The air-dried and powdered fruiting bodies of R. foetens (7.2 g) were extracted with 80% MeOH two times at room temperature and filtered. The filtrate was evaporated under vacuum to afford a methanolic extract (1.3 g), which was suspended in H2O, and then partitioned with n-hexane, CHCl3 and n-BuOH, successively, yielding n-hexane (90 mg), CHCl3 (120 mg) and n-BuOH fractions (150 mg). Each fraction was evaluated for cytotoxicity against A549, SK-OV-3, SK-MEL-2 and HCT-15 cell lines using the sulforhodamine B assay. It was found that the n-hexane and CHCl3 soluble fractions were of moderate cytotoxicity against the tumor cell lines. The n-hexane soluble fraction (90 mg) was separated on a Sephadex LH-20 (Pharmacia, Uppsala, Sweden) column with CH2Cl2-MeOH (1:1) as the eluent to give three fractions (H1–H3). Fraction H2 (12 mg) was further purified by normal-phase preparative HPLC using a solvent of n-hexane-EtOAc (1:1) at a flow rate of 2.0 ml min−1 (Apollo Silica 5 μm column (Alltech, Nicholasville, KY, USA), 250 × 10 mm) to give compound 4 (5 mg). The CHCl3 soluble fraction (120 mg) was separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) as the eluent to give two fractions (C1–C2). Fraction C2 (25 mg) was further purified by RP-C18 preparative HPLC (Econosil RP-18 10 μ column (Alltech), 250 × 10 mm; 60% MeOH) to afford compounds 2 (3 mg) and 5 (3 mg). The n-BuOH soluble fraction (150 mg) showing weak cytotoxicity was also separated on a Sephadex LH-20 column with CH2Cl2-MeOH (1:1) as the eluent to give four fractions (B1–B4). Fraction B3 (30 mg) was further purified by RP-C18 preparative HPLC (Econosil RP-18 10 μ column, 250 × 10 mm; 60% MeOH) to give compounds 1 (4 mg) and 3 (3 mg).

Compound 1 was obtained as a colorless oil. Its molecular formula was determined to be C15H22O4 from the [M+H]+ peak at m/z 267.1590 (calcd. for C15H23O4, 267.1596) in the positive-ion high-resolution FAB-MS spectrum. The IR spectrum of 1 showed a broad hydroxyl band at 3388 cm−1 and a γ-lactone carbonyl absorption at 1751 cm−1. Its UV spectrum revealed end absorption λmax 217 nm with low extinction. The physico-chemical properties of 1 are summarized in Supplementary Information. The 1H- and 13C-NMR spectral data of 1 are shown in Table 1.

Table 1 1H- and 13C-NMR data of 1 and 3
Full size table

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