Revealing Novel Angiotensin‐Converting Enzyme (ACE)‐Inhibitory Peptides From Mackerel Hydrolysates and Potential Antihypertensive Mechanism Using Virtual Screening and Network Pharmacology Analysis

Angiotensin-converting enzyme-inhibitory (ACE-I) peptides derived from marine proteins have received widespread attention for their high efficiency and safety in the management of hypertension. In this study, the mackerel (Scomber japonicus) muscle papain hydrolysate (MMPH) was isolated via ultrafiltration and gel chromatography. Then peptides with ACE-I activity were screened using LC-MS/MS and virtual screening. Ten novel ACE-I peptides were identified from MMPH. In which, APFLAG showed the greatest inhibitory effects in vitro with an IC₅₀ value of 69.45 ± 2.93 µM, followed by FDHKKFF (212.85 ± 3.19 µM) and LFPKFA (259.05 ± 3.72 µM). Molecular docking studies revealed that all these three peptides bind to S₁ and S₂ pockets, while APFLAG and LFPKFA interacted with the zinc (Zn)-binding domain residues and only FDHKKFF interacted with the S₁' pocket of ACE. Furthermore, the best peptide, APFLAG, inhibited ACE competitively and noncompetitively. Network pharmacology analysis suggested that APFLAG regulates blood pressure by targeting the renin-angiotensin system (RAS) and modulating renin secretion via ACE and renin. Additionally, APFLAG promotes vasodilation and lowers blood pressure by regulating vascular smooth muscle cells through the activation of tyrosine-protein kinase SRC (SRC) and signal transducer and activator of transcription 3 (STAT3). These findings underscore the multifaceted anti-hypertensive mechanisms of APFLAG and highlight its potential for therapeutic management of hypertension. Practical Application: Preparation of mackerel muscle hydrolysate with strong ACE-I activity by papain hydrolysis, ultrafiltration, and gel chromatography. The oligopeptides APFLAG, FDHKKFF, and LFPKFA exhibited excellent ACE-I activity. Producing bioactive peptides is a promising strategy to realize high-value applications of mackerel muscle protein.