Identification, Characterization and Multi-Target Mechanism of Novel ACE Inhibitory Peptides from Idesia polycarpa Seed Cake Protein with Ultrasound-Assisted Extraction

Figures

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  Figure 1. Structural characterization of ultrasound-extracted protein and isolation of gastrointestinal-stable ACE inhibitory peptide fractions from its enzymatic hydrolysate. “Control” refers to conventional alkaline-extracted protein without ultrasonic treatment, and “Optimized” refers to protein extracted by optimized ultrasound-assisted extraction. All data are expressed as mean ± standard deviation from three independent biological replicates. where *** and **** indicates an extremely significant difference between the two indicated groups (p < 0.001 and p < 0.0001). The same significance notation applies to all subsequent figures unless otherwise specified. (A) FTIR spectrum of the extracted protein; (B) DSC thermogram of the extracted protein; (C) ACE inhibitory rates (%) of positive control, negative control and ultrafiltration fractions; (D) Sephadex G-25 gel filtration elution profile of fraction CP3; (E) ACE inhibitory rates (%) of individual peaks from gel filtration separation; (F) ACE inhibitory stability of fraction CP3-H3 after in vitro simulated gastrointestinal digestion.

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  Figure 2. Amino acid composition of IPM raw material and its enzymatic hydrolysate fractions. (A) Idesia polycarpa meal raw material; (B) crude enzymatic hydrolysate (CP); (C) CP1 fraction (Mw > 10 kDa); (D) CP2 fraction (3 kDa < Mw < 10 kDa); (E) CP3 fraction (Mw < 3 kDa); (F) proportions of hydrophobic amino acids (HAAs) and aromatic amino acids (AAAs) in each ultrafiltration fraction. The symbol “**” indicates a significant difference between groups (p < 0.05), while “ns” indicates no significant difference between groups (p > 0.05).

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  Figure 3. Sequence identification of ACE inhibitory peptides derived from IPMP. (A) MS base peak chromatogram of the CP3-H3 fraction obtained by LC-MS/MS; (B) peptide length distribution based on amino acid residue number of identified sequences; (C) molecular weight distribution of identified peptides; (D) amino acid composition profile of identified peptides based on residue counts.

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  Figure 4. 2D structures and MS/MS spectra of the eight potential antihypertensive peptides.

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  Figure 5. Network pharmacology analysis. (A) Venn diagram showing the intersection between potential targets of CP3-H3 peptides and antihypertensive targets; (B) PPI network of intersecting antihypertensive targets for CP3-H3 peptides; (C) interaction network of core targets; (D) integrated peptide–target–disease–pathway network for CP3-H3 peptides.

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  Figure 6. GO functional and KEGG pathway enrichment analysis. (A) Bar plot of GO functional enrichment for key antihypertensive targets of CP3-H3 (top 10 terms per category); (B) chord plot of enriched GO biological process BP terms (top 50); (C) chord plot of enriched GO cellular component CC terms (top 50); (D) chord plot of enriched GO molecular function MF terms (top 50). (E) Bubble plot of KEGG pathway enrichment for core antihypertensive targets of CP3-H3 (top 20 pathways).

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  Figure 7. Molecular docking analysis. (A) Binding mode visualization of eight novel IPM-derived ACE inhibitory peptides with human angiotensin-converting enzyme. Peptides are presented in stick mode, and the ACE active pocket is shown in surface mode. (B) Heatmap of binding free energies from 64 molecular docking simulations between eight IPM-derived ACE inhibitory peptides and eight core antihypertensive targets. The values in the heatmap represent binding free energy in kcal/mol; more negative values indicate stronger binding affinity between the peptide and the target. (C) Binding mode visualization of high-affinity peptide–target complexes with binding free energy ≤ −10 kcal/mol, including representative complexes of WDW-MMP9, WNWD-MMP9, WDW-PTGS2, and WDW-MMP2.

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  Figure 8. In vitro ACE inhibitory activity validation of synthetic WNWD peptide. All data are expressed as mean ± standard deviation from three independent biological replicates. (A) ACE inhibitory rates of captopril (positive control, 10 mM), deionized water (negative control) and synthetic WNWD peptide at 10 mM concentration; (B) dose–response curve and half-maximal inhibitory concentration (IC50) value of synthetic WNWD peptide against ACE. *** and **** indicates an extremely significant difference between the two indicated groups (p < 0.001 and p < 0.0001).