A pentapeptide RA5 with ACE inhibitory activity, its preparation method and application

Abstract

This invention discloses a pentapeptide RA5 with ACE inhibitory activity, its preparation method, and its applications, belonging to the field of small molecule peptide technology. The amino acid sequence of the pentapeptide RA5 is RVVVA, and it can be prepared by solid-phase synthesis and enzymatic hydrolysis. The pentapeptide RA5 was identified from the protein hydrolysate of *Euphorbia milii* and exhibits potential interaction with ACE. At a concentration of 0.1 mg / mL, the ACE inhibition rate is 86.88%, which is significantly increased (p<0.01) and extremely significantly increased (p<0.001) compared to VPP and IPP (at the same concentration, the ACE inhibition rates are 82.32% and 78.00%, respectively). The pentapeptide RA5 has a stronger antihypertensive function than VPP and IPP and can be used to prepare antihypertensive drugs or functional foods that assist in lowering blood pressure.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of small molecule peptides, in particular to a pentapeptide RA5 with ACE inhibitory activity and a preparation method and application thereof. BACKGROUND

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

[0003] Food-derived ACE inhibitory peptides are praised as a new generation of functional antihypertensive ingredients due to their safe source, easy absorption and low side effects. Among them, valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) derived from milk proteins are the most in-depth studied and are the most representative ACE inhibitory peptides, which can be used as positive controls for evaluating the activity of new ACE inhibitory peptides.

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

[0005] Eucheuma (Latin name: Eucheuma denticulatum) is a major economic seaweed rich in protein, but its resources have not been maximally utilized. Precise mining of high-activity ACE inhibitory peptides from Eucheuma proteolytic liquid will greatly enhance the added value of Eucheuma. SUMMARY

[0006] The purpose of the present application is to provide a small molecule peptide with novel sequence structure, strong ACE inhibitory activity, identified from Eucheuma proteolytic liquid, as well as a preparation method and application thereof.

[0007] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows:

[0008] A pentapeptide RA5 with ACE inhibitory activity, the amino acid sequence of the pentapeptide RA5 is RVVVA.

[0009] The preparation method of the aforementioned five-peptide RA5 with ACE inhibitory activity adopts a solid-phase synthesis method, uses Fmoc-protected amino acids as raw materials, and selects polystyrene resin as a solid-phase carrier to synthesize the five-peptide RA5.

[0010] The preparation method of the aforementioned five-peptide RA5 with ACE inhibitory activity adopts an enzymatic hydrolysis method, and specifically includes the following steps: (1) placing the eucheuma into water, adding yeast after the temperature is increased to 40 DEG C, and performing enzymatic hydrolysis at the temperature for 2 hours; (2) continuously increasing the temperature, adding alkaline protease and neutral protease after the temperature is increased to 48 DEG C, and performing enzymatic hydrolysis at the temperature for 2 hours; (3) continuously increasing the temperature, adding papain after the temperature is increased to 58 DEG C, and performing enzymatic hydrolysis at the temperature for 3 hours; (4) continuously increasing the temperature, keeping the temperature at 85 DEG C for 30 minutes; and (5) allowing the enzymatic hydrolysate to stand and precipitate, centrifuging the supernatant, and performing spray drying on the centrifuged supernatant to obtain eucheuma protein peptides containing the five-peptide RA5; wherein the mass ratio of the eucheuma, the yeast, the alkaline protease, the neutral protease, and the papain is 100:1:3:2:2.

[0011] The aforementioned five-peptide RA5 with ACE inhibitory activity is applied to the preparation of a blood pressure-lowering drug or a blood pressure-lowering functional food.

[0012] The five-peptide RA5 provided by the application is identified from eucheuma protein enzymatic hydrolysate, and is found to have potential interaction with ACE through molecular docking. The ACE inhibition rate of the five-peptide RA5 is 86.88%, which is significantly increased (p<0.01) and extremely significantly increased (p<0.001) compared with the ACE inhibition rates of the classical ACE inhibitory peptides VPP and IPP (the ACE inhibition rates are 82.32% and 78.00% respectively under the same concentration). The ACE inhibitory activity of the five-peptide RA5 is stronger, and the five-peptide RA5 has stronger blood pressure-lowering function than VPP and IPP, and can be applied to the preparation of a blood pressure-lowering drug or a blood pressure-lowering functional food. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a schematic diagram of the binding mode of the five-peptide RA5 and ACE;

[0014] Figure 2 is a graph of the ACE inhibition rate detection results of the five-peptide RA5, VPP, and IPP. DETAILED DESCRIPTION

[0015] The application is specifically introduced below in combination with the drawings and specific embodiments.

