A tuna multi-target antihypertensive peptide and its application

Abstract

This invention provides a tuna multi-target antihypertensive peptide and its applications. The amino acid sequence of the peptide is one or more of FAPF, VSFP, FQPSF, and FFLP, and it is a bioactive peptide possessing ACE2 upregulation, ET-1 downregulation, and ACE inhibitory activities. The tuna multi-target antihypertensive peptide provided by this invention is prepared from yellowfin tuna scraps. This bioactive peptide has excellent biocompatibility, small molecular weight, and high activity, making it easy to isolate, purify, and synthesize artificially. This invention utilizes Ang-II-induced HUVEC cells to isolate and purify a novel bioactive peptide with ACE2 upregulation, ET-1 downregulation, and ACE inhibition from tuna hydrolysate. This overcomes the problem of blood pressure reversal after long-term use of single-target ACE inhibitory peptides and can synergistically exert a better antihypertensive effect with ACE inhibitory peptides.

Description

Technical Field

[0001] This invention belongs to the field of bioactive product manufacturing, and specifically relates to a tuna multi-target antihypertensive peptide and its application. Background Technology

[0002] Hypertension is a significant risk factor for cardiovascular and cerebrovascular diseases, and medication is a crucial means of controlling it. Hypertensive patients often require long-term, even lifelong, medication. On the one hand, long-term medication use can easily lead to symptoms such as fatigue, arrhythmia, elevated blood pressure, rapid heart rate, elevated blood sugar, dyslipidemia, and elevated uric acid. Simultaneously, long-term medication use and potential discomfort may create a psychological burden for patients, easily leading to resistance and affecting medication adherence. This is particularly unsuitable for intervention in young and middle-aged individuals with hypertension. For this population, there is a need to develop healthier and safer antihypertensive products. Antihypertensive peptides, as bioactive peptides with antihypertensive effects, generally have better biocompatibility and lower toxicity, with minimal impact on liver and kidney function, and do not accumulate toxicity in the body, making long-term use relatively safe.

[0003] The renin-angiotensin system is a crucial system for blood pressure regulation, and angiotensinase (ACE) is its primary target. Current research on antihypertensive peptides primarily focuses on ACE-inhibiting peptides. However, many peptides with high in vitro ACE-inhibiting activity fail to exert their antihypertensive effects in in vivo studies, such as the egg-derived hexapeptide TNGIIR. Furthermore, long-term use of ACE-inhibiting peptides can activate alternative pathways, leading to a rebound in blood pressure, a phenomenon known as "Ang-II escape."

[0004] With the deepening research into the pathogenesis of hypertension, it has been discovered that multiple targets and systems in the human blood pressure regulation system have synergistic effects. The ACE2 / Ang (1-7) / Mas receptor (MasR) axis is a new direction in the study of blood pressure-lowering systems. ACE2 is a homologous metalloproteinase of ACE, capable of hydrolyzing single amino acid residues in Ang-I (1-10) or Ang-II (1-8), thereby producing Ang-(1-9) and Ang-(1-7). Ang-(1-7) can bind to MasR and angiotensin II receptor (AT1), thereby inhibiting the interaction between Ang-II and AT1 receptors. In addition, Ang-(1-7) exerts vasodilatory effects through the kallikrein-kinin system, the nitric oxide-endothelin system, and other mechanisms. Endothelin-1 (ET-1) is a polypeptide composed of 21 amino acids, mainly synthesized and secreted by vascular endothelial cells. ET-1 binds to the ET-A receptor on vascular smooth muscle cells, activating downstream signaling pathways and leading to increased intracellular calcium ion concentration. This induces vascular smooth muscle contraction, thereby increasing vascular resistance and raising blood pressure. Studies have shown that the synthesis and release of endothelin-1 (ET-1) are significantly elevated in hypertensive patients, and its release is significantly positively correlated with blood pressure levels. Furthermore, ET-1 participates in vascular remodeling by promoting cell proliferation and hypertrophy; long-term effects can lead to thickening of the blood vessel wall and narrowing of the lumen, thus exacerbating the pathological progression of hypertension.

