Pentapeptide RA5 with ACE (angiotensin converting enzyme) inhibitory activity as well as preparation method and application of pentapeptide RA5

Abstract

The invention discloses a pentapeptide RA5 with ACE inhibitory activity and a preparation method and application thereof, and belongs to the technical field of small molecule peptides. The amino acid sequence of the pentapeptide RA5 is RVVVA, and the pentapeptide RA5 can be prepared through a solid-phase synthesis method and an enzymolysis method. The pentapeptide RA5 is identified from eucheuma proteolysis liquid and has potential interaction with ACE, under the concentration of 0.1 mg / mL, the ACE inhibition rate is 86.88%, and compared with VPP and IPP (under the same concentration, the ACE inhibition rates are 82.32% and 78.00% respectively), the ACE inhibition rates are remarkably increased (plt; 0.01) and an extremely significantly increased (plt; the pentapeptide RA5 has a higher blood pressure lowering function than VPP and IPP, and can be used for preparing blood pressure lowering drugs or functional foods assisting in lowering blood pressure.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to a pentapeptide RA5 with ACE inhibitory activity, its preparation method, and its applications. Background Technology

[0002] Hypertension is one of the leading causes of cardiovascular problems and death worldwide, with the highest incidence rate. The renin-angiotensin system plays a central role in blood pressure regulation, and angiotensin-converting enzyme (ACE) is a key rate-limiting enzyme within it. Therefore, ACE has become a core drug target for clinical treatment of hypertension. Chemically synthesized ACE inhibitors (such as captopril) are widely used clinically with proven efficacy, but long-term use can cause some side effects. Therefore, researchers need to continuously explore safer and gentler alternatives derived from natural foods.

[0003] Dietary ACE inhibitory peptides are hailed as a new generation of functional antihypertensive ingredients due to their safe origin, easy absorption, and low side effects. Among them, valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) derived from milk proteins have been studied most extensively and are landmark ACE inhibitory peptides that can be used as positive controls for evaluating the activity of novel ACE inhibitory peptides.

[0004] Currently, the research and development of food-derived ACE inhibitory peptides faces two major bottlenecks: the first is the activity bottleneck, as there are few active peptides with significantly better ACE inhibitory activity than VPP and IPP; the second is the screening efficiency bottleneck, as traditional screening strategies rely on repeated separation, purification and activity verification of protease hydrolysates, which is time-consuming, labor-intensive and prone to missing key active peptides with low content but high activity, making it difficult to achieve targeted discovery of new sequence structures, low abundance and high activity peptides.

[0005] Eucheuma denticulatum, a major economic seaweed, is rich in protein, yet its resources have not been fully utilized. Precisely extracting highly active ACE-inhibiting peptides from Eucheuma denticulatum protease hydrolysate will greatly enhance its added value. Summary of the Invention

[0006] The purpose of this invention is to provide a small molecule peptide with a novel sequence structure and strong ACE inhibitory activity, which is identified from the protein hydrolysate of Euphorbia milii, as well as the preparation method and application of the small molecule peptide.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A pentapeptide RA5 with ACE inhibitory activity, wherein the amino acid sequence of the pentapeptide RA5 is RVVVA.

[0008] The aforementioned method for preparing the pentapeptide RA5 with ACE inhibitory activity employs a solid-phase synthesis method, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize the pentapeptide RA5 in a solid phase.

[0009] The aforementioned method for preparing the pentapeptide RA5 with ACE inhibitory activity is an enzymatic hydrolysis method, as follows: (1) Place the Euphorbia lactea in water, heat it to 40°C, add yeast, and hydrolyze it at this temperature for 2 hours; (2) Continue to heat it to 48°C, add alkaline protease and neutral protease, and hydrolyze it at this temperature for 2 hours; (3) Continue to heat it to 58°C, add papain, and hydrolyze it at this temperature for 3 hours; (4) Continue to heat it to 85°C and maintain it for 30 minutes; (5) Let the hydrolysate stand to precipitate, take the supernatant and centrifuge it, and spray dry the supernatant after centrifugation to obtain Euphorbia lactea protein peptide, which contains the pentapeptide RA5; wherein, by mass, the ratio of Euphorbia lactea, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2.

[0010] The aforementioned pentapeptide RA5 with ACE inhibitory activity is used in the preparation of antihypertensive drugs or functional foods that help lower blood pressure.

