Structural and Functional Profiling of Water-Extracted Polypeptides from

Low-molecular-weight polypeptides (<3 kDa) were prepared from Periplaneta americana via enzymatic hydrolysis and ultrafiltration, yielding 3.53 ± 0.01 mg/g of peptide-rich extract. The extract was primarily composed of peptides, proteins, polysaccharides, phenolics, and flavonoids. HPLC-MS analysis identified 1402 peptide sequences, 80.51% of which were below 1000 Da, predominantly consisting of tri-, tetra-, and octapeptides. Monosaccharide profiling detected D-(+)-galactose, and quantitative assays determined the contents of total phenolics (12.28 mg/g), flavonoids (15.50 mg/g), proteins (85.84 mg/g), and total sugars (17.62 mg/g). The biological activities of the extract were systematically evaluated. The peptide fraction inhibited hyaluronidase activity by 58% at 5 mg/mL, suggesting protection of extracellular matrix integrity. In HaCaT keratinocytes, it promoted cell proliferation by 62.6%, accelerated scratch wound closure by 54%, upregulated Wnt-10b and β-catenin expression, and reduced intracellular ROS levels under oxidative stress. In LPS-stimulated RAW 264.7 macrophages, the extract decreased TNF-α, IL-6, and IL-1β production by 30%, 25%, and 28%, respectively, reduced MDA levels by 35.2%, and enhanced CAT and SOD activities by 12.3% and 60.3%. In vivo, complete closure of full-thickness skin wounds in mice was achieved by day 14. Safety evaluations using the chick chorioallantoic membrane assay and human patch tests confirmed the extract to be non-irritating and non-toxic. These findings highlight