The Journal of Supercritical Fluids 146 (2019) 55–64

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The Journal of Supercritical Fluids

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

Antioxidant activity and fatty acid profile of yacon leaves extracts obtained T
by supercritical CO2 + ethanol solvent
Pollyanna N. Cruz, Damian L. Fetzer, Wanderlei do Amaral, Eriel F. de Andrade,
⁎
Marcos L. Corazza , Maria L. Masson
Department of Chemical Engineering, Federal University of Paraná, CEP 81531-990, Curitiba, PR, Brazil

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: Extraction efficiency of bioactive compounds from yacon leaves using supercritical CO2 + ethanol
Smallanthus sonchifolius (scCO2+EtOH) fluid extraction and a conventional method were investigated. scCO2+EtOH runs were per-
Supercritical fluid extraction formed under the following conditions: 30–70 °C, 15–25 MPa and varying the ethanol (co-solvent) to solid (raw
Soxhlet extraction material) mass ratio (MR). All extractions were evaluated in terms of overall extraction yield (X0), fatty acid
Extraction curves
composition, total phenolic content (TPC) and antioxidant activity (AA). Results indicated that scCO2+EtOH
Phenolic content
presented the highest extraction yield 5.22 wt% at 70 °C/25 MPa/MR 3:1. High values of TPC and AA were found
in all extracts. Major unsaturated fatty acids found in yacon leaves extracts were gamma-linolenic (ω-6), eico-
sapentaenoic (ω-3) and linoleic (ω-6) acids. The highest ω-6/ω-3 fatty acids ratios were found in the extracts
obtained using scCO2+EtOH.

1. Introduction fructooligosaccharides (FOS), inulin and phenolic compounds [2–4].

Yacon (Smallanthus sonchifolius) is a perennial plant which belongs
to the Asteraceae family, native to the Andes and now it is cultivated in
several regions of world [1]. Studies suggest that the yacon roots pre-
sent functional value for their content of bioactive substances such as
Yacon leaves are used in the food industry and in folk medicine
mainly to produce teas due to its various phytochemical properties,
such as antimicrobial [5], anti-inflammatory [6], antioxidant [2,7] and
anti-diabetic [8,9]. These properties have been associated to the pre-
sence of bioactive compounds in leaves such as phenols (gallic acid,

⁎
Corresponding author.
E-mail address: corazza@ufpr.br (M.L. Corazza).

https://doi.org/10.1016/j.supflu.2019.01.007
Received 7 November 2018; Received in revised form 8 January 2019; Accepted 9 January 2019
Available online 09 January 2019
0896-8446/ © 2019 Published by Elsevier B.V.
P.N. Cruz et al. The Journal of Supercritical Fluids 146 (2019) 55–64

