Characterization and Cytotoxicity Assessment of the Lipophilic Fractions of Different Morphological Parts of Acacia dealbata

Abstract

Acacia dealbata biomass, either from forest exploitation or from the management of invasive species, can be a strategic topic, namely as a source of high-value compounds. In this sense, the present study aimed at the detailed characterization of the lipophilic components of different morphological parts of A. dealbata and the evaluation of their cytotoxicity in cells representative of different mammals’ tissues. The chemical composition of lipophilic extracts from A. dealbata bark, wood and leaves was evaluated using gas chromatography-mass spectrometry (GC–MS). Terpenic compounds (representing 50.2%–68.4% of the total bark and leaves extracts, respectively) and sterols (60.5% of the total wood extract) were the main components of these extracts. Other constituents, such as fatty acids, long-chain aliphatic alcohols, monoglycerides, and aromatic compounds were also detected in the studied extracts. All the extracts showed low or no cytotoxicity in the different cells tested, demonstrating their safety profile and highlighting their potential to be used in nutraceutical or pharmaceutical applications. This study is therefore an important contribution to the valorization of A. dealbata, demonstrating the potential of this species as a source of high value lipophilic compounds.

Full text

International Journal of
Molecular Sciences
Article
Characterization and Cytotoxicity Assessment of
the Lipophilic Fractions of Different Morphological
Parts of Acacia dealbata
Cátia S. D. Oliveira 1, Patrícia Moreira 2, Judite Resende 1,3 , Maria T. Cruz 2,4,
Cláudia M. F. Pereira 2,5 , Artur M. S. Silva 3, Sónia A. O. Santos 1and
Armando J. D. Silvestre 1, *
1CICECO—Aveiro Institute of Materials, University of Aveiro, 3810-193 Aveiro, Portugal;
cs.oliveira@ua.pt (C.S.D.O.); judite.resende@ua.pt (J.R.); santos.sonia@ua.pt (S.A.O.S.)
2CNC—Center for Neuroscience and Cellular Biology, University of Coimbra, 3004-504 Coimbra, Portugal;
patriciaraquel_jm@hotmail.com (P.M.); trosete@ff.uc.pt (M.T.C.); claudia.mf.pereira@gmail.com (C.M.F.P.)
3LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal;
artur.silva@ua.pt
4Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal
5Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal
*Correspondence: armsil@ua.pt
Received: 10 February 2020; Accepted: 3 March 2020; Published: 6 March 2020


Abstract:
Acacia dealbata biomass, either from forest exploitation or from the management of invasive
species, can be a strategic topic, namely as a source of high-value compounds. In this sense, the present
study aimed at the detailed characterization of the lipophilic components of different morphological
parts of A. dealbata and the evaluation of their cytotoxicity in cells representative of different mammals’
tissues. The chemical composition of lipophilic extracts from A. dealbata bark, wood and leaves was
evaluated using gas chromatography-mass spectrometry (GC–MS). Terpenic compounds (representing
50.2%–68.4% of the total bark and leaves extracts, respectively) and sterols (60.5% of the total wood
extract) were the main components of these extracts. Other constituents, such as fatty acids, long-chain
aliphatic alcohols, monoglycerides, and aromatic compounds were also detected in the studied extracts.
All the extracts showed low or no cytotoxicity in the different cells tested, demonstrating their safety profile
and highlighting their potential to be used in nutraceutical or pharmaceutical applications. This study is
therefore an important contribution to the valorization of A. dealbata, demonstrating the potential of this
species as a source of high value lipophilic compounds.
Keywords:
Acacia dealbata; forest biomass; biorefinery; lipophilic compounds; GC–MS analysis;
cytotoxicity; nutraceutical applications; pharmaceutical applications
1. Introduction
Acacia dealbata (silver wattle or mimosa) is a woody legume, introduced in Europe in the 18th century,
which then invaded Atlantic and Mediterranean climates, from Portugal to Italy [
1
–
4
]. Soil disturbances,
fires, and climate changes, leading to increasing areas susceptible to colonization [
3
] have probably
contributed to A. dealbata invasion of many regions. Although this species produces wood suitable
for good quality pulp fibers [
5
–
7
], its industrial applications have been mainly focused on wood for
furniture [
8
], gum, or as a substitute of gum arabic, on bark for tanning production [
7
,
9
–
11
], and on
flowers for absolute oil production, and in the flavor and perfumes industry [
12
]. In addition, A. dealbata
has been traditionally used to control soil erosion, and as an ornamental species [13].
Int. J. Mol. Sci. 2020,21, 1814; doi:10.3390/ijms21051814 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020,21, 1814 2 of 16
Notwithstanding, the invading behavior of A. dealbata turned it into a major concern for the forestry
sector, particularly for the pulp and paper industry and the respective Eucalyptus species plantations [
14
].
Actually, A. dealbata has been considered a residue for pulp industry, being left in the forest or used
essentially an energy source [
9
,
15
,
16
]. Thus, the valorization of this species for high value applications
is of major importance for countries where pulp and paper industry has a high impact. In fact,
this industry is increasingly on the alert for waste reduction and recovery of residual forest biomass.
So far, only a few studies have been focused on searching innovative ways to add value to
A. dealbata, demonstrating that it is particularly interesting as a source of high-value compounds [
8
,
15
,
17
].
Nevertheless, these studies have only focused on specific families [
8
,
15
], specific tree parts [
17
], or even
neglected the quantification of the compounds detected [
8
]. In fact, while different phytosterols
were identified and quantified in A. dealbata wood and bark [15], only two terpenic compounds were
reported as constituents of this species, namely lupenone in leaves, flowers and seeds and lupeol
in leaves [
8
], and in both cases without quantitative data. Some fatty acids, long-chain aliphatic
alcohols, and monoglycerides were also identified in A. dealbata bark [
17
], but their presence in other
parts of the tree as well as their abundance remain unknown.
Additionally, the cytotoxicity of A. dealbata lipophilic extracts has not been exploited so far,
although a wide range of biological properties have been already reported for some of its constituents,
such as the anti-inflammatory, antidiabetic, antiviral, and anticancer activities of lupenone [
18
] and
anti-inflammatory, antiproliferative, and anticarcinogenic activities associated to spinasterol and
22,23-dihydrospinasterol [
19
–
22
], which were identified as the major sterols in A. dealbata bark and
wood [15].
In this vein, the detailed knowledge about the lipophilic composition of different morphological
parts of A. dealbata, envisaging their integrated exploitation, remains scarce, particularly concerning
all the important families of lipophilic compounds, like fatty acids, long-chain aliphatic alcohols,
sterols, terpenic compounds, or monoglycerides.
In order to fill this gap, this work aims at a systematic study concerning the lipophilic composition
of the bark, wood, and leaves of A. dealbata, by gas chromatography-mass spectrometry analysis
(GC–MS). Additionally, and in order to evaluate the potential exploitation of A. dealbata lipophilic
fractions for pharmaceutical (in oral or topical applications) and nutraceutical purposes, the cytotoxicity
in different cell lines representative of different tissues and organs, such as the brain, innate immune
system, skin, lung, and liver is also disclosed.
2. Results and Discussion
2.1. Lipophilic Extractives Yield of A. dealbata Bark, Wood and Leaves
The dichloromethane extracts from A. dealbata bark, wood and leaves presented distinct extraction
yields. A. dealbata leaves showed the highest yield, accounting for 6.2
±
0.22%, followed by bark extract
(2.3 ±0.25%), while wood presented the lowest yield, accounting for 0.30 ±0.01%.
The extraction yields obtained for A. dealbata bark and wood are in accordance with those already
reported in the literature for these morphological parts (2.00
±
0.06 and 0.36
±
0.03%, respectively) [
15
].
Although no data have been reported so far concerning the lipophilic extraction yield of A. dealbata
leaves, the value obtained is higher than that reported for the hexane extract of A. sinulata leaves [
23
].
2.2. Chemical Characterization of the Lipophilic Extract
The chemical composition of the dichloromethane extracts of the three morphological parts of
A. dealbata was studied by GC–MS analysis. Six main families of lipophilic compounds were identified
and quantified, namely fatty acids, long-chain aliphatic alcohols (LCAAs), terpenic compounds, sterols,
monoglycerides and aromatic compounds (Figure 1). The identification and detailed quantification of
the main lipophilic components are summarized in Table 1. The total contents of identified compounds
were 976.3 mg kg−1dw in wood, 6872.4 mg kg−1dw in bark, and 21394.5 mg kg−1dw in leaves.

