J. of Supercritical Fluids 86 (2014) 4 14

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Extract a (leaves trasuperc ts

Tbata T b,Pedro Ma Department o , Brazb Chemical, Bio ampin

a r t i c l

Article history:Received 19 AReceived in re18 November 2013Accepted 19 November 2013

Keywords:Eugenia uniora L.Supercritical extractionPhenolic compSequential extFixed bed extrAntioxidants

ractin in

using supercritical CO2 (non-polar) as solvent in a rst step, followed by ethanol (polarity: 5.2) andwater (polarity: 9.0) in a second and third steps, respectively. All extracts were evaluated for globalextraction yield, concentration and yield of both polyphenols and total avonoids and antioxidant activ-ity by DPPH method (in terms of EC50). The nature of the solvent signicantly inuenced the process,since the extraction yield increased with solvent polarity. The aqueous extracts presented higher globalextraction yield (22%), followed by ethanolic (16%) and supercritical extracts (5%). The study pointed out

1. Introdu

Eugenia the Myrtaceical origin, and can be Caribbean. the Northefruit with h

E. uniotic propertimedicine. Ahypotensivcrobial andcompoundshyperglyce

CorresponE-mail add

0896-8446/$ http://dx.doi.ooundsractionactor

that the sequential extraction process is the most effective in terms of global extraction yield and yieldof polyphenols and total avonoids, because it produced the more concentrated extracts on phenoliccompounds, since the supercritical ethanolic extract presented the highest phenolics content (240.5 mgGAE/g extract) and antioxidant capacity (EC50 = 9.15 g/mL). The most volatile fraction from the super-critical extract, which is similar to the essential oils obtained by steam distillation or hydrodistillation,presented as major compounds the germacrenos D and B + bicyclogermacrene (40.75%), selina-1,3,7(11)-trien-8-one + selina-1,3,7(11)-trien-8-one epoxide (27.7%) and trans-caryophyllene (14.18%).

2013 Elsevier B.V. All rights reserved.

ction

uniora L., also known as pitanga, is a perennial tree ofae family native to South America [1]. Despite its trop-

its cultivation is already widespread in many countriesfound in some Asian countries, in the United States andIn Brazil, although it is native to South and Southeast,ast is the only region that commercially exploits thisigh economic potential [2,3].ra leaves are known for their numerous therapeu-es, having been used for a long time in the popularmong the medicinal applications, the leaves are use ase, antigout and stomachic agent, in addition to antimi-

hyperglycemic activities [1]. Studies have shown that present in the leaves have anti-inammatory [4], anti-mia and hypertriglyceridemia [5], antimicrobial and

ding author. Tel.: +55 19 3521 4030; fax: +55 19 3521 4027.ress: cabral@fea.unicamp.br (F.A. Cabral).

antifungal [6,7], larvicide (Aedes aegypti) [8], antihypertensive [2]cytotoxic [9] and antioxidant properties [10].

Amat et al. [11] studied the diuretic action of aqueous extractsof E. uniora leaves in mice, demonstrating the use of the plantas a hypotensive agent in the popular medicine. Rattmann et al.[12] reported the use of a avonoid-rich fraction obtained from E.uniora leaves for treatment of inammatory diseases.

The benets attributed to E. uniora are due to the secondarymetabolites present in its leaves, including phenolic compoundssuch as avonoids, terpenes, tannins, anthraquinones and essentialoils [13]. The composition of the extracts from E. uniora leaves bydifferent extraction methods is reported by several studies [14,15].The phenolic compounds, also known as polyphenols, are consid-ered naturally occurring antioxidants and represent an importantgroup of bioactive compounds in food. These substances are presentin all plant foods, but the kind and levels vary greatly depending onthe plant, genetic factors and environmental conditions [16].

Polyphenols can be classied into simple phenols, phenolic acids(benzoic acid, cinnamic acid and its derivatives), avonoids (antho-cyanins, avonols and their derivatives) and tannins [17]. Suchcompounds possess a number of pharmacological properties which

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.supu.2013.11.014ion of phenolic compounds from pitangby sequential extraction in xed bed exritical CO2, ethanol and water as solven

. Garmusa, Losiane C. Paviania, Carmen L. Queiroga

. Magalhesb, Fernando A. Cabrala,

f Food Engineering, State University of Campinas UNICAMP, 13083-862 Campinas, SPlogical and Agricultural Pluridisciplinary Research Center (CPQBA), State University of C

e i n f o

ugust 2013vised form

a b s t r a c t

With the goal of maximizing the extnia uniora L.), a sequential extractio/ locate /supf lu

Eugenia uniora L.)ctor using

ilas UNICAMP, 13083-970 Campinas, SP, Brazil

on yield of phenolic compounds from pitanga leaves (Euge-xed bed was carried out in three steps at 60 C and 400 bar,

T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14 5

make them act on biological systems. Consequently, many of theseproperties act efciently in preventing certain diseases in humans[2,18].

In recent years, studies on the extraction of polyphenols fromnatural souvery importnolic compoextraction utechniques nols [19].

The supnatural pro(CO2). The ide (scCO2)vapor (steamnent optionsubstances pounds, asupercriticalow cost, nohas good ex[20,21].

Martineuniora by step and etcompared wprocess propolyphenol

This studto obtain thextracts onbed extractof increasinethanol in the results and phenolobtained by(convention

2. Materia

2.1. Charac

Pitanga leld at the Research Celation ovenlocation of [22]; howeleaves, whileaves.

A mean from the m24270, accdiameter d(0.83 mm), 11.3% (0.191.50 0.01 etry (Quantmoisture coAOCS Ca 23KF Thermop

To charasity of themethod [25

from the real density of the sample and the apparent density of thebed as described by Rahman et al. [26] was 0.70 0.01.

