Sea buckthorn flavonoid IGRh and its metabolites delay Caenorhabditis elegans aging via SIR-2.1/DAF-16-mediated IIS and autophagy-lysosomal pathways.

<h4>Background</h4>Isorhamnetin-3-O-glucoside-7-O-rhamnoside (IGRh) is a characteristic flavonoid in sea buckthorn leaves, yet its anti-aging potential and underlying mechanisms remain largely unexplored.<h4>Purpose</h4>This study aimed to systematically evaluate the anti-aging effects of IGRh and elucidate its mechanistic basis using Caenorhabditis elegans.<h4>Methods</h4>Lifespan, healthspan(definition), stress resistance, and metabolic phenotypes were assessed in wild-type, mutant, and transgenic C. elegans strains. IGRh metabolites were profiled in vivo using UHPLC-QTOF-MS, and their interactions with SIR-2.1 were analyzed through molecular docking and molecular dynamics simulations.<h4>Results</h4>IGRh significantly extended lifespan and improved key aging-related phenotypes, including motility, intestinal barrier integrity, muscle structure, and lipid homeostasis, without impairing reproduction. IGRh also enhanced tolerance to heat and oxidative stress by elevating superoxide dismutase (SOD) and catalase (CAT) activities and reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Mechanistically, IGRh upregulated SIR-2.1 expression and promoted nuclear translocation of the transcription factors DAF-16 and HSF-1, thereby upregulating antioxidant and proteostasis(definition)-related genes. IGRh further stimulated the autophagy(definition)-lysosomal system and mitochondrial quality control, and its longevity-promoting effects were abolished in sir-2.1, daf-16, hlh-30, bec-1, clk-1, and mev-1 mutants. Metabolomic profiling revealed that IGRh was rapidly transformed into bioactive metabolites, including isorhamnetin, isorhamnetin-3-O-glucoside (I3G), quercetin-3-O-glucoside (Q3G), protocatechuic acid, and vanillic acid, several of which persisted in vivo. Q3G showed the most favorable docking score and the highest structural stability within the SIR-2.1 binding pocket. Functional assays further demonstrated that Q3G significantly delayed aging and enhanced stress resistance in C. elegans in a sir-2.1-dependent manner. Together with the CETSA results, these findings support Q3G as a key candidate bioactive metabolite contributing to the SIR-2.1-dependent anti-aging effects of IGRh.<h4>Conclusion</h4>IGRh and its metabolites delay aging in C. elegans by mediating SIR-2.1/DAF-16 signaling and modulating downstream IIS and autophagy-lysosomal pathways. These findings provide mechanistic insight into sea buckthorn leaf flavonoids and support their potential development as natural anti-aging interventions.