Abstract: Food-derived antihypertensive peptides play a positive role in regulating blood pressure. In this study, angiotensin converting enzyme (ACE) inhibitory peptides from the hydrolysate of pumpkin seed protein with alcalase for 4 h was separated by membrane filtration and identified structurally by mass spectrometry (MS). The action mechanism of the ACE inhibitory peptides was studied by kinetic analysis of the inhibitory effect and molecular docking. In addition, ACE inhibition assay and spontaneously hypertensive rats (SHRs) were used to evaluate the antihypertensive activity of the protein hydrolysate, its fractions separated by membrane filtration and the peptides present in the fraction with molecular mass less than 1 kDa. The results showed that the fraction with molecular mass less than 1 kDa had good ACE inhibitory activity, with an inhibition rate of 46.22% at a concentration of 1 mg/mL. The systolic blood pressure of SHRs decreased by 21.42 mm Hg at 6 hours after oral administration with this faction at a dose of 100 mg/kg mb. Nine ACE inhibitory peptides were identified, namely LLV, LVF, LTPL, SVLF, LLPQ, MLPL, LLPGF, VLLPE and RFPLL, and RFPLL, LLPGF, MLPL and LVF had good ACE inhibitory activity, with a half maximal inhibitory concentration less than 1 mmol/L. RFPLL and LVF had relatively better antihypertensive activity, decreasing the systolic blood pressure of SHRs by 37.0 and 22.2 mm Hg, and decreasing the diastolic blood pressure by 17.0 and 11.2 mm Hg at 6 hours after gavage administration at a dose of 30 mg/kg mb, respectively. RFPLL, LLPGF, MLPL and LVF could dock at the active center of ACE very well. RFPLL was a mixed inhibitor of ACE. ACE could be inhibited by MLPL competitively, while LLPGF and LVF could be non-competitive inhibitors of ACE. Therefore, pumpkin seed protein hydrolysates with molecular mass less than 1 kDa are promising ingredients of functional foods with antihypertensive activity, and RFPLL and LVF can be used for the development of antihypertensive functional foods or drugs.

Key words: pumpkin seed peptide; angiotensin converting enzyme; antihypertensive activity; inhibitory kinetics; molecular docking