2.2. Chemicals

usey W2). E828ationion w

reag.0% w(Braica (

(97.0etec lhidrome

tract

niortractnt so

dioxthanoe of the onessuof 1.5

thro apprtracthe ste ofe frombmitt a ed oimat

comressxtraced

essuts.

globweenetwe

perim

expE, UN

appath (llectistato, EUsupe

in tor, aressu

was matrces have attracted special interest. The extraction is aant step in the isolation, identication and use of phe-unds and there is no one single extraction method. Thesing organic solvent and supercritical uid extractionare more commonly used for the isolation of polyphe-

ercritical uid most commonly used for extraction ofducts from foods or pharmaceuticals is carbon dioxideextraction processes using supercritical carbon diox-

as a substitute for some organic solvents and water distillation or hydrodistillation) are one of the promi-

s for obtaining natural extracts containing bioactive(antioxidants, essential oils, carotenoids, phenolic com-vonoids and others). Among the advantages of usingl carbon dioxide extraction processes stand out itsn-toxicity, non-ammability, as well as it is inert andtraction capacity due to its higher penetration power

z-Correa et al. [22] obtained natural extracts from E.two-step extraction processes, using scCO2 in the rsthanol or water in the second conventional step. Whenith conventional extraction (one-step), the two-stepduced aqueous and ethanolic extracts with highers content and more active avonoids.y aimed to use the sequential extraction in xed bede maximum extraction yield and more concentrated

phenolic compounds. A sequential extraction in xedor was conducted using the following solvents in orderg polarity: supercritical carbon dioxide in the rst step,the second step and water in the third step. Then,for global extraction yield, composition of essential oilics content of the extracts were compared with those

one-step process using water and ethanol as solventsal and xed bed methods).

l and methods

terization of raw material

eaves (E. uniora) were collected from an experimentalChemical, Biological and Agricultural Pluridisciplinaryntre (CPQBAUNICAMP) and dried in a forced air circu-

at 42 C for 3 days. The collection was made in the samethe sample used in the study of Martinez-Correa et al.ver, the present sample consisted of very young plantle Martinez-Correa et al. studied well-developed plant

particle diameter of 0.336 0.003 mm was calculatedeans of the materials retained on the Tyler meshesording to ASAE procedures [23]. The geometric meanistribution of the particles of E. uniora was: 2.5%25.5% (0.59 mm), 35.1% (0.38 mm), 14.0% (0.27 mm),

mm) and 9.0% (0.09 mm). The particle density ofg/cm3 was determined at 25 C by helium gas pycnom-achrome Ultrapyc 1200e Automatic pycnometer). Thentent determined by Karl-Fisher method, according to55 [24] (Metrohm 701 KF Titrino equipped with 832rep oven) was 6.0 0.1%.cterize the supercritical extraction, the apparent den-

particle bed determined according to the Uquiche] was 0.46 0.01 g/cm3, and the bed porosity calculated

CO2plied b113 C/1lot AE8Corporextract

Theide (95xodo Dinmnitritefrom V2-picridichlor

2.3. Ex

E. ubed exdifferecarbonwith eschem

In tand prscCO2owedple forrst exunder ow raresiduwas su60 C asame bapprox

For(high pvious eperformlow prsolven

Theter betratio b

2.4. Ex

TheEXTRA

Theated b(5), cothermoSupelcin the locatedextracthigh ptor (5)groundd in the experiments was 99.5% pure and sup-hite Martins Gases Industriais (Campinas, Brazil, lotthanol (99.8% v/v) was purchased from xodo (Brazil,

RA), ultra pure water from a Milli-Q system (Millipore, EUA) and hexane (98.5% v/v) used in the supercriticalas purchased from Synth (Brazil, lot 135524).ents sodium carbonate (99.5% w/w) sodium hydrox-/w) and ethyl acetate (99.5% v/v) were obtained from

zil). The FolinCiocalteau reagent was purchased fromBrazil), aluminum chloride (99.0% w/w) and sodium% w/w) was from Ecibra (Brazil), gallic acid (99.0% w/w)(Brazil), the (+)-catechin (98.0% w/w) and 1,1-diphenyl-azyl (DPPH) were purchased from SigmaAldrich andthane (99.0% v/v) from Merck (Germany).

ion procedures

a leaves were subjected to extraction processes in xedor (high pressure extraction) in three steps using threelvents. The rst step was performed with supercriticalide, while the second and third steps were carried outl and water, respectively. Fig. 1 presents an illustrativehe extractions.e-step process, the operating conditions of temperaturere were 60 C and 400 bar with an average ow rate of

L/min (2.475 g/min). The CO2 in the supercritical stateugh a xed bed containing approximately 7.0 g of sam-oximately 6 h. In the second step, the residue from thetion (Residue 1) was subjected to a further extractioname operating conditions, using ethanol as solvent at a

0.5 mL/min (0.395 g/min) for a total period of 6 h. The the supercritical and ethanolic extraction (Residue 2)

ted to a third extraction, in which water at 400 bar andow rate of 0.5 mL/min (0.5 g/min) owed through thef particles. The average extraction time at this stage wasely 6 h.parison, one-step processes were performed in xed bedure) using ethanol and water as solvents, without pre-tion with scCO2 (Fig. 2). Conventional extractions wereto complement the ndings (Fig. 3), i.e. extractions atre (atmospheric pressure), using ethanol and water as

al extraction yield was used as a comparative parame- the different extraction methods, which expresses theen the mass of dry extract and the mass of raw material.

ental extraction in xed bed

eriments were performed in an experimental unit (LabICAMP, Brazil) as shown in Fig. 4.

aratus consisted mainly of a CO2 cylinder (1), refriger-2), high-pressure pump (3), supply tank (4), extractoron ask (7), gas ow meter (9) volume totalizer (10),ic bath (11), trap adsorvent Porapak-Q (80/100 mesh,A) (8) used only to capture the volatile compoundsrcritical extraction, two Bourdon pressure gauges, onehe supply tank and the other on the inlet side of the

peristaltic pump (6) used to inject the hexane, and are pump for ethanol/water (12). In all cases, the extrac-

packed by hand with approximately 7.0 g of dry anderial. Glass beads (6 mesh) were used to ll the empty

6 T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14

Fig. 1. Flow diagram of the sequential extraction process in xed bed extractor. First step: supercritical extract (SC) and volatile supercritical extract (SC-V); second step:ethanolic extract after supercritical extraction (SCE); third step: aqueous extract after supercritical and ethanolic extraction (SCA). The ow rates of solvents were adjustedat 25 C and 0.93 bar; the solvent densities in these conditions were CO2 = 1.65 g/L; ethanol = 0.79 g/mL and water = 1.0 g/mL.

