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J. of Supercritical Fluids 86 (2014) 4– 14

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

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

xtraction of phenolic compounds from pitanga (Eugenia uniflora L.)eaves by sequential extraction in fixed bed extractor usingupercritical CO2, ethanol and water as solvents

ábata T. Garmusa, Losiane C. Paviania, Carmen L. Queirogab,edro M. Magalhãesb, 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, Brazil

r t i c l e i n f o

rticle history:eceived 19 August 2013eceived in revised form8 November 2013ccepted 19 November 2013

eywords:ugenia uniflora L.upercritical extractionhenolic compounds

a b s t r a c t

With the goal of maximizing the extraction yield of phenolic compounds from pitanga leaves (Euge-nia uniflora L.), a sequential extraction in fixed bed was carried out in three steps at 60 ◦C and 400 bar,using supercritical CO2 (non-polar) as solvent in a first 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 flavonoids and antioxidant activ-ity by DPPH method (in terms of EC50). The nature of the solvent significantly influenced 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 outthat the sequential extraction process is the most effective in terms of global extraction yield and yield

equential extractionixed bed extractorntioxidants

of polyphenols and total flavonoids, 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%).

. Introduction

Eugenia uniflora L., also known as pitanga, is a perennial tree ofhe Myrtaceae family native to South America [1]. Despite its trop-cal origin, its cultivation is already widespread in many countriesnd can be found in some Asian countries, in the United States andaribbean. In Brazil, although it is native to South and Southeast,he Northeast is the only region that commercially exploits thisruit with high economic potential [2,3].

E. uniflora leaves are known for their numerous therapeu-ic properties, having been used for a long time in the popular

edicine. Among the medicinal applications, the leaves are use asypotensive, antigout and stomachic agent, in addition to antimi-robial and hyperglycemic activities [1]. Studies have shown that

ompounds present in the leaves have anti-inflammatory [4], anti-yperglycemia and hypertriglyceridemia [5], antimicrobial and

∗ 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.2013.11.014

© 2013 Elsevier B.V. All rights reserved.

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. uniflora 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 flavonoid-rich fraction obtained from E.uniflora leaves for treatment of inflammatory diseases.

The benefits attributed to E. uniflora are due to the secondarymetabolites present in its leaves, including phenolic compoundssuch as flavonoids, terpenes, tannins, anthraquinones and essentialoils [13]. The composition of the extracts from E. uniflora 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 classified into simple phenols, phenolic acids(benzoic acid, cinnamic acid and its derivatives), flavonoids (antho-cyanins, flavonols and their derivatives) and tannins [17]. Suchcompounds possess a number of pharmacological properties which

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ake them act on biological systems. Consequently, many of theseroperties act efficiently in preventing certain diseases in humans2,18].

In recent years, studies on the extraction of polyphenols fromatural sources have attracted special interest. The extraction is aery important step in the isolation, identification and use of phe-olic compounds and there is no one single extraction method. Thextraction using organic solvent and supercritical fluid extractionechniques are more commonly used for the isolation of polyphe-ols [19].

The supercritical fluid most commonly used for extraction ofatural products from foods or pharmaceuticals is carbon dioxideCO2). The extraction processes using supercritical carbon diox-de (scCO2) as a substitute for some organic solvents and waterapor (steam distillation or hydrodistillation) are one of the promi-ent options for obtaining natural extracts containing bioactiveubstances (antioxidants, essential oils, carotenoids, phenolic com-ounds, flavonoids and others). Among the advantages of usingupercritical carbon dioxide extraction processes stand out itsow cost, non-toxicity, non-flammability, as well as it is inert andas good extraction capacity due to its higher penetration power20,21].

Martinez-Correa et al. [22] obtained natural extracts from E.niflora by two-step extraction processes, using scCO2 in the firsttep and ethanol or water in the second conventional step. Whenompared with conventional extraction (one-step), the two-steprocess produced aqueous and ethanolic extracts with higherolyphenols content and more active flavonoids.

This study aimed to use the sequential extraction in fixed bedo obtain the maximum extraction yield and more concentratedxtracts on phenolic compounds. A sequential extraction in fixeded extractor was conducted using the following solvents in orderf increasing polarity: supercritical carbon dioxide in the first step,thanol in the second step and water in the third step. Then,he results for global extraction yield, composition of essential oilnd phenolics content of the extracts were compared with thosebtained by one-step process using water and ethanol as solventsconventional and fixed bed methods).

. Material and methods

.1. Characterization of raw material

Pitanga leaves (E. uniflora) were collected from an experimentaleld at the Chemical, Biological and Agricultural Pluridisciplinaryesearch Centre (CPQBA–UNICAMP) and dried in a forced air circu-

ation oven at 42 ◦C for 3 days. The collection was made in the sameocation of the sample used in the study of Martinez-Correa et al.22]; however, the present sample consisted of very young planteaves, while Martinez-Correa et al. studied well-developed planteaves.