protocatecuic acid, chlorogenic acid, caffeic acid and ferrulic acid)
[2,7] and sesquiterpene lactones (enhydrin, uvedalin, sonchifolin and
polimatin B) [10,11].
In previous studies reported in the literature, extracts from yacon
leaves were obtained by different extraction methods, such as Soxhlet
extraction with methanol and ethyl acetate solvents [7,8], infusion and
decoction with water solvent [12,6–9] maceration with methanol sol-
vent [5,6], ultrasonic with ethanol solvent [13] and solid-phase ex-
traction [14]. The quality of natural extracts and consequently their
biological activity is related to their composition which depends on the
extraction process, the solvent used, the origin of the raw material as
well as its storage condition and the pre-treatment applied [15–17].
Therefore, alternative methods to obtain natural bioactive substances,
such as supercritical fluid extraction, have been studied [18].
The supercritical fluid extraction (SFE) is based on the use of sol-
vents above their critical point and enables the continuous modulation
of the solvation power and the selectivity of the solvent [19]. Carbon
dioxide (CO2) has been the commonly used solvent in SFE because, in
contrast to organic solvents, it is non-toxic, inexpensive and volatile,
with moderate critical pressure and temperature. Besides moderate
temperature, the absence of light and oxygen during the extraction
process avoids degradation reactions of bioactive substances. In addi-
tion, aiming to overcome the drawback associated to the lower solu-
bility of polar and high molar mass compounds in CO2, polar co-sol-
vents have been proposed to increase the solvation power of the fluid
phase during SFE [20,21] and enhance the extraction efficiency.
To the best of our knowledge no data has been reported in the lit-
erature about the use of yacon leaves as a raw material in the pro-
duction of biologically active extracts using supercritical technology.
Therefore, the present work aims to evaluate the use of two extraction
methods to obtain yacon leaves extracts: Soxhlet, a conventional
method, and SFE with CO2 associated to ethanol in a gas-expanded li-
quid (GLX) like techology, a green technology. The results were com-
pared in terms of process extraction yield (wt%), total phenolic content
(TPC), and antioxidant activity (AA) by DPPH and ABTS assays.
Moreover, gas chromatography was carried out for the identification
and relative quantification of the fatty acids present in the extrats.
2. Materials and methods
2.1. Sample preparation
Yacon leaves samples were obtained from a local producer in São
José dos Pinhais, Paraná state, Brazil (coordinates: 25°37′14″ latitude,
49°07′40″ W, altitude of 906 m). All samples were dried at 40 °C for
24 h in a forced air oven and moisture content reduced from 76.94% to
final moisture of 8.43%, determined according to the 925.09 AOAC
method [22].
Dried samples were ground in a domestic food processor for 10 s
and the average particle diameter (dp) was calculated based on mean
size distribution as described by Gomide [23], and resulted in dp =
0.2 mm. The samples were packed in low-density polyethylene bags
and sealed under vaccum, and then stored at −18 °C until their use.
2.2. Raw material characterization
the free volume of a solid, by varying the gas pressure in a chamber of
known volume. The bed porosity was determined by the ratio between
the apparent and the real densities (Eq. (2)) as follows:
ρa
ε=1−
ρr (2)
where ρa is the apparent density and ρr is the real density.
2.3. Supercritical fluid extraction (SFE)
Extractions using scCO2 and scCO2+co-solvent were carried out in a
laboratory scale unit. The main dimensions of the unit are: a 62.4 mL
stainless steel extraction cell with 1.9 cm bed diameter, 22 cm bed
height with controlled pressure and temperature, as described in pre-
vious studies [24,25]. The extraction system consists of a jacketed-ex-
tractor coupled to a ultrathermostatized bath, a needle valve for the
CO2 flow control, a syringe-type pump (Teledyne ISCO 500D), pressure
and temperature sensors. Supercritical extractions were performed
using carbon dioxide (White Martins S.A., 99.5% of purity in the liquid
phase) as the solvent and ethanol (EtOH), as well as hidrated ethanol (a
EtOH-H2O mixture) (1/1, v/v) as co-solvents, which were the most
efficient liquid solvents evaluated in previous tests. The co-solvent
amount was added and mixed to the raw material (mass of co-solvent to
mass of solids in grams), and thereafter the wetted raw material was
loaded into the extraction vessel prior to the extractions. Therefore, the
experimental procedure used was: around 10 g of the raw material was
soaked (wetted) with EtOH or EtOH-H2O mixture and right after the
mixture was loaded into the extractor and thus the extractor was filled
with the CO2, and once the extractor has been filled with the sample/
(EtOH or EtOH-H2O mixture) and CO2 at the desired pressure and
temperature, the static extraction time started (a confinement period).
At the end of the static period (which as fixed in 60 min), the dynamic
extraction was started using a compressed CO2 flow rate around of
2.0 ± 0.2 mL min−1 (controlled and measured by the syringe pump at
10 °C and the pressure of the extraction vessel), where both pressure
and temperature were controlled all the time.
A full factorial design with 2 levels and 3 variables (23) (runs 1–11)
was used with a triplicate of central point (Table 1), in order to evaluate
the influence of temperature (30, 50 and 70 °C), pressure (15, 20 and
25 MPa) and ethanol to solid (raw material) mass ratio (1:1, 2:1 and
3:1) on the extraction yields and antioxidant activites. Afterwards,
additional experiments (runs 12–13) were performed altering the EtOH
as the liquid solvent for the EtOH-H2O mixture under the best condi-
tions of the previous experimental design.
The overall extraction curves (extraction kinetics) of yacon leaves
were evaluated by collecting and weighing the extract samples in amber
vials (cooled at around 0.1 MPa and 25 °C) at intervals of 5 min. At the
samples collected were dried in an air circulation oven to remove
ethanol. The dried extract amounts were gravimetrically quantified for
each sampling, stored at −18 °C and protected from light prior to
Table 1
Factors and levels evaluated for the experimental design of extraction using
scCO2+EtOH.
Run Temperature (ºC) Pressure (MPa) Mass Ratio (mc:ms)

The apparent density was estimated considering the amount of raw 1 30 (-1) 15 (-1) 1:1 (-1)
2 70 (1) 15 (-1) 1:1 (-1)
material (m) needed to fill the total volume of the extraction vessel (Vc),
3 30 (-1) 25 (1) 1:1 (-1)
as the following equation (Eq. (1)): 4 70 (1) 25 (1) 1:1 (-1)
m 5 30 (-1) 15 (-1) 3:1 (1)
ρa = 6 70 (1) 15 (-1) 3:1 (1)
Vc (1)
7 30 (-1) 25 (1) 3:1 (1)
The real density of yacon leaves was determined in the Institute of 8 70 (1) 25 (1) 3:1 (1)
9 50 (0) 20 (0) 2:1 (0)
Chemistry/UNICAMP, Campinas, Brazil by the technique of picnometry
10 50 (0) 20 (0) 2:1 (0)
in helium gas. Helium gas penetrates the pores of the plant matrix due 11 50 (0) 20 (0) 2:1 (0)
to its low surface tension without changing its characteristics determing

