Purification, Characterization, and Bioactivity of Angiotensin-Converting Enzyme Inhibitory Peptides from Enzymatic Hydrolysate Cicada Chrysalis Protein

Abstract

In this study, Cicada chrysalis protein (CCP) was enzymatically hydrolyzed using alkaline protease 6L, followed by ultrafiltration and preparative high-performance liquid chromatography (prep-HPLC) for fractionation. The optimal peptide fractions were identified through nano-HPLC-MS/MS, leading to the discovery of four novel angiotensin-converting enzyme (ACE) inhibitory peptides: FRGF, SPRPW, GPKLF, and SYRF. The IC₅₀ values for these peptides were determined to be 1.716 mg/mL, 0.34 mg/mL, 0.118 mg/mL, and 0.026 mg/mL, respectively. Molecular interaction analysis revealed that these peptides primarily bind to key residues within the ACE active site via hydrogen bonding. Isothermal titration calorimetry (ITC) further confirmed the binding of all peptides to the ACE active site, revealing a compensatory relationship between entropy and enthalpy during the binding process. Notably, GPKLF and SYRF were shown to significantly increase nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) while reducing endothelin-1 (ET-1) secretion. By inhibiting the ACE/AT1R axis and ET-1 expression, while simultaneously activating the ACE2/Ang1-7 axis and NO signaling pathway, these bioactive peptides help restore the balance of the renin-angiotensin system and enhance endothelial function, thereby exerting their protective effects on cardiovascular health. The results of this study suggest that CPP may serve as a promising source of ACE inhibitory peptides, offering a theoretical foundation for the development of functional foods with antihypertensive properties.

Note:
Funding declaration: This work was supported financially by the National Natural Science Foundation of China (No. 32172197), the Innovative Project of the State Key Laboratory of Food Science and Technology (SKLF-ZZB-202307), and program of “Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province”, China.

Conflict of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Keywords: Cicada chrysalis protein；ACE inhibitory peptides；Molecular Docking, HUVECs