Metabolites produced by an endophyte Alternaria alternata isolated from Maytenus hookeri

Abstract

Endophytes are bacteria or fungi that live in the intercellular spaces of the tissues of host plants without causing discernible manifestation of disease [1]. Recently, endophytes have been recognized as important sources of a variety of structurally novel and biologically active secondary metabolites, including terpenoids, steroids, alkaloids, and isocoumarin derivatives. In the course of our search for new biologically active secondary metabolites from endophytic microorganisms residing in Maytenus hookeri (Celastraceae) [2, 3], we studied chemical substances from an endophytic fungus Alternaria alternata of Maytenus hookeri and isolated and characterized nine compounds, alternariol (1), alternariol monomethyl ether (2), 5 -epialtenuene (3), altenuene (4), uridine (5), adenosine (6), ACTG toxin-E (7), ergosta-4,6,8,22-tetraen-3-one (8), and ergosta7,24(28)-dien-3-ol (9) from the ethyl acetate–methanol–acetic acid extract of the solid-state fermentations of this fungus. Among them, compounds 5 and 6 were isolated for the first time from this genus Alternaria, both 8 and 9 first being obtained from the species. In this paper, we describe the isolation and structure elucidation of these compounds 1–9. With the use of column chromatography over silica gel, and Sephadex LH-20 and reversed-phase RP-18, as well as preparative TLC on silica gel, further separation of methanol and petroleum ether–soluble parts of the ethyl acetate–methanol– acetic acid extract of the cultures of A. alternata afforded nine compounds 1–9. Compound 1 was isolated as a white powder. ESI-MS (negative mode) showed a molecular ion at m/z 257 [M – H]–. From the 13C NMR (DEPT) spectra, the molecular formula was deduced as C14H10O5, with 10 degrees of unsaturation. The 13C NMR and DEPT indicated 14 skeletal C atoms, including one methyl, four methines, and nine quarternary C atoms. In the 1H NMR spectrum, the downfield signals appeared at 6.88 (1H, d, J = 1.69 Hz, H-4), 6.94 (1H, d, J = 2.36 Hz, H-5 ), 6.99 (1H, d, J = 2.48 Hz, H-3 ), and 7.44 (1H, d, J = 1.71 Hz, H-6), which suggested it was a biphenyl-type compounds. The above spectral data were in agreement with values reported in the literature [4] for 3,4 ,5-trihydroxy-6 -methyldibenzo-pyrone. Compound 2, white powder, C15H12O5 was identified as alternariol monomethyl ether by comparison of physicochemical data and spectral data (EI-MS, 1H NMR and 13C NMR) with those reported in the literature [4, 5].