A hexapeptide DE6 with ACE inhibitory activity and preparation method and application thereof

Abstract

The application discloses a hexapeptide DE6 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 hexapeptide DE6 is DGVFPE, and the hexapeptide DE6 can be prepared by a solid-phase synthesis method and an enzymatic hydrolysis method. The hexapeptide DE6 is identified from a spirulina platensis protease hydrolysate, has potential interaction with ACE, and has an ACE inhibition rate of 89.81% at a concentration of 0.1 mg / mL. Compared with VPP and IPP (the ACE inhibition rates of VPP and IPP are 82.32% and 78.00% respectively at the same concentration), the ACE inhibitory activity is extremely significantly increased (p<0.001), and the hexapeptide DE6 can be used for preparing antihypertensive drugs or antihypertensive functional foods.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of small molecule peptides, and particularly relates to a hexapeptide DE6 with ACE inhibitory activity and a preparation method and application thereof. BACKGROUND

[0002] Hypertension is one of the main risk factors for cardiovascular problems and death with the highest incidence worldwide. In the blood pressure regulation mechanism, the renin-angiotensin system occupies a core position. Angiotensin-converting enzyme (ACE) becomes an important driving factor for elevated blood pressure by catalyzing the conversion of angiotensin I to potent vasoconstrictor angiotensin II and simultaneously degrading the vasodilator bradykinin. Therefore, specific inhibition of ACE activity has become a key target for clinical intervention of hypertension.

[0003] ACE, as a zinc-containing dipeptidyl peptidase, has irreplaceable clinical value in the field of hypertension treatment. Although chemical synthesis of ACE inhibitors such as captopril has been widely used, it may induce some adverse reactions. Under this background, screening of ACE inhibitors with high efficiency and low toxicity from natural products has become a research hotspot. Among them, food-derived active peptides obtained by proteolysis have unique potential in blood pressure regulation and cardiovascular protection due to their good biocompatibility, low metabolic burden and high safety threshold. Valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) are typical ACE inhibitory peptides and are often used as reference substances to evaluate the activity of new ACE inhibitory peptides. 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 ACE inhibitory activity significantly better than that of VPP and IPP; the second is the screening efficiency bottleneck, and traditional screening strategies rely on repeated separation, purification and activity verification of proteolysis 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.

[0004] Marine organisms have unique living environments, and their protein amino acid sequences often have special structures that land organisms do not have, making them an excellent source for discovering novel bioactive peptides. Spirulina platensis belongs to Cyanophyta, Cyanophyceae, Oscillatoriaceae and Spirulina, and has the characteristics of high protein content (60-70%) and complete essential amino acid composition. Spirulina platensis protein is rich in aspartic acid and glutamic acid, and is an excellent raw material for preparing various functional bioactive peptides. Systematic exploration of Spirulina platensis protein and mining of peptides with specific ACE inhibitory activity from it is an effective way to realize the high-value utilization of this marine resource. SUMMARY

[0005] The application aims to provide a small molecule peptide with novel sequence structure and strong ACE inhibitory activity identified from a protein enzymatic hydrolysate of Spirulina platensis, and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned target, the application adopts the following technical scheme:

[0007] A hexapeptide DE6 with ACE inhibitory activity, the amino acid sequence of the hexapeptide DE6 is DGVFPE.

[0008] The preparation method of the aforementioned hexapeptide DE6 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 hexapeptide DE6.

[0009] The preparation method of the aforementioned hexapeptide DE6 with ACE inhibitory activity adopts an enzymatic hydrolysis method, and specifically as follows: (1) the Spirulina platensis is put into water, and after being heated to 40 DEG C, yeast is added, and enzymatic hydrolysis is carried out at the temperature for 2 hours; (2) continue to heat, after being heated to 48 DEG C, alkaline protease and neutral protease are added, and enzymatic hydrolysis is carried out at the temperature for 2 hours; (3) continue to heat, after being heated to 58 DEG C, papain is added, and enzymatic hydrolysis is carried out at the temperature for 3 hours; (4) continue to heat, after being heated to 85 DEG C, keep for 30 minutes; (5) the enzymatic hydrolysate is left to precipitate, the supernatant is taken out and centrifuged, and the supernatant after centrifugation is spray-dried to obtain a Spirulina platensis protein peptide, which contains the hexapeptide DE6; wherein the mass ratio of the use amount of the Spirulina platensis, the yeast, the alkaline protease, the neutral protease and the papain is 100:1:3:2:2.

[0010] The aforementioned hexapeptide DE6 with ACE inhibitory activity is applied to preparation of a blood pressure lowering drug or a blood pressure lowering functional food.

[0011] The hexapeptide DE6 provided by the application is identified from a protein enzymatic hydrolysate of Spirulina platensis, and it is found through molecular docking that the hexapeptide DE6 has potential interaction with ACE, and it is found through in-vitro ACE inhibitory activity test that the ACE inhibition rate of the hexapeptide DE6 is 89.81%, which is extremely significantly increased (p<0.001) compared with the classic ACE inhibitory peptides VPP and IPP (the ACE inhibition rates are 82.32% and 78.00% respectively under the same concentration), and the hexapeptide DE6 can be used for preparation of a blood pressure lowering drug or a blood pressure lowering functional food. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is a schematic diagram of the binding mode of the hexapeptide DE6 and ACE;

[0013] Figure 2The graph shows the ACE inhibition rate of hexapeptides DE6, VPP, and IPP. *** indicates p < 0.001. Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0015] I. Preparation of Spirulina platensis protein peptides

[0016] The method for preparing Spirulina platensis protein peptides specifically includes the following steps:

[0017] (1) Put 100g of Spirulina platensis into 1000mL of water, heat to 40℃ and add 1g of yeast, and enzymatically hydrolyze at this temperature for 2h;

[0018] (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.

