MDGC-MS analysis of essential oils from Protium heptaphyllum (Aubl. ) and their antifungal activity against Candida specie / Análise MDGC-MS de óleos essenciais de Protium heptaphyllum (Aubl. ) e sua atividade antifúngica contra espécies de Candida

ABSTRACT Protium heptaphyllum is found in the Amazon region, and in various Brazilian states and South American countries. Also Known as almecega, it produces an oil resin used in traditional medicine as analgesic, anti-inflammatory, cicatrizant and expectorant, it is rich in pentacyclic triterpenes and essential oil. The main objective of this study was to analyze the chemical composition of P. heptaphyllumresin (OEPh) over different extraction times and to evaluate their antifungal activity against Candida species, obtained from gardeners with onychomycosis, using the disk diffusion method. The OEPh was obtained by hydrodistillation and analyzed by Multidimensional Gas Chromatography coupled with Mass Spectrometry (MDGC / MS). Candida species were obtained from lesions on the nails of horticulturist from a community garden in the city of Teresina, Piauí, Brazil. The antifungal activity in concentrations of 1000 µg/L, 500 µg/L and 250 µg/L, PROTOCOL M44-A2 (CLSI 2009) OEPh was tested. The main constituents identified were: l-limonene, α-terpineol, p-cineol, o-cymene and α-phellandrene, however, its composition varies significantly with extraction time. All species, except C. rugosa, were inhibited with halo (≥ 14 mm) at 1000 μg / L. C. krusei is naturally resistant to the drug fluconazole, but when tested with OEPh the clinical species (case 9) demonstrated sensitivity in three dilutions (halo ≤ 10 ≥ 14) and the standard strain was inhibited at concentration of 1000 μg/Lg / L (halo 14mm). A similar situation also occurred with the standard strain of C. parapsilosis (halo ≥ 11mm). OEPh has considerable antifungal activity, which merits further investigation for alternative clinical applications, since this species is widely distributed in our community, and it presents good yields, and also has important therapeutic applications.

RESUMO Protium heptaphyllum é encontrada na região amazônica, em vários estados do Brasil e países da América do Sul. Conhecida como almecega produz uma resina oleosa usada na medicina popular como analgésica, antiinflamatória, cicatrizante e expectorante, é rica em triterpenos pentaciclicos e óleo essencial. O objetivo principal do presente trabalho foi analisar a composição química do óleo essencial da resina P. heptaphyllum (OEPh) em diferentes tempo de extração e avaliarsuaatividade antifúngica contra espécies de Candida, isoladas de horticultores com onicomicoses, por método de disco-difusão. O OEPh foi obtido por hidrodestilação, analisado por Cromatografia Gasosa Multidimensinal Acoplada a Espectrometria de Massas (MDGC/MS). As espécies de Candida foram obtidas de lesões nas unhas de horticultores de uma horta comunitária na cidade de Teresina, Piauí, Brasil. Testou-se a atividade antifúngica do OEPhnas concentrações de 1000 μg/L, 500 μg/L e 250 μg/L, protocolo M44-A2 (CLSI 2009). Os principais constituintes identificados foram l- limoneno, α-terpineol, p-cineol, o-cimeno e α-felandreno, entretanto, sua composição varia significativamente em decorrência do tempo de extração. Todas as espécies, exceto a C. rugosa, foram inibidas com halo ( Χ ≥ 14 mm) na concentração de 1000 μg/L. C. krusei é naturalmente resistente ao fármaco fluconazol, mas quando testado com OEPh,a espécie clínico (caso 9) demonstrou sensibilidade nas três diluições (halo Χ ≤ 10 ≥ 14) e a cepa padrão foi inibida na concentração de 1000 μg/L (halo Χ 14mm). Fato semelhante também ocorreu com a cepa padrão de C. parapsilosis (halo Χ ≥ 11mm). O OEPh possui atividade antifúngica considerável, merecendo uma investigação mais aprofundada para aplicações clínicas alternativas, uma vez que esta espécie é amplamente distribuída em nossa comunidade, apresenta bom rendimento e, ainda, aplicações terapêuticas importantes.

Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species

Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods.