Comparative Phytochemical and Biological Evaluation of Walnut Septum and Pellicle Extracts in Chemical, Computational, and Cell-Based Models

Background: Walnut (Juglans regia L.) processing generates polyphenol-rich by-products, including the septum (WS) and pellicle (WP). This study comparatively characterized the phytochemical composition of WS and WP from Romania and investigated their antioxidant and preliminary immunomodulatory properties using chemical, computational, and cell-based approaches. Methods: Extracts were characterized by targeted LC–QTOF–MS/MS, and antioxidant capacity was evaluated using ABTS, DPPH, and FRAP assays. Major phenolic compounds were docked against Kelch-like ECH-associated protein 1 (KEAP1), followed by 100 ns molecular dynamics simulations. Cytocompatibility was evaluated in Caco-2, Jurkat, and THP-1 cells, and cellular antioxidant activity was assessed in H2O2-stimulated Caco-2 cells using the DCFH-DA assay. Immunomodulatory potential was investigated following lipopolysaccharide (LPS) stimulation of the same cell lines. Results: WSLE contained 25 quantified compounds, with ellagic acid, catechin, procyanidins, and quercitrin among the major quantified constituents, whereas WP contained 13, with higher ellagic acid, gallic acid, and castalagin levels. WPE showed greater cell-free antioxidant capacity, while both extracts attenuated the H2O2-induced increase in intracellular DCF fluorescence in Caco-2 cells. Simulations suggested favorable KEAP1 interactions, with quercitrin and catechin predicted to form the most stable complexes. LPS induced an insufficient cytokine response in Caco-2 and Jurkat cells. In THP-1 cells, IL-8 was the only responsive marker, and both extracts reduced its LPS-induced secretion. Conclusions: The extracts showed distinct polyphenolic profiles and redox-related cellular activity. Their effects on IL-8 secretion in THP-1 cells and predicted KEAP1 binding are preliminary and hypothesis-generating; Nrf2 activation was not measured, and validation in physiologically relevant in vivo and clinical models is required to establish the biological relevance and potential applications of these extracts.