![Simplified scheme of the SFE installation. Reprinted with permission from [25]. Copyright 2021, Elsevier.](https://www.researchgate.net/publication/353001481/figure/fig2/AS:11431281388056971@1745155754486/Simplified-scheme-of-the-SFE-installation-Reprinted-with-permission-from-25-Copyright_Q320.jpg)
Access to this full-text is provided by MDPI.
Content available from Processes
This content is subject to copyright.
processes
Article
Extraction of Added-Value Triterpenoids from Acacia dealbata
Leaves Using Supercritical Fluid Extraction
Vítor H. Rodrigues , Marcelo M. R. de Melo, Inês Portugal and Carlos M. Silva *
Citation: Rodrigues, V.H.; de Melo,
M.M.R.; Portugal, I.; Silva, C.M.
Extraction of Added-Value
Triterpenoids from Acacia dealbata
Leaves Using Supercritical Fluid
Extraction. Processes 2021,9, 1159.
https://doi.org/10.3390/pr9071159
Academic Editor: Juan
Francisco García Martín
Received: 21 June 2021
Accepted: 2 July 2021
Published: 3 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
CICECO—Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, Campus Universitário de
Santiago, 3810-193 Aveiro, Portugal; vitorhrodrigues@ua.pt (V.H.R.); marcelo.melo@ua.pt (M.M.R.d.M.);
inesport@ua.pt (I.P.)
*Correspondence: carlos.manuel@ua.pt
Abstract:
Forestry biomass is a by-product which commonly ends up being burnt for energy gen-
eration, despite comprising valuable bioactive compounds with valorisation potential. Leaves of
Acacia dealbata were extracted for the first time by supercritical fluid extraction (SFE) using different
conditions of pressure, temperature and cosolvents. Total extraction yield, individual triterpenoids
extraction yields and concentrations were assessed and contrasted with Soxhlet extractions using
solvents of distinct polarity. The extracts were characterized by gas chromatography coupled to mass
spectrometry (GC-MS) and target triterpenoids were quantified. The total extraction yields ranged
from 1.76 to 11.58 wt.% and the major compounds identified were fatty acids, polyols, and, from
the triterpenoids family, lupenone,
α
-amyrin and
β
-amyrin. SFE was selective to lupenone, with
higher individual yields (2139–3512
mg kg−1
leaves
) and concentrations (10.1–12.4 wt.%) in comparison
to Soxhlet extractions, which in turn obtained higher yields and concentrations of the remaining
triterpenoids.
Keywords:
Acacia dealbata; GC-MS; leaves; lupenone; supercritical fluid extraction; Soxhlet extrac-
tion; triterpenoids
1. Introduction
The genus Acacia is widespread through the Portuguese landscape, consisting of three
main species: Acacia dealbata,Acacia longifolia and Acacia melanoxylon [
1
]. A. dealbata was
introduced for dune erosion protection as well as ornamental and wood supply purposes
during the 19th and 20th century [
2
]. Currently, it is considered a plague due to its fast
growth and dominance over the natural flora [
1
,
3
]. From 2005 to 2015, the occupied area of
Acacia species increased 4000 ha in Portugal, corresponding to an estimated total arboreal
biomass growth of 2 Mt [
4
]. The removal of these trees generates forest biomass that, under
the Renewable Energy Directive II of the European Union Commission [
5
], can be utilized
for the production of liquid and gaseous biofuels. However, it is a common practice to
leave these residues in the forest for soil remediation.
The research towards A. dealbata biomass extraction has focused on several morphological
parts, namely wood [
6
–
10
], bark [
6
–
8
,
10
–
14
], flowers [
11
,
15
–
20
] and leaves [
6
,
8
,
11
,
17
,
21
,
22
].
The explored extraction methods so far consist of solid-liquid extraction with organic
solvents, such as dichloromethane, ethanol, methanol, hexane, acetone and some hydroal-
coholic mixtures. Extraction of essential oils by steam distillation has been applied only
to flowers [
16
]. Besides these conventional methods, there are few works on greener and
more innovative extraction procedures, such as the work of Borges et al. [
22
], who applied
microwave and ultrasound-assisted extraction to the leaves, and Lopez-Hortas et al. [
16
],
using microwave hydrodiffusion to obtain the flower essential oil. One alternative tech-
nique for the extraction of vegetable biomass is supercritical fluid extraction (SFE) [
23
].
It is mainly employed with carbon dioxide (CO
2
) as solvent due to its low cost, safety,
Processes 2021,9, 1159. https://doi.org/10.3390/pr9071159 https://www.mdpi.com/journal/processes
Processes 2021,9, 1159 2 of 15
availability and low critical point conditions, which allows extraction at near room tem-
peratures [
24
]. The manipulation of temperature and pressure allows the tuning of CO
2
properties (density, viscosity and diffusivity) that maximize the desired responses, such as
total extraction yield or the selective uptake of target chemical families or compounds. For
example, triterpenic acids in the case of Eucalyptus globulus leaves [
25
,
26
] and bark [
27
–
29
],
triterpenes from Vitis vinifera leaves [
30
], friedelin from Quercus cerris cork [
31
] and sterols
from Eichhornia crassipes [32].
The phytochemistry of A. dealbata biomass (bark, leaves, wood, flowers and seeds) in-
cludes several families of compounds, such as alkaloids [
8
], amines [
33
], phenolics [
8
,
10
,
13
,
15
],
polysaccharides [
9
], chalcone glycosides [
18
], steryl glucosides [
7
], tannins [
10
,
11
,
13
], caf-
feic acid esters [
12
], sterols [
6
] and triterpenes [
6
,
17
]. SFE is a proven technology for the
selective removal of triterpenes and sterols from many vegetable matrices [
23
,
30
–
32
,
34
,
35
].
For instance, compounds such as lupenone, lupeol, lupenyl palmitate, lupenyl cinnamate,
squalene,
β
-amyrone,
α
-amyrin,
β
-amyrin and 22,23-dihydrospinasterol have already
been identified and quantified [
6
,
17
]. These have been reported for several potential
bioactive properties, namely anti-inflammatory, anti-virus, anti-diabetes, anti-cancer and
antiproliferative, among others [
36
–
45
] which may explain the association of Acacia species
with traditional medicine practices [
46
,
47
]. The wide range of biological activities poten-
tiates the interest for multiple applications of the extracts, for example, to obtain active
pharmaceutical ingredients or for incorporation in nutraceuticals, food, animal feed and
cosmetic products.
This work focuses on the SFE of triterpenoids from Acacia dealbata leaves under
different experimental conditions of pressure, temperature and cosolvents content, and its
comparison with conventional Soxhlet extraction using organic solvents of distinct polarity.
The extracts were characterized by gas chromatography coupled to mass spectrometry
(GC-MS) and triterpenoids contents were determined. To the best of our knowledge, this is
the first time SFE is applied to Acacia dealbata leaves aiming for the extraction of potential
bioactive compounds.
2. Materials and Methods
2.1. Chemicals
Carbon dioxide (CO
2
, purity 99%) was supplied by Air Liquide (Algés, Portugal).
Dichloromethane (purity 99.98%), n-hexane (purity 99%) and ethanol (purity 99.5%) were
supplied by Fisher Scientific (Leicestershire, UK). Ethyl acetate (purity 99%) was sup-
plied by VWR International (Fontenay-sous-Bois, France). Pyridine (purity 99.5%), tetra-
cosane (purity 99%) N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, purity 98%) and
chlorotrimethylsilane (TMSCl, purity 99%) were supplied by Sigma Aldrich (Madrid,
Spain). Betulinic, oleanolic and ursolic acids (purity 98%) were supplied by AK Scientific
(Union City, CA, USA).
2.2. Acacia Dealbata Biomass
The A. dealbata leaves were supplied by RAIZ—Forest and Paper Research Institute
(Eixo, Portugal). The leaves were collected from 8-year-old Acacia dealbata trees located in
Porto/Valongo (Portugal) region during the winter season. The leaves (see Figure 1) were
manually separated from the branches and dried at 35
◦
C for 72 h in a forced convection
oven, reducing their moisture content from 65.6 to 4.5 wt.%.
Processes 2021,9, 1159 3 of 15
Processes 2021, 9, x FOR PEER REVIEW 3 of 16
Figure 1. Oven-dried Acacia dealbata leaves.
