon Dioxide: International Thermo-dynamic Table of the Fluid State, vol. 3, Pergamon Press, New York, 1976, p.266.

32] M. Nguyen, D. Francis, S. Schwartz, Thermal isomerisation susceptibilityof carotenoids in different tomato varieties, J. Sci. Food Agric. 81 (2001)910–917.

33] A.Z. Mercadante, Carotenoids in foods: sources and stability during process-ing and storage, in: C. Socaciu (Ed.), Food Colorants: Chemical and FunctionalProperties, CRC Press, Boca Raton, 2007, pp. 213–240.