Collection of the samples
The macromycetes (age 3 – 5 days), were collected near Plovdiv, Bulgaria, in October 2005.
The fresh fruiting bodies of macromycetes (A. pseudopratensis – 105 g; A. placomyces – 245 g) were cut into small pieces and consecutively extracted with ethanol, ethanol-chloroform (1:1) and chloroform. The extracts were combined, water was added and the chloroform layers were removed and evaporated in vacuo to yield 1.88 g of dry residue for A. placomyces and 0.6 g for A. pseudopratensis.
Isolation and analysis of volatile compounds
A portion of the chloroform extract (about 300 mg) was subjected to a four-hour distillation-extraction on Lickens-Nickerson apparatus . The volatile compounds were extracted from the distillate with diethyl ether (yields of volatiles in Table 1), and analysed by GC/MS on a GC Hewlett Packard 6890 + MS 5973 (Hewlett Packard, Palo Alto, California, USA), with a HP5-MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness, Agilent Technologies, Wilmington, Delaware, USA). The ion source was set at 250°C and the ionisation voltage at 70 eV. The temperature was programmed from 40 – 280°C at a rate of 6°C min-1, and a helium carrier gas was used used.
Analysis of polar compounds
A portion of the n-butanol extract (5 mg) was dissolved in 50 μL of dry pyridine, before the addition of 75 μL of bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). The mixture was heated at 80°C for 30 min and analyzed by GC/MS. The silylated extract was investigated by GC/MS using the same instrument described above, with a capillary column HP-5 (23 m × 0.2 mm, 0.5 μL film thickness, Agilent Technologies, Wilmington, Delaware, USA). A helium carrier gas was used. The temperature programmed at 100 – 315°C at a rate of 5°C min-1, with a 10 min hold at 315°C.
Identification of compounds
Identification was carried out by searching commercial library databases. Some components remained unidentified, however, owing to both a lack of authentic samples and library spectra of the corresponding compounds.
Isolation and identification of sterols
The chloroform extracts was subjected to column chromatography on silica gel with an n-hexane – acetone gradient (30:1 – 5:1) to produce several fractions. The third set of fractions, after further purification by preparative TLC (silica gel G, n-hexane – acetone 10:1), yielded sterol mixtures: 23 mg from A. placomyes and 7.9 mg from A. pseudopratensis, which were analysed by GC-MS. A gas chromatograph (Hewlett Packard 5890) linked to a mass spectrometer (Hewlett Packard 5972) with a capillary column SPB-50 (30 m × 0.32 mm, 0.25 μm film thickness) was employed. A helium carrier gas was used with a temperature programme set at 270°C – 290°C at a rate of 4°C min-1 with a 20 min hold. The ion source was set at 250°C with the ionisation voltage at 70 eV.
From the fourth set of preparative TLC fractions (silica gel, n-hexane – methyl ethyl ketone 10:1) obtained from both species 5α,8α-epidioxi-24(ξ)-methylcholesta-6,22-diene-3β-ol was isolated (10.4 mg from A. pseudopratensis and 81 mg from A. placomyces) and characterised through comparison of its EIMS, 1H- and 13C-NMR spectra with literature data .