[0011] The advantages of this invention are as follows: The pentapeptide RA5 provided by this invention was identified from the protein hydrolysate of Euphorbia milii. Molecular docking revealed that pentapeptide RA5 has a potential interaction with ACE. In vitro ACE inhibitory activity tests showed that the ACE inhibition rate of pentapeptide RA5 was 86.88%, which is significantly increased (p<0.01) and extremely significantly increased (p<0.001) compared with the classic ACE inhibitory peptides VPP and IPP (at the same concentration, the ACE inhibition rates are 82.32% and 78.00%, respectively). Pentapeptide RA5 has stronger ACE inhibitory activity and a stronger antihypertensive function than VPP and IPP, and can be used to prepare antihypertensive drugs or functional foods that help lower blood pressure. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the binding mode between the pentapeptide RA5 and ACE; Figure 2 This is a graph showing the ACE inhibition rate of the pentapeptides RA5, VPP, and IPP. Detailed Implementation

[0013] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0014] I. Preparation of Euphorbia lactea protein peptides The method for preparing Euphorbia lactea protein peptides specifically includes the following steps: (1) Put 100g of Euphorbia milii into 1000mL of water, heat it to 40℃ and add 1g of yeast, and enzymatically hydrolyze it at this temperature for 2h; (2) Continue heating until the temperature reaches 48°C. Then add 3g of alkaline protease and 2g of neutral protease and hydrolyze at this temperature for 2 hours. (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours. (4) Continue to heat up to 85℃ and hold for 30 minutes; (5) Let the enzyme hydrolysate stand to precipitate, take the supernatant and centrifuge at 8000 rpm for 30 min, and spray dry the supernatant after centrifugation to obtain a powdered product, which is the Kirin vegetable protein peptide.

[0015] II. Obtaining the polypeptide sequence from the protein peptide of *Euphorbia milii*. The obtained *Euphorbia lactea* protein peptides were analyzed by LC-MS / MS, and the results were analyzed using mass spectrometry software to obtain several polypeptide sequences.

[0016] The LC-MS / MS determination conditions are as follows: (1) Liquid chromatography method: The chromatographic column is C18, 3μm, 250mm×75μm (Eksigent). The mobile phase A is water, 0.1% formic acid, and the mobile phase B is acetonitrile, 0.1% formic acid. The flow rate is 300nL / min, the injection volume is 1μL, and the chromatographic gradient is 70min. The specific elution gradient is as follows: 0-55min, phase A decreases uniformly from 95% to 65%; 55-63min, phase A decreases uniformly from 65% to 50%; 63-64min, phase A decreases uniformly from 50% to 0%; 64-70min, phase A is maintained at 0%.

[0017] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, primary resolution of 120,000, AGC set to 310, scan range of 110-2000 m / z. MIPS mode is peptide, valence state 1-6 is selected, secondary resolution is 17,500, separation window is 1.6 m / z.

[0018] III. Screening peak area > 1.00 × 10 8 Active peptides with ≤6 amino acids From the several polypeptide sequences obtained above, 20 sequences with peak areas > 1.00 × 10⁻⁶ were finally selected. 8 The screening results for active peptides with ≤6 amino acids are shown in Table 1.

[0019] Table 1. Peak area of ​​protein peptides from *Euphorbia milii* > 1.00 × 10⁻⁶ 8 Active peptides with ≤6 amino acids

[0020] IV. Screening for bioactive peptides with strong ACE binding ability Using Discovery Studio software, the active peptide sequences in Table 1 were molecularly docked with ACE. Before docking, the 2D structure of the active peptides was converted into a 3D structure by minimizing energy, and active peptide sequences with strong binding ability to ACE were screened.

[0021] The 3D structure of ACE can be downloaded from the RCSB protein database (PDB ID: 1O8A). The docking result is expressed as a docking score; the higher the docking score, the stronger the binding ability of the active peptide to ACE, and the more likely it is to inhibit ACE activity.

[0022] The molecular docking results of the above 20 bioactive peptides with ACE are shown in Table 2.

[0023] Table 2. Predicted Interactions Between 20 Bioactive Peptides and ACE

[0024] V. Molecular docking analysis Among the 20 bioactive peptides listed in Table 2, RVVVA (denoted as pentapeptide RA5, SEQ ID NO: 5) had the highest docking score, at 92.3779 kcal / mol. Therefore, RVVVA (pentapeptide RA5) was selected for further molecular docking analysis.

