Phytochemical Analysis and Antimicrobial Activity of Myrcia tomentosa (Aubl.) DC. Leaves

This work describes the isolation and structural elucidation of compounds from the leaves of Myrcia tomentosa (Aubl.) DC. (goiaba-brava) and evaluates the antimicrobial activity of the crude extract, fractions and isolated compounds against bacteria and fungi. Column chromatography was used to fractionate and purify the extract of the M. tomentosa leaves and the chemical structures of the compounds were determined using spectroscopic techniques. The antibacterial and antifungal activities were assessed using the broth microdilution method. The phytochemical investigation isolated 11 compounds: α-bisabolol, α-bisabolol oxide B, α-cadinol, β-sitosterol, n-pentacosane, n-tetracosane, quercetin, kaempferol, avicularin, juglanin and guaijaverin. The crude ethanolic extract and its fractions were tested against 15 bacteria and 9 yeasts. The crude extract inhibited the in vitro growth of yeasts at concentration of 4 to 32 μg/mL. The hexane, dichloromethane, ethyl acetate and aqueous fractions inhibited Candida sp. at concentrations of 4 to 256 μg/mL, whereas the Cryptococcus sp. isolates were inhibited only by the hexane and dichloromethane fractions in minimal inhibitory concentrations (MICs) at 16 to 64 μg/mL. The flavonoid quercetin-3-O-α-arabinofuranose (avicularin) was the most active compound, inhibiting Candida species in concentrations of 2 to 32 μg/mL. The MIC values suggest potential activity of this plant species against yeast.