Int. J. Mol. Sci. 2020,21, 1814 3 of 16
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 16
Figure 1. The major families of lipophilic compounds identified in DCM extracts of A. dealbata bark,
wood and leaves. Abbreviations: FA, fatty acids; LCAA, long-chain aliphatic alcohols; T, terpenic
compounds; ST, sterols; MG, monoglycerides and AR, aromatic compounds.
Terpenic compounds were the main family present in the bark and leaves extracts, with contents
ranging from 3450.8 mg kg
−1
dw in bark to 14635.3 mg kg
−1
dw in leaves (representing 50.2% and
68.4% of the total content of identified compounds in bark and leaves, respectively). In opposition,
terpenic compounds were not found in the wood extract. Actually, the lipophilic extract of this
morphological part was shown to be mainly composed of sterols, with a content of 590.4 mg kg
−1
dw,
representing 60.5% of the total content of identified compounds in wood.
Table 1. Chemical composition of dichloromethane extracts from bark, wood and leaves lipophilic of
A. dealbata.
Rt
(min)
Compound mg g
−1
of Extract mg kg
−1
of dw
Bark Wood Leaves Bark Wood Leaves
Fatty acids 46.2 95.1 28.3 1060.0 290.0 1747.4
Saturated fatty acids 41.2 55.4 23.0 946.1 168.7 1415.9
24.12 Dodecanoic acid 0.1 0.3 0.2 2.9 0.9 12.9
29.26 Tetradecanoic acid 0.1 0.3 0.4 2.7 0.9 24.0
31.65 Pentadecanoic acid 0.1 0.5 n.d. 1.7 1.4 n.d.
33.95 Hexadecanoic acid 5.4 22.9 6.3 124.3 69.7 389.1
36.15 Heptadecanoic acid 0.1 1.1 0.1 3.0 3.3 7.2
38.26 Octadecanoic acid 0.8 8.4 1.0 17.4 25.6 59.4
40.28 Nonadecanoic acid 0.1 0.6 n.d. 1.4 1.7 n.d.
42.25 Eicosanoic acid 0.4 2.3 0.7 8.4 6.9 41.5
44.14 Heneicosanoic acid 0.2 1.6 0.3 3.5 5.0 21.4
45.96 Docosanoic acid 1.6 4.6 0.9 36.5 14.1 55.1
47.73 Tricosanoic acid 0.5 2.8 0.6 12.6 8.5 37.2
49.43 Tetracosanoic acid 6.1 6.2 1.3 141.0 18.8 80.0
51.10 Pentacosanoic acid 0.6 1.7 n.d. 13.9 5.1 n.d.
52.90 Hexacosanoic acid 3.7 2.2 1.4 85.9 6.6 86.3
54.80 Heptacosanoic acid 1.1 n.d. n.d. 24.5 n.d. n.d.
0
50
100
150
200
250
300
350
Bark Wood Leaves
mg g-1 of extract
FA
LCAA
T
ST
MG
AR
Others
Figure 1.
The major families of lipophilic compounds identified in DCM extracts of A. dealbata bark,
wood and leaves. Abbreviations: FA, fatty acids; LCAA, long-chain aliphatic alcohols; T, terpenic
compounds; ST, sterols; MG, monoglycerides and AR, aromatic compounds.
Table 1.
Chemical composition of dichloromethane extracts from bark, wood and leaves lipophilic
of A. dealbata.
Rt(min) Compound mg g−1of Extract mg kg−1of dw
Bark Wood Leaves Bark Wood Leaves
Fatty acids 46.2 95.1 28.3 1060.0 290.0 1747.4
Saturated fatty acids 41.2 55.4 23.0 946.1 168.7 1415.9
24.12 Dodecanoic acid 0.1 0.3 0.2 2.9 0.9 12.9
29.26 Tetradecanoic acid 0.1 0.3 0.4 2.7 0.9 24.0
31.65 Pentadecanoic acid 0.1 0.5 n.d. 1.7 1.4 n.d.
33.95 Hexadecanoic acid 5.4 22.9 6.3 124.3 69.7 389.1
36.15 Heptadecanoic acid 0.1 1.1 0.1 3.0 3.3 7.2
38.26 Octadecanoic acid 0.8 8.4 1.0 17.4 25.6 59.4
40.28 Nonadecanoic acid 0.1 0.6 n.d. 1.4 1.7 n.d.
42.25 Eicosanoic acid 0.4 2.3 0.7 8.4 6.9 41.5
44.14 Heneicosanoic acid 0.2 1.6 0.3 3.5 5.0 21.4
45.96 Docosanoic acid 1.6 4.6 0.9 36.5 14.1 55.1
47.73 Tricosanoic acid 0.5 2.8 0.6 12.6 8.5 37.2
49.43 Tetracosanoic acid 6.1 6.2 1.3 141.0 18.8 80.0
51.10 Pentacosanoic acid 0.6 1.7 n.d. 13.9 5.1 n.d.
52.90 Hexacosanoic acid 3.7 2.2 1.4 85.9 6.6 86.3
54.80 Heptacosanoic acid 1.1 n.d. n.d. 24.5 n.d. n.d.
56.83 Octacosanoic acid 5.3 n.d. 5.4 120.5 n.d. 334.1
58.87 Nonacosanoic acid 3.1 n.d. n.d. 72.0 n.d. n.d.
61.10 Triacontanoic acid 8.2 n.d. 4.3 188.9 n.d. 267.5
65.98 Dotriacontanoic acid 3.7 n.d. n.d. 84.9 n.d. n.d.
Unsaturated fatty acids 4.2 38.7 5.4 95.7 118.0 331.5
33.34 Hexadec-9-enoic acid 0.1 0.2 0.1 1.3 0.7 6.1