56
P.N. Cruz et al.
analysis.
2.4. Soxhlet extraction (SE)
Soxhlet extractions (SE) were performed according to the 920.39C
method of AOAC [26], using 5 g of dried sample to 150 mL of solvent
for six hours. The solvents used were: ethyl acetate (EtOAc), ethanol
(EtOH), methanol (MetOH) and ethanol-water (EtOH-H2O), with po-
larities of 4.3, 5.2, 6.6 and 7.1, respectively [27]. The procedure was
performed in triplicate.
2.4.1. Solvent-solute separation
All extracts obtained from SE were concentrated at 47 °C in a rotary
evaporator (Ika HB 10, Germany), supplied with cooling and vacuum
control (440 mmHg) in order to obtain the crude extracts. The extracts
were then stored in sealed amber glass bottles at −18 °C. The yields of
all extractions were determined by the ratio between the mass of crude
extract and the mass of raw material used. The results are presented as
average ± standard deviation.
2.5. Total phenolic content (TPC)
TPC was determined according to the Folin–Ciocalteu method [28].
The reaction mixture was composed by 0.1 mL of extract solutions
(concentration of 1667 mg/L), 7.9 mL of distilled water, 0.5 mL of Fo-
lin–Ciocalteu reagent (a mixture of phosphomolybdate and phospho-
tungstate) and 1.5 mL of 20% sodium carbonate, placed in amber flasks.
The flasks were agitated, left in the dark for 2 h and then the absorbance
was measured at 765 nm. Gallic acid was used as standard to plot the
calibration curve (concentration range from 0 to 500 mg/mL,
R2 = 0.99). All analyses were carried out in triplicate and the results
were expressed as milligrams of gallic acid equivalent per gram of ex-
tract (mg GAE/g).
2.6. Antioxidant activity (AA)
2.6.1. DPPH
The free radical scavenging of the yacon leaves extracts was eval-
uated using the 1,1-diphenyl-2-picrylhydrazil (DPPH) method as de-
scribed by Mensor et al. [29]. Briefly, each extract was weighed, diluted
in its respective solvent and fractioned in order to prepare different
extract concentrations. Following this, a 0.3 mM DPPH solution was
added to each extract fraction. After 30 min at room temperature, the
absorbance values were measured at 517 nm and converted into per-
centage of antioxidant activity (AA%). The DPPH results were ex-
pressed as the effective concentration at 50% (EC50), that is the con-
centration of solution required to decrease the absorbance of the test
solution in 50% compared to a blank solution and expressed in μg of
extract/ mL. The EC50 values were calculated from the linear regression
of the AA% curves obtained for the different extract concentrations.
AA% as well as the EC50 for all extracts were obtained considering the
average of triplicate assays.
2.6.2. ABTS•+
This assay was carried out based on the procedure described by Re
et al. [30] with modifications. Trolox (6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid) was used as standard. The absor-
bance was measured at 734 nm 6 min after the initial mixing of the
samples with the ABTS solution [2,2-azino-bis-(3-ethylbenzotiazoline-
6-sulfonic acid)]. Results are presented by the average of triplicate as-
says and expressed as Trolox equivalent antioxidant capacity (TEAC)
(μM concentration of a Trolox solution which AA is equivalent to the
activity of 1.0 mg/mL of sample solution).
57
The Journal of Supercritical Fluids 146 (2019) 55–64
2.7. Fatty acid composition (FAC)
FAC of the yacon leaves extracts was determined by preparation of
methyl esters according to the method described by O´Fallon et al. [31]
with modifications. 100 μL of extract was used, to wich 0.7 mL of 10 M
KOH solution and 5.3 mL of methanol were added. After vortexing the
test tubes were incubated in a water bath at 55 °C during 90 min.
Subsequently, 580 μL of 12 M sulfuric acid solution was added and the
tubes were agitated again and incubated in a water bath at 55 °C during
90 min. Finally, 3 mL of hexane was added and the mixture was cen-
trifuged to remove the supernatant. The fatty acid methyl esters
(FAMEs) were identified by gas chromatography (GC) (AOCS, Oficial
Method Ce 2–66 [32]), using a Varian (Santa Clara, CA 95051, USA) gas
chromatograph (model CP 3900) with flame ionization detector and
equipped with a fused silica capillary column (CP-SIL 88 CB; 100 m
x 0.25 mm x 0.2 μm). The operating conditions of GC-FID were:
Split = 1:100, column Flow = 2.0 ml/min, detector temperature =
300 °C, injector temperature = 260 °C, oven = 140–240 °C (40 min;
2 °C/min); carrier gas = nitrogen. FAMEs samples were diluted in n-
hexane (chromatographic grade) and injected in GC-FID. The FAMEs
were identified by comparison with retention times of the standard
FAMEs mixture (Supelco, MIX FAME 37, St. Louis, MO 63103, USA).
Each fatty acid was quantified as percentage of total methyl ester peak
areas.
2.8. Statistical analysis
Results were statistically evaluated by analysis of variance (ANOVA)
at 95% level of confidence using the Statistica 7.0 software (Statsoft
Inc., USA) in order to identify significant differences between X0, TPC
and antioxidant activity values. In addition, the responses analyzed in
the factorial design (23) were performed using the same software by
using the response surface methodology.
3. Results and discussion
3.1. Extraction yields (X0)
Dried and ground yacon leaves presented apparent and real den-
sities of 160 kg m−3 and 1440 kg m−3, respectively, resulting in a bed
porosity of 0.89. Table 2 shows the extraction yields results (X0) besides
the polarity index of each solvent used and the CO2 densities at dif-
ferent temperature/pressure combinations applied. The SE yield with
ethanol was considered as the reference to calculate the extraction
percent (%) of all extractions.
When EtOH-H2O was used as solvent in SE (run 14), a yield value of
19.39 wt% was achieved followed by MetOH and EtOH solvents with
12.61 and 7.29 wt%, respectively. In the SE method, the temperature
conditions, the use of fresh solvent, the solvent cycle and also the sol-
vent/solute interactions contribute to the extraction of the raw mate-
rials, i.e., higher temperatures reduce the surface tension and viscosity
of solvent, wich easily reaches the matrix spaces that contain the solutes
of interest enchancing their solubilization and also increasing the
number of components soluble in the solvent phase [33]. This fact
justifies the high extraction yields attained by this extraction method.
The influence of the solvent polarity in SE also needs to be taken
into account. Higher yields were obtained when high (EtOH-H2O) and
intermediate (EtOH) polarity solvents were used, suggesting that the
vegetable matrix is mainly formed by components from high to inter-
mediate polarity.
Andrade et al. [7] presented an extraction yield of 25% for extracts
of yacon leaves obtained by SE with methanol for 72 h. This value is
higher than the results hereby obtained with the same extraction
method, however, the extraction time used in present work is much
lower than in the work of Andrade et al. [7]. In addition, studies have
shown the need for reduction of organic solvents (hexane, methanol,
P.N. Cruz et al. The Journal of Supercritical Fluids 146 (2019) 55–64

