J.

of Supercritical Fluids 55 (2011) 998–1006

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journal homepage: www.elsevier.com/locate/supflu

Extracts from pitanga (Eugenia uniflora L.) leaves: Influence of extraction process
on antioxidant properties and yield of phenolic compounds
Hugo A. Martinez-Correa a,b,∗ , Pedro M. Magalhães c , Carmen L. Queiroga c , Camila A. Peixoto a ,
Alessandra L. Oliveira d , Fernando A. Cabral a
a
Departamento de Engenharia de Alimentos, Universidade Estadual de Campinas, 13083-862 Campinas, SP, Brazil
b
Departamento de Ingenieria, Universidad Nacional de Colombia, Cr 32 Chapinero via Candelaria, Palmira, Colombia
c
CPQBA - UNICAMP, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil
d
Departamento de Engenharia de Alimentos, Universidade de São Paulo, 13635-900 Pirassununga, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Different extraction processes were employed to extract the polyphenolic compounds from pitanga
Received 26 May 2010 (Eugenia uniflora L.) leaves: a one-step process using water, ethanol or supercritical CO2 as solvents,
Received in revised form 31 August 2010 and a two-step process using supercritical CO2 followed by either water or ethanol. The total polyphe-
Accepted 3 September 2010
nolic compounds, total flavonoids and antioxidant activity were determined in all the extracts obtained.
The process performance was evaluated with respect to three variables: global extraction yield, con-
Keywords:
centration and yield of both polyphenols and flavonoids in the extracts. For the one-step extraction,
Eugenia uniflora L.
the results showed that the extraction yield increased with solvent polarity. For the two-step process,
Supercritical extraction
Phenolic compounds
the results suggested that water was more efficient in extracting the phenolic compounds from E. uni-
Antioxidants flora when the matrix was previously extracted with scCO2 . With respect to the antioxidant activity, the
ethanolic extracts obtained from both processes, using either the DPPH radical scavenging method or the
␤-carotene bleaching method, presented high antioxidant activities.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction of natural origin over those of synthetic origin is due to fac-

Eugenia uniflora L. (local name in Brazil: pitanga) is a peren-
nial tree of the Myrtaceae family, possessing 100 genera and 3600
species [1]. With appropriate soil conditions and nutrients, the tree
can reach up to 6 m, being very common in South America countries
and currently cultivated in Central America, The Caribbean, trop-
ical areas of the United States, Southeastern Asia, China, India, Sri
Lanka, Madagascar, South Africa, Israel and some countries of The
Mediterranean. Ethanolic extracts of pitanga leaves possess high
antioxidant activity [2] and studies have shown that pitanga may
be efficient in preventing some human diseases [3].
Antioxidants are important in retarding oxidation processes in
fats and oils. However, in biological systems, the antioxidants are
of great importance in the prevention of diseases of live organ-
isms, since they inhibit the initiation and propagation of oxidative
chain reactions, which are responsible for the generation of reactive
oxygen species, thus preventing the development of degenera-
tive diseases and cancer [4]. The superiority of the antioxidants
∗ Corresponding author at: Departamento de Ingenieria, Universidad Nacional de
Colombia, Cr 32 Chapinero via Candelaria, Palmira, Colombia. Tel.: +57 2 2717008;
fax: +57 2 2717008.
E-mail address: hamartinezc@palmira.unal.edu.co (H.A. Martinez-Correa).
tors such as tolerance, safety, atoxicity and the absence of side
effects, which contribute to increasing consumer preference [5].
Most antioxidants isolated from higher plants are polyphenols,
which present anti-oxidative features besides holding antibacte-
rial, anti-carcinogenic, anti-inflammatory and antiviral properties
[6]. Extraction processes employing supercritical carbon dioxide
(scCO2 ) take advantage of the ability of some chemicals to act as
excellent solvents at temperature and pressure above critical point.
The solvent is non-toxic and safe (GRAS) as well as non-flammable
and of low cost.
It has been observed [7,8] that when combining supercriti-
cal extraction in a sequential manner with conventional solvent
extraction, it can produce extracts with higher antioxidant activity.
The aim of the present study was to compare the different pro-
cesses for extracting phenolic compounds with antioxidant activity
from pitanga leaves. Thus a one-step extraction process was car-
ried out in order to obtain supercritical and conventional extracts:
supercritical carbon dioxide, aqueous (A) and ethanolic (E) extracts
at atmospheric pressure, and a two-step process was also stud-
ied, where in a first stage the extraction was carried out using
supercritical carbon dioxide (SC process), and in a second stage
the residue from the first stage was re-extracted with both water
(SCA) and ethanol (SCE) at atmospheric pressure. The total polyphe-
nol content and total flavonoids as well as the antioxidant activity,