Int. J. Mol. Sci. 2020,21, 1814 4 of 16
Table 1. Cont.
Rt(min) Compound mg g−1of Extract mg kg−1of dw
Bark Wood Leaves Bark Wood Leaves
37.38 Octadeca-9,12-dienoic acid 1.7 31.9 1.7 39.6 97.2 104.4
37.42 Octadeca-9,12,15-trienoic acid 0.4 1.2 1.9 9.4 3.7 116.2
37.58 cis-Octadec-9-enoic acid 1.6 4.1 1.5 36.3 12.6 89.6
37.73 trans-Octadec-9-enoic acid 0.4 1.3 0.2 9.2 3.8 15.1
w-Hydroxyacids 0.8 1.1 n.d 18.2 3.3 n.d.
52.01 22-Hydroxydocosanoic 0.8 1.1 n.d. 18.2 3.3 n.d.
Long-chain aliphatic alcohols 47.2 14.2 30.7 1082.5 43.7 1891.5
22.02 Dodecan-1-ol 0.01 0.5 0.02 0.3 1.4 1.4
24.73 Tridecan-1-ol 0.1 1.4 0.05 1.2 4.2 3.1
27.35 Tetradecan-1-ol 0.04 2.2 n.d. 0.9 6.8 n.d.
29.85 Pentadecan-1-ol 0.7 2.0 n.d. 15.9 6.2 n.d.
32.19 Hexadecan-1-ol 0.04 0.6 0.1 0.8 1.8 8.1
36.63 Octadecan-1-ol 0.1 0.4 0.1 2.0 1.4 3.1
40.71 Eicosan-1-ol 0.1 n.d. n.d. 2.0 n.d. n.d.
44.50 Docosan-1-ol 0.2 0.3 n.d. 4.3 1.2 n.d.
46.30 Tricosan-1-ol 0.2 n.d. n.d. 3.7 n.d. n.d.
48.05 Tetracosan-1-ol 3.8 n.d. n.d. 86.5 n.d. n.d.
49.73 Pentacosan-1-ol 0.9 n.d. n.d. 19.8 n.d. n.d.
51.43 Hexacosan-1-ol 11.6 n.d. 3.0 265.5 n.d. 184.1
53.21 Heptacosan-1-ol 1.5 n.d. n.d. 34.3 n.d. n.d.
55.15 Octacosan-1-ol 9.6 1.8 4.2 221.0 5.6 261.0
57.17 Nonacosan-1-ol 2.3 n.d. n.d. 53.2 n.d. n.d.
59.25 Triacontan-1-ol 10.8 5.0 16.8 247.9 15.2 1035.2
63.71 Dotricontan-1-ol 5.4 n.d. 6.4 123.3 n.d. 395.6
Terpenic compounds 150.5 n.d. 237.4 3450.8 n.d. 14,635.3
29.00 Neophytadiene n.d. n.d. 2.6 n.d. n.d. 158.8
37.02 Phytol n.d. n.d. 5.5 n.d. n.d. 340.6
48.89 Squalene n.d. n.d. 28.4 n.d. n.d. 1747.6
56.66 β-Amyrone n.d. n.d. 20.4 n.d. n.d. 1256.5
57.57 Lupenone 56.4 n.d. 112.7
1293.7
n.d. 6946.8
58.36 β-Amyrin n.d. n.d. 26.1 n.d. n.d. 1606.8
58.97 α-Amyrin n.d. n.d. 41.8 n.d. n.d. 2578.2
60.52 Lupenyl acetate 94.0 n.d. n.d.
2157.1
n.d. n.d.
Sterols 21.1 193.7 10.3 484.2 590.4 635.7
58.39 Spinasterol 8.6 94.6 n.d. 198.3 288.5 n.d.
58.69 Sitostanol n.d. 7.0 n.d. n.d. 21.2 n.d.
59.63 22,23-Dihydrospinasterol 12.5 92.1 10.3 285.8 280.7 635.7
Monoglycerides 30.2 6.0 0.8 691.6 18.4 49.4
45.14 1-Monohexadecenoin n.d. n.d. 0.3 n.d. n.d. 16.7
45.36 1-Monohexadecanoin 0.1 2.8 0.5 3.4 8.4 32.7
47.55 2-Monolinolein n.d. 0.5 n.d. n.d. 1.6 n.d.
48.13 1-Monolinolein n.d. 2.8 n.d. n.d. 8.4 n.d.
55.92 1-Monodocosanoin 6.5 n.d. n.d. 149.5 n.d. n.d.
60.06 1-Monotetracosanoin 23.5 n.d. n.d. 538.7 n.d. n.d.
Aromatic compounds 1.3 8.8 1.8 29.0 26.9 112.0
Aromatic aldehydes 0.2 5.8 0.1 4.4 17.6 9.1
14.86 4-Hydroxybenzaldehyde 0.1 0.3 0.1 1.3 0.8 7.3
19.75 Vanillin 0.1 2.2 n.d. 2.5 6.8 n.d.
24.42 Syringaldehyde 0.02 1.4 0.03 0.6 4.2 1.8
24.53 2,5-Hydroxybenzaldehyde n.d. 0.8 n.d. n.d. 2.5 n.d.
28.02 Coniferaldehyde n.d. 0.5 n.d. n.d. 1.5 n.d.

