# 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

## TL;DR

This study identifies three polyphenols from the Chaga mushroom that show antioxidant and anti-inflammatory effects, potentially useful for treating gout and oxidative stress-related conditions.

## Contribution

The study reveals the novel anti-inflammatory mechanisms of three I. obliquus polyphenols via the MyD88/TLR4/NF-κB pathway in gout-related inflammation.

## Key findings

- Osmundacetone (OS) showed the strongest xanthine oxidase inhibition among the tested polyphenols.
- All tested polyphenols reduced oxidative stress and pro-inflammatory mediators in MSU-induced macrophages.
- The compounds downregulated TLR4, MyD88, and NF-κB expression, indicating anti-inflammatory effects.

## Abstract

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 (IC50), superoxide anion (O2−) 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 (IC50 = 4.91 mM), followed by PAH (IC50 = 5.92 mM) and PA (IC50 = 26.53 mM); OS exerted dual redox effects by scavenging O2− and suppressing XO-mediated O2− 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.