Bioactivity-Guided Isolation of Anti-Inflammatory Constituents of the Rare Mushroom Calvatia nipponica in LPS-Stimulated RAW264.7 Macrophages

Calvatia species, generally known as puffball mushrooms, are used both as sources of food and as traditional medicine. Among the Calvatia genus, Calvatia nipponica (Agaricaceae) is one of the rarest species. Using bioassay-guided fractionation based on anti-inflammatory effects, five alkaloids (1 – 5), two phenolics (6 and 7), and a fatty acid methyl ester (8) were isolated from the fruiting bodies of C. nipponica. Compound 8 was identified from C. nipponica for the first time, and all isolates (1 – 8) were tested for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compound 7 showed mild inhibition while compound 8 significantly inhibited NO production with an IC₅₀ value of 27.50 ± 0.08 μm. The mechanism of NO inhibition of compound 7 was simulated by molecular docking analysis against nitric oxide synthase (iNOS), which revealed the interactions of 7 with the key amino acid residue and the heme in the active site. With the most potent inhibition against LPS-induced inflammation, compound 8 was further investigated with respect to its mechanism of action, and the activity was found to be mediated through the inhibition of iNOS and COX-2 expression.