Int. J. Mol. Sci. 2020,21, 1814 5 of 16
Table 1. Cont.
Rt(min) Compound mg g−1of Extract mg kg−1of dw
Bark Wood Leaves Bark Wood Leaves
31.89 Sinapaldehyde n.d. 0.6 n.d. n.d. 1.8 n.d.
Aromatic acids 0.7 2.1 0.8 15.9 6.4 47.7
10.98 Benzoic acid n.d. n.d. 0.1 n.d. n.d. 7.0
23.24 p-Hydroxybenzoic acid 0.1 0.04 0.1 1.3 0.1 8.4
26.85 Vanillic acid 0.2 1.2 0.2 5.6 3.8 12.4
26.97 Homovanillic acid 0.1 0.1 n.d. 1.2 0.3 n.d.
30.18 Syringic acid 0.3 0.7 n.d. 7.8 2.2 n.d.
31.09 p-Coumaric acid n.d. n.d. 0.3 n.d. n.d. 19.8
Other aromatic compounds 0.4 1.0 0.9 8.8 2.9 55.3
16.07 Resorcinol 0.1 n.d. 0.1 2.8 n.d. 6.8
21.75 Tyrosol 0.02 0.1 0.8 0.5 0.3 48.5
23.62 Vanillyl alcohol 0.2 0.4 n.d. 5.5 1.2 n.d.
27.76 p-Coumaric alcohol n.d. 0.5 n.d. n.d. 1.4 n.d.
Others 3.2 2.3 37.7 74.4 6.9 2323.2
13.44 Glycerol 0.9 1.5 5.5 20.2 4.6 339.7
15.57 trans-Erythronoic
acid-γ-lactone 0.2 0.5 0.4 4.2 1.6 21.6
17.01 cis-Erythronoic acid-γ-lactone 0.2 0.2 0.4 3.8 0.7 25.7
54.54 α-Tocopherol 2.0 n.d. 31.4 46.1 n.d. 1936.1
Total 299.6 320.2 347.1
6872.4
976.3
21,394.5
Terpenic compounds were the main family present in the bark and leaves extracts, with contents
ranging from 3450.8 mg kg
−1
dw in bark to 14,635.3 mg kg
−1
dw in leaves (representing 50.2% and
68.4% of the total content of identified compounds in bark and leaves, respectively). In opposition,
terpenic compounds were not found in the wood extract. Actually, the lipophilic extract of this
morphological part was shown to be mainly composed of sterols, with a content of 590.4 mg kg
−1
dw,
representing 60.5% of the total content of identified compounds in wood.
2.2.1. Fatty Acids
Fatty acids represent between 8.2% (leaves) and 29.7% (wood) of the total lipophilic components
extracted from the different morphological parts of A. dealbata. Total saturated fatty acids accounted
for 168.7 mg kg
−1
dw in wood, 946.1 mg kg
−1
dw in bark and 1415.9 mg kg
−1
dw in leaves. Saturated
fatty acids were found at higher contents than unsaturated ones, accounting for 81.0% and 89.3% of
the total fatty acids content in leaves and bark, respectively. The chain length of fatty acids ranged
from 12 to 32 carbon atoms. Hexadecanoic acid was the most abundant saturated fatty acid found
in leaves and wood, accounting for 389.1 and 69.7 mg kg
−1
dw, respectively, whereas triacontanoic
acid was the most abundant saturated fatty acid in bark, accounting for 188.9 mg kg−1dw.
Considerable amounts of unsaturated fatty acids were found in bark, wood and leaves, with
contents ranging from 95.7 to 331.5 mg kg
−1
dw. These components represented about 40.7% of
the total fatty acids detected in wood, accounting 118.0 mg kg
−1
dw. Octadeca
−
9,12-dienoic acid,
an
ω
-6 fatty acid, was the most abundant unsaturated fatty acid detected in bark and wood, accounting
for, respectively 39.6 and 97.2 mg kg
−1
dw, while the
ω
-3 fatty acid octadeca-9,12,15-trienoic acid was
the major unsaturated fatty acid observed in leaves, accounting for 116.2 mg kg−1dw.
Unsaturated acids, despite being essential for human health, are not synthesized by the body.
These compounds are associated with the prevention and/or treatment of chronic and acute diseases
such as cardiovascular disease, cancer, osteoporosis and immune disorders [
24
]. Their abundance
in A. dealbata lipophilic extracts thus highlights the potential of this fraction to be exploited
in nutraceutical applications.

Int. J. Mol. Sci. 2020,21, 1814 6 of 16
So far, only a limited number of fatty acids were identified in A. dealbata, and particularly
in the bark, namely hexadecenoic, octadeca-9,12-dienoic, docosanoic, tetracosanoic, octacosanoic and
triacontanoic acids [
17
]. All the remaining fatty acids were identified here for the first time as bark
constituents. In addition, fatty acids identified in wood and leaves are reported here for the first time.
2.2.2. Long-Chain Aliphatic Alcohols
Long-chain aliphatic alcohols (LCAA) were also detected in all extracts and represented
a significative fraction of the total content of identified lipophilic compounds. The highest LCAA
contents were found in leaves and bark (1891.5 and 1082.5 mg kg
−1
dw, respectively), while
a considerably lower amount was observed in wood (43.7 mg kg
−1
dw). Triacontan-1-ol was
the major LCAA in wood and leaves, with contents of 15.2 mg kg
−1
dw and 1035.2 mg kg
−1
dw,
respectively. The bark extract showed the highest number of LCAA, presenting chain lengths from
12 to 32 carbon
atoms. Hexacosan-1-ol was the most abundant LCAA in this morphological part,
reaching up to 265.5 mg kg
−1
dw. Although no information has been reported so far regarding the LCAA
content in the different morphological parts of this tree, it was suggested in a previous study [
17
] that
hexacosan-1-ol was the major LCAA of A. dealbata bark DCM extract, which is in accordance with
the findings of the present study.
With the exception of tetracosan-1-ol, hexacosan-1-ol, octacosan-1-ol and triacontan-1-ol, which
were previously identified in A. dealbata bark [
17
], all the other LCAA (Table 1) are reported here for
the first time in this morphological part. Considering wood and leaves fractions, to the best of our
knowledge, this is the first study reporting the LCAA profile in these morphological parts of A. dealbata.
2.2.3. Terpenic Compounds
Terpenic compounds were the main family identified in bark and leaves lipophilic extracts,
accounting for about 50.2% and 68.4% of the total content of identified lipophilic compounds,
respectively (Figure 1). Terpenic compounds were absent in the wood extract, while two triterpenic
compounds were found in the bark lipophilic extract in relatively high amounts, namely lupenone
(1293.7 mg kg
−1
dw) and lupenyl acetate (2157.1 mg kg
−1
dw) (Figure 2). Interestingly, this component
was only present in the bark extract. Seven terpenic compounds were identified in the leaves extract,
corresponding to a total content of 14,635.3 mg kg
−1
dw. Among those, lupenone was the most abundant
compound of this family in leaves (6946.8 mg kg
−1
dw), followed by
α
-amyrin (2578.2 mg kg
−1
dw)
and squalene (1747.6 mg kg
−1
dw). From the identified terpenic compounds, only lupenone has been
previously reported as constituent of A. dealbata leaves [8].
Plant-derived triterpenic compounds have been associated with a wide variety of biological
activities. Lupenone, present in bark and leaves extracts (56.4 and 112.7 mg g
−1
extract, respectively),
has been described as a potential therapeutic agent in inflammation, diabetes, virus infection, cancer
and Chagas disease [
18
]. Lupenyl acetate, identified in the bark extract (94.0 mg g
−1
extract), has been
reported to possess
in vitro
and
in vivo
anti-inflammatory activity [
25
,
26
], highlighting the potential
of A. dealbata lipophilic fraction for high value applications.

