Antioxidant, anti-inflammatory and cytoprotective potential of enzymatic protein hydrolysate from <i>Moringa oleifera</i> Lam. leaves in lipopolysaccharide-stimulated RAW 264.7 macrophages

Moringa oleifera leaves are rich in proteins and represent a promising source of bioactive peptides with potential health benefits. This study aimed to evaluate the antioxidant, cytotoxic, cytoprotective and anti-inflammatory effects of protein concentrate extracted from M. oleifera leaves using alkaline extraction (non-hydrolyzed protein) and enzymatic protein hydrolysis with Alcalase® and Neutrase®. Protein content and degree of hydrolysis (DH) were determined by Bradford and o-phthaldialdehyde assays. Antioxidant and cytotoxic activities were analyzed using DPPH and MTT assays, respectively, while cytoprotective and anti-inflammatory activities were evaluated in RAW 264.7 macrophages. The optimal protein extraction condition was 0.5 M sodium hydroxide (NaOH) with a solid-to-solvent ratio of 1:5 w/v, which yielded the highest protein content. Hydrolysis with Alcalase® and Neutrase® for 6 h produced the highest DH values. Protein hydrolysates showed greater antioxidant activity than the non-hydrolyzed protein. Neither the hydrolysates nor the non-hydrolyzed protein exhibited cytotoxicity toward RAW 264.7 cells, and both effectively protected the cells from H2O2-induced oxidative stress. The strongest cytoprotective effects were observed in Alcalase® and Neutrase® hydrolysates, which increased cell viability to 86.46% and 82.87%, respectively. Furthermore, the Alcalase® hydrolysate significantly inhibited NO, TNF-α and IL-6 production in a dose-dependent manner without causing cytotoxicity. These results demonstrate that enzymatic hydrolysis enhances the biological properties of M. oleifera leaf proteins, leading to improved antioxidant, cytoprotective and anti-inflammatory activities. Therefore, M. oleifera leaf protein hydrolysates, particularly those produced with Alcalase®, hold potential as functional ingredients or therapeutic agents for mitigating oxidative stress and inflammation-related diseases.