Integrated Analysis of Pithecellobium dulce Leaf Fractions for Ex Vivo Antioxidant, DNA Damage Protective, and Afzelin-Mediated Anti-Inflammatory Potentials via In Vivo and In Silico Approaches.

As medicinal plants offer promising alternatives to synthetic drugs, this study explored the ex vivo antioxidant and in vitro anti-inflammatory capacities of Pithecellobium dulce leaf extracts, alongside the in vivo anti-inflammatory potential of its bioactive flavonoid, afzelin, using experimental and computational approaches. In erythrocytes, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) induced a time-dependent increase in hemolysis, malondialdehyde (MDA) formation, and depletion of cytosolic glutathione (GSH) and superoxide dismutase (SOD). The chloroform fraction (CHF) and ethyl acetate fraction (EAF) mitigated hemolysis and lipid peroxidation, enhanced GSH and SOD levels, lowered MDA levels compared with the AAPH group, and also protected PUC19 plasmid DNA from oxidative damage. In anti-inflammatory assays, EAF and CHF inhibited heat-induced red blood cell (RBC) lysis by 57.31% ± 0.80% and 51.39% ± 0.27% and egg albumin denaturation by 39.74% ± 0.77% and 37.20% ± 2.16%, respectively, compared with 61.52% ± 0.58% for acetylsalicylic acid. Afzelin (50 mg/kg body weight) reduced carrageenan-induced paw edema by 56.16%, versus 70.83% for diclofenac sodium. Bioinformatics analysis identified 38 potential anti-inflammatory protein targets of afzelin, of which 11 were further examined via molecular docking. Docking results revealed favorable binding energies for all targets, with the highest affinity observed for JAK3 (-9.60 kcal/mol) compared to native ligands. These computational results suggest that afzelin can form stable, energetically favorable interactions with multiple key proinflammatory proteins. Overall, these findings indicate that P. dulce leaf extracts possess significant antioxidant and anti-inflammatory activities, and afzelin showed predicted interactions with multiple kinase and nonkinase inflammation-associated proteins.