Int. J. Mol. Sci. 2020,21, 1814 7 of 16
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 16
Figure 2. Chemical structures of the major constituents identified in the A. dealbata bark, wood and
leaves lipophilic extracts.
2.2.4. Sterols
Considerable amounts of sterols were observed in the three morphological parts of A. dealbata,
with contents ranging from 484.2 mg kg
−1
dw in bark to 635.7 mg kg
−1
dw in leaves. Wood extract
showed to be particularly rich in sterols (Figure 1), with this family representing 60.5% of the total
compounds identified. Two Δ
7
sterols were identified in bark and wood, namely 22,23-
dihydrospinasterol (285.8 and 280.7 and mg kg
−1
dw, respectively) and spinasterol (198.3 and 288.5
and mg kg
−1
dw, respectively) (Figure 2). In addition, sitostanol was also identified in wood,
accounting for 21.2 mg kg
−1
dw.
From a qualitative point of view, the sterols identified in bark and wood are in accordance with
previous reports [15]. However, some differences can be pointed out from a quantitative perspective.
Actually, sterols contents observed for bark and wood (484.2 and 590.4 mg kg
−1
dw, respectively)
were considerably higher than those reported before, following a similar extraction procedure (259.4
and 381 mg kg
−1
dw in bark and wood, respectively) [15]. These differences may arise from the
variability caused by geographic origin or edaphoclimatic conditions. Regarding leaves extract, only
22,23-dihydrospinasterol was detected, however at a considerable high content, namely 635.7 mg kg
−1
dw. As far as we know, this is the first and only sterol identified in A. dealbata leaves.
Spinasterol has been reported to show various biological properties, including anticarcinogenic,
anti-inflammatory, antitumor, antiulcerogenic activities, and antinociceptive effects [19,21,27–30].
2.2.5. Monoglycerides
Specific monoglycerides were found in all morphological parts of A. dealbata. Bark extract
showed the highest percentage of monoglycerides corresponding to 10.1% of the total content of
identified lipophilic compounds, while leaves and wood extracts showed relatively low amounts
Figure 2.
Chemical structures of the major constituents identified in the A. dealbata bark, wood and
leaves lipophilic extracts.
2.2.4. Sterols
Considerable amounts of sterols were observed in the three morphological parts of A. dealbata, with
contents ranging from 484.2 mg kg
−1
dw in bark to 635.7 mg kg
−1
dw in leaves. Wood extract showed
to be particularly rich in sterols (Figure 1), with this family representing 60.5% of the total compounds
identified. Two
∆7
sterols were identified in bark and wood, namely 22,23-dihydrospinasterol (285.8 and
280.7 and mg kg
−1
dw, respectively) and spinasterol (198.3 and 288.5 and mg kg
−1
dw, respectively)
(Figure 2). In addition, sitostanol was also identified in wood, accounting for 21.2 mg kg−1dw.
From a qualitative point of view, the sterols identified in bark and wood are in accordance
with previous reports [
15
]. However, some differences can be pointed out from a quantitative
perspective. Actually, sterols contents observed for bark and wood (484.2 and 590.4 mg kg
−1
dw,
respectively) were considerably higher than those reported before, following a similar extraction
procedure (259.4 and 381 mg kg
−1
dw in bark and wood, respectively) [
15
]. These differences may
arise from the variability caused by geographic origin or edaphoclimatic conditions. Regarding leaves
extract, only 22,23-dihydrospinasterol was detected, however at a considerable high content, namely
635.7 mg kg−1dw. As far as we know, this is the first and only sterol identified in A. dealbata leaves.
Spinasterol has been reported to show various biological properties, including anticarcinogenic,
anti-inflammatory, antitumor, antiulcerogenic activities, and antinociceptive effects [19,21,27–30].
2.2.5. Monoglycerides
Specific monoglycerides were found in all morphological parts of A. dealbata. Bark extract
showed the highest percentage of monoglycerides corresponding to 10.1% of the total content of
identified lipophilic compounds, while leaves and wood extracts showed relatively low amounts
(0.2% and 1.9% of the total content of identified compounds). Further, 1-monodocasanoin and
1-monotetracosanoin were the major monoglycerides found in the bark, representing 149.5 mg kg
−1
dw