2.3. Soxhlet Extraction
The leaves of A. dealbata were extracted with n-hexane, dichloromethane, ethyl
acetate and ethanol (Table 1). In each assay, extraction of the leaves (ca. 3 g) was performed
with 180 mL of each solvent for 6 h. The produced extracts were evaporated to dryness in
a rotary evaporator, weighed for the determination of total extraction yield (, wt.%)
and analyzed by GC-MS to evaluate triterpenoids individual yields (, mgkg
) and
concentrations (, wt.%), as follows:
=
× 100 (1)
=
×10
(2)
=
× 100 (3)
where is the mass of dry leaves, corresponds to extract mass free of
solvent and is the mass of triterpenoids measured by GC-MS.
Table 1. List of Soxhlet and SFE assays with the respective operating conditions.
Run Method Solvent T
(°C)
P
(bar)
()
SX1 Soxhlet n-Hexane 68.5 * 1 -
SX2 Dichloromethane 39.6 * 1 -
SX3 Ethyl acetate 77.1 * 1 -
SX4 Ethanol 78.4 * 1 -
SFE1 SFE CO2 40 200 840.6 [48]
SFE2 CO2 80 200 594.9 [48]
SFE3 CO2 60 300 830.4 [48]
SFE4 CO2:Ethanol (95:5 wt.%) 80 300 764.6 [49]
SFE5 CO2:Ethyl acetate (95:5 wt.%) 80 300 761.4 [50]
* boiling point of the pure solvents.
Figure 1. Oven-dried Acacia dealbata leaves.
2.3. Soxhlet Extraction
The leaves of A. dealbata were extracted with n-hexane, dichloromethane, ethyl acetate
and ethanol (Table 1). In each assay, extraction of the leaves (ca. 3 g) was performed with
180 mL of each solvent for 6 h. The produced extracts were evaporated to dryness in a
rotary evaporator, weighed for the determination of total extraction yield (
ηTotal
, wt.%)
and analyzed by GC-MS to evaluate triterpenoids individual yields (
ηi
,
mg kg−1
leaves
) and
concentrations (Ci, wt.%), as follows:
ηTotal =mextract
mdry leaves
×100 (1)
ηi=mi
mdry leaves
×106(2)
Ci=mi
mextract
×100 (3)
where
mdry leaves
is the mass of dry leaves,
mextract
corresponds to extract mass free of
solvent and miis the mass of triterpenoids measured by GC-MS.
Table 1. List of Soxhlet and SFE assays with the respective operating conditions.
Run Method Solvent T
(◦C)
P
(bar)
ρf
(kg m−3)
SX1 Soxhlet n-Hexane 68.5 * 1 -
SX2 Dichloromethane 39.6 * 1 -
SX3 Ethyl acetate 77.1 * 1 -
SX4 Ethanol 78.4 * 1 -
SFE1 SFE CO240 200 840.6 [48]
SFE2 CO280 200 594.9 [48]
SFE3 CO260 300 830.4 [48]
SFE4 CO2:Ethanol (95:5 wt.%) 80 300 764.6 [49]
SFE5 CO2:Ethyl acetate (95:5 wt.%) 80 300 761.4 [50]
* boiling point of the pure solvents.
Processes 2021,9, 1159 4 of 15
2.4. Supercritical Fluid Extraction
The SFE assays were performed in a lab scale Spe-ed SFE unit, a model of Helix
SFE System-Applied Separations, Inc., (Allentown, PA, USA) schematically presented in
Figure 2. In each run, ca. 25 g of leaves were loaded into the extractor while the supercritical
fluid flowed upwards at constant flow rate (
QCO2
) of 12
g min−1
for 6 h. The experimental
conditions of pressure, temperature and cosolvent content are presented in Table 1(runs
SFE1 to SFE3). The detailed procedure is described elsewhere [
25
]. The total extraction
yield, the individual compound concentrations and respective yields were determined
according to Equations (1)–(3), respectively.
Processes 2021, 9, x FOR PEER REVIEW 4 of 16
2.4. Supercritical Fluid Extraction
The SFE assays were performed in a lab scale Spe-ed SFE unit, a model of Helix SFE
System-Applied Separations, Inc., (Allentown, PA, USA) schematically presented in
Figure 2. In each run, ca. 25 g of leaves were loaded into the extractor while the
supercritical fluid flowed upwards at constant flow rate (
) of 12 gmin for 6 h. The
experimental conditions of pressure, temperature and cosolvent content are presented in
Table 1 (runs SFE1 to SFE3). The detailed procedure is described elsewhere [25]. The total
extraction yield, the individual compound concentrations and respective yields were
determined according to Equations (1)–(3), respectively.
Figure 2. Simplified scheme of the SFE installation. Reprinted with permission from [25]. Copyright
2021, Elsevier.
For the runs, SFE4 and SFE5 ethanol and ethyl acetate were added as cosolvent to
modify the supercritical fluid polarity and the solubility of solutes. In these runs, the
cosolvent was fed to the pre-heating vessel using a HPLC pump, as presented in Figure 2.
The densities of the supercritical fluids (), both pure and modified supercritical
carbon dioxide (SC-CO
2
), at each experimental condition are presented in Table 1. They
were obtained using the equation of state of Pitzer and Schreiber for pure SC-CO
2
[48],
Falco and Kiran for SC-CO
2
modified with ethyl acetate [50] and Pöhler and Kiran for SC-
CO
2
modified with ethanol [49].
2.5. Gas Chromatography Coupled to Mass Spectrometry
The extracts were analyzed by GC–MS using a Trace Gas Chromatograph Ultra
equipped with a DB-1 J&W capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness)
and coupled with a Thermo DSQ mass spectrometer. The extracts were prepared and
analyzed following a procedure previously published [28,29]. For the quantification of
individual triterpenoids in the extracts, tetracosane and pure betulinic, oleanolic and
ursolic acids were selected as internal and external standards, respectively. The
identification of the compounds was performed with the aid of reverse match factors (RSI)
from Wiley 9 library, which defines thresholds of mass spectral match: 900 and above is
considered excellent; 800–900 is considered good, 700–800 is considered fair, and below
700 is considered a poor match [51].
Figure 2.
Simplified scheme of the SFE installation. Reprinted with permission from [
25
]. Copyright
2021, Elsevier.
For the runs, SFE4 and SFE5 ethanol and ethyl acetate were added as cosolvent to
modify the supercritical fluid polarity and the solubility of solutes. In these runs, the
cosolvent was fed to the pre-heating vessel using a HPLC pump, as presented in Figure 2.
The densities of the supercritical fluids (
ρf
), both pure and modified supercritical
carbon dioxide (SC-CO
2
), at each experimental condition are presented in Table 1. They
were obtained using the equation of state of Pitzer and Schreiber for pure SC-CO
2
[
48
],
Falco and Kiran for SC-CO
2
modified with ethyl acetate [
50
] and Pöhler and Kiran for
SC-CO2modified with ethanol [49].
2.5. Gas Chromatography Coupled to Mass Spectrometry
The extracts were analyzed by GC–MS using a Trace Gas Chromatograph Ultra
equipped with a DB-1 J&W capillary column (30 m
×
0.32 mm i.d., 0.25
µ
m film thickness)
and coupled with a Thermo DSQ mass spectrometer. The extracts were prepared and
analyzed following a procedure previously published [
28
,
29
]. For the quantification of
individual triterpenoids in the extracts, tetracosane and pure betulinic, oleanolic and ursolic
acids were selected as internal and external standards, respectively. The identification of the
compounds was performed with the aid of reverse match factors (RSI) from Wiley 9 library,
which defines thresholds of mass spectral match: 900 and above is considered excellent;
800–900 is considered good, 700–800 is considered fair, and below 700 is considered a poor
match [51].
Processes 2021,9, 1159 5 of 15
3. Results and Discussion
3.1. Total Extraction Yield
The total extraction yield (
ηTotal
, Equation (1)) measures the total extract amount
produced independently of its composition. The results obtained with Soxhlet and SFE can
be visualized in Figure 3. Concerning Soxhlet extraction, the
ηTotal
values increased with
the polarity of the solvent and ranged from 3.60 to 11.58 wt.% for dichloromethane and
ethanol, respectively. The second highest value (7.97 wt.%) was obtained by ethyl acetate,
and n-hexane achieved an equivalent value to dichloromethane (3.64 wt.%). Literature
results on Soxhlet extractions of A. dealbata leaves present unequal yield scores. For instance,
Oliveira et al. [
6
] obtained a
ηTotal
of 6.2 wt.% with dichloromethane, almost double of the
value obtained in this work, which may be due to the particle size reduction performed.
On the other hand, Luís et al. [
8
] obtained a
ηTotal
of 6.75 wt.% with ethanol, which is
considerably lower than the value obtained in this work, and may be due to a different
time of extraction, as it was stopped as soon as the extraction solvent became colorless.