0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2010.09.001
 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006
determined by both DPPH scavenging and the ␤-carotene bleaching
method, were determined in the extracts.
2. Materials and methods
2.1. Raw material characterization
Pitanga leaves (E. uniflora) were collected from an experimental
field at the Chemical, Biological and Agricultural Pluridisciplinary
Research Centre (CPQBA, Campinas, Brazil). They were dried at
room temperature and then milled in a knife mill (Marconi, model
MA 340, Brazil). The sample was protected from light and stored at
−10 ◦ C prior to further use. A mean particle diameter of 0.817 mm
was calculated from the means of the materials retained on the fol-
lowing sieves: Tyler meshes 12 (0.01%), 16 (0.92%), 24 (23.27%), 48
(55.47%) and 60 (20.34%) using the ASAE procedure [9] employing
sieves in a vibratory sieve system (Model 1868, Bertel, SP, Brazil).
A particle density of 1.48 ± 0.04 g/cm3 was determined by helium
gas pycnometry (Micromeritics, Accu Pyr II 1340 V1.02). The mois-
ture content determined by the Karl Fisher method (Methom 701
KF Tritino equipped with 832 KF Thermoprep) was 10.2 ± 0.2%. For
the supercritical extraction, the apparent density of the particle bed
was 0.312 ± 0.047 g/cm3 , determined according to the Uquiche et
al. method [10]. The bed porosity was calculated from the real den-
sity of the sample and the apparent density of the bed according to
the method described by Rahman [11], the result being 0.789. Leaf
samples ground before and after the supercritical extraction were
analyzed by scanning electronic microscopy (LEO 440i) in order to
determine their structure.
2.2. Chemicals
The CO2 employed in the experiments was 99.5% pure and
supplied by White Martins Gases Industriais (Campinas, Brazil).
Ethanol, 99.5% (w/w) was obtained from Synth (Brazil), and ultra
pure water from a Milli Q system (Millipore Corporation, USA).
Anhydrous sodium carbonate and anhydrous sodium hydroxide
were from Carlo Erba (Italy), the Folin–Ciocalteau reagent from
Dinâmica (Brazil), aluminum chloride, sodium nitrite, chloroform
and l(+)-ascorbic acid from Ecibra (Brazil), gallic acid (99%) from
Vetec (Brazil), (+)-catechin, 1,1-diphenyl-2-picrilhidrazyl (DPPH),
linoleic acid and Tween 40® from Sigma–Aldrich (Germany) and
␤-carotene from Fluka (USA).Extraction procedures
2.3.1. One-step extraction
The samples were extracted by two different processes: one-
step and two-step. The solvents used in the one-step process were
supercritical carbon dioxide at 40 MPa and 60 ◦ C (SC), ethanol at
atmospheric pressure and 25 ◦ C (E), and water at atmospheric pres-
sure and 60 ◦ C (A) (Fig. 1).
2.3.2. Two-step extraction
In the two-step process, the sample was first extracted with
supercritical carbon dioxide (40 MPa, 60 ◦ C). The residue from this
step was then extracted at atmospheric pressure in the second step,
either using ethanol (atmospheric pressure, 25 ◦ C) (SCE) or water
(atmospheric pressure, 60 ◦ C) (SCA) (Fig. 1, dashed line).
The ethanolic extracts (E, SCE) were obtained from 3 g of pre-
pared leaves (or residual mass from SC process) and 10 mL of
ethanol in a shaker (Marconi, Brazil) at 25 ◦ C for 42 h. The mix-
ture was filtered and the residual solid extracted again with 10 mL
of ethanol and centrifuged (Jouan, BR4i, France) at 5000 rpm, 25 ◦ C
for 5 min. The aqueous extracts (A, SCA) were obtained using 3 g
of plant (or residual mass from SC process) and 60 mL of ultra pure
water at 60 ◦ C in a thermostatic bath (Tecnal, Brazil) for 10 min, then
being centrifuged for 10 min at 10,000 rpm (Jouan, BR4i, France),
999
Fig. 1. One- and two-step extraction process scheme.
and finally obtained the extract to leave through filtration system
[12].
The experimental apparatus used for the Supercritical Fluid
extractions is shown in Fig. 2. It consisted of a 50 mL equilibrium cell
(5) (stainless steel AISI 316, Suprilab, Campinas, Brazil) immersed
in a water bath controlled by a heater (11) (Suprilab, Campinas,
Brazil) to within ±0.1 ◦ C. Approximately 7 × 10−3 kg of sample was
used to form a fixed bed of particles. The CO2 from the supply
tank (1) was cooled to the liquid state using a refrigerated bath
(2) (model 12101-30, Cole Parmer Instrument Company, Vernon
Hills, IL, USA) and then compressed into the extractor using a high-
pressure pump (3) (Model AA100S, Eldex Laboratories Inc., Napa,
CA, USA). The trap (8) was prepared by packing Porapak-Q (80–100
mesh, Waters Corporation, Milford, MA) between silanized glass
wool plugs in the glass tube (0.5 cm id × 50 mm). After extraction,
all the tubing in the process line was washed with ethanol using a
peristaltic pump (model L/S 77910 Cole Parmer Instruments com-
pany) (6) to recover the extract deposited in it. The ethanol was then
evaporated off at 45 ◦ C in a rotary evaporator (Marconi, MA120,