A mean particle diameter of 0.336 ± 0.003 mm was calculatedrom the means of the materials retained on the Tyler meshes4–270, according to ASAE procedures [23]. The geometric meaniameter distribution of the particles of E. uniflora was: 2.5%0.83 mm), 25.5% (0.59 mm), 35.1% (0.38 mm), 14.0% (0.27 mm),1.3% (0.19 mm) and 9.0% (0.09 mm). The particle density of.50 ± 0.01 g/cm3 was determined at 25 ◦C by helium gas pycnom-try (Quantachrome Ultrapyc 1200e Automatic pycnometer). Theoisture content determined by Karl-Fisher method, according toOCS Ca 23–55 [24] (Metrohm 701 KF Titrino equipped with 832

F Thermoprep oven) was 6.0 ± 0.1%.

To characterize the supercritical extraction, the apparent den-ity of the particle bed determined according to the Uquicheethod [25] was 0.46 ± 0.01 g/cm3, and the bed porosity calculated

itical Fluids 86 (2014) 4– 14 5

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

CO2 used in the experiments was 99.5% pure and sup-plied by White Martins Gases Industriais (Campinas, Brazil, lot113 C/12). Ethanol (99.8% v/v) was purchased from Êxodo (Brazil,lot AE8828RA), ultra pure water from a Milli-Q system (MilliporeCorporation, EUA) and hexane (98.5% v/v) used in the supercriticalextraction was purchased from Synth (Brazil, lot 135524).

The reagents sodium carbonate (99.5% w/w) sodium hydrox-ide (95.0% w/w) and ethyl acetate (99.5% v/v) were obtained fromÊxodo (Brazil). The Folin–Ciocalteau reagent was purchased fromDinâmica (Brazil), aluminum chloride (99.0% w/w) and sodiumnitrite (97.0% w/w) was from Ecibra (Brazil), gallic acid (99.0% w/w)from Vetec (Brazil), the (+)-catechin (98.0% w/w) and 1,1-diphenyl-2-picrilhidrazyl (DPPH) were purchased from Sigma–Aldrich anddichloromethane (99.0% v/v) from Merck (Germany).

2.3. Extraction procedures

E. uniflora leaves were subjected to extraction processes in fixedbed extractor (high pressure extraction) in three steps using threedifferent solvents. The first step was performed with supercriticalcarbon dioxide, while the second and third steps were carried outwith ethanol and water, respectively. Fig. 1 presents an illustrativescheme of the extractions.

In the one-step process, the operating conditions of temperatureand pressure were 60 ◦C and 400 bar with an average flow rate ofscCO2 of 1.5 L/min (2.475 g/min). The CO2 in the supercritical stateflowed through a fixed bed containing approximately 7.0 g of sam-ple for approximately 6 h. In the second step, the residue from thefirst extraction (Residue 1) was subjected to a further extractionunder the same operating conditions, using ethanol as solvent at aflow rate of 0.5 mL/min (0.395 g/min) for a total period of 6 h. Theresidue from the supercritical and ethanolic extraction (Residue 2)was submitted to a third extraction, in which water at 400 bar and60 ◦C at a flow rate of 0.5 mL/min (0.5 g/min) flowed through thesame bed of particles. The average extraction time at this stage wasapproximately 6 h.

For comparison, one-step processes were performed in fixed bed(high pressure) using ethanol and water as solvents, without pre-vious extraction with scCO2 (Fig. 2). Conventional extractions wereperformed to complement the findings (Fig. 3), i.e. extractions atlow pressure (atmospheric pressure), using ethanol and water assolvents.

The global extraction yield was used as a comparative parame-ter between the different extraction methods, which expresses theratio between the mass of dry extract and the mass of raw material.

2.4. Experimental extraction in fixed bed

The experiments were performed in an experimental unit (LabEXTRAE, UNICAMP, Brazil) as shown in Fig. 4.

The apparatus consisted mainly of a CO2 cylinder (1), refriger-ated bath (2), high-pressure pump (3), supply tank (4), extractor(5), collection flask (7), gas flow meter (9) volume totalizer (10),thermostatic bath (11), trap adsorvent Porapak-Q (80/100 mesh,Supelco, EUA) (8) used only to capture the volatile compoundsin the supercritical extraction, two Bourdon pressure gauges, onelocated in the supply tank and the other on the inlet side of the

extractor, a peristaltic pump (6) used to inject the hexane, and ahigh pressure pump for ethanol/water (12). In all cases, the extrac-tor (5) was packed by hand with approximately 7.0 g of dry andground material. Glass beads (6 mesh) were used to fill 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 fixed 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 flow 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.

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ig. 2. Flow diagram of the sequential extraction process in fixed bed extractor (onhe aqueous extraction: aqueous extract in fixed bed (ALF); the flow rates of solveethanol = 0.79 g/mL and �water = 1.0 g/mL.

paces of the extractor to avoid preferential pathways for CO2. Theperating conditions were adjusted to 400 bar and 60 ◦C for allxperiments. When the conditions were achieved, a period of halfn hour was adopted as static time to stabilize the system, start-ng the extraction by flowing CO2 at a rate of 1.5 L/min throughhe bed, and collecting the extract in the collection flask (7). Theaseous CO2 that left the collector flask (7) was drained through arap-Poropak Q (8), and led to the flow meter (9) and volume total-zer (10) to quantify the carbon dioxide used in the process. Thextracts were collected at predetermined time intervals in order tobtain the extraction curves (extraction yield as a function of theass of solvent). After each supercritical extraction, the tubing in

he process line was washed with hexane using a peristaltic pump6).