Borges et al. [
22
] obtained a
ηTotal
of 13 wt.% using water and 16 h of Soxhlet extraction,
which can compare with the value obtained with ethanol (11.58 wt.%), the closest solvent
in terms of polarity. However, it is noteworthy that extraction with ethanol (6 h at 78.4
◦
C)
would be more energy efficient than with water (16 h at 100 ◦C).
Processes 2021, 9, x FOR PEER REVIEW 5 of 16
3. Results and Discussion
3.1. Total Extraction Yield
The total extraction yield (, Equation (1)) measures the total extract amount
produced independently of its composition. The results obtained with Soxhlet and SFE
can be visualized in Figure 3. Concerning Soxhlet extraction, the values increased
with the polarity of the solvent and ranged from 3.60 to 11.58 wt.% for dichloromethane
and ethanol, respectively. The second highest value (7.97 wt.%) was obtained by ethyl
acetate, and n-hexane achieved an equivalent value to dichloromethane (3.64 wt.%).
Literature results on Soxhlet extractions of A. dealbata leaves present unequal yield scores.
For instance, Oliveira et al. [6] obtained a of 6.2 wt.% with dichloromethane, almost
double of the value obtained in this work, which may be due to the particle size reduction
performed. On the other hand, Luís et al. [8] obtained a of 6.75 wt.% with ethanol,
which is considerably lower than the value obtained in this work, and may be due to a
different time of extraction, as it was stopped as soon as the extraction solvent became
colorless. Borges et al. [22] obtained a of 13 wt.% using water and 16 h of Soxhlet
extraction, which can compare with the value obtained with ethanol (11.58 wt.%), the
closest solvent in terms of polarity. However, it is noteworthy that extraction with ethanol
(6 h at 78.4 °C) would be more energy efficient than with water (16 h at 100 °C).
Figure 3. Total extraction yields () obtained by Soxhlet using n-hexane, dichloromethane (DCM), ethyl acetate (EA),
ethanol (E) and SFE at different conditions.
Regarding SFE assays, the results varied from 1.76 to 3.26 wt.% for SFE1 (200
bar, 40 °C, no cosolvent) and SFE5 (300 bar, 80 °C, 5 wt.% ethanol), respectively. Three
experimental parameters were tested in these runs: pressure, temperature and cosolvent
addition. Setting SFE1 (200 bar, 40 °C, no cosolvent) as the reference run, the effect of
temperature on can be assessed by comparison with run SFE2 (200 bar, 80 °C, no
cosolvent), where an increase of 38% was observed. Even though higher temperatures
lower the solvent power due to SC-CO2 density decrease (840.6 kg m−3 for SFE1 and 594.6
kg m−3 for SFE2), they increase the vapour pressure of solutes (i.e., their solubility in the
supercritical solvent). These opposing effects can lead to different results from system to
Figure 3.
Total extraction yields (
ηTotal
) obtained by Soxhlet using n-hexane, dichloromethane (DCM), ethyl acetate (EA),
ethanol (E) and SFE at different conditions.
Regarding SFE assays, the
ηTotal
results varied from 1.76 to 3.26 wt.% for SFE1 (200 bar,
40
◦
C, no cosolvent) and SFE5 (300 bar, 80
◦
C, 5 wt.% ethanol), respectively. Three experi-
mental parameters were tested in these runs: pressure, temperature and cosolvent addition.
Setting SFE1 (200 bar, 40
◦
C, no cosolvent) as the reference run, the effect of temperature on
ηTotal
can be assessed by comparison with run SFE2 (200 bar, 80
◦
C, no cosolvent), where an
increase of 38% was observed. Even though higher temperatures lower the solvent power
due to SC-CO
2
density decrease (840.6 kg m
−3
for SFE1 and 594.6 kg m
−3
for SFE2), they
increase the vapour pressure of solutes (i.e., their solubility in the supercritical solvent).
These opposing effects can lead to different results from system to system, and in this case,
the solubility enhancement effect was prevalent. Moving to Run SFE3 (300 bar, 60
◦
C, no
Processes 2021,9, 1159 6 of 15
cosolvent), this assay was performed at more 100 bar and less 20
◦
C of SFE2, but yielded
an identical
ηTotal
(2.37 wt.%). This occurs possibly because the higher SC-CO
2
density in
Run 3 (830.4 kg m
−3
, i.e., 40% more than in SFE2) compensated the thermal penalization
on the vapor pressure side of solutes. Furthermore, the addition of cosolvents was tested
at 300 bar and 80
◦
C in runs SFE4 (5 wt.% of ethanol) and SFE5 (5 wt.% of ethyl acetate).
The results were identical for both runs (3.24–3.26 wt.%), almost doubling the
ηTotal
value
of SFE1. The different cosolvents did not significantly affect the fluid densities between
each other (764.6 kg m
−3
for SFE4 and 761.4 kg m
−3
for SFE5) and imposed an increase of
only 2.4% in relation to pure SC-CO
2
under the same
P−T
conditions (746.2 kg m
−3
) [
48
].
Hence, the yield gain in runs SFE4 and SFE5 was attributed to a greater affinity of the
solute to solvent mixtures of higher polarity.
Overall, it is clear that Soxhlet extraction produces higher
ηTotal
, especially when
employing high polarity solvents (i.e., ethanol or ethyl acetate). When employing low
polarity solvents (i.e., dichloromethane or n-hexane), the
ηTotal
values are analogous to
those of modified SC-CO2and higher than those of SFE without entrainers.
3.2. Volatile Extractives
The extracts were analyzed by GC-MS, and the chromatograms of runs SX2 (A), SX4
(B) and SFE1 (C) are presented in Figure 4A–C. These are representative of the Soxhlet ex-
tractions with low and higher polarity solvents, and SFE runs, respectively. It is possible to
observe that the chromatograms of runs SX2 and SFE1 (Figure 4A,C) are considerably simi-
lar, as the same peaks appear in both, which in turn confirms the affinity of dichloromethane
and SC-CO
2
to similar solutes. On the contrary, SX4 (Figure 4B) stands out due to the
proliferation of peaks in the region on the left of the internal standard—i.e., at retention
time (Rt) lower than 38.76 min—and also due to the very sharp peak at Rt = 27.55 min,
identified as myo-inositol (whose structure is disclosed in Figure 5). The latter forced a
rescaling of the relative absorbance axis to a comparable range (from 100% in SX2 and SFE1
to 6% in SX4). All but one of the identified compounds in these peaks encompassed RSI
scores comprehended between 700 and 942, which correspond to a matching quality from
fair to excellent. In fact, the only exception to this was
α
-amyrin, whose RSI score was 691.
The full list of identified compounds for all Soxhlet extractions and SFE runs is
reported in Table 2, altogether with the respective retention times and maximum RSI scores
within the analyzed extracts. As previously observed in Figure 4B, run SX4 shows more
peaks in the left half of the chromatogram, and that observation can be confirmed in Table 2,
also for runs SX3 (ethyl acetate) and SX4 (ethanol), specifically amid retention times of 10.77
to 44.37 min. The said peaks consist mainly of polyols (P) and monosaccharides (M). After
these come the fatty acids (FA), long-chain aliphatic alcohols (LCAA) and triterpenoids
(TT), although these were also found in every extract. Furthermore, SX1 (n-hexane) and
SX2 (dichloromethane) present a very similar pattern of detected compounds, as well as
the runs SFE1-SFE5. This corroborates what was observed in the analysis of Figure 4A,C.
Such similarities show that the polarity of the solvent strongly influences the compounds
extracted, and that, in the case of SFE, the modification of SC-CO
2
with 5 wt.% of ethanol
or ethyl acetate was not enough to lead to the extraction of volatile compounds with higher
affinity to polar organic solvents.
Processes 2021,9, 1159 7 of 15
Processes 2021, 9, x FOR PEER REVIEW 7 of 16
Figure 4. Chromatograms of the extracts from runs (A) SX2 (dichloromethane), (B) SX4 (ethanol) and (C) SFE1 (200, bar
40 °C). Internal standard (tetracosane) appears at 38.7 min.
Table 2. List of identified compounds and respective retention times and reverse match factors (RSI) for all Soxhlet and
SFE runs.
