Polypeptide with effects of reducing blood pressure and resisting oxidation as well as related product and application thereof

Abstract

The invention discloses a polypeptide with effects of reducing blood pressure and resisting oxidation as well as a related product and application thereof, and relates to the technical field of biology. The polypeptide with angiotensin converting enzyme inhibitory activity and antioxidant activity is obtained through screening, and the amino acid sequence of the polypeptide is AVPYPQRDMP. In-vitro activity evaluation results show that the polypeptide not only has strong ACE inhibitory activity, but also can effectively scavenge free radicals; the ACE inhibition IC50 value is 258.46 mu M, the free radical scavenging TEAC value is 1.47 mu mol TE / mu mol, and compared with similar polypeptides, the polypeptide has better ACE inhibition effect and free radical scavenging effect. In-vivo antihypertensive evaluation is carried out through a spontaneous hypertension rat model, it is also shown that the compound has a good antihypertensive effect, and the compound can be used for preparing products related to antihypertensive and / or antioxidation.

Description

A polypeptide with blood pressure lowering and antioxidant effects, and related products and applications. Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a polypeptide with blood pressure-lowering and antioxidant effects, as well as related products and applications. Background Technology

[0002] Hypertension is a common chronic disease affecting health, characterized by persistently elevated arterial blood pressure. It is a significant risk factor for cardiovascular and cerebrovascular diseases such as coronary heart disease, stroke, and heart failure. In the medical field, hypertension is defined as a systolic blood pressure ≥140 mmHg and / or a diastolic blood pressure ≥90 mmHg. Blood pressure in the human body is mainly controlled by multiple systems, including the renin-angiotensin-aldosterone system and the vascular endothelium.

[0003] Angiotensin-converting enzyme (ACE) plays a crucial role in blood pressure regulation: on the one hand, it catalyzes the conversion of angiotensin I into the potent vasoconstrictor angiotensin II; on the other hand, it accelerates the inactivation of bradykinin, a vasodilator, and promotes aldosterone secretion, leading to increased peripheral vascular resistance. Simultaneously, hypertension-induced endothelial damage exacerbates oxidative stress, creating a vicious cycle of oxidative stress and hypertension. Based on these pathological mechanisms, inhibiting ACE activity and improving oxidative stress levels have become dual targets for hypertension intervention. Hypertension treatment often employs chemically synthesized ACE inhibitors such as captopril, enalapril, and lisinopril. However, long-term use of these chemically synthesized drugs may lead to potential side effects such as kidney damage, allergic reactions, and rashes. Bioactive peptides, due to their high activity, safety, and ease of metabolism in the human body, are becoming an important area of ​​research and development for novel ACE inhibitors.

[0004] Currently, the market lacks bioactive peptides with blood pressure-lowering and / or antioxidant functions.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide with blood pressure lowering and antioxidant effects, as well as related products and applications.

[0007] The present invention is implemented as follows: In a first aspect, embodiments of the present invention provide a polypeptide having an amino acid sequence as shown in SEQ ID NO:1.

[0008] In a second aspect, embodiments of the present invention provide a biological material selected from any of the following: (I) an isolated nucleic acid molecule encoding the polypeptide described in the foregoing embodiments; (II) a carrier containing the nucleic acid molecule; and (III) a recombinant cell containing the carrier.

[0009] Thirdly, embodiments of the present invention provide a method for preparing polypeptides, comprising: artificially synthesizing the polypeptides described in the foregoing embodiments or culturing the recombinant cells described in the foregoing embodiments.

[0010] Fourthly, embodiments of the present invention provide a composition whose active ingredient includes the polypeptide described in the foregoing embodiments.

[0011] Fifthly, embodiments of the present invention provide the use of the polypeptides, biomaterials, or compositions described in the foregoing embodiments in the preparation of compositions that help maintain healthy blood pressure levels.

[0012] In a sixth aspect, embodiments of the present invention provide the use of the polypeptides, biomaterials, or compositions as described in the foregoing embodiments in the preparation of angiotensin-converting enzyme inhibitors or in the preparation of compositions for treating diseases related to abnormal angiotensin-converting enzyme activity.