[0005] Screening for multi-target antihypertensive peptides offers a potential strategy for addressing the "Ang-II escape" phenomenon, reducing related side effects, and enhancing the antihypertensive efficacy of bioactive peptides. However, current research primarily focuses on the screening and purification of angiotensin-converting enzyme (ACE) inhibitory peptides, with limited research and purification of other target peptides such as ACE2 and ET-1. Summary of the Invention

[0006] This invention utilizes computational screening methods, combined with an Ang-II-induced HUVEC cell model and in vitro ACE enzyme inhibition experiments, and employs ion chromatography, high-performance liquid chromatography-reverse column chromatography, and high-performance liquid chromatography-tandem mass spectrometry to obtain four peptide sequences with multiple targets of ACE2 downregulation, ET-1 upregulation, and ACE inhibition from tuna minced meat enzymatic hydrolysate. Their amino acid sequences are FAPF (SEQ ID NO:1), VSFP (SEQ ID NO:2), FQPSF (SEQ ID NO:3), or FFLP (SEQ ID NO:4).

[0007] Furthermore, these four polypeptide sequences were synthesized in a solid-phase manner, and their ACE2 downregulation, ET-1 upregulation, and ACE inhibitory activities were verified using an Ang-II-induced HUVEC cell model and in vitro ACE enzyme inhibition experiments. The presence of one, two, three, or all four sequences all demonstrated significant multi-target activity.

[0008] Furthermore, FQPSF possesses ACE2 upregulation, ET-1 downregulation, and ACE inhibitory activity;

[0009] FFLP exhibits ACE2 upregulation, ET-1 downregulation, and ACE inhibitory activity;

[0010] VSFP possesses ET-1 downregulation and ACE inhibitory activity;

[0011] FAPF exhibits ACE2 upregulation activity;

[0012] The polypeptide provided by this invention is used to prepare antihypertensive products.

[0013] The beneficial effects of this invention are as follows:

[0014] 1) The bioactive peptides provided by this invention are derived from tuna mince, thus the bioactive peptides have good biosafety, and have small molecular weight and high activity, which not only facilitates separation and purification, but also facilitates artificial synthesis.

[0015] 2) The active peptides FAPF, FQPSF and FFLP provided by this invention can significantly increase ACE2 expression in Ang-II induced HUVEC cells, among which 50 μM FQPSF can restore ACE2 expression to normal levels;

[0016] 3) The active peptides provided by this invention can significantly reduce Ang-II-induced ET-1 expression in HUVEC cells, and 200 μM of FFLP and FQPSF can restore ET-1 expression to normal levels.

[0017] 4) The active peptides provided by this invention also have significant ACE inhibitory activity, among which FQPSF, FFLP, and VSFP have an ACE inhibition IC50 value. 50 The values ​​were 17.70 ± 0.40 μM, 91.90 ± 1.80 μM, and 141.50 ± 3.53 μM, respectively.

[0018] 5) The active peptides provided by this invention can work synergistically with multiple targets to exert a blood pressure-lowering effect, overcoming the shortcomings of single-target ACE inhibitory peptides.

[0019] The active peptides provided by this invention can be biosynthesized and developed into novel multi-target antihypertensive drugs. Attached Figure Description

[0020] Figure 1 : Cation chromatogram of the peptide hydrolysate of the present invention;

[0021] Figure 2 : The effect of the cationic chromatographically separated components of the present invention on ACE2 production in HUVEC cells;

[0022] Figure 3 : The effect of the cationic chromatographically separated components of the present invention on ET-1 production in HUVEC cells;

[0023] Figure 4 : Liquid chromatogram of the target cation peak component of the hydrolysate of the present invention;

[0024] Figure 5 : The effect of the liquid chromatography-separated components of this invention on ACE2 production in HUVEC cells;

[0025] Figure 6 : The effect of the liquid chromatography-separated components of this invention on ET-1 production in HUVEC cells;

[0026] Figure 7 : The effect of the synthetic peptide of this invention on ACE2 production in HUVEC cells;

[0027] Figure 8 : The effect of the synthetic peptide of the present invention on ET-1 production in HUVEC cells. Detailed Implementation