Rt (min) Compound Family RSI SX1 SX2 SX3 SX4 SFE1 SFE2 SFE3 SFE4 SFE5
10.77 Glycerol P 909 - + + + - - - - -
18.67 Erythritol P 942 - - + + - - - - -
20.6 5-Hydroxypipecolic acid CA 770 - - - + - - - - -
24.17 Xylitol P 800 - - + + - - - - -
24.79 Ribitol P 887 - - + + - - - - -
27.55 myo-Inositol P 803 - - + + - - - - -
28.19 Tyramine A 890 - - - + - - - - -
28.79 D-Mannose M 803 - - + + - - - - -
30.13 Mannitol P 851 - - + + - - - - -
31.27 Glucose M 827 - - + + - - - - -
Figure 4.
Chromatograms of the extracts from runs (
A
) SX2 (dichloromethane), (
B
) SX4 (ethanol) and (
C
) SFE1 (200, bar
40 ◦C). Internal standard (tetracosane) appears at 38.7 min.
Processes 2021, 9, x FOR PEER REVIEW 8 of 16
44.37 Docosanoic acid FA 729 + + - - + + + + +
46.09 Squalene TT 833 - - - - + + + + +
47.76 Pentacosanoic acid FA 829 + + + + + + + + +
48.61 Hexadecanoic acid FA 705 + + + - - - - - -
50.98 Octacosanoic acid FA 849 + + + + + + + + +
51.82 Octacosan-1-ol LCAA 811 + - + - - - - - -
52.66 4’-OH,5-OH,7-Di-O-Glucoside F 688 + + + + + + + + +
52.86 α-Amyrone TT 786 + + - - + + + + +
53.06 β-Amyrone TT 738 + + + + + + + + +
53.66 Lupenone TT 843 + + + + + + + + +
54.92 β-Amyrin TT 755 + + + + + + + + +
55.16 α-Amyrin TT 691 + + + + + + + + +
56.28 Lupenyl acetate TT 742 + + - - + + + + +
* P—polyol; CA—carboxylic acid; A—amine; M—monosaccharide; FA—fatty acid; TT—triterpenoid; LCAA—long-chain aliphatic
alcohol; F—flavonoid.
As observed in Figure 4B, myo-inositol (see Figure 5) peak has an area of different
magnitude from the targeted triterpenoids, thirteen times higher (run SX4) than the
internal standard. Even though it is a known constituent of plant and animal cells, the
results obtained demonstrate the potential of ethanol and ethyl acetate, and potentially
other polar organic solvents, for the production of myo-inositol-rich extracts. According to
the literature, this compound and its derivatives have been identified and quantified in
several plant species [52–56], including Acacia trees, such as in Acacia pennata and Acacia
farnesiana leaves [57], and Acacia mangium and Acacia maidenii seeds [58]. Moreover, myo-
inositol plays an important role in several cell functions, such as growth, development
and reproduction, among others [59]. As a dietary supplement, it can be beneficial for
human disorders associated with insulin resistance, such as polycystic ovary syndrome,
gestational diabetes mellitus or metabolic syndrome, and the prevention or treatment of
some diabetic complications, namely, neuropathy, nephropathy and cataract [59]. Even
though this represents a promising result for the valorisation of Acacia dealbata biomass,
the main focus of this work is the triterpenoid fraction attainable by SFE, which leaves
myo-inositol out of the work scope. Nevertheless, it might open the way to sequential
extraction strategies.
Figure 5. Structural formula of myo-inositol.
The main triterpenoids (TT) identified in the produced extracts were squalene, α-
amyrone, β-amyrone, lupenone, β-amyrin, α-amyrin and lupenyl acetate. These contain
30 carbon atoms, except for lupenyl acetate which has 32, and all of these compounds are
interrelated by known biosynthesis pathways, as summarized in Figure 6. Here, it can be
seen that squalene is the prime precursor, having been originated by successive
condensation reactions of the isomers of isopentenyl diphosphate and dimethylallyl
Figure 5. Structural formula of myo-inositol.
Processes 2021,9, 1159 8 of 15
Table 2.
List of identified compounds and respective retention times and reverse match factors (RSI) for all Soxhlet and
SFE runs.
Rt (min) Compound Family RSI SX1 SX2 SX3 SX4 SFE1 SFE2 SFE3 SFE4 SFE5
10.77 Glycerol P 909 - + + + - - - - -
18.67 Erythritol P 942 - - + + - - - - -
20.6 5-Hydroxypipecolic
acid CA 770 - - - + - - - - -
24.17 Xylitol P 800 - - + + - - - - -
24.79 Ribitol P 887 - - + + - - - - -
27.55 myo-Inositol P 803 - - + + - - - - -
28.19 Tyramine A 890 - - - + - - - - -
28.79 D-Mannose M 803 - - + + - - - - -
30.13 Mannitol P 851 - - + + - - - - -
31.27 Glucose M 827 - - + + - - - - -
44.37 Docosanoic acid FA 729 + + - - + + + + +
46.09 Squalene TT 833 - - - - + + + + +
47.76 Pentacosanoic acid FA 829 + + + + + + + + +
48.61 Hexadecanoic acid FA 705 + + + - - - - - -
50.98 Octacosanoic acid FA 849 + + + + + + + + +
51.82 Octacosan-1-ol LCAA 811 + - + - - - - - -
52.66 4’-OH,5-OH,7-Di-O-
Glucoside F688+++++++++
52.86 α-Amyrone TT 786 + + - - + + + + +
53.06 β-Amyrone TT 738 + + + + + + + + +
53.66 Lupenone TT 843 + + + + + + + + +
54.92 β-Amyrin TT 755 + + + + + + + + +
55.16 α-Amyrin TT 691 + + + + + + + + +
56.28 Lupenyl acetate TT 742 + + - - + + + + +
P—polyol; CA—carboxylic acid; A—amine; M—monosaccharide; FA—fatty acid; TT—triterpenoid; LCAA—long-chain aliphatic alcohol;
F—flavonoid.
As observed in Figure 4B, myo-inositol (see Figure 5) peak has an area of different
magnitude from the targeted triterpenoids, thirteen times higher (run SX4) than the internal
standard. Even though it is a known constituent of plant and animal cells, the results
obtained demonstrate the potential of ethanol and ethyl acetate, and potentially other
polar organic solvents, for the production of myo-inositol-rich extracts. According to
the literature, this compound and its derivatives have been identified and quantified in
several plant species [
52
–
56
], including Acacia trees, such as in Acacia pennata and Acacia
farnesiana leaves [
57
], and Acacia mangium and Acacia maidenii seeds [
58
]. Moreover, myo-
inositol plays an important role in several cell functions, such as growth, development
and reproduction, among others [
59
]. As a dietary supplement, it can be beneficial for
human disorders associated with insulin resistance, such as polycystic ovary syndrome,
gestational diabetes mellitus or metabolic syndrome, and the prevention or treatment of
some diabetic complications, namely, neuropathy, nephropathy and cataract [
59
]. Even
though this represents a promising result for the valorisation of Acacia dealbata biomass,
the main focus of this work is the triterpenoid fraction attainable by SFE, which leaves
myo-inositol out of the work scope. Nevertheless, it might open the way to sequential
extraction strategies.
The main triterpenoids (TT) identified in the produced extracts were squalene,
α
-
amyrone,
β
-amyrone, lupenone,
β
-amyrin,
α
-amyrin and lupenyl acetate. These contain
30 carbon atoms, except for lupenyl acetate which has 32, and all of these compounds
are interrelated by known biosynthesis pathways, as summarized in Figure 6. Here, it
can be seen that squalene is the prime precursor, having been originated by successive
condensation reactions of the isomers of isopentenyl diphosphate and dimethylallyl diphos-
phate [
60
–
62
]. Eventually, squalene can be oxidized to 2,3-oxidosqualene, which in turn
is the direct precursor of tricyclic, tetracyclic or pentacyclic triterpenoids. When under
the chair-chair-chair conformation, 2,3-oxidosqualene can undergo cyclization reactions
forming the tetracyclic dammarenyl cation, which, after ring expansions, originates diverse
Processes 2021,9, 1159 9 of 15
skeletons of pentacyclic triterpenoids, such as the lupane, oleanane and ursane types (see
Figure 6). The latter three types are the precursors of lupeol,
β
-amyrin and
α
-amyrin,
respectively [
60
–
63
]. Upon undergoing further rearrangements, and/or oxidation, sub-
stitution or glycosylation reactions, other triterpenoids are generated, namely lupenone,
β
-amyrone and
α
-amyrone (as the ketone versions of lupeol,
β
-amyrin and
α
-amyrin,
respectively), and lupenyl acetate (as the acetylated version of lupeol). To conclude, the
majority of these compounds were also identified in previous extraction works of A. deal-
bata biomass [
6
,
17
], especially in the leaves, bark and other external parts, since they are
thought to provide protection against insects and microbes [63].