After the soluble compounds were extracted by scCO2, the CO2ow was stopped and the system was depressurized. Then, ethanol

ig. 3. Flow diagram of the conventional extraction process (three steps). The ethanolic each step.

process). For the ethanolic extraction: ethanolic extract in fixed bed (ELF); and forere adjusted at 25 ◦C and 0.93 bar; the solvent densities in these conditions were:

at 0.5 mL min was flowed through the bed of particles using thepump for ethanol/water (12) and the same operational condi-tions were adjusted (60 ◦C and 400 bar), considering a stabilizationperiod of half an hour. The extracts were collected in the collectionflask (7) at predetermined time intervals to obtain the curve withliquid ethanol. In the third step of the sequential extraction, thewater was pumped at flow rate of 0.5 mL/min through the pump(12) until the process conditions were achieved. After the stabiliza-tion time of the system, the extraction took place and samples wereobtained in fixed time intervals. All extractions were performed intriplicate.

2.5. One-step extraction in fixed bed using water or ethanol

For this stage, an experimental procedure similar to thatdescribed in Section 2.4 (Fig. 4) was adopted. The extractor was

xtracts (EC) or 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

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acked with 7.0 g of plant and water or ethanol was drained at.5 mL/min through the bed of particles by using the pump forthanol/water (12).

.6. Conventional aqueous or ethanolic extraction

The conventional extracts were obtained according to Cseket al. [27]. About 4.5 g of dried and crushed sample were mixedith 50 mL of solvent (water or ethanol) in a balance cell, in which

he mixture was stirred with the aid of a magnetic stirrer and theemperature adjusted at 60 ◦C using a thermostatic bath connectedo the cell. A reflux condenser was attached to the system, whoseurpose was to prevent the loss of desirable compounds by evap-ration during extraction, and the temperature was controlled by

thermostatic bath at 5 ◦C. The mixture remained stirring for 2 h,nd then was vacuum filtered. The filtrate was reserved and theolid residue extracted with a further 50 mL of solvent, repeatinghe process twice. Thus, each new filtrate was mixed to the pre-ious one, constituting the extract. The total extraction time was

h.The solvent of the ethanolic extracts was evaporated in rota-

vaporator at 50 ◦C under vacuum of 600 mm Hg (Marconi, MA-058,razil), and in a vacuum oven (Marconi, MA 030-12, Brazil; pumparconi, model MA-057-13, SP, Brazil) under the same conditions

ntil constant weight. The water of the extracts was removed byreeze-drying (lyophilizer Liobras, model L101, SP, Brazil) to obtainhe dried extracts.

.7. Extract composition

All extracts were analyzed for total phenolics content, totalavonoids and antioxidant activity, as well as the profile of ter-ene compounds present in the volatile fraction retained in theorapak-Q trap.

action apparatus.

2.7.1. Determination of polyphenols and total flavonoidsThe total polyphenols content was determined using the

Folin–Ciocalteu reagent, according to Singleton et al. [28], and theresults were expressed as equivalents of gallic acid (mg GAE/gdry extract). The absorbance was measured at 750 nm using aspectrophotometer (UV-VIS lambda 40, Perkin Elmer, USA) andthe results were calculated by a standard curve of gallic acid(0–125 mg/L).

The total flavonoids were determined using the methodologydescribed by Zhishen et al. [29], and the results were expressedin catechin equivalents (mg CE/g dry extract). The absorbance wasmeasured at 510 nm using a spectrophotometer (UV-VIS lambda40, PerkinElmer, USA) and the results were calculated by a standardcurve of catechin (0–125 mg/L). All determinations were carried outin triplicate.

2.7.2. Analysis of the more volatile fraction by GC–MSThe volatile fractions from the supercritical extraction (SC)

retained by the Porapak-Q trap were analyzed by gas chromatog-raphy (Agilent 6890N) coupled to a mass detector (MSD 5975).The chromatograph was equipped with a HP-5MS capillary col-umn (30 m × 0.25 mm × 0.25 �m) and stripping gas of helium at1.0 mL/min. The column was programmed from 60 ◦C to 240 ◦C at3 ◦C/min, and from 240 ◦C to 280 ◦C at 4 ◦C/min. The temperatures ofthe injector and detector were 220 ◦C and 290 ◦C, respectively. ThePorapak-Q cartridge was eluted with 15.0 mL of dichloromethaneto extract the volatile compounds. Then, the solution containingthe volatiles was concentrated on a rotary evaporator at 40 ◦Cand 100 mm Hg vacuum. The dried extract was dissolved in ethylacetate (20.0 mg/mL) and an aliquot of 1.0 �L was injected in the

equipment (GC–MS). The compounds were identified by compar-ing the mass spectra obtained in the present study with those inliterature using the CG/EM system database NIST library, and bycomparing with 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 flavonoids and antioxidant activity of the extracts.

