molecules Article Phytochemical Analysis and Antimicrobial Activity of Myrcia tomentosa (Aubl.) DC. Leaves Fabyola Amaral da Silva Sa 1,3, Joelma Abadia Marciano de Paula 2, Pierre Alexandre dos Santos 3, Leandra de Almeida Ribeiro Oliveira 3, Gerlon de Almeida Ribeiro Oliveira 4, Luciano Morais Liao 4 , Jose Realino de Paula 3,* and Maria do Rosario Rodrigues Silva 1,* 1 Institute of Tropical Pathology and Public Health, Federal University of Goias, Goiânia 74605-050, Brazil; [email protected] 2 Unit of Exact and Technologic Sciences, Goias State University, Anápolis 75132-400, Brazil; [email protected] 3 Faculty of Pharmacy, Federal University of Goias, Goiânia 74605-170, Brazil; [email protected] (P.A.d.S.); [email protected] (L.d.A.R.O.) 4 Chemistry Institute, Federal University of Goiás, Goiânia 74690-900, Brazil; [email protected] (G.d.A.R.O.); [email protected] (L.M.L.) * Correspondence: [email protected] (J.R.d.P.); [email protected] (M.d.R.R.S.); Tel.: +55-62-3209-6127 (M.d.R.R.S.); Fax: +55-62-3209-6363 (M.d.R.R.S.) Academic Editor: Isabel C. F. R. Ferreira Received: 23 May 2017; Accepted: 29 June 2017; Published: 4 July 2017 Abstract: 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. Keywords: myrtaceae; antifungal activity; avicularin; Candida sp.; phytochemistry; flavonoid 1. 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 [1–3]. 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 [4–6]. 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 Molecules 2017, 22, 1100; doi:10.3390/molecules22071100 www.mdpi.com/journal/molecules Molecules 2017, 22, 1100 2 of 10 pharmacognosticMolecules 2017, 22, 1100 or phytochemical studies. Several biological activities have been described for2 ofthe 10 species of this genus, such as the inhibition of thyroid peroxidase, anti-obesity, hypolipidemic, hypoglycemic,of Brazil [7,8]. antimicrobial, Despite its frequentantioxidant, citation antifu inngal, floristic anti-inflammatory, or phytosociological anti-nociceptive surveys [4, 9and,10], hepatoprotectiveM. tomentosa is underreported activities [11–17]. in pharmacognosticThese activities are or often phytochemical attributed to studies. the presence Several of secondary biological metabolites,activities have such been as described their essential for the species oils, but of this with genus, few such properties as the inhibition related to of thyroidtheir non-volatile peroxidase, compoundsanti-obesity, [6]. hypolipidemic, hypoglycemic, antimicrobial, antioxidant, antifungal, anti-inflammatory, anti-nociceptiveBecause of the and scarcity hepatoprotective of studies about activities M. tomentosa [11–17]., These the aim activities of this arework often is to attributed realize the to first the bioassay-guidedpresence of secondary isolation metabolites, of the extract such and as theirstructural essential elucidation oils, but of with the compounds, few properties so relatedas to verify to their the antimicrobialnon-volatile compounds activity of these [6]. extracts and compounds against certain bacterial and fungal pathogens. Because of the scarcity of studies about M. tomentosa, the aim of this work is to realize the first 2. Results and Discussion bioassay-guided isolation of the extract and structural elucidation of the compounds, so as to verify the antimicrobialThe chemical activity structures of these of extracts the compounds and compounds were elucidated against certain using bacterial Nuclear and Magnetic fungal Resonance pathogens. (NMR) data with 2D experiments Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear2. Results and DiscussionMultiple Bond Correlation (HMBC), Gas Chromatography/Mass Spectrometry (GC/MS)The chemicaland comparison structures with of the the literature compounds (copies were of elucidated the origin usingal spectra Nuclear can be Magnetic obtained Resonance from the (NMR)corresponding data withauthor). 