plants

Article
Exploring the Phytochemicals of Acacia melanoxylon R. Br.
Diana Alves 1 , Sidónio Duarte 1 , Pedro Arsénio 2 , Joana Gonçalves 3 , Cecília M. P. Rodrigues 3 ,
Ana Lourenço 1, * and Patrícia Máximo 1, *

1 LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, NOVA University,
2829-516 Caparica, Portugal; dfl.alves@campus.fct.unl.pt (D.A.); sidonioduarte88@gmail.com (S.D.)
2 Linking Landscape, Environment, Agriculture and Food (LEAF)—Research Center, Associated Laboratory
TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda,
1349-017 Lisbon, Portugal; arseniop@isa.ulisboa.pt
3 Faculty of Pharmacy, Research Institute for Medicines (iMed.ULisboa), Universidade de Lisboa, Av. Prof.
Gama Pinto, 1649-003 Lisbon, Portugal; joanalmg@hotmail.com (J.G.); cmprodrigues@ff.ulisboa.pt (C.M.P.R.)
* Correspondence: ana.lourenco@fct.unl.pt (A.L.); psm@fct.unl.pt (P.M.)

Abstract: Invasive species are currently a world menace to the environment, although the study of
their chemistry may provide a means for their future beneficial use. From a study of Portuguese
Acacia melanoxylon R. Br. five known compounds were isolated: lupeol, 3β-Z-coumaroyl lupeol,
3β-E-coumaroyl lupeol (dioslupecin A), kolavic acid 15-methyl ester and vomifoliol (blumenol A).
Their structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry, and as
a result some corrections are made to their previous 13 C NMR assignments. Cytotoxicity of 3β-E-
coumaroyl lupeol (dioslupecin A) and kolavic acid 15-methyl ester was evaluated against HCT116



human colorectal cancer cells although biological activity was not evident.



Citation: Alves, D.; Duarte, S.; Keywords: invasive species; Acacia melanoxylon; terpenoids; HCT116 human colorectal cancer
Arsénio, P.; Gonçalves, J.; Rodrigues, cells; cytotoxicity
C.M.P.; Lourenço, A.; Máximo, P.
Exploring the Phytochemicals of
Acacia melanoxylon R. Br. Plants 2021,
10, 2698. https://doi.org/10.3390/ 1. Introduction
plants10122698
Plant invasive species are one of the great threats to biodiversity since they establish
and supersede native species, leading occasionally to the extinction of the latter, by dis-
Academic Editors: Barbara Sladonja
rupting the biotic and abiotic balance of the invaded ecosystem. Apart from this ecological
and Danijela Poljuha
impact, they also have a socio-economic impact by influencing human health, infrastruc-
tures and local economies [1,2]. These species have long been a concern in the Portuguese
Received: 12 November 2021
Accepted: 2 December 2021
territory [3–5] and elsewhere, which resulted in the publication of legal regulations to
Published: 8 December 2021
prevent and manage the introduction and spread of invasive alien plants, both at the
national [6] and European level [7,8]. The management of this problem in Europe costs
Publisher’s Note: MDPI stays neutral
millions of euros [9] and, since eradication is rarely achieved, actions end up frequently
with regard to jurisdictional claims in
with periodic growth control and containment of these species [10]. The use of invasive
published maps and institutional affil- species as a source of chemicals or pharmaceuticals allows a rational use of resources that
iations. can mitigate the cost of their control, turning a useless and abundant natural good into
an added value resource [11]. Most likely, the prevalence of invasive species over endemic
ones relies on the bioactivity of the metabolites they produce as invaders, that are surely
responsible for their ease of expansion and dominance of the new habitat [11,12].
Copyright: © 2021 by the authors.
Bearing this in mind, we began our studies on the chemistry of invasive Acacia species.
Licensee MDPI, Basel, Switzerland.
Acacia species, mainly from Australia, settled in the Mediterranean area and conquered
This article is an open access article
significant lands. In Portugal they are distributed throughout the country, preferentially
distributed under the terms and on acid substrates. Originally, they were introduced as a source for wood, as an erosion
conditions of the Creative Commons preventer, for reforestation purposes and for ornamental reasons and the perfume industry,
Attribution (CC BY) license (https:// among others [13,14]. Abiotic and biotic factors favored their establishment and with time
creativecommons.org/licenses/by/ some of their uses deteriorated and economic value lowered, leading to an increase in
4.0/). their abundance and to an invasion condition [14–16]. The management and control of