[0013] In a seventh aspect, embodiments of the present invention provide the use of the polypeptides, biomaterials, or compositions as described in the foregoing embodiments in the preparation of compositions that contribute to antioxidant activity.

[0014] Eighthly, embodiments of the present invention provide the use of the polypeptides, biomaterials, or compositions as described in the foregoing embodiments in the preparation of compositions that help maintain healthy blood pressure levels and help with antioxidant effects.

[0015] This invention offers the following advantages: It screened and obtained a polypeptide with angiotensin-converting enzyme (ACE) inhibitory and antioxidant activities, with the amino acid sequence AVPYPQRDMP. In vitro activity evaluation showed that this polypeptide not only possesses strong ACE inhibitory activity but also effectively scavenges free radicals; the IC50 value for ACE inhibition was 258.46 μM, and the TEAC value for free radical scavenging was 1.47 μmol TE / μmol, demonstrating better ACE inhibition and free radical scavenging effects compared to similar polypeptides. In vivo blood pressure-lowering effects were also demonstrated in a spontaneously hypertensive rat model, indicating its good antihypertensive effect, making it suitable for preparing products related to blood pressure reduction and / or antioxidant activity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 shows the mass spectrum of peptide AVPYPQRDMP; Figure 2 shows the high-performance liquid chromatography (HPLC) separation of peptide AVPYPQRDMP for purity identification; Figure 3 shows the ACE inhibitory activity assay of peptide AVPYPQRDMP; Figure 4 shows the in vivo antihypertensive activity assay of peptide AVPYPQRDMP. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] The term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. Amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0020] The term "pharmaceuticalally acceptable excipient" means that the excipient meets requirements for safety, stability, and suitability for formulation.

[0021] The term “treatment” includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0022] The inventors of this application obtained a novel bioactive peptide with dual functions of antioxidation and blood pressure reduction (amino acid sequence as shown in SEQ ID NO:1, AVPYPQRDMP) by screening casein through a targeted hydrolysis process. The bioactive peptide has been verified to have a good blood pressure reduction effect in vivo, and has the advantages of high safety, low allergenicity, high stability and easy absorption, providing a new option for the development of products with blood pressure reduction and antioxidant activity.

[0023] On one hand, embodiments of the present invention provide a polypeptide (polypeptide 1) whose amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0024] In an optional embodiment, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0025] On the other hand, embodiments of the present invention provide a biological material selected from any of the following: (I) an isolated nucleic acid molecule encoding the polypeptide described in any of the foregoing embodiments; (II) a carrier containing the nucleic acid molecule; (III) a recombinant cell containing the carrier.

[0026] In optional embodiments, the vector is an expression vector or a cloning vector. It can be an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged as a virus) into host cells via transformation, transfection, or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0027] In optional embodiments, the recombinant cells can be prokaryotic cells, eukaryotic cells, or bacteriophages. The aforementioned prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*; insect cells such as *Ardisia crenata*; mammalian cells such as BHK cells, CHO cells, COS cells, NSO cells, 293 series cells, HepG2, HEK293 cell lines, Huh7 cells, and myeloma cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0028] On the other hand, embodiments of the present invention provide a method for preparing polypeptides, which includes: artificially synthesizing the polypeptides described in any of the foregoing embodiments or culturing the recombinant cells described in any of the foregoing embodiments.

[0029] On the other hand, embodiments of the present invention provide a composition whose active ingredient includes the polypeptide described in any of the foregoing embodiments.

[0030] In an optional embodiment, the active ingredient further includes at least one of a polypeptide (polypeptide 2) with an amino acid sequence as shown in SEQ ID NO:2 (ALPQY) and a polypeptide (polypeptide 3) with an amino acid sequence as shown in SEQ ID NO:3 (LDAYPSGAW).

[0031] In an optional embodiment, the active ingredients include polypeptide 1 and polypeptide 2.

[0032] In an optional embodiment, the active ingredients include polypeptide 1 and polypeptide 3.

[0033] In an optional embodiment, the active ingredient includes: polypeptide 1, polypeptide 2, and polypeptide 3.

[0034] In an optional embodiment, the composition further includes excipients.

[0035] In an optional embodiment, the excipient is a pharmaceutically acceptable excipient.

