Abstract Sporothrix spp. are the major dimorphic fungus associated with a type of subcutaneous mycosis, sporotrichosis. The limitation of antifungal availability and the past reports of in vitro resistance of Sporothrix spp. clinical isolates makes it important to search for new compounds with antifungal activities. In this study, we therefore evaluate the in vitro activities of complexes coordinated with Co(II) and cobalt chloride hexahydrate against clinical isolates of Sporothrix spp. Broth microdilution test was performed as per M38-A2 from CLSI (2008) in duplicate for 31 clinical isolates of Sporothrix spp. (27 S. brasiliensis e 04 S. schenckii stricto sensu). The antifungal activities of the complexes coordinated with Co(II) and cobalt chloride hexahydrate were detected at a concentration range of 32-128 µg/mL for all isolates. None of the compounds demonstrated any cytotoxicity (to macrophage cells) at the concentration of 200 µg/mL. The activity against Sporothrix spp. recorded in this study instigate the continuity of experimental studies with Co(II) to search for the mechanisms of antifungal action as well as to evaluate its interaction with the commercial antifungal drugs.

Resumo Duroia saccifera (Rubiaceae) ocorre na floresta Amazônica. Seus extratos apresentaram atividade antibacteriana. Para obter maiores quantidades de substâncias ativas, foram utilizados segmentos de folhas de plântulas in vitro de D. saccifera como explantes para indução de calos; os calos foram multiplicados através de múltiplos subcultivos, depois secos e extraídos com hexano, seguido por acetato de etila (AcOEt) e metanol (MeOH). Como D. macrophylla foi relatada como produtora de substâncias antimicobacterianas, os extratos de calos foram testados contra Mycobacterium tuberculosis (cepa H37Rv). O extrato AcOEt dos calos foi ativo, com uma concentração inibitória mínima (CIM) de ≤ 25 mg mL-1, IC90de 19,5 mg mL-1 e concentração bactericida mínima (MBC) de 200 mg mL-1. O extrato AcOEt foi analisado por cromatografia em camada delgada comparativa (CCDC) e Ressonância Magnética Nuclear (RMN) para determinar o seu perfil químico e mostrou ser rico em terpenos. O fracionamento cromatográfico do extrato AcOEt conduziu ao isolamento de uma mistura de dois esteroides, β-sitosterol e estigmasterol (na proporção de 2:1), os quais foram identificados por análises de RMN de 1H e bidimensionais. Pelo levantamento bibliográfico realizado, este é o primeiro relato da cultura de células in vitro de Duroia saccifera, da atividade antituberculose do extrato de calos e do isolamento de β-sitosterol e estigmasterol da cultura de células vegetais in vitro da espécie.

Abstract Duroia saccifera (Rubiaceae) occurs in the Amazon rainforest and their extracts showed antibacterial properties. To obtain greater quantities of active substances, leaf segments from in vitro D. saccifera seedlings were used as explants for calli induction; calli were multiplied via multiple subcultures, dried and extracted with hexane followed by ethyl acetate (EtOAc) and methanol (MeOH). As D. macrophylla had been reported to produce antimycobacterial substances, we assayed calli extracts against Mycobacterium tuberculosis (H37Rv strain). Calli EtOAc extract was active, with a minimal inhibitory concentration (MIC) of ≤ 25 mg mL-1, IC90of 19.5 mg mL-1 and minimal bactericidal concentration (MBC) of 200 mg mL-1. EtOAc extract was analyzed by Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) to determine its chemical profile, and was found to be rich in terpenes. Chromatographic fractionation of the EtOAc extract yielded a mixture of two sterols, β-sitosterol and stigmasterol (in proportion of 2:1), which were identified by 1H and 13C NMR analysis. As far as we know, this is the first report of Duroia saccifera in vitro cell culture, antituberculosis activity of calli extract and β-sitosterol and stigmasterol isolation from in vitro plant cell culture.