[0028] This invention provides a multi-target antihypertensive peptide from tuna, and provides the effects of the above peptide on ACE2 and ET-1 protein expression in Ang-II-induced HUVEC cells, as well as the assay of the peptide's ACE inhibitory activity. This invention utilizes an Ang-II-induced HUVEC cell model and ACE enzyme activity inhibition experiments to isolate and purify a novel multi-target antihypertensive peptide with ACE2 upregulation, ET-1 downregulation, and ACE inhibition activities from tuna hydrolysate. This overcomes the problem of blood pressure reversal after long-term use of single-target ACE inhibitory peptides and can synergistically exert a better antihypertensive effect with ACE inhibitory peptides.

[0029] The bioactive peptides provided by this invention are obtained by a method comprising the following steps:

[0030] (1) Enzymatic hydrolysis of yellowfin tuna mince: After homogenizing the tuna mince, protease was added for enzymatic hydrolysis. Under the optimal enzymatic hydrolysis conditions, the hydrolysis time was 4 hours. After inactivating the enzyme by boiling water bath for 10 minutes, the supernatant was collected by filtration and freeze-dried to obtain crude peptide samples.

[0031] (2) Isolation and purification of multi-target antihypertensive peptides from yellowfin tuna: Preliminary separation was performed using a cation exchange column. The effect of each component on the expression of ACE2 and ET-1 proteins in Ang-II induced HUVEC cells was determined. The components that resulted in the highest expression level of ACE2 and the lowest expression level of ET-1 protein were collected. The components were further separated and purified using a liquid semi-preparative C-18 column. The effect of each liquid chromatographic peak component on the expression of ACE2 and ET-1 proteins in Ang-II induced HUVEC cells was determined. The components that resulted in the highest expression level of ACE2 and the lowest expression level of ET-1 protein were collected. The peptide sequences contained in the components were analyzed by high performance liquid chromatography-tandem mass spectrometry to obtain peptide sequences with ACE2 upregulation and ET-1 downregulation activities, including one or more of FAPF, VSFP, FQPSF, and FFLP.

[0032] The bioactive peptides of this invention can be prepared by artificial synthesis.

[0033] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1: Preparation of bioactive peptides with ACE2 upregulation and ET-1 downregulation

[0035] After homogenizing the minced yellowfin tuna, the material-to-liquid ratio was adjusted to 1:3-1:15. The mixture was boiled to denature the protein, cooled, and then protease was added. The mixture was hydrolyzed for 4 hours, and then the enzyme was inactivated by boiling in a water bath for 10 minutes. The supernatant was collected by filtration and freeze-dried to obtain a mixed peptide sample.

[0036] (1) Cation chromatography separation and purification of multi-target antihypertensive peptides from yellowfin tuna: Preliminary separation was performed using a cation chromatography column. The effect of each component at a concentration of 100 μg / ml on the expression of ACE2 and ET-1 proteins in Ang-II-induced HUVEC cells was determined. Among them, the expression level of ACE2 in HUVEC cells was the highest after treatment with component F2, and the expression level of ET-1 protein was the lowest. Component F2 was collected for further purification, such as... Figure 1 , Figure 2 , Figure 3 As shown;

[0037] (2) Separation and purification of multi-target antihypertensive peptides from yellowfin tuna using liquid semi-preparative chromatography: The F2 fraction was further separated and purified using a liquid semi-preparative C-18 column. The effect of each liquid chromatographic peak fraction at a concentration of 50 μg / ml on the expression of ACE2 and ET-1 proteins in Ang-II-induced HUVEC cells was determined. Among them, the expression level of ACE2 in HUVEC cells treated with the F2-5 fraction was the highest, and the expression level of ET-1 protein was the lowest. Figures 4-6As shown; F2-5 fractions were collected to obtain peptide sequences with ACE2 upregulation and ET-1 downregulation activities, namely FAPF, VSFP, FQPSF and FFLP.

[0038] Example 2: Effect of peptides on Ang-II-induced ACE2 protein expression in HUVEC cells

[0039] HUVEC cell culture: Cell culture medium formulation: 10% fetal bovine serum, 1% penicillin-drug antibiotics, 89% DMEM high glucose medium; Culture temperature: 37℃; CO2 concentration: 5%.

