Angiotensin-I-Converting Enzyme (ACE)-Inhibitory Peptides from the Collagens of Monkfish (Lophius litulon) Swim Bladders: Isolation, Characterization, Molecular Docking Analysis and Activity Evaluation

Abstract

The objective of this study was to isolate and characterize collagen and angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) peptides from the swim bladders of monkfish (Lophius litulon). Therefore, acid-soluble collagen (ASC-M) and pepsin-soluble collagen (PSC-M) with yields of 4.27 ± 0.22% and 9.54 ± 0.51%, respectively, were extracted from monkfish swim bladders using acid and enzyme methods. The ASC-M and PSC-M contained Gly (325.2 and 314.9 residues/1000 residues, respectively) as the major amino acid, but they had low imino acid content (192.5 and 188.6 residues/1000 residues, respectively) in comparison with collagen from calf skins (CSC) (216.6 residues/1000 residues). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and ultraviolet (UV) absorption spectrums of ASC-M and PSC-M illustrated that they were mainly composed of type I collagen. Subsequently, three ACEi peptides were isolated from a PSC-M hydrolysate prepared via a double-enzyme system (alcalase + neutrase) and identified as SEGPK (MHP6), FDGPY (MHP7) and SPGPW (MHP9), with molecular weights of 516.5, 597.6 and 542.6 Da, respectively. SEGPK, FDGPY and SPGPW displayed remarkable anti-ACE activity, with IC₅₀ values of 0.63, 0.94 and 0.71 mg/mL, respectively. Additionally, a molecular docking assay demonstrated that the affinities of SEGPK, FDGPY and SPGPW with ACE were -7.3, -10.9 and -9.4 kcal/mol, respectively. The remarkable ACEi activity of SEGPK, FDGPY and SPGPW was due to their connection with the active pockets and/or sites of ACE via hydrogen bonding, hydrophobic interaction and electrostatic force. Moreover, SEGPK, FDGPY and SPGPW could protect HUVECs by controlling levels of nitric oxide (NO) and endothelin-1 (ET-1). Therefore, this work provides an effective means for the preparation of collagens and novel ACEi peptides from monkfish swim bladders, and the prepared ACEi peptides, including SEGPK, FDGPY and SPGPW, could serve as natural functional components in the development of health care products to control hypertension.

Keywords: angiotensin-I-converting enzyme (ACE); collagen; molecular docking analysis; monkfish (Lophius litulon); peptides; swim bladder.

Figure 1

SDS-PAGE patterns of ASC-M and PSC-M from swim bladders of monkfish (L. litulon). Lane 1: CSC (type I collagen); lane 2: PSC-M; lane 3: ASC-M; lane 4: marker protein.

Figure 2

UV spectrums of ASC-M and PSC-M from swim bladders of monkfish (L. litulon).

Figure 3

The ACEi activity of hydrolysates from ASC-M and PSC-M from monkfish (L. litulon). ^a–d Values with same letters indicate no significant difference (p > 0.05).

Figure 4

The ACEi activity of fractions from PSC-MH prepared via ultrafiltration membranes. ^a–d Values with same letters indicate no significant difference (p > 0.05).

Figure 5

Separation of MH-I and ACEi activity of isolated peptides. (A) Chromatogram of MH-I purified with Sephadex G-25 column. (B) ACEi activity of fractions (MH-Ia to MH-Ic) from MH-I at 2.5 mg/mL. (C) HPLC profile of MH-Ib at 220 nm. (D) ACEi activity of peptides (MHP-1 to MHP-12) from MH-Ib at 1.5 mg/mL. ^a–f Values with same letters indicate no significant difference (p > 0.05).

Figure 6

Mass spectrums of three isolated ACEi peptides from PSC-MH. (A) MHP6 (SEGPK); (B) MHP7 (FDGPY); (C) MHP9 (SPGPW).

Figure 7

Molecular docking results of MHP6 (SEGPK) (A), MHP7 (FDGPY) (B) and MHP9 (SPGPW) (C) with ACE. (A1,A2): Two- and three-dimensional details of ACE and MHP6 (SEGPK) interaction; (B1,B2): Two- and three-dimensional details of ACE and MHP7 (FDGPY) interaction; (C1,C2): Two- and three-dimensional details of ACE and MHP9 (SPGPW) interaction.

Figure 8

Effects of MHP6, MHP7 and MHP9 on viability (A), NO level (B) and ET-1 production (C) of HUVECs. Compared with control group: ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001; compared with NE group: * p < 0.05, ** p < 0.01 and *** p < 0.001.