Processes 2021, 9, x FOR PEER REVIEW 9 of 16
diphosphate [60–62]. Eventually, squalene can be oxidized to 2,3-oxidosqualene, which in
turn is the direct precursor of tricyclic, tetracyclic or pentacyclic triterpenoids. When
under the chair-chair-chair conformation, 2,3-oxidosqualene can undergo cyclization
reactions forming the tetracyclic dammarenyl cation, which, after ring expansions,
originates diverse skeletons of pentacyclic triterpenoids, such as the lupane, oleanane and
ursane types (see Figure 6). The latter three types are the precursors of lupeol, β-amyrin
and α-amyrin, respectively [60–63]. Upon undergoing further rearrangements, and/or
oxidation, substitution or glycosylation reactions, other triterpenoids are generated,
namely lupenone, β-amyrone and α-amyrone (as the ketone versions of lupeol, β-amyrin
and α-amyrin, respectively), and lupenyl acetate (as the acetylated version of lupeol). To
conclude, the majority of these compounds were also identified in previous extraction
works of A. dealbata biomass [6,17], especially in the leaves, bark and other external parts,
since they are thought to provide protection against insects and microbes [63].
Figure 6. Scheme representative of synthesis pathways of triterpenoids from squalene, namely the lupane, oleanane and
ursane series.
3.3. Triterpenoid Extraction Yields
The individual and total triterpenoid extraction yields of Soxhlet and
SFE assays are presented in Figure 7A,B, respectively. Regarding Soxhlet extractions
(Figure 7A), lupenone was the most extracted compound of the four organic solvents
tested, with values ranging from 2114 to 2994 mgkg
for n-hexane and ethyl
acetate extraction, respectively. It was followed by α-amyrin, with yields from 1249 to
2851 mgkg
for dichloromethane and ethyl acetate, respectively. Although
was generally lower than , this difference is especially evident in the
Figure 6.
Scheme representative of synthesis pathways of triterpenoids from squalene, namely the lupane, oleanane and
ursane series.
3.3. Triterpenoid Extraction Yields
The individual
(ηi)
and total triterpenoid
(ηTotal TT)
extraction yields of Soxhlet
and SFE assays are presented in Figure 7A,B, respectively. Regarding Soxhlet extrac-
tions (Figure 7A), lupenone was the most extracted compound of the four organic sol-
vents tested, with
ηlupenone
values ranging from 2114 to 2994
mg kg−1
leaves
for n-hexane
and ethyl acetate extraction, respectively. It was followed by
α
-amyrin, with yields from
1249 to 2851
mg kg−1
leaves
for dichloromethane and ethyl acetate, respectively. Although
ηβ−amyrin
was generally lower than
ηα−amyrin
, this difference is especially evident in the
dichloromethane Soxhlet extract, where the latter yielded circa 2.7 times more. In turn,
ethyl acetate attenuated the two amyrin yields, with their ratio falling to ca. 1.6. For
extracts produced with more polar solvents, the two amyrin yields approached the values
of
ηlupenone
. This was translated to the magnitude of
ηTotal TT
, which incremented from
the minimum of 4908
mg kg−1
leaves
for n-hexane to 8201
mg kg−1
leaves
for ethyl acetate and
Processes 2021,9, 1159 10 of 15
to 6259
mg kg−1
leaves
for ethanol. The remaining triterpenoids did not differ significantly
between organic solvents, and their individual yields were markedly low.
Processes 2021, 9, x FOR PEER REVIEW 10 of 16
dichloromethane Soxhlet extract, where the latter yielded circa 2.7 times more. In turn,
ethyl acetate attenuated the two amyrin yields, with their ratio falling to ca. 1.6. For
extracts produced with more polar solvents, the two amyrin yields approached the values
of . This was translated to the magnitude of , which incremented from
the minimum of 4908 mgkg
for n-hexane to 8201 mgkg
for ethyl acetate and to
6259 mgkg
for ethanol. The remaining triterpenoids did not differ significantly be-
tween organic solvents, and their individual yields were markedly low.
Figure 7. Individual and total triterpenoid (Total TT) extraction yields in: (A) Soxhlet extracts using different organic sol-
vents, and (B) SFE at different conditions of temperature, pressure, and ethanol (E) or ethyl acetate (EA) content as modi-
fiers.
Concerning uptake of triterpenoids by SFE (see Figure 7B), lupenone confirmed its
leading individual yield also in this separation method, which varied from 2139 to 3512
mgkg
for SFE1 (200 bar, 40 °C, no cosolvent) and SFE4 (300 bar, 80 °C, 5 wt.% of
ethanol), respectively. None of the remaining triterpenoids surpassed 500 mgkg
Figure 7.
Individual and total triterpenoid (Total TT) extraction yields in: (
A
) Soxhlet extracts using different organic
solvents, and (
B
) SFE at different conditions of temperature, pressure, and ethanol (E) or ethyl acetate (EA) content
as modifiers.
Concerning uptake of triterpenoids by SFE (see Figure 7B), lupenone confirmed
its leading individual yield also in this separation method, which varied from 2139 to
3512
mg kg−1
leaves
for SFE1 (200 bar, 40
◦
C, no cosolvent) and SFE4 (300 bar, 80
◦
C, 5 wt.%
of ethanol), respectively. None of the remaining triterpenoids surpassed 500
mg kg−1
leaves
threshold. The increase of temperature from 40
◦
C to 80
◦
C of runs SFE1 and SFE2,
Processes 2021,9, 1159 11 of 15
respectively, improved the
ηlupenone
by 41% and, if contrasted with the 38% increase
verified in the
ηTotal
(recall Figure 3), represents a proportional increase. Furthermore, run
SFE3 (300 bar, 60
◦
C, no cosolvent) shows that even though it produced a similar
ηTotal
of SFE2, the joint
P−T
change decreased the lupenone uptake to 2879
mg kg−1
leaves
. This
confirms that the favorable effect of temperature on solubility prevailed over the loss of
SC-CO
2
density. With the employment of ethanol and ethyl acetate at 300 bar and 80
◦
C
(runs SFE4 and SFE5), higher lupenone yields were obtained, namely 3512
mg kg−1
leaves
and 3273
mg kg−1
leaves
, respectively. This suggests that the joint optimization of
P
,
T
and
cosolvent content can be determinant to potentiate the removal of this compound by SFE.
The low yields for the other triterpenoids explain the lower
ηTotal TT
obtained by SFE.
These were only slightly inferior to the Soxhlet extractions with n-hexane and dichloromethane,
but substantially lower in relation to ethyl acetate and ethanol, whose
ηTotal TT
values scored,
respectively, 74% and 33% higher than of SFE4 (the richest in
ηTotal TT
with 4719
mg kg−1
leaves
).
3.4. Triterpenoid Concentration in Extracts
To assess the effect of distinct extraction methods and operating conditions on the
selectivity of the identified triterpenoids, their individual concentrations were determined
and are presented in Figure 8A,B for Soxhlet and SFE assays, respectively.
Regarding the Soxhlet extracts (Figure 8A), the concentration trend does not fol-
low those of total or individual triterpenoid yields. The highest extract concentration of
lupenone amounted 7.7 wt.% and occurred for dichloromethane assay (SX2), followed
by n-hexane (SX1) with 5.8 wt.%, ethyl acetate (SX3) with 3.8 wt.%, and finally, ethanol
(SX4) with 2.2 wt.%. Even though
ηTotal TT
and
ηlupenone
of dichloromethane and n-hexane
Soxhlet extraction were lower than those of ethyl acetate and ethanol, they resulted in
higher
Clupenone
. This is due to the fact that, as discussed previously (see Table 2), the
more polar solvents are able to coextract other families of compounds, thus diluting the
content of triterpenoids and fading the selectivity towards them, namely to lupenone. The
same can be observed for the remaining triterpenoids. Another interesting result is the
levelled scores of
Cα−amyrin
among the four organic solvents studied, ranging from 1.89 to
3.57 wt.%, which were far from expected given the significantly higher
ηα−amyrin
obtained
with ethyl acetate and ethanol (see Figure 7A). The observed yield/concentration nuances
are ultimately reflected in the
CTotal TT
response, which was as low as 10.3% and 5.4 wt.%
for ethyl acetate and ethanol, respectively, against 13.4 and 14.4 wt.% for dichloromethane
and n-hexane, respectively. As a result, the use of the two non-polar solvents are better
choices than ethanol or ethyl acetate when seeking a higher selectivity to lupenone, despite
having lower yields of this compound as counterpart.
