Leaf Senescence Reshapes Phytochemistry and Bioactivity in Carica papaya: Implications for the Traditional Use of Aged Leaf Extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaf preparations of Carica papaya L. (Caricaceae) are widely used in traditional medicine across Africa, Asia, the Caribbean, and Australia for the management of inflammatory conditions, dengue fever, asthma, infections, wound healing, and, in some traditions, cancer. Ethnopharmacological reports further indicate that aged or senescent leaves are sometimes preferred over fresh material for extract preparation, yet the scientific basis for this practice remains largely unexplored. AIM OF THE STUDY: This study aims to investigate how leaf senescence influences the phytochemical composition and biological activities of C. papaya leaf extracts (PLE) and to evaluate whether these changes support the traditional preference for aged leaf material. MATERIAL AND METHODS: Methanolic extracts and traditionally inspired aqueous leaf juice and decoction preparations were characterized by HPLC-DAD-MS and UHPLC-VWD-HRMS/MS. Feature-based molecular networking was applied to visualize compositional differences. Antioxidative activity was evaluated using ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl assays and by measuring intracellular reactive oxygen species levels. Wound-healing potential was assessed in scratch assays using rat epithelial and alveolar cells. Immunomodulatory effects were investigated using a THP-1-Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter system and pro-inflammatory cytokine quantification. Lipid mediator profiling in polarized macrophages was performed using a UHPLC-QTRAP setup. Antiviral activity was assessed in dengue virus serotype 2-infected Huh7 cells and chikungunya virus-infected Vero cells using plaque assay. Antifungal activity was evaluated against five pathogenic fungi. RESULTS: Senescent extracts showed enrichment of flavonoid-associated compounds, while extracts derived from senescent and withered leaves additionally contained phyllobilins (PB). Senescent PLE exhibited superior antioxidative effects. Withered extracts demonstrated the most pronounced wound-healing activity. Green PLE significantly reduced NF-κB activation and modestly decreased interleukin 6, interleukin 8, and tumor necrosis factor α release, whereas senescent and withered extracts more strongly reduced the formation of specific 5-lipoxygenase-derived lipid mediators by macrophages. Only minimal antiviral effects and no antifungal effects were observed under the tested conditions. CONCLUSION: Leaf senescence substantially reshapes the phytochemical composition and bioactivity of PLE. Whereas fresh and aged leaves differentially modulate inflammatory pathways, aged leaves display superior antioxidant and wound-healing properties. These findings provide experimental support for the ethnopharmacological preference for aged leaf material and highlights the importance of plant developmental stage in ethnopharmacological research and standardization of herbal preparations.