Anti-Inflammatory Phenolic Metabolites from the Edible Fungus Phellinus baumii in LPS-Stimulated RAW264.7 Cells
The edible fungus Phellinus baumii Pilat (Hymenochaetaceae) has been used in Korean traditional medicines for strengthening health and prolonging life. An extract of the fruiting bodies of P. baumii was subjected to bioassay-guided fractionation based on its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The resulting fractions were chemically investigated, leading to isolation of three phenolic compounds (1-3), a sesquiterpene (4), two steroids (5-6), a fatty
... ids (5-6), a fatty acid (7) , and a cerebroside (8). Spectroscopic analyses including 1D and 2D NMR spectroscopy and LC/MS were used to determine their chemical structures. Compounds 2, 4, 5, 7 and 8 were identified in P. baumii for the first time. Since all compounds were isolated from active fractions with anti-inflammatory activity, their ability to inhibit LPS-stimulated nitric oxide (NO) production in RAW264.7 cells were evaluated in vitro. Compounds 1, 2, 3, 5 and 7 inhibited LPS-stimulated NO production, and compounds 1-3 had IC 50 values <10 µM. Treatment of LPS-stimulated RAW264.7 cells with compounds 1-3 inhibited phosphorylation of IKKα and IκBα. In addition, treatment of compounds 1-3 reduced LPS-induced increases of nuclear factor-kappa B (NF-κB) p65, iNOS and COX-2 protein expressions. Collectively, compounds 1-3 inhibited NF-κB-dependent inflammation in RAW264.7 cells. Thus, P. baumii is a potential source of natural anti-inflammatory agents, and active compounds 1-3 could be promising lead compounds for the development of novel anti-inflammatory agents. , and hypoglycemic properties [7, 10,     . Although many chemical analyses have been published on Phellinus fungi such as P. linteus, the chemical constituents and significant metabolites of P. baumii have been relatively uninvestigated. Previous studies have demonstrated that polysaccharides isolated from P. baumii can inhibit tumor growth and metastasis [7, 15] , and other reports have noted the nuclear factor-kappa B (NF-κB) inhibitory effects of phenolic compounds and anti-influenza activities of polyphenols isolated from P. baumii [10, 16] . In our continuing efforts to discover bioactive secondary metabolites from Korean wild mushrooms       , we have found that an ethanolic (EtOH) extract of the fruiting bodies of P. baumii can inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In this study, we used a bioactivity-guided isolation technique to identify metabolites from this EtOH extract with anti-inflammatory activity in LPS-stimulated RAW264.7 cells. Our chemical analysis led to the isolation and identification of three phenolic compounds (1-3), a sesquiterpene (4), two steroids (5-6), a fatty acid (7) , and a cerebroside (8) with anti-inflammatory activity from the active fractions. Herein, we report the bioactivity-guided isolation and structural elucidation of compounds 1-8, along with their anti-inflammatory activities and underlying mechanisms of action. Results & Discussion Bioactivity-Guided Fractionation for Anti-Inflammatory Effects Dried and chopped fungal material was extracted with 60% aqueous EtOH at room temperature and then filtered. The filtrate was evaporated under reduced pressure with a rotavapor to obtain a crude EtOH extract. In our screening test, the EtOH extract inhibited NO production in a dose-dependent manner in LPS-stimulated RAW264.7 cells, with an IC 50 value of 56.9 ± 1.2 µg/mL, and no significant cell death was observed up to the concentration of 100 µg/mL (Figure 1 ).