Plants 2021, 10, 2698. https://doi.org/10.3390/plants10122698 https://www.mdpi.com/journal/plants

Plants 2021, 10, 2698 2 of 11

Acacia invasions includes various steps, from risk assessment to containment, eradication
and ecosystem restoration actions [13,15,17]. Some of the containment and control actions
include mechanical removal (ring-barking, hand-pulling or cutting) followed by chemical
control (with glyphosate), or biological control [18].
A. melanoxylon R. Br. (Australian blackwood) is a 15 m tree with evergreen leaves
and pale-yellow flowers arranged in a globular head of 10–12 mm diameter. Flowering
occurs in Portugal from February to June, and its fruits are brownish red pods. The seeds,
encircled by an orange funicle, remain viable in the ground for more than 50 years, are
dispersed by birds, wind, water, or rodents, and germinate after a space opening and/or
fire occurrence. This species also propagates vegetatively, forming vigorous sprouts from
the stump and roots [19].
Previous studies on the chemistry of A. melanoxylon include the isolation of hyperoside
(quercetin-3-D-galactoside) from the flowers [20], dihydroflavonoids [21], hydroxyfla-
vans [22], leucoanthocyanidins [23–25], a pyrogallol A-ring proanthocyanidin dimer [26],
[4-O-4]-linked biflavanoids [27] from the heartwood, and acamelin (a furanoquinone) and
a benzoquinone from undisclosed parts [28]. Studies from Portuguese invasive species
include the identification of ∆7 phytosterols, phytosteryl glucosides and long-chain n-alkyl
caffeates by GC-EIMS from the dichloromethane extracts of wood and bark [29,30], as
well as the antimicrobial activity of aqueous, ethanolic and methanolic leaf extracts, that
showed no interesting results [31]. Other references to bioactivity of extracts of this species
can be found in the recent review by Correia et al. on the biomass valorization of Acacia
species [32].
Following our interest in the chemistry of invasive species, in this study we report
on the isolation of five compounds: lupeol 1 [33], 3β-Z-coumaroyl lupeol 2 [34,35], 3β-
E-coumaroyl lupeol (dioslupecin A) 3 [36,37], kolavic acid 15-methyl ester 4 [38–40] and
vomifoliol (blumenol A) 5 [41–43] from a Portuguese invasive A. melanoxylon and demon-
strate the utility of investigating its phytoconstituents, and as corollary of invasive species
in general.
For compound 3, previous biological activity studies regard the cytotoxicity studies
on KB, COLO-205, HEPA-3B, and HELA cell lines and showed no activity [37].
For compound 4, biological activity studies have been performed, namely inhibitory
(Trypanosoma brucei) [38], and antimicrobial (Escherichia coli, Proteus sp., Streptococcus au-
reus and Candida albicans) [39] activities, as well as cytotoxicity (AGP01, HCT116, MCF07,
NIHOVCAR, SKAMELL4 and SF295 cell lines) and anti-inflammatory activities [40]. Al-
though antimicrobial activities and cytotoxicity were not observed, compound 4 showed
high lipoxygenase inhibition activity when compared to standard quercertin and inhib-
ited the production of IL-6 [40]. It also exhibited an inhibitory activity on the growth of
Trypanosoma brucei with respect to the clinically used antitrypanosomal agents suramin and
melarsoprol and showed a strong and selective inhibitory activity on the GAPDH enzyme
of T. brucei [38].
For compound 5, previous anticancer studies showed no significant activity (HIF-1
and NF-κB activities in reporter assays, and in cytotoxicity against A549, MDA-MB-231,
MCF-7, KB, KB-VIN, HT29, A498, PC3 and PACA2 cell lines) [43–45]. Antimicrobial
(Micrococcus tetragenus, Escherichia coli, Staphylococcus albus, Bacillus cereus, Staphylococcus
aureus, Micrococcus luteus, Bacillus subtilis, Pseudomonas aeruginosa, methicillin-resistant
Staphylococcus aureus, Vibrio parahemolyticus and Candida albicans), DPPH free radical scav-
enging, acetylcholinesterase inhibitory and brine shrimp larvicidal activities also showed
no results [42,46].

