Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21

Contents lists available at ScienceDirect

Food and Bioproducts Processing

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

Recovery of phenolic compounds from citrus

by-products using pressurized liquids — An
application to orange peel

Francisco Manuel Barrales a , Paula Silveira a ,

Paula de Paula Menezes Barbosa b , Amanda Roggia Ruviaro c ,
Bruno Nicolau Paulino d , Glaucia Maria Pastore b ,
Gabriela Alves Macedo c , Julian Martinez a,∗
a College of Food Engineering, Food Engineering Department, University of Campinas, UNICAMP, 13083-862,
Campinas, SP, Brazil
b College of Food Engineering, Food Science Department, University of Campinas, UNICAMP, 13083-862, Campinas,

SP, Brazil
c College of Food Engineering, Food and Nutrition Department, University of Campinas, UNICAMP, 13083-862,

Campinas, SP, Brazil

d Faculty of Pharmaceutical Sciences, Federal University of Amazonas, 69077-000, Manaus, AM, Brazil

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

Article history: Citrus industry residues are sources of phenolic compounds, which may be extracted by
Received 13 December 2017 pressurized liquid extraction (PLE) and volatile compounds, extractable by supercritical fluid
Received in revised form 25 July 2018 extraction (SFE). PLE and SFE are fast and allow using non-toxic solvents and moderate
Accepted 9 August 2018 temperatures. Therefore, the objective of this work was to extract volatile and phenolic
Available online 18 August 2018 compounds from orange peel by SFE and PLE. The raw material was orange peel with-
out (Lot 1) and with (Lot 2) previous supercritical CO2 extraction performed at 40 ◦ C and
Keywords: 35 MPa. The volatile profile was evaluated in the SFE extract by HS-SPME-CG-MS. PLE sol-
Industrial by-products vents were absolute ethanol and mixtures of ethanol and water (75% and 50% ethanol, v/v)
Orange peel at the temperatures 45, 55, and 65 ◦ C and pressure of 10 MPa. Global yield, total phenolic
Phenolic compounds content (TPC), antioxidant capacity by DPPH and FRAP methods, total reducing sugars and
Flavonoids the concentration of the major phenolic compounds by HPLC were evaluated in the extracts.
Antioxidant capacity Temperature and ethanol concentration had significant effects on all responses. The main
Extraction kinetics volatile compound found in the SFE extract was ␣-Terpineol, followed by d-Limonene. The
major phenolic compound was hesperidin, which highest recovery (19.3 ± 0.9 mg/g dry peel)
was achieved with 75% ethanol at 65 ◦ C from Lot 1. At the same condition, high TPC and
antioxidant capacity were also achieved. The three-line spline and two-site kinetic mod-
els provided good adjustments to the PLE curves, being able to describe their behavior. PLE
using water and ethanol can be applied to recover phenolics from a large variety of fruit
by-products.
© 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