Extraction Extracta Globalyield (%)

Total polyphenols Total flavonoids Antioxidantactivity

Concentration(mg GAE/gextract)

Yield (mgGAE/g leaves)

Concentration(mg CE/gextract)

Yield (mg CE/gleaves)

EC50

(�g/mL)

Sequential fixed bed (3 steps)

SC 5 ± 1g 32.70 ± 0.03f 1.8 ± 0.3e 153 ± 4a 8 ± 2b,c >200SC-V 0.31 ± 0.03h 26.0 ± 0.2g 0.08 ± 0.01e 64 ± 1b 0.20 ± 0.02d >200SCE 16 ± 1f 240.5 ± 0.2a 38 ± 2c 27.8 ± 0.3d 4 ± 1c,d 9.15SCA 22.0 ± 0.4d 233.8 ± 0.5b 51 ± 1b 20 ± 1e 4.4 ± 0.3c,d 25.41SC + SCE + SCA 43 ± 2a – 91 ± 3a – 17 ± 3a –

Fixed bed (1 step)ELF 20 ± 1d,e 163.5 ± 0.4c 32 ± 2d 40 ± 2c 8 ± 1b,c 16.19ALF 33.3 ± 0.4b 152.2 ± 0.2d 51 ± 1b 15 ± 1e 5.0 ± 0.4c 15.72

Conventional (1 step)EC 18 ± 2e,f 151 ± 1d 28 ± 3d 61 ± 2b 11 ± 2b 12.71AC 27 ± 1c 108.7 ± 0.2e 30 ± 1d 20 ± 2e 6 ± 1c 19.58

Gallic acid Standard – 1000 – – – 2.09

Values represent the mean of triplicate assays ± standard deviation. Different letters represent statistically significant differences (p < 0.05).a Supercritical (SC); volatile supercritical (SC-V); sequential ethanolic (SCE); sequential aqueous (SCA); ethanolic fixed bed (ELF); aqueous fixed bed (ALF); conventional

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thanolic (EC); conventional aqueous (AC); and accumulated yield (SC + SCE + SCA).

eries (C8–C20) with the quantitative analysis made by peak areaormalization.

.8. Antioxidant activity by DPPH assay

The antioxidant activity was determined according to therocedure described by Mensor et al. [30] using the 1,1-difenil--picrilhidrazyl (DPPH) reagent. Starting from the stock solution1 mg dried extract/mL ethanol), solutions with final concentra-ions of 5–150 �g/mL, were prepared. Aliquots of 2.5 mL of eachxtract solution were transferred to test tubes protected from theight. One millilitre of a recently prepared ethanolic solution ofPPH (0.3 mM) (Sigma–Aldrich Chemie, Alemanha, lote S4869-48) was added to each tube, and the mixture was stirred and

eft to react for 30 min at room temperature. Then, the absorbanceAbssample) was measured at 517 nm using a spectrophotometerUV-VIS lambda 40, Perkin Elmer, EUA). The blank was preparedy mixing 1 mL ethanol and 2.5 mL extract, and the negativeontrol by mixing 1 mL DPPH solution and 2.5 mL ethanol. Thebsorbance of both solutions was measured at the same wave-engh (Absblank, Abscontrol). The spectrophotometer was calibratedo zero absorbance using ethanol (99.5%), and gallic acid as the posi-ive control (standard). The antioxidant activity (AA) was calculatedrom the following equation:

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The extract concentration responsible for a 50% decrease in thenitial activity of the DPPH (EC50, �g/mL) was calculated by lin-ar and non-linear regression of the AA (%) curves obtained for allxtract concentrations.

.9. Statistical analysis

Data were analyzed by analysis of variance (ANOVA). Compar-sons between means by Tukey’s test at 5.0% significance level wereerformed to identify the treatments with the best responses. Theoftware Statistica® version 7.0 (StatSoft, USA) was used for thistep.

3. Results and discussion

3.1. Extraction yields

Table 1 shows the values obtained for global extraction yield,concentration and yield of both polyphenols and total flavonoidsand antioxidant activity of the extracts obtained by supercritical,ethanolic and aqueous extraction in one-step process (conventionalor fixed bed) and sequential extraction in three steps. The resultsare averages of experimental values performed in triplicate. Tukey’stest (p < 0.05) was performed to determine whether there is signif-icant difference between the average results.

The extraction using scCO2, ethanol and water showed sig-nificant differences regarding the global extraction yield, whichindicates the influence of the nature of the solvent used in theprocess. The extraction yield tended to increase with the solventpolarity. The aqueous extracts showed higher yields, followed bythe ethanolic extracts, since the water is more polar than ethanol.As expected, the global extraction yield of 43% obtained in thesequential extraction (SCE + SC + SCA) is significantly higher thanthe extraction yields of 18% and 27% obtained in the one-step pro-cess using ethanol or water, respectively. When the extraction stepsare compared separately, the supercritical ethanolic extract (SCE)with 16% of global yield was lower than the yield of 20% obtained inthe one-step extraction in fixed bed with ethanol (ELF). The ethanolalso extracted the soluble compounds in the scCO2, which werefractionated on the sequential extraction, considering that the sumSC + SCE was of the order of 21%. The prior extraction with scCO2promoted a change in the structure of the solid matrix due to the useof high pressure and subsequent depressurization. These changesmodify the interactions between the solute and the solid matrix andmay facilitate the extraction. The best yield obtained in the one-step process was 33% in the aqueous extraction in fixed bed (ALF),followed by 27% in the conventional aqueous extraction (AC), indi-cating the positive effect of high pressures in the global extractionyield. The same effect was not observed when ethanol was used assolvent. The yield of 18 ± 2% in the conventional ethanolic extrac-tion (CE) showed no significant difference as compared to 20 ± 1%in the ethanolic extraction in fixed bed (ELF).