[0036] In an optional embodiment, the excipients include any one or more of the following: carrier, pH adjuster, humectant, emulsifier, thickener, antioxidant, preservative, stabilizer, chelating agent, and colorant.

[0037] In optional embodiments, the composition is in the form of powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0038] In optional embodiments, the composition may be applied to humans (e.g., infants, children, adolescents, the elderly, etc.) or animals (e.g., mammals).

[0039] On the other hand, embodiments of the present invention provide the use of polypeptides, biomaterials, or compositions as described in any of the foregoing embodiments in the preparation of compositions that help maintain healthy blood pressure levels.

[0040] In an optional embodiment, the composition that helps maintain healthy blood pressure levels may be a health supplement, food, or food additive.

[0041] On the other hand, embodiments of the present invention provide the use of polypeptides as described in any of the foregoing embodiments, or biomaterials as described in the foregoing embodiments, or compositions as described in the foregoing embodiments, in the preparation of angiotensin-converting enzyme inhibitors or in the preparation of compositions for treating diseases related to abnormal angiotensin-converting enzyme activity.

[0042] In an optional embodiment, the diseases associated with abnormal angiotensin-converting enzyme activity include at least one of hypertension, heart failure, and diabetic nephropathy.

[0043] On the other hand, embodiments of the present invention provide the use of polypeptides, biomaterials, or compositions as described in any of the foregoing embodiments in the preparation of compositions that contribute to antioxidant activity.

[0044] Furthermore, embodiments of the present invention provide the use of polypeptides, biomaterials, or compositions as described in any of the foregoing embodiments in the preparation of compositions that help maintain healthy blood pressure levels and that contribute to antioxidant effects.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] In the examples, the peptides AVPYPQRDMP, ALPQY, and LDAYPSGAW used were all derived from casein. After targeted hydrolysis, separation, purification, and screening, peptide fragments were obtained and their amino acid sequences were identified. Subsequently, they were prepared using solid-phase synthesis technology based on the sequences.

[0047] Example 1, Synthesis and Characterization of Peptides (1) Solid-phase synthesis of peptides: First, the C-terminal amino acid (Pro) was immobilized on the resin, and then Met, Asp, Arg, Gln, Pro, Tyr, Pro, Val, and Ala were added sequentially. Before each addition of a new amino acid, the protecting group of the previous amino acid was removed. The deprotection-condensation process was repeated until all amino acids were sequentially linked. The synthesized crude peptide was cut off from the resin and the side chain protecting groups were removed to obtain the crude peptide of AVPYPQRDMP.

[0048] (2) Peptide purification steps (1) The synthesized crude peptide was purified by high performance liquid chromatography (HPLC) using a Sinochrom ODS-BP column with a size of 4.6×250 mm and a diameter of 5 μm. Mobile phase A was water containing 0.1% (v / v) trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 0.1% (v / v) TFA. The flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the injection volume was 5 μL.

[0049] (3) Characterization of the peptide The purified peptide was characterized by LC-MS and HPLC. Figure 1 shows the mass spectrum of the peptide AVPYPQRDMP. The experimental results show that the peptide AVPYPQRDMP was successfully synthesized in Example 1. The chromatographic analysis results are shown in Table 1, and Figure 2 shows that the purity of the peptide AVPYPQRDMP is 97.95%.

[0050] Table 1 Chromatographic Analysis Results

[0051] Example 2: Determination of the antioxidant activity of the peptide. ABTS reacts with potassium persulfate to generate relatively stable blue-green cationic free radicals ABTS• + Antioxidants will react with ABTS• + A reaction occurs, causing the reaction system to decolorize. This is achieved through ABTS• + The antioxidant capacity of a substance is reflected by detecting changes in absorbance at the maximum absorption wavelength of free radicals, 734 nm. 50 μL of 40 μM sample solution was added to a 96-well microplate, followed by rapid addition of 150 μL of ABTS using a pipette. + The solution and reaction mixture were incubated at 30°C for 30 min, and the absorbance at 734 nm was measured. 50 μL of PBS solution and different concentrations of Trolox solution were used instead of the above sample solution as the control group and standard group, respectively. The effects of Trolox on ABTS• in the sample and standard groups were calculated. + The clearance rate was further calculated, and the TEAC value of the sample was expressed as μmol TE / μmol sample. The results are shown in Table 2.