Fig. 2. Flow d ). Forthe aqueous e sted aethanol = 0.79 g

spaces of thoperating cexperimentan hour waing the extthe bed, angaseous COtrap-Poropaizer (10) toextracts weobtain the emass of solthe process(6).

After theow was sto

L mfor

Fig. 3. Flow deach step.iagram of the sequential extraction process in xed bed extractor (one-step processxtraction: aqueous extract in xed bed (ALF); the ow rates of solvents were adju/mL and water = 1.0 g/mL.

e extractor to avoid preferential pathways for CO2. Theonditions were adjusted to 400 bar and 60 C for all

at 0.5 mpump s. When the conditions were achieved, a period of halfs adopted as static time to stabilize the system, start-raction by owing CO2 at a rate of 1.5 L/min throughd collecting the extract in the collection ask (7). The2 that left the collector ask (7) was drained through ak Q (8), and led to the ow meter (9) and volume total-

quantify the carbon dioxide used in the process. There collected at predetermined time intervals in order toxtraction curves (extraction yield as a function of the

vent). After each supercritical extraction, the tubing in line was washed with hexane using a peristaltic pump

soluble compounds were extracted by scCO2, the CO2pped and the system was depressurized. Then, ethanol

tions were period of haask (7) at liquid ethawater was (12) until thtion time ofobtained intriplicate.

2.5. One-st

For thisdescribed i

iagram of the conventional extraction process (three steps). The ethanolic extracts (EC) o the ethanolic extraction: ethanolic extract in xed bed (ELF); and fort 25 C and 0.93 bar; the solvent densities in these conditions were:

in was owed through the bed of particles using theethanol/water (12) and the same operational condi-

adjusted (60 C and 400 bar), considering a stabilizationlf an hour. The extracts were collected in the collectionpredetermined time intervals to obtain the curve withnol. In the third step of the sequential extraction, thepumped at ow rate of 0.5 mL/min through the pumpe process conditions were achieved. After the stabiliza-

the system, the extraction took place and samples were xed time intervals. All extractions were performed in

ep extraction in xed bed using water or ethanol

stage, an experimental procedure similar to thatn Section 2.4 (Fig. 4) was adopted. The extractor was

r aqueous extracts (AC) are constituted of a mixture of extracts from

T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14 7

ppara

packed wit0.5 mL/minethanol/wa

2.6. Conven

The conet al. [27]. with 50 mLthe mixturetemperaturto the cell. purpose waoration dura thermostaand then wsolid residuthe processvious one, c6 h.

The solvevaporator Brazil), andMarconi, muntil constafreeze-dryinthe dried ex

2.7. Extract

All extraavonoids pene compPorapak-Q

Deter totioca

wertractpho

sultsFig. 4. Supercritical extraction a

h 7.0 g of plant and water or ethanol was drained at through the bed of particles by using the pump forter (12).

tional aqueous or ethanolic extraction

ventional extracts were obtained according to Cseke

2.7.1. The

FolinCresultsdry exspectrothe reAbout 4.5 g of dried and crushed sample were mixed of solvent (water or ethanol) in a balance cell, in which

was stirred with the aid of a magnetic stirrer and thee adjusted at 60 C using a thermostatic bath connectedA reux condenser was attached to the system, whoses to prevent the loss of desirable compounds by evap-ing extraction, and the temperature was controlled bytic bath at 5 C. The mixture remained stirring for 2 h,as vacuum ltered. The ltrate was reserved and thee extracted with a further 50 mL of solvent, repeating

twice. Thus, each new ltrate was mixed to the pre-onstituting the extract. The total extraction time was

ent of the ethanolic extracts was evaporated in rota-at 50 C under vacuum of 600 mm Hg (Marconi, MA-058,

in a vacuum oven (Marconi, MA 030-12, Brazil; pumpodel MA-057-13, SP, Brazil) under the same conditionsnt weight. The water of the extracts was removed byg (lyophilizer Liobras, model L101, SP, Brazil) to obtaintracts.

composition

cts were analyzed for total phenolics content, totaland antioxidant activity, as well as the prole of ter-ounds present in the volatile fraction retained in thetrap.

(0125 mg/The tota

described bin catechin measured a40, PerkinEcurve of catin triplicate

2.7.2. AnalyThe vol

retained byraphy (AgilThe chromumn (30 m1.0 mL/min3 C/min, anthe injectorPorapak-Q to extract tthe volatileand 100 mmacetate (20equipmenting the maliterature ucomparing tus.

mination of polyphenols and total avonoidsal polyphenols content was determined using thelteu reagent, according to Singleton et al. [28], and thee expressed as equivalents of gallic acid (mg GAE/g). The absorbance was measured at 750 nm using atometer (UV-VIS lambda 40, Perkin Elmer, USA) and

were calculated by a standard curve of gallic acid

L).l avonoids were determined using the methodologyy Zhishen et al. [29], and the results were expressedequivalents (mg CE/g dry extract). The absorbance wast 510 nm using a spectrophotometer (UV-VIS lambdalmer, USA) and the results were calculated by a standardechin (0125 mg/L). All determinations were carried out.

sis of the more volatile fraction by GCMSatile fractions from the supercritical extraction (SC)

the Porapak-Q trap were analyzed by gas chromatog-ent 6890N) coupled to a mass detector (MSD 5975).atograph was equipped with a HP-5MS capillary col-

0.25 mm 0.25 m) and stripping gas of helium at. The column was programmed from 60 C to 240 C atd from 240 C to 280 C at 4 C/min. The temperatures of

and detector were 220 C and 290 C, respectively. Thecartridge was eluted with 15.0 mL of dichloromethanehe volatile compounds. Then, the solution containings was concentrated on a rotary evaporator at 40 C

Hg vacuum. The dried extract was dissolved in ethyl.0 mg/mL) and an aliquot of 1.0 L was injected in the

(GCMS). The compounds were identied by compar-ss spectra obtained in the present study with those insing the CG/EM system database NIST library, and bywith the retention indexes reported for the n-alkane

8 T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14

Table 1Global extraction yield, concentration and yield of polyphenols and total avonoids and antioxidant activity of the extracts.

