Active peptide with antihypertensive activity and preparation method and application thereof

Abstract

The invention discloses an active peptide with antihypertensive activity and a preparation method and application thereof, and relates to the technical field of dairy products. A novel active peptide is obtained through screening, and the amino acid sequence of the novel active peptide is LDAYPGSAW. Through in-vitro activity evaluation, the polypeptide not only has relatively strong ACE inhibitory activity, but also has relatively strong oxidation resistance, the IC50 value for inhibiting ACE is 62.37 [mu] M, and the TEAC value for scavenging free radicals is 2.10 [mu] mol TE / [mu] mol. In-vivo blood pressure reduction evaluation is carried out through a spontaneous hypertension rat model, and it is shown that the compound also has an excellent blood pressure reduction effect in vivo and can be applied to preparation of products related to blood pressure reduction and / or oxidation resistance.

Description

An bioactive peptide with antihypertensive activity, its preparation method and application Technical Field

[0001] This invention relates to the field of dairy technology, and more specifically, to an active peptide with antihypertensive activity, its preparation method, and its application. Background Technology

[0002] Hypertension is one of the most common chronic diseases affecting health in recent years. Its main clinical feature is persistently elevated arterial blood pressure, and 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, bioactive peptides with blood pressure-lowering and / or antioxidant effects are still very scarce in the market.

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

[0006] The purpose of this invention is to provide an active peptide with antihypertensive activity, its preparation method, and its application.

[0007] The present invention is achieved as follows: In a first aspect, embodiments of the present invention provide the use of an active peptide or a combination of polypeptides containing said active peptide in the preparation of a composition which helps maintain a healthy blood pressure level, wherein the amino acid sequence of said active peptide is shown in SEQ ID NO:1.

[0008] In a second aspect, embodiments of the present invention provide the use of an active peptide or a combination of polypeptides containing the active peptide in the preparation of angiotensin-converting enzyme inhibitors or compositions for treating diseases related to abnormal angiotensin-converting enzyme activity, wherein the amino acid sequence of the active peptide is shown in SEQ ID NO:1.

[0009] Thirdly, embodiments of the present invention provide the use of an active peptide or a combination of polypeptides containing the active peptide in the preparation of a composition that contributes to antioxidation, wherein the amino acid sequence of the active peptide is shown in SEQ ID NO:1.

[0010] Fourthly, embodiments of the present invention provide the use of an active peptide or a combination of peptides containing the active peptide in the preparation of compositions that help maintain healthy blood pressure levels and help with antioxidation, wherein the amino acid sequence of the active peptide is shown in SEQ ID NO:1.

[0011] Fifthly, embodiments of the present invention provide a composition whose active ingredient contains: an active peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0012] This invention has the following beneficial effects: A novel bioactive peptide with the amino acid sequence LDAYPSGAW was obtained through screening. In vitro activity evaluation showed that this peptide not only possesses strong ACE inhibitory activity but also strong antioxidant properties, inhibiting the IC50 of ACE. 50 The concentration was 62.37 μM, and the TEAC value for scavenging free radicals was 2.10 μmolTE / μmol. In vivo evaluation using a spontaneously hypertensive rat model showed excellent blood pressure-lowering effects, suggesting its potential application in the preparation of products related to blood pressure reduction and / or antioxidant activity. Attached Figure Description

[0013] 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.

[0014] Figure 1 shows the mass spectrum of peptide LDAYPSGAW; Figure 2 shows the high performance liquid chromatography separation of peptide LDAYPSGAW for purity identification; Figure 3 shows the ACE inhibitory activity assay of peptide LDAYPSGAW; Figure 4 shows the in vivo antihypertensive activity assay of peptide LDAYPSGAW. Detailed Implementation

[0015] 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.

[0016] The inventors of this application obtained an active peptide (amino acid sequence as shown in SEQ ID NO:1, LDAYPSGAW) from casein through a multi-enzyme directed hydrolysis process, followed by separation, purification, and identification. Verification has shown that this active peptide possesses excellent ACE inhibitory activity and antioxidant properties, and also exhibits a good blood pressure-lowering effect in vivo. It boasts advantages such as high safety, low allergenicity, high stability, and easy absorption, providing a new option for developing foods or health foods with auxiliary blood pressure maintenance functions.

[0017] 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.

[0018] The term "pharmaceutical acceptable" means that the form of the compound must meet requirements for safety, stability, and suitability for formulation.

[0019] On one hand, embodiments of the present invention provide the use of an active peptide or a combination of polypeptides containing the active peptide in the preparation of a composition that helps maintain healthy blood pressure levels, wherein the amino acid sequence of the active peptide (polypeptide 1) has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0020] In an optional embodiment, the amino acid sequence of the active peptide is shown in SEQ ID NO:1.

[0021] In an optional embodiment, the polypeptide combination further includes: a polypeptide (polypeptide 2) with an amino acid sequence as shown in SEQ ID NO:2 (ALPQY) and / or a polypeptide (polypeptide 3) with an amino acid sequence as shown in SEQ ID NO:3 (AVPYPQRDMP).