The SFE results (see Figure 8B) show higher
Clupenone
than any of the Soxhlet extracts,
ranging from 10.1 to 12.4 wt.%, for runs SFE5 (300 bar, 80
◦
C, 5 wt.% of ethyl acetate) and
SFE2 (200 bar, 80
◦
C, no cosolvent), respectively. Once again, one can observe that the
discussed trends on
ηTotal TT
and
ηlupenone
are not verified for the concentration values.
Accordingly, even though run SFE2 showed the highest selectivity towards lupenone, it
only attained the third highest
ηlupenone
(see Figure 7B), and the same applies to run SFE1
(200 bar, 40
◦
C, no cosolvent). In turn, the inclusion of polar modifiers in runs SFE4 (300 bar,
80
◦
C, 5 wt.% of ethanol) and SFE5 (300 bar, 80
◦
C, 5 wt.% of ethyl acetate) created the
same penalization observed in Soxhlet: lower
Clupenone
was attained despite the higher
ηTotal TT
and
ηlupenone
scores (see Figures 3and 7B). As a result, the SFE results show that
pure SC-CO
2
was the most selective to lupenone, but the method was not able to selectively
coextract other triterpenoids. In terms of
CTotal TT
, similarly to what was observed for the
triterpenoid yield (see Figure 7B), the values follow the lupenone trend since the remaining
triterpenoids were extracted in significantly lower amounts. Accordingly, the maximum
was attained by run SFE2 (200 bar, 80
◦
C, no cosolvent), where
CTotal TT
is worth 18.0 wt.%.
The individual contribution of other triterpenoids in the extract for this score did not
surpass 2 wt.%.
Processes 2021,9, 1159 12 of 15
Processes 2021, 9, x FOR PEER REVIEW 12 of 16
worth 18.0 wt.%. The individual contribution of other triterpenoids in the extract for this
score did not surpass 2 wt.%.
Figure 8. Plots of the concentrations of individual triterpenoids identified in (A) Soxhlet extracts and respective solvents,
and (B) SFE at different conditions of temperature, pressure, and addition of ethanol (E) and ethyl acetate (EA) as modifi-
ers.
Overall, the attained SFE results show that the method can selectively extract lupe-
none from the leaves of A. dealbata, even though similar triterpenoids are only coextracted
on a negligible basis. Furthermore, lupenone therapeutic potential for inflammation, vi-
rus, infection, diabetes, cancer, and treatment of Chagas disease [36] justifies its valorisa-
tion in an industrial process for pharmaceutical and food applications.
4. Conclusions
Soxhlet extraction of Acacia dealbata leaves provided higher total extraction yields
than SFE, and the yield increased with the polarity of the organic solvent. In turn, SFE
yields were favored by increasing temperature and pressure, and by the addition of polar
Figure 8.
Plots of the concentrations of individual triterpenoids identified in (
A
) Soxhlet extracts and respective solvents,
and (
B
) SFE at different conditions of temperature, pressure, and addition of ethanol (E) and ethyl acetate (EA) as modifiers.
Overall, the attained SFE results show that the method can selectively extract lupenone
from the leaves of A. dealbata, even though similar triterpenoids are only coextracted on
a negligible basis. Furthermore, lupenone therapeutic potential for inflammation, virus,
infection, diabetes, cancer, and treatment of Chagas disease [
36
] justifies its valorisation in
an industrial process for pharmaceutical and food applications.
4. Conclusions
Soxhlet extraction of Acacia dealbata leaves provided higher total extraction yields
than SFE, and the yield increased with the polarity of the organic solvent. In turn, SFE
yields were favored by increasing temperature and pressure, and by the addition of polar
cosolvents. In both cases the main triterpenoids extracted were squalene,
β
-amyrone,
α
-
amyrone,
β
-amyrin,
α
-amyrin, lupenyl acetate and lupenone, with the latter exhibiting the
highest content. Overall, Soxhlet extracts exhibited higher amounts of total triterpenoids
in comparison to SFE extracts. Interestingly SFE selectively extracted lupenone, reaching
higher contents than those obtained with Soxhlet.
Processes 2021,9, 1159 13 of 15
Even though the increase of pressure and temperature combined with the addition
of cosolvents favored the SFE total yield, the same effect was not so evident for lupenone
yield. In fact, pure CO
2
attained a lupenone yield comparable to the maximum value
achieved at high pressure with SC-CO
2
modified with ethanol. Furthermore, the highest
lupenone concentration was also obtained for pure CO2extracts.
Considering the anti-inflammatory, anti-virus, anti-diabetes, and anti-cancer proper-
ties of lupenone, as well as its potential for the treatment of Chagas disease, one may state
that SFE contributes to the valorisation of A. dealbata leaves in the production of lupenone-
enriched extracts for the pharmaceutical, nutraceutical or food industries.
Globally, this work envisions SFE technology as a tool to address the current challenges
associated to the management of A. dealbata spread, namely by pointing to biorefinery
opportunities for its leaves. Even though the present study indicates that high selectivity
to lupenone may be achieved by SFE, optimization of the extraction conditions and a
preliminary economic evaluation of the process are highly recommended.
Author Contributions:
Conceptualization, C.M.S.; writing—original draft preparation, V.H.R. and
M.M.R.d.M.; methodology, V.H.R. and M.M.R.d.M.; investigation, V.H.R.; formal analysis, M.M.R.d.M.,
I.P. and C.M.S.; writing—review and editing, I.P. and C.M.S.; supervision, I.P. and C.M.S.; funding
acquisition, C.M.S.; resources, C.M.S. All authors have read and agreed to the published version of
the manuscript.
Funding:
This work was developed within the scope of the project CICECO-Aveiro Institute of
Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC
and, when appropriate, co-financed by FEDER under the PT2020 Partnership Agreement. Authors
want to thank Project inpactus—innovative products and technologies from eucalyptus, Project N
◦
21874 funded by Portugal 2020 through European Regional Development Fund (ERDF) in the frame of
COMPETE 2020 n
◦
246/AXIS II/2017. Authors want to thank the funding from Project AgroForWealth
(CENTRO-01-0145-FEDER-000001), funded by Centro2020, through FEDER and PT2020.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Lorenzo, P.; Gonzalez, L.O.; Reigosa, M.J. The genus Acacia as invader: The characteristic case of Acacia dealbata Link in Europe.
Ann. For. Sci. 2010,67, 101. [CrossRef]
2.
Kull, C.A.; Shackleton, C.M.; Cunningham, P.J.; Ducatillon, C.; Dufour-Dror, J.-M.; Esler, K.J.; Friday, J.B.; Gouveia, A.C.; Griffin,
A.R.; Marchante, E.; et al. Adoption, use and perception of Australian acacias around the world. Divers. Distrib.
2011
,17, 822–836.
[CrossRef]
3.
Le Maitre, D.C.; Gaertner, M.; Marchante, E.; Ens, E.-J.; Holmes, P.M.; Pauchard, A.; O’Farrell, P.J.; Rogers, A.M.; Blanchard, R.;
Blignaut, J.; et al. Impacts of invasive Australian acacias: Implications for management and restoration. Divers. Distrib.
2011
,17,
1015–1029. [CrossRef]
4. Instituto da Conservação da Natureza e das Florestas. Inventário Florestal Nacional (IFN6)–Principais Resultados; 2019.
5.
European Commission Directive (EU) 2018/2002 of the European Parliament and of the Council of 11 December 2018 amending
Directive 2012/27/EU on energy efficiency. Off. J. Eur. Union 2018,61, 1–230.
6.
Oliveira, C.S.D.; Moreira, P.; Resende, J.; Cruz, M.T.; Pereira, C.M.F.; Silva, A.M.S.; Santos, S.A.O.; Silvestre, A.J.D. Characterization
and Cytotoxicity Assessment of the Lipophilic Fractions of Different Morphological Parts of Acacia dealbata.Int. J. Mol. Sci.
2020
,
21, 1814. [CrossRef]
7.
Freire, C.; Coelho, D.S.C.; Santos, N.M.; Silvestre, A.J.D.; Neto, C. Identification of
∆
7 phytosterols and phytosteryl glucosides in
the wood and bark of several Acacia species. Lipids 2005,40, 317–322. [CrossRef]
8.
Luís, A.; Gil, N.; Amaral, M.E.; Duarte, A.P. Antioxidant activities of extracts from Acacia melanoxylon,Acacia dealbata and Olea
europaea and alkaloids estimation. Int. J. Pharm. Pharm. Sci. 2012,4, 225–231.