Int. J. Mol. Sci. 2020,21, 1814 8 of 16
and
538.7 mg kg−1dw
, respectively, and 1-monohexadecanoin was the only monoglyceride found
in all extracts. With the exception of 1-monotetracosanoin, previously reported as constituent of
A. dealbata bark [
17
], all other monoglycerides found were identified here for the first time in the different
morphological parts of A. dealbata.
2.2.6. Aromatic Compounds
Several aromatic compounds were identified in A. dealbata bark, wood and leaves (Table 1),
with contents ranging between 26.9 mg kg
−1
dw in wood and 112.0 mg kg
−1
dw in leaves. Tyrosol
was the dominant aromatic compound observed in leaves extract (48.5
mg kg−1dw
), accounting
for 43.3% of the total aromatic compounds content. Other abundant aromatic compounds were
verified in leaves lipophilic extract, namely vanillic and p-coumaric acids (12.4 and 19.8 mg kg
−1
dw,
respectively). Vanillin was the major aromatic compound (6.8 mg kg
−1
dw) of wood lipophilic extract
while syringic acid was the most abundant aromatic compound observed in the bark lipophilic extract
(
7.8 mg kg−1dw
). To the best of our knowledge, all these aromatic compounds are reported here for
the first time as components of A. dealbata, with exception of syringic and p-coumaric acids that were
already described in the literature as constituents of a mixture of aerial parts (wood, bark, and leaves)
of A. dealbata [31].
2.2.7. Other Components
Finally, other compounds were also identified in high amounts in bark and leaves lipophilic
extracts, such as glycerol (20.2 mg kg
−1
dw in bark and 339.7 mg kg
−1
dw in leaves) and
α
-tocopherol
(46.1 mg kg−1dw in bark and 1936.1 mg kg−1dw in leaves).
2.3. Cytotoxity Evaluation of A. dealbata Lipophilic Extracts
In order to evaluate the safety of lipophilic extracts from bark, wood and leaves of A. dealbata, their
cytotoxicity was evaluated by the MTT assay, in cell lines representative of different tissues and organs
(brain, innate immune system, skin, lung and liver), namely non-differentiated and differentiated
neuronal cells (N2A), microglia (BV
−
2), macrophages (Raw 264.7), fibroblasts (NIH/3T3), keratinocytes
(HaCaT), lung cells (A549), and hepatocytes (HepG2). Regarding non-differentiated neuronal cells
(Figure 3A), after 24 h treatment with the lipophilic extracts, all tested concentrations of leaves extracts
were devoid of toxicity, and bark and wood extracts did not exhibit cytotoxicity at doses bellow
50
µ
g mL
−1
and 6.3
µ
g mL
−1
, respectively. In differentiated neuronal cells (Figure 3B), an absence
of toxicity was observed after 24 h treatment with the lipophilic extracts at concentrations bellow
50
µ
g mL
−1
for leaves and for bark, and 6.3
µ
g mL
−1
for wood. In the case of microglia cells, which are
the brain-resident immune cells (Figure 3C), non-toxic effects of the lipophilic extracts were observed
at 24 h for concentrations bellow 6.3
µ
g mL
−1
for leaves and for wood and 3.2
µ
g mL
−1
for bark.
In macrophages (Figure 3D), which are cells of the peripheral immune system, no significant toxicity
was observed for all tested concentrations of wood extracts and non-toxic effects were detected 24 h
after cells exposure to the leaves and bark lipophilic extracts at concentrations bellow 6.3
µ
g mL
−1
and
25
µ
g mL
−1
, respectively. Lipophilic extracts of A. dealbata were also tested in skin cells: fibroblasts
and keratinocytes, representative of the dermis and epidermis, respectively. In fibroblasts (Figure 3E),
absence of toxicity was found after 24-h incubation with these extracts at concentrations bellow
50 µg mL−1and 12.5 µg mL−1for leaves and for wood, respectively, while no significant toxicity was
observed for bark extract at any tested dose. In the case of keratinocytes (Figure 3F), lipophilic extracts
induced significant toxicity in concentrations above 25
µ
g mL
−1
for leaves, 6.3
µ
g mL
−1
for bark and
12.5
µ
g mL
−1
for wood, after 24 h treatment. In lung cells (Figure 3G), non-toxic effects of the lipophilic
extracts were observed at 24 h for concentrations bellow 25 µg mL−1for leaves, 6.3 µg mL−1for bark,
and 3.2
µ
g mL
−1
for wood. Finally, lipophilic extracts were shown to be safe in hepatocytes (Figure 3H),
which are the main liver cells. Indeed, after 24 h of incubation, significant toxicity of wood extracts
was detected only for concentrations above 50 µg mL−1.

Int. J. Mol. Sci. 2020,21, 1814 9 of 16
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 16
incubation, significant toxicity of wood extracts was detected only for concentrations above 50 µg
mL
−1
.
Figure 3. Cont.

Int. J. Mol. Sci. 2020,21, 1814 10 of 16
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 16
Figure 3. Effect of lipophilic extracts from A. dealbata on non-differentiated (A) and differentiated (B)
neuronal N2A, microglia BV-2 (C), macrophages Raw 264.7 (D), fibroblasts NIH/3T3 (E),
keratinocytes HaCaT (F), lung A549 (G) and liver hepatocyte HepG2 (H) cells viability. Cells were
treated for 24 h at 37 °C with concentrations of 0–50 µg mL
-1
of lipophilic extracts obtained from bark,
wood and leaves of A. dealbata and then viability was evaluated by the MTT assay. The results
expressed as percentage (%) of control represent the mean ± SEM of at least 3 independent
experiments performed in triplicate. Statistical analysis was made by one-way analysis of variance
(ANOVA) followed by Dunnett’s multiple comparison test. * p < 0.05, **p < 0.01, ***p < 0.001, **** p <
0.0001 significantly different compared to control.
Figure 3.
Effect of lipophilic extracts from A. dealbata on non-differentiated (
A
) and differentiated
(
B
) neuronal N2A, microglia BV-2 (
C
), macrophages Raw 264.7 (
D
), fibroblasts NIH/3T3 (
E
), keratinocytes
HaCaT (
F
), lung A549 (
G
) and liver hepatocyte HepG2 (
H
) cells viability. Cells were treated for 24 h
at 37
◦
C with concentrations of 0–50
µ
g mL
−1
of lipophilic extracts obtained from bark, wood and
leaves of A. dealbata and then viability was evaluated by the MTT assay. The results expressed as
percentage (%) of control represent the mean
±
SEM of at least 3 independent experiments performed
in triplicate. Statistical analysis was made by one-way analysis of variance (ANOVA) followed by
Dunnett’s multiple comparison test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 significantly
different compared to control.
For the first time, a cytotoxicity screening of lipophilic extracts obtained from bark, wood and
leaves of A. dealbata was performed in several mammalian cell lines representing brain, immune
system, skin, lung and liver cells, unveiling its safe doses. The results obtained with the lipophilic
extracts from leaves and bark are very promising regarding its future incorporation in oral or topic

