Endophytic fungi are eukaryotes colonized in plants and do not cause any significant disease at any time during their colonization period [1]. In addition, they always play a vital role in assisting host plants in chemical defense by producing bioactive metabolites, especially host-specific compounds [2]. Many endophytic fungi have a large number of secondary metabolites encoding biosynthetic pathways, but only a small number of metabolites have been isolated in the laboratory. Natural chemical diversity can be changed systematically by changing cultivation parameters to limit strains. To increase the diversity of available secondary fungal metabolites, methods have been developed to activate silent gene sequences in fungi [3, 4]. One such method, the OSMAC (One Strain-Many Compounds) strategy developed by Zeeck in the early 2000s proved to be a simple and effective method [5]. In this strategy, silenced gene sequences are activated by changing culture conditions, including medium composition, medium phase, and temperature [6, 7].

Previously, we found that the endophytic fungus Aspergillus fumigatus from the stem of Astragalus membranaceus associated with potato dextrose agar (PDA) media can produce structurally diverse indole diketopiperazines [8]. In fact, many microorganisms harbor significant numbers of secondary metabolite-encoding biosynthetic pathways, but only a few metabolites have been detected in the laboratory. Thus, the application of the OSMAC approach to this strain using three different media resulted in the production of eight indole diketopiperazines and five quinazolinone alkaloids, in addition to three helvolic acid derivatives.

The endophytic fungus A. membranaceus was grown on potato dextrose agar at 28°C for 6 days. Three pieces (diameter 0.5 cm) of mycelial agar plugs were inoculated into Erlenmeyer flasks (250 mL) containing 100 mL Czapek′s medium (sucrose 30 g/L, KH2PO4 1.0 g/L, MgSO4 7H2O 0.5 g/L, NaNO3 3.0 g/L, KCl 0.5 g/L, and FeSO4 0.01 g/L). The flasks were incubated at 28°C on a rotary shaker (150 rpm) for 7 days to obtain the fungus seed. The seed liquids were inoculated separately in three different media, PDB (potato dextrose broth), sterilized rice, and SP solid medium (sucrose 20 g/L, peptone 30 g/L, KH2PO4 0.5 g/L, MgSO4 0.5 g/L, and agar 15 g/L). Fermentation was performed in 500 mL Erlenmeyer flasks, each Erlenmeyer flask containing 200 mL of culture medium at 28°C. PDB fermentation (80 L) was maintained on a rotary shaker at 140 rpm for 12 days, and SP fermentation (30 L) was kept for 20 days, while sterilized rice fermentation (15 L) was maintained for 25 days. The extracts of the three fermentations were analyzed by HPLC eluted with a methanol gradient, and the chromatograms were detected by a UV detector.

The PDB culture broth was extracted with ethyl acetate three times. The culture afforded a dark brown crude extract, which was subjected to silica gel column chromatography eluting successively with ethyl acetate–methanol gradient to yield nine fractions (Frs. A–I). Fractions B and C containing indole diketopiperazines alkaloids were detected and chromatographed on a silica gel column with a gradient of ethyl acetate in petroleum ether separately. Fraction B1 was chromatographed over a silica gel column eluting with petroleum ether/ethyl acetate and Sephadex LH-20 column (100 g) eluting with methanol to afford compounds 1 and 2. Fraction B2 was chromatographed over a silica gel column eluting with petroleum ether–ethyl acetate and then preparative TLC to afford compounds 3 and 4. Fraction C was separated by column chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give six subfractions (Subfrs. C1–6). Subfraction C2, eluted with methanol, was separated by semipreparative reversed-phase HPLC (H2O–MeOH, 1:4, 2.5 mL/min) to yield compounds 5–7. This procedure afforded the pure compound 8 from Subfr. C3.

figure a

The SP medium was extracted with ethyl acetate three times. The crude extract was then subjected to a silica gel column and eluted with a chloroform–methanol gradient to give six fractions (Frs. A–F). Fraction B containing indole DKP alkaloids and Fr. C containing helvolic acid derivatives were detected. Fraction B was chromatographed on silica gel column with a gradient of ethyl acetate in petroleum ether to give four subfractions (Subfrs. B1–4). Subfraction B1 was chromatographed on MCI gel eluted with a MeOH–H2O mixture and further purified on Sephadex LH-20, silica gel, and PTLC to afford compounds 1 and 3. Fraction C was chromatographed on a silica gel column with a gradient of ethyl acetate in petroleum ether to give three subfractions (Subfrs. C1–3). Subfraction C1 was chromatographed over a silica gel column eluting with petroleum ether–ethyl acetate to give two subfractions (Subfrs. C1-1 and C1-2). Subfraction C1-1 was chromatographed on a Sephadex LH-20 column eluting with methanol to afford compound 9. Subfraction C1-2 was recrystallized and afforded compound 10. Subfraction C2 was chromatographed over a silica gel column eluting with petroleum ether–ethyl acetate and then PTLC to afford compound 11.

