Valorization and Optimization of Agro-Food By-Products from Punica granatum Bark and Juglans regia Husk Combined with Syzygium aromaticum for Enhanced Antioxidant and Anti-Inflammatory Activities: A D-Optimal Mixture Design Approach

Background: The sustainable valorization of agri-food processing by-products has emerged as a scientifically promising strategy for the development of natural functional ingredients rich in bioactive compounds, contributing simultaneously to the circular bioeconomy and the formulation of health-promoting food and nutraceutical products. Aims: The present study aimed to optimize a multi-component plant-derived formulation comprising Syzygium aromaticum (clove), Punica granatum bark (pomegranate), and Juglans regia husk (walnut) employing a D-optimal ABCD mixture design. Materials and Methods: A ten-run D-optimal ABCD mixture design was employed to optimize the proportions of three plant-derived powders — Syzygium aromaticum (clove), Punica granatum bark, and Juglans regia husk — with TPC, TFC, and DPPH radical scavenging activity as response variables. The optimized formulation was characterized by LC-MS/MS for phenolic compound profiling. Antioxidant capacity was assessed using DPPH, ABTS, FRAP, and ferrous ion chelating assays. Anti-inflammatory activity was evaluated by BSA denaturation inhibition, and cytotoxicity by hemolytic activity assay on human erythrocytes. Results: The optimal formulation comprised 58% pomegranate bark, 30% clove and 12% walnut husk. LC-MS/MS revealed that cloves was the richest source of acid (7,947.87 ± 255.68 µg/g) and quinic acid (5,235.29 ± 79.53 µg/g), pomegranate bark was characterized by catechin, epicatechin, and citric acid, and walnut husk by catechin, quercetin, and citric acid. The optimized mixture revealed a more enriched phenolic profile than the individual extracts, with substantial levels of rutin, kaempferol, naringenin, catechin, and epicatechin concentrations, suggesting synergistic phytochemical interactions. IC₅₀ values for DPPH, ABTS, ferrous iron chelating and BSA denaturation inhibition were 50.97 ± 0.31, 94.84 ± 1.34, 71.15 ± 0.72, and 4.33 ± 0.15 µg/mL, respectively; EC₅₀ for ferric reducing power was 170.83 ± 0.58 µg/ml. Hemolytic activity was negligible (13.38%) at the highest concentration tested (5 mg/mL), confirming erythrocyte biocompatibility. Conclusions: The present study demonstrates that D-optimal mixture design constitutes an effective methodological strategy for the optimization of multi-component plant formulations, enabling the identification of synergistic combinations with enhanced phytochemical composition and biological activities.