Extraction Extracta Globalyield (%)

Total polyphenols Total avonoids Antioxidantactivity

Concentration(mg GAE/gextract)

Yield (mGAE/g le

Sequential

.8 08 38 51 91

Fixed bed (132 51

Conventiona28 30

Gallic acid

Values represe atistica Supercritic us (SC

ethanolic (EC)

series (C8Cnormalizati

2.8. Antioxi

The antprocedure 2-picrilhidr(1 mg driedtions of 5extract solulight. One mDPPH (0.3 m348) was aleft to react(Abssample) (UV-VIS lamby mixing control by absorbancelengh (Absbto zero absotive controlfrom the fo

AA (%) =[

A

The extrinitial activear and nonextract con

2.9. Statisti

Data weisons betweperformed software Ststep.

ults

tract

le 1 tratitioxilic and bedrage

< 0.0iffer

extt difes ths. Thy. Thanolectetial ractiing epar

6% ofxed bed (3 steps)

SC 5 1g 32.70 0.03f 1SC-V 0.31 0.03h 26.0 0.2g 0.SCE 16 1f 240.5 0.2a SCA 22.0 0.4d 233.8 0.5b SC + SCE + SCA 43 2a

step)ELF 20 1d,e 163.5 0.4cALF 33.3 0.4b 152.2 0.2d

l (1 step)EC 18 2e,f 151 1d AC 27 1c 108.7 0.2e Standard 1000

nt the mean of triplicate assays standard deviation. Different letters represent stal (SC); volatile supercritical (SC-V); sequential ethanolic (SCE); sequential aqueo; conventional aqueous (AC); and accumulated yield (SC + SCE + SCA).

20) with the quantitative analysis made by peak areaon.

dant activity by DPPH assay

ioxidant activity was determined according to thedescribed by Mensor et al. [30] using the 1,1-difenil-azyl (DPPH) reagent. Starting from the stock solution

extract/mL ethanol), solutions with nal concentra-150 g/mL, were prepared. Aliquots of 2.5 mL of eachtion were transferred to test tubes protected from theillilitre of a recently prepared ethanolic solution ofM) (SigmaAldrich Chemie, Alemanha, lote S4869-

dded to each tube, and the mixture was stirred and for 30 min at room temperature. Then, the absorbancewas measured at 517 nm using a spectrophotometerbda 40, Perkin Elmer, EUA). The blank was prepared1 mL ethanol and 2.5 mL extract, and the negativemixing 1 mL DPPH solution and 2.5 mL ethanol. The

of both solutions was measured at the same wave-lank, Abscontrol). The spectrophotometer was calibratedrbance using ethanol (99.5%), and gallic acid as the posi-

(standard). The antioxidant activity (AA) was calculated

3. Res

3.1. Ex

Tabconcenand anethanoor xeare avetest (picant d

Thenicanindicatprocespolaritthe ethAs expsequenthe extcess usare comwith 1llowing equation:

control (Asample Ablank

)Acontrol

100]

(1)

act concentration responsible for a 50% decrease in theity of the DPPH (EC50, g/mL) was calculated by lin--linear regression of the AA (%) curves obtained for allcentrations.

cal analysis

re analyzed by analysis of variance (ANOVA). Compar-en means by Tukeys test at 5.0% signicance level wereto identify the treatments with the best responses. Theatistica version 7.0 (StatSoft, USA) was used for this

the one-stealso extracfractionatedSC + SCE wapromoted aof high premodify the may facilitastep procesfollowed bycating the pyield. The ssolvent. Thtion (CE) shin the ethan

The glotion was hiet al. [22] u(400 bar angaves)

Concentration(mg CE/gextract)

Yield (mg CE/gleaves)

EC50(g/mL)

0.3e 153 4a 8 2b,c >2000.01e 64 1b 0.20 0.02d >2002c 27.8 0.3d 4 1c,d 9.151b 20 1e 4.4 0.3c,d 25.413a 17 3a 2d 40 2c 8 1b,c 16.191b 15 1e 5.0 0.4c 15.723d 61 2b 11 2b 12.711d 20 2e 6 1c 19.58

2.09

ally signicant differences (p < 0.05).A); ethanolic xed bed (ELF); aqueous xed bed (ALF); conventional

and discussion

ion yields

shows the values obtained for global extraction yield,on and yield of both polyphenols and total avonoidsdant activity of the extracts obtained by supercritical,d aqueous extraction in one-step process (conventional) and sequential extraction in three steps. The results

s of experimental values performed in triplicate. Tukeys5) was performed to determine whether there is signif-ence between the average results.raction using scCO2, ethanol and water showed sig-ferences regarding the global extraction yield, whiche inuence of the nature of the solvent used in thee extraction yield tended to increase with the solvente aqueous extracts showed higher yields, followed byic extracts, since the water is more polar than ethanol.d, the global extraction yield of 43% obtained in theextraction (SCE + SC + SCA) is signicantly higher thanon yields of 18% and 27% obtained in the one-step pro-thanol or water, respectively. When the extraction stepsed separately, the supercritical ethanolic extract (SCE)

global yield was lower than the yield of 20% obtained in

p extraction in xed bed with ethanol (ELF). The ethanolted the soluble compounds in the scCO2, which were

on the sequential extraction, considering that the sums of the order of 21%. The prior extraction with scCO2

change in the structure of the solid matrix due to the usessure and subsequent depressurization. These changesinteractions between the solute and the solid matrix andte the extraction. The best yield obtained in the one-s was 33% in the aqueous extraction in xed bed (ALF),