[0022] In an optional embodiment, the polypeptide combination includes polypeptide 1 and polypeptide 2.

[0023] In an optional embodiment, the polypeptide combination includes polypeptide 1 and polypeptide 3.

[0024] In an optional embodiment, the polypeptide combination includes polypeptide 1, polypeptide 2, and polypeptide 3.

[0025] In optional embodiments, the composition may be a food or health product.

[0026] On the other hand, embodiments of the present invention provide the use of an active peptide or a combination of polypeptides containing the active peptide described in any of the foregoing embodiments in the preparation of angiotensin-converting enzyme inhibitors or compositions for treating diseases related to abnormal angiotensin-converting enzyme activity, wherein the amino acid sequence of the active peptide has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0027] In an optional embodiment, the diseases associated with abnormal angiotensin-converting enzyme activity include at least one of hypertension, heart failure, and diabetic nephropathy.

[0028] On the other hand, embodiments of the present invention provide the use of active peptides or polypeptide combinations containing said active peptides as described in any of the foregoing embodiments in the preparation of compositions that contribute to antioxidation, wherein the amino acid sequence of said active peptide has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0029] On the other hand, embodiments of the present invention provide the use of active peptides or polypeptide combinations containing said active peptides as described in any of the foregoing embodiments in the preparation of compositions that help maintain healthy blood pressure levels and help with antioxidation, wherein the amino acid sequence of said active peptide has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0030] In optional embodiments, the aforementioned at least 80% can be 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0031] Furthermore, embodiments of the present invention also provide a composition whose active ingredient contains the active peptides described in the foregoing embodiments.

[0032] In optional embodiments, the active ingredient further includes: a polypeptide (polypeptide 2) with an amino acid sequence as shown in SEQ ID NO:2 and / or a polypeptide (polypeptide 3) with an amino acid sequence as shown in SEQ ID NO:3.

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

[0034] In an optional embodiment, the active ingredient includes the active peptide and the polypeptide 3.

[0035] In an optional embodiment, the active ingredient includes: the active peptide, the polypeptide 2, and the polypeptide 3.

[0036] In an optional embodiment, the composition further includes excipients.

[0037] In optional embodiments, the excipients include any one or more of the following: carrier, pH adjuster, humectant, emulsifier, thickener, antioxidant, preservative, stabilizer, chelating agent, and colorant.

[0038] In optional embodiments, the carrier includes at least one of a food-grade carrier and a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is, but is not limited to, any pharmaceutically acceptable excipient, carrier, adjuvant, additive, surfactant, desiccant, or diluent. A pharmaceutically acceptable carrier can be solid, semi-solid, or liquid.

[0039] In optional embodiments, the composition may be in the form of powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0040] In optional embodiments, the composition may be applied to humans (e.g., infants, children, adolescents, the elderly, etc.) or animals (e.g., mammals).

[0041] In optional embodiments, the composition is an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0042] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0043] 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.

[0044] Example 1. I. Peptide Sequence (1) Solid-phase synthesis of peptides: First, the C-terminal amino acid (Trp) was immobilized on the resin, and then Ala, Gly, Ser, Pro, Tyr, Ala, Asp, and Leu were added sequentially. Before adding each 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 LDAYPSGAW.

[0045] (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.

[0046] (3) Characterization of the peptide: The purified peptide was characterized by LC-MS and HPLC. Figure 1 shows the mass spectrum of the peptide LDAYPSGAW. The experimental results show that the peptide LDAYPSGAW was successfully synthesized in Example 1.

[0047] Figure 2 shows that the purity of the polypeptide LDAYPSGAW is 95.12%.

[0048] Table 1 Peptide Sequences

[0049] Example 2: Determination of the antioxidant activity of peptides. ABTS reacts with potassium persulfate to generate relatively stable blue-green cationic free radicals ABTS•+, while antioxidants react with ABTS•+, causing the reaction system to decolorize. The antioxidant capacity of the substance is reflected by detecting the change in absorbance at the maximum absorbance wavelength of ABTS•+ free radicals, 734 nm. 50 μL of 40 μM sample solution was added to a 96-well microplate, followed by the rapid addition of 150 μL of ABTS•+ solution using a pipette. The reaction mixture was 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 scavenging rate of ABTS•+ by Trolox in the samples and standards was calculated, and the TEAC value of the samples was further calculated, expressed as μmol TE / μmol sample. The results are shown in Table 2. Experimental results showed that the TEAC value of the peptide LDAYPSGAW was 2.10 μmolTE / μmol, indicating that it has strong antioxidant capacity.