9.
Yáñez, R.; Gómez, B.; Martínez, M.; Gullón, B.; Alonso, J.L.; Estévez, B.G. Valorization of an invasive woody species, Acacia
dealbata, by means of Ionic liquid pretreatment and enzymatic hydrolysis. J. Chem. Technol. Biotechnol.
2014
,89, 1337–1343.
[CrossRef]
10.
Yildiz, S.; Gürgen, A.; Can, Z.; Tabbouche, S.A.; Kiliç, A.O. Some bioactive properties of Acacia dealbata extracts and their
potential utilization in wood protection. Drewno 2018,61, 81–97. [CrossRef]
11.
Devi, S.R.; Prasad, M.N.V. Tannins and related polyphenols from ten common Acacia species of India. Bioresour. Technol.
1991
,36,
189–192. [CrossRef]
12.
Freire, C.S.R.; Silvestre, A.J.D.; Neto, C.P. Demonstration of long-chain n-alkyl caffeates and
∆
7-steryl glucosides in the bark of
Acacia species by gas chromatography-mass spectrometry. Phytochem. Anal. 2007,18, 151–156. [CrossRef]
Processes 2021,9, 1159 14 of 15
13.
Lisperguer, J.; Saraiva, Y.; Vergara, E. Structure and thermal behavior of tannins from Acacia dealbata bark and their reactivity
toward formaldehyde. J. Chil. Chem. Soc. 2016,61, 3188–3190. [CrossRef]
14.
Neiva, D.M.; Luís, Â.; Gominho, J.; Domingues, F.; Duarte, A.P.; Pereira, H. Bark residues valorization potential regarding
antioxidant and antimicrobial extracts. Wood Sci. Technol. 2020,54, 559–585. [CrossRef]
15.
Casas, M.P.; Conde, E.; Ribeiro, D.; Fernandes, E.; Domínguez, H.; Torres, M.D. Bioactive properties of Acacia dealbata flowers
extracts. Waste Biomass Valoriz. 2020,11, 2549–2557. [CrossRef]
16.
López-Hortas, L.; Falqué, E.; Domínguez, H.; Torres, M.D. Microwave hydrodiffusion and gravity versus conventional distillation
for Acacia dealbata flowers. Recovery of bioactive extracts for cosmetic purposes. J. Clean. Prod. 2020,274. [CrossRef]
17. Pereira, F.B.M.; Domingues, F.M.J.; Silva, A.M.S. Triterpenes from Acacia dealbata.Nat. Prod. Lett. 1996,8, 97–103. [CrossRef]
18. Imperato, F. A chalcone glycoside from Acacia dealbata.Phytochemistry 1982,21, 480–481. [CrossRef]
19.
Rodolphe, P.; Katharina, B.; Meierhenrich, U.J.; Elise, C.; Georges, F.; Nicolas, B. Chemical composition of french mimosa absolute
oil. J. Agric. Food Chem. 2010,58, 1844–1849. [CrossRef]
20.
Soto, M.L.; Parada, M.; Falqué, E.; Domínguez, H. Personal-care products formulated with natural antioxidant extracts. Cosmetics
2018,5, 13. [CrossRef]
21.
Silva, E.; Fernandes, S.; Bacelar, E.; Sampaio, A. Antimicrobial activity of aqueous, ethanolic and methanolic leaf extracts from
Acacia spp. and Eucalyptus nicholii.Afr. J. Tradit. Complement. Altern. Med. 2016,13, 130–134. [CrossRef]
22.
Borges, A.; José, H.; Homem, V.; Simões, M. Comparison of Techniques and Solvents on the Antimicrobial and Antioxidant
Potential of Extracts from Acacia dealbata and Olea europaea.Antibiotics 2020,9, 48. [CrossRef]
23.
de Melo, M.M.R.; Oliveira, E.L.G.; Silvestre, A.J.D.; Silva, C.M. Supercritical fluid extraction of vegetable matrices: Applications,
trends and future perspectives of a convincing green technology. J. Supercrit. Fluids 2014,92, 115–176. [CrossRef]
24.
Reverchon, E.; Marco, I. De Supercritical fluid extraction and fractionation of natural matter. J. Supercrit. Fluids
2006
,38, 146–166.
[CrossRef]
25.
Rodrigues, V.H.; de Melo, M.M.R.; Portugal, I.; Silva, C.M. Extraction of Eucalyptus leaves using solvents of distinct polarity.
Cluster analysis and extracts characterization. J. Supercrit. Fluids 2018,135, 263–274. [CrossRef]
26.
Rodrigues, V.H.; de Melo, M.M.R.; Portugal, I.; Silva, C.M. Supercritical fluid extraction of Eucalyptus globulus leaves. Experimental
and modelling studies of the influence of operating conditions and biomass pretreatment upon yields and kinetics. Sep. Purif.
Technol. 2018,191, 173–181. [CrossRef]
27.
Domingues, R.M.A.; de Melo, M.M.R.; Oliveira, E.L.G.; Neto, C.P.; Silvestre, A.J.D.; Silva, C.M. Optimization of the supercritical
fluid extraction of triterpenic acids from Eucalyptus globulus bark using experimental design. J. Supercrit. Fluids
2013
,74, 105–114.
[CrossRef]
28.
de Melo, M.M.R.; Oliveira, E.L.G.; Silvestre, A.J.D.; Silva, C.M. Supercritical fluid extraction of triterpenic acids from Eucalyptus
globulus bark. J. Supercrit. Fluids 2012,70, 137–145. [CrossRef]
29.
Domingues, R.M.A.; Oliveira, E.L.G.; Freire, C.S.R.; Couto, R.M.; Simões, P.C.; Neto, C.P.; Silvestre, A.J.D.; Silva, C.M. Supercritical
fluid extraction of Eucalyptus globulus bark-A promising approach for triterpenoid production. Int. J. Mol. Sci.
2012
,13, 7648–7662.
[CrossRef]
30.
de Melo, M.M.R.; Carius, B.; Simões, M.M.Q.; Portugal, I.; Saraiva, J.; Silva, C.M. Supercritical CO2 extraction of V. vinifera leaves:
Influence of cosolvents and particle size on removal kinetics and selectivity to target compounds. J. Supercrit. Fluids
2020
,165,
104959. [CrossRef]
31.
de Melo, M.M.R.; Vieira, P.G.; ¸Sen, A.; Pereira, H.; Portugal, I.; Silva, C.M. Optimization of the supercritical fluid extraction of
Quercus cerris cork towards extraction yield and selectivity to friedelin. Sep. Purif. Technol. 2020,238, 116395. [CrossRef]
32.
Martins, P.F.; de Melo, M.M.R.; Sarmento, P.; Silva, C.M. Supercritical fluid extraction of sterols from Eichhornia crassipes biomass
using pure and modified carbon dioxide. Enhancement of stigmasterol yield and extract concentration. J. Supercrit. Fluids
2016
,
107, 441–449. [CrossRef]
33.
Evans, C.S.; Qureshi, M.Y.; Bell, E.A. Free amino acids in the seeds of Acacia species. Phytochemistry
1977
,16, 565–570. [CrossRef]
34.
Domingues, R.; Guerra, A.; Duarte, M.; Freire, C.; Neto, C.; Silva, C.; Silvestre, A. Bioactive Triterpenic Acids: From Agroforestry
Biomass Residues to Promising Therapeutic Tools. Mini Rev. Org. Chem. 2014,11, 382–399. [CrossRef]
35.
de Melo, M.M.R.; Domingues, R.M.A.; Silvestre, A.J.D.; Silva, C.M. Extraction and purification of triterpenoids using supercritical
fluids: From lab to exploitation. Mini Rev. Org. Chem. 2014, 362–381. [CrossRef]
36.
Xu, F.; Huang, X.; Wu, H.; Wang, X. Beneficial health effects of lupenone triterpene: A review. Biomed. Pharmacother.
2018
,103,
198–203. [CrossRef]
37. Saleem, M. Lupeol, a novel anti-inflammatory and anti-cancer dietary triterpene. Cancer Lett. 2009,285, 109–115. [CrossRef]
38.
Meneses-Sagrero, S.E.; Navarro-Navarro, M.; Ruiz-Bustos, E.; Del-Toro-Sánchez, C.L.; Jiménez-Estrada, M.; Robles-Zepeda, R.E.
Antiproliferative activity of spinasterol isolated of Stegnosperma halimifolium (Benth, 1844). Saudi Pharm. J.
2017
,25, 1137–1143.