2. Results and Discussion

2.1. Compound Identification
From the dichloromethane extract (17.63 g) of A. melanoxylon R. Br. (1250 g) collected at
Peninha, Sintra, Portugal, lupeol 1 [33] (purified, <0.1 mg), 3β-Z-coumaroyl lupeol 2 [34,35]
(purified, 15.6 mg), 3β-E-coumaroyl lupeol (dioslupecin A) 3 [36,37] (purified, 39.0 mg),
Plants 2021, 10, 2698 3 of 11

and kolavic acid 15-methyl ester 4 [38–40] (purified 111.1 mg) were isolated (Figure 1).
The study of the alkaloid content of this species led instead to the isolation of vomifoliol
(blumenol A) 5 [41–43] (unpurified, 5.4 mg). Alkaloids were detected by TLC but their
isolation was not achieved due to the low content in this species (this may be related to
the time of harvest, since the accumulation of alkaloids is known to be seasonal, or due to
other biotic or abiotic factors).

Figure 1. Lupeol 1, 3β-Z-coumaroyl lupeol 2, 3β-E-coumaroyl lupeol (dioslupecin A) 3, kolavic acid

15-methyl ester 4 and vomifoliol (blumenol A) 5 isolated from A. melanoxylon.

The use of 1D and 2D NMR allowed the structure determination of all five compounds,
further confirmed by mass spectrometry and comparison with literature data (Figure 1).
Compound 1 was identified by its characteristic H-3 (δ 3.19 ppm, dd, J = 5.0 Hz,
J = 11.2 Hz), aliphatic methyl groups (δ 0.76 ppm, 0.79 ppm, 0.83 ppm, 0.94 ppm, 0.97 ppm
and 1.03 ppm), CH2 -29 (δ 4.56 ppm, sl and δ 4.56 ppm, sl) and Me-30 (δ 1.68 ppm) resonances
(Figure S1), all in agreement with literature values [33]. Comparison of the NMR spectra of
2 (Figures S2–S8) and 3 (Figures S9–S14) with that of 1 (Figure S1) allowed the recognition
of a lupeol base skeleton substituted at C-3 with a coumaroyl unit—the structures of 3β-
Z-coumaroyl lupeol 2 [34,35] and 3β-E-coumaroyl lupeol (dioslupecin A) 3 [36,37] were
proposed, based on the J value couplings of the substituent’s double bond (12.7 Hz and
Plants 2021, 10, 2698 4 of 11

15.9 Hz, respectively). Further confirmation came from comparison of the 1 H and 13 C
resonance values with those of the literature [34–37].
For compound 3 corrections are made for the literature [36,37] resonances of the aliphatic
methyl groups, based on HMBC and NOESY correlations (Table 1): HMBC between δ 0.89
and δ 0.92 with δ 81.2 (C-3) clearly indicates the presence of Me-23 and Me-24, distinguished
by NOESY with H-3 (for δ 0.89); HMBC of δ 0.88 with δ 50.3 (C-9) and δ 37.1 (C-10) assigns
Me-25; HMBC of δ 1.03 with δ 34.2 (C-7), δ 40.8 (C-8), δ 42.8 (C-14) and δ 50.3 (C-9) assigns
Me-26; HMBC of δ 0.95 with δ 27.4 (C-15) and δ 42.8 (C-14) assigns Me-27; finally, HMBC of δ
0.79 with δ 35.5 (C-16), δ 43.0 (C-17) and δ 48.2 (C-18) assigns Me-28.

Table 1. 1 H and 13 C resonances of the aliphatic methyl groups of 3β-E-coumaroyl lupeol 3 (CDCl3 ,
400 and 100 MHz).

1H (δ ppm) 13 C (δ ppm) *
Me-23 0.89 28.0
Me-24 0.92 16.6
Me-25 0.88 16.2
Me-26 1.03 15.9
Me-27 0.95 14.5
Me-28 0.79 18.0
* assigned by HSQC.

