LAUSR

Lichens are symbiotic associations between fungi and photobionts, which can be algae or cyanobacteria. They are slow growing, stress-tolerant organisms, which provide a habitat for a variety of bacteria, including actinomycetes [1]. Recent studies have shown that most of the lichen-associated actinomycetes have broad spectrum antibacterial activity, of which Streptomyces are most prominent [2]. However, up to now, there are only a few bioactive substances reported from lichen-associated actinomyces, including antibaterial BE-24566B [3] and uncialamycin [4], cytotoxic cladoniamides [5] and angucycline [6], and antiproliferative cyaneodimycin [7]. Our current study is aimed to investigate the bioactive secondary metabolites from lichen-associated actinomycetes. Two Streptomyces strains YIM 130597 and YIM 130932 isolated from the lichen Punctelia rudecta in Yunnan Province, P. R. China, were found to exhibit obvious antibacterial activity. We have studied the secondary metabolites of these two strains and obtained ten compounds. The strains YIM 130597 and YIM 130932 were cultured in 2000 mL Erlenmeyer flasks (× 200) containing 400 mL of fermentation medium (peptone, 2 g; glucose, 20 g; starch, 5 g; yeast extact, 2 g; NaCl, 4 g; K2HPO4, 0.5 g; MgSO4·7 H2O, 0.5 g; CaCO3, 2 g; H2O, 1 L; pH 7.8) at 200 rpm at 28°C for 7 days on a rotary shaker. The culture broth was filtered, and the filtrate was concentrated in vacuum to a volume of 20 L and then successively extracted four times with an equal volume of EtOAc. Compounds 1–6 were obtained from the culture broth of the strain YIM 130932. The EtOAc phase was evaporated to yield 8 g crude extracts, which was purified by silica gel column chromatography with CHCl3–MeOH (10:0 to 0:10) to yield six fractions. Compound 3 (100 mg) was crystallized from fraction 2. Fraction 3 was subjected to column chromatography on silica gel with petroleum ether–EtOAc (8:2) and RP-18 silica gel with MeOH–H2O (3:7) to yield compounds 4 (10 mg) and 5 (6 mg). Fraction 4 was purified by column chromatography on silica gel with a gradient of MeOH in CHCl3 to give compound 6 (5 mg). Fraction 5 was submitted to column chromatography on silica gel with petroleum ether–acetone (6:4) and RP-18 silica gel eluting with MeOH–H2O (1:1) to yield compounds 1 (2 mg) and 2 (1 mg). Compounds 7–10 were isolated from the fermentation broth of the strain YIM 130597. The EtOAc extract (24 g) was chromatographed on a silica gel column with a mixture of CHCl3–MeOH (10:0 to 0:10) gradiently, yielding four fractions. Fraction 1 was subjected to column chromatography on silica gel with petroleum ether–EtOAc (9:1, 8:2, 7:3) to yield three subfractions. Subfraction 1.1 was chromatographed on an RP-18 silica gel column with MeOH–H2O (4:6) to give compound 8 (3 mg). Compound 9 (8 mg) was obtained by crystallization in the mixture of CHCl3–MeOH from fraction 1.2. Fraction 2 was subjected to column chromatography on silica gel with CHCl3–MeOH (30:1–20:1) to yield two subfractions. Subfraction 2.1 was purified by Sephadex LH-20 with MeOH to afford compound 10 (20 mg). Fraction 2.2 was subjected to column chromatography on silica gel with petroleum ether–EtOAc (8:2) to give compound 7 (1 mg). The structures of the isolated compounds were confirmed by comparison of their spectroscopic data with published data. They were determined to be 7-O-methylkoninginin D (1) [8], koninginin E (2) [9], cyclo-(Gly-Phe) (3) [10], cyclo-(Phe-Tyr) (4) [11], cyclo(Leu-Tyr) (5) [12], cyclo-(Ala-Leu) (6) [13], 4-methoxy-5-(methylthio)-[2,2′-bipyridine]-6-carbonitrile (7) [14], 7-methoxy-5-(pyridin-2-yl)isothiazolo[4,5-b]pyridine (8) [15], turnagainolide B (9) [16], and campesterol (10) [17].