[CrossRef]
39.
Lou-Bonafonte, J.M.; Martínez-Beamonte, R.; Sanclemente, T.; Surra, J.C.; Herrera-Marcos, L.V.; Sanchez-Marco, J.; Arnal, C.;
Osada, J. Current Insights into the Biological Action of Squalene. Mol. Nutr. Food Res. 2018,62, 1–16. [CrossRef]
40.
Aragão, G.F.; Carneiro, L.M.V.; Junior, A.P.F.; Vieira, L.C.; Bandeira, P.N.; Lemos, T.L.G.; de B. Viana, G.S. A possible mechanism
for anxiolytic and antidepressant effects of alpha- and beta-amyrin from Protium heptaphyllum (Aubl.) March. Pharmacol.
Biochem. Behav. 2006,85, 827–834. [CrossRef]
Processes 2021,9, 1159 15 of 15
41.
Aragão, G.F.; Cunha Pinheiro, M.C.; Nogueira Bandeira, P.; Gomes Lemos, T.L.; de Barros Viana, G.S. Analgesic and anti-
inflammatory activities of the isomeric mixture of alpha- and beta-amyrin from protium heptaphyllum (Aubl.) March. J. Herb.
Pharmacother. 2007,7, 31–47. [CrossRef] [PubMed]
42.
da Silva Júnior, W.F.; Lima Bezerra de Menezes, D.; Calvarho de Oliveira, L.; Scherer Koester, L.; Olveira de Almeida, P.D.; Lima,
E.S.; Pereira de Azevedo, E.; da Veiga Júnior, V.F.; Neves de Lima, Á.A. Inclusion complexes of
β
and HP
β
-cyclodextrin with
α
,
β
amyrin and in vitro anti-inflammatory activity. Biomolecules 2019,9, 241. [CrossRef]
43.
Holanda Pinto, S.A.; Pinto, L.M.S.; Cunha, G.M.A.; Chaves, M.H.; Santos, F.A.; Rao, V.S. Anti-inflammatory effect of
α
,
β
-Amyrin,
a pentacyclic triterpene from Protium heptaphyllum in rat model of acute periodontitis. Inflammopharmacology
2008
,16, 48–52.
[CrossRef]
44.
Oliveira, F.A.; Vieira-Júnior, G.M.; Chaves, M.H.; Almeida, F.R.C.; Florêncio, M.G.; Lima, R.C.P., Jr.; Silva, R.M.; Santos, F.A.; Rao,
V.S.N. Gastroprotective and anti-inflammatory effects of resin from Protium heptaphyllum in mice and rats. Pharmacol. Res.
2004
,
49, 105–111. [CrossRef]
45.
Okoye, N.N.; Ajaghaku, D.L.; Okeke, H.N.; Ilodigwe, E.E.; Nworu, C.S.; Okoye, F.B.C. Beta-Amyrin and alpha-Amyrin acetate
isolated from the stem bark of Alstonia boonei display profound anti-inflammatory activity. Pharm. Biol.
2014
,52, 1478–1486.
[CrossRef] [PubMed]
46.
Jæger, D.; O’Leary, M.C.; Weinstein, P.; Møller, B.L.; Semple, S.J. Phytochemistry and bioactivity of Acacia sensu stricto (Fabaceae:
Mimosoideae). Phytochem. Rev. 2019,18, 129–172. [CrossRef]
47.
Subhan, N.; Burrows, G.E.; Kerr, P.G.; Obied, H.K. Phytochemistry, Ethnomedicine, and Pharmacology of Acacia.Stud. Nat. Prod.
Chem. 2018,57, 247–326. [CrossRef]
48.
Pitzer, K.S.; Schreiber, D.R. Improving equation-of-state accuracy in the critical region; equations for carbon dioxide and
neopentane as examples. Fluid Phase Equilib. 1988,41, 1–17. [CrossRef]
49.
Pöhler, H.; Kiran, E. Volumetric properties of carbon dioxide + ethanol at high pressures. J. Chem. Eng. Data
1997
,42, 384–388.
[CrossRef]
50.
Falco, N.; Kiran, E. Volumetric properties of ethyl acetate + carbon dioxide binary fluid mixtures at high pressures. J. Supercrit.
Fluids 2012,61, 9–24. [CrossRef]
51.
Gujar, A.; Anderson, T.; Cavagnino, D.; Patel, A. Comparative analysis of mass spectral matching for confident compound
identification using the Advanced Electron Ionization source for GC-MS. Thermoscientific 2018,10598, 1–7.
52.
Zuluaga, A.M.; Mena-García, A.; Soria Monzón, A.C.; Rada-Mendoza, M.; Chito, D.M.; Ruiz-Matute, A.I.; Sanz, M.L. Microwave
assisted extraction of inositols for the valorization of legume by-products. LWT 2020,133, 109971. [CrossRef]
53.
Zuluaga, A.M.; Mena-García, A.; Chito-Trujillo, D.; Rada-Mendoza, M.; Sanz, M.L.; Ruiz-Matute, A.I. Development of a
microwave-assisted extraction method for the recovery of bioactive inositols from lettuce ( Lactuca sativa) byproducts. Electrophore-
sis 2020,41, 1804–1811. [CrossRef] [PubMed]
54.
Mena-García, A.; Rodríguez-Sánchez, S.; Ruiz-Matute, A.I.; Sanz, M.L. Exploitation of artichoke byproducts to obtain bioactive
extracts enriched in inositols and caffeoylquinic acids by Microwave Assisted Extraction. J. Chromatogr. A
2020
,1613, 460703.
[CrossRef]
55.
Ruiz-Aceituno, L.; García-Sarrió, M.J.; Alonso-Rodriguez, B.; Ramos, L.; Sanz, M.L. Extraction of bioactive carbohydrates from
artichoke (Cynara scolymus L.) external bracts using microwave assisted extraction and pressurized liquid extraction. Food Chem.
2016,196, 1156–1162. [CrossRef] [PubMed]
56.
Chóez-Guaranda, I.; Ruíz-Barzola, O.; Ruales, J.; Manzano, P. Antioxidant activity optimization and GC-MS profile of aqueous
extracts of Vernonanthura patens (Kunth) H. Rob. leaves. Nat. Prod. Res. 2020,34, 2505–2509. [CrossRef]
57.
Somsub, W.; Kongkachuichai, R.; Sungpuag, P.; Charoensiri, R. Effects of three conventional cooking methods on vitamin C,
tannin, myo-inositol phosphates contents in selected Thai vegetables. J. Food Compos. Anal. 2008,21, 187–197. [CrossRef]
58.
Warren, C.R.; Aranda, I.; Cano, F.J. Responses to water stress of gas exchange and metabolites in Eucalyptus and Acacia spp. Plant
Cell Environ. 2011,34, 1609–1629. [CrossRef]
59.
Croze, M.L.; Soulage, C.O. Potential role and therapeutic interests of myo-inositol in metabolic diseases. Biochimie
2013
,95,
1811–1827. [CrossRef] [PubMed]
60.
Pütter, K.M.; van Deenen, N.; Müller, B.; Fuchs, L.; Vorwerk, K.; Unland, K.; Bröker, J.N.; Scherer, E.; Huber, C.; Eisenreich, W.; et al.
The enzymes OSC1 and CYP716A263 produce a high variety of triterpenoids in the latex of Taraxacum koksaghyz.Sci. Rep.
2019
,9,
5942. [CrossRef] [PubMed]
61.
Yendo, A.C.A.; De Costa, F.; Gosmann, G.; Fett-Neto, A.G. Production of plant bioactive Triterpenoid saponins: Elicitation
strategies and target genes to improve yields. Mol. Biotechnol. 2010,46, 94–104. [CrossRef]
62.
Fukushima, E.O.; Seki, H.; Ohyama, K.; Ono, E.; Umemoto, N.; Mizutani, M.; Saito, K.; Muranaka, T. CYP716A Subfamily
Members are Multifunctional Oxidases in Triterpenoid Biosynthesis. Plant Cell Physiol.
2011
,52, 2050–2061. [CrossRef] [PubMed]
63.
Brendolise, C.; Yauk, Y.K.; Eberhard, E.D.; Wang, M.; Chagne, D.; Andre, C.; Greenwood, D.R.; Beuning, L.L. An unusual plant
triterpene synthase with predominant
α
-amyrin—Producing activity identified by characterizing oxidosqualene cyclases from
Malus ×domestica.FEBS J. 2011,278, 2485–2499. [CrossRef] [PubMed]
Available via license: CC BY
Content may be subject to copyright.