On purification and analysis, isomerization of the double bond was observed as

described previously for coumaroyl esters [47]: the final 1 H NMR spectrum of the sample
of 2 is composed of a 2.0:1.0 mixture of E and Z isomers (Figure S15), also present in the
chromatogram of the GC-FID analysis (1.6:1.0, different ratios accountable by different
isomerization times, Figure S16). Even the E isomer, compound 3, obtained in pure form,
seems to equilibrate in CDCl3 to a 4.4:1.0 mixture of E and Z forms (Figure S17).
For compound 4, a detailed analysis of the NMR data led to the proposed structure,
confirmed by the [M − H2 O + H]+ and [M − H]− ions present at m/z 331.3 u and m/z 347.2
u in the positive and negative ESI-MS spectra, respectively (Figures S18–S25). Analysis
of the spectra and comparison of the NMR resonances with those of the literature [38–40]
allowed us to make some corrections to previously assigned 13 C NMR values that should
be interchanged: (a) C-2 and C-7: interchanged based on HMBC with H-3 and Me-17,
respectively; (b) C-6 and C-11: interchanged based on COSY of H-12 with H-11 (C-11,
assigned by HSQC); (c) Me-19 and Me-20: interchanged based on HMBC with C-11; (d) C-5
and C-9: interchanged based on HMBC correlations of Me-19 and Me-20, respectively;
and (e) C-15 and C-18: interchanged based on HMBC of the OMe group (confirmed by
HMBC of H-14). Table 2 lists the full 13 C NMR assignment and the 1 H NMR outstanding
resonances (full 1 H NMR assignment can be found in the literature [38–40]).

Table 2. 13 C assignment and the 1 H outstanding resonances of kolavic acid 15-methyl ester 4 (CDCl3 ,
125 and 500 MHz). s singlet, d duplet, t triplet.

1H 13 C 1H 13 C

1 16.8 11 36.2
2 24.4 12 34.4
3 6.80 t 3.8 142.2 13 161.5
4 137.5 14 5.68 d 1.0 114.9
5 36.3 15 167.3
6 36.8 16 2.18 d 1.2 19.2
7 28.6 17 0.77 d 6.9 15.9
8 37.8 18 172.7
9 40.3 19 1.24 s 33.4
10 45.4 20 0.78 s 18.0
COOMe 3.69 s 50.8
Plants 2021, 10, 2698 5 of 11

Finally, for compound 5, the proposed structure based on NMR analysis was confirmed
by the [M − H2 O + H]+ , [M + H]+ , [M + Na]+ and [M − H]− ions present at m/z 207.1 u,
m/z 225.1 u, m/z 247.1 u, and m/z 223.0 u in the positive and negative ESI-MS spectra,
respectively (Figures S26–S32). Comparison of the 1 H and 13 C NMR resonances with
those of the literature [41–43] confirm the structure, and allows us to correct the 13 C NMR
δ values of the ∆7 double bond that must be interchanged: C-7 δ 129.0 ppm and C-8 δ
135.7 ppm (ascertained by HSQC).

2.2. Cytotoxicity Evaluation

Cytotoxicity studies were performed for kolavic acid 15-methyl ester 4 and a sample
of 3β-E-coumaroyl lupeol (dioslupecin A) 3. The fact that compounds 2 and 3 equilibrate
when in solution prevented us from testing both compounds separately. Since lupeol 1 and
vomifoliol (blumenol A) 5 were isolated in small amounts their biological testing was also
not performed.
To determine cellular toxicity, preliminary assays were performed with compounds 3
and 4 incubated in HCT116 cells for 72 h. Compound 3 showed to decrease cell viability to
64% only at the highest concentration tested (243 µM). However, at the same concentration,
compound 4 decreased cell viability to 3%.
Therefore, only the IC50 of compound 4 was determined by the MTS metabolism
assay, in order to evaluate its potential cytotoxic activity. In fact, compound 4 showed to
have a relatively low cytotoxicity against the HCT116 colon cancer cell line, with an IC50
value of 176.3 µM (95% CI = 163.8 to 189.8 µM, Figure 2). This might suggest its use as
an anti-inflammatory agent [40]. Nonetheless, additional studies using other cell lines are
required to discard completely the cytotoxic effect of this compound. 5-Fluorouracil (5-FU),
a cytotoxic agent in colon cancer treatment, was used as a positive control (IC50 value of
2.763 µM; 95% CI = 2.539 to 3.007 µM, Figure 3).