Brazil). The global extraction yields were calculated as the ratio of
the total mass of extract (extraction + cleaning process) to the initial
mass of raw material. For all the supercritical extractions the con-
ditions were established at 40 MPa, 60 ◦ C and a CO2 mass flow rate
of 4.10−5 kg/s. Two fractions of supercritical extract were obtained,
an extract fraction (SC), collected in a glass flask (7) and a more
volatile fraction (FV), which was stripped by CO2 in the depressur-
ization process and captured in the Porapak trap Q (8). The other
equipment used were: CO2 tank (4), gas flow meter (model 32908-
69 Cole Parmer Instrument Company, Vernon Hills, IL, USA) (9) and
volume totalizer (model G1, LAO Indústria Ltda, Osasco, SP, Brazil)
(10).
2.4. Extract composition
2.4.1. Determination of the total polyphenol content (TPC)
The total polyphenol content was quantified using the
Folin–Ciocalteu reagent, according to the procedure of Singleton et
al. [13]. 1 mL of diluted extract was transferred to a 25 mL volumet-
ric flask containing 9 mL of ultra pure water. The Folin–Ciocalteu
reagent (1 mL) was added and mixed. After 5 min, 10 mL of sodium
carbonate (7%) were added and the volume completed with ultra
pure water. After 90 min of incubation at 23 ◦ C in the dark, the
absorbance was measured at 750 nm in a spectrophotometer
(UV–VIS lambda 40, Perkin Elmer, USA), and the result calculated
using a pre-prepared gallic acid calibration curve (0–100 mg/L). The
blank was prepared using the same procedure with 1 mL of ultra
pure water in the place of the 1 mL extract. The determinations
of the extracts and of the calibration curve were carried out with
1000 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006
Fig. 2. Supercritical CO2 extraction apparatus.
three replicates, and the results expressed as equivalents of gallic
acid (mg GAE/g dry extract).
2.4.2. Determination of total flavonoid contents (TF)
The total flavonoids were measured as described by Zhishen et
al. [14]. An aliquot (1 mL) of adequately diluted extract or of the
aqueous catechin solutions (0–100 mg/L) was added to a 10 mL
volumetric flask containing 4 mL of ultra pure water, and 0.3 mL
sodium nitrite (5%) added. After 5 min, 0.3 mL of aluminum chloride
solution (10%) was added, and after a further 6 min, 2 mL of sodium
hydroxide solution (1M) was added and the volume completed
with ultra pure water. The absorbance was measured against ultra
pure water as the blank at 510 nm, using a UV/VIS spectrophotome-
ter (UV–VIS lambda 40, Perkin Elmer, USA). The determinations of
the extracts and of the calibration curve were carried out with three
replicates, and the results were expressed in catechin equivalent
(mg CE/g dry extract).
2.4.3. Analysis of the more volatile fraction by GC–MS
The volatile fraction from the supercritical extraction (FV)
that was retained by the Porapak-Q trap, was analyzed by GC
(gas chromatography). The volatiles trapped in the Porapak-
Q were eluted with 1 mL of ethyl acetate and analyzed for
identification by gas chromatography coupled to mass spec-
trometry (GC 6890N, Agilent 5975), with an HP-5MS capillary
column (30 m × 0.25 mm × 0.25 ␮m) and stripping gas of helium
at 1 mL/min. The column was programmed from 60 to 246 ◦ C at
3 ◦ C/min and the temperatures of the injector and detector were
220 ◦ C and 250 ◦ C, respectively. The compounds were identified
by comparing the mass spectra obtained with those in the liter-
ature [15], using the CG/EM system database – Wiley library and
NIST; and by comparing with the retention indexes reported for the
n-alkane series (C9–C30, C32).
2.5. Antioxidant activity
2.5.1. DPPH spectrophotometric assay
The antioxidant activity was determined according to the
procedure described by Mensor et al. [16] using the 1,1-diphenyl-
2-picrilhidrazyl (DPPH) reagent. Starting from the stock solution
(1 mg/mL) of ethanolic extract, solutions with final concentrations
of from 5 to 250 ␮g/mL were prepared. Aliquots of 2.5 mL of each
extract solution were transferred to test tubes protected from the
light, and 1 mL of a recently prepared ethanolic solution of DPPH
(0.28 mM) added to each, mixed well and left to react in the dark for
30 min at room temperature. The absorbance (Abssample ) was then
measured at 517 nm using a spectrophotometer (UV–VIS lambda
40, Perkin Elmer, USA). The blank was prepared by mixing 1 mL
ethanol with 2.5 mL extract, and the negative control by mixing
1 mL of DPPH solution and 2.5 mL of ethanol, and the absorbance of
these solutions also measured (Absblank , Abscontrol ). The spectropho-
tometer was zeroed with ethanol (99.5%) and (+)-catechin, gallic
acid and l(+)-ascorbic acid were used as the positive controls (stan-
dards). The tests were carried out in triplicate and the antioxidant
activity (AA) was calculated from Eq. (1).
 