∗
Corresponding author.
E-mail address: julian@unicamp.br (J. Martinez).
https://doi.org/10.1016/j.fbp.2018.08.006
0960-3085/© 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
10 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21
1. Introduction
Brazil is the third world’s fruit producer and the first in citrus fruits.
Among the citrus, oranges are the most important. 45% of the total
orange crop in Brazil is destined to produce juice and essential
oil. These processes generate about 17 million ton/year of bagasse
(FAOSTAT, 2015). The citrus bagasse is mostly composed by peel, which
is usually recovered for pellets production for animal feed. However,
the citrus bagasse still contains some residual essential oil (Kimball,
1999) and phenolic compounds with several biological activities (Devi
et al., 2015; Lee et al., 2012; Yumnam et al., 2014).
Among the phenolic compounds found in citrus, flavonoids and
hydroxybenzoic acids (gallic and ellagic acids) have received special
attention. Flavonoids are attractive because of their beneficial effects
on human health, which include action against colon (Bartoszewski
et al., 2014; Park et al., 2008), breast (Bailón-Moscoso et al., 2016),
lung (Hsu et al., 2012; Lee et al., 2012) and liver (Yumnam et al.,
2014) cancers, among others. On the other hand, several biological
activities have also been attributed to hydroxybenzoic acids, such
as antioxidant effect (Balasundram et al., 2006), inhibition of human
immunodeficiency viruses (Nutan et al., 2017) and anti-inflammatory
effect (Karimi-Khouzani et al., 2017).
Phenolic compounds are currently produced by chemical synthesis
or extracted from natural sources. The usually applied processes are
solid–liquid extractions using organic solvents in reflux systems at high
temperatures. However, novel methods to extract phenolic compounds
from solid substrates have been investigated, such as ultrasound-
assisted extraction, microwave-assisted extraction, supercritical fluid
and pressurized fluid extractions (Martins et al., 2011). Supercritical
fluid extraction (SFE) and pressurized liquid extraction (PLE) are green
methods that allow using environmentally friendly solvents, like CO2 ,
ethanol, and water. However, when CO2 is used as solvent the abil-
ity to extract polar compounds is weak, thus requiring the addition of
another solvent to modify the polarity (Benelli et al., 2010; Espinosa-
Pardo et al., 2017; Pasquel-Reátegui et al., 2014). In this sense, PLE has
emerged as a powerful method to recover polar molecules from fruits
and their by-products, such as phenolic compounds, using ethanol and
water as solvents (García-Mendoza et al., 2017; Machado et al., 2017,
2015; Pereira et al., 2016; Viganó et al., 2016b).
Given this context, the present work aimed to extract volatile and
phenolic compounds from orange peel by SFE, using CO2 , and by PLE,
using ethanol and water as solvents at different temperatures, and
compare its efficiency to hot reflux extraction (Soxhlet) and ultrasound
assisted extraction.
2. Material and methods
The raw material used in this work was orange peel donated by
an orange juice industry located in Matão – SP, southeastern
Brazil. This orange peel is usually discarded by the industry
after the processing of juice and essential oil.
2.1. Pre-treatment of orange peel
Lot 1 was produced by drying the orange peel in an air circu-
lation oven (model 320 SE, Fanem, SP – Brazil) at 50 ◦ C for 24 h,
and then milling it in a knife mill (model MA 340, Marconi, SP
– Brazil). Part of Lot 1 was extracted with supercritical CO2 to
remove the essential oil remaining in the orange peel, produc-
ing a defatted orange peel (Lot 2). Lots 1 and 2 were stored at
−18 ◦ C in hermetic flasks and protected from light until the
extractions.
2.1.1. Characterization of the orange peel
The proximate composition of the orange peel was analyzed
to characterize the material used in this work. The moisture
of the orange peel was determined before and after drying,
and before (Lot 1) and after supercritical CO2 extraction (Lot
2) (method 934.06). Crude protein was quantified by the semi-
micro-Kjeldahl procedure (method 970.22), ash by incineration
at 550 ◦ C (method 942.05), and the total lipids were determined
by Soxhlet extraction using petroleum ether (method 963.15)
for both lots (AOAC, 1997). The analyses mentioned in this
section were performed in triplicate.
2.1.2. Supercritical fluid extraction (SFE)
Supercritical CO2 extraction was performed in a laboratory
scale SFE unit described by Viganó et al. (2016a), in order to
remove the essential oil that remained in the orange peel after
the industrial processing. For the extraction, a 50 ml column
was filled with the orange peel from Lot 1. CO2 (White Mar-
tins, Campinas, Brazil) with 99.0% purity was used as solvent
at 40 ◦ C and 35 MPa. The solvent to feed (S/F) ratio was 32 kg
CO2 /kg dry peel. Each extraction was performed with 30 g of
the material from Lot 1, and glass wool was used as filtering
material. A stabilization time of 20 min was used to ensure
temperature homogeneity inside the extraction column. The
extract was collected in a glass flask after decompression in
the micrometer valve. The defatted orange peel after SFE (Lot
2) was transferred to a hermetic flask, stored in the dark at
−18 ◦ C until being subjected to PLE.
The global extraction yield for SFE (X0 ) was calculated
according to Eq. (1)
mextract
X0 = × 100 (1)
F
where: mextract = extract mass (kg); F = mass of dry peel (kg).
2.1.3. Volatile organic compounds profile
The volatile organic compounds (VOCs) profile of the SFE
extract was characterized by headspace solid-phase microex-
traction combined with gas chromatography coupled to mass
spectrometry (HS-SPME-GC–MS), performed at the Labora-
tory of Bioflavors (DCA-FEA-UNICAMP, Campinas, Brazil). This
methodology used a manual holder and SPME fibers pur-
chased from Supelco (Bellafonte, PA, USA).
2.1.3.1. Extraction of volatile organic compounds.
Three different fiber coatings for SPME analysis,
Carboxen/Polydimethylsiloxane (CAR/PDMS, 75 ␮m),
Polydimethylsiloxane (PDMS, 100 ␮m) and Polydimethyl-
siloxane/Divinylbenzene (PDMS/DVB, 50/30 ␮m), and two
volatilization temperatures (45 and 65 ◦ C) were evaluated for
optimum extraction of VOCs present in the sample. Before
use, all fibers were conditioned at 250 ◦ C for 30 min in CG-–MS
injector to avoid possible contamination. The SFE extract
was dissolved in ethyl acetate (J.T. Baker, EEUU) in a concen-
tration of 10 mg/mL, and an aliquot of 100 ␮L was placed in
a 100 mL vial, sealed with a screw-capped top containing a
Teflon-lined septum. The sealed system was incubated at the
corresponding temperature (45 or 65 ◦ C), for an equilibrium
time of 15 min, and then the fiber was exposed inside the vial
for 15 min at constant temperature for extraction of VOCs.
2.1.3.2. GC–MS analysis. After the extraction step, the man-
ual holder containing the SPME fiber was inserted into
the GC injector port at 250 ◦ C for 10 min for desorption
of VOCs. GC–MS analyses were performed in an Agilent
7890A (Agilent Technologies) gas chromatograph system cou-
pled to an Agilent 5975C triple-axis mass detector (Agilent
 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21
Fig. 1 – Flowchart of the pressurized liquid extraction (PLE) unit; V-1, V-2 and V-3—blocking valves; SV—safety valve;
MV—micrometer valve; P—HPLC pump; HB—heating bath; I-1—pressure indicator; I-2—temperature indicator;
EC—extraction column (10 ml).
Technologies). Compounds were separated in a DB-Wax col-
umn (30 m × 0.25 mm × 0.25 ␮m). Helium was the carrier gas
at a flow rate of 1.0 mL/min. The adopted temperature pro-
gram was: initial temperature 40 ◦ C (2 min hold time), then
ramped at 10 ◦ C/min to 60 ◦ C (2 min hold time), then ramped
at 2 ◦ C/min to 190 ◦ C and then ramped at 5 ◦ C/min to 230 ◦ C
(hold time: 15 min). The injector temperature was set at 250 ◦ C
in splitless mode. Ion source temperature was 230 ◦ C, and the
interface temperature was set at 230 ◦ C. The MS was scanned
in the range of 45–650 atomic mass units at 70 eV. Compounds
were identified using NIST 14.0 database and Linear Retention
Index (LRI) calculated with a series of n-alkanes (C7 –C40 ).
2.2. Pressurized liquid extraction (PLE)
PLE was carried out to obtain polar bioactive compounds from
the orange peel, according to the method described by Viganó
et al. (2016b). A laboratory scale PLE unit, schematized in the
flow diagram of Fig. 1, was used for the experiments.
2.2.1. Extraction conditions
The influence of temperature and solvent composition on the
global extraction yield, total phenolic compounds, antioxidant
capacity and major phenolics quantification was evaluated.
In order to choose the solvent to be used in PLE, the char-
acteristics of the target compounds and those of the solid
matrix must be considered. The target compounds are pheno-
lics from orange peel, which consist of glycosylated flavonoids
and hydroxybenzoic acids, polar substances with affinity to
water, ethanol and their mixtures (Espinosa-Pardo et al., 2017;
Madeira et al., 2014; Nakajima et al., 2016). On the other hand,
the orange peel contains a great amount of pectin. When
mixed with water at high temperature, pectin may form a
gel that clogs the equipment’s pipes, interrupting the extrac-
tion process. Thus, pure water is not recommended as solvent
for substrates with high pectin content. A preliminary exper-
iment was performed in test tubes, mixing 2 g of orange peel
(Lot 1) with 10 ml of mixtures of ethanol/water, at concentra-
tions of 0% (deionized water), 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, and 99,5% (absolute ethanol), simulating the
solvent/feed (S/F) ratio of the PLE process. The tubes were
incubated at 65 ◦ C for 40 min. After this period, the possible
formation of solid gel was verified in each tube. Ethanol con-
11
centrations of 50% and above did not present gel formation.
Therefore, 50% and 99,5% were adopted as the limits of ethanol
concentration in the solvent. On the other hand, high tem-
peratures may promote degradation of phenolic compounds.
Therefore, the maximum temperature used in this work was
65 ◦ C.
Thus, the PLE solvents were mixtures of ethanol and deion-
ized water with different concentrations and temperatures, in
a Box-Behnken experimental design, as shown in Table 3.
2.2.2. Experimental procedure
A 10 ml stainless steel extraction cell was used for the PLE
experiments. Each extraction was performed using 2 g of
orange peels from Lots 1 or 2 at 10 MPa at the correspond-
ing solvent and temperature, for 40 min, with a flow rate of
2.37 g/min, resulting in a S/F ratio of 47 kg solvent/kg sample,
in dynamic operation. After the extraction, the mixture of sol-
vent and extract was decompressed in a micrometer valve and
collected in dark flasks with hermetical closure. The final vol-
ume was measured, the extracts were stored at −18 ◦ C and
protected from light for further analyses.
2.2.3. PLE kinetics and modeling
The PLE kinetics was investigated by performing experiments
as described in Section 2.2.2, but collecting extract fractions at
determined times (5, 10, 15, 20, 30 and 40 min). The PLE kinetics
were performed at 65 ◦ C for the three solvents used in this
work (50%, 75%, and 99,5% ethanol). After the extraction, the
volume of each fraction was measured and the global yield and
phenolic content were determined, as described in Sections
2.4 and 2.6. The extractions were performed in duplicates and
the extracts analyzed in triplicate. Two mathematical models
were adjusted to the experimental data, as described in the
following sections.
2.2.3.1. Spline model. Three stages can be identified along the
PLE kinetics. First, the CER stage (constant extraction rate),
where convection is the main extraction mechanism; next,
the FER stage (falling extraction rate), where both convection
and intraparticle diffusion are important; and finally the DC
(diffusion-controlled) stage, in which only diffusion controls
the extraction. The identification of these stages in the PLE
curve is important to define the extraction time, since about
12 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21
70–90% of the extractable material is usually recovered until
the end of the CER period. Interrupting the process at the CER
time allows reducing the total extraction time, and therefore