[0050] Table 2. TEAC values ​​of peptide LDAYPSGAW for scavenging ABTS free radicals

[0051] Example 3: Antioxidant Activity Determination of Peptide Combinations LDAYPSGAW+AVPYPQRDMP and ALPQY+LDAYPSGAW+AVPYPQRDMP The antioxidant activity (ABTS free radical scavenging rate) of peptides LDAYPSGAW, ALPYPQRDMP, and LDAYPSGAW+AVPYPQRDMP was determined. The experimental procedures of Example 2 were repeated, except that when determining the mixed peptide (LDAYPSGAW+AVPYPQRDMP), 25 μL of 40 μM peptide LDAYPSGAW and peptide ALPYPQRDMP sample solutions were added to each well of a 96-well microplate; and when determining the mixed peptide (ALPQY+LDAYPSGAW+AVPYPQRDMP), 16.7 μL of 40 μM peptide ALPQY, peptide LDAYPSGAW, and peptide ALPYPQRDMP sample solutions were added to each well of a 96-well microplate. The results are shown in Table 3.

[0052] Experimental results showed that the free radical scavenging rate of peptide LDAYPSGAW was 59.79%, that of peptide AVPYPQRDMP was 48.88%, that of peptide combination LDAYPSGAW+AVPYPQRDMP was 54.34%, and that of peptide combination ALPQY+LDAYPSGAW+AVPYPQRDMP was 55.39%.

[0053] Table 3. ABTS free radical scavenging rates of different peptides (combinations)

[0054] Example 4: 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 hippuryl-histyl-leucine (HHL) to produce hippuric acid (HA). 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 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%.

[0055] Figure 3 shows the ACE inhibitory activity of the nonapeptide LDAYPSGAW at different concentrations, IC50 50 The value is 62.37 μM.

[0056] Example 5: ACE inhibitory activity assay of peptide combination LDAYPSGAW+AVPYPQRDMP and peptide combination ALPQY+LDAYPSGAW+AVPYPQRDMP. The ACE inhibitory activities of peptides LDAYPSGAW, AVPYPQRDMP and LDAYPSGAW+AVPYPQRDMP were measured respectively. The experimental steps of Example 4 were repeated. When measuring single peptides, 20 μL of 500 μM sample was added to a 96-well microplate. When measuring mixed peptides (LDAYPSGAW+AVPYPQRDMP), 10 μL of 500 μM peptide LDAYPSGAW and peptide AVPYPQRDMP sample solutions were added to each well of a 96-well microplate. When measuring mixed peptides (ALPQY+LDAYPSGAW+AVPYPQRDMP), 6.7 μL of 500 μM peptide ALPQY, peptide LDAYPSGAW, and peptide AVPYPQRDMP sample solutions were added to each well of a 96-well microplate. The results are shown in Table 4.

[0057] The experimental results showed that the ACE inhibitory activity of peptide LDAYPSGAW was 70.24%, that of peptide AVPYPQRDMP was 32.89%, that of peptide combination LDAYPSGAW+AVPYPQRDMP was 55.75%, and that of peptide combination ALPQY+LDAYPSGAW+AVPYPQRDMP was 60.51%.

[0058] Table 4. ACE inhibitory activity of different peptides (combinations)

[0059] Example 6: 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 day / night cycle, room temperature controlled at 22 ± 3 °C, and relative humidity at 50%–70%. After 7 days of acclimatization, rats were divided into three groups based on their baseline blood pressure and body weight: a model group (saline), a positive control group (captopril, 10 mg / kg·bw / d), and a peptide LDAYPSGAW group (30 mg / kg·bw / d), 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 °C 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.

[0060] 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 LDAYPSGAW peptide group, blood pressure continued to decrease after gavage, with the largest decrease in systolic blood pressure at 6 hours, a reduction of 26.66 mmHg. These results demonstrate that the antihypertensive peptide of the present invention also has a good blood pressure-lowering effect in vivo.

[0061] 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. The use of an active peptide or a combination of peptides containing said active peptide in the preparation of a composition that helps maintain healthy blood pressure levels, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The polypeptide combination further includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO:2 and / or a polypeptide with an amino acid sequence as shown in SEQ ID NO:

3.

3. The use of an active peptide or a polypeptide combination containing said active peptide as described in claim 1 or 2 in the preparation of angiotensin-converting enzyme inhibitors or compositions for treating diseases related to abnormal angiotensin-converting enzyme activity, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

4. The application according to claim 3, characterized in that, The diseases associated with abnormal angiotensin-converting enzyme activity include at least one of hypertension, heart failure, and diabetic nephropathy.

5. The use of an active peptide or a polypeptide combination containing said active peptide as described in claim 1 or 2 in the preparation of a composition that contributes to antioxidant activity, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

6. The use of an active peptide or a polypeptide combination containing said active peptide as described in claim 1 or 2 in the preparation of a composition that helps maintain healthy blood pressure levels and has antioxidant properties, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

7. A composition, characterized in that, Its active ingredient contains active peptides, the amino acid sequence of which is shown in SEQ ID NO:

1.

8. The composition according to claim 7, characterized in that, The active ingredient further includes: a polypeptide with an amino acid sequence as shown in SEQ ID NO:2 and / or a polypeptide with an amino acid sequence as shown in SEQ ID NO:

3.

9. The composition according to claim 8, characterized in that, The composition also includes excipients.

10. The composition according to claim 9, characterized in that, The excipients include any one or more of the following: carrier, pH adjuster, humectant, emulsifier, thickener, antioxidant, preservative, stabilizer, chelating agent, and colorant.

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