Details of the producing organism, Phellinus sp. KACC93057P, were described in a previous paper.9 The producing organism was stationary cultured at 25 °C for 14 days in a tissue culture bottle (500 ml) containing 120 ml of YGM medium (yeast extract 1%, glucose 0.4% and malt extract 0.4%). In total, 2 l of the culture broth of Phellinus sp. KACC93057P were filtered to separate the broth filtrate and the mycelium cake. The mycelium cake was extracted with 0.5 l of 80% aqueous acetone. The acetone extract was filtered, and the filtrate was concentrated under reduced pressure to remove acetone. The resulting residue was extracted twice with ethyl acetate and then subjected to chromatography on a Sephadex LH-20 column where MeOH was used as the elution solvent. This was followed by ODS column chromatography where the product was eluted with a gradient of increasing methanol (40–90%) in water. The antioxidant fractions, which eluted with 70∼80% aqueous MeOH, were combined and chromatographed on a Sephadex LH-20 column with 70% aqueous MeOH and then, finally purified by reversed-phase thin layer chromatography (TLC) developed with 70% aqueous MeOH to give 1 (2.5 mg) and 2 (2.2 mg).

The 1H and 13C NMR spectra of 1b were very similar to those of 1a. In the 1H NMR spectrum, signals due to a 1,2,4-trisubstituted benzene, a trans-disubstituted double bond at δ 6.50 (d, J=16.0 Hz) and 7.24 (d, J=16.0 Hz), and one sp2 methine singlet at δ 6.01 were evident, and these signals were in good agreement with those of the hispidin skeleton. Other proton signals including H-3, H-4 and H-9, however, showed significantly different chemical shift values from those of 1a. In the 13C NMR spectrum, the appearance of a carbon at δ 98.8 (C-2) in 1b instead of the carbonyl carbon (δ 210.1, C-9) in 1a was evident. The cross-peaks of H-4 with H-3 and H-9 in the 1H–1H COSY spectrum indicated the presence of a partial structure that was consistent with a propyl group, as shown in Figure 2. The structure of 1b was also determined through the interpretation of the HMBC spectrum, which showed long-range correlations from H-10 to C-4a and from H-9 to C-2 and C-3. By the process of elimination, C-2 must be connected to C-8a by an ethereal linkage. Therefore, the structure of 1b was determined to be the cyclic hemiketal form of the open chain type 1b, as shown in Figure 1.

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