Figure 2. Dose-response curve in HCT116 cells upon incubation with compound 4 for 72 h. These
results are representative of at least three independent experiments.

Figure 3. Dose-response curve in HCT116 cells upon incubation with 5-FU for 72 h. These results are
representative of at least three independent experiments.
Plants 2021, 10, 2698 6 of 11

3. Materials and Methods

3.1. General Experimental Procedures
Silica gel 60 for chromatography (Merck 7734 or Merck 109385) was used for column
chromatography (gravity and flash, respectively) and TLC was performed on Silica gel 60
Merck 5744 (0.5 mm) or 5554 TLC plates employing 254 nm and/or 366 nm UV-lamp for
visualization. Molybdophosphoric acid or Munier Dragendorff reagent were used for addi-
tional spot development [48]. NMR spectra were recorded on a 400 MHz (100 MHz for 13 C)
Brucker Avance III 400 or 500 MHz (125 MHz for 13 C) Bruker AVANCE Neo 500 spectrom-
eter (Fällanden, Switzerland) at 25 ◦ C using standard pulse programs. Residual solvent
signals were used for calibration (1 H: δ(CHCl3 ) = 7.26 ppm, 13 C: δ(CDCl3 ) = 77.00 ppm).
GC-FID was performed on GC Agilent 6890 operated at the following conditions: carrier
gas Helium, split ratio 1:30; Column: ZB1HTinferno, L: 15 m, ϕ: 0.25 mm, df: 0.10 µm,
injector temperature 280 ◦ C; detector temperature 280 ◦ C; temperature program 100 ◦ C,
hold for 0 min, increase 10 ◦ C/min to 320 ◦ C, hold for 20 min.
ESI-MS spectra were performed on a Thermo orbitrap Qexactive focus apparatus with
direct inlet by a Thermo vanquish apparatus.

3.2. Plant Collection and Preparation

Branches and leaves of A. melanoxylon R. Br. were collected at Peninha, Sintra, Portugal
(38◦ 460 1000
N 9◦ 270 3300 W), on 15 December 2019 and a voucher specimen (LISI032915, Patrícia
Máximo and Ana Lourenço, s.n., collected on 1 March 2020) was deposited at Herbário João
de Carvalho e Vasconcellos (LISI), School of Agriculture (ISA), University of Lisbon. After air
drying at room temperature in the dark, and milling, 1850 g were obtained.

3.3. Extraction and Isolation

3.3.1. Dichloromethane Extract
An amount of 1250 g of A. melanoxylon R. Br. was defatted with n-hexane and extracted
with 2.5 L of dichloromethane (DCM), at room temperature for 5 h 30 min. After this time the
plant material was separated and re-extracted with another 2.5 L of dichloromethane, at room
temperature, for 18 h. The gathered filtrates were evaporated to yield 17.63 g of extract.
The extract was chromatographed on gravity column (diameter 15.0 cm, height
25 cm) with mixtures of n-hexane/ethyl acetate, ethyl acetate, two mixtures of ethyl ac-
etate/methanol and methanol to yield 7 fractions (DCM1 (8/2), DCM2 (7/3), DCM3 (6/4),
DCM4 (1/1), DCM5 (EtOAc), DCM6 (10% and 20% of MeOH), DCM7 (MeOH)).
The fraction DCM1 (4.13 g) was purified by flash chromatography with a mixture of
n-hexane/ethyl acetate 9/1 to yield six fractions, two of them further purified: DCM1E and
DCM1F. The fraction DCM1E (638.0 mg) was purified by flash column chromatography
with mixtures of n-hexane/ethyl acetate 85/15 to yield the fraction DCM1E1 (586.0 mg).
This fraction was further purified by flash column chromatography with mixtures of
n-hexane/ethyl acetate 85/15, 8/2, and ethyl acetate to yield 4 fractions. One of them,
DCM1E1B (385.0 mg), was purified twice by thin layer chromatography using mixtures
of n-hexane/ethyl acetate 8/2 and 85/15 to yield fraction DCM1E1Bp1p, compound 1
(15.1 mg). Another, DCM1E1C (30.0 mg) was purified by thin layer chromatography using
a mixture of n-hexane/ethyl acetate 8/2 to yield DCM1E1Cp1, compound 2 (23.1 mg).
Another, DCM1E1D (64.0 mg) gave a mixture of compounds 2 and 3. The fraction DCM1F
(574.0 mg) was purified by flash column chromatography with a mixture of n-hexane/ethyl
acetate 85/15 to yield fraction DCM1F2 (393.0 mg) that, after purification by thin layer
chromatography using a mixture of n-hexane/ethyl acetate 8/2, yielded fraction DCM1F2p,
compound 3 (57.0 mg).
The fraction DCM2 (2.91 g) was purified by flash column chromatography with
mixtures of n-hexane/ethyl acetate 8/2 and 7/3 to yield three fractions; one of them,
DCM2B (1.00 g), was further purified by flash column chromatography using mixtures of
n-hexane/ethyl acetate 8/2 and 7/3, and ethyl acetate to yield six fractions; one of them,
DCM2B5 yielded compound 4 (168.8 mg).
Plants 2021, 10, 2698 7 of 11

