Anti-Inflammatory Phenolic Metabolites from the Edible Fungus Phellinus baumii in LPS-Stimulated RAW264.7 Cells

Abstract

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 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 IC₅₀ 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.

Keywords: Hymenochaetaceae; NF-kappaB; NO; Phellinus baumii; anti-inflammation; bioactivity-guided isolation.

Figure 1

Anti-inflammatory effects of the EtOH extract of P. baumii and its fractions (hexane (HX) and dichloromethane (DCM) fractions): inhibition of lipopolysaccharide (LPS)-induced NO production in RAW264.7 mouse macrophages. (A–C) Inhibitory effects of the EtOH extract of P. baumii (A) and its hexane (B) and dichloromethane (C) fractions on LPS-induced NO production in RAW264.7 mouse macrophages. The cells were pretreated with the indicated concentrations of samples for 2 h and then exposed to 1 μg/mL LPS for 24 h. * p < 0.05 compared to the LPS-treated value.

Figure 2

Chemical structures of compounds 1–8.

Figure 3

Anti-inflammatory effects of the isolated compounds: inhibition of LPS-induced NO production in RAW264.7 mouse macrophages. Inhibitory effects of isolated compounds 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), 7 (G), and 8 (H) on LPS-induced NO production in RAW264.7 mouse macrophages. The cells were pretreated with the indicated concentrations of samples for 2 h and then exposed to 1 μg/mL LPS for 24 h. * p < 0.05 compared to the LPS-treated value.

Figure 4

Effects of compounds 1, 2 and 3 on the expression of proteins associated with inhibition of NF-κB in LPS-stimulated RAW264.7 mouse macrophages (A). Each bar graph presents the densitometric quantification of western blot bands (B). * p < 0.05 compared to the LPS-treated value.