27% in the conventional aqueous extraction (AC), indi-ositive effect of high pressures in the global extractioname effect was not observed when ethanol was used ase yield of 18 2% in the conventional ethanolic extrac-owed no signicant difference as compared to 20 1%olic extraction in xed bed (ELF).

bal yield of 5% obtained in the supercritical extrac-gher than the 3.5% yield reported by Martinez-Correander the same conditions of temperature and pressured 60 C). The global yields obtained in the conventional

T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14 9

Fig. 5. Kineticsupercritical (Saqueous (AC).

extractionsauthors [2218% and 27ventional acaused partmean diam

In superated into tw(SC-V) was CO2 in the of 0.31% obas the yieldas the 0.1%sure values50 C and 6Martinez-Cstudied E. ufound yieldwhich are ences may s and yields of the sequential extraction (a) using scCO2 in a rst step (b), ethanol in a seC-V); sequential ethanolic (SCE); sequential aqueous (SCA); ethanolic xed bed (ELF); aq

were also much higher than those obtained by the], who obtained 8% and 20% of extraction yields, versus% obtained in the present study for ethanolic and con-queous extraction, respectively. This difference may beially by the use of young leaves and lower geometriceter of the particles.critical extraction with scCO2, the extract was fraction-o parts (SC and SC-V), where the most volatile portioncaptured on Porapak-Q trap for not being stripped bydepressurization process. The global extraction yieldtained in the SC-V is of the same order of magnitude

reported in the literature for supercritical extraction, yield obtained by Peixoto et al. [31], who used pres-

between 100 and 300 bar and temperatures between0 C, as well as 0.47% of extraction yield obtained byorrea et al. [22] using 400 bar and 60 C. Several authorsniora volatile oil obtained by hydrodistillation and

values comparable to the SC-V supercritical extracts,of the order 0.54% [31] and 0.74% [32]. These differ-be attributed to the extraction methods, plant origin,

age and deture, altitudothers [33]

3.2. Kinetic

As can tion (super60 C repreof extract/1mass of so(S/F). In th01 and 02)a single extformed.

Fig. 5b sof the supewhile 90% glast 3 h of exprimarily bcond step (c) and water in a third step (d). Supercritical (SC); volatileueous xed bed (ALF); conventional ethanolic (EC); and conventional

velopment, seasonality, pluviometric index, tempera-e, nutrients, conditions of collection, storage, among

.

s of sequential extraction

be seen in Fig. 5a, the curves of sequential extrac-critical + ethanolic + aqueous) obtained at 400 bar andsent the cumulative percentage yield of the extract (g00 g raw material) as a function of the ratio of thelvent accumulated by the mass of the raw materiale three extraction steps, two extraction curves (runs

and a total extraction (run 03) were built to obtainract, in which the chemical determinations were per-

hows that the global yield was 70% during the rst hourrcritical extraction (SC), which corresponds to S/F = 20,lobal yield was obtained in the rst 3 h (S/F = 60). In thetraction, in which the rate of mass transfer is controlled

y diffusion phenomena inside the solid particle, only the

10 T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14

Fig. 6. Kinetics and extraction yields with ethanol (a) and water (b) at 400 bar and 60 C (xed bed) of E. uniora extracts.

last 10% of the compounds were extracted. Given the above, 3 h(60 S/F) of extraction would be sufcient.

For sequential ethanolic extraction (SCE), as shown in Fig. 5c,about 50% of the total yield was obtained during the rst hour,using appro15 S/F, abouof SCE extracomplete dglobal yieldFig. 5a and dmass of extwas about 9

The yielshow that ssion periodsequential aethanolic ex

3.3. Kinetic

The kinexed bed (Fig. 6a andextracts weand 02) anfunction of

It is obsecient for eexperimentdepends onearly stagestion proceswhich corre

stage, in the following 2 h (70 g solvent) the yield reached nearly80% of the total.

In the early stages of the aqueous extraction (Fig. 6b), differentregions are observed: the rst one is dependent on the solubility

tes etrol

35 ged, wes as exally

ncen

valua) anract an bnd al phed theA > A

lowupern (SClic coextraed by

seqg o

was ano

ows

Fig. 7. Concen(SCE); sequentximately 5 S/F. After 4 h extraction using approximatelyt 90% yield was reached. The arrangement of the pointsction curves indicates the possibility of not reaching theepletion of the solute in the bed along the 6 h, even for a

of 16%. The sequential aqueous extraction curves (SCA),, showed good reproducibility of the experiments. Theract after 3 h of extraction using approximately 14 S/F0% of the total yield.ds obtained during the sequential extraction kineticsmaller extraction periods could be used, once the diffu-

(DC) was achieved after 3 h for the supercritical (SC) andqueous extraction (SCA) and from 4 h for the sequentialtraction (SCE).

s of ethanolic/aqueous extraction in xed bed

tics of ethanolic (ELF) and aqueous extraction (ALF) in400 bar and 60 C) in one-step process are shown in

b, respectively. Fig. 6 shows two replicates, in whichre collected at predetermined time intervals (runs 01d a point (one extraction) in run 03, expressed as athe mass of extract.rved in Fig. 6a that the extraction period was not suf-xhaustion of the bed, which indicates that most of theal points were located in the intermediate region, which

the solutes solubility and internal diffusivity. In the (1-h extraction or 25 g solvent) of the ethanolic extrac-s (Fig. 6a), the yield reached an average value of 10%,sponded to approximately 60% of global yield. After this

of soluare contion orobtainindicataqueoupractic

3.4. Co

The(Fig. 7the ext

It c(SCE) aof totaalloweand SC

Thein the sfractiophenotrated follow

Theof 51 mvalue the ething shtration (a) and yield (b) of phenolics and total avonoids of pitanga (E. uniora) leaves (rmial aqueous (SCA); ethanolic xed bed (ELF); aqueous xed bed (ALF); conventional ethaxtracted, and the intermediate and the third regionsled by the diffusivity. In the rst 6 points (1-h extrac-

solvent) an average of 18% of accumulated yield washich corresponds to nearly 50% of the total yield. This

high rate of mass transfer in the early stages of thetraction. After 3 h (90 g solvent), the curve tends to aconstant value.