[0019] (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours.

[0020] (4) Continue to heat up to 85℃ and hold for 30 minutes;

[0021] (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 Spirulina platensis protein peptide.

[0022] II. Obtaining the polypeptide sequence from Spirulina platensis protein peptides

[0023] The previously obtained Spirulina platensis protein peptides were analyzed by LC-MS / MS mass spectrometry. The mass spectrometry results were analyzed using mass spectrometry analysis software to obtain several peptide sequences.

[0024] The LC-MS / MS determination conditions are as follows:

[0025] (1) Liquid chromatography method: The chromatographic column is C18, 3μm, 250mm×75μm (Eksigent). The mobile phase A is water containing 0.1% formic acid and the mobile phase B is acetonitrile containing 0.1% formic acid. The flow rate is 300nL / min and the injection volume is 1μL. 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%.

[0026] (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.

[0027] III. Screening peak area ≥ 1.00 × 10 8 Active peptides with ≤6 amino acids

[0028] From the several polypeptide sequences obtained above, 22 peak areas ≥ 1.00 × 10⁻⁶ were finally selected. 8 The screening results for bioactive peptides with ≤6 amino acid counts are shown in Tables 1-1 and 1-2.

[0029] Table 1-1 High-abundance bioactive peptides in Spirulina platensis protein peptides (Part 1)

[0030]

[0031] Table 1-2 High-abundance bioactive peptides in Spirulina platensis protein peptides (II)

[0032]

[0033] IV. Screening for bioactive peptides with strong ACE binding ability

[0034] Using Discovery Studio software, the active peptide sequences in Tables 1-1 and 1-2 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.

[0035] 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.

[0036] The molecular docking results of the above 22 active peptides with ACE are shown in Tables 2-1 and 2-2.

[0037] Table 2-1 Predicted results of interactions between bioactive peptides and ACE (Part 1)

[0038]

[0039] Table 2-2 Predicted results of interactions between bioactive peptides and ACE (Part II)

[0040]

[0041] V. Molecular docking analysis

[0042] Among the 22 bioactive peptides mentioned above, DGVFPE (denoted as hexapeptide DE6, SEQ ID NO: 5) had the highest docking score, at 103.2490 kcal / mol. Therefore, DGVFPE (hexapeptide DE6) was selected for further molecular docking analysis.

[0043] Analysis revealed that the binding mode of hexapeptide DE6 to ACE is as follows: Figure 1 As shown, the molecular docking is as follows:

[0044] The hexapeptide DE6 forms two salt bridge interactions, seven HH bond interactions, six CH bond interactions, and four electrostatic interactions with ACE. Fifteen amino acid residues are involved in the interaction between the hexapeptide DE6 and ACE.

[0045] VI. Evaluation of the ACE inhibitory activity of hexapeptide DE6

[0046] 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 hexapeptide DE6 (purity >90%).

[0047] The hexapeptide DE6, VPP (positive control), or IPP (positive control) obtained by solid-phase synthesis were dissolved in ultrapure water to prepare DE6 solution, VPP solution, and IPP solution with a concentration of 0.1 mg / mL, respectively.

[0048] Sample group: Take 10 μL of DE6 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.

[0049] 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.

[0050] 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.

[0051] The formula for calculating the ACE inhibition rate is as follows:

[0052]

[0053] Where A represents the hippuric acid content in the control group and B represents the hippuric acid content in the sample group.

[0054] Calculations showed that at a concentration of 0.1 mg / mL, the ACE inhibition rates of hexapeptide DE6, VPP, and IPP were 89.81±2.3%, 82.32±1.2%, and 78.00±1.4%, respectively. (See the comparison below.) Figure 2 .

[0055] Depend on Figure 2 It can be seen that, at the same concentration, the ACE inhibition rate of hexapeptide DE6 was significantly increased compared with the positive controls VPP and IPP (p<0.001).

[0056] In summary, hexapeptide DE6 has stronger ACE inhibitory activity than VPP and IPP, and can be used to prepare antihypertensive drugs or functional foods that help lower blood pressure.

[0057] 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 hexapeptide DE6 with ACE inhibitory activity, characterized in that, The amino acid sequence of the hexapeptide DE6 is DGVFPE.

2. The method for preparing the hexapeptide DE6 with ACE inhibitory activity as described in claim 1, characterized in that, Hexapeptide DE6 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 hexapeptide DE6 with ACE inhibitory activity as described in claim 1, characterized in that, The enzymatic hydrolysis method is used, as detailed below: (1) Place Spirulina platensis in water, heat to 40°C, add yeast, and enzymatically hydrolyze 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 Spirulina platensis protein peptide, which contains hexapeptide DE6; The ratio of Spirulina platensis, yeast, alkaline protease, neutral protease and papain by mass is 100:1:3:2:

2.

4. The use of the hexapeptide DE6 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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