[0025] Analysis revealed that the binding mode of the pentapeptide RA5 to ACE is as follows: Figure 1 As shown, the molecular docking is as follows: The pentapeptide RA5 forms one salt bridge interaction, 14 HH bond interactions, 4 CH bond interactions and 5 electrostatic interactions with ACE. Thirteen amino acid residues are involved in the interaction between the pentapeptide RA5 and ACE.

[0026] VI. Evaluation of the ACE inhibitory activity of the pentapeptide RA5 A solid-phase synthesis method was adopted, using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier to synthesize the pentapeptide RA5 (purity >90%).

[0027] The pentapeptide RA5, VPP (positive control), or IPP (positive control) obtained by solid-phase synthesis was dissolved in ultrapure water to prepare RA5 solution, VPP solution, and IPP solution with a concentration of 0.1 mg / mL, respectively.

[0028] Sample group: Take 10 μL of RA5 solution, VPP solution or IPP solution, mix with 30 μL of malourethramide leucine solution (4 mM), incubate at 37℃ for 3 min, then add 20 μL of ACE solution (0.1 U / mL), incubate at 37℃ for 30 min, and finally add 20 μL of hydrochloric acid (1 M) to terminate the reaction.

[0029] Control group: Take 10 μL of PBS buffer and mix it with 30 μL of kilotinib histidine leucine solution (4 mM). Incubate at 37°C for 3 min, then add 20 μL of ACE solution (0.1 U / mL) and incubate at 37°C for 30 min. Finally, add 20 μL of hydrochloric acid (1 M) to terminate the reaction.

[0030] The concentration of hippuric acid in the reaction system was determined by high-performance liquid chromatography (HPLC). A C18 column (5 μm, 4.6 mm × 250 mm) was used. The mobile phase A was 0.4% (v / v) aqueous acetic acid, and the mobile phase B was HPLC-grade methanol. Isocratic elution conditions were A:B = 67:33 (v / v), flow rate 1.0 mL / min at 25 °C, injection volume 20 μL per sample, and run time 30 min. The column was equilibrated with the mobile phase for at least 30 min before injection. The detection wavelength was 254 nm.

[0031] The formula for calculating the ACE inhibition rate is as follows:

[0032] Where A represents the hippuric acid content in the control group and B represents the hippuric acid content in the sample group.

[0033] Calculations showed that at a concentration of 0.1 mg / mL, the ACE inhibition rates of pentapeptides RA5, VPP, and IPP were 86.88±3.5%, 82.32±1.2%, and 78.00±1.4%, respectively. (See the comparison below.) Figure 2 .

[0034] Depend on Figure 2 It can be seen that, at the same concentration, compared with the positive controls VPP and IPP, the ACE inhibition rate of pentapeptide RA5 was significantly increased (p<0.01) and extremely significantly increased (p<0.001), respectively.

[0035] In summary, pentapeptide RA5 has a stronger blood pressure-lowering function than VPP and IPP, and can be used to prepare blood pressure-lowering drugs or functional foods that can help lower blood pressure.

[0036] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the protection scope of this invention.

Claims

1. A pentapeptide RA5 having ACE inhibitory activity, characterized in that, The amino acid sequence of the pentapeptide RA5 is RVVVA.

2. A process for the preparation of the pentapeptide RA5 having ACE inhibitory activity as claimed in claim 1, characterized in that, The pentapeptide RA5 is synthesized by solid phase synthesis method, using Fmoc-protected amino acids as raw materials and polystyrene resin as solid phase carrier.

3. A process for the preparation of the pentapeptide RA5 having ACE inhibitory activity as claimed in claim 1, characterized in that, The enzymatic method is as follows: (1) Put the Eucheuma into water, and then add yeast after heating to 40℃, and enzymatically hydrolyze for 2 hours at the temperature; (2) Continue to heat, and then add alkaline protease and neutral protease after heating to 48℃, and enzymatically hydrolyze for 2 hours at the temperature; (3) Continue to heat, and then add papain after heating to 58℃, and enzymatically hydrolyze for 3 hours at the temperature; (4) Continue to heat, and then keep the temperature at 85℃ for 30 minutes; (5) Let the enzymatic hydrolysate stand and precipitate, take the supernatant and centrifuge, and then spray dry the centrifuged supernatant to obtain Eucheuma protein peptide, which contains the pentapeptide RA5; The mass ratio of Eucheuma, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:

2.

4. Application of the pentapeptide RA5 with ACE inhibitory activity in claim 1 in the preparation of a blood pressure lowering drug or a food with auxiliary blood pressure lowering function.

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