The global yield of 5% obtained in the supercritical extrac-tion was higher than the 3.5% yield reported by Martinez-Correaet al. [22] under the same conditions of temperature and pressure(400 bar and 60 ◦C). The global yields obtained in the conventional

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

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ig. 5. Kinetics and yields of the sequential extraction (a) using scCO2 in a first stepupercritical (SC-V); sequential ethanolic (SCE); sequential aqueous (SCA); ethanoliqueous (AC).

xtractions were also much higher than those obtained by theuthors [22], who obtained 8% and 20% of extraction yields, versus8% and 27% obtained in the present study for ethanolic and con-entional aqueous extraction, respectively. This difference may beaused partially by the use of young leaves and lower geometricean diameter of the particles.In supercritical extraction with scCO2, the extract was fraction-

ted into two parts (SC and SC-V), where the most volatile portionSC-V) was captured on Porapak-Q trap for not being stripped byO2 in the depressurization process. The global extraction yieldf 0.31% obtained in the SC-V is of the same order of magnitudes the yield reported in the literature for supercritical extraction,s the 0.1% yield obtained by Peixoto et al. [31], who used pres-ure values between 100 and 300 bar and temperatures between0 ◦C and 60 ◦C, as well as 0.47% of extraction yield obtained byartinez-Correa et al. [22] using 400 bar and 60 ◦C. Several authors

tudied E. uniflora volatile oil obtained by hydrodistillation andound yield values comparable to the SC-V supercritical extracts,hich are of the order 0.54% [31] and 0.74% [32]. These differ-

nces may be attributed to the extraction methods, plant origin,

thanol in a second step (c) and water in a third step (d). Supercritical (SC); volatile bed (ELF); aqueous fixed bed (ALF); conventional ethanolic (EC); and conventional

age and development, seasonality, pluviometric index, tempera-ture, altitude, nutrients, conditions of collection, storage, amongothers [33].

3.2. Kinetics of sequential extraction

As can be seen in Fig. 5a, the curves of sequential extrac-tion (supercritical + ethanolic + aqueous) obtained at 400 bar and60 ◦C represent the cumulative percentage yield of the extract (gof extract/100 g raw material) as a function of the ratio of themass of solvent accumulated by the mass of the raw material(S/F). In the three extraction steps, two extraction curves (runs01 and 02) and a total extraction (run 03) were built to obtaina single extract, in which the chemical determinations were per-formed.

Fig. 5b shows that the global yield was 70% during the first hour

of the supercritical extraction (SC), which corresponds to S/F = 20,while 90% global yield was obtained in the first 3 h (S/F = 60). In thelast 3 h of extraction, in which the rate of mass transfer is controlledprimarily by diffusion phenomena inside the solid particle, only the

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

water

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Fig. 6. Kinetics and extraction yields with ethanol (a) and

ast 10% of the compounds were extracted. Given the above, 3 h60 S/F) of extraction would be sufficient.

For sequential ethanolic extraction (SCE), as shown in Fig. 5c,bout 50% of the total yield was obtained during the first hour,sing approximately 5 S/F. After 4 h extraction using approximately5 S/F, about 90% yield was reached. The arrangement of the pointsf SCE extraction curves indicates the possibility of not reaching theomplete depletion of the solute in the bed along the 6 h, even for alobal yield of 16%. The sequential aqueous extraction curves (SCA),ig. 5a and d, showed good reproducibility of the experiments. Theass of extract after 3 h of extraction using approximately 14 S/Fas about 90% of the total yield.

The yields obtained during the sequential extraction kineticshow that smaller extraction periods could be used, once the diffu-ion period (DC) was achieved after 3 h for the supercritical (SC) andequential aqueous extraction (SCA) and from 4 h for the sequentialthanolic extraction (SCE).

.3. Kinetics of ethanolic/aqueous extraction in fixed bed

The kinetics of ethanolic (ELF) and aqueous extraction (ALF) inxed bed (400 bar and 60 ◦C) in one-step process are shown inig. 6a and b, respectively. Fig. 6 shows two replicates, in whichxtracts were collected at predetermined time intervals (runs 01nd 02) and a point (one extraction) in run 03, expressed as aunction of the mass of extract.

It is observed in Fig. 6a that the extraction period was not suf-cient for exhaustion of the bed, which indicates that most of thexperimental points were located in the intermediate region, which

epends on the solutes solubility and internal diffusivity. In thearly stages (1-h extraction or 25 g solvent) of the ethanolic extrac-ion process (Fig. 6a), the yield reached an average value of 10%,hich corresponded to approximately 60% of global yield. After this

ig. 7. Concentration (a) and yield (b) of phenolics and total flavonoids of pitanga (E. unifloSCE); sequential aqueous (SCA); ethanolic fixed bed (ELF); aqueous fixed bed (ALF); conv

(b) at 400 bar and 60 ◦C (fixed bed) of E. uniflora extracts.

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 first one is dependent on the solubilityof solutes extracted, and the intermediate and the third regionsare controlled by the diffusivity. In the first 6 points (1-h extrac-tion or 35 g solvent) an average of 18% of accumulated yield wasobtained, which corresponds to nearly 50% of the total yield. Thisindicates a high rate of mass transfer in the early stages of theaqueous extraction. After 3 h (90 g solvent), the curve tends to apractically constant value.