The rice medium was extracted with acetone four times. The extract was then scattered in 50% MeOH and further extracted with chloroform five times. The chloroform portion was subjected to silica gel column chromatography eluting successively with ethyl acetate–methanol gradient to yield nine fractions (Frs. A–I). Fractions B and C containing indole DKP alkaloids and Fr. C containing quinazolinone alkaloids were detected. Fraction B was separated by column chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give nine subfractions (Subfrs. B1–9). Subfraction B2 was separated by silica gel column chromatography with elution with petroleum ether–ethyl acetate to give compounds 1, 2, and 12. Subfraction B3 was separated by silica gel column chromatography with elution with petroleum ether–ethyl acetate and then recrystallized from petroleum ether–ethyl acetate to give compounds 3 and 6. This procedure afforded the pure compound 7 from Fr. C2. Fraction D was subjected to repeated chromatographic purifications using ethyl acetate–methanol and Sephadex LH-20 to give ten subfractions (Subfrs. D1–10); then compounds 13–16 were obtained from Subfrs. D2 and D3 similarly.

The compounds were subjected to characterization using spectroscopic analysis and identified as fumitremorgin B (1) [9], cyclotryprostatins B (2) [10], verruculogen (3) [11], tryprostatin B (4) [12], tryprostatin A (5) [12], cyclotryprostatins A (6) [10], cyclotryprostatins C (7) [10], cyclotryprostatins D (8) [10], helvolic acid (9) [13], hydrohelvolic acid (10) [14], helvolic acid methyl ester (11) [15], fumitremorgin C (12) [16, 17], fumiquinazoline A (13) [18], fumiquinazoline C (14) [18], fumiquinazoline D (15) [18], and fumiquinazoline J (16) [19], by comparison of their spectral data with the reported data in the literature.

Helvolic acid (9), white needle crystals, mp 214–216°C. HR-ESI-MS m/z 591.2936 [M + Na]+. 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 7.31 (1H, d, J = 10.1, H-1), 5.89 (1H, d, J = 8.2, H-16), 5.85 (1H, d, J = 9.7, H-2), 5.23 (1H, s, H-6), 5.11 (1H, s, H-24), 2.79 (1H, d, J = 12.0, H-5), 2.78 (1H, d, J = 12.0, H-4), 2.63 (1H, s, H-9), 2.57 (1H, s, H-13), 2.48 (2H, m, H-22), 2.41 (1H, m, H-12b), 2.21 (1H, m, H-15b), 2.14 (1H, m, H-23b), 2.11 (3H, s, H-33), 2.10 (1H, m, H-23a), 1.95 (3H, s, H-31), 1.89 (1H, m, H-15a), 1.87 (1H, m, H-11b), 1.81 (1H, m, H-12a), 1.69 (3H, s, H-27), 1.61 (3H, s, H-26), 1.57 (1H, m, H-11a), 1.45 (3H, s, H-19), 1.28 (3H, d, J = 6.5, H-28), 1.18 (3H, s, H-29), 0.93 (3H, s, H-18). 13C NMR (100 MHz, CDCl3, δ, ppm): 157.1 (C-1), 127.8 (C-2), 201.4 (C-3), 40.4 (C-4), 47.3 (C-5), 73.8 (C-6), 208.8 (C-7), 52.7 (C-8), 41.8 (C-9), 38.2 (C-10), 23.9 (C-11), 25.9 (C-12), 49.5 (C-13), 46.6 (C-14), 40.7 (C-15), 73.5 (C-16), 147.7 (C-17), 18.0 (C-18), 27.5 (C-19), 130.3 (C-20), 173.8 (C-21), 28.6 (C-22), 28.3 (C-23), 122.8 (C-24), 132.9 (C-25), 17.7 (C-26), 25.7 (C-27), 13.1 (C-28), 18.3 (C-29), 170.1 (C-30), 20.5 (C-31), 168.8 (C-32), 20.7 (C-33).

