Anais da Academia Brasileira de Ciências (2017) 89(4): 2817-2824 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201720170339 www.scielo.br/aabc | www.fb.com/aabcjournal Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. FLAVIANE G. PEREIRA1, RONALDO MARQUETE2, LEVY T. DOMINGOS3, MARCO E.N. ROCHA4, ANTONIO FERREIRA-PEREIRA3, ELISABETH MANSUR5 and DAVYSON L. MOREIRA4 1Programa de Pós-Graduação em Biotecnologia Vegetal e Bioprocessos, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Edifício do Centro de Ciências da Saúde, Bloco K, 2º andar, sala K 032, Cidade Universitária, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil 2Fundação Instituto Brasileiro de Geografia e Estatística, Diretoria de eociências,G Avenida República do Chile, 500, 15º andar, Centro, 20031-170 Rio de Janeiro, RJ, Brazil 3Instituto de Microbiologia Professor Paulo de Góes, Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Edifício do Centro de Ciências da Saúde, Bloco I, 1º andar, sala I044, Cidade Universitária, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil 4Fundação Oswaldo Cruz, Departamento de Produtos Naturais, Instituto de Tecnologia Fármacos, Farmanguinhos, Rua Sizenando Nabuco, 100, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil 5Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Rua São Francisco Xavier, 524, sala 505 PHLC, Maracanã, 20550-013 Rio de Janeiro, RJ, Brazil Manuscript received on May 15, 2017; accepted for publication on August 7, 2017 ABSTRACT Casearia genus (Salicaceae) is found in sub-tropical and tropical regions of the world and comprises about 160-200 species. It is a medicinal plant used in South America, also known as “guaçatonga”, “erva-de- tiú”, “cafezinho-do-mato”. In Brazil, there are about 48 species and 12 are registered in the State of Rio de Janeiro, including Casearia sylvestris Sw. There are many studies related to the chemical profile and cytotoxic activities of extracts from these plants, although few studies about the antifungal potential of the essential oil have been reported. In this work, we have studied the antifungal properties of the essential oil of C. sylvestris leaves, as well as of their fractions, against four yeasts (Saccharomyces cerevisae, Candida albicans, C. glabrata and C. krusei) for the first time. The chemical analysis of the essential oil revealed a very diversified (n = 21 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%). These sesquiterpenes included α-humulene (17.8%) and α-copaene (8.5%) and the oxygenated sesquiterpene spathulenol (11.8%) were also identified. Monoterpenes were not identified. The fractions are mainly composed of oxygenated sesquiterpenes, and the most active fraction is rich in the sesquiterpene 14-hydroxy -9-epi-β-caryophyllene. This fraction was the most effective in inhibiting the growth of three yeast strains. Key words: Candida sp., Casearia genus, essential oil, medicinal plant, Saccharomyces cerevisae. Correspondence to: Flaviane Gomes Pereira E-mail: [email protected] An Acad Bras Cienc (2017) 89 (4) 2818 FLAVIANE G. PEREIRA et al. INTRODUCTION Fungal diseases are a menacing problem in the world, affecting mainly immunocompromised Casearia genus belongs to Salicaceae family, and patients (Cannon et al. 1995, 2009). The genus it is distributed in sub-tropical and tropical regions of the world, comprising 200 species. The genus Candida is the most common agent diagnosed is found in the Americas, Africa (rarely), Asia in invasive fungal infections, with high rates and Australia (Sleumer 1980, Mosaddik et al. of morbidity and mortality. This pathogen is 2004, Breteler 2008). In Brazil, there are about 48 frequently isolated from patients in critical species, 24 of which are endemic, and 12 species are health condition, and is among leading causes of registered in the State of Rio de Janeiro (Marquete bloodstream and catheter-associated urinary tract and Vaz 2007, Marquete and Mansano 2010). infections (Lockhart 2014). Candida albicans Casearia sylvestris Sw. can be found throughout strains are found in healthy individuals, including the Brazilian territory. It is popularly known as C. glabrata, C. tropicalis and C. krusei, albeit “guaçatonga”, “tiú” or “erva-de-lagarto”, and has they are also opportunistic pathogens (Procop and medicinal properties according to ethnobotanical surveys (Bratti et al. 2013, Pinto et al. 2013, Tomazi Roberts 2004, Pfaller et al. 2008). Resistance of et al. 2014). This plant is also included in the public Candida yeast to current medications, including health system (SUS) of Brazil and widely used to the class of azoles, has been reported (Pfaller and treat different diseases. According to Sato et al. Diekema 2004, Pfaller 2012), and one of the causes (1996), the chemical components found in essential of this resistance is the overexpression of plasma oils and extracts from leaves of C. sylvestris