the solvent consumption (Meireles, 2008).
The identification of the three stages of the PLE kinetics was
performed through the Spline model, described by Meireles
(2008), by fitting three straight lines to the experimental data.
Each straight line represents an extraction stage. The three
lines Spline model is presented in Eqs. (2)–(4).
y = b0 + b1 t for t ≤ tCER
y = b0 − tCER b2 + (b1 + b2 ) t for tCER < t ≤ tFER
y = b0 − tCER b2 − tFER b3 + (b1 + b2 + b3 ) t for tFER < t
where: y: response variable; bi (for i = 0, 1–3): linear coefficients
of the lines; t: time; tCER : CER period time; tFER : FER period time.
The three-line Spline model was fitted to the experimen-
tal data using the PROC NLIN procedure in the software SAS
University Edition, as described by Rodrigues et al. (2003).
From the adjusted parameters (bo ; b1 –b3 ; tCER ; tFER ), the
global yield and TPC at tCER and tFER were calculated using Eq.
(4) (yCER and yFER ). Next, Eq. (5) was used to calculate how much
yCER and yFER represent in terms of y at 40 min of extraction
time, which was called recovery.
Recovery (%) = yt ∗100⁄y40
where yt represents the response (global yield or TPC) at the
time tCER or tFER .
2.2.3.2. Two-site kinetic desorption model. The PLE kinetics
was fitted with the empirical two-site kinetic desorption
model described by Kubatova et al. (2002). This model con-
siders that a fraction of the extractable compounds is near
the particle surface, being easily accessible to the solvent, and
thus desorbed at a fast rate (washing process). The remaining
fraction of extractable compounds is located inside the parti-
cles of the vegetable matrix, so it is hardly accessible, being
desorbed at a slower rate (slow diffusion). Eq. (6) describes the
model.
−k1 t −k2 t
yt = y∞ ∗ [1 − f ∗ e − (1 − f ) ∗ e ] (6)
where yt is the response variable at the time t, y∞ is the
response variable at saturation; f is the fraction of extractable
material of easy access, t is the extraction time, k1 is the
first-order rate constant describing the desorption of the easy
access material, and k2 is the first-order rate constant describ-
ing the slow diffusion. The constants k1 and k2, as well as f and
y∞, are the model’s adjustable parameters.
This model was fitted to the experimental data using the
Microsoft EXCEL Solver, minimizing the mean relative per-
cent deviation (MRPD), which was calculated using Eq. (7) as
described by Dias et al. (2017).
100  yi,exp − yi,cal
n
MRPD (%) = | |
n yi,exp
i=1
where n is the number of experimental data of the kinetics,
yi,exp is the experimental data i, and yi,cal is the predicted data
i.
2.3. Low-pressure extractions
Low-pressure extractions were applied to be compared to PLE.
The performed methods were Soxhlet and ultrasound assisted
extraction.
2.3.1. Soxhlet
In a Soxhlet apparatus, 5 g of Lot 1 were extracted in trip-
licate for six hours using absolute ethanol as solvent. After
extraction, the solvent was removed on a rotatory evaporator
(2) under vacuum at 40 ◦ C, to determine the extracted solid mass.
The extract was then dissolved in 100 ml of ethanol, stored at
(3) −18 ◦ C and protected from light in a hermetic flask for further
analyses.
(4)
2.3.2. Ultrasound-assisted extraction (UAE)
Both lots of orange bagasse were submitted to UAE with
ethanol 50% (v/v), according to the method described by
Nakajima et al. (2016). Briefly, 2 g of bagasse (Lot 1 or 2) were
mixed with 113 ml of the solvent (ethanol 50% (v/v)), to achieve
the same S/F ratio used in PLE, and treated in an ultrasonic
bath at 30 ◦ C for 15 min. Next, the samples were transferred to
a shaker at 200 rpm for 15 min. A Whatman N◦ 1 filter was used
to remove the solids, and the extract was taken to evaporation
under vacuum at 40 ◦ C to remove ethanol. The extract was
stored in hermetic flasks and protected from light at −18 ◦ C.
(5) 2.4. Global yield (X0 )
The global yield accounts all the extracted solids. For each
experiment, a 10 ml aliquot of each extract was dried in an
air convection oven at 60 ◦ C until constant weight. Next, the
global yield (X0 ) was calculated according to Eq. (8), as the ratio
between the total solid extract mass (msolids ), corrected to the
total extraction volume (vextract ), and the sample mass (F) in
dry basis.
msolids × vextract ⁄10ml
X0 = × 100 (8)
F
2.5. Total reducing sugar content (TRS)
The orange peel is composed mainly of carbohydrates that
may be present in the forms of cellulose, fiber, and sugars.
As sugars are soluble in water, when water and its mixtures
with ethanol are used to extract phenolic compounds, sug-
ars are the main contaminant in the extracts. Therefore, it is
important to evaluate the sugar amount extracted with those
solvents to decide which extraction technique leads to the best
product. The total reducing sugars (TRS) were determined only
in the extracts obtained from Lot 1, since sugars are insoluble
in supercritical CO2 (Brunner, 2005).
The TRS content in the extracts was determined by the
dinitrosalicylic acid method (DNS), described by Miller (1959).
First, the DNS reagent was prepared by mixing 1.4133 g of
DNS with 2.64 g of NaOH, 1.1067 g of sodium metabisulfite,
and 1.01 ml of phenol melted at 50 ◦ C, in 200 ml of deion-
ized water. Next, a potassium sodium tartrate solution was
(7) prepared, diluting 15.1 g of potassium sodium tartrate tetrahy-
drate in 1 l of deionized water. The extracts’ concentration
was adjusted to 1 mg solids/ml through dilution in deion-
ized water. Dilutions of glucose with concentrations from 0.01
to 0.1 mg/ml were prepared as calibration standards. Next,
the reaction was performed in test tubes, mixing 1 ml of the
 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21
extracts, deionized water (blank) or the calibration curve dilu-
tions with 1 ml of the DNS reagent. The tubes were stirred for
10 s and heated for 5 min at 100 ◦ C in boiling water. After that,
the tubes were refrigerated for 5 min in an ice bath. Next, 16 ml
of the tartrate solution were added to each tube and stirred.
Finally, the absorbance was read at a wavelength of 540 nm in
a UV–vis spectrophotometer (Hach, DR/4000U, Colorado, USA).
The absorbance of the samples was converted into TRS using a
calibration curve plotted from the concentrations of the stan-
dards and their absorbances. TRS values were expressed in mg
Glucose/g dried peel (DP).
2.6. Total phenolic content
The total phenolic content (TPC) of the extracts was deter-
mined by the Folin–Ciocalteu method described by Singleton
et al. (1999), with modifications. First, dilutions of gallic acid
with concentrations from 0.01 to 0.075 mg/ml were prepared
as calibration standards. Next, the reaction was performed
in test tubes, mixing 0.5 ml of the extracts, deionized water
(blank) or the calibration curve dilutions, and 0.5 ml of
Folin–Ciocalteu reagent (Dinâmica, São Paulo, Brazil). After
3 min, 0.5 ml of sodium bicarbonate (Synth, São Paulo, Brazil)
and 3.5 ml of deionized water were added. The tubes were
stirred for 10 s and let to rest in the dark for two hours at room
temperature. Finally, the absorbance was read at a wavelength
of 760 nm in a UV–vis spectrophotometer (Hach, DR/4000U,
Colorado, USA). The TPC was calculated as gallic acid equiva-
lent (GAE)/g dried peel (DP).
2.7. Antioxidant capacity
2.7.1. DPPH
The in vitro antioxidant capacity was determined by the DPPH
(2,2-Diphenyl-1-picrylhydrazyl) method (Rufino et al., 2006)
with modifications. Solutions of the extracts in ethanol were
prepared, with concentrations of 1 mg/ml: a DPPH solution
(60 ␮M) in ethanol and Trolox solutions in ethanol (50, 100, 200,
400, 600, 800, 1000 and 1200 ␮M). In the dark, a 0.1 ml aliquot
of the sample solution, ethanol (control) or the Trolox solution
(to plot the standard curve) was added into a test tube, then
3.9 ml of the DPPH solution was added and the mixture was
stirred for 10 s. The mixtures were let reacting in the dark at
room temperature for 40 min. Next, the absorbance of the mix-
ture was determined at 515 nm in a UV–vis spectrophotometer
(Hach, DR/4000U, Colorado, USA), using ethanol as blank. The
antioxidant capacities of the extracts were expressed using
the values of the standard curve absorbance, as equivalent
units of Trolox (mg TE/g DP).
2.7.2. FRAP
The ferric ion reduction capacity of the extracts was evaluated
using the method proposed by Benzie and Strain (1996), with
modifications. Solutions of 15 mg solids/ml of the extracts
were prepared using deionized water. Next, Trolox (Sigma-
Aldrich, São Paulo, Brazil) solutions from 50 to 1200 ␮M of were
prepared using deionized water to plot the standard curve. The
FRAP reagent was obtained immediately before the analysis,
by mixing a 10 mM solution of TPTZ (2,4,6-tris(2-pyridyl)-1,3,5-
triazine) (Sigma-Aldrich, São Paulo, Brazil) dissolved in 40 mM
of HCl (Synth, São Paulo, Brazil), with sodium acetate buffer
0.3 M (Synth, São Paulo, Brazil) and a 20 mM FeCl3 ·6H2 O solu-
tion (Dinâmica, São Paulo, Brazil), at the proportion 1/10/1
(v/v/v). The reactions were performed transferring 90 ␮l of the
13
samples, standard curve solution or deionized water (Blank) to
270 ␮l of deionized water and 2.7 ml of the FRAP reagent. After
stirring for 10 s, the tubes were kept in the dark for 30 min
in a thermostatic bath at 37 ◦ C. Finally, the absorbance of the
mixture was determined at 595 nm in a UV–vis spectropho-
tometer (Hach, DR/4000U, Colorado, USA). The antioxidant
capacity of the extracts was expressed as equivalent units of
Trolox (mg TE/g DP), using the values of the standard curve
absorbance.
2.8. Identification and quantification of phenolic
compounds
The identification and quantification of the phenolic com-
pounds in the extracts were performed by high-performance
liquid chromatography (HPLC) at the Bioprocesses Laboratory
(LB-DEPAN-FEA-UNICAMP, Campinas, Brazil). In preparation
for chromatography, ethanol was removed from the extracts
by evaporation under vacuum at 40 ◦ C. The extracts were
properly diluted in methanol (J.T. Baker, EEUU) 70% (v/v) and
then filtered using a Millex filter (HV PVDF, 0.45 nm, Millipore,
Massachusetts, EEUU). The filtered extracts were injected
into a Dionex-Ultimate 3000 chromatograph equipped with a
reverse phase column C18 (150 nm × 4.6 mm i.d., Waters, Mas-
sachusetts, EEUU), coupled to a diode array detector, at 210,
260, 280 and 330 nm, according to the method described by
Madeira et al. (2014). The eluents were water (0.1% formic
acid) (A) and methanol (0.1% formic acid) (B). The elution was
0.6 ml/min, at 30 ◦ C, and the gradient was as follows: 0 min,
90% A; 5 min, 90% A; 80 min, 20% A; 85 min, 90% A. The data
processing was performed using the Chromeleon software
(version 6.8). The compound identification was based on their
retention times and UV spectra (at 280 nm). Standards solu-
tions of hesperidin, naringin, gallic acid, ellagic acid, narirutin,
tangeritin, naringenin, hesperetin and diosmetin were used
to plot the standard curves and to quantify the flavonoids
and hydroxybenzoic acids, using the diode arrange detector
at 280 nm.
3. Results and discussion
3.1. Characterization of the orange peel and SFE
The characterization of the orange peel was performed in
terms of proximate composition and particle characterization,
as shown in Table 1.
The SFE yield was 0.48 ± 0.02%, significantly lower than that
obtained by Benelli et al. (2010) for orange peel (1.37 ± 0.09%),
although obtained at different conditions (50 ◦ C and 30 MPa).
Higher temperature increases the vapor pressure of the
volatile compounds, such as the terpenes present in the essen-
tial oil. Nonetheless, Benelli’s work used an S/F ratio of 340 kg
CO2 /kg sample, much higher than that of the present work
(32 kg CO2 /kg sample), which could explain the discrepancy
in global yield. Furthermore, differences in the raw material,
such as orange subspecies, could account for this behavior, as
well as the drying processes.
The VOCs profile of the extract is presented in Table 2.
CAR/PDMS fiber extracted greater percentage, approximately
99% of the Key Volatile Compounds (KVC) at 45 ◦ C, while
pdms and pdms/dvb coatings extracted around 97% of
KVC in the same conditions. In contrast, the extraction
at 65 ◦ C was less efficient, recovering around 88% of the
KVC of the extract. Thus, the CAR/PDMS coating was
14 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21