Table 2
Experimental conditions and results of yacon leaves extraction.
Run Extraction method T (ºC) P (MPa) MR (mc:ms)1 CO2 density (kg m−3) Extraction time (min) Extraction yield2 (wt%) Extraction percent3 (wt%)

0 SFE – CO2 50 20 – 784 60 0.27 3.70

1 SFE – CO2+ EtOH 30 15 1:1 847 30 2.33 31.96
2 70 15 1:1 506 30 3.82 52.40
3 30 25 1:1 922 65 2.32 31.82
4 70 25 1:1 737 30 3.76 51.58
5 30 15 3:1 847 55 4.32 59.26
6 70 15 3:1 506 30 5.14 70.51
7 30 25 3:1 922 60 3.52 48.29
8 70 25 3:1 737 35 5.22 71.60
9 50 20 2:1 784 35 3.76 51.58
10 50 20 2:1 784 35 3.73 51.17
11 50 20 2:1 784 30 3.70 50.75
12 SFE – CO2+ EtOH-H2O 30 25 3:1 922 65 3.83 52.54
13 70 25 3:1 737 55 5.11 70.09
Baixa pressão Solvents Polarity Index
14 Soxhlet EtOH-H2O 7.1 360 19.39 ± 0.34 265.98
15 MetOH 6.6 360 12.61 ± 0.74 172.98
16 EtOH 5.2 360 7.29 ± 0.06 100
17 EtOAc 4.3 360 7.05 ± 0.27 96.70

1
Ethanol to raw material (solids) mass ratio (wt/wt).
2
(Mass of extract by the mass of dried material fed) × 100.
3
(Mass of extract obtained at the end of total time of extraction by mass extracted with Soxhlet ethanol) × 100.

methylene chloride, etc.) usage, which due to their toxicity have un-
desirable effects on the environment and on the quality and safety of
food and other end products. This concern has surpassed the academic
circles, which is evident from the EU environmental policy and legis-
lation for the period 2010–2050, where the reduction hazardous sol-
vents in industry is one of the priorities. For that reason, extraction
technologies that use safe and non-toxic solvents (such are water,
carbon dioxide, and ethanol) have been introduced and applied for the
extraction of various materials and compounds [19].
A preliminary test was performed using pure scCO2 at 50 °C and
20 MPa (Run 0) in this work, however a very low extraction yield of
0.27% was observed, indicationg that the pure CO2 is an inefficient for
yacon leaves extraction. Therefore, EtOH and EtOH-H2O were used,
based in the results achieved from the Soxhlet extraction, as promissing
alternatives associated to scCO2 aiming to improve the extraction
yields.
From Table 2, it can be seen that the presence of EtOH and EtOH- Fig. 1. Pareto chart showing the effects of process parameters on the yields.
H2O significantly enhanced the yacon leaves extraction when compared
to pure scCO2, leading to extraction yields over 5%. Such yields are still variables revealed positive effects, e.g., increasing the mass ratio and
lower than the results obtained by SE, however it is worth mentioning temperature, the extraction yield consequently increases. This is in
that the amount of EtOH and EtOH-H2O used with scCO2 in SFE is much good agreement with previous findings in the literature by Cruz et al.
lower than the amount used in SE. Furthermore, SFE extracts are ob- [35], Mazzutti et al. [36], Fetzer et al. [37] and Rodriguez et al. [38] for
tained (5 times) faster than the SE extracts. different raw materials.
When using the raw material wetted with organic solvent (EtOH or The addition of a co-solvent such as EtOH or EtOH-H2O to scCO2
EtOH-H2O, in this case), the scCO2 acts as a carrier for the organic reduces the process selectivity but improves the extraction yield by
solvent, minimizing the resistance to mass transfer and improving the increasing the solubility of polar compounds from the matrix [39].
contact between the solutes and the organic solvent. This behavior re- The extraction performance at isobaric conditions is mainly affected
sults in high yields using small amounts of organic solvent. Runs 12 and by temperature due to two factors: the vapor pressure of the solute and
13 shows that when the mixture scCO2+EtOH was modified to the solvent density. In fact, an increase in temperature leads to an in-
scCO2+EtOH-H2O the yield increased at lower temperature (30 °C) but crease in the vapor pressure of the solute, enhancing its solubility in the
decreased at higher temperature (70 °C). According to Pourmortazavi solvent; nevertheless, the solvent density decreases when the tem-
and Hajimirsadeghi [34] the water in excess acts as a solvent and perature is increased [40].
competes with the scCO2 for some substances, decreasing the recovery Eq. (3) represents the yield behavior model of yacon leaves extracts
of extract. obtained from scCO2+EtOH.
The highest extraction yield (5.22 wt%) was achieved at 70 °C/ (3)
Y = 0.98 + 0.03T + 0.75MR
25 MPa/MR 3:1 corresponding to the highest values of temperature,
o
pressure and mass ratio, respectively. The lowest yield (2.32 wt%) was Where, T is the temperature ( C) and MR states for the solids to ethanol
achieved at 30 °C/25 MPa/MR 1:1. These results are presented by the mass ratio.
Pareto chart (Fig. 1) which illustrates the influence of the independent According to Box and Wetz [41] and Neto et al. [42], for a regres-
variables on yield, showing that mass ratio and temperature are the sion to be not only statistically significant but also useful for predictive
most statistically significant variables in the extraction process. Both means, the value of Fcalculated must be at least 4–5 times greater than
Ftabulated. Taking this into account, results obtained in this work led to a