can efflux pumps (Holmes et al. 2008, Sanglard and be related to their uses in folk medicine as an Bille 2002, Rogers and Barker 2003, Cannon et al. antiseptic, antimicrobial and antiviral (Esteves et 2009). al. 2005, Pereira et al. 2016). In view of these facts, studies of biologically The secondary metabolites of Casearia are active molecules from plants which can play mainly diterpenes, specially the clerodane type (Kanokmedhakul et al. 2007, Carvalho et al. 2009, an important role as an alternative antifungal Ferreira et al. 2010). The essential oils of this compound or in association with antifungal drugs, genus are rich in monoterpenes and sesquiterpenes are highly recommended. It is important to note that (Tininis et al. 2006, Sousa et al. 2007, Silva et al. both extracts and essential oils of several species 2008, Pereira et al. 2017), and some of the main have been shown to possess potent antifungal compounds already identified are α-zingiberene, activity (Cruz et al. 2007, Kalidindi et al. 2015). α-humulene, caryophyllene, and bicyclogermacrene Development of new therapeutic approaches (Esteves et al. 2005, Bou et al. 2013). may contribute for the treatment of diseases caused In spite of the several phytochemical studies by fungi because the resistance to existing drugs on C. sylvestris, the chemical profile and the is a serious complication for patients (Pfaller et antifungal activity of essential oils have not been al. 2005, Pfaller and Diekema 2007). This study studied so far. It seems sesquiterpenes may play an important role as antifungal agents, and considering shows that sesquiterpenes with basic caryophyllene the possibility of further studies, non-oxygenated skeleton derived from C. sylvestris might be used sesquiterpenes such α-humulene found in Casearia in association with the current antifungal drugs to species can be investigated (Pereira et al. 2017). treat Candida infections. An Acad Bras Cienc (2017) 89 (4) ANTIFUNGAL ACTIVITIES OF C. sylvestris 2819 MATERIALS AND METHODS Aldrich), performed using the same column and conditions adopted in the GC analysis for the Casearia sylvestris Sw. (Salicaceae) was essential oils, and with the equation proposed by collected in Tijuca National Park (S22°57’05.04” Vandendool and Kratz (1963). W43°17’10.09”), Rio de Janeiro, Brazil (SISBIO The parameters adopted for HP-5MS were license n. 38765-1/CGEN license n. 010105/2014- (5% diphenyl and 95% dimethylpolysiloxane), 0). Plant identification was performed by Dr. column (30 m × 0.32 mm i.d. × 0.25 μm particle Ronaldo Marquete, and the herbarium voucher size), temperature programming from 60 to 240°C, was deposited in the Botanical Garden Herbarium with increase of 3°C/min, using synthetic air and of Rio de Janeiro with registration number RB hydrogen as the carrier gases, with a flow rate of 570651. 1 ml/min and injection volume of 1μl. For GC- Fresh leaves of C. sylvestris (1.5 kg) were cut MS, the parameters used for HP-5MS were (5% to small pieces and submitted to hydrodistillation diphenyl and 95% dimethylpolysiloxane), column in a modified Clevenger-type apparatus for two (30 m × 0.32 mm i.d. × 0.25 μm particle size), hours. Essential oil was directly separated from the temperature programming from 60 to 240°C, with aqueous phase yielding 1.2% (w/v), transferred to increase of 3°C/min, using helium as the carrier o amber flasks and kept at low temperature (-20 C) gas, with a flow rate of 1 ml/min and injection until analysis. The sample was subjected to analysis volume of 1μl (Pereira et al. 2017). by gas chromatography coupled to flame ionization For the phytochemical analysis of the major detector (HP-Agilent 6890 GC-FID) and by gas essential oil compound, the extraction of essential chromatography coupled to mass spectrometry (HP oil of C. sylvestris fresh leaves yielded around 20 Agilent GC 6890 – MS 5973), at the Analytical mL of a pure, clear, fluid and yellowish collored Platform of Institute of Pharmaceutical Technology oil. Part of the oil (2 mL) was submitted to a silica (Farmanguinhos) Oswaldo Cruz Foundation (Rio gel chromatographic column, which was eluted de Janeiro, Brazil), as described previously (Pereira by n-hexane, ethyl acetate and methanol and their et al. 2016). Briefly, the essential oil was diluted mixtures, using an increasing polarity gradient. The in dichloromethane (1.0 mg/ml) and analyzed by chromatographic running was very slow as well GC-MS to obtain the mass spectra and to perform as the increasing polarity of solvent systems, and chemical characterization. Concomitantly, another 99 fractions were obtained. After analysis by TLC sample of essential oil (0.5 mg/ml) was analyzed by plates under UV wavelength, the fractions were GC-FID for quantification of chemical constituents reunited according to their similarity, resulting into and to determine the retention indices (RI).