Table 1 – Proximate composition of the orange peel.

Analysis Value Unit

Moisture (fresh sample) 0.875 ± 0.006 kg/kg fresh peel

Analysis Value Unit

Lot 1 Lot 2

Moisture 0.048 ± 0.003 0.037 ± 0.005 kg/kg dry peel

Protein 0.0753 ± 0.0001 0.0765 ± 0.0001 kg/kg dry peel
Total lipid content 0.0097 ± 0.0008 0.0049 ± 0.0002 kg/kg dry peel
Ash 0.0390 ± 0.0003 0.0396 ± 0.0003 kg/kg dry peel
Carbohydrate 0.828 0.842 kg/kg dry peel

Values expressed as mean ± standard deviation.

Table 2 – Volatile compounds profile of the SFE extract: Influence of the type of HS-SPME fiber coatings on the extraction
efficiency of the volatile compounds.
SPME fiber coating CAR/PDMS PDMS PDMS/DVB LRI-exp LRI-ref LRI reference
Temperature 45 ◦ C 65 ◦ C 45 ◦ C 65 ◦ C 45 ◦ C 65 ◦ C
Compound

␣-Terpineol 76.20 71.00 67.32 66.88 71.79 70.61 1705 1704 Pozo-Bayón et al. (2007)
d-Limonene 18.80 9.52 25.70 13.34 22.31 9.93 1189 1192 Lopes et al. (2004)
␣-Copaene 0.87 1.27 0.64 1.04 0.71 0.76 1478 1475 Lopes et al. (2004)
Caryophyllene 0.82 1.45 0.67 0.76 0.86 0.61 1579 1576 Lopes et al. (2004)
␤-Cubebene 0.70 1.45 0.36 1.83 0.98 1.43 1575 1560 Stashenko et al. (1995)
Eremophilene 0.50 1.92 0.49 1.70 0.46 1.57 1713 1732 Guo et al. (2008)
Linalool 0.43 0.19 0.51 0.45 0.17 0.44 1551 1552 Lopes et al. (2004)
␤-Cadinene 0.42 1.71 0.60 2.32 n.d 2.21 1752 1769 Miyazawa et al. (2016)
Total of KVC 98.74 88.51 97.54 88.32 97.29 87.56
Minor compounds 1.26 11.49 2.46 11.68 2.71 12.44
Total 100 100 100 100 100 100

KVC: Key Volatile Compounds; PDMS: polydimethylsiloxane; PDMS/CAR: polydimethylsiloxane/carboxen; PDMS/DVB: polydimethylsilox-
ane/divinylbenzene. LRI-exp: experimental Linear Retention Index, calculated for the black fiber at 45 ◦ C; LRI-ref: reference for Linear Retention
Index. n.d.: not detected.
The bold values indicates the condition chosen to represent the volatile composition of the extract.

