Phellinins B and C, new styrylpyrones from the culture broth of Phellinus sp.

Many fungi have the ability to synthesize pigments including carotenoids, quinones and styrylpyrones. Among the Aphyllophorales, the styrylpyrone pigments are strictly restricted to Hymenochaetaceae including Phellinus and Inonotus.¹ It is interesting to note that several mushrooms, which belong to the genera Phellinus and Inonotus and produce styrylpyrone pigments, have been used as traditional medicines for the treatment of gastrointestinal cancer, liver or heart diseases, and stomach ailments without the presence of toxic side effects.² Recently, the number of novel styrylpyrones with different biological activities from the fruiting body and mycelial culture of Phellinus and Inonotus spp. has been rising sharply.^{3, 4, 5, 6, 7} Previously, we reported on four major antioxidants, hispidin, hypholomine B, 3,14′-bihispidinyl and 1,1–distyrylpyrylethan isolated from the culture broths of fungi Phellinus linteus and Inonotus xeranticus.⁸ Based on the findings of this study, it was suggested that these pigments might have an important role in the biological activity of these fungi. In addition, we recently identified new prenylated styrylpyrones, phellinins A1 and A2, isolated from the culture broth of Phellinus sp. KACC93057P⁹ and suggested that these were a new group of a natural polyketide–isoprenoid hybrid compounds. In a continuous search for styrylpyrone compounds from the culture broth of Phellinus sp. KACC93057P, we isolated new antioxidants, phellinins B (1) and C (2).¹⁰ Phellinin B consisted of a mixture of inseparable isomers with an open chain (1a), trans-hemiketal (1b) and cis-hemiketal (1c) at an approximate equal ratio and phellinin C was obtained as isomeric pairs of trans-ketal (2a) and cis-ketal (2b), as shown in Figure 1. In this paper, the isolation, structure determination and biological properties of compounds 1 and 2 are described.

Figure 1

Structures of phellinins B (1) and C (2).

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).

Compound 1 was obtained as a yellow powder and a single peak in the HPLC profile. However, three sets of signals with the same intensities in the ¹H NMR spectrum suggested that 1 was a mixture of three inseparable components, 1a, 1b and 1c, which were present in an approximate equal ratio. Thus, extensive spectroscopic analyses were carried to determine the structure of these three inseparable compounds. The absorption peaks in the IR spectrum of 1 suggested the presence of hydroxyl (3444 cm⁻¹) and conjugated carbonyl (1651 cm⁻¹) groups and its UV absorption maxima at 204, 258 and 375 nm revealed the presence of a styrylpyrone moiety.⁴ The EI-MS spectrum of 1 contained an ion peak at m/z 330 [M]⁺, and the molecular formula was determined to be C₁₈H₁₈O₆ by HR-EI-MS, suggesting that 1a–1c had the same molecular composition. The ¹H NMR and ¹H-¹H COSY spectra of 1a indicated the presence of a 1,2,4-trisubstituted benzene moiety at δ 6.61 (d, J=8.4 Hz), 6.92 (dd, J=2.0, 8.4 Hz), and 7.00 (d, J=2.0 Hz), a trans-disubstitued double bond unit at δ 6.55 (d, J=16.0 Hz) and 7.24 (d, J=16.0 Hz), and a partial structure by the COSY connectivity of δ 3.58 (H-7) to δ 3.01/2.86 (H-8) and δ 1.20 (H-11), as depicted in Figure 2. The chemical structure of 1 was established with the aid of the HMBC spectrum. The styrylpyrone moiety was assigned by the long range correlations of H-5 to C-3, C-6 and C-1′; H-1′ to C-6; H-2′ to C-6, C-1′, C-4′ and C-8′; H-4′ to C-6′ and C-8′; H-7′ to C-3′ and C-5′; and H-8′ to C-2′, C-4′ and C-6′, and all of the carbon chemical shifts were identical with the corresponding carbons for hispidin compounds, a representative styrylpyrone.⁴ The HMBC correlations from H-11 to C-7 and C-8, H-8 to C-10 and H-10 to a ketone carbonyl of C-9 revealed the presence of a pentanone moiety in 1a. This pentanone substructure was substituted to C-3 of the styrylpyrone moiety by the HMBC correlation of H-11 to C-3, as shown in Figure 2. Therefore, the structure of compound 1a was determined to be a new styrylpyrone that displayed antioxidant activity.

Figure 2

Two-dimensional NMR correlations of compounds 1 and 2.

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.