tration and yield of polyphenols and avonoids

es reported in Table 1 concerning the concentrationd the yield (Fig. 7b) of polyphenols and avonoids ofare compared in Fig. 7.e observed in Fig. 7a that the supercritical ethanolicqueous (SCA) extracts presented the highest contentnolics, indicating that the prior extraction with scCO2

concentration of phenolic compounds (SCE > ELF > ECLF > AC).est concentration of phenolic compounds was obtainedcritical extract (SC) (32.7 mg/g) followed by the volatile-V) (26 mg/g). These results indicate that most of thempounds have polar character, since the more concen-cts were obtained primarily in the ethanolic extracts,

the aqueous extracts and supercritical extracts.uential aqueous extract (SCA) had the highest yieldf total polyphenols per gram of leaves, and the sameobtained in the ALF extract, which was higher thanlic extracts (SCA = ALF > SCE > ELF = EC = AC). This nd-the greatest advantage of the three-step sequential). Supercritical (SC); volatile supercritical (SC-V); sequential ethanolicnolic (EC); and conventional aqueous (AC).

T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14 11

extraction versus one-step aqueous extraction (xed bed or con-ventional). The sequential extraction enabled to obtain threeextracts: 5% of supercritical extract, 16% of concentrated etha-nolic extract (240.3 mg GAE/g), and 22% of concentrated aqueousextract (23GAE/g leaveleaves in thless concenalso correspless concenGAE/g, whic

The effeextraction aand with hagainst neaconventionand concenora leavesthat the seqstep aqueoucompounds

The cheavonoids tions were 153 mg/g foequal to thenolic extracextraction.

An impoextractionsobtained ucontents. Itcal extractiolater stagesextracts wh

Compariwith scCO2whose behaand Paula eiment. Howgroup of phbe lower orto the standand total areference sconcentratithe polyphe

The quatechniques.the most exto that metstituents ofnot providereducing caings supporstandardizenols and tot

The besttion, whichone-step excompoundsistics. Besidthe solvent plant may aThe three-sethanolic o

step-process, possibly due to the preference of these compoundsfor the scCO2 one-step extraction. The accumulated yield in thesequential extraction was higher than the one-step extraction.Therefore, the combination of extraction processes may be con-

thecati

resua extred wple

wer p the

nal encens ex

rtine extion iora e

totaby aqressed afoncelic anird sd of den the anlics atinezts thas sophen

tioxi

rea) witting sorba

[37xtratratiendiationresentudied n-V) textra

resu increum riticaaine

s usihis mof efftractivityidantt ofrisonordin1], v3.8 mg GAE/g), which corresponds to a yield of 51 mgs in the aqueous extract, and 91 mg GAE per gram ofe total extract (SC + SCE + SCA), as compared to 33% of atrated aqueous extract containing 163 mg GAE/g, whichonds to 51 mg GAE/g leaves, or against 27% of a muchtrated conventional aqueous extract containing 108 mgh corresponds to a yield lower than 30 mg GAE/g leaves.ct of the prior supercritical extraction in the sequentialllowed to obtain more concentrated ethanolic extracts,igher yield of polyphenols, being 38 mg GAE/g leavesrly 30 mg GAE/g leaves obtained in xed bed (ELF) oral extraction (CE). The analyses of the data on the yieldtration of phenolic compounds of the extracts of E. uni-

obtained from different extraction processes proveduential extraction (SCE + SC + SCA) followed by the one-s extraction (ALF) are effective methods to extract these.mical characterization of the extracts regarding thecontent (Fig. 7a) showed that the highest concentra-obtained in the supercritical extracts, with values ofr SC and 64 mg/g for SC-V. The latter was statistically

avonoids content obtained in the conventional etha-tion (CE), followed by the ethanolic and nally aqueous

rtant point to be noted is that despite the aqueous presented the best global extraction yields, the extractssing water as solvent showed the lowest avonoids

is possible to extract more avonoids in the supercriti-n; consequently, these compounds are decreased in the, presenting lower extraction yields in the SCE and SCAen compared to ELF, EC, ALF and AC one-step extraction.ng the data in Fig. 7a, it was observed that the extractionpresented higher avonoids than polyphenols contents,vior was also observed by Martinez-Correa et al. [22]t al. [34], who used the same standard of this exper-ever, it is known that avonoids are included in theenolic compounds, thus the avonoids content should

equal to that of phenolics. These results may be relatedards used to assess the concentration of polyphenolicsvonoids in the extract. If the reducing capacity of the

ubstance is not precisely the same of the extract, theon calculated from the standard curve will not reectnolics or avonoids in the sample.ntication of polyphenols is performed by a variety of

Although the technique using the FolinCiocalteu istensively used, some limitations have been attributedhod, once the reagents can be reduced by other con-

the extract. Thus, the FolinCiocalteu method does an accurate result of polyphenolics content, but thepacity of the sample under study [35,36]. These nd-t the need for a different analytical approach, and aim to

and validate the methods for quantication of polyphe-al avonoids in supercritical extraction.

yield of avonoids was obtained by sequential extrac- was about 50240% higher than those obtained in thetractions. The sequential extraction enables to remove

of the avonoids family having different character-es the solubility of the compounds vary according topolarity, the interactions with other constituents of thelso affect the degree of extraction of these substances.tep process allowed one to obtain more concentratedr aqueous extracts in terms of avonoids than the one

sideredquanti

TheuniorcompaThe samhad loclose toventioThe coaqueou

MacriticalextractE. uniobtainlowed (high pobtainmore cethanoand thmetho

Whtion arphenoby Marsuggeswater in poly

3.5. An

The(DPPHpromothe ab517 nmof the econcen

Depthe relmay prange sexhibitand SCof the

ThevaluesmaximsupercAA remproces

In tterms the extial actantioxpendencompa

Accet al. [4 most effective method for obtaining avonoids for theon of these compounds.lts of polyphenols and avonoids contents in the E.racts obtained by different extraction methods wereith the results reported by Martinez-Correa et al. [22].