selected for characterization of the major volatile components
present in the SFE extract. These results are highlighted in
Table 2.
The volatile profile obtained in this work is different
from those found for fresh orange peel oil by other authors
(Badee et al., 2011; Mira et al., 1999; Razzaghi et al., 2019).
The major compound found in this work was ␣-Terpineol,
followed by d-Limonene, while the major component in
orange peel oil is usually d-Limonene. The previous pro-
cesses, cold press for juice and oil extraction, and drying
might have favored the loss of volatile compounds by evap-
oration along with water. As the ␣-Terpineol boiling point is
higher than that of d-Limonene (219 and 176 ◦ C respectively),
the evaporation rate of d-Limonene is higher, decreasing its
concentration in the peel. A similar change in the volatile
compounds extracted from orange pomace was presented
by Benelli et al. (2010). These results valorize even more
the extracted oil, since ␣-Terpineol is a stable monoter-
pene alcohol, widely used in the fragrance industry and has
greater market value than d-Limonene, which is a less stable
monoterpene.
Although 0.48% of essential oil recovered seems to be a
small percentage, the orange industry scale must be consid-
ered. Brazilian orange processing industries discard 17 million
tons/year of orange peel, which represents a potential recovery
of 16,000 tons of essential oil per year. Therefore, this pro-
cess must be considered for further evaluations of economic
feasibility.
3.2. Global yield and total reducing sugars
The global yields obtained for PLE, UAE, and Soxhlet are pre-
sented in Table 3. There are significant differences among the
extraction methods performed in this work. PLE achieved the
highest global yields, which might be explained by the reduc-
tion of the liquid’s surface tension due to the high pressure
applied. High pressure improves the penetration of the solvent
inside the solid pores, enhancing the contact between solvent
and solute and thus intensifying the extraction (Mustafa and
Turner, 2011).
The increase of PLE temperature had a positive effect on
the global yield at all the evaluated ethanol concentrations. As
temperature increases, intermolecular forces like hydrogen,
van der Waals, and dipole-dipole bonds are reduced. There-
fore, the activation energy for the desorption of extractable
compounds decreases. Moreover, the solvent’s viscosity and
surface tension decrease as temperature rises, improving the
penetration capacity of the solvent in the solid substrate
(Machado et al., 2015; Viganó et al., 2016b).
Regarding the solvent composition, the addition of water to
ethanol increased the global yield, as usually noted with mix-
tures of solvents. The solvent characteristics determine which
type of compound may be dissolved. Therefore, the solute’s
 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21 15

Table 3 – Global yield, total phenolic content, antioxidant capacity determined by DPPH and FRAP, and total reducing
sugars for the extracts obtained from orange peel.
Experimental T(◦ C) Lot 1 Lot 2
condition
%Ethanol Global TPC (mg DPPH (mg FRAP (mg TRS (mg Global TPC (mg DPPH (mg FRAP (mg
yield (%) GAE/g DP) TE/g DP) TE/g DP) Glucose/g yield (%) GAE/g DP) TE/g DP) TE/g DP)
DP)

99.5 45 14.1 ± 0.8i 1.6 ± 0.3lm 1.3 ± 0.1gh 86 ± 9f 92 ± 5ef 13.7 ± 0.7i 1.4 ± 0.1m 1.0 ± 0.1gh 58 ± 2g
55 14.6 ± 0.3i 2.8 ± 0.2k 1.9 ± 0.1g 87 ± 8f 99 ± 6de 14.8 ± 0.2i 2.4 ± 0.3kl 1.5 ± 0.1gh 83 ± 9f
65 15.7 ± 0.2hi 4.6 ± 0.2ij 1.8 ± 0.1g 123 ± 6e 110 ± 4cd 14.9 ± 0.6i 4.0 ± 0.1j 1.8 ± 0.1g 98 ± 2f
75 45 30.7 ± 0.8ef 11.2 ± 0.1d 3.7 ± 0.2f 155 ± 9d 121 ± 1c 30.2 ± 0.3f 11.1 ± 0.6de 3.6 ± 0.1f 168 ± 4cd
55 33 ± 1cd 13.3 ± 0.8c 4.0 ± 0.2ef 200 ± 8b 135 ± 7b 32.6 ± 0.3de 12.7 ± 0.1c 4.0 ± 0.1ef 185 ± 5c
65 34.8 ± 0.3bc 14.9 ± 0.7b 4.6 ± 0.3de 255 ± 9a 136 ± 6b 33.9 ± 0.3bcd 15.9 ± 0.2a 5.1 ± 0.3abc 240 ± 5a
50 45 35 ± 2bc 9.6 ± 0.9g 4.5 ± 0.6de 203 ± 9b 162 ± 2a 35.6 ± 0.5b 10.0 ± 0.2fg 4.9 ± 0.2bcd 200 ± 8b
55 35 ± 1bcd 10.1 ± 0.1fg 5.6 ± 0.6a 215 ± 9b 151 ± 2a 38.3 ± 0.4a 10.8 ± 0.4def 5.3 ± 0.2abc 207 ± 3b
65 38.5 ± 0.9a 10.3 ± 0.5efg 5.5 ± 0.6ab 241 ± 3a 161 ± 4a 40.1 ± 0.3a 10.3 ± 0.5defg 5.3 ± 0.3ab 203 ± 9b
UAE 21.9 ± 0.5g 5.5 ± 0.1hi 4.3 ± 0.1def 88 ± 2f 65 ± 2h 21.5 ± 0.3g 6.0 ± 0.1h 4.5 ± 0.1cde 88 ± 3f
Soxhlet 18 ± 1h 5.8 ± 0.1h 1.70 ± 0.04gh 33.4 ± 0.4h 82 ± 2fg

Values expressed as mean ± standard deviation. Values followed by different letters in the same columns indicate statistical difference by the
Tukey’s test with 95% of confidence. GAE: gallic acid equivalent; DP: dry peel; TE: trolox equivalent. 99.5% ethanol corresponds to absolute
ethanol.

polarity must be similar to that of the solvent (Mustafa and
Turner, 2011). Hence, ethanol will be able to dissolve mod-
erately polar compounds, whereas water can dissolve those
of high polarity. The mixture of these two solvents is able
to dissolve moderate to high polarity compounds, depend-
ing on the concentration. This behavior may be positive when
greater global yields need to be achieved. However, a signif-
icant part of the extract may not be formed by the target
compounds, leading to a product with a high proportion of
undesirable molecules, which will require further purification
stages (Pereira et al., 2016).
Table 3 also shows the TRS content of the extracts obtained
from Lot 1. The TRS behavior was similar to that of the
global yield, showing a Pearson’s correlation coefficient of 0.93.
Sugars have high solubility in water and low in ethanol. There-
fore, as the water concentration increases, greater TRS was
achieved. It was also verified that high temperatures increase
the TRS extraction. Both effects had been reported by Alavi
et al. (2014) in a study of fructose solubility in ethanol–water
and methanol–water mixtures.
3.3. Total phenolic content (TPC)
compounds. However, with ethanol concentration below 75%,
there was a reduction of TPC, with no change in the global
yield. Therefore, the increase of water content in the solvent
may have promoted the extraction of other compounds from
the orange peel, which can be considered as contaminants in
the extract, such as sugars, as shown in Table 3, and other
short-chain carbohydrates. Similar behavior was observed by
Viganó et al. (2016a, 2016b) and Machado et al. (2015) for the
PLE of passion fruit and blackberry bagasses, respectively.
A similar by-product of orange processing was used by
Espinosa-Pardo et al. (2017), who extracted phenolic com-
pounds by supercritical CO2 extraction with ethanol as
cosolvent. They achieved a TPC of 0.57 mg GAE/g dry peel in
the best condition. Ethanol modified the solvent’s polarity,
enhancing the extraction of phenolic compounds, although
with low concentration and amount. Nevertheless, PLE was
more efficient than SFE with ethanol as polarity modifier to
recover phenolics from orange peel since, as shown in Table 3,
the process in this work resulted in greater TPCs. Further-
more, PLE reduces the solvent consumption, since the S/F ratio
used in this work was 47 kg solvent/kg sample, against 124 kg
solvent/kg sample applied by Espinosa-Pardo et al. (2017).