Final purification of the compounds for biological testing was achieved by flash
chromatography for 4 (white solid, 111.1 mg, n-hexane/ethyl acetate 8/2) or thin layer
chromatography for 1 (white solid, <1 mg, n-hexane/ethyl acetate 8/2), 2 (white solid,
3.5 mg, n-hexane/ethyl acetate 8/2), 3 (white solid, 27.8 mg, n-hexane/ethyl acetate 7/3),
and for the mixture of 2 and 3 (white solids, 12.1 and 11.2 mg, respectively, n-hexane/ethyl
acetate 8/2). On performing spectroscopic analysis isomerization of 2 and 3 was observed
for some of the samples.

3.3.2. Vomifoliol 5 Extraction

Acidic extraction of the plant was performed for the isolation of alkaloids. However,
a co-extraction compound, vomifoliol (blumenol A) 5, was isolated instead. An amount of
500 g of A. melanoxylon R. Br. was extracted with 1.55 l of HCl 0.5 M, at room temperature,
for 40 min. After centrifugation the supernatant was concentrated and basified with
NH4 OH 1M. This solution was applied in an isolute® HM-N (Biotage, Uppsala, Sweden)
column and after elution with dichloromethane and evaporation, the extract Amx (80.4 mg)
was obtained.
This extract was purified by thin layer chromatography using a mixture of dichloromethane/
methanol/NH4 OH 95/5/1 to yield 3 fractions. Of these, Amx-2 (11.8 mg) was purified
by thin layer chromatography using a mixture of dichloromethane/methanol/NH4 OH
95/5/1 to yield fraction Amx-2.2B, compound 5 (5.4 mg).

3.4. Compound Characterization

Compound 1 (unpurified)—1 H NMR spectrum, see Supplementary Materials, Figure S1.
Compound 2 (unpurified)—NMR spectra, see Supplementary Materials Figures S2–S8.
Compound 3 (unpurified)—NMR spectra, see Supplementary Materials Figures S9–S14.
Mixture 1.0:2.0 of compounds 2 and 3—1 H NMR spectrum, see Supplementary Materials
Figure S15. Mixture 1.0:1.6 of compounds 2 and 3—GC-FID chromatogram, see Supple-
mentary Materials Figure S16. Mixture 1.0:4.4 of compounds 2 and 3—1 H NMR spectrum,
see Supplementary Materials Figure S17. Compound 4—NMR spectra and ESI-MS spectra,
see Supplementary Materials Figures S18–S25. Compound 5 (unpurified)—NMR spectra
and ESI-MS spectra, see Supplementary Materials Figures S26–S32.

3.5. Cell Culture and Treatments

HCT116 human colon carcinoma cells, commonly used in drug screens, were grown
in McCoy’s 5A modified medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and 1% antibiotic/antimycotic solution (Gibco, Life Technologies, Paisley, UK).
Cells were cultured at 37 ◦ C under a humidified atmosphere of 5% CO2 .
For cell viability experiments, HCT116 cells were seeded in 96-well plates, at a concen-
tration of 5 × 103 cells/well and allowed to adhere for 24 h. Then, cells were exposed to
the test compound, compound 4, previously prepared in sterile DMSO. In order to plot
a dose–response curve, cells were exposed to this compound in a range of concentrations
between 0.04 µM and 4000 µM for 72 h. 5-Fluorouracil (5-FU), a cytotoxic agent used in
colon cancer treatment, was used as a positive control and DMSO was used as vehicle
control. Data are representative of three independent experiments.