[Abscontrol (Abssample − Absblank )]
AA(%) = × 100 (1)
Abscontrol
The extract concentration responsible for a 50% decrease in the
initial activity of the DPPH (EC50, ␮g/mL) was calculated by non-
linear regression of the AA(%) curves obtained for all the extract
concentrations.
2.5.2. ˇ-Carotene bleaching assay
The ␤-carotene bleaching (DBC) method is based on the spec-
trophotometric monitoring of the oxidation products produced
from the degradation of linoleic acid. The methodology was based
on the methods of Marco [17] and Miller [18], in which 5 mL of
dry emulsion (␤-carotene and linoleic acid) were transferred to
a test tube and 0.2 mL of the extracts diluted in ethanol added
(200 ␮g/mL), as also for the standard solutions. The control solution
was prepared in the same way substituting the extracts for 0.2 mL
ethanol. The sample and control tubes were submitted to thermal
self-oxidation for 180 min at 50 ◦ C. The absorbance values of the
samples, standards and control were measured at 464 nm (UV–VIS
spectrophotometer lambda 40, Perkin Elmer, USA) at intervals of
30 min against a blank prepared with 5 mL emulsion without the
␤-carotene plus 0.2 mL ethanol. The antioxidant activity (AA) was
calculated as the percentage of inhibition in relation to the control
(100% oxidation) using Eq. (2):
 
