Purification and identification of antitumor peptides from the enzymatic hydrolysate of rapeseed protein

Rapeseed protein, an abundant by-product of the vegetable oil industry, is rich in essential amino acids and represents a promising source of antitumor peptides. However, the purification, characterization and mechanistic studies of antitumor peptides derived from rapeseed protein remain limited.

Rapeseed protein was hydrolyzed using four proteases (alkaline protease, compound proteinase, flavourzyme and trypsin), followed by ultrafiltration and purification using gel filtration and semi-preparative HPLC. Purified peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and antitumor activities were evaluated against HepG2, MKN-28, A549 and MCF-7 cells using the 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (i.e. MTT) assay. At 1 mg mL^-1, the < 1 kDa fraction showed the strongest inhibitory effects, with inhibition rates of 55.22% ± 0.45%, 60.54% ± 0.84%, 65.33% ± 0.84% and 62.78% ± 1.57%, respectively. Five novel peptides (DHHAPQL, GVIRPPL, NDGNQPL, THPGVAQ and VTDGEAH) were identified. Among them, GVIRPPL exhibited the highest activity, with IC₅₀ values ranging from 1.14 to 1.69 mm. Mechanistic analysis showed that the reactive oxygen species (ROS) level of HepG2 cells treated with 2.0 mm rapeseed antitumor peptide GVIRPPL increased by 50.6% and reduced mitochondrial membrane potential by 53.7%, indicating that rapeseed antitumor peptides can exert antitumor effects by inducing ROS production and interfering with mitochondrial membrane potential, at the same time as having no obvious cytotoxicity to normal human hepatocytes.

This study systematically isolated and identified novel antitumor peptides from rapeseed protein hydrolysates and further explored their antitumor activity and mechanisms of action, providing a scientific basis for the high-value utilization of rapeseed protein and the development of antitumor bioactive components for functional food applications. © 2026 Society of Chemical Industry.