2D The experiments compounds Heteronuclear isolated from Singlethe leaves Quantum of M. tomentosa Coherence (Figure (HSQC) 1) were and identifiedHeteronuclear as α-bisabolol Multiple (Mt-1 Bond) [18], Correlation α-bisabolol (HMBC), oxide B ( GasMt-2 Chromatography/Mass) [19], α-cadinol (Mt-3) [19], Spectrometry β-sitosterol (GC/MS)(Mt-4) [20,21], and n comparison-pentacosane with (Mt-5 the) [19], literature n-tetracosane (copies of(Mt-6 the) original[19], quercetin spectra (Mt-7 can be) [22], obtained kaempferol from (theMt-8 corresponding) [23], avicularin author). (Mt-9 The) [22,24], compounds juglanin isolated (Mt-10) from [25] and the leavesguaijaverin of M. ( tomentosaMt-11) [26].(Figure 1) were identifiedThe sesquiterpenes as α-bisabolol ( Mt-1(α-bisabolol,)[18], α-bisabolol bisabolol oxide B oxide B (Mt-2 and)[19 α],-cadinol),α-cadinol (theMt-3 hydrocarbons)[19], β-sitosterol (n- (pentacosaneMt-4)[20,21 ],andn-pentacosane n-tetracosane), (Mt-5 the)[ steroid19], n-tetracosane (β-sitosterol) (Mt-6 and)[ the19 ],flavonoi quercetinds (quercetin, (Mt-7)[22], kaempferol, kaempferol (guaijaverin)Mt-8)[23], avicularin were isolated (Mt-9 and)[22 identified,24], juglanin for the (Mt-10 first )[time25] in and this guaijaverin species. (Mt-11)[26]. OH HO H OH H H O H Mt-3 Mt-2 Mt-1 R1 R2 HO O R3 OH O H3C CH3 7-11 HO Mt-4 n Mt-7:R1=OH; R2=OH; R3= OH Mt-8:R1=H; R2=OH; R3= OH Mt-5:n=22 Mt-9: R1=OH; R2=OH; R3= arabinofuranose Mt-6:n=23 Mt-10:R1=H; R2=OH; R3= arabinofuranose Mt-11:R1=OH; R2=OH; R3= arabinopyranose Figure 1. ChemicalChemical structures structures of of compounds compounds isolated isolated from from the the leaves leaves of ofMyrciaMyrcia tomentosa tomentosa. (Mt-1.(Mt-1) α-) bisabolol.α-bisabolol. (Mt-2 (Mt-2) α)-cadinol.α-cadinol. (Mt-3 (Mt-3) α) -bisabololα-bisabolol oxide oxide B. B. ( (Mt-4Mt-4)) β-sitosterol.-sitosterol. ( (Mt-5Mt-5)) n-tetracosane. (Mt-6) nn-pentacosane.-pentacosane. ( (Mt-7Mt-7)) Quercetin. ( (Mt-8Mt-8)) Kaempferol. ( (Mt-9Mt-9)) Avicularin. Avicularin. ( (Mt-10Mt-10)) Juglanin. (Mt-11) Guaijaverin. Guaijaverin. The study sesquiterpenes biomonitored (α -bisabolol,the fractions bisabolol of M. tomentosa B oxide which and allowedα-cadinol), the isolation the hydrocarbonsof substances (thatn-pentacosane were responsible and n-tetracosane), for the antimicrobial the steroid activity (β-sitosterol) of this and plant the flavonoidsspecies. The (quercetin, results showed kaempferol, the antimicrobialguaijaverin) were activity isolated by screening and identified the crude for theextract first and time its in fractions. this species. AccordingThe study biomonitoredto Holetz et al. the [27] fractions and other of authors,M. tomentosa such whichas Ayres allowed et al. [28] the and isolation Regasini of substances et al. [29], thethat MIC were values responsible below 100 for theμg/mL antimicrobial have good activityantimicrobial of this activity; plant species. an MIC Thefrom results 100 to showed 500 μg/mL the antimicrobial activity by screening the crude extract and its fractions. Molecules 2017, 22, 1100 3 of 10 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