(Ac0 − Act ) − (Aa0 − Aat )
AA(%) = × 100 (2)
(Ac0 − Act )
where “c” represents the control and “a” the sample, respectively,
and A0 and At are the absorbance values at 0 and t minutes of the test
solution. Solutions (200 ␮g/mL) of BHT (=98% HPLC, USA), quercetin
(≥99.0% USA) and ascorbic acid (>99%, Ecibra, Brazil) were used as
positive controls (standards).
 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006 1001

2.6. Supercritical extraction modeling

In order to describe the behavior of the extraction curve with

scCO2 , three kinetic models were used: the desorption model [19],
the empirical model presented by Naik et al. [20] and the model
presented by Sovová et al. [21].
The Sovová et al. model [21] ignores the effects of the mass trans-
fer resistance in the solvent. The model simulates the extraction
process of two groups of substances from the following expression:

e = e1[1 − exp(−k1)] + k2q (3)

where “e” represents the extraction yield (w/w), and “q” the sol-
vent/raw material ratio (w/w), and e1, k1 and k2 are the adjustable
parameters of the model.
The desorption model proposed by Tan and Liou [19] describes
the mass transfer during the extraction as a process dominated by
desorption of the solutes, in which the extraction rate is propor-
tional to the concentration of the solute in the solid phase. The
model ignores the effects of axial dispersion and of intra-particle
diffusion:
A
mext = [1 − exp(−kd B)][exp(−kd t) − 1]
kd
1 − ε s
A = QCO2 X0
ε 
εHS
B=
QCO2
where H represents the length of the bed (m), S the cross-sectional
area (m2 ),  the solvent density (kg/m3 ), QCO2 the CO2 mass flow
rate (kg/s), X0 is the initial mass of solute in the inert solid matrix,
s the particle density of the solid (kg/m3 ), ε the porosity of the
bed, kd the adjustable parameter and “e” represents the desorption
constant.
The Naik et al. model [20] empirically describes the evolution of
the extraction process using the following expression:
e∞
e=
b+t
where e represents the yield (kg extract/kg raw material), t the time
of extraction (min), and e∞ and b the adjustable parameters of the
model, e∞ represents the value for extractable solute contained in
the raw material.
2.7. Statistical analysis
The statistical analyses were carried out using Statistica® 7.0
(Statsoft, USA). A one-way ANOVA and post hoc Tukey test (p < 0.05)
were carried out to verify significant differences, and the results
were expressed as the mean ± SD. All the extraction procedures
and chemical analysis were carried out in triplicate.
3. Results and discussion
3.1. Supercritical CO2 extraction
In Fig. 3 the supercritical extraction curves (triplicate) of the
pitanga leaves are shown at 40 MPa and 60 ◦ C, which was expressed
in terms of the mass of supercritical extract SC as a function of time,
on the run 3 was collected a total extract in a single flask . The
values calculated using the kinetic models of Sovová et al. [21], Tan
and Liou [19] and Naik et al. [20] are also shown, and their fitted
parameters can be seen in Table 1.
In the early stages (Fig. 3) of the supercritical extraction process
(0–60 min), a high extraction rate can be observed, but after this
phase the curve quickly attained a constant value, which would
(4) Fig. 3. Kinetics of supercritical extraction of E. uniflora L.
(5) indicate that the plant matrix was nearly exhausted of CO2 soluble
compounds. The kinetics of the extraction process indicates that
the soluble compounds are easily removable and present low mass
(6)
transfer resistance and high solubility.
In the modeling of the extraction kinetics, the value of the total
fraction of extractable solute in the inert solid matrix for the Tan
and Liou model was considered equal to the yield in free base of
the solute obtained at the end of the extraction curve [22], and the
value calculated for the porosity of the bed was 0.789.
A comparison between the values for the mass extracted
obtained experimentally, and the values calculated by the mod-
els and using the fitted parameters presented in Table 1, show that
the desorption model [19] did not describe the behavior appro-
priately, overestimating the values calculated, indicating that the
(7)
phenomenon describing the process is more important than the
solute desorption phenomenon occurring during extraction with
scCO2 . These observations agree with that reported in other stud-
ies [23]. The models of Naik and Sovová presented low values of
average absolute relative deviation (AARD), and consequently they
described the experimental behavior well, in which about 90% of
the substances soluble in CO2 were quickly extracted (60 min), indi-
cating high solvent accessibility in this step. In the following period,
the solutes showing greater interaction with the solid matrix were
extracted [21,24].
3.2. Analysis of the volatile fraction by GC–MS
The yield of the more volatile fraction captured in the Porapak-
Q trap during the supercritical extraction of the E. uniflora (dry
base) was 0.46%. Peixoto et al. [25] reported yields to the order
of 0.1% in the extraction of the more volatile fraction captured in
Porapak-Q during extraction with scCO2 (100–300 bar, 50–60 ◦ C).
Melo et al. [26] reported values between 0.5 and 1% in the extraction
of volatile oil from different leaf samples by the steam distilla-
tion method. Galhiane et al. [27] and Peixoto et al. [25] reported
yields of 0.416% and 0.54% using the hydro-distillation method. The
results of the GC–MS analysis presented in Table 2 show the major
compounds present: selina-1,3-7(11)-trien-8-one (23.45%) and
selina-1,3-7(11)-trien-8-epoxide (17.49%) [28]. In the sequence,
the next compounds were germacrene D and trans-Caryophyllene.
According to Weyerstahl et al. [29], this sample of E. uniflora would
correspond to chemotype I, which possesses a predominance of
1002 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006

Table 1
Fitted parameters and average deviation obtained for the models.

Model

Tan and Liou Naik et al. Sovová et al.

Parameters kd (min−1 ) × 10−3 32.30 e∞ (g/g) × 10−3 31.90 e1 (g/g) × 10−3 29.59
b (min) 9.18 k1 (g/g) × 10−2 24.34
k2 (g/g) × 10−4 0.49

AARD (%)a 22.54 0.69 0.61

a
Average absolute relative deviation, AARD = [((mass ext)exp − (mass ext)calc )/n] × 100.

Table 2
Chemical composition of the volatile fraction extract from E. uniflora leaves obtained by GC–MS analysis.

Peak Compound RT (min) RIa RIb ID Relative %

1 ␤-Myrcene 6.63 991 990 NIST-MS 0.71

2 Copaene 21.48 1375 1375 NIST-MS 1.66
3 ␤-Elemene 22.17 1391 1389 MS 1.46
4 Trans-Caryophyllene 23.26 1418 1419 MS 9.63
5 – 23.86 1433 1.01
6 ␣-Humulene 24.60 1451 1452 MS 0.815
7 d-Germacrene 25.74 1480 1485 MS 11.62
8 – 26.35 1495 9.01
9 – 26.67 1503 2.20
10 ␦-Cadinene 27.41 1522 1522 MS 1.10
11 28.68 1556 14.07
12 Spatulenol 29.44 1575 1578 NIST-MS 1.21
13 Selina-1,3,7(11)-trien-8-one 31.50 1631 1634c c
17.49
14 Selina-1,3,7(11)-trien-8-epoxide 36.62 1745 1748c c
23.45
15 – 38.85 1839 2.13
16 – 44.90 2027 1.65