3.4. Concentration and yield of polyphenols and flavonoids

The values reported in Table 1 concerning the concentration(Fig. 7a) and the yield (Fig. 7b) of polyphenols and flavonoids ofthe extract are compared in Fig. 7.

It can be observed in Fig. 7a that the supercritical ethanolic(SCE) and aqueous (SCA) extracts presented the highest contentof total phenolics, indicating that the prior extraction with scCO2allowed the concentration of phenolic compounds (SCE > ELF > ECand SCA > ALF > AC).

The lowest concentration of phenolic compounds was obtainedin the supercritical extract (SC) (32.7 mg/g) followed by the volatilefraction (SC-V) (26 mg/g). These results indicate that most of thephenolic compounds have polar character, since the more concen-trated extracts were obtained primarily in the ethanolic extracts,followed by the aqueous extracts and supercritical extracts.

The sequential aqueous extract (SCA) had the highest yield

of 51 mg of total polyphenols per gram of leaves, and the samevalue was obtained in the ALF extract, which was higher thanthe ethanolic extracts (SCA = ALF > SCE > ELF = EC = AC). This find-ing shows the greatest advantage of the three-step sequential

ra) leaves (rm). Supercritical (SC); volatile supercritical (SC-V); sequential ethanolicentional ethanolic (EC); and conventional aqueous (AC).

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xtraction versus one-step aqueous extraction (fixed bed or con-entional). The sequential extraction enabled to obtain threextracts: 5% of supercritical extract, 16% of concentrated etha-olic extract (240.3 mg GAE/g), and 22% of concentrated aqueousxtract (233.8 mg GAE/g), which corresponds to a yield of 51 mgAE/g leaves in the aqueous extract, and 91 mg GAE per gram of

eaves in the total extract (SC + SCE + SCA), as compared to 33% of aess concentrated aqueous extract containing 163 mg GAE/g, whichlso corresponds to 51 mg GAE/g leaves, or against 27% of a muchess concentrated conventional aqueous extract containing 108 mgAE/g, which corresponds to a yield lower than 30 mg GAE/g leaves.

The effect of the prior supercritical extraction in the sequentialxtraction allowed to obtain more concentrated ethanolic extracts,nd with higher yield of polyphenols, being 38 mg GAE/g leavesgainst nearly 30 mg GAE/g leaves obtained in fixed bed (ELF) oronventional extraction (CE). The analyses of the data on the yieldnd concentration of phenolic compounds of the extracts of E. uni-ora leaves obtained from different extraction processes provedhat the sequential extraction (SCE + SC + SCA) followed by the one-tep aqueous extraction (ALF) are effective methods to extract theseompounds.

The chemical characterization of the extracts regarding theavonoids content (Fig. 7a) showed that the highest concentra-ions were obtained in the supercritical extracts, with values of53 mg/g for SC and 64 mg/g for SC-V. The latter was statisticallyqual to the flavonoids content obtained in the conventional etha-olic extraction (CE), followed by the ethanolic and finally aqueousxtraction.

An important point to be noted is that despite the aqueousxtractions presented the best global extraction yields, the extractsbtained using water as solvent showed the lowest flavonoidsontents. It is possible to extract more flavonoids in the supercriti-al extraction; consequently, these compounds are decreased in theater stages, presenting lower extraction yields in the SCE and SCAxtracts when compared to ELF, EC, ALF and AC one-step extraction.

Comparing the data in Fig. 7a, it was observed that the extractionith scCO2 presented higher flavonoids than polyphenols contents,hose behavior was also observed by Martinez-Correa et al. [22]

nd Paula et al. [34], who used the same standard of this exper-ment. However, it is known that flavonoids are included in theroup of phenolic compounds, thus the flavonoids content shoulde lower or equal to that of phenolics. These results may be relatedo the standards used to assess the concentration of polyphenolicsnd total flavonoids in the extract. If the reducing capacity of theeference substance is not precisely the same of the extract, theoncentration calculated from the standard curve will not reflecthe polyphenolics or flavonoids in the sample.

The quantification of polyphenols is performed by a variety ofechniques. Although the technique using the Folin–Ciocalteu ishe most extensively used, some limitations have been attributedo that method, once the reagents can be reduced by other con-tituents of the extract. Thus, the Folin–Ciocalteu method doesot provide an accurate result of polyphenolics content, but theeducing capacity of the sample under study [35,36]. These find-ngs support the need for a different analytical approach, and aim totandardize and validate the methods for quantification of polyphe-ols and total flavonoids in supercritical extraction.

The best yield of flavonoids was obtained by sequential extrac-ion, which was about 50–240% higher than those obtained in thene-step extractions. The sequential extraction enables to removeompounds of the flavonoids family having different character-stics. Besides the solubility of the compounds vary according to

he solvent polarity, the interactions with other constituents of thelant may also affect the degree of extraction of these substances.he three-step process allowed one to obtain more concentratedthanolic or aqueous extracts in terms of flavonoids than the one

itical Fluids 86 (2014) 4– 14 11

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-sidered the most effective method for obtaining flavonoids for thequantification of these compounds.

The results of polyphenols and flavonoids contents in the E.uniflora extracts obtained by different extraction methods werecompared with the results reported by Martinez-Correa et al. [22].The sample used in the present study, comprised of younger leaves,had lower polyphenols content, but the extraction yield was veryclose to the sample studied by the authors in supercritical (SC), con-ventional ethanolic (EC) and conventional aqueous (AC) extraction.The concentration and yield of flavonoids were higher, except in theaqueous extract.

