Previously reported peptides derived from napin of rapeseed (Brassica napus) have been shown to inhibit DPP-IV in silico. In the present study, napin extracted from rapeseed was hydrolyzed by commercial enzymes and filtered by an ultrafiltration membrane. The napin hydrolysate was then purified by a Sephadex G-15 gel-filtration column and preparative RP-HPLC. A two-enzyme-combination approach with alcalase and trypsin was the most favorable in terms of the DPP-IVinhibitory activity (IC 50 = 0.68 mg/mL) of the napin hydrolysate. Three peptides and one modified peptide (pyroglutamate mutation at the N-terminus) were identified using HPLC-triple-TOF-MS/MS. DPP-IV-inhibitory activity and the types of enzyme inhibition were also determined. Meanwhile, key residues associated with the interactions between the selected peptides and DPP-IV were investigated by molecular docking. IPQVS has key amino acid residues (Tyr547, Glu205, and Glu206) that are consistent with Diprotin A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of DPP-IV.

Studies on antioxidant peptides extracted from foodstuff sources have included not only experiments to elucidate their chemical characteristics but also to investigate their bioavailability and intracellular mechanisms. This study was designed to clarify the absorption and antioxidative activity of YWDHNNPQIR (named RAP), which is derived from rapeseed protein using a Caco-2 cell transwell model. Results showed that 0.8% RAP (C = 0.2 mM, t = 90 min) could maintain the original structure across the Caco-2 cell monolayers via the intracellular transcytosis pathway, and the apparent drug absorption rate (P) was (6.6 ± 1.24) × 10 cm/s. Three main fragments (WDHNNPQIR, DHNNPQIR, and YWDHNNPQ) and five modified peptides derived from RAP were found in both the apical and basolateral side of the Caco-2 cell transwell model. Among these new metabolites, WDHNNPQIR had the greatest antioxidative activity in Caco-2 cells apart from the DPPH assay. With a RAP concentration of 200 μM, there were significant differences in four antioxidative indicators (T-AOC, GSH-Px, SOD, and MDA) compared to the oxidative stress control (P < 0.05). In addition, RAP may also influence apoptosis of the Caco-2 cells, which was caused by AAPH-induced oxidative damage.

WDHHAPQLR (RAP) is an antioxidative peptide derived from rapeseed protein. Although the health benefits from RAP, due to its antioxidant activities, have been determined by chemical methods, a systematic assessment regarding the absorption, metabolism, and antioxidation processes of RAP is still lacking attention. Hence, Caco-2 cell monolayer models and animal experiments were used to evaluate the absorption and bioavailability of RAP. As expected, RAP could be absorbed by intestinal epithelial cells, and the Papp was 0.82 ± 0.19 × 10 cm/s. Three main fragments, RAP, DHHAPQLR, and WDHHAP were transported by the paracellular pathway, and QLR was transported by PepT1. An important modified product of RAP (EGDHHAPQLR) was found to contribute to the elimination of intracellular reactive oxygen species. The absolute bioavailability of RAP was 3.56%, and three degradation products of RAP were also detected in rat serum. More importantly, RAP exerts its antioxidant activity by inhibiting the apoptosis of oxidative stress cells. RAP could downregulate the expression of Bax and caspase-3 and upregulate the expression of Bcl-2 in HO-induced HUVECs (human umbilical vein endothelial cells). In general, using in vitro and in vivo experimental models, the in vivo absorption and transformation processes of RAP and its antioxidative molecular mechanisms by inhibiting apoptosis of cells were revealed.