RT: retention time, RI: retention index, ID: identification, (–) not identified. NIST-MS: identification by comparison with NIST mass spectrum. MS: identification by comparison
of mass spectrum and retention times from Adams [15].
a
Calculated retention index.
b
Retention index from Adams [15].
c
Retention index from Costa et al. [28].

these two selina sesquiterpenes. These compounds were also found
in the volatile fraction of supercritical extracts pitanga leaves by
Morais et al. [30] and by Ogunwande et al. [31].
3.3. Extraction yields
Table 3 shows the values obtained for global yield for the three
types of extraction: supercritical (SC), ethanolic (E) and aqueous
(A), in the one- and two-step processes.
The results of the different extraction processes showed that the
characteristics of the solvent influenced the global extraction yield.
The one-step process global yield increased with increasing polar-
ity of the solvent, showing significant differences in the following
decreasing order: aqueous extraction (A), ethanolic extraction (E)
and supercritical extraction (SC).
The two-step process extraction with prior extraction with
scCO2 promoted a significant improvement in the accumulated
global yield for the two steps when the second extraction was done
with water, going from 20 to 25% of global yield, but when the
second extraction was done with ethanol, the accumulated global
yield was practically equal to the global yield obtained with the
one-step extraction with ethanol. This behavior can be explained
by two phenomena. The first is the nature of the solid matrix and
its interactions with the solutes, since many apolar compounds and
others of low polarity are extracted by scCO2 , altering the interac-
tions between the solutes and the solid matrix. These results agree
with other studies published in the literature [32,33], which sug-
gest that a previous extraction of lipophilic components with scCO2
could promote changes in the interactions between the solutes
retained and the residual matrix, which are extracted more eas-

Table 3
Global yield, extraction yield and concentrations of total polyphenolics and total flavonoids in the extracts.

Process Global yielda Total polyphenolics Total flavonoids

X0 (%) Concentration (mg GAE/g) TPC yield (mg/g leaves) Concentration (mg CE/g) TF yield (mg/g leaves)

