Secondary Metabolites of Endophytic Fungus Trichoderma sp. YM 311505 of Azadirachta indica

Abstract

Endophytic fungi colonize internal tissues of plants without resulting in overt damage while the tissue is alive. They are important sources to provide a wide variety of structurally unique and biologically potent natural products [1]. In our previous search for secondary metabolites of endophytic fungi from Azadirachta indica, some bioactive and/or new compounds were isolated [2–4]. Trichoderma species have long been studied as biological control agents of phytopathogens and are well known for their ability to produce a wide range of antibiotic substances and for their ability to parasitize other fungi [5]. In our ongoing work, we have studied the secondary metabolites of Trichoderma sp. YM 311505 from A. indica and obtained nine compounds. The fungal strain Trichoderma sp. YM 311505 was isolated from the fruit of A. indica collected in Yuanjiang Country, Yunnan Province, P. R. China. It was classified as a Trichoderma species by its morphological characteristics and ITS rDNA sequence analysis. The strain was deposited in Yunnan Institute of Microbiology, Kunming, P. R. China. The fungus was cultured in 500 mL Erlenmeyer flasks ( 750) containing 200 mL of PDB medium at 200 rpm at 28 C for 6 days on a rotary shaker. The culture broth was filtered to remove mycelia. The filtrate was concentrated in vacuum to 30 L and then extracted with EtOAc (5 30 L). The combined extracts were evaporated to yield 164.5 g of the crude extract, which was subjected to column chromatography on silica gel eluting with petroleum–acetone (9:1–3:7) to afford nine fractions (I–IX). Repeated chromatography of fraction II on silica gel with petroleum ether–EtOAc (5:1) and CHCl3–acetone (95:5) afforded compounds 1 (30 mg), 5 (50 mg), and 6 (4 mg). Fraction III was then subjected to column chromatography on RP-18 silica gel with acetone–H2O (8:2) to give compound 2 (28 mg). Fraction IV was subjected to repeated column chromatography on silica gel with petroleum ether–EtOAc (2:1) and RP-18 silica gel with acetone–H2O (8:2) to afford compound 9 (14 mg). Fraction V was submitted to column chromatography on silica gel with CHCl3–acetone (3:1) to give compound 3 (18 mg) and 8 (11 mg). Fraction VI was subjected to column chromatography on silica gel with petroleum ether–acetone (3:1) and RP-18 silica gel with acetone–H2O (1:1) to yeild compound 4 (10 mg). Fraction VIII was chromatographed on silica gel with CHCl3–MeOH (9:1) to give compound 7 (5 mg).