[0040] Human umbilical vein endothelial cells (HUVECs) were collected at a rate of 3 × 10⁶ cells per well. 5 Cells were cultured at a density of 1000 cells / well in 6-well plates for 12 hours, followed by treatment with different concentrations of sample for 4 hours. The treated cells were then incubated with angiotensin II (10 μM) for 12 hours. Cells were subsequently collected and lysed, and cellular proteins were extracted for analysis of ACE2 production.

[0041] Cellular protein concentrations were quantified using a (BCA) protein assay kit and subsequently standardized.

[0042] The concentration of ACE2 protein in different samples was determined using the Shanghai Tongwei ACE2 content assay kit, and the effects of different sample treatments on ACE2 protein expression were analyzed.

[0043] Experimental results: such as Figure 7 As shown, compared with the model group, FAPF, FQPSF, and FFLP can all increase ACE2 protein expression, among which 50 μM FQPSF can restore ACE2 expression to normal levels.

[0044] Example 3: Effect of peptides on Ang-II-induced ET-1 protein expression in HUVEC cells

[0045] Human umbilical vein endothelial cells (HUVECs) were collected at a rate of 3 × 10⁶ cells per well. 5 Cells were cultured at a density of 1000 cells / well in 6-well plates for 12 hours, followed by treatment with different concentrations of sample for 4 hours. The treated cells were then incubated with angiotensin II (10 μM) for 12 hours. The supernatant was then collected to assess endothelin-1 (ET-1) levels according to the kit instructions.

[0046] (1) The concentration of ET-1 protein in different samples was determined using the Shanghai Tongwei ET-1 content assay kit, and the effect of different sample treatments on ET-1 protein expression was analyzed.

[0047] (2) Experimental results: such as Figure 8As shown, VFSP, FQPSF, and FFLP can all reduce ET-1 expression, with 200 μM FFLP and FQPSF restoring ET-1 expression to normal levels.

[0048] Example 4: Assay of Peptide ACE Inhibitory Activity

[0049] (1) ACE and HHL were dissolved in 50 mM HEPES buffer (pH 8.3) containing 0.3 M NaCl. 80 µL of peptide solution was mixed with 80 µL of 5 mM HHL and preheated at 37 °C for 5 min. Then, 80 µL of preheated ACE (1 mg / mL) was added, and the mixture was incubated at 37 °C for 60 min. The reaction was then terminated by adding 240 µL of 1 M HCl solution. Hippuric acid generated from HHL was analyzed by RP-HPLC using 28% acetonitrile (containing 0.1% TFA) as the mobile phase at a flow rate of 0.3 mL / min. The detection wavelength was 228 nm, and the column type was Zorbax SB-C18 (4.6 × 100 mm, 3.5 μM, Agilent, USAM002835). The IC50 of the sample was... 50 The value is defined as the peptide concentration required to achieve 50% ACE activity inhibition in the reaction. The molecular weights of the synthesized peptides and their ACE inhibition IC50 values ​​are shown in Table 1.

[0050] (2) Experimental results: VSFP, FQPSF, and FFLP have high ACE inhibitory activity, among which FQPSF has the highest ACE inhibition IC50 value. 50 The value is 17.70±0.40μM.

[0051] Table 1: Molecular weight of synthetic peptides and ACE inhibition IC50 50 Data table

[0052] peptide sequence molecular weight <![CDATA[ACE inhibitor IC 50 (μM)]]> FAPF 480.55 883.90 ± 65.91 μM VSFP 448.51 141.50 ±3.53 μM FQPSF 624.88 17.70 ± 0.40 μM FFLP 522.63 91.90 ± 1.80 μM

[0053] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A polypeptide derived from yellowfin tuna, characterized in that, The amino acid sequence of the polypeptide is VSFP.

2. The use of the polypeptide according to claim 1 in the preparation of antihypertensive products.

3. The application as described in claim 2, characterized in that, The peptide is used to prepare products that downregulate ET-1 and inhibit ACE activity.

4. A pressure-reducing product, characterized in that, The blood pressure-lowering product contains the polypeptide described in claim 1.

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