One-step SC 3.5 ± 0.1 51 ± 7 1.7 ± 0.3 63 ± 1 2.2 ± 0.1

0.47 ± 0.01b
A 20 ± 2 138.9 ± 0.5 28 ± 2 38 ± 1 7.8 ± 0.7
E 8.0 ± 0.1 407 ± 4 32.7 ± 0.7 46.5 ± 0.3 3.73 ± 0.07
Two-step SCA 21 ± 2 291 ± 15 61 ± 3 47.1 ± 0.9 9.9 ± 0.9
SCE 4.2 ± 0.3 504 ± 15 21 ± 2 57 ± 4 2.4 ± 0.4
SC + SCAc 25 ± 2 63 ± 3 12 ± 1
SC + SCEc 8.1 ± 0.4 23 ± 2 4.6 ± 0.5
a
Global yield (%, d.b.).
b
Volatile fraction yield (captured on Porapak-Q). TPC (mg GAE/g), TF (mg CE/g), TPC yield and TF yield represent the extraction yields of TPC and TF (mg/g leaves). The
results are expressed as the mean ± SD.
c
Accumulated global yield (%, d.b.).
 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006
Fig. 4. Microstructures of E. uniflora L. leaves (500×) (a) before and (b) after supercritical extraction.
ily with water than with ethanol, because of its polarity. Another
phenomenon that can alter the matrix is the depressurization after
supercritical extraction, since a large pressure difference can pro-
mote physicals alterations in the matrix structure, improving the
efficiency of aqueous extraction (SCA). Fig. 4 shows images obtained
by scanning electronic microscopy (SEM) of the sample of pitanga
leaves, which visually show evidence of changes in the microstruc-
ture of the solid matrix due to contact with the scCO2. These results
agree with those found by Stamenic et al. [34].
3.4. Total polyphenol (TPC) and Total flavonoid (TF) contents
The TPC contents in the E. uniflora extracts ranged from 51 to
504 mg GAE/g, showing higher TPC concentrations in the ethano-
lic extracts (Table 3). The one-step process is dependent on the
solvent polarity, where the polyphenolic substances were found
in the following decreasing order of concentration: ethanolic
extracts > aqueous extracts > supercritical ones. Fig. 5 presents the
concentration values and extraction yields for the polyphenolic and
flavonoid components.
The two-step process allowed one to obtain more concentrated
aqueous and ethanolic extracts in terms of the polyphenolic com-
pounds than the single step process (SCA > A and SCE > E). The global
extraction yield was higher in the second step, maintaining the
same order of magnitude as the one-step aqueous extraction yield,
so there was a considerable increase in TPC yield in the aqueous
extract after the supercritical extraction (SCA). The TPC yield ranged
from 1.7 to 61 mg per gram of leaves. In both processes – one and
Fig. 5. Polyphenolics and flavonoids—concentrations and extraction yields.
1003
two-steps – the extracts obtained with ethanol (SCE, E) showed a
higher polyphenolic content than the extract obtained in the SC
process. There was a considerable increase in concentration from
407 to 504 mg GAE/g for conventional ethanolic extraction (E) and
ethanolic extraction after supercritical extraction (SCE), respec-
tively, however, due to the drastic reduction in overall yield from 8
to 4.2%, the TPC yield decreased from 33 to 21 mg GAE/g of leaves.
These results agree with the trends observed in the polyphenolic
contents of extracts of both black tea and black mate tea [35].
The total flavonoid contents (TF) ranged from 38 to 63 mg CE/g
(Table 3). For the one-step process, the flavonoid concentration was
highest in the supercritical extracts, followed by the ethanol extract
and finally the aqueous extract. However, when considering the
extraction yield, the result was reversed, the water extraction (A)
being the highest, followed by the ethanol (E) and finally by the
supercritical (SC) extractions. This occurred due to the large differ-
ence in global extraction yields. The TF increased from 47 to 57 mg
CE/g when comparing the one-step ethanolic extract (E) with the
two-step ethanolic process (SCE), although reduced yields, decreas-
ing from 3.73 to 2.4 mg/g leaves were observed.
The prior supercritical CO2 extraction had a positive influence
on the SCA process, producing greater TPC and FT extractions than
in the SCE process (Fig. 5), due to the higher global yield in the
SCA process as compared to the SCE process. Thus, the use of
supercritical extraction followed by aqueous extraction is useful
if the goal is to extract the maximum amounts of polyphenolic and
flavonoid compounds, whereas an ethanolic extraction after the
supercritical extraction is indicated if the goal is to obtain more con-
centrated extracts of the polyphenolic and flavonoid compounds.
These results suggest the presence of polyphenolic compounds
with different polarities in this plant matrix, most of them with
polar characteristics, as indicated by the difference between the SC
and SCE/SCA extracts.
3.5. Antioxidant activity
3.5.1. DPPH assay
The reaction of the free radical 1,1-diphenyl-2-picrilhidrazyl
(DPPH) with antioxidant species inhibits and stabilizes the rad-
ical, promoting color changes from purple to yellow, which, in
turn, decreases the absorbance, which was monitored using a spec-
trophotometer at 517 nm [36]. In this method the antioxidant
activity is presented in terms of EC50 . Fig. 6a shows the activity
of DPPH scavenging as a function of the extract concentration and
standard compounds concentration (Fig. 6b). Table 4 shows the
values for the calculated EC50s.
1004 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006

Fig. 6. DPPH scavenging activity of E. unifora L. extracts (a) and of some standard compounds (b).

Table 4
Calculated values for EC50 (␮g/mL) for the extracts of E. uniflora and for some standard compounds.

Extract SC A E SCE SCA

>200 15 ± 3 6.2 ± 0.3 5.2 ± 0.1 15 ± 2

Standard Catechin 5.40 ± 0.02, galic acid 1.87, ascorbic acid 10.9 ± 0.6

All the extracts studied, with the exception of SC, exhibited
scavenging activity of the DPPH• radical (in terms of EC50 ), pre-
senting high activity (Fig. 6a). It is worth mentioning that the high
activity of the aqueous extracts (A, SCA), with EC50 values around
15 ␮g/mL, and of the ethanolic extracts (E, SCE), with EC50 val-
ues of 6 and 5 ␮g/mL for the one-step and two-step extraction
processes, respectively, are close to the EC50 value of pure cate-
chin (5.40 ± 0.02 ␮g/mL) and better than that of pure ascorbic acid
(10.9 ± 0.6 ␮g/mL). Such EC50 values agree with the values reported
in other studies [37].
The effect of the solvents in the one-step scheme on the antiox-
idant activity of the extracts, showed a tendency to increase the
antioxidant capacity (low value for EC50 ) when using polar solvents
(water and ethanol). The high value for the EC50 of the SC extract
(>200 ␮g/mL) was due to the non-polar extract obtained with SC-
CO2 [38,39]. A prior extraction with scCO2 favored the production
of extracts with greater antioxidant activity when using ethanol in
the 2nd stage of extraction.
The antioxidant activity of the E. uniflora extracts could be
attributed to the presence of different compounds with antioxi-
dant properties. The correspondence between the total phenol and
antioxidant activity (EC50 values) was low (R2 = 0.49), this behavior
agreeing with that reported in other studies [40,41]. This fact can
be explained if one considers these natural extracts as a complex
mixture of phenolic acids, flavonoids and polyphenolics, produc-
ing a response for antioxidant activity linked to possible synergistic
effects amongst these different compounds [42,43] or the presence
of oxygenated substances that act as pro-oxidants [44].
in terms of the percent inhibition at 120 min (AA120), calculated
using Eq. (2), and the results are shown in Table 5.
The ethanolic extracts (E, SCE) obtained from E. uniflora pre-
sented high antioxidant activity, followed by the supercritical
extract (SC), with behavior similar to that of quercetin solutions and
BHT. The aqueous extracts (A, SCA) presented lower activity when
compared with the standards. In addition it was observed that the
antioxidant activity decreased quickly with time. In this assay, the
polar antioxidants remained in the aqueous phase, and were there-
fore less effective in protecting the lipids, a phenomenon known as
“polar paradox” [45,46]. This phenomenon can explain the results
obtained for ascorbic acid, which is a polar antioxidant but nev-
ertheless did not present antioxidant activity in this assay (Fig. 7).
The SC extract presented better antioxidant activity in the DBC test
than in the DPPH test, a behavior typical of phenolic compounds
[47].
It was observed that the ethanolic extracts presented high
AA120 values of 46.3% and 34.5% for the E and SCE extracts, respec-
tively, closely matching the activity of commercial antioxidant
solutions (BHT, AA120: 46 5%), followed by the supercritical extract

