Fulvifomes xylocarpicola, T. Hatt, Sakayaroj & E. B. G. Jones, T. Hatt, Sakayaroj & E. B. G. Jones

Isaka, Masahiko, Palasarn, Somporn, Sakayaroj, Jariya, Srichomthong, Kitlada, Nithithanasilp, Sutichai & Sappan, Malipan, 2021, Limonoids from fruiting bodies of the wood-rot basidiomycete Fulvifomes xylocarpicola associated with the mangrove tree Xylocarpus granatum, Phytochemistry (112555) 181, pp. 1-6 : 5-6

4.5. LCMS analysis of the extracts from the fruiting body of F. xylocarpicola and the bark of X. granatum

In the collection of F. xylocarpicola , after stripping off the mushroom, the plant side (bark) of the mushroom-bonded surface was scrapped to collect small crops ( Fig. S1 ). Dried crops from plant bark (24 g) were cut into small pieces and extracted with CH 2 Cl 2 (400 ml, room temperature, 7 days; twice), and combined CH 2 Cl 2 solution was concentrated under reduced pressure to obtain a CH 2 Cl 2 extract (303 mg). The plant residue was then extracted with MeOH (500 ml, room temperature, 7 days; twice), and combined MeOH solution was concentrated under reduced pressure. The residue was dissolved in EtOAc (600 ml) and washed with H 2 O (30 ml). The organic layer was concentrated under reduced pressure to obtain a MeOH extract (417 mg). These extracts and the extracts from F. xylocarpicola fruiting body (described above) were subjected to LC-MS analysis for detection of fulvifomin A (1), the major limonoid in the mushroom.

LC-MS analysis was performed using Bruker MicrOTOF mass spectrometer connected to Agilent 1200 Series HPLC system. HPLC : column, Dionex Acclaim 120 C18, 4.6 × 150 mm, 3 μm; mobile phase, MeCN/ H 2 O +0.05% formic acid, gradient from 5:95 to 95:5 over 50 min; flow rate, 0.3 ml/min; UV detection, 210 nm. ESI-TOF (positive ion mode) mass trace was obtained for identification of fulvifomin A (1): HPLC retention time, 44.9 min; ESI-MS, [M+H] + = 571 (Fig. S29). A 20 μl portion from 0.5 mg /ml MeOH solution of each extract was injected for the LC-MS. Compound 1 was detected in the extracts from the fungus, but, not in the extracts from the plant (Fig. S30 and Fig. S31).

4.6. Fermentation of F. xylocarpicola , extraction, and 1 H NMR spectroscopic analysis

Four strains of F. xylocarpicola , which have been preserved at the BIOTEC Culture Collection (BCC 42654, BCC 47460, BCC 47461, and BCC 47478), were fermented using three different liquid media: malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g /l, maltose 1.8 g /l, dextrose 6.0 g/l), potato dextrose broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l), and peptone yeast glucose medium (PYGM; bacteriological peptone 5.0 g/l, yeast extract 20.0 g/l, glucose 10.0 g/l, KH 2 PO 4 1.0 g/l, MgSO 4 ⋅7H 2 O 0.5 g /l). Each strain was fermented in a 1000 ml Erlenmeyer flask containing 250 ml of the liquid media under static conditions until good mycelial growth (19–32 days). Among 12 cultures, there were 4 cases of insufficient mycelial growth, and such cultures were discarded. Each culture was filtered to separate broth (filtrate) and mycelia (residue). The broth was extracted with EtOAc (250 ml), and the organic layer was concentrated under reduced pressure to obtain a broth extract (10–49 mg). The wet mycelia were macerated in MeOH (100 ml, room temperature, 2 days), then filtered. The filtrate was defatted by partitioning with hexane (50 ml). The aqueous MeOH (bottom) layer was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 ml), washed with H 2 O (50 ml), and concentrated under reduced pressure to obtain a mycelial extract (3–16 mg). The broth and mycelial extracts were subjected to 1 H NMR spectroscopic analysis. The spectra strongly suggested that none of the extracts contained any limonoids in detectable quantity.

Figures

Fig. 1. Structures of compounds 1–5.