Novel angiotensin-converting enzyme inhibitory peptides from tuna byproducts-milts: Preparation, characterization, molecular docking study, and antioxidant function on H2O2-damaged human umbilical vein endothelial cells

Abstract

To prepare peptides with high angiotensin-converting enzyme (ACE) inhibitory (ACEi) activity, Alcalase was screened from five proteases and employed to prepare protein hydrolysate (TMH) of skipjack tuna (Katsuwonus pelamis) milts. Subsequently, 10 novel ACEi peptides were isolated from the high-ACEi activity TMH and identified as Tyr-Asp-Asp (YDD), Thr-Arg-Glu (TRE), Arg-Asp-Tyr (RDY), Thr-Glu-Arg-Met (TERM), Asp-Arg-Arg-Tyr-Gly (DRRYG), Ile-Cys-Tyr (ICY), Leu-Ser-Phe-Arg (LSFR), Gly-Val-Arg-Phe (GVRF), Lys-Leu-Tyr-Ala-Leu-Phe (KLYALF), and Ile-Tyr-Ser-Pro (IYSP) with molecular weights of 411.35, 404.41, 452.45, 535.60, 665.69, 397.48, 521.61, 477.55, 753.91, and 478.53 Da, respectively. Among them, the IC₅₀ values of ICY, LSFR, and IYSP on ACE were 0.48, 0.59, and 0.76 mg/mL, respectively. The significant ACEi activity of ICY, LSFR, and IYSP with affinities of -7.0, -8.5, and -8.3 kcal/mol mainly attributed to effectively combining with the ACEi active sites through hydrogen bonding, electrostatic force, and hydrophobic interaction. Moreover, ICY, LSFR, and IYSP could positively influence the production of nitric oxide (NO) and endothelin-1 (ET-1) secretion in human umbilical vein endothelial cells (HUVECs) and weaken the adverse impact of norepinephrine (NE) on the production of NO and ET-1. In addition, ICY, LSFR, and IYSP could provide significant protection to HUVECs against H₂O₂ damage by increasing antioxidase levels to decrease the contents of reactive oxide species and malondialdehyde. Therefore, the ACEi peptides of ICY, LSFR, and IYSP are beneficial functional molecules for healthy foods against hypertension and cardiovascular diseases.

Keywords: angiotensin-I-converting enzyme (ACE); antihypertensive function; antioxidant activity; milt; peptide; skipjack tuna (Katsuwonus pelamis).

Figure 1

Effects of protease species and hydrolysis time on ACEi activities of protein hydrolysates from skipjack tuna milts at 2.5 mg/ml.

Figure 2

ACEi rates of ultrafiltration peptide fractions (TMH-I to TMH-IV) of protein hydrolysates (TMH) of skipjack tuna milts at a concentration of 1.0 mg/mL. ^a−cValues with same letters indicated no significant difference (P > 0.05).

Figure 3

Chromatogram profiles of TMH-I isolated by Sephadex G-25 (A) and ACEi rates of prepared subfractions (GH-1–GH-4) from TMH-I at a concentration of 1.0 mg/mL (B). ^a−dValues with the same letters indicated no significant difference (P > 0.05).

Figure 4

Elution profile of subfraction GH-3 by RP-HPLC using a gradient of acetonitrile containing 0.06% trifluoroacetic acid at 214 nm.

Figure 5

Mass spectrogram of 10 ACEi peptides (TP1–TP10) from protein hydrolysate of skipjack tuna milts (TMH). (A) TP1, (B) TP2, (C) TP3, (D) TP4, (E) TP5, (F) TP6, (G) TP7, (H) TP8, (I) TP9, and (J) TP10.

Figure 6

Molecular docking results of TP6, TP7, and TP10 with ACE. (A1) 2-D details of ACE and TP6 interaction. (A2) 3-D interaction details for TP6; (B1) 2-D details of ACE and TP7 interaction. (B2) 3-D interaction details for TP7; (C1) 2-D details of ACE and TP10 interaction. (C2) 3-D interaction details for TP10.

Figure 7

Cell viability of HUVECs treated with TP6, TP7, and TP10 for 24 h, respectively.

Figure 8

Contents of nitric oxide (NO) (A) and endothelin-1 (ET-1) (B) of HUVECs treated with TP6, TP7, and TP10 for 24 h, respectively. The cell group treated with captopril (Cap) was designed as the positive control. ***P < 0.001 vs. control; ^###P < 0.001 and ^##P < 0.01 vs. captopril; ^ΔΔΔP < 0.001 vs. norepinephrine (NE).

Figure 9

Effects on the viability of different H₂O₂ concentration (100–600 μM)-treated HUVECs (A) and TP6-, TP7-, and TP10-treated H₂O₂-damaged HUVECs (B). (A) ^a−fValues with same letters indicate no significant difference (P > 0.05); (B) ***P < 0.001 vs. control; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. model; ^ΔP < 0.05 and ^ΔΔΔP < 0.001 vs. GSH.

Figure 10

Effects of TP6, TP7, and TP10 on the ROS (A) and MDA (B) levels of H₂O₂-damaged HUVECs. ***P < 0.001 and **P < 0.01 vs. control; ^###P < 0.001 vs. model; ^ΔP < 0.05 and ^ΔΔΔP < 0.001 vs. GSH.

Figure 11

Effects of TP6, TP7, and TP10 on the SOD (A) and GSH-Px (B) levels of H₂O₂-damaged HUVECs. All values are means ± SD (n = 3). ***P < 0.001 and **P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. model; ^ΔΔP < 0.01 and ^ΔΔΔP < 0.001 vs. GSH.