Introduction
Infectious diseases are of great interest in the scientific community because some microorganisms cause severe morbidity and can be lethal. Plant species are a potential reservoir for the discovery of new drugs [1][2][3].
Among the plants of the Brazilian Cerrado, the Myrtaceae family has a great representation, and several species are used as ornaments, wood, food and medicines [4][5][6]. Myrcia tomentosa (Aubl.) DC. is a species of the Myrtaceae family and popularly known as "goiaba-brava". It is a species native from the Brazilian Cerrado and can be found from Panama to the southeast of Brazil [7,8]. Despite its frequent citation in floristic or phytosociological surveys [4,9,10], M. tomentosa is underreported in pharmacognostic or phytochemical studies. Several biological activities have been described for the species of this genus, such as the inhibition of thyroid peroxidase, anti-obesity, hypolipidemic, hypoglycemic, antimicrobial, antioxidant, antifungal, anti-inflammatory, anti-nociceptive and hepatoprotective activities [11][12][13][14][15][16][17]. These activities are often attributed to the presence of secondary metabolites, such as their essential oils, but with few properties related to their non-volatile compounds [6].
Because of the scarcity of studies about M. tomentosa, the aim of this work is to realize the first bioassay-guided isolation of the extract and structural elucidation of the compounds, so as to verify the antimicrobial activity of these extracts and compounds against certain bacterial and fungal pathogens.
The sesquiterpenes (α-bisabolol, bisabolol B oxide and α-cadinol), the hydrocarbons (npentacosane and n-tetracosane), the steroid (β-sitosterol) and the flavonoids (quercetin, kaempferol, guaijaverin) were isolated and identified for the first time in this species.  The study biomonitored the fractions of M. tomentosa which allowed the isolation of substances that were responsible for the antimicrobial activity of this plant species. The results showed the antimicrobial activity by screening the crude extract and its fractions.
The study biomonitored the fractions of M. tomentosa which allowed the isolation of substances that were responsible for the antimicrobial activity of this plant species. The results showed the antimicrobial activity by screening the crude extract and its fractions.
According to Holetz et al. [27] and other authors, such as Ayres et al. [28] and Regasini et al. [29], the MIC values below 100 µg/mL have good antimicrobial activity; an MIC from 100 to 500 µg/mL represents moderate antimicrobial activity; an MIC from 500 to 1000 µg/mL represents weak activity; an MIC above 1000 µg/mL suggests that the substance is inactive.
The antimicrobial activity against bacteria and yeasts using the crude ethanolic extract and fractions of M. tomentosa showed that the crude extract was inactive or weakly active for most tested bacteria; however, for the yeasts of the genus Candida and Cryptococcus neoformans species complex, the MIC range was 4-32 µg/mL. For the fractions, the antibacterial activity was moderate for some Gram-positive bacteria, and the ethyl acetate and aqueous fractions showed MICs of 125-500 µg/mL against P. aeruginosa (Table 1). Although the constituents of this plant, such as sesquiterpens, exhibit well-known antibacterial activity, the fractions isolated from the M. tomentosa leaves showed poor activity against bacteria. The MIC values ranged of 4 to >1024 µg/mL against the studied yeasts. The polar fractions ethyl acetate fraction (EAF) and aqueous fraction (AF) and non-polar fractions hexane fraction (HF) and dichloromethane fraction (DF) were effective against Candida with low MIC values, which ranged from 4 to 256 µg/mL. Cryptococcus neoformans species complex was particularly inhibited by the non-polar fractions (HF and DF) of the leaf extract, with MIC values ranging of 16 to 64 µg/mL as shown in Table 1.
The main compounds obtained from HF and DF were identified as isolated sesquiterpenes (α-bisabolol, α-bisabolol oxide B and α-cadinol) or a mixture of these sesquiterpenes. Terpenes are a class of secondary metabolites with important functions in the interaction between a plant and its environment, as frequently implied in the defensive functions against herbivores and pathogens in the  [30]. In this study, the identified sesquiterpenes (α-bisabolol, bisabolol B oxide and α-cadinol), which were earlier reported on for their antimicrobial activity [31][32][33][34], were inactive in tested concentration against Candida sp. and showed onyl moderate activity (128 µg/mL) in the mixture. These results suggest that these sesquiterpenes partially contributed to the antifungal activity of this fraction, but also that other compounds are necessary for the fractions to exhibit good activity.
The compounds and mixtures of compounds of this plant evaluated against yeasts of the genus Candida, are showed in Tables 1 and 2. Interesting results were obtained with avicularin isolated from polar fraction ethyl acetate. This substance showed a good antifungal activity (2-32 µg/mL) for all Candida strains; thus, it is mainly responsible for the activity of the fraction and probably the crude extract. The flavonoid guaijaverin showed moderate activity for 55.5% of isolates and the juglanin showed similar activity for 44.4% of the isolates. Similar results were found by Metwally et al. [35] that reported good activity of avicularin and guajaverin against C. albicans.
The flavonoids and mixture flavonoids of this plant evaluated against yeasts of the genus Candida are showed in Table 2.
Several researchers, such as Kuete [36] and Martins et al. [37] associated the antimicrobial activity of aromatic plants with phenolic compounds. These compounds are mainly induced by fungal membrane damage with a consequent increase in cellular permeability [38]. Salazar-Aranda et al. [39] also suggested a structure-activity relationship where the hydroxylation pattern of the B or C ring of the flavonoid can determine its degree of antifungal activity.
Additionally, the results observed by Holetz et al. [27], Domingues et al. [40], Paula et al. [41] and Correia et al. [42] showed good activity against yeasts of other species of the Myrtaceae family, suggesting that compounds of M. tomentosa, as avicularin can be used as a potential antifungal.
In conclusion, the phytochemical analysis of M. tomentosa amplifies the chemical knowledge of the genus since the reported studies are mainly related to essential oils. In addition, the results of this study prove the antifungal activity of the ethanolic extract of M. tomentosa and its fractions and show that the flavonoid quercetin-3-O-α-arabinofuranose (avicularin) is mainly responsible for this biological activity and a potential source of new antifungal alternatives.

General Procedures
The 1 H and 13 C one-dimensional and two-dimensional NMR spectra were obtained in deuterated chloroform (CDCl 3 ) or deuterated methanol (CD 3 OD) on a Bruker Avance 500 MHz instrument (500 MHz for 1 H and 125 MHz for 13 C-NMR). The chemical shifts are expressed in δ values (ppm) with tetramethylsilane (TMS, δ = 0.0 ppm) as an internal reference. The coupling constants (J) were measured in Hertz (Hz). To process and analyze the spectra, the TopSpin ACD/Labs 12.0 programs were used.
The gas chromatography coupled to mass spectrometry (GC/MS) was performed on the chromatograph QP2010 (Shimadzu), which was equipped with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 mM) using helium as the carrier gas. The injection volume was 1 µL, and the ionization energy was 70 eV. The parameters in the identification of chemical constituents are the presence of the molecular ion peak, basic peak, visual comparison with the spectra provided by the specter equipment [19], and fragmentation pattern in relation to the described mass spectra in the literature.
High-performance liquid chromatography (HPLC) was performed for the polar fractions using a Waters instrument e2596 with a quaternary pump, the diode array detector (DAD) 2998 and 2.0 Enpower data-processing system. The column was a Zorbax XDB C18 column (25 cm × 4.6 mm × 5 um), the flow was 1 mL/min, the temperature was 25 • C, and the injection volume was 10 µL. The mobile phase consisted of methanol and acidified water with 2% glacial acetic acid in different proportions. The samples were pre-filtered using a 0.45-µM Millex ® membrane (Millipore, Cork, Ireland) and a mobile-phase PVDF membrane of 0.45 micrometre (Millipore, Cork, Ireland).
The semipurified polar samples were submitted to a Sepacore preparative chromatograph (Buchi) with the peristaltic pump model C-615. A Sepacore ® C-18 column (9 cm × 10 mm) with a flow rate of 10 mL/min was used. The samples were previously filtered through a 0.45-µm Millex ® membrane (Millipore, Cork, Ireland) and chromatographed with the mobile phase, which consisted of methanol and purified water in different proportions.