Martinez-Correa et al. [22] reported the positive effect of super-critical extraction prior to ethanolic or conventional aqueousextraction in the concentration of polyphenols and flavonoids in theE. uniflora extracts. The same was observed in the present study toobtain total phenolics when using the supercritical extraction fol-lowed by aqueous extraction and ethanolic extraction in fixed bed(high pressure). The conventional ethanolic and aqueous extractsobtained after the supercritical extraction by the authors [22] weremore concentrated in polyphenols and total flavonoids than theethanolic and aqueous extracts obtained in fixed bed in the secondand third step of sequential extraction (this study used the samemethod of determining polyphenols described by the authors [22]).

When the results obtained by the three-step sequential extrac-tion are analyzed, the global extraction yield and the yield ofphenolics and total flavonoids are higher than the values reportedby Martinez-Correa et al. [22] using two- and one-step process. Thissuggests the use of sequential extraction using scCO2, ethanol andwater as solvents as an efficient strategy for obtaining extracts richin polyphenols and flavonoids.

3.5. Antioxidant activity (DPPH)

The reaction of free radical 1,1-diphenyl-2-picrylhydrazyl(DPPH) with antioxidant species inhibits and stabilizes the radical,promoting color changes from purple to yellow, which decreasesthe absorbance that was monitored using a spectrophotometer at517 nm [37]. Fig. 8 shows the antioxidant activity (AA) as a functionof the extract concentration (0–150 �g/mL) and gallic acid standardconcentration (0–5 �g/mL).

Depending on the extract studied and its chemical composition,the relationship between AA and the antioxidant content (extract)may present a linear or non-linear behavior in the concentrationrange studied, determined by the nature of the extracts. All extractsexhibited non-linear behavior, except the supercritical extract (SCand SC-V) that presented almost linear behavior, which is typicalof the extracts obtained using solvents of low polarity [38].

The results of DPPH scavenging capacity showed that the AAvalues increased rapidly with the extract concentration to reach amaximum value of approximately 95% for all extracts except thesupercritical (SC and SC-V). After reaching this maximum value,AA remained practically constant. It is worth mentioning that theprocess using ethanol exhibited better DPPH scavenging capacity.

In this method, the antioxidant activity (AA) is represented interms of effective concentration (EC50, �g/mL) which is defined asthe extract concentration responsible for 50% decrease in the ini-tial activity of DPPH [39]. The EC50 provides a direct comparison ofantioxidant activity between different substances, since it is inde-

pendent of the concentration of the sample [40]. Fig. 9 shows acomparison between the EC50 calculated values.

According to the antioxidant activity described by Reynertsonet al. [41], very active extracts exhibit EC50 < 50 �g/mL, moderately

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

F percrb aqueo

ai

eeTmi

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F(((

this study and the findings of Martinez-Correa et al. [22].The correlation between the total phenolics content and EC50

values showed increased antioxidant activity with increasing thetotal phenolics content (mg GAE/g), the last point on the graph

ig. 8. DPPH scavenging activity of E. uniflora extracts. Supercritical (SC); volatile sued (ELF); aqueous fixed bed (ALF); conventional ethanolic (EC); and conventional

ctive extracts from 50 to 100, slightly active from 100 to 200, andnactive extracts present EC50 > 200.

The final EC50 values measured by DPPH assay showed that thextracts exhibited high activity, with values well below 50 �g/mL,xcept for the supercritical extract that exhibited EC50 > 200 �g/mL.he extract after the supercritical extraction (SCE) stands out as theost active, presenting the lowest EC50 value of 9.15 �g/mL, which

s related to its high phenolics content.The prior extraction with scCOs was positive, because it

ncreased the antioxidant activity of the ethanolic extracts,ndicating that the supercritical extraction has removed the non-olar compounds with low antioxidant activity by DPPH assayEC50 > 200 �g/mL for the SC and SC-V extracts) from the vegetal

atrix, and thus, concentrating the residue obtained in the super-ritical extraction to substances having polar character and highntioxidant activity.

The ethanolic extracts presented high antioxidant activity, fol-owed by the aqueous and supercritical extracts. In this regard,

artinez-Correa et al. [22] measured the antioxidant activity of E.niflora extracts by DPPH and BCB (�-carotene bleaching) meth-ds. The latter evaluates the ability of an extract to inhibit thexidation process of �-carotene. The ethanolic extracts presentedigh antioxidant activity by both methodologies. The supercriti-

al extract (SC) had higher antioxidant activity than the aqueousxtracts when measured by BCB method, but was inactive when thePPH assay was performed (in accordance with the experimentalbservations of this study). In the aqueous extracts, the antioxidant

ig. 9. Antioxidant activity of E. uniflora extracts EC50 (�g/mL) values. SupercriticalSC); volatile supercritical (SC-V); sequential ethanolic (SCE); sequential aqueousSCA); ethanolic fixed bed (ELF), aqueous fixed bed (ALF), conventional ethanolicEC) and conventional aqueous (AC).

itical (SC-V); sequential ethanolic (SCE); aqueous sequential (SCA); ethanolic fixedus (AC).

activity measured by DPPH assay was higher than that obtained byBCB method. These results can be due to the compounds responsi-ble for the antioxidant activity are not necessarily the same for eachmethod, since the extracts are complex mixtures of compounds,thus different interactions may occur (synergistic or antagonistic)[22].

