Pomegranate fruit extract modulates oxidative stress and lifespan in Caenorhabditis elegans: chemical profiling, reproducible survival effects, and DAF-16/SKN-1 involvement

Aging is closely associated with chronic oxidative stress, motivating interest in plant-derived antioxidant products. Previous studies have already shown that pomegranate preparations can extend Caenorhabditis elegans lifespan and engage DAF-16/SKN-1-related stress responses. Here, we chemically characterized the specific crude pomegranate fruit extract (PFE) used in our assays and examined whether a defined 24-h exposure beginning at the L4 stage produced biological effects that persisted after PFE withdrawal. Gas chromatography-mass spectrometry (GC-MS/MS) quantified 15 carbohydrate-related constituents, while UPLC-Q Exactive Orbitrap-MS provided putative annotations for 18 phenolic and related constituents. Three independent wild-type lifespan experiments showed reproducible survival extension after the 24-h pulse. PFE also reduced reactive oxygen species and lipofuscin, increased antioxidant-enzyme activities, promoted DAF-16 and SKN-1 nuclear localization, and altered expression of daf-2 , daf-16 , sod-3 , gst-4 , skn-1 , and gcs-1 . PFE did not increase mean lifespan in daf-16 (mgDf50) or skn-1 (zu67) single mutants. In an additional IIS genetic assay, 100 mg/L PFE did not further extend the lifespan of the long-lived daf-2 (e1370) mutant, consistent with a non-additive effect and convergence on reduced DAF-2/IIS signaling. RNA-seq identified 941 differentially expressed genes enriched in spliceosome-, mTOR(definition)-, neuroactive ligand-, and longevity-related categories. Network pharmacology identified candidate compound-target associations involving aging- and oxidative-stress-related signaling pathways, while molecular docking yielded binding energies ranging from −2.60 to −5.19 kcal/mol, indicating modest predicted interactions between selected PFE-derived compounds and DAF-16, SKN-1, or AKT-1. Together, the data support a reproducible antioxidant and lifespan-associated response to PFE involving DAF-2/IIS, DAF-16, and SKN-1.