3.6. Viability Assays

Cell viability of cells treated with compound 4 was evaluated using the CellTiter
96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) ac-
cording to the manufacturer’s instructions. This colorimetric assay is based in the capacity
of metabolic active cells to convert 3-(4,5-dimethylthiazo-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) to formazan, a dye that is soluble in
cell culture media. Formazan is quantified by measuring the absorbance at 490 nm and
correlates with the amount of living cells in culture. Absorbance was measured using
the GloMax-Multi+ microplate multimode reader (Promega, Madison, WI, USA) and the
Plants 2021, 10, 2698 8 of 11

best-fit IC50 value, from at least three independent experiments, was calculated using the
log (inhibitor) versus response (variable slope) function from GraphPad Prism software
(version 8.0.2; San Diego, CA, USA).

4. Conclusions
In this study, five already known compounds were isolated and as a result some
corrections are made to their previous 13 C NMR assignments. Cytotoxicity against HCT116
cells was evaluated for two of them, although no positive results were obtained.
As for the utility of the study of the chemistry of invasive species, here illustrated
by two extracts of A. melanoxylon, we can propose that they can be used as a source of
bioactive metabolites. Based on the literature and our own experimental results, lupeol
derivatives 2 and 3 show no anticancer activity. Previous reports on vomifoliol (blumenol
A) 5 for diverse bioactivities also showed no results. Nonetheless, kolavic acid 15-methyl
ester 4, the most abundant metabolite, is not cytotoxic and has previously been recognized
as a bioactive naturally occurring trypanocide that may contribute to anti-inflammatory
effects. A. melanoxylon can thus be considered a source for this metabolite.
We further add that it would be interesting to perform a detailed phytochemical
study of bark samples of this species in general—ring-barking is presently one of the
control measures for this species. Although Freire et al. [29,30] showed the presence of ∆7
phytosterols and phytosteryl glucosides in the dichloromethane extracts of the bark by
GC-EIMS, some of them with interesting reported bioactivities, many compounds may
have escaped this screening; furthermore, although the antimicrobial bioactivity of the
more polar ethanolic and aqueous extracts of the bark of this species is not noteworthy [49],
again a detailed phytochemical approach could provide pure metabolites for which many
more activities could be considered.
To conclude, although more studies are needed, this paper demonstrates that studying
the chemistry of invasive species might provide a utility for this natural and abundant
good that is currently underexplored.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/plants10122698/s1. Figure S1—1H NMR spectrum of Lupeol 1 (unpurified) (CDCl3, 400 MHz);
Figure S2—1H NMR spectrum of 3β-Z-coumaroyl lupeol 2 (unpurified) (CDCl3, 400 MHz); Figure S3—13C
NMR spectrum of 3β-Z-coumaroyl lupeol 2 (unpurified) (CDCl3, 100 MHz); Figure S4—DEPT135 spectrum
of 3β-Z-coumaroyl lupeol 2 (unpurified) (CDCl3 ); Figure S5—HSQC spectrum of 3β-Z-coumaroyl
lupeol 2 (unpurified) (CDCl3 ); Figure S6—HMBC spectrum of 3β-Z-coumaroyl lupeol 2 (unpurified)
(CDCl3 ); Figure S7—COSY spectrum of 3β-Z-coumaroyl lupeol 2 (unpurified) (CDCl3); Figure S8—NOESY
spectrum of 3β-Z-coumaroyl lupeol 2 (unpurified) (CDCl3); Figure S9—1 H NMR spectrum of 3β-E-
coumaroyl lupeol (dioslupecin A) 3 (unpurified) (CDCl3 , 400 MHz); Figure S10—13 C NMR spectrum
of 3β-E-coumaroyl lupeol (dioslupecin A) 3 (unpurified) (CDCl3, 100 MHz); Figure S11—DEPT135 spec-
trum of 3β-E-coumaroyl lupeol (dioslupecin A) 3 (unpurified) (CDCl3); Figure S12—HSQC spectrum of
3β-E-coumaroyl lupeol (dioslupecin A) 3 (unpurified) (CDCl3 ); Figure S13—HMBC spectrum of
3β-E-coumaroyl lupeol (dioslupecin A) 3 (unpurified) (CDCl3 ); Figure S14—NOESY spectrum of
3β-E-coumaroyl lupeol (dioslupecin A) 3 (unpurified) (CDCl3 ); Figure S15—1 H NMR spectrum of
the mixture of 3β-Z-coumaroyl lupeol 2 and 3β-E-coumaroyl lupeol 3 (1.0:2.0) (CDCl3 , 500 MHz);
Figure S16—GC-FID chromatogram of the mixture of 3β-Z-coumaroyl lupeol 2 and 3β-E-coumaroyl
lupeol 3 (1.0:1.6); Figure S17—1 H NMR spectrum of the mixture of 3β-Z-coumaroyl lupeol 2 and
3β-E-coumaroyl lupeol 3 (1.0:4.4) (CDCl3 , 500 MHz); Figure S18—1 H NMR spectrum of kolavic
acid 15-methyl ester 4 (CDCl3 , 500 MHz); Figure S19—13 C NMR spectrum of kolavic acid 15-methyl
ester 4 (CDCl3 , 125 MHz); Figure S20—DEPT135 spectrum of kolavic acid 15-methyl ester 4 (CDCl3 );
Figure S21—HSQC spectrum of kolavic acid 15-methyl ester 4 (CDCl3 ); Figure S22—HMBC spectrum
of kolavic acid 15-methyl ester 4 (CDCl3 ); Figure S23—COSY spectrum of kolavic acid 15-methyl ester
4 (CDCl3 ); Figure S24—NOESY spectrum of kolavic acid 15-methyl ester 4 (CDCl3 ); Figure S25—ESI-
MS spectra of kolavic acid 15-methyl ester 4; Figure S26—1 H NMR spectrum of vomifoliol (blumenol
A) 5 (unpurified) (CDCl3 , 500 MHz); Figure S27—13 C NMR spectrum of vomifoliol (blumenol A) 5
(unpurified) (CDCl3 , 125 MHz); Figure S28—DEPT135 spectrum of vomifoliol (blumenol A) 5 (un-
purified) (CDCl3 ); Figure S29—HSQC spectrum of vomifoliol (blumenol A) 5 (unpurified) (CDCl3 );
Plants 2021, 10, 2698 9 of 11

