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.
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.
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.
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 λmax(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.
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).
Fiasson, J. L. Distribution of styrylpyrones in the basidiocarps of various Hymenochaetaceae. Biochem. Syst. Ecol. 10, 289–296 (1982).
Zaidman, B. Z., Tassin, M., Mahajna, J. & Wasser, S. P. Medicinal mushroom modulators of molecular targets as cancer therapeutics. Appl. Microbiol. Biotechnol. 67, 453–468 (2005).
Lee, I. K., Seok, S. J., Kim, W. K. & Yun, B. S. Hispidin derivatives from the mushroom Inonotus xeranticus and their antioxidant activity. J. Nat. Prod. 69, 299–301 (2006).
Lee, I. K. & Yun, B. S. Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus. Bioorg. Med. Chem. 15, 3309–3314 (2007).
Lee, I. K., Kim, Y. S., Jang, Y. W., Jung, J. Y. & Yun, B. S. New antioxidant polyphenols from the medicinal mushroom Inonotus obliquus. Bioorg. Med. Chem. Lett. 17, 6678–6681 (2007).
Lee, Y. G. et al. Src kinase-targeted anti-inflammatory activity of davallialactone from Inonotus xeranticus in lipopolysaccharide-activated RAW264.7 cells. Br. J. Pharmacol. 154, 852–863 (2008).
Kim, S. D. et al. The mechanism of anti-platelet activity of davallialactone: involvement of intracellular calcium ions, extracellular signal-regulated kinase 2 and p38 mitogen-activated protein kinase. Eur. J. Pharmacol. 584, 361–367 (2008).
Jung, J. Y. et al. Antioxidant polyphenols from the mycelial culture of the medicinal fungi Inonotus xeranticus and Phellinus linteus. J. Appl. Microbiol. 104, 1824–1832 (2008).
Lee, I. K., Seo, G. S., Jeon, N. B., Kang, H. W. & Yun, B. S. Phellinins A1 and A2, new styrylpyrones from the culture broth of Phellinus sp. KACC93057P: I. Fermentation, taxonomy, isolation and biological properties. J. Antibiot. 62, 631–634 (2009).
Yun, B. S., Lee, I. K., Kim, Y. S., Jung, J. Y. & Jang, Y. W. Polyphenol antioxidants from the culture broth of genera Phellinus and Inonotus Korea Patent (10-0831757 B1) (2008).
Castleberry, B., Valente, E. J. & Eggleston, D. S. Open-cyclic warfarin isomerism: 5-hydroxywarfarin. J. Crystallogr. Spectrosco. Res. 20, 583–593 (1990).
This work was supported by a grant (20080401-034-069) from the BioGreen 21 Program of the Rural Development Administration (RDA), Republic of Korea.
About this article
Cite this article
Lee, I., Jung, J., Kim, Y. et al. Phellinins B and C, new styrylpyrones from the culture broth of Phellinus sp.. J Antibiot 63, 263–266 (2010). https://doi.org/10.1038/ja.2010.25
- antioxidant activity
- phellinins B and C
- Phellinus sp.
Gold-Katalyse für die Heterocyclenchemie: eine repräsentative Fallstudie zu Naturstoffen der Pyron-Reihe
Angewandte Chemie (2018)
Angewandte Chemie International Edition (2018)
Gold- or Silver-Catalyzed Syntheses of Pyrones and Pyridine Derivatives: Mechanistic and Synthetic Aspects
Chemistry - A European Journal (2016)
Sesquiterpenes from the Fruiting Bodies of <i>Ramaria formosa</i> and Their Human Neutrophil Elastase Inhibitory Activity
CHEMICAL & PHARMACEUTICAL BULLETIN (2015)