Extraction and Purification
The air-dried and powdered leaves of M. tomentosa (50 g) were extracted with 95% ethanol by maceration (1:5 w/v) at room temperature. The crude ethanolic extract was filtered and concentrated on a rotary evaporator at a temperature below 40 • C. Fifty grams of dried extract were solubilized in 200 mL MeOH/H 2 O (7:3) and subjected to a liquid/liquid extraction with solvents of increasing polarity (hexane, dichloromethane, and ethyl acetate). These fractions were concentrated on a rotary evaporator at 40 • C and maintained at the exhaust hood until the solvent was completely removed. The MeOH/H 2 O residual was lyophilized, which resulted in an aqueous fraction. The resulting fractions were named hexane fraction (HF), dichloromethane fraction (DF), ethyl acetate fraction (EAF) and aqueous fraction (AF).

Microbial Strains
The studied microorganisms are as follows: reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus roseus, Micrococcus luteus, Bacillus cereus, Bacillus subtilis, Escherichia coli, Enterobacter cloacae, Enterobacteraerogenes, Enterobacteraerogenes, Pseudomonas aeruginosa, Serratia marcescens, Salmonella sp., Candida albicans, Candida parapsilosis and Cryptococcus neoformans species complex, obtained from the American Type Culture Collection (ATCC) standard strains, except of theclinical isolates of Pseudomonas aeruginosa (SPM1), C. albicans (2, 3, 48, 111, 138, Molecules 2017, 22, 1100 7 of 10 181), C. parapsilosis (11) and C. neoformans species complex (L1, L2), which belong to the collection of the Laboratory of Medical Bacteriology and Laboratory of Mycology at the Institute of Tropical Pathology and Public Health from Federal University of Goiás, Goiânia, GO, Brazil (IPTSP-UFG). They were maintained at 4 • C. Prior to testing, the bacteria were transferred to Casoy agar (Difco) and incubated at 37 • C for 24 h; while the fungi were transferred to Sabouraud agar (Difco) and incubated at room temperature for 24-48 h for Candida spp. and 48-72 h for the C. neoformans species complex.

In Vitro Susceptibility Testing
The in vitro activity of the ethanolic leaf extract, their fractions and compounds of M. tomentosa was evaluated using the broth microdilution method, as described in the Clinical and Laboratory Standards Institute (CLSI) documents M07-A8 for bacteria and M27-A3 and M27-S4 for yeasts [43][44][45].
In the antibacterial test, 200 µL of plant extract in initial concentration of 2000 µg/mL was diluted in Mueller-Hinton broth and 10% dimethyl sulfoxide (DMSO). Serial two-fold dilutions were conducted in 96-well microplates for the final concentrations of 1000 to 1.95 µg/mL of the extract or fractions. A volume of 5 µL containing 10 4 UFC/mL of microbial inoculum was added to each well plate and incubated at 35 • C for 18-20 h. The bacterial growth was visualized by adding 0.5% triphenyl tetrazolium chloride to each well and analyzed after 30 min of incubation. The appearance of reddish color was considered as proof of bacterial growth.
In the antifungal activity, the samples were diluted in an RPMI 1640 medium with L-glutamine without bicarbonate, which was then buffered with 0.165 M MOPS (morpholine propane sulfonic acid) and 5-10% DMSO. Serial two-fold dilutions were conducted in 96-well microplates for the final concentrations of 1024 to 1 µg/mL of the extract or fractions and 128 to 0.125 µg/mL for each compound. A volume of 100 µL of microbial inoculum at a concentration of 10 3 UFC/mL was added to each well and incubated at 35 • C for 48 h for Candida sp. and at room temperature for 72 h for the Cneoformans species complex. Fungal growth was checked visually and the MIC was defined as the lowest concentration, which resulted in total inhibition of visible microorganism growth.
The tests were performed in duplicatein two independent experiments. Vancomycin