Identification of novel angiotensin I-converting enzyme inhibitory peptides from walnut (Juglans sigillata Dode) protein hydrolysates and their vascular endothelial protective properties: In silico and in vitro studies

Abstract

The objective of this study was to explore novel functional peptides derived from walnut protein isolate (WPI) that exhibited gastrointestinal stability, angiotensin I-converting enzyme (ACE1) inhibitory activity, and endothelial protective properties. Among the 81 detected peptides, 36 peptides with Glu, Leu, Pro, and Asp as the predominant residues were predicted to exhibit ACE1 inhibitory activity. Four promising peptides (TLEPT, TLEPN, VYLEQ, and VLSPQ) were selected for further evaluation based on predicted bioactivity and abundance. In vitro assays confirmed significant ACE1 inhibitory activity, with VYLEQ and VLSPQ demonstrating exceptional potency, exhibiting IC₅₀ values of 27.88 ± 2.45 and 104.75 ± 7.62 μM, respectively. Molecular docking and dynamics simulations further revealed that VYLEQ displayed stronger binding affinity to ACE1 (-8.8 kcal/mol) compared to VLSPQ (-8.4 kcal/mol), facilitated by hydrogen bonding, van der Waals forces, and interactions with Zn²⁺ at the enzyme's active site, contributing to enhanced stability of the ACE1-VYLEQ complex. Moreover, the EA.hy926 cell assay showed that both peptides promoted nitric oxide (NO) release and inhibited endothelin-1 (ET-1) secretion. Molecular docking revealed that both peptides interact with key proteins that regulate NO and ET-1 mainly through van der Waals forces and hydrogen bonding. These findings suggest their potential benefit in antihypertensive effects. The findings of this study have the potential to increase the economic value of skim walnut meal, and to provide a theoretical foundation for the development of novel functional foods that may have a beneficial effect on the health of patients suffering from hypertension.

Keywords: ACE1 inhibitory peptides; Gastrointestinal digestion; Human endothelial cell; Molecular docking; Molecular dynamics simulation; Walnut protein isolate.

Graphical abstract

Fig. 1

The amino acid profile of peptides with potential ACE1 inhibitory activity was predicted by AHTPeptideFusion deep learning. A is the distribution of amino acids; B is the relative abundance of amino acids.

Fig. 2

Results of the inhibition rate of ACE1 activity by the four peptides. All data are expressed as the mean ± SD (n = 3). Different letters indicated that the difference was statistically significant (p < 0.05).

Fig. 3

Results of the molecular docking poses and combined state of the ACE1 inhibitory peptides VYLEQ (A) and VLSPQ (B) with ACE1. Numbers 1–3 represent map of the 3D binding site, hydrogen bond interactions in 3D images, and intermolecular interaction forces in 2D images, respectively.

Fig. 4

The MD simulation results of VLSPQ and VYLEQ with ACE1. A is the root mean square deviations (RMSD, nm); B is the radius of gyration (Rg, nm); C is the total solvent accessible surfACE1 area (Total SASA, nm²); D is the number of hydrogen bonds (H-bonds number) of ACE1.

Fig. 5

Effect of different concentrations of VYLEQ and VLSPQ on nitric oxide (NO) and endothelin-1 (ET-1) release from EA.hy926 cells. All data are expressed as the mean ± SD (n = 3). Different letters in the same index indicated that the difference was statistically significant (p < 0.05).

Fig. 6

Interaction analysis of peptides VYLEQ and VLSPQ with eNOS (A), ECE-1 (B) and Fufin (C), respectively. Numbers 1–3 represent the 3D binding site image, intermolecular interaction forces in 2D images with VYLEQ, and intermolecular interaction forces in 2D images with VLSPQ, respectively.