Int. J. Mol. Sci. 2020,21, 1814 11 of 16
pharmaceutical formulations, since there is a lack of toxicity in cells of the liver, namely hepatocytes
(HepG2), cells of the epidermis, keratinocytes (HaCaT), and the dermis, fibroblasts (NIH/3T3). In fact,
Acacia is used in several cosmetic products, which are applied to different parts of the body, however
the available data in the literature is considered insufficient to support the safety of the Acacia-derived
cosmetics [
32
]. The absence of leaves extracts’ toxicity towards epithelial alveolar cells supports their
potential administration by inhalation. These extracts also displayed low toxicity in non-differentiated
and differentiated neuronal cells, and although the pharmacological and medicinal properties of
A. dealbata have not been studied yet, different species of Acacia are used for the treatment of
convulsions and dizziness [
33
]. Additionally, the wood lipophilic extract did not exhibit cytotoxicity
on macrophages for all the concentrations tested, suggesting that these extracts are not deleterious to
the innate immune system. These findings encourage the in-depth evaluation of the bioactive effects of
A. dealbata lipophilic extracts, as well as the signaling pathways and molecular targets modulated by
the extracts. In fact, the anti-inflammatory activity of wood extracts from other Acacia species have
been reported, and is believed to be due to the antioxidant properties, although no direct associations
have been made yet [
34
]. This study highlights the safe bioactive doses of A. dealbata lipophilic extracts
emphasizing its therapeutic value that should be more extensively investigated for future applications
in the pharmaceutical/nutraceutical industry.
3. Materials and Methods
3.1. Reagents
Dichloromethane (p.a.,
≥
99% purity) was supplied by Fisher Scientific (Thermo Fisher Scientific,
Waltham, Massachusetts, USA). Pyridine (p.a.,
≥
99.5% purity), N,O-bis(trimethylsilyl)trifluroacetamide
(99% purity), trimethylchlorosilane (99% purity), tetracosane (99% purity), hexadecanoic acid
(
≥
99% purity), nonadecan-1-ol (99% purity), syringic acid (99% purity), vanillin (99% purity) and
stigmasterol (95% purity) were supplied by Sigma Chemical Co (Madrid, Spain). Betulonic acid (95%
purity) was purchased from Chemos GmbH (Regenstauf, Germany).
Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640 Medium, sodium bicarbonate, sodium
pyruvate, non-essential amino acid derivatives, L-glutamine, glucose, phenol red, trypsin-EDTA
solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), retinoic acid and
dimethyl sulfoxide (DMSO) were supplied by Sigma-Aldrich (Lisbon, Portugal). Fetal bovine serum
(FBS), penicillin, and streptomycin were purchased from Gibco (Carlsbad, CA, USA).
3.2. Sample Collection
Bark, wood, and leaves samples, representative of harvesting biomass residues, were sampled from
a 8-year-old A. dealbata tree, in September 2018, randomly selected from a property of “The Navigator
Company”, Quinta Rei (GPS coordinates 41
◦
12
0
50” N, 8
◦
29
0
34” W), region of Porto/Valongo, Portugal.
Samples were air dried until a constant weight was achieved and grounded in order to select the fraction
with a granulometry lower than 1/2 mm prior to extraction.
3.3. Characterization of Lipophilic Extracts
3.3.1. Lipophilic Compounds Extraction
Three ground samples (nearly 10 g) of each fraction (bark, wood and leaves) of A. dealbata tree
were Soxhlet extracted during 8 h with dichloromethane (DCM), a fairly selective solvent to recover
lipophilics from biomass [
15
,
35
]. The solvent was evaporated to dryness, the extracts were weighed,
and the results are expressed as a percentage of dry weight (dw) biomass.

Int. J. Mol. Sci. 2020,21, 1814 12 of 16
3.3.2. GC–MS Analysis
Previous to GC–MS analysis, 20 mg samples of each dried extract were dissolved
in 250
µ
L of pyridine containing 0.6 mg of tetracosane (internal standard), and then 250
µ
L
of N,O-bis(trimethylsilyl)trifluoroacetamide and 50
µ
L of trimethylchlorosilane were added and
the mixture at 70
◦
C for 30 min. By adding these last two reagents, the hydroxyl and carboxyl
groups of the components of the extracts were converted into trimethylsilyl (TMS) ethers and esters,
respectively [15,36].
The derivatized extracts were analyzed by GC–MS using a Trace Gas Chromatograph (2000 series)
equipped with a Thermo Scientific DSQ II mass spectrometer (Waltham, Massachusetts, USA).
Compounds were separated in a DB-1 J&W capillary column (30 m
×
0.32 mm inner diameter,
0.25
µ
m film thickness, Santa Clara, California, USA), using helium as the carrier gas (35 cm s
−1
).
The temperature program was as follows: initial temperature, 80
◦
C for 5 min; temperature rate, 4
◦
C
min
−1
up to 260
◦
C; temperature rate, 2
◦
C min
−1
up to 285
◦
C which was kept for 8 min. The injector
and the transfer-line temperatures were, respectively 250
◦
C and 290
◦
C, while the split ratio was 1:33.
The mass spectrometer was operated in the electron impact mode with energy of 70eV, and the data
were collected at a rate of 1 scan s
−1
over a range of m/z33–700. The ion source was maintained
at 250 ◦C [37].
Compounds were identified by comparing their mass spectra (MS) with a mass spectral library
(Wiley-NIST Mass Spectral Library, 2014), by comparing their MS fragmentation profiles with literature
data [
15
,
17
,
37
] and, in some cases confirmed based on the characteristic retention times under the same
experimental conditions [36–38] and/or by injection of standards.
For semi-quantitative analysis, GC–MS calibration was performed with pure reference standards,
representative of the main families of lipophilic compounds present in the extracts (hexadecanoic acid,
nonadecan-1-ol, betulonic acid, stigmasterol, syringic acid and vanillin), relative to the internal standard
(tetracosane). Response factors were determined by averaging six GC–MS runs. Three derivatized
extracts were prepared from each morphological part and injected in duplicate. The results
presented correspond to the average of the concordant values obtained with a variation of less
than 5% (either between injections of the same aliquots and between triplicate extracts of the same
morphological part).
3.4. Cytotoxicity Evaluation of Lipophilic Extracts
3.4.1. Cell Culture
The mouse neuroblastoma (N2A, from ATCC CCL
−
131), human keratinocyte (HaCaT, from CLS,
Cell Lines Service, Eppelheim, Germany), mouse fibroblast (NIH/3T3, from ATCC CRL-1658) and
human lung carcinoma (A549, from ATCC CCL-185) cell lines were cultured with Dulbecco’s Modified
Eagle’s Medium (DMEM) (#D5648), supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS), 1% (v/v) antibiotic solution (10,000 U mL
−1
penicillin, 10,000
µ
g mL
−1
streptomycin), 3.7 g L
−1
sodium bicarbonate, and 1 mM sodium pyruvate. The N2A cell line culture medium was additionally
supplemented with 1% (v/v) non-essential amino acids. The mouse leukaemic macrophage cell line
(Raw 264.7, from ATCC TIB
−
71) was cultured in DMEM supplemented with 10% (v/v) non-inactivated
FBS, 1% (v/v) antibiotic solution, 1.5 g L
−1
sodium bicarbonate and 1 mM sodium pyruvate. The human
hepatocellular carcinoma cell line (HepG2, from ATCC HB-8065) was cultured with DMEM (#D5030),
supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) antibiotic solution, 1.5 g L
−1
sodium
bicarbonate, 1 mM sodium pyruvate, 4 mM L-glutamine, 1 g L
−1
glucose and phenol red. The mouse
microglia cell line (BV-2, from ICLC ATL03001, Interlab Cell Line Collection) was cultured in RPMI-1640
medium (#R4130) supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) antibiotic solution and
2 g L
−1
sodium bicarbonate. Cells were cultured in 75 cm
2
flasks and maintained in a humidified
5% CO
2
–95% air atmosphere at 37
◦
C, and the medium was changed every 2–3 days. Cultures were
passaged by trypsinization with Trypsin-EDTA solution 1x when cells reached 70–80% confluence