used in the present study, comprised of younger leaves,olyphenols content, but the extraction yield was very

sample studied by the authors in supercritical (SC), con-thanolic (EC) and conventional aqueous (AC) extraction.tration and yield of avonoids were higher, except in thetract.z-Correa et al. [22] reported the positive effect of super-raction prior to ethanolic or conventional aqueousn the concentration of polyphenols and avonoids in thextracts. The same was observed in the present study tol phenolics when using the supercritical extraction fol-ueous extraction and ethanolic extraction in xed bed

ure). The conventional ethanolic and aqueous extractster the supercritical extraction by the authors [22] werentrated in polyphenols and total avonoids than thed aqueous extracts obtained in xed bed in the second

tep of sequential extraction (this study used the sameetermining polyphenols described by the authors [22]).e results obtained by the three-step sequential extrac-alyzed, the global extraction yield and the yield ofnd total avonoids are higher than the values reported-Correa et al. [22] using two- and one-step process. Thise use of sequential extraction using scCO2, ethanol andlvents as an efcient strategy for obtaining extracts richols and avonoids.

dant activity (DPPH)

ction of free radical 1,1-diphenyl-2-picrylhydrazylh antioxidant species inhibits and stabilizes the radical,color changes from purple to yellow, which decreasesnce that was monitored using a spectrophotometer at]. Fig. 8 shows the antioxidant activity (AA) as a functionct concentration (0150 g/mL) and gallic acid standardon (05 g/mL).ng on the extract studied and its chemical composition,ship between AA and the antioxidant content (extract)t a linear or non-linear behavior in the concentrationed, determined by the nature of the extracts. All extractson-linear behavior, except the supercritical extract (SChat presented almost linear behavior, which is typicalcts obtained using solvents of low polarity [38].lts of DPPH scavenging capacity showed that the AAased rapidly with the extract concentration to reach a

value of approximately 95% for all extracts except thel (SC and SC-V). After reaching this maximum value,d practically constant. It is worth mentioning that theng ethanol exhibited better DPPH scavenging capacity.ethod, the antioxidant activity (AA) is represented in

ective concentration (EC50, g/mL) which is dened as concentration responsible for 50% decrease in the ini-

of DPPH [39]. The EC50 provides a direct comparison of activity between different substances, since it is inde-

the concentration of the sample [40]. Fig. 9 shows a between the EC50 calculated values.g to the antioxidant activity described by Reynertsonery active extracts exhibit EC50 < 50 g/mL, moderately

12 T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14

Fig. 8. DPPH scavenging activity of E. uniora extracts. Supercritical (SC); volatile supercritical (SC-V); sequential ethanolic (SCE); aqueous sequential (SCA); ethanolic xedbed (ELF); aqueous xed bed (ALF); conventional ethanolic (EC); and conventional aqueous (AC).

active extracts from 50 to 100, slightly active from 100 to 200, andinactive ext

The naextracts exhexcept for thThe extractmost activeis related to

The priincreased indicating tpolar comp(EC50 > 200 matrix, andcritical extrantioxidant

The ethalowed by tMartinez-Cuniora extods. The laoxidation phigh antioxcal extract extracts whDPPH assayobservation

Fig. 9. Antioxi(SC); volatile s(SCA); ethano(EC) and conve

activity measured by DPPH assay was higher than that obtained byethod. These results can be due to the compounds responsi-

the antioxidant activity are not necessarily the same for eachd, since the extracts are complex mixtures of compounds,ifferent interactions may occur (synergistic or antagonistic)

an be observed in Table 1 that the EC50 values obtainedh case are strongly linked to the total phenolics contentsed as mg gallic acid equivalents per gram of dry extractE/g). The parameter is indicative of antioxidant activity: the

the vng eH as

ts, usdy a

corr showhenoracts present EC50 > 200.l EC50 values measured by DPPH assay showed that theibited high activity, with values well below 50 g/mL,e supercritical extract that exhibited EC50 > 200 g/mL.

after the supercritical extraction (SCE) stands out as the, presenting the lowest EC50 value of 9.15 g/mL, which

its high phenolics content.or extraction with scCOs was positive, because itthe antioxidant activity of the ethanolic extracts,hat the supercritical extraction has removed the non-ounds with low antioxidant activity by DPPH assayg/mL for the SC and SC-V extracts) from the vegetal

thus, concentrating the residue obtained in the super-action to substances having polar character and high

activity.nolic extracts presented high antioxidant activity, fol-he aqueous and supercritical extracts. In this regard,orrea et al. [22] measured the antioxidant activity of E.racts by DPPH and BCB (-carotene bleaching) meth-tter evaluates the ability of an extract to inhibit therocess of -carotene. The ethanolic extracts presentedidant activity by both methodologies. The supercriti-(SC) had higher antioxidant activity than the aqueousen measured by BCB method, but was inactive when the

was performed (in accordance with the experimentals of this study). In the aqueous extracts, the antioxidant

BCB mble formethothus d[22].

It cin eacexpres(mg GAlower spondiby DPPextracthis stu

Thevaluestotal pdant activity of E. uniora extracts EC50 (g/mL) values. Supercriticalupercritical (SC-V); sequential ethanolic (SCE); sequential aqueouslic xed bed (ELF), aqueous xed bed (ALF), conventional ethanolicntional aqueous (AC).

Fig. 10. Relatphenolics conof this study,Points in termlic acid > SCE >study of Martialue, the higher the antioxidant activity of the corre-xtract. Fig. 10 was built to correlate the EC50 activitysay with the total phenolics content of pitanga leavesing the EC50 versus concentration (mg GAE/g) values ofnd the ndings of Martinez-Correa et al. [22].elation between the total phenolics content and EC50ed increased antioxidant activity with increasing thelics content (mg GAE/g), the last point on the graphionship between antioxidant activity (in terms of EC50) and totaltent (mg GAE/g) in ethanolic and aqueous extracts of E. uniora

as compared to the data reported by Martinez-Correa et al. [22].s of phenolics content showed the following descending order: gal-

ELF > ALF > EC > AC for this study and gallic acid > SCE > EC > AC for thenez-Correa et al. [22].

