RESUMEN
RATIONALE: Although the fruiting-body of the fungi of the genus Xylaria shows a great variety of morphological characteristics, their mycelial forms are always very similar, imposing difficulties for their identification. Intact cell mass spectrometry (ICMS) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be a fast and reliable strategy to support the differentiation/identification of Xylaria species in those cases where fruit-bodies are not available. METHODS: Many experimental parameters such as sample preparation and culture media are crucial for filamentous fungi analysis by MALDI-TOFMS. For the purposes of this study, we used four matrices (CHCA, DHB, FA and SA) with five different concentrations (0.1, 0.3, 0.5, 1.0 and 2.5%) of TFA in the matrix, the influence of six different culture media (solid and liquid), and three mycelium peptide/protein extraction protocols (acid, basic and thymol-supported solution) to optimize the sample preparation of the endophytic fungus X. arbuscula. RESULTS: It was observed that sinapinic acid (30 mg/mL) dissolved in acetonitrile/0.1% TFA and PDA were the best matrix solution and culture medium, respectively, for the ICMS of X. arbuscula. The formic acid and ammonium bicarbonate (AB) protocols provided similar mass spectra; however, a higher number of peaks were observed using AB extraction. Mass spectra obtained from different thymol-containing solutions (EtOH/aqueous 0.1% TFA and ACN/aqueous 0.1% TFA) show increasing peak abundances at m/z 3000-6500. CONCLUSIONS: X. arbuscula could be analyzed by ICMS. However, an extraction step was required to provide suitable MALDI mass spectra. Formic acid-, AB- and thymol-containing solutions were demonstrated to be good cocktails for the extraction of peptide/protein biomarkers from these fungi.
Asunto(s)
Proteínas Fúngicas/análisis , Micelio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Xylariales/química , Bicarbonatos/química , Fraccionamiento Químico/métodos , Ácidos Cumáricos/química , Medios de Cultivo/química , Formiatos/química , Proteínas Fúngicas/aislamiento & purificación , Micelio/clasificación , Péptidos/análisis , Péptidos/aislamiento & purificación , Timol/química , Ácido Trifluoroacético/química , Xylariales/clasificaciónRESUMEN
Trachyloban-19-oic acid (1) is a diterpene very abundant in nature and its structural modification can furnish new bioactive compounds. Biotransformation of 1 by fungus Syncephalastrum racemosum provided three derivatives, two hydroxylated products (2-3) and one product of rearrangement (4). Products 3 and 4 have never been reported so far, to the best of our knowledge. Structure of 3 was formed after oxidation and rearrangement of compound 2. Compounds 1-4 were evaluated for inhibition of acetylcholinesterase, enzyme linked to the symptomatic control of Alzheimer's disease. All the compounds presented inhibitory activity higher than starting material 1, and product 3 presented IC50â¯=â¯0.06⯵M, which is about six times higher than activity found for galanthamine (IC50â¯=â¯0.38⯵M), the positive control used in this assay.