Ultrasound-Assisted Enzymatic Hydrolysates from Common Bean and Pumpkin Seed Proteins: Antioxidant and Anti-Inflammatory Properties

Chronic low-grade inflammation, a key driver of diabetes and fatty liver disease, is present in obesity, which affects 2.1 billion adults as of 2021. Plant-derived bioactive peptides have emerged as promising alternatives to treat inflammation in these pathological processes. This study evaluated the effect of pre- and post-ultrasound-assisted enzymatic hydrolysis on bioactive peptide production and antioxidant activity from common bean (Phaseolus vulgaris L.) and pumpkin (Cucurbita argyoesperma) seed proteins. Pre-treated hydrolysates were fractionated by molecular weight (<3 kDa and 3-10 kDa) and evaluated for their anti-inflammatory properties by measuring nitric oxide and Reactive Oxygen Species in three treatment schemes (pre-, co-, and post-treatment) in an obesity/inflammatory macrophage model. Ultrasound pre-treatment achieved a higher degree of hydrolysis (peptide production) compared to post-treatment, with corresponding increases in antioxidant activity as measured by the ABTS and ORAC assays. All hydrolysate fractions demonstrated dose-dependent inhibition of pro-inflammatory markers. Fractions administered as a co-treatment showed the strongest anti-inflammatory effect, reducing Nos-2 and COX-2 mRNA expression, as well as secreted levels of pro-inflammatory cytokines (TNF-α, IL-6, MCP-1). These findings indicate that ultrasound treatment, mainly as pre-treatment, represents an effective strategy for producing bioactive peptide hydrolysates with anti-inflammatory properties in vitro that warrant deeper investigation.