In Vitro Antioxidant and Anti-inflammatory Effects of Protaetia brevitarsis Larvae Hydrolysates in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Protaetia brevitarsis larvae protein (PBP) has been investigated as a health-functional-food resource, but its enzymatic hydrolysates remain underexplored.In this study, three PBP hydrolysates were prepared using Alcalase ® (PB-A), Protamex ® (PB-P), and their combination (PB-AP), and their antioxidant and anti-inflammatory properties were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Enzymatic hydrolysis significantly increased the degree of hydrolysis and soluble solids content relative to nonhydrolyzed PBP, with PB-AP yielding the highest values, indicating an synergistic effect of dual-enzyme treatment.All hydrolysates showed increased total polyphenol content and enhanced 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid)/trolox equivalent antioxidant capacity radical-scavenging activity, again most pronounced for PB-AP.In LPS-stimulated macrophages, all three hydrolysates promoted proliferation to a comparable extent and attenuated LPS-induced cytotoxicity, with PB-AP showing the greatest protective effect.LPS-induced upregulation of Tnf-α, Il-1β, and Nos2 and the resulting nitric oxide production were suppressed by all hydrolysates, with PB-AP producing the strongest suppression, while the anti-inflammatory gene Il-10 was reciprocally upregulated, most markedly by PB-AP.Consistent with these transcriptional effects, PB-AP most potently inhibited LPS-induced phosphorylation of nuclear factor of kappa-light-chain-enhancer of activated B cells and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation were reduced without reaching statistical significance.These findings indicate that enzymatic hydrolysis enhances the antioxidant and anti-inflammatory bioactivity of PBP, and that dual-enzyme (Alcalase ® + Protamex ® ) treatment produces a synergistic enhancement beyond either enzyme alone-supporting PB-AP as a promising candidate ingredient for health functional foods targeting oxidative stress-and inflammation-related conditions.