The total phenolic content of the extracts is presented in
Table 3. The TPC obtained by Soxhlet extraction, UAE, and
PLE using pure ethanol as solvent did not show significant
differences at a confidence level of 95%, and were the lowest
among all of the extracts. However, PLE using absolute ethanol
achieved the same TPC as UAE and Soxhlet in a shorter time.
The high pressure and temperature employed in PLE improved
the extraction of phenolic compounds, achieving the highest
yield with 75% ethanol at 65 ◦ C. At this condition, the TPC
were 14.9 ± 0.7 and 15.9 ± 0.2 mg GAE/g DP for Lots 1 and 2,
respectively.
TPC increases with temperature in PLE, which may be con-
sequence of the reduction of the surface tension, a factor
that controls the penetration of the solvent into the solid
pores. Moreover, higher temperature increases the phenolic
compounds’ diffusivity into the solvent, favoring their trans-
port. Regarding the ethanol concentration, the presence of
water in the solvent improved its capacity to extract phenolic
3.4. Antioxidant capacity of the extracts
The antioxidant capacity of the extracts, obtained by DPPH
and FRAP, are presented in Table 3. Both methods showed the
superiority of PLE over Soxhlet and UAE in terms of antiox-
idant capacity. In order to evaluate the influence of the TPC
over the antioxidant capacity, the Pearson’s correlation coeffi-
cient was calculated. The Pearson’s coefficient obtained were
0.83 and 0.97 for DPPH and FRAP respectively, indicating the
strong correlation between the TPC and the antioxidant capac-
ity of the extracts. Therefore, the antioxidant capacity can be
attributed to the phenolic compounds extracted from orange
peel.
3.4.1. DPPH
The best extraction conditions, considering the DPPH antiox-
idant capacity, were PLE with 50% ethanol at 55 and 65 ◦ C,
with 5.3 ± 0.2 and 5.6 ± 0.6 mg TE/mg DP, with no significant
16 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21
difference at a confidence level of 95%. The increase of tem-
perature had a positive effect on the antioxidant capacity, as
well as on TPC. The DPPH antioxidant capacity also increased
with the reduction of ethanol concentration in the solvent.
This behavior is different from that of TPC. The DPPH reagent
could have reacted with another compound present in the
extracts, which was not identified, such as carotenoids or vita-
min C (Kimball, 1999), both antioxidant substances that may
be found in orange peels.
3.4.2. FRAP
The best condition considering the FRAP antioxidant capacity
was PLE with 75% ethanol at 65 ◦ C, coinciding with the best
condition for TPC, in which the antioxidant capacities were
255 ± 9 mg TE/mg DP and 240 ± 5 mg TE/mg DP for Lots 1 and
2, respectively. These values are not significantly different at a
confidence level of 95%. Temperature had a positive effect on
FRAP antioxidant capacity. Regarding the ethanol concentra-
tion, the addition of water to the solvent was positive up to 75%
ethanol, due to the changes in the solvent’s polarity and den-
sity. However, when the ethanol concentration was reduced to
50%, the antioxidant capacity of the extracts decreased, indi-
cating that undesired compounds may have been extracted
under those conditions.
3.5. Identification and quantification of phenolic
compounds
The extracts obtained from orange peel were analyzed
through HPLC, in order to tentatively identify and quan-
tify the extracted flavonoids and hydroxybenzoic acids. Nine
phenolic compounds were identified in the extracts, as pre-
sented in Fig. 2. However, two of them showed response areas
below the quantification limit. Table 4 shows the phenolic
compounds identified in the extracts, with their correspond-
ing concentrations on dry extract basis, and Table 5 shows
Fig. 2 – Representative HPLC-UV–vis (280 nm) chromatogram of the phenolic compounds identified in the extracts obtained
from orange peel, and structures of the two major phenolic compounds. 1–Gallic acid; 2—narirutin; 3—naringin;
4—hesperidin; 5—ellagic acid; 6—naringenin; 7—hesperitin; 8—diosmetin; 9—tangeritin.
only the quantified phenolic compounds, with concentra-
tions expressed on dry peel basis. The phenolics found in
all the extracts were hesperidin, narirutin, tangeritin, narin-
genin, hesperetin and diosmetin, although the last could not
be quantified. Among the identified phenolics, hesperidin was
the most abundant, corresponding to the typical flavonoid
composition of citrus peel (Rafiq et al., 2016). The phenolics
concentration were consistent with those found in the litera-
ture for the same raw material (Lachos-Perez et al., 2018).
Among the evaluated extraction methods, PLE achieved the
highest yields of glycosylated flavonoids, such as hesperidin,
naringin, and narirutin, but the deglycosylated phenolics
did not follow this behavior. The sugar molecules linked to
flavonoids enhance their extraction using a polar solvent.
However, the different temperature and ethanol concentra-
tions used in each experiment, presented in Tables 4 and 5,
affected the proportion in which each phenolic compound was
extracted. Therefore, the process conditions not only affected
the phenolics concentration, but also modified the pheno-
lics profile. On the other hand, the deglycosylated phenolics
were obtained in greater amounts by Soxhlet extraction, using
ethanol at high temperature (about 78 ◦ C). However, in the
Soxhlet extraction the solvent is continuously recirculated for
six hours. Therefore, the comparison between Soxhlet and PLE
should consider the number of solvent cycles, as well as the
solvent volume of each cycle, to establish the S/F ratio.
Both temperature and ethanol concentration in the sol-
vent influenced the phenolic compounds’ extraction by PLE.
Temperature had a positive effect — as it increases, a greater
TPC is achieved. The effects of increased diffusivity caused by
temperature and the reduction of the solvent’s surface ten-
sion accelerate the desorption of phenolic compounds from
the vegetal matrix. Regarding the ethanol concentration, the
water added to the solvent enhanced the recovery of glyco-
sylated flavonoids. However, when the ethanol concentration
was 75%, the hesperidin concentration was not significantly
 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21 17

Table 4 – Concentrations of the phenolic compounds identified by HPLC in the extracts obtained from orange peel,
expressed on dry extract basis.
Ethanol T Hesperidin Naringin Gallic acid Narirutin Tangeritin Naringenin Hesperitin Ellagic Diosmetin
(%) (◦ C) (mg/g DE) (mg/g DE) (mg/g DE) (mg/g DE) (mg/g DE) (mg/g DE) (mg/g DE) acid

