Phytochemical investigation and antimicrobial activity of an endophytic fungus Phoma sp.

2.1 General experimental procedure

Ultraviolet (UV) spectra were recorded in methanol on a Hitachi U-3200 spectrophotometer. Infra Red (IR) spectra were measured on Shimadzu-8900 spectrophotometer. EI-MS and HR-EI-MS were carried out using MAT 8200 and Micromass LCT mass spectrometers, in m/z. The ¹H NMR spectra were recorded on a Bruker AMX-500 spectrometer using TMS as an internal reference. The chemical shifts are reported in ppm (δ) while the coupling constants (J) in Hertz. The ¹³C NMR spectra were recorded at 125 MHz on the same instrument. Column chromatography (CC) was carried out using silica gel (70–230 and 230–400 mesh; E-Merck, Darmstadt, Germany) and Aluminium sheets precoated with silica gel 60 F 254 (0.2 mm thick; E-Merck) were used for TLC to check the purity of the compounds and were visualized under UV light (254 and 366 nm) followed by ceric sulphate as the spray reagent. Microbiological methods and culture conditions are as described previously (Höller et al., 2000; Schulz et al., 1995).

2.2 Identification, culture, extraction, and isolation

The endophytic fungus Phoma sp., (internal strain No. 7133), was isolated from Senecio kleinii from Gomera, and was cultivated on biomalt solid agar medium (12 L, 5% w/v) at room temperature for 28 days. The endophytic fungus was identified by Dr. Siegfried Draeger and a voucher specimen (TUB-7133) was deposited in the culture collection of the Institute of Microbiology, Technical University of Braunschweig, Germany. The cultures were extracted with ethyl acetate to afford a residue (4.3 g). The extract was separated into three fractions by column chromatography on silica gel with a gradient of n-hexane/ethyl acetate (90:10, 50:50, 0:100) as the eluent. The sub-fraction F₁ was further purified by silica gel column chromatography (CC) and preparative TLC with n-hexane/ethyl acetate (10:1 to 5:1) to give pure compounds 1 (4.5 mg), 2 (7.0 mg), 3 (5.3 mg), and 4 (4.3 mg).

2.3 Bioactivity test-agar diffusion test

Tests for antifungal, antibacterial, and antialgal activities were performed as previously described (Schulz et al., 1995). The test organisms for the agar diffusion and screening tests were bacteria B. megaterium de Bary (gram positive) and Escherichia coli (Migula) Castellani & Chalmers (gram negative), the fungi U. violacea (Pers.) Roussel (Ustomycetes), Mycotypha microspora Fenner (Zygomycetes), E. repens Corda (Ascomycetes) and Fusarium oxysporum Schltdl. (Deuteromycetes) and the alga Chlorella fusca Shih Krauss (Chlorophyceae), where the inhibition of C. fusca is usually correlated with broader antialgal activity (Schulz et al., 1995). Compounds 1–4 were dissolved in acetone at a concentration of 1 mg/mL. Fifty microlitres of the solutions (50 μg) was pipetted onto a sterile filter disc (Schleicher & Schuell, 9 mm), which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organism (Schulz et al., 1995).

3.1 Structure elucidation

The ethyl acetate extract of endophytic fungus Phoma sp. was chromatographed on silica gel to give four compounds 1–4. These four compounds were identified as viz., sclerodin (1) (Ayer et al., 1986), 8,9-dihydro-3,5,7-trihydroxy-1,8,8,9-tetramethyl-5-(2-oxopropyl)-4H-phenaleno[1,2-b]furan-4,6(5H)-dione (2) (Ayer et al., 1986), atrovenetinone (3) (Ayer et al., 1986), and sclerodione (4) (Ayer et al., 1986) (Fig. 1) and their structures were confirmed by a comparison of their spectral data to the literature.

3.2 Antimicrobial activity

Antibacterial, antifungal and antialgal properties of the four pure isolated compounds viz., sclerodin (1), 8,9-dihydro-3,5,7-trihydroxy-1,8,8,9-tetramethyl-5-(2-oxopropyl)-4H-phenaleno[1,2-b]furan-4,6(5H)-dione (2), atrovenetinone (3) and sclerodione (4) are compiled in Table 1. The isolated compounds 1–4 were tested in an agar diffusion assay for their antifungal, antibacterial, and antialgal properties towards Chlorella fusca, U. violacea, E. repens, M. microspora, F. oxysporum, E. coli, and B. megaterium.

Sclerodin (1) which has a pyran-2,6-dione group showed moderate algicidal activity towards C. fusca and sclerodione (4) which has cyclopent-1,2-dione showed moderate antifungal activity towards E. repens. On the other hand 8,9-dihydro-3,5,7-trihydroxy-1,8,8,9-tetramethyl-5-(2-oxopropyl)-4H-phenaleno[1,2-b]furan-4,6(5H)-dione (2) and atrovenetinone (3) showed moderate antifungal activities towards M. microspora. Moreover compounds 1 and 4 were not active against M. microspora. Atrovenetinone (3) displayed moderate antifungal activity towards F. oxysporum while compounds 1, 2, and 4 were not active against F. oxysporum. It is noteworthy that compound 3 has a cyclohex-4-ene-1,2,3-trione group while compounds 1, 2, and 4 do have said group in their structures. On the other hand compound 2 has a propyl-2-one and hydroxyl group instead of oxygen which is present in sclerodin (1). Moreover atrovenetinone (3) has a third carbonyl group instead of oxygen which is present in sclerodin (1). In addition 8,9-dihydro-3,5,7-trihydroxy-1,8,8,9-tetramethyl-5-(2-oxopropyl)-4H-phenaleno[1,2-b]furan-4,6(5H)-dione (2) and atrovenetinone (3) showed moderate antibacterial activity against B. megaterium. Furthermore atrovenetinone (3) showed good antifungal activity towards E. repens. Interestingly atrovenetinone (3) and sclerodione (4) showed very strong antifungal activity towards U. violacea while compounds 1 and 2 were not active against U. violacea. It is noteworthy that this compound has a cyclohex-4-ene-1,2,3-trione group while compound 4 has cyclopentane-1,2-dione group in their structures. It is important to note that none of these compounds was active against E. coli. Compounds 1 and 2 were not active against E. repens and U. violacea.