Int. J. Mol. Sci. 2020,21, 1814 13 of 16
and were sub-cultured over a maximum of ten passages. Raw 264.7 and BV-2 cells were passaged by
detaching the cells with a cell scraper.
3.4.2. Cell Viability Assay
For the evaluation of cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reduction assay was performed. RAW 264.7, non-differentiated N2A and HepG2, HaCaT, A549,
NIH/3T3 and BV-2 cells were seeded in 96-well plates at a density of 9.6
×
10
4
, 2.5
×
10
4
, 2
×
10
4
, 1.6
×
10
4
, 1
×
10
4
and 8
×
10
3
cells/well, respectively, and allowed to adhere for 24 h. For the differentiation of
neuronal processes, N2A cells were plated in 24-well plates at a density of 2
×
10
4
cells/well and 24 h later
were treated with 10
µ
M retinoic acid in DMEM with 1% (v/v) FBS, during 48 h, with medium refreshed
every 24 h. Stock solutions of lipophilic extracts from bark, wood, and leaves of A. dealbata were prepared
in DMSO and stored at
−
20
◦
C. On the day of experiment, culture medium was replaced by freshly
prepared exposure media [DMEM supplemented with 1% (v/v) FBS or DMEM with 10
µ
M retinoic
acid and 1% (v/v) FBS in the case of differentiated N2A cells and RPMI-1640 medium supplemented
with 2% (v/v) FBS in the case of BV-2 cells]. Each plate also included a solvent control [0.2% (v/v)
DMSO prepared in exposure medium]. Dose-response curves were obtained incubating the cells with
0–50
µ
g mL
−1
lipophilic extracts from bark, wood and leaves of A. dealbata for 24 h at 37
◦
C. After
the incubation period, the medium was removed and a fresh solution of MTT (0.5 mg L
−1
) prepared
in Krebs medium (pH 7.4) was added. The different cell lines were incubated with MTT at 37
◦
C
during 30 min
(Raw 264. 7 cells)
, 1 h (HepG2 cells), 2 h (N2A, A549, and HaCaT cells), 3 h (BV-2),
or 4 h (NIH/3T3 cells). After incubation, the MTT solution was removed and the formed formazan
crystals were dissolved in DMSO. The absorbance was measured at 570 nm in a spectrophotometer
(SLT spectra II) after 10 min shaking. The results were expressed as percentage of the absorbance value
obtained in control, which was considered 100% and were graphically presented as a percentage of cell
viability versus the lipophilic extracts concentration (µg mL−1).
3.4.3. Statistical Analysis
The results are presented as the mean
±
standard error of the mean (SEM) of the indicated number
of experiments. Normality of the data distribution was assessed by the D’Agostino & Pearson and
Shapiro-Wilk normality tests. Statistical comparisons between groups were performed by one-way
analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Significance was
accepted at pvalues <0.05. All statistical calculations were performed using GraphPad Prism software
(8.0.2, GraphPad Software Inc., San Diego, CA, USA).
4. Conclusions
The present study highlight, for the first time, the detailed chemical composition of the lipophilic
fraction of different morphological parts of A. dealbata, envisaging their integrated exploitation.
The amount and composition of dichloromethane extractives of A. dealbata bark, wood and leaves
differ significantly. Terpenic compounds represented the major lipophilic family present in bark and
leaves extracts, whereas sterols were the dominant components in the wood lipophilic fraction. Leaves
extract showed the highest content of lipophilic compounds, with lupenone as the major compound,
accounting for 32.5% of the total lipophilic extract. The bark extract was demonstrated to be rich
in long-chain aliphatic alcohols and monoglycerides, while the wood extract presented the highest
fatty acids content.
All lipophilic extracts of A. dealbata exhibited none or low cytotoxicity at the tested doses in different
mammalian cell lines representing brain, immune system, skin, lung, and liver cells, highlighting
the potential of these fractions to be further exploited for oral or topic pharmaceutical applications.
The present study thus represents a valuable contribution to promote the economic exploitation
of this forest by-product through in-depth knowledge of its chemical composition and safety.
These findings encourage the evaluation of the bioactivity of non-toxic doses of these lipophilic extracts

Int. J. Mol. Sci. 2020,21, 1814 14 of 16
of A. dealbata in order to obtain scientific support for their application in the nutraceutical industry.
Additionally, uniformization of procedures to characterize these fractions are an important issue to
make possible the comparison between biomass from different geographical origins. Future studies
concerning the search for alternative extraction methodologies and solvents, such as supercritical carbon
dioxide extraction or extraction with deep eutectic solvents are also crucial to allow the exploitation of
this natural resource.
Author Contributions:
Conceptualization, C.M.F.P., S.A.O.S. and A.J.D.S.; Formal analysis, C.S.D.O. and J.R.;
Investigation, C.S.D.O.; Methodology, C.S.D.O., P.M. and J.R.; Supervision, M.T.C., C.M.F.P., A.M.S.S., S.A.O.S.
and A.J.D.S.; Writing—original draft, C.S.D.O. and P.M.; Writing—review & editing, M.T.C., C.M.F.P., A.M.S.S.,
S.A.O.S. and A.J.D.S. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was carried out under the 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. The authors thank FCT/MEC for the financial
support to CICECO—Aveiro Institute of Materials (UIDB/50011/2020 & UIDP/50011/2020), LAQV-REQUIMTE
(UIDB/50006/2020), and CNC.IBILI Consortia (UIDB/04539/2020), and when appropriate the co-financing support
by FEDER, under the PT2020 Partnership Agreement. S
ó
nia A. O. Santos and Judite Resende thank the project
“AgroForWealth: Biorefining of agricultural and forest by-products and wastes: integrated strategic for valorization
of resources towards society wealth and sustainability” (CENTRO-01-0145-FEDER-000001), for the contract and
research grant, respectively.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study;
in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish
the results.
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