Lot 1 99.5 45 17 ± 3ghi Nd Nd 4 ± 3c 0.3 ± 0.2b 0.5 ± 0.3bcd 0.3 ± 0.1de Nd *

55 36 ± 6def Nd 1.1 ± 0.1b 7.6 ± 0.5abc 0.43 ± 0.01ab 0.52 ± 0.06bcd 0.4 ± 0.1cd Nd *
65 41 ± 6cd Nd Nd 6 ± 2abc 0.3 ± 0.1b 0.4 ± 0.2bcd 0.3 ± 0.1de Nd *
75 45 26 ± 5fg 0.10 ± 0.03a Nd 5 ± 1abc 0.17 ± 0.02b 0.26 ± 0.03cd 0.12 ± 0.02e * *
55 58 ± 2a Nd Nd 7.0 ± 0.3abc 0.240 ± 0.02b 0.30 ± 0.01bcd 0.319 ± 0.004de Nd *
65 58 ± 3a 0.4 ± 0.3a Nd 9 ± 1abc 0.32 ± 0.01b 0.48 ± 0.02bcd 0.27 ± 0.02de Nd *
50 45 12 ± 1hij 0.062 ± 0.009a Nd 3.1 ± 0.7c 0.092 ± 0.001b 0.118 ± 0.003d 0.123 ± 0.001e Nd *
55 29 ± 1ef 0.089 ± 0.009a Nd 6.0 ± 0.4abc 0.213 ± 0.006b 0.26 ± 0.01cd 0.27 ± 0.01de * *
65 34 ± 1def 0.073 ± 0.008a 0.34 ± 0.02c 2.4 ± 0.3c 0.154 ± 0.001b 0.193 ± 0.006d 0.12 ± 0.02e * *
Lot 2 99.5 45 42 ± 1cd Nd Nd 14 ± 2ab 1.09 ± 0.03a 0.91 ± 0.02ab 0.85 ± 0.06b Nd *
55 49 ± 1abc Nd 2.5 ± 0.4a 15 ± 9a 1.1 ± 0.7a 0.9 ± 0.5abc 0.4 ± 0.2cd Nd *
65 55 ± 2ab Nd Nd 7.1 ± 0.9abc 0.5 ± 0.2ab 0.4 ± 0.1bcd 0.32 ± 0.07de Nd *
75 45 37 ± 1de 0.22 ± 0.04a 0.37 ± 0.05c 5.4 ± 0.3abc 0.78 ± 0.09ab 0.7 ± 0.1bcd 0.64 ± 0.06bc * *
55 47 ± 1bc 0.13 ± 0.03a 0.32 ± 0.1c 11 ± 1abc 0.38 ± 0.03ab 0.42 ± 0.04bcd 0.23 ± 0.02de * *
65 54 ± 1ab 0.226 ± 0.005a 0.42 ± 0.03c 14 ± 2ab 0.54 ± 0.03ab 0.54 ± 0.09bcd 0.260 ± 0.002de * *
50 45 26 ± 1fg 0.113 ± 0.005a Nd 2.9 ± 0.3c 0.171 ± 0.005b 0.215 ± 0.001cd 0.189 ± 0.001de * *
55 29 ± 1ef 0.11 ± 0.01a Nd 2.7 ± 0.1c 0.19 ± 0.04b 0.23 ± 0.04cd 0.201 ± 0.001de * *
65 34 ± 1def 0.13 ± 0.04a Nd 4.2 ± 0.1c 0.36 ± 0.04b 0.40 ± 0.07bcd 0.35 ± 0.06de * *
Soxhlet 18 ± 1gh Nd 1.3 ± 0.2b 11 ± 1abc 1.1 ± 0.1a 1.38 ± 0.08a 1.37 ± 0.06a * *
UAE Lot 1 7.1 ± 0.7j Nd 0.153 ± 0.004d 5.0 ± 0.4bc 0.26 ± 0.03b 0.249 ± 0.001cd 0.24 ± 0.02de Nd *
UAE Lot 2 7.7 ± 0.8ij Nd 0.154 ± 0.001d 5.5 ± 0.1abc 0.24 ± 0.03b 0.250 ± 0.006cd 0.24 ± 0.02de Nd *

Concentrations expressed as mg of compound/g dry extract. Values presented as mean ± standard deviation; DE: dry extract; *: compound
identified under the quantification limit. Nd: not detected. Means followed by different letters in the same column are significantly different by
the Tukey’s test (95% confidence). 99.5% ethanol corresponds to absolute ethanol.

Table 5 – Concentrations of the phenolic compounds identified by HPLC in the extracts obtained from orange bagasse,
expressed on dry peel basis.
Ethanol T Hesperidin Naringin Gallic acid Narirutin Tangeritin Naringenin Hesperitin
(%) (◦ C) (mg/g DP) (mg/g DP) (mg/g DP) (mg/g DP) (mg/g DP) (mg/g DP) (mg/g DP)

Lot 1 99.5 45 2.5 ± 0.5ij Nd Nd 0.6 ± 0.5g 0.029 ± 0.009e 0.036 ± 0.005f 0.032 ± 0.002f
55 5.3 ± 0.9gh Nd 0.16 ± 0.02bc 1.1 ± 0.1efg 0.063 ± 0.002de 0.08 ± 0.01def 0.053 ± 0.002cdef
65 6.2 ± 0.8fg Nd Nd 1.0 ± 0.4efg 0.064 ± 0.003cde 0.10 ± 0.01def 0.06 ± 0.01cdef
75 45 9 ± 2de 0.03 ± 0.01a Nd 1.8 ± 0.4bcdef 0.061 ± 0.008de 0.09 ± 0.01def 0.043 ± 0.006ef
55 17.7 ± 0.6a Nd Nd 2.3 ± 0.1efg 0.035 ± 0.001e 0.043 ± 0.002f 0.0467 ± 0.0005def
65 19.3 ± 0.9a 0.1 ± 0.1a Nd 3.0 ± 0.5ab 0.107 ± 0.005bcde 0.16 ± 0.01bc 0.092 ± 0.006cd
50 45 4.4 ± 0.3ghi 0.019 ± 0.003a Nd 1.1 ± 0.2fg 0.0283 ± 0.0003e 0.036 ± 0.001f 0.0370 ± 0.0004f
55 10.1 ± 0.1de 0.031 ± 0.003a Nd 2.1 ± 0.2bcde 0.074 ± 0.002cde 0.091 ± 0.005def 0.092 ± 0.003c
65 13,1 ± 0.1bc 0.025 ± 0.003a 0.12 ± 0.01cd 0.9 ± 0.1efg 0.053 ± 0.0004de 0.066 ± 0.002ef 0.041 ± 0.007f
Lot 2 99.5 45 2.8 ± 0.1ij Nd Nd 1.0 ± 0.1efg 0.073 ± 0.002cde 0.061 ± 0.001ef 0.057 ± 0.004cdef
55 5.3 ± 0.1gh Nd 0.27 ± 0.04a 1.6 ± 0.9defg 0.12 ± 0.08bcd 0.052 ± 0.002ef 0.035 ± 0.008f
65 8.2 ± 0.3ef Nd Nd 1.1 ± 0.1efg 0.07 ± 0.03cde 0.07 ± 0.01ef 0.056 ± 0.001cdef
75 45 11.1 ± 0.3cd 0.020 ± 0.004a 0.035 ± 0.005d 1.6 ± 0.1cdefg 0.073 ± 0.008cde 0.11 ± 0.04cde 0.061 ± 0.005cdef
55 15.4 ± 0.6b 0.04 ± 0.01a 0.104 ± 0.004cd 3.44 ± 0.4bc 0.12 ± 0.01bcd 0.14 ± 0.01bcd 0.075 ± 0.005cdef
65 18.6 ± 0.4a 0.077 ± 0.002a 0.14 ± 0.1c 4.8 ± 0.7a 0.18 ± 0.01ab 0.18 ± 0.03b 0.088 ± 0.001cde
50 45 9.3 ± 0.4de 0.040 ± 0.002a Nd 1.1 ± 0.1cdefg 0.061 ± 0.002de 0.0765 ± 0.0002def 0.0675 ± 0.0001cdef
55 11.1 ± 0.2cd 0.045 ± 0.005a Nd 1.0 ± 0.1bcdef 0.07 ± 0.01cde 0.09 ± 0.01def 0.077 ± 0.001cdef
65 13.6 ± 0.1b 0.05 ± 0.1a Nd 1.7 ± 0.1bcd 0.14 ± 0.01abc 0.16 ± 0.03bc 0.14 ± 0.02b
Soxhlet 3.4 ± 0.2hij Nd 0.23 ± 0.04ab 2.0 ± 0.2bcde 0.21 ± 0.01a 0.25 ± 0.02a 0.23 ± 0.04a
UAE Lot 1 1.6 ± 0.1j Nd 0.033 ± 0.001d 1.1 ± 0.1efg 0.056 ± 0.007de 0.0544 ± 0.0002ef 0.053 ± 0.004cdef
UAE Lot 2 1.6 ± 0.1j Nd 0.034 ± 0.001d 1.2 ± 0.1efg 0.053 ± 0.007de 0.054 ± 0.001ef 0.053 ± 0.004cdef

Concentrations expressed as mg of compound/g dry bagasse. Values presented as mean ± standard deviation; DP: dry peel; Nd: not detected.
Means followed by different letters in the same column are significantly different by the Tukey’s test (95% confidence). 99.5% ethanol corresponds
to absolute ethanol.

different from that obtained with 50% ethanol, as can be
observed in Table 5. With higher hesperidin concentration,
as expressed in Table 4, the best PLE condition was ethanol
75% at 65 ◦ C. Both lots showed similar behavior, with little or
no significant difference, indicating that the supercritical CO2
extraction prior to PLE did not extract phenolic compounds. In
fact, polar molecules like flavonoids and hydroxybenzoic acids
have very low solubility in CO2 . Moreover, the sugar molecule
linked to the phenolics limits their solvation in supercritical
CO2 , in which carbohydrates are not soluble (Brunner, 2005).
The peels of citrus fruits have similar composition in terms
of carbohydrates, pectin and phenolic compounds (Rafiq et al.,
18 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21

Fig. 3 – Experimental and modeled global yield kinetic curves of PLE from orange peel (Lot 1) at 65 ◦ C, using 99.5, 75, and
50% ethanol in water.