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P.N. Cruz et al. The Journal of Supercritical Fluids 146 (2019) 55–64

Fig. 2. (a) Response surface plot showing the effects of the interaction between mass ratio and temperature on the extraction yields and (b) contour curve plot
showing the effects of interaction between mass ratio and temperature on the extraction yields.

significant predictive model, since a good fit (r2 = 0.97) was reached
with Fcalculated (68.30) 15.70 times greater than Ftabulated (4.35) ob-
tained in Fisher distribution table, using α = 0.05, DFregression = 3 and
DFresidual = 7 [42]. Since the model is statistically significant and pre-
dictive, it is possible to perform a response surface in order to easily
identify the regions presenting the highest yields. The Mass Ratio versus
Temperature response surface, as well as the contour curve plot (Fig. 2)
illustrates that the highest yield is located at 70 °C and 3:1 mass ratio.
The kinetic curves for the yacon leaves extraction using
scCO2+EtOH and scCO2+EtOH-H2O described in Table 2 are pre-
sented in Fig. 3.
It can be seen in Fig. 3 that the highest initial extraction rate was
achieved at 70 °C/15 MPa/MR 1:1 (run 2) and after 10 min the highest
extraction rate was achieved at 70 °C/15 MPa/MR 3:1 (run 6). Never-
theless, after 20 min the highest extraction rate was attained at 70 °C/
25 MPa/MR 3:1 (run 8) then keeping this highest extraction rate until
the end of the process. Run 8 provided the most efficient condition,
since the yacon leaves extract was obtained with a high initial extrac-
tion rate, the highest extraction yield (5.22 wt%) and also the highest
extraction percentage (71.60 wt%).
Most curves demonstrate an unconventional characteristic when
compared to extraction using pure supercritical fluid. The three ex-
traction periods named constant extraction rate (CER), falling extrac-
tion rate (FER) and diffusive controlled period (DCP) were observed
only for extractions performed at 30 °C/25 MPa/MR 1:1 (run 3) and at
30 °C/25 MPa/MR 3:1 (run 7). For the other runs, the initial extraction
rate increased and then the extract recovery stagneted. This behavior
indicates that in the very first minutes of extraction the process was
driven by interactions between co-solvent (ethanol) and solute mole-
cules and thus the mass transfer decreased as the co-solvent con-
centration into the extractor also decreased. The same behavior is si-
milar to that observed by Fetzer et al. [37] on their study of
supercritical extraction using scCO2 with co-solvent.
The results of extraction yield demonstrate the good performance of
scCO2 with co-solvent on the recovery of yacon leaves extracts though
the maximum yield had been achieved by the conventional method. In
addition to the good performance of the GLX technique in the extrac-
tion yield, it is worth mentioning the high quality of extracts obtained,
such as strong antioxidant activity, presence of essential fatty acids and
high ω-6/ω-3 fatty acids ratios were found mainly in the extracts

Fig. 3. (a) Experimental kinetic curves for yacon leaves extraction using scCO2+EtOH: (□) 30 °C/15 MPa/MR 1:1, (○) 70 °C/15 MPa/MR 1:1, (◊) 30 °C/25 MPa/MR
1:1, (×) 70 °C/25 MPa/MR 1:1, solid-filled symbols are the replicate at the central point (50 °C/ 20 MPa/MR 2:1) and (b) experimental kinetic curves for yacon leaves
extraction using scCO2+EtOH: ( ) 30 °C/15 MPa/MR 3:1, ( ) 70 °C/15 MPa/MR 3:1, ( ) 30 °C/25 MPa/MR 3:1, ( ) 70 °C/25 MPa/MR 3:1 and using
scCO2+EtOH-H2O: ( ) 30 °C/25 MPa/MR 3:1, ( ) 70 °C/25 MPa/MR 3:1.

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P.N. Cruz et al. The Journal of Supercritical Fluids 146 (2019) 55–64

Table 3
Total phenolic content (TPC) and antioxidant activities (AA) of yacon leaves extracts obtained by different extraction methods.
Run Extraction methods1 Solvent2 TPC5 (mg EAG/gextract) EC50 – DPPH3,5(μg/mL) ABTS5 (μM TEAC/gextract)4