Figure S30—HMBC spectrum of vomifoliol (blumenol A) 5 (unpurified) (CDCl3 ); Figure S31—COSY

spectrum of vomifoliol (blumenol A) 5 (unpurified) (CDCl3 ); Figure S32—ESI-MS spectra of vomifo-
liol (blumenol A) 5 (unpurified).
Author Contributions: Conceptualization, P.M.; methodology, P.M., A.L., P.A. and C.M.P.R.; investi-
gation, D.A., S.D. and J.G.; resources A.L., P.A. and C.M.P.R.; writing—original draft preparation,
P.M.; writing—review and editing, P.M., A.L., P.A., J.G. and C.M.P.R.; supervision, P.M., A.L., P.A.
and C.M.P.R.; funding acquisition, A.L., P.A. and C.M.P.R. All authors have read and agreed to the
published version of the manuscript.
Funding: This work has received funding from FEDER through COMPETE 2020 under the Pro-
gramme grant LISBOA-01-0145-FEDER-016405, and from National Funds through FCT under the
Programme grant SAICTPAC/0019/2015 and PTDC/MED-FAR/29097/2017.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This work was supported by the Associate Laboratory for Green Chemistry-LAQV
which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).
GC-FID data obtained by the Analysis Laboratory, LAQV REQUIMTE—Chemistry department,
FCT NOVA—PORTUGAL. FCT/MCTES is also acknowledged for supporting the National Por-
tuguese NMR Network (ROTEIRO/0031/2013-PINFRA/22161/2016, co-financed by FEDER through
COMPETE 2020, POCI, PORL, and FCT through PIDDAC).
Conflicts of Interest: The authors declare no conflict of interest.

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