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. 2022 Feb 27;20(3):176.
doi: 10.3390/md20030176.

Preparation, Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle

Shuo-Lei Zheng  1 , Qian-Bin Luo  2 , Shi-Kun Suo  1 , Yu-Qin Zhao  1 , Chang-Feng Chi  2 , Bin Wang  1
Affiliations

Preparation, Identification, Molecular Docking Study and Protective Function on HUVECs of Novel ACE Inhibitory Peptides from Protein Hydrolysate of Skipjack Tuna Muscle

Shuo-Lei Zheng et al. Mar Drugs. .

Abstract

To prepare bioactive peptides with high angiotensin-I-converting enzyme (ACE)-inhibitory (ACEi) activity, Alcalase was selected from five kinds of protease for hydrolyzing Skipjack tuna (Katsuwonus pelamis) muscle, and its best hydrolysis conditions were optimized using single factor and response surface experiments. Then, the high ACEi protein hydrolysate (TMPH) of skipjack tuna muscle was prepared using Alcalase under the optimum conditions of enzyme dose 2.3%, enzymolysis temperature 56.2 °C, and pH 9.4, and its ACEi activity reached 72.71% at 1.0 mg/mL. Subsequently, six novel ACEi peptides were prepared from TMPH using ultrafiltration and chromatography methods and were identified as Ser-Pro (SP), Val-Asp-Arg-Tyr-Phe (VDRYF), Val-His-Gly-Val-Val (VHGVV), Tyr-Glu (YE), Phe-Glu-Met (FEM), and Phe-Trp-Arg-Val (FWRV), with molecular weights of 202.3, 698.9, 509.7, 310.4, 425.6, and 606.8 Da, respectively. SP and VDRYF displayed noticeable ACEi activity, with IC50 values of 0.06 ± 0.01 and 0.28 ± 0.03 mg/mL, respectively. Molecular docking analysis illustrated that the high ACEi activity of SP and VDRYF was attributed to effective interaction with the active sites/pockets of ACE by hydrogen bonding, electrostatic force, and hydrophobic interaction. Furthermore, SP and VDRYF could significantly up-regulate nitric oxide (NO) production and down-regulate endothelin-1 (ET-1) secretion in HUVECs after 24 h treatment, but also abolish the negative effect of 0.5 μM norepinephrine (NE) on the generation of NO and ET-1. Therefore, ACEi peptides derived from skipjack tuna (K. pelamis) muscle, especially SP and VDRYF, are beneficial components for functional food against hypertension and cardiovascular diseases.

Keywords: angiotensin-I-converting enzyme (ACE) peptide; endothelin-1 (ET-1); molecular docking; nitric oxide (NO); skipjack tuna (Katsuwonus pelamis) muscle.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Effects of papain (55 °C, pH 7.0), pepsase (37.5 °C, pH 2.0), Alcalase (55 °C, pH 9.5), Neutrase (55 °C, pH 7.0), and trypsase (37.5 °C, pH 7.8) with enzyme dose of 2% (w/w) for 3h on the ACE inhibitory (ACEi) activity of protein hydrolysates from skipjack tuna muscle. a–c Values with same letters indicate no significant difference (p > 0.05).
Figure 2
Figure 2
Effects of different hydrolysis conditions on ACEi activity of protein hydrolysates from skipjack tuna muscle. (A) pH; (B) enzyme dose (%); (C) temperature (°C). a–d Values with same letters indicate no significant difference (p > 0.05).
Figure 3
Figure 3
Response surface graph for ACEi rate as a function of (A) temperature and pH, (B) enzyme dose and pH, and (C) enzyme dose and temperature during the hydrolysis of skipjack tuna muscle with Alcalase.
Figure 4
Figure 4
ACEi rates of ultrafiltration fractions (TMPH-I, TMPH-II, TMPH-III and TMPH-IV) of TMPH at 0.5 mg/mL. a–d Values with same letters indicate no significant difference (p > 0.05).
Figure 5
Figure 5
Chromatogram profile of TMPH-I isolated by Sephadex G-25 (A) and the ACEi rates of prepared subfractions (IA-IE) from TMPH-I at 0.5 mg/mL (B). a–e Values with same letters indicate no significant difference (p > 0.05).
Figure 6
Figure 6
Elution profiles of subfraction ID by RP-HPLC using a gradient of acetonitrile containing 0.06% trifluoroacetic acid at 254 nm and 280 nm.
Figure 7
Figure 7
Mass spectrogram of six ACEi peptides (TMAP1-TMAP6) from protein hydrolysate of skipjack tuna muscle (STPM). (A) TMAP1; (B) TMAP2; (C) TMAP3; (D) TMAP4; (E) TMAP5; (F) TMAP6.
Figure 8
Figure 8
Molecular docking results of TMAP1 and TMAP2 with ACE. (A1) 2D details of ACE and TMAP1 interaction. (A2) 3D interaction details for TMAP1. (B1) 2D details of ACE and TMAP2 interaction. (B2) 3D interaction details for TMAP2.
Figure 9
Figure 9
The cell viability of HUVECs treated with TMAP1 and TMAP2 for 24 h.
Figure 10
Figure 10
The production of nitrico xide (NO) of HUVECs treated with TMAP1 and TMAP2 for 24 h. Cell group treated with captopril (Cap) was designed as a positive control. ### p < 0.001 vs. control group; *** p < 0.001 vs. norepinephrine (NE) group.
Figure 11
Figure 11
The endothelin-1 (ET-1) secretion of HUVECs treated with TMAP1 and TMAP2 for 24 h. Cell group treated with captopril (Cap) was designed as a positive control. ### p < 0.001 and ## p < 0.01 vs. control group; *** p < 0.001 vs. norepinephrine (NE) group.
Figure 12
Figure 12
Flow diagram of purifying ACEi peptides from protein hydrolysate (TMPH) of skipjack tuna muscle prepared using Alcalase.
See this image and copyright information in PMC

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