1 SFE 30 °C/15 MPa CO2 + EtOH (1e:1s) 29.34 ± 4.24efg 189 ± 11efgh 97.0 ± 0.2f
2 SFE 70 °C/15 MPa 37.34 ± 7.07cde 178.1 ± 0.6gh 167.0 ± 2.2d
3 SFE 30 °C/25 MPa 28.34 ± 0.1fg 270.9 ± 2.1a 109.1 ± 2.0f
4 SFE 70 °C/25 MPa 38.84 ± 0.71cd 236.5 ± 5.4b 197.6 ± 0.2c
5 SFE 30 °C/15 MPa CO2 + EtOH (3e:1s) 29.09 ± 0.35efg 243 ± 2b 160.2 ± 0.4d
6 SFE 70 °C/15 MPa 39.59 ± 0.35bcd 202.5 ± 3.0cde 195.4 ± 0.4c
7 SFE 30 °C/25 MPa 30.68 ± 1.04efg 260.5 ± 0.3a 158.4 ± 0.04d
8 SFE 70 °C/25 MPa 39.59 ± 0.35bcd 189 ± 2efgh 189.1 ± 1.2c
9 SFE 50 °C/30 MPa CO2 + EtOH (2e:1sa) 32.34 ± 0.71defg 191.1 ± 2.9efg 163.3 ± 0.6d
10 SFE 50 °C/20 MPa 34.09 ± 1.06def 212.9 ± 1.8cd 162.8 ± 0.07d
11 SFE 50 °C/20 MPa 32.68 ± 1.61defg 185 ± 4fgh 161.8 ± 0.01d
12 SFE 30 °C/25 MPa CO2+EtOH-H2O (3e:1s) 16.60 ± 1.06h 273.4 ± 5.8a 132.9 ± 0.03e
13 SFE 70 °C/25 MPa 26.59 ± 1.77fg 215.4 ± 2.8c 166.6 ± 1.4d
14 SE EtOH-H2O 69 ± 0.76a 159.7 ± 2.1i 221.9 ± 2.1b
15 MetOH 42.51 ± 2.31bc 196.8 ± 1.7def 282.7 ± 0.3a
16 EtOH 47.59 ± 1.06b 148 ± 1i 269.0 ± 0.5a
17 EtOAc 25.34 ± 3.53g 174.95 ± 1.3h 163.9 ± 0.9d

1
SFE: supercritical fluid extraction, SE: Soxhlet extraction.
2
EtOH: ethanol, EtOH-H2O: ethanol-water, MetOH: methanol, EtOAC: ethyl acetate.
3
Effective concentration (EC) to obtain 50% of AA.
4
Trolox equivalent antioxidant capacity (TEAC) in μM/g of extract.
5
Equal letters in the same column indicates no significant difference at level of 5% (p < 0.05).

obtained at high pressure. These results will be detailed in Section 3.2 obtained using scCO2+EtOH.
and 3.3.
TPC = 19.65 + 0.24T (4)

3.2. Total phenolic content (TPC) and antioxidant activity (AA)
Table 3 presents the total phenolic content (TPC) and the anti-
oxidant activity (AA) found in yacon leaves extracts. The antioxidant
activity was evaluated by two radical scavenging methods: DPPH• and
ABTS•+. These assays are popular for determination of the antioxidant
activity of foodstuff for the procedures are fast, simple, reproducible
and sensitive [43].
The highest TPC achieved by scCO2 + co-solvent was 39.59 mg
GAE/gextract at 70 °C/15 MPa/MR 3:1 and 70 °C/25 MPa/MR 3:1. On
the other hand, the lowest TPC was 28.34 mg GAE/gextract obtained at
30 °C/25 MPa/MR 1:1. These results can be explained by Pareto chart
(Fig. 4) which represents the influence of the independent variables on
TPC, showing that the temperature is the most influential parameter on
total phenolics extraction. The temperature has a positive effect,
showing that higher temperatures lead to higher TPC extraction. Ro-
driguez et al. [38] also found high TPC values on burdock extracts
obtained by SFE at 70 °C.
Eq. (4) represents the TPC behavior model of yacon leaves extracts
Fig. 4. Pareto chart showing the effect of process parameters on TPC of extracts
obtained using scCO2+EtOH.
Where, T is the extraction temperature (oC)
ANOVA analysis for the TPC showed that the results obtained led to
a significant predictive model since a good fit (r2 = 0.97). The Fcalculated
(64.45) was 14.82 times greater than the Ftabulated (4.35) value from
Fisher's distribution table, with α = 0.05, DFRegression = 3 and
DFResidue = 7 [42].
As the model is statistically significant and predictive, it is possible
to build a response surface in order to evaluate the regions having the
most prominent TPC values. From the response surface plot and the
contour curve plot for TPC varying both temperature and mass ratio
(Fig. 5) it was verified that 70 °C and 3:1 provided the highest TPC
value.
Regarding the SE, the highest TPC was achieved when using EtOH-
H2O as solvent (69 mg GAE/gextract) followed by EtOH (47.59 mg GAE/
gextract). The good performance of EtOH-H2O and EtOH suggests the
presence of medium to high polarity components in yacon leaves due to
the polarity of the solvents (7.1 for EtOH-H2O and 5.2 for EtOH).
Andrade et al. [7] reported TPC values of 42.2 mg GAE/gextract for
yacon leaves extracts obtained by decoction with water. This result is
similar to that found in the present work by SE with EtOH and MetOH
but lower than the value obtained with EtOH-H2O.
A high antioxidant activity for the DPPH method is indicated by a
low EC50 value, therefore, the smaller the EC50 the greater the anti-
oxidant activity of the sample. The best EC50 values were achieved by
SE with EtOH (148 μg/mL) and with EtOH-H2O (159.7 μg/mL).
The antioxidant activities from extracts obtained using scCO2 + co-
solvent showed no significant effect of temperature, pressure or mass
ratio. The best EC50 values were attained at 70 °C/15 MPa/MR 1:1
(178.1 μg/mL) and 50 °C/20 MPa/MR 2:1 (185 μg/mL). According to
Campos et al. [44] extracts with EC50 values bellow 250 μg/mL are
considered as good antioxidants. Therefore, all extraction runs pre-
sented good AA except the run 3 (270 μg/mL). The extracts obtained by
SE with MetOH and EtOH provided the highest results for the ABTS
method (282.7 and 269 μM TEAC/gextract respectively).
Regarding the GLX, the highest ABTS values were 197.6 and 195.4
μM TEAC/gextract obtained at 70 °C/25 MPa/MR 1:1 and at 70 °C/
15 MPa/MR 3:1, respectively. These results may be explained by the
Pareto chart (Fig. 6) that illustrates the influence of the independent