[0052] Table 2. TEAC values ​​of peptide AVPYPQRDMP for scavenging ABTS free radicals

[0053] Experimental results showed that the TEAC value of the peptide AVPYPQRDMP was 1.47 μmolTE / μmol, indicating that it has strong antioxidant capacity.

[0054] Example 3: Antioxidant Activity Assay of Peptide Combination ALPQY+AVPYPQRDMP The antioxidant activity (ABTS free radical scavenging rate) of peptides ALPQYPQRDMP and ALPQY+AVPYPQRDMP was determined. The experimental steps of Example 2 were repeated, except that 25 μL of 40 μM peptide ALPQY and peptide ALPYPQRDMP sample solutions were added to each of the 96-well microplates when measuring the mixed peptides. The results are shown in Table 3.

[0055] Experimental results showed that the free radical scavenging rate of the peptide AVPYPQRDMP was 48.88%, and the free radical scavenging rate of the peptide combination ALPQY+AVPYPQRDMP was 53.02%.

[0056] Table 3. ABTS free radical scavenging rates of different peptides (combinations)

[0057] Example 4: Antioxidant Activity Determination of the Peptide Combination ALPQY+LDAYPSGAW+AVPYPQRDMP The antioxidant activity (ABTS free radical scavenging rate) of peptides ALPQY+LDAYPSGAW+AVPYPQRDMP was determined separately. The experimental steps of Example 2 were repeated, except that 16.7 μL of 40 μM peptide ALPQY, peptide LDAYPSGAW, and peptide ALPYPQRDMP sample solutions were added to each of the 96-well microplates when measuring the mixed peptides. The results are shown in Table 4.

[0058] Experimental results showed that the free radical scavenging rate of the peptide AVPYPQRDMP was 48.88%, and the free radical scavenging rate of the peptide combination ALPQY+LDAYPSGAW+AVPYPQRDMP was 55.39%.

[0059] Table 4. ABTS free radical scavenging rates of different peptides (combinations)

[0060] Example 5: Determination of ACE Inhibitory Activity of Peptides. The ACE inhibitory activity of peptides was assessed by measuring the peak area of ​​HA at 228 nm using high-performance liquid chromatography (HPLC) based on the in vitro cleavage of hippuric acid (HA) by ACE simulating hippuryl-histyl-leucine (HHL). A gradient concentration solution was prepared by mixing 30 μL of substrate (HHL, 7.5 mM) with 20 μL of pure peptide sample. An equal volume of sodium borate buffer was used as a blank. The mixture was incubated at 37°C for 10 min, and then 30 μL of ACE solution (0.05 U / mL) was added to initiate the reaction. After reacting at 37°C for 30 min, 20 μL of HCl (0.1 M) was added to terminate the reaction. The ACE inhibitory activity of the sample was calculated. The half-maximal inhibitory concentration (IC50) was determined. 50 () represents the peptide concentration corresponding to an ACE inhibitory activity of 50%.

[0061] Figure 3 shows the ACE inhibitory activity of the decapeptide AVPYPQRDMP at different concentrations, IC50, and ACE inhibitory activity. 50 The value is 258.46 μM.

[0062] Example 6: ACE Inhibitory Activity Assay of the Peptide Combination ALPQY+AVPYPQRDMP The ACE inhibitory activities of peptides ALPQY+AVPYPQRDMP were determined separately. The experimental steps of Example 5 were repeated. For single peptide assays, 20 μL of 500 μM sample was added to a 96-well microplate; for mixed peptide assays, 10 μL of 500 μM peptide ALPQY and peptide ALPYPQRDMP sample solutions were added to each 96-well microplate. The results are shown in Table 5.

[0063] Experimental results showed that the ACE inhibitory activity of the peptide AVPYPQRDMP was 32.89%, and the ACE inhibitory activity of the peptide combination ALPQY+AVPYPQRDMP was 68.40%.