[0016] I. Preparation of eucheuma protein peptides

[0017] The method for preparing the eucheuma protein peptides specifically includes the following steps:

[0018] (1) Put 100 g of Eucheuma into 1000 mL of water, heat to 40℃, then add 1 g of yeast, and enzymatically hydrolyze at this temperature for 2 h;

[0019] (2) Continue to heat, heat to 48℃, then add 3 g of alkaline protease and 2 g of neutral protease, and enzymatically hydrolyze at this temperature for 2 h;

[0020] (3) Continue to heat, heat to 58℃, then add 2 g of papain, and enzymatically hydrolyze at this temperature for 3 h;

[0021] (4) Continue to heat, heat to 85℃, and keep for 30 min;

[0022] (5) Let the enzymatic hydrolysate stand and precipitate, take the supernatant and centrifuge at 8000 rpm for 30 min, and spray dry the supernatant after centrifugation to obtain a powdery product, which is Eucheuma protein peptide.

[0023] II. Obtain polypeptide sequences in Eucheuma protein peptide

[0024] The Eucheuma protein peptide obtained above is subjected to mass spectrometry determination by LC-MS / MS, and the results of mass spectrometry determination are analyzed by mass spectrometry analysis software to obtain a plurality of polypeptide sequences.

[0025] The LC-MS / MS determination conditions are as follows:

[0026] (1) Liquid phase method: the chromatographic column is C18, 3 μm, 250 mm x 75 μm (Eksigent), the mobile phase A is water, 0.1% formic acid, the mobile phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nL / min, the injection volume is 1 μL, the chromatographic gradient is 70 min, and the specific elution gradient is: 0-55 min, A phase from 95% uniformly reduced to 65%; 55-63 min, A phase from 65% uniformly reduced to 50%, 63-64 min, A phase from 50% uniformly reduced to 0; 64-70 min, keep 0% A phase.

[0027] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, first order resolution is 120000, AGC is set to 310, scan range is 110-2000 m / z. MIPS mode is peptide, selecting valence state 1-6, second order resolution is 17500, separation window is 1.6 m / z.

[0028] III. Screen active peptides with peak area >1.00 x 10 8 and amino acid number ≤6

[0029] From the plurality of polypeptide sequences obtained above, 20 polypeptide sequences with peak area >1.00 x 108 The screening results for active peptides with ≤6 amino acids are shown in Table 1.

[0030] Table 1. Peak area of ​​protein peptides from *Euphorbia milii* > 1.00 × 10⁻⁶ 8 Active peptides with ≤6 amino acids

[0031]

[0032] IV. Screening for bioactive peptides with strong ACE binding ability

[0033] 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.

[0034] 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.

[0035] The molecular docking results of the above 20 bioactive peptides with ACE are shown in Table 2.

[0036] Table 2. Predicted Interactions Between 20 Bioactive Peptides and ACE

[0037]

[0038] V. Molecular docking analysis

[0039] 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.

[0040] 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:

[0041] 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.

[0042] VI. Evaluation of the ACE inhibitory activity of the pentapeptide RA5

[0043] 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%).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The formula for calculating the ACE inhibition rate is as follows:

[0049]

[0050] Where A represents the hippuric acid content in the control group and B represents the hippuric acid content in the sample group.

[0051] 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 .

[0052] 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.

[0053] 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.

[0054] 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 with ACE inhibitory activity, characterized in that, The amino acid sequence of the pentapeptide RA5 is RVVVA.

2. The method for preparing the pentapeptide RA5 with ACE inhibitory activity as described in claim 1, characterized in that, The pentapeptide RA5 was synthesized in a solid-phase manner using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier.

3. The method for preparing the pentapeptide RA5 with ACE inhibitory activity as described in claim 1, characterized in that, The enzymatic hydrolysis method is used, as detailed below: (1) Put the seaweed into water, heat it to 40°C, add yeast, and enzymatically hydrolyze it at this temperature for 2 hours; (2) Continue heating until the temperature reaches 48°C. Then add alkaline protease and neutral protease and hydrolyze at this temperature for 2 hours. (3) Continue to heat up to 58°C and add papain. At this temperature, enzymatically hydrolyze for 3 hours. (4) Continue to heat up to 85℃ and hold for 30 minutes; (5) Let the enzymatic hydrolysate stand to precipitate, take the supernatant and centrifuge, and spray dry the supernatant after centrifugation to obtain the Kirinia protein peptide, which contains the pentapeptide RA5; The ratio of the amounts of Euphorbia lactea, yeast, alkaline protease, neutral protease, and papain, by mass, is 100:1:3:2:

2.

4. The use of the pentapeptide RA5 with ACE inhibitory activity as described in claim 1 in the preparation of antihypertensive drugs or functional foods that assist in lowering blood pressure.

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