T.T. Garmus et al. / J. of Supercritical Fluids 86 (2014) 4 14 13

Table 2Chemical composition of the volatile fraction extract from Eugenia uniora L. leaves.

Peak Compound MM Relative (%) RIa RIb

1 -Copaene 204 1.77 1374 13762 3 5 6 7 8 9

10 11 12 13 14 15 16 17 18

RI, retention ina Calculatedb Retention

being the g1.87 as repo

3.6. Analysi

The volacritical extspectrometical compoleaves is sh

The voextraction selina-1,3-7trans-caryobicyclogerm(12.0%). Thfrom the pidentied 1extract.

Martinevolatile supand 400 baselina-1,3-7trien-8-oneand trans-ccal extractsto 300 bar abetween 1.4as C15H20Otion (SC-V)compoundsC15H20O2.

Differenpounds notuniora, su[45]. Thesemethod an[46], the tefrom othersposition. Tht in chemselina.

clus

s stuethoateris havnolicy shoin exciate

ctivittotal

wled

autht andss Fa

nces

. Auriacob-Elemene 204 Trans-caryophyllene 204 NI 202 -Humulene 204 Allo-aromadendrene 204 Germacrene D 204 Bicyclogermacrene 204 NI 204 -Cadinene 204 -Cadinene 204 Germacrene B 204 Spathulenol 220 Selina-1,3,7(11)-trien-8-one 216 -Muurolol 222 Selina-1,3,7(11)-trien-8-one-epoxide 232 NI 234

Total

dex; MM, molecular mass; NI, not identied. retention index.index from Adams [42,43].

allic acid pure standard, EC50 = 2.09 in this study, andrted by Martinez-Correa et al. [22].

s of the more volatile fraction by GCMS

tile fraction (SC-V) of the extracts obtained by super-raction was assessed by gas chromatographymassry (GCMS) for analysis of their constituents. The chem-sition of the volatile fraction extract from E. unioraown in Table 2.latile fraction recovered during the supercriticalassessed by GCMS showed as major compounds(11)-trien-8-one (15.7%), germacrene D (15.0%) andphyllene (14.2%), followed by germacrene B (13.5%),acrene (12.4%) and selina-1,3-7(11)-trien-8-epoxide

ese compounds were also found in the essential oillant obtained by hydrodistillation [7,9]. In total, we6 compounds corresponding to 92.5% of the volatile

z-Correa et al. [22] obtained supercritical (SC) andercritical extracts (SC-V) from E. uniora at 60 C

r. The SV-V extracts presented as major compounds

4. Con

Thitive mraw mextractof pheactivitto obtabe assodant ato the

Ackno

Thesuppor(Proce

Refere

[1] M.Tfarm(11)-trien-8-epoxide (15.5%) and selina-1,3-7(11)- (11.6%) followed by germacrene B, germacrene D,aryophyllene. Peixoto et al. [31] obtained supercriti-

from E. uniora leaves at pressures varying from 100t 50 and 60 C. The SC extracts showed global yields5% and 3.17%, with the presence of major compounds

2, curzerene and germacrene B. The most volatile frac- showed global yield close to 0.1%, as well as major

such as germacrene, curzerene B, germacrene D and

t researchers have reported the presence of other com- identied in the present study in the essential oil of E.ch as curzerene [44], furanodiene and furanoelemene

differences may be associated with the extractiond the chemotype of the plant. According to ANVISArm chemotype applies to aromatic plant that differs

of the same species due to its different chemical com-erefore, the E. uniora sample of this study wouldotype I, with a predominance of sesquiterpenes of

62 (2003[2] A.E. Cons

taceae) a(2002) 57

[3] M. VizzotAgropecuimprensa

[4] E.E.S. Schuation ofEthnopha

[5] I. Arai, S.Y. Momoglycemia(1999) 30

[6] F.B. HoleScreeningment of 102710

[7] F.N. VictoSilva, S. dora L. an50 (2012

[8] F.G. Famuniora in

[9] I.A. OgunW.N. Set1.67 1391 139014.18 1419 14191.05 1433 1.10 1452 14540.62 1459 1461

15.00 1481 148012.26 1496 14943.34 1504 1.08 1515 15131.18 1522 1524

13.49 1556 15561.73 1577 1576

15.72 1631 16340.76 1643 1645

11.98 1753 17470.48 1840

97.41

ions

dy showed that the sequential extraction is an effec-d for obtaining differentiated extracts using the sameal. The three-step process was more efcient to obtaining high global extraction yield and high concentration

compounds of interest. The analysis of the antioxidantwed that the processes using ethanol as solvent enabledtracts exhibiting high antioxidant activity, which couldd to the presence of phenolic compounds. The antioxi-y by DPPH assay measured as EC50 could be correlated

phenolics content.

gments

ors wish to thank CNPq and Fapesp for their nancial for the scholarship awarded to Tbata Tayara Garmus

pesp No. 2011/14309-8).

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Extraction of phenolic compounds from pitanga (Eugenia uniflora L.) leaves by sequential extraction in fixed bed extractor...1 Introduction2 Material and methods2.1 Characterization of raw material2.2 Chemicals2.3 Extraction procedures2.4 Experimental extraction in fixed bed2.5 One-step extraction in fixed bed using water or ethanol2.6 Conventional aqueous or ethanolic extraction2.7 Extract composition2.7.1 Determination of polyphenols and total flavonoids2.7.2 Analysis of the more volatile fraction by GCMS

2.8 Antioxidant activity by DPPH assay2.9 Statistical analysis

3 Results and discussion3.1 Extraction yields3.2 Kinetics of sequential extraction3.3 Kinetics of ethanolic/aqueous extraction in fixed bed3.4 Concentration and yield of polyphenols and flavonoids3.5 Antioxidant activity (DPPH)3.6 Analysis of the more volatile fraction by GCMS

4 ConclusionsAcknowledgmentsReferences