[0064] Table 5. ACE inhibitory activity of different peptides (combinations)

[0065] Example 7: ACE Inhibitory Activity Assay of the Peptide Combination ALPQY+LDAYPSGAW+AVPYPQRDMP The ACE inhibitory activities of peptides ALPQY+LDAYPSGAW+AVPYPQRDMP were determined separately. The experimental steps of Example 5 were repeated. When measuring single peptides, 20 μL of 500 μM sample was added to a 96-well microplate; when measuring mixed peptides, 6.7 μL of 500 μM peptide ALPQY, peptide LDAYPSGAW, and peptide ALPYPQRDMP sample solutions were added to each 96-well microplate. The results are shown in Table 6.

[0066] Experimental results showed that the ACE inhibitory activity of the peptide AVPYPQRDMP was 32.89%, and the ACE inhibitory activity of the peptide combination ALPQY+LDAYPSGAW+AVPYPQRDMP was 60.51%.

[0067] Table 6. ACE inhibitory activity of different peptides (combinations)

[0068] Example 8: Determination of the in vivo antihypertensive activity of the peptide. Twenty-four 10-week-old male SHR rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal room was maintained with a 12 / 12-hour light / dark cycle, room temperature controlled at 22±3℃, and relative humidity at 50%–70%. After 7 days of acclimatization, rats were divided into a model group (physiological saline), a positive control group (captopril, 10 mg / kg·bw / d), and a peptide AVPYPQRDMP group (30 mg / kg·bw / d) based on their baseline blood pressure and body weight, with six rats in each group. Before each blood pressure measurement, the rats were gently placed on a rat support and administered gastric perfusion sequentially according to their SHR rat numbers. The temperature was then adjusted to 37±1℃ using a non-invasive blood pressure monitor, and the rats were allowed to acclimatize until their blood pressure stabilized. Systolic blood pressure was measured at 0 h, 2 h, 4 h, 6 h, and 8 h.

[0069] The results are shown in Figure 4. The results indicate that the blood pressure in the model group administered saline by gavage did not decrease, while the blood pressure in the positive control group administered captopril by gavage showed a significant decreasing trend, reaching its lowest value at 8 hours, a reduction of 31.43 mmHg. In the peptide AVPYPQRDMP group, blood pressure continued to decrease after gavage, with the largest decrease in systolic blood pressure at 2 hours, a reduction of 18.38 mmHg. These results demonstrate that the antihypertensive peptide of the present invention also has a good blood pressure-lowering effect in vivo.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A biomaterial, characterized in that, It is selected from any one of the following: (I) an isolated nucleic acid molecule encoding the polypeptide of claim 1; (II) a vector containing the nucleic acid molecule; (III) a recombinant cell containing the vector.

3. A method for preparing polypeptides, characterized in that, It includes: The polypeptide of claim 1 is synthesized artificially or the recombinant cells of claim 2 are cultured.

4. A composition, characterized in that, Its active ingredients include the polypeptide described in claim 1.

5. The composition according to claim 4, characterized in that, The active ingredient further includes at least one of a polypeptide with an amino acid sequence as shown in SEQ ID NO:2 and a polypeptide with an amino acid sequence as shown in SEQ ID NO:

3.

6. The composition according to claim 5, characterized in that, The composition further includes excipients; the excipients include: The carrier, pH adjuster, humectant, emulsifier, thickener, antioxidant, preservative, stabilizer, chelating agent and colorant are any one or more of these.

7. The use of the polypeptide of claim 1 or the biomaterial of claim 2 in the preparation of compositions that help maintain healthy blood pressure levels.

8. The use of the polypeptide of claim 1 or the biomaterial of claim 2 in the preparation of angiotensin-converting enzyme inhibitors or in the preparation of compositions for treating diseases related to abnormal angiotensin-converting enzyme activity; wherein the diseases related to abnormal angiotensin-converting enzyme activity include: At least one of hypertension, heart failure, and diabetic nephropathy.

9. The use of the polypeptide of claim 1 or the biomaterial of claim 2 in the preparation of compositions that contribute to antioxidant activity.

10. The use of the polypeptide of claim 1 or the biomaterial of claim 2 in the preparation of compositions that help maintain healthy blood pressure levels and have antioxidant properties.

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