It can be observed in Table 1 that the EC50 values obtainedin each case are strongly linked to the total phenolics contentexpressed as mg gallic acid equivalents per gram of dry extract(mg GAE/g). The parameter is indicative of antioxidant activity: thelower the value, the higher the antioxidant activity of the corre-sponding extract. Fig. 10 was built to correlate the EC50 activityby DPPH assay with the total phenolics content of pitanga leavesextracts, using the EC50 versus concentration (mg GAE/g) values of

Fig. 10. Relationship between antioxidant activity (in terms of EC50) and totalphenolics content (mg GAE/g) in ethanolic and aqueous extracts of E. unifloraof this study, as compared to the data reported by Martinez-Correa et al. [22].Points in terms of phenolics content showed the following descending order: gal-lic acid > SCE > ELF > ALF > EC > AC for this study and gallic acid > SCE > EC > AC for thestudy of Martinez-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 uniflora L. leaves.

Peak Compound MM Relative (%) RIa RIb

1 �-Copaene 204 1.77 1374 13762 �-Elemene 204 1.67 1391 13903 Trans-caryophyllene 204 14.18 1419 14195 NI 202 1.05 1433 –6 �-Humulene 204 1.10 1452 14547 Allo-aromadendrene 204 0.62 1459 14618 Germacrene D 204 15.00 1481 14809 Bicyclogermacrene 204 12.26 1496 1494

10 NI 204 3.34 1504 –11 �-Cadinene 204 1.08 1515 151312 �-Cadinene 204 1.18 1522 152413 Germacrene B 204 13.49 1556 155614 Spathulenol 220 1.73 1577 157615 Selina-1,3,7(11)-trien-8-one 216 15.72 1631 163416 �-Muurolol 222 0.76 1643 164517 Selina-1,3,7(11)-trien-8-one-epoxide 232 11.98 1753 174718 NI 234 0.48 1840 –

Total 97.41

R

b1

3

csil

estb(fie

vastactbatcC

pu[m[fpfis

I, retention index; MM, molecular mass; NI, not identified.a Calculated retention index.b Retention index from Adams [42,43].

eing the gallic acid pure standard, EC50 = 2.09 in this study, and.87 as reported by Martinez-Correa et al. [22].

.6. Analysis of the more volatile fraction by GC–MS

The volatile fraction (SC-V) of the extracts obtained by super-ritical extraction was assessed by gas chromatography–masspectrometry (GC–MS) for analysis of their constituents. The chem-cal composition of the volatile fraction extract from E. unifloraeaves is shown in Table 2.

The volatile fraction recovered during the supercriticalxtraction assessed by GC–MS showed as major compoundselina-1,3-7(11)-trien-8-one (15.7%), germacrene D (15.0%) andrans-caryophyllene (14.2%), followed by germacrene B (13.5%),icyclogermacrene (12.4%) and selina-1,3-7(11)-trien-8-epoxide12.0%). These compounds were also found in the essential oilrom the plant obtained by hydrodistillation [7,9]. In total, wedentified 16 compounds corresponding to 92.5% of the volatilextract.

Martinez-Correa et al. [22] obtained supercritical (SC) andolatile supercritical extracts (SC-V) from E. uniflora at 60 ◦Cnd 400 bar. The SV-V extracts presented as major compoundselina-1,3-7(11)-trien-8-epoxide (15.5%) and selina-1,3-7(11)-rien-8-one (11.6%) followed by germacrene B, germacrene D,nd trans-caryophyllene. Peixoto et al. [31] obtained supercriti-al extracts from E. uniflora leaves at pressures varying from 100o 300 bar at 50 and 60 ◦C. The SC extracts showed global yieldsetween 1.45% and 3.17%, with the presence of major compoundss C15H20O2, curzerene and germacrene B. The most volatile frac-ion (SC-V) showed global yield close to 0.1%, as well as majorompounds such as germacrene, curzerene B, germacrene D and15H20O2.

Different researchers have reported the presence of other com-ounds not identified in the present study in the essential oil of E.niflora, such as curzerene [44], furanodiene and furanoelemene45]. These differences may be associated with the extraction

ethod and the chemotype of the plant. According to ANVISA46], the term chemotype applies to aromatic plant that differs

rom others of the same species due to its different chemical com-osition. Therefore, the E. uniflora sample of this study wouldt in chemotype I, with a predominance of sesquiterpenes ofelina.

4. Conclusions

This study showed that the sequential extraction is an effec-tive method for obtaining differentiated extracts using the sameraw material. The three-step process was more efficient to obtainextracts having high global extraction yield and high concentrationof phenolic compounds of interest. The analysis of the antioxidantactivity showed that the processes using ethanol as solvent enabledto obtain extracts exhibiting high antioxidant activity, which couldbe associated to the presence of phenolic compounds. The antioxi-dant activity by DPPH assay measured as EC50 could be correlatedto the total phenolics content.

Acknowledgments

The authors wish to thank CNPq and Fapesp for their financialsupport and for the scholarship awarded to Tábata Tayara Garmus(Process Fapesp No. 2011/14309-8).

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