3.5.2. ˇ-Carotene bleaching assay

␤-Carotene (DBC) bleaching is induced by the presence of the
peroxyl radical formed from the oxidative degradation of linoleic
acid, reducing the amount of ␤-carotene in the solution and there-
fore the absorbance [36]. Fig. 7 shows the results of the DBC
test for each extract during 180 min. The results are presented as
the absorbance differential (abs), calculated by subtracting the
absorbance of the control from that of the corresponding sample,
the value for (abs) being directly proportional to the antioxidant
activity of the sample. For a better comparison of the antioxidant
activities of the extracts, in this assay the results were expressed Fig. 7. Antioxidant activity by the ␤-carotene bleaching method (DBC).
 H.A. Martinez-Correa et al. / J. of Supercritical Fluids 55 (2011) 998–1006
Table 5
Antioxidant activity (%) for the different extracts determined by the DBC method (t = 120 min) AA120 .
Plant SC A E
E. uniflora 23 ± 9 10.2 ± 0.5
Standards BHT 46 ± 5, quercetin 57 ± 9, ascorbic acid 6.3 ± 0.8
(SC), the aqueous extract (A) and the supercritical aqueous extract
(SCA) with 23 ± 9, 10.2 ± 0.5 and 5 ± 2%, respectively. It is worth
mentioning that in this test the supercritical extract presented
antioxidant activity, whereas it did not in the previous test with
DPPH.
In both methodologies for the evaluation of antioxidant activ-
ity, the ethanolic extracts (E and SCE) presented high responses.
However it should be stressed that the compounds responsible for
the antioxidant activity in each of the methods are not necessar-
ily the same ones, since the extracts are mixtures of compounds
that may present either antagonistic or synergist interactions. The
non-polarity of the extracts obtained in the supercritical processes
can explain the differences obtained in the DPPH test and in the
DBC test. The DBC activity test may be dependent on the polarity
of the extract, because it is a emulsified substrate. Thus, non-polar
extracts may have high antioxidant activity in emulsified systems,
because they are concentrated at the interface water/lipid, protect-
ing the emulsion and inhibiting the oxidation of ␤-carotene [46].
The low capacity scavenging activity of the DPPH radical of the SC
extract, can be explained by structural factors such as location and
number of hydroxyl groups in the structure of phenolic compounds
[48,49].
4. Conclusions
This study showed that both the one-step and two-step extrac-
tion processes allowed for the recovery of extracts from E. uniflora
with different compositions and functionality. For the one-step
and two-step processes, the recovery of the TPC and TF depended
mainly on the nature of the solvent and on its polarity and on dif-
ferences in the solid matrix. A prior supercritical CO2 extraction
had a positive influence on the SCE process, producing high TPC
and FT contents. The use of supercritical extraction (first stage) fol-
lowed by aqueous or ethanolic extraction (second stage) depended
on the objective of the process: maximum extraction of TPC and TF
or more concentrated extracts of these substances. In addition, the
volatile fractions are likely to be captured in the Porapak-Q trap
during the first extraction. Antioxidant activity analyses showed
that the processes involving ethanol as the solvent allowed one to
obtain extracts with high antioxidant activity.
Acknowledgments
The authors wish to thank Fapesp for their financial support and
CAPES for the scholarship awarded to Hugo Alexander Martinez-
Correa.
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