Fig. 4 – Experimental and modeled TPC kinetic curves of PLE from orange peel (Lot 1) at 65 ◦ C, using 99.5, 75, and 50%
ethanol in water.

2016). Therefore, similar trends and difficulties, as those dis-
cussed in this section, are expected for PLE from other citrus,
although the phenolics profile and concentrations may change
according to the species.
3.6. PLE kinetics
PLE kinetics were performed at 65 ◦ C, since this temperature
achieved the best performance regarding global yield, TPC,
antioxidant capacity and phenolics quantification for all sol-
vents (99.5, 75, and 50% ethanol). Figs. 3 and 4 show the
experimental and modeled PLE curves for global yield and TPC,
respectively.
3.6.1. Spline model
The three-line Spline model successfully described the PLE
kinetics behavior, determining the CER and FER times, which
are presented in Table 6. In all the kinetics the three extraction
periods, CER, FER, and DC, were identified.
As expected, a great part of the extractable material was
recovered in the CER period, as shown in Table 6 in terms
of global yield and TPC. This type of behavior is usual
in PLE, making possible to extract from 70 to 90% of the
extractable material during the CER period, depending on
 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21 19

Table 6 – Estimated parameters of the three-line Spline model and the two site kinetic desorption model for the PLE of
orange peel (Lot 1) at 10 MPa and 65 ◦ C.
Model Response X0 TPC

% Ethanol 99.5 75 50 99.5 75 50

Three-lines spline tCER (min) 5.3 5.4 5.7 5.4 5.3 6.1
YCER 9.50 27.83 34.86 2.51 12.67 7.23
RecoveryCER (%) 70.36 82.97 81.08 67.82 82.53 65.59
tFER (min) 18.1 17.8 17.2 23.8 16.7 17.7
YFER 11.85 31.60 40.35 3.31 14.19 9.45
RecoveryFER (%) 87.75 94.21 93.87 89.52 92.46 85.76
R2 >0.999 >0.999 >0.999 >0.999 >0.999 >0.999
Two site f 0.551 0.726 0.708 0.484 0.761 0.549
k1 (min−1 ) 0.618 0.513 0.405 0.547 0.499 0.256
k2 (min−1 ) 0.047 0.069 0.076 0.028 0.039 0.028
R2 >0.999 >0.999 >0.999 >0.999 >0.999 >0.999
MRPD (%) 0.094 0.033 0.087 2.423E-09 0.141 0.190

tCER : CER period time; tFER : FER period time; YCER and YFER : global yield (X0 ; %) or total phenolic concentration (TPC; mg GAE/g DP) at the end
of tCER and tFER , respectively; y∞ : extract response at saturation; f: fast desorption fraction; k1 : rate constant for washing; k2 : rate constant for
diffusion; R2 : determination factor; MRPD: mean relative percent deviation.

the vegetable matrix (Meireles, 2008). Moreover, Viganó et al. 4. Conclusions

(2016b) obtained similar results for PLE of phenolics from
passion fruit rinds, making it possible to propose a shorter
extraction time, resulting on a reduction of the solvent con-
sumption. FER time increased the recovery of extractable
material in about 10–15%, and of TPC in about 10–22%, depend-
ing on the condition. At the best extraction condition (65 ◦ C
using ethanol 75%), 94.21% of the total extractable material
and 92.46% of the TPC were extracted until the end of the
FER period, which was below 20 min. Therefore, a reduction
in the extraction time to 20 min can be suggested to obtain
almost the same amount of product and reduce the solvent
consumption.
The solvent composition did not change the extension of
CER and FER periods. However, the extraction rates changed
due to the difference in solubility resulting from the solvent
compositions.
3.6.2. Two-site kinetic desorption model
The proximity of the experimental and modeled data in
Figs. 3 and 4 reveals the accuracy of this model to describe the
PLE process. In all the PLE curves the fast and slow parts of the
kinetics were clearly identified. The different solvents affected
the solubility of the extractable materials, varying the values
of y∞ , and both external (k1 ) and internal (k2 ) mass transfer
mechanisms were important, as observed in Table 6.
Higher water concentrations in the solvent lead to higher
values of y∞ , reinforcing how the mixture of water and ethanol
improves the solvation power. As the driving force for the
desorption from the solid to the solvent is the concentration
gradient between the solid matrix and the extraction fluid,
higher values of y∞ increase the global yield. The highest value
of k1 was obtained with the ethanol concentration of 99.5%, in
which the lowest value of k2 is observed. In this case, the wash-
ing stage is almost instantaneous, followed by a long diffusion
period (Fig. 4).
Regarding TPC, y∞ increases only from 99.5 to 75% ethanol
and then decreases to 50% ethanol, showing the selectivity of
the solvent. Regarding the mass transfer rates, the behavior
for k1 was similar to that described for global yield. However,
k2 presented differences. The highest k2 was obtained at 75%
ethanol, leading to a faster diffusion period.
Phenolic compounds were extracted from orange peel by PLE,
UAE, and Soxhlet. Nine flavonoids, being three of them glyco-
sylated, and two hydroxybenzoic acids were identified in the
extracts by HPLC.
Supercritical CO2 was able to extract the essential oil
remaining in the orange peel, which showed to have a high
proportion of the valuable ␣-Terpineol in its volatile fraction.
Moreover, SFE did not affect the recovery of the phenolic com-
pounds by PLE, UAE, and Soxhlet at the performed conditions.
However, the operational costs of this step must be consid-
ered before deciding about its possible use in a sequential
process.
PLE has shown better performance than UAE and Soxhlet
to extract glycosylated flavonoids from orange peel, achiev-
ing a greater recovery of these bioactive compounds in a
shorter time and using moderate temperatures. The phenolic
yields indicate that they are resistant to high tempera-
tures. Moreover, the deglycosylated flavonoids have shown
more affinity to the less-polar solvent at high tempera-
ture, which suggests evaluating even higher temperatures
in PLE.
Two mathematical models were adjusted to the PLE kinet-
ics and helped describing the three extraction stages of the
process. Moreover, the models’ parameters indicate that the
process was controlled by the external mass transfer of
the solute to solvent and internal diffusion. The high yield
and TPC of the CER period suggest that the PLE process
can be performed for 20 min with a remarkable economy of
solvent.
The conclusions concerning the extraction mechanisms,
solvent composition and process temperature may guide fur-
ther investigations on PLE of phenolics applied to other raw
materials. The results presented in this work indicate that
not only citrus peel, but also by-products from other fruits’
processing, can be used as raw material to recover phenolic
compounds with proven biological activities. Further works
should focus on purification processes, in order to remove the
sugars present in the extracts. Packed column fractionation
may be an alternative to separate the sugars in a process that
could be integrated to PLE.
20 Food and Bioproducts Processing 1 1 2 ( 2 0 1 8 ) 9–21
Acknowledgments
The authors wish to thank CAPES, CNPq (Ph.D. fellowship
140883/2015-0) and FAPESP (2015/11932-7) for the financial
support.
Appendix A. Supplementary data
Supplementary data associated with this arti-
cle can be found, in the online version, at
https://doi.org/10.1016/j.fbp.2018.08.006.
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