New Insights into the Structural Profiling of Polyphenol Aggregates from Pu-erh Tea and Their Anti-Inflammatory Activity <i>via</i> the TLR4/MyD88/NF-κB Signaling Pathway and Gut Microbiota

Pu-erh tea polyphenol aggregates (PTPAs), macromolecular complexes analogous to theabrownin formed during microbial fermentation, are key bioactive components in this tea. Although their bioactivities are documented, critical gaps persist regarding both precise structural features and molecular mechanisms underlying the anti-inflammatory efficacy across biological models. To address these limitations, we systematically analyzed PTPAs using integrated methods, including alkaline hydrolysis, acid hydrolysis, and benzyl mercaptan-mediated degradation. The anti-inflammatory effects of PTPAs were then evaluated in LPS-induced RAW264.7 cells and DSS-induced colitis mice. Structural characterization revealed that the backbone of PTPAs consists primarily of catechin, epicatechin, and epicatechin gallate units, with flavan-3-ols, flavonols, and anthocyanins as the terminal units. Moreover, phenolic acids and amino acids are potentially integral components of PTPAs. Mechanistically, we demonstrated that PTPAs exert anti-inflammatory effects by inhibiting TLR4/MyD88-mediated NF-κB signaling pathway activation, thereby suppressing downstream inflammatory mediators including NO, TNF-α, IL-1β, and IL-6. Notably, in DSS-induced colitis, PTPAs demonstrated multimodal therapeutic effects by preserving intestinal barrier integrity, modulating systemic inflammation, and reshaping the gut microbiota composition. This study provides the first comprehensive evidence linking the structural complexity of PTPAs to pleiotropic anti-inflammatory actions <i>via</i> gut-immune axis regulation, elucidating Pu-erh tea's phytochemical basis and therapeutic potential.