Bioactive metabolites from Alternaria brassicicola ML-P08, an endophytic fungus residing in Malus halliana

A total of 48 strains were isolated from the normal tissues of Malus halliana and the EtOAc extracts of their cultures were subjected to primary antimicrobial screening against four test bacteria and three fungi. As a result, 22 strains exhibited antimicrobial activity against at least one test microbe. Among them, Alternaria brassicicola ML-P08 showing strong activity (MICs: 0.31-2.50 mg/ml) was selected for further investigation on its secondary metabolites. Bioassay-guided fractionation of the EtOAc extract of its liquid culture afforded seven compounds, which were identified as alternariol (1), alternariol 9-methyl ether (2), altechromone A (3), herbarin A (4), cerevisterol (5), 3 beta,5 alpha-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6) and 3 beta-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (7), respectively, by spectral means (MS, IR, (1)H- and (13)C-NMR). In vitro antimicrobial assay showed that compound 3 was substantially active against Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Candida albicans with the MICs of 3.9, 3.9, 1.8, and 3.9 mu g/ml, respectively. Compound 4 also showed pronounced antifungal activity against Trichophyton rubrum and C. albicans with MICs of both 15.6 mu g/ml. In addition, compound 1 exhibited strong xanthine oxidase inhibitory activity with the IC(50) of 15.5 mu M, comparable to that of positive control, allopurinol (IC(50): 10.7 mu M).