Metabolomic Profiling and Mechanistic Insights Into the Antioxidant and Anti‐Inflammatory Effects of Traditional Fermented Mustard From Mizoram

Fermented Brassica-based foods generate diverse bioactive metabolites through microbial transformation. This study examined two traditionally fermented mustard leaf products from Northeast India-Ṭam-um (TUM) and Ṭam-um tengkhang (TUTM)-for their chemical composition and anti-inflammatory potential. Methanolic extracts of TUM and TUTM were analyzed using ultra-high-performance liquid chromatography coupled with Q-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). Antioxidant and anti-inflammatory activities were evaluated through cell-free assays and in LPS-stimulated RAW 264.7 macrophages. Gene and protein expression of inflammatory mediators were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Molecular docking was performed between the major identified metabolite, 11-(methylsulfinyl) undecyl glucosinolate, and murine tumor necrosis factor-alpha (TNF-α) and IL-6. Metabolomic profiling revealed glucosinolates as key constituents, dominated by 11-(methylsulfinyl) undecyl glucosinolate. Both extracts showed high reactive oxygen species (ROS)-scavenging activity and upregulated SOD2 expression. They also suppressed nitric oxide production and reduced TNF-α, IL-6, and inducible nitric oxide synthase (iNOS) expression. Western blot results indicated inhibition of phosphorylated p38 and SAPK/JNK, suggesting modulation of the mitogen-activated protein kinase (MAPK) pathway, whereas nuclear factor-kappa B (NF-κB) remained largely unaffected. Docking results showed stronger binding of the major glucosinolate to TNF-α than epigallocatechin gallate and comparable affinity for IL-6. Fermented mustard extracts demonstrated significant antioxidant and anti-inflammatory effects, primarily through modulation of MAPK signaling rather than NF-κB suppression. These findings highlight their potential as functional, redox-active foods with specific molecular targets.