LAUSR

Gliotoxin, displaying this typical bridged disulfide, was described in 1943 [1]. It is one of the better known members of the epipo-lythiodioxopiperazine (ETP) class of fungal metabolites. The structure of the ETP class of fungal products is characterized by the bridged disulfide piperazinedione six-membered ring that appears to be necessary for most of the biological properties of these compounds [2]. Endophytic fungi are eukaryotic organisms that live inside plant tissues and are usually specific at the host species level [3, 4]. Endophytic fungi show high potential as sources of novel antimicrobial, antiviral, anticancer, antioxidant, and insecticidal compounds [5]. In the course of our search for biologically active metabolites from endophytic fungi from Chinese medicinal plants, a subculture of an isolate of Penicillium sp., obtained from roots of Eucommia ulmoides, was cultivated on potato dextrose agar (PDA). Eucommia ulmoides Oliv. (Eucommiaceae), known as “Du-Zhong,” is commonly used for the treatment of hypertension, rheumatoid arthritis, lumbago, and ischialgia in traditional Chinese medicine [6]. An ethyl acetate extract of the culture showed significant antimicrobial activity. This prompted us to carry out secondary metabolite studies on this fungus, which resulted in the isolation of seven gliotoxin analogues. Herein we describe the isolation, structural elucidation, and antimicrobial activities of the secondary metabolites. The fungal strain ER16-5 was isolated from the roots of Eucommia ulmoides, collected in the Qinling Mountains, Shaanxi Province, China, on September 1, 2016. The fungus was identified as a member of the genus Penicillium by DNA amplification and sequencing of the ITS region. The fungus was identified as Penicillium sp. The fungal strain has been preserved at the Applied Biotechnology Institute of Shanxi Datong University, Shanxi Province, China. Starter cultures were maintained on PDA medium at 28°C for 7 days. Plugs of agar supporting mycelial growth were cut and transferred aseptically to 1000 mL Erlenmeyer flasks containing 400 mL of liquid Czapek medium at 28°C on a rotary shaker set to 120 r/min for 15 days. The fungal culture (60 L) was filtered through cheesecloth. The filtrate was concentrated to 10 L below 60°C and then extracted five times with ethyl acetate (15 L). The dried mycelium (55°C, 95 g) was extracted three times with methanol (4 L). All extracts were concentrated at reduced pressure to afford 22.1 g of a crude extract. The crude extract was separated into eight fractions (A–H) on silica gel (500 g), with use of gradients of ethyl acetate–methanol (1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 0:1). Fraction B was separated by silica gel column chromatography (110 g) eluting with petroleum ether–ethyl acetate (1:0, 50:1, 25:1) to give three subfractions (Subfrs. B1–3). Subfraction B1 (1.29 g) was chromatographied over a silica gel column (30 g) eluting with petroleum ether–ethyl acetate and Sephadex LH-20 column (100 g) eluting with methanol to afford compound 1 (35 mg) together with compound 2 (47 mg). Subfraction B2 (0.76) was chromatographied over a silica gel column (60 g) eluting with petroleum ether–ethyl acetate (100:1) and then PTLC to afford compounds 3 (21 mg) and crude 4. The crude compound 4 was then recrystallized from ethyl acetate–methanol to give the pure compound 4 (9 mg). Fraction C was separated by column chromatography on silica gel (50 g) with a gradient of ethyl acetate in petroleum ether to give five subfractions (Subfrs. C1–5). Subfraction C2 that eluted with methanol was separated by semipreparative reversed-phase HPLC (H2O–MeOH, 1:3, 2 mL/min) to yield compounds 5 (16 mg), 6 (8 mg), and 7 (5 mg). The secondary metabolites were identified as gliotoxin (1) [7], bisdethiobis(methylthio)gliotoxin (2) [8], dehydroxybisdethiobis(methylthio)gliotoxin (3) [9], dehydrogliotoxin (4) [10], 6-deoxy-5a,6-didehydrogliotoxin (5) [11], didehydrobisdethiobis(methylthio)gliotoxin (6) [12, 13], and bis-N-norgliovietin (7) [13] by comparison of their spectral data with the reported data in the literature.