Exploring the Phytochemicals of Acacia melanoxylon R. Br.

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 13C 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.


Introduction
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 disrupting the biotic and abiotic balance of the invaded ecosystem. Apart from this ecological impact, they also have a socio-economic impact by influencing human health, infrastructures and local economies [1,2]. These species have long been a concern in the Portuguese territory [3][4][5] and elsewhere, which resulted in the publication of legal regulations to 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 millions of euros [9] and, since eradication is rarely achieved, actions end up frequently with periodic growth control and containment of these species [10]. The use of invasive species as a source of chemicals or pharmaceuticals allows a rational use of resources that 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].
Bearing this in mind, we began our studies on the chemistry of invasive Acacia species. Acacia species, mainly from Australia, settled in the Mediterranean area and conquered significant lands. In Portugal they are distributed throughout the country, preferentially on acid substrates. Originally, they were introduced as a source for wood, as an erosion preventer, for reforestation purposes and for ornamental reasons and the perfume industry, among others [13,14]. Abiotic and biotic factors favored their establishment and with time some of their uses deteriorated and economic value lowered, leading to an increase in their abundance and to an invasion condition [14][15][16]. The management and control of 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].
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 aureus and Candida albicans) [39] activities, as well as cytotoxicity (AGP01, HCT116, MCF07, NIHOVCAR, SKAMELL4 and SF295 cell lines) and anti-inflammatory activities [40]. Although antimicrobial activities and cytotoxicity were not observed, compound 4 showed high lipoxygenase inhibition activity when compared to standard quercertin and inhibited 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 3 corrections are made for the literature [36,37] resonances of the aliphatic methyl groups, based on HMBC and NOESY correlations (Table 1)  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 CDCl 3 to a 4.4:1.0 mixture of E and Z forms ( Figure S17).  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][39][40]).  [41][42][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).

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).
ESI-MS spectra were performed on a Thermo orbitrap Qexactive focus apparatus with direct inlet by a Thermo vanquish apparatus.

Plant Collection and Preparation
Branches and leaves of A. melanoxylon R. Br. were collected at Peninha, Sintra, Portugal (38 •  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).

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 NH 4 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.

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% CO 2 .
For cell viability experiments, HCT116 cells were seeded in 96-well plates, at a concentration of 5 × 10 3 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.

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) according 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 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).

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.