60
P.N. Cruz et al.
Fig. 5. (a) Response surface plot and (b) contour curve plot showing the effects of interaction between mass ratio and temperature on TPC of extracts obtained using
scCO2+EtOH.
Fig. 6. Pareto chart showing the effects of the process parameters on the ABTS
antioxidant assay of extracts obtained using scCO2+EtOH.
variables on the ABTS method, showing that the temperature and the
mass ratio are the most influential variables on antioxidant extraction.
The temperature and mass ratio had positive effects, that is when both
variables rise the antioxidant activity of the extract increases. This fact
is explained by increasing the polarity of the solvent due to the ethanol
addition which increases the solvation power of the polar compounds
emphasizing the antioxidant activity of the extracts [45].
Eq. (5) represents the behavior model for the antioxidant method
ABTS of yacon leaves extracts obtained using scCO2+EtOH.
ABTS = 2.21T + 70.90MR − 0.58T × MR − 0.13P × MR
Where, T is the temperature (ºC), P is the pressure (MPa) and MR is the
solid to ethanol mass ratio.
ANOVA analysis for ABTS showed that the results obtained led to a
significant predictive model since a good fit (r2 = 0.99). The Fcalculated
(72.76) was 11.81 times greater than the Ftabulated (6.16) value from
Fisher's distribution table, with α = 0.05, DFRegression = 6 and
DFResidue = 4 [42].
The temperature x mass ratio response surface plot and the contour
curve plot for ABTS (Fig. 7) show that the best temperature range and
the best mass ratio for antioxidants recovery are 60–70 °C and 3:1, re-
spectively.
The results of TPC and antioxidant assays for yacon leaves extracts
showed from medium to high antioxidant activity whatever the
The Journal of Supercritical Fluids 146 (2019) 55–64
extraction method employed. According to Valentová and Ulrichová [2]
and Andrade et al. [7] the antioxidant activity of yacon leaves extracts
is influenced by phenolics and flavonoids content, also known as nat-
ural antioxidants. Gallic acid, protocatecuic acid, chlorogenic acid,
caffeic acid, ferrulic acid, rutin and myricetin were the compounds
found in yacon leaves extracts.
These results indicate the good efficiency of the scCO2+EtOH
mixture in obtaining antioxidant extracts. This property of scCO2 was
previously reported by several authors who have evaluated different
raw materials such as tamarind, butia and passion fruit seeds
[34,45,46], orange and grape pomaces [47,48], and medicinal plants
such as Arctium lappa, Plantago major and Plantago lanceolata
[35,49,50].
3.3. Fatty acid composition
The fatty acid composition found in yacon leaves extracts is detailed
in Table 4. It can be noted the presence of saturated fatty acids (SAFA)
with relative concentration ranging from 36.29% for extract obtained
using scCO2+EtOH at 50 °C/20 MPa/ MR 2:1 (9) to 71.05% obtained at
70 °C/15 MPa/MR 1:1 (2). The most representative SAFA in yacon
leaves extracts are behenic and palmitic acids.
The relative concentration of unsaturated fatty acids (UFA) obtained
using scCO2+EtOH ranged from 31.6% at 30 °C/15 MPa/MR 3:1 (5), to
63.71% at 50 °C/20 MPa/MR 2:1 (9). According to results shown in
Table 4, gamma-linolenic (ω-6), eicosapentaenoic (ω-3) and linoleic (ω-
6) acids were the major UFAs, specially gamma-linolenic due to its high
content found in all samples whatever the extraction method used.
(5) In the GLX technique, it can be observed that at lower MR (1:1) the
pressure had a greater influence on the extractions of linoleic and γ-
linolenic acids. However, using higher MR values, results showed that
the temperature presented higher effect on the extraction of these
compounds. In addition, when analyzing the effect of the mass ratio on
the extraction, increasing the ethanol amount added to the raw material
the extraction of these unsaturated acids increased. The extracts ob-
tained at 50 °C/20 MPa/MR 2:1 and by SE with ethanol presented the
highest values of γ-linolenic acid with 39.26 and 38.95%, respectively.
The ω-3 and ω-6 groups are not synthesized in the human body then
they must be obtained by means of the feeding, and among the UFAs,
linolenic acids are known as able to reduce the levels of serum coles-
terol, triglycerides and LDL-cholesterol, decreasing the risk of arter-
iolosclerosis, cancer and allergic diseases [51]. According to FAO/WHO
[52] for health maintenance a ω-6/ω-3 ratio in the human diet must be
61
P.N. Cruz et al. The Journal of Supercritical Fluids 146 (2019) 55–64

Fig. 7. (a) Response surface plot and (b) contour curve plot showing the effects of interaction between mass ratio and temperature on ABTS antioxidant assay of
extracts obtained using scCO2+EtOH.

