Three Polyphenolic Compounds from Inonotus obliquus: Antioxidant Activity, Xanthine Oxidase Inhibition, and Regulatory Effects on MyD88/TLR4/NF-κB Pathway in MSU-Induced RAW 264.7 Macrophages.

BACKGROUND: Inonotus obliquus (Chaga), a medicinal and edible macrofungus abundant in bioactive polyphenols, is a potential source of natural antioxidants and anti-inflammatory agents for functional foods. This study aimed to evaluate the antioxidant capacity of three key polyphenols (osmundacetone [OS], protocatechuic aldehyde [PAH], protocatechuic acid [PA]) from I. obliquus and decipher their anti-inflammatory mechanisms via the MyD88/TLR4/NF-κB pathway in a gout-related model. METHODS: Antioxidant activity was assessed by xanthine oxidase (XO) inhibition (IC(50)), superoxide anion (O(2)(-)) scavenging, and structure-activity relationship (SAR) analysis; in a monosodium urate (MSU)-induced acute gout cell model, reactive oxygen species (ROS), nitric oxide (NO), lactate dehydrogenase (LDH), superoxide dismutase (SOD), pro-inflammatory cytokines (TNF-α, IL-1β) were quantified, and MyD88/TLR4/NF-κB pathway proteins were analyzed by Western blot. RESULTS: OS showed the strongest XO inhibition (IC(50) = 4.91 mM), followed by PAH (IC(50) = 5.92 mM) and PA (IC(50) = 26.53 mM); OS exerted dual redox effects by scavenging O(2)(-) and suppressing XO-mediated O(2)(-) generation, with its conjugated C=C-carbonyl system and PAH's aldehyde group enhancing XO binding. All polyphenols and I. obliquus crude extract significantly reduced ROS, NO, LDH, and cytokines (p < 0.05), increased SOD, and downregulated TLR4, MyD88, and NF-κB expression. CONCLUSIONS: I. obliquus-derived polyphenols exhibit obvious antioxidant and xanthine oxidase inhibitory effects, and regulate oxidative stress, pro-inflammatory mediators, and the MyD88/TLR4/NF-κB signaling pathway in monosodium urate-stimulated RAW 264.7 inflammatory macrophages, supporting their development as natural functional food ingredients and potential candidates for gout-related and oxidative stress-associated inflammatory cellular disorders.