Hydrohelvolic acid (10), white needle crystals. HR-ESI-MS m/z 609.3044 [M + Na]+. 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 7.31 (1H, d, J = 10.0, H-1), 5.87 (1H, d, J = 9.8, H-2), 5.86 (1H, d, J = 8.2, H-16), 5.23 (1H, s, H-6), 2.79 (1H, d, J = 12.0, H-5), 2.78 (1H, d, J = 12.0, H-4), 2.62 (1H, s, H-9), 2.58 (1H, s, H-13), 2.51 (1H, m, H-22a), 2.42 (1H, m, H-12b), 2.41 (1H, m, H-22b), 2.25 (1H, m, H-15b), 2.11 (3H, s, H-33), 1.95 (3H, s, H-31), 1.91 (1H, m, H-11b), 1.89 (1H, m, H-15a), 1.81 (1H, m, H-12a), 1.59 (1H, m, H-11a), 1.49 (2H, m, H-23), 1.48 (2H, m, H-24), 1.45 (3H, s, H-19), 1.28 (3H, d, J = 6.5, H-28), 1.22 (3H, s, H-26), 1.20 (3H, s, H-27), 1.18 (3H, s, H-29), 0.92 (3H, s, H-18). 13C NMR (100 MHz, CDCl3, δ, ppm): 157.3 (C-1), 127.8 (C-2), 201.5 (C-3), 40.4 (C-4), 47.2 (C-5), 73.8 (C-6), 208.8 (C-7), 52.6 (C-8), 41.7 (C-9), 38.2 (C-10), 23.9 (C-11), 25.9 (C-12), 49.4 (C-13), 46.6 (C-14), 40.6 (C-15), 73.5 (C-16), 147.8 (C-17), 18.0 (C-18), 27.5 (C-19), 130.3 (C-20), 174.3 (C-21), 28.5 (C-22), 28.2 (C-23), 43.1 (C-24), 71.2 (C-25), 29.4 (C-26), 29.2 (C-27), 13.1 (C-28), 18.4 (C-29), 170.1 (C-30), 20.5 (C-31), 168.9 (C-32), 20.8 (C-33).

Helvolic acid methyl ester (11), white needle crystals, mp 221.0–223.0°C. HR-ESI-MS m/z 583.3284 [M + H]+. 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 7.30 (1H, d, J = 10.1, H-1), 5.89 (1H, d, J = 8.2, H-16), 5.85 (1H, d, J = 9.7, H-2), 5.23 (1H, s, H-6), 5.11 (1H, s, H-24), 3.71 (3H, s, H-34), 2.79 (1H, d, J = 12.0, H-5), 2.78 (1H, d, J = 12.0, H-4), 2.63 (1H, s, H-9), 2.57 (1H, s, H-13), 2.46 (2H, m, H-22), 2.41 (1H, m, H-12b), 2.21 (1H, m, H-15b), 2.14 (1H, m, H-23b), 2.11 (3H, s, H-33), 2.10 (1H, m, H-23a), 1.95 (3H, s, H-31), 1.89 (1H, m, H-15a), 1.87 (1H, m, H-11b), 1.81 (1H, m, H-12a), 1.69 (3H, s, H-27), 1.61 (3H, s, H-26), 1.57 (1H, m, H-11a), 1.45 (3H, s, H-19), 1.28 (3H, d, J = 6.5, H-28), 1.18 (3H, s, H-29), 0.93 (3H, s, H-18). 13C NMR (100 MHz, CDCl3, δ, ppm): 157.1 (C-1), 127.8 (C-2), 201.4 (C-3), 40.4 (C-4), 47.3 (C-5), 73.8 (C-6), 208.8 (C-7), 52.7 (C-8), 41.8 (C-9), 38.2 (C-10), 23.9 (C-11), 25.9 (C-12), 49.5 (C-13), 46.6 (C-14), 40.7 (C-15), 73.5 (C-16), 147.7 (C-17), 18.0 (C-18), 27.5 (C-19), 130.3 (C-20), 172.9 (C-21), 28.8 (C-22), 28.3 (C-23), 122.8 (C-24), 132.9 (C-25), 17.7 (C-26), 25.7 (C-27), 13.1 (C-28), 18.3 (C-29), 170.1 (C-30), 20.5 (C-31), 168.8 (C-32), 20.7 (C-33), 53.6 (C-34).

The OSMAC approach was applied to produce a variety of metabolites, using the fungus A. fumigatus from A. membranaceus as a microbial producer. The metabolite production of A. fumigatus depended not only on the culture medium, but also on the sources of nutrients in PDB, SP, and rice medium. Diverse metabolites were produced by A. fumigatus simply by changing sources of potato, sugar, and rice in the culture media. The present study suggests that endophytic fungi are potentially suitable microorganisms for the OSMAC method, which could enhance the chemical diversity of secondary metabolites.