comprised between 5–10. Fig. 8 shows the ω-6/ω-3 fatty acids ratio
found in the different extraction methods.
The results revealed a good nutritional value for the extracts, mainly
for a natural ω-6 source. The best ω-6/ω-3 fatty acids ratio were found
in the extracts obtained using scCO2+EtOH at 50 °C/20 MPa/MR 2:1,
SFE at 70 °C/25 MPa/MR 3:1 and by SE with ethyl acetate with values
of 6.15, 5.54 and 4.68, respectively.
Table 4 shows that although several extraction techniques have
been carried out as well as different solvents and operating conditions
have been also tested, the amount of each fatty acid in different extracts
showed little variation.
4. Conclusions
In this work, scCO2+co-solvent was used to obtain extracts of yacon
leaves (Smallanthus sonchifolius), where the addition of ethanol en-
hanced the extraction efficiency. The best extraction condition in terms
of global yield was achieved at the highest temperature and the highest
mass ratio of ethanol investigated, 70 °C and 3:1, respectively, in-
dicating that these parameters are that most relevant ones on super-
critical extraction of yacon leaves. For the SE extractions, the highest
yield was achieved by using the ethanol-water mixture as solvent.
The results of TPC and antioxidant assays demonstrate a moderate

Table 4
Relative composition profile (% peak area) of yacon leaves extracts obtained by different.
Fatty acids Cx:y Extraction methods and conditions1

scCO2+EtOH Soxhlet extraction

1 2 3 4 5 6 7 8 9 10 11 12 13

Lauric acid C 12:0 – – – – – – – – – – 7.42 – –

Myristic acid C 14:0 – – – – – – – 4.63 – – – 21.45 –
Pentadecanoic acid C 15:0 0.41 – – – – – – – – – – – –
Palmitic acid C 16:0 16.24 27.85 21.6 18.72 1.16 18.77 15.31 19.42 15.63 30.92 29.23 – 17.16
Palmitoleic acid C 16:1 0.38 – 0.63 0.15 – – – 3.48 2.55 – 3.22 – 2.35
Margaric acid C 17:0 – – – – – – – – – – – 20.96 –
Stearic acid C 18:0 1.15 1.83 2.83 2.92 – 2.82 3.49 3.51 3.11 9.38 5.29 – 3.17
Oleic acid C 18:1 n9 0.66 – 4.52 5.15 – 2.83 2.87 2.86 2.18 4.33 3.23 – 2.19
Linoleic acid C 18:2 n6 1.14 2.21 5.90 7.85 0.55 11.00 6.34 10.10 11.47 12.16 8.91 – 8.95
Gama-Linolenic acid C18:3 n6 2.72 6.64 20.7 21.45 3.01 35.95 13.04 30.87 39.26 19.62 18.99 38.95 25.89
Arachidic acid C 20:0 – – – – – – – – – – – 18.64 –
Eicosatrienoic acid C 20:3 n6 1.93 – – – 1.92 – – – – – – – –
Di-homo-alpha-linolenic C 20:3 n3 – – – – 0.76 – – – – 23.59 – – –
Eicosapentaenoic acid C 20:5 n3 2.46 5.41 6.02 10.07 1.10 11.05 17.49 7.40 8.25 – 23.71 – 7.45
Heneicosanoic acid C 21:0 – – – – 0.58 – – – – – – – –
Behenic acid C 22:0 36.13 41.37 37.7 33.69 66.66 17.58 41.46 17.73 17.55 – – – 32.84
Eicosadienoic acid C 22:2 7.8 – – – – – – – – – – – –
Docosahexadienoic acid C22:6 n3 22.10 – – – 0.58 – – – – – – – –
Nerve acid C 24:1 6.88 14.7 – – 23.69 – – – – – – – –
SAFA2 – 53.93 71.05 62.1 55.33 68.4 39.17 60.26 45.29 36.29 40.3 41.94 61.05 53.17
UFA3 – 46.07 28.95 37.9 44.67 31.6 60.83 39.74 54.71 63.71 59.7 58.06 38.95 46.83

1
1: 30 °C/15 MPa/MR 1:1; 2: 70 °C/15 MPa/MR 1:1; 3: 30 °C/25 MPa/MR 1:1; 4: 70 °C/25 MPa/MR 1:1; 5: 30 °C/15 MPa/MR 3:1; 6: 70 °C/15 MPa/MR 3:1; 7:
30 °C/25 MPa/MR 3:1; 8: 70 °C/25 MPa/MR 3:1; 9: 50 °C/20 MPa/MR 2:1; 10: SE ethanol-water 11: SE methanol; 12: SE etanol; 13: SE ethyl acetate.
2
SAFA: saturated fatty acids.
3
UFA: unsaturated fatty acids.

62
P.N. Cruz et al.
to strong antioxidant activity whatever the extraction method em-
ployed except for the run 3 which was carried out at 30 °C/25 MPa/MR
1:1.
All the extracts presented similar fatty acids composition and the
extracts carried out at 50 °C/20 MPa/MR 2:1 and at 70 °C/15 MPa/MR
3:1 showed the highest levels of unsaturated fatty acids. The GLX at
50 °C/20 MPa/MR 2:1 and SE with ethanol presented the highest levels
of gamma-linolenic acid. Moreover, other important fatty acids such as
oleic, linoleic and eicosapentaenoic acids were also identified in the
extracts.
This work has revealed that SFE is a promising technology for ob-
taining bioactive compounds from yacon leaves, because although
higher extraction yileds were found with the conventional technique,
the SFE techique demands lower amount of EtOH and EtOH-H2O, sorter
times of process are possible, provides extracts with high antioxidant
activities, presence of important fatty acids and high ω-6/ω-3 fatty
acids ratio were observed in the results obtained. Thus, these extracts
can be used to develop interesting formulations to pharmaceutical,
cosmetic and functional food industries.
Acknowledgments
The authors wish to thank CAPES (Brazilian agency) for the fi-
nancial support.
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