The ¹H NMR and ¹³C NMR spectra of 1c were almost the same as those of 1b, except for a slight difference in the chemical shifts of H-3, H-4, H-9 and H-10. Analysis of the ¹H–¹H COSY and HMBC spectra of 1c indicated that its plenary structure was the same as that of 1b. This result suggested that 1c was an isomer of 1b in the cyclic hemiketal moiety. The relative stereochemistry was determined by comparing their ¹H NMR chemical shifts with warfarin analogs, which showed that the methyl protons of H-9 and H-10 were shifted downfield and upfield, respectively, in the trans form.¹¹ Thus, compounds 1b and 1c were determined to be (2S*,4R*)-trans-7-(3,4-dihydroxystyryl)-3,4-dihydro-2-hydroxy-2,4-dimethylpyrano[4,3-b]pyran-5(2H)-one and (2S*,4S*)-cis-7-(3,4-dihydroxystyryl)-3,4-dihydro-2-hydroxy-2,4-dimethylpyrano[4,3-b]pyran-5(2H)-one, respectively. The complete ¹H and ¹³C NMR assignments of 1a–1c are described in Table 1.

Table 1 ¹H and ¹³C NMR spectral data of phellinin B (1) in CD₃OD

Compound 2 was obtained as a yellow powder and contained a pair of peaks with similar intensity in the HPLC profile. It is interesting to note that after each peak was purified by preparative HPLC they continually converted back to a pair of peaks until they equilibrated. This indicates that compound 2 was a mixture of two inseparable isomers (2a and 2b). Thus, the structure of compound 2 was determined as a mixture. Their molecular formulas were determined to be C₁₉H₂₀O₆ by HR-EI-MS (found m/z 344.1261 [M]⁺, calculated m/z 344.1260 [M]⁺). Bands because of the presence of hydroxyl (3444 cm⁻¹) and conjugated carbonyl (1635 cm⁻¹) groups were observed in the IR spectrum. The UV absorption maxima at λ_max(MeOH)(log ɛ) 204 (4.23), 258 (3.86) and 375 (4.36) nm suggested that 2 was also a styrylpyrone analog. Analysis of the ¹H NMR data and ¹H–¹H COSY spectra of 2 provided two sets of signals that had a 1:1 ratio intensity, which suggested that the two compounds in 2 had an isomeric relationship similar to 1b and 1c, as shown in Figure 1. The ¹H NMR spectrum of 2 was very similar to 1b and 1c except for the appearance of a methyl singlet at δ 3.26 and 3.35 in addition to those of 1b and 1c. The structure of 2a was determined by the HMBC spectrum, which showed critical correlations from H-11 to C-2, as shown in Figure 2. Other correlations from the HMBC spectrum were well-matched to the corresponding cross-peaks for compound 1b. ¹H NMR and ¹H–¹H COSY spectra of 2b were very similar to those of 2a except for the difference in chemical shift values of H-3, H-4, H-10 and H-11. Analysis of the HMBC spectrum revealed that the plenary structure of compound 2b was identical to 2a. This result indicates that compounds 2a and 2b have a diastereomeric relationship and the cyclic ketal forms of compounds 1b and 1c, respectively. Therefore, 2a was determined to be (2S,4R)-trans-7-(3,4-dihydroxystyryl)-3,4-dihydro-2-methoxy-2,4-dimethylpyrano[4,3-b]pyran-5(2H)-one and 2b was determined to be (2S,4S)-cis-7-(3,4-dihydroxystyryl)-3,4-dihydro-2-methoxy-2,4-dimethylpyrano[4,3-b]pyran-5-(2H)-one. The ¹H NMR spectral data of 2a and 2b are described in Table 2.

Table 2 ¹H NMR spectral data of phellinins C (2) in CD₃OD

Compounds 1 and 2 were isolated as a mixture of inseparable isomers, respectively, and thus we assessed the free radical scavenging efficacy, a main property of antioxidants, of the mixture by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical, ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical anion and superoxide radical cation scavenging assay methods.⁸ For DPPH and ABTS radical scavenging activity, results were expressed in terms of trolox equivalent antioxidant capacity (TEAC, IC₅₀ of μM compound/IC₅₀ of μM trolox). The free radical scavenging activities of the compound mixtures are presented in Table 3. In these assays, all compounds were shown to be capable of scavenging DPPH, ABTS and superoxide radicals, in a concentration-dependent manner. Compound 1 was found to be approximately 5–10 times higher than 2, which contained a methoxyl group, and all of the compounds tested showed potent activity comparable with the positive control, caffeic acid and BHA (butylated hydroxyanisole).

Table 3 Free radical scavenging activity of phellinins B and C