A pentapeptide ES5 with ACE inhibitory activity and preparation method and application thereof

Abstract

The application discloses a pentapeptide ES5 with ACE inhibitory activity and a preparation method and application thereof, and belongs to the technical field of biotechnology.The amino acid sequence of the pentapeptide ES5 is ERQFS, and the pentapeptide ES5 can be prepared by a solid-phase synthesis method and an enzymatic hydrolysis method.The pentapeptide ES5 is identified from a protease hydrolysate of red algae (real Gracilaria) agar residue, has potential interaction with ACE, and has an ACE inhibition rate of 90.84% at a concentration of 0.1 mg / mL; compared with VPP and IPP (the ACE inhibition rates are 82.32% and 78.00% respectively at the same concentration), the ACE inhibitory activity is extremely significantly increased (p<0.001); and the pentapeptide ES5 can be used for preparing antihypertensive drugs or antihypertensive functional food.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to a pentapeptide ES5 with ACE inhibitory activity, its preparation method, and its applications. Background Technology

[0002] Hypertension, a globally prevalent chronic metabolic disease, faces increasingly serious challenges in its prevention and treatment. In the renin-angiotensin system, which regulates blood pressure, angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I into the pressor substance angiotensin II and degrades the antihypertensive substance bradykinin, forming a dual pathway for blood pressure regulation. Therefore, ACE has become a key target for hypertension intervention. While currently widely used ACE inhibitors (such as captopril) effectively control blood pressure, they have adverse reactions such as dry cough and edema. Studies have shown that ACE inhibitors cause cough in 5.3%-10.1% of cases. Therefore, researchers are shifting their focus to the development of dietary ACE inhibitory peptides.

[0003] ACE-inhibiting peptides derived from proteins of different origins exhibit significant structural differences after enzymatic hydrolysis. Wu Jingna et al. reported in "Screening and Stability Evaluation of Angiotensin-Converting Enzyme Inhibiting Peptides from *Nanochloropsis oculata*" that the nonapeptide LVLLFLFGE, present in the enzymatic hydrolysis products of *Nanochloropsis oculata*, can interact with ACE through hydrogen bonds and possesses ACE-inhibiting activity. Lin et al. reported in "Identification of novel ACE inhibitory peptides from *Nanochloropsis oculata* through peptidomics, in silico screening and molecular docking" that the ACE-inhibiting peptide GGPPFTVF, isolated from the enzymatic hydrolysis products of *Nanochloropsis oculata*, possesses excellent ACE-inhibiting activity. Xiang et al. reported in "From garlic proteins to bioactive peptides: A computer-aided screening for saltiness-enhancers and angiotensin I-converting enzyme inhibitors with mechanical and kinetic analysis" that the heptapeptide ASTCMAR, derived from garlic enzymatic hydrolysis, possesses ACE-inhibiting activity. In their paper "From Sea to Lab: Angiotensin I-Converting Enzyme Inhibition by Marine Peptides-Mechanisms and Applications", Jo et al. compiled and analyzed ACE inhibitory peptide sequences from fish, mollusks, algae, and sponges. These ACE inhibitory peptides differed significantly in sequence from the ACE inhibitory peptides involved in this invention.

[0004] Valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) are typical ACE inhibitory peptides and are often used as references for evaluating the activity of novel ACE inhibitory peptides. Research and development of food-derived ACE inhibitory peptides face two major bottlenecks: first, the activity bottleneck, as there are relatively few active peptides with significantly superior ACE inhibitory activity compared to VPP and IPP; second, the screening efficiency bottleneck, as traditional screening strategies rely on repeated separation, purification, and activity verification of proteolytic products, which is time-consuming, labor-intensive, and prone to missing key active peptides with low abundance but high activity, making it difficult to achieve targeted discovery of novel sequence structures and low-abundance, high-activity peptides.

[0005] Marine organisms, due to their unique habitats, often possess proteins with amino acid sequences that are not found in terrestrial organisms, making them an excellent source for discovering novel bioactive peptides. Currently, the protein resources of agar residue, a byproduct of red algae processing, are not being effectively utilized. During agar extraction, approximately 60%-70% of the algal protein remains in the residue and is directly discarded. These proteins provide ideal raw materials for the preparation of ACE-inhibiting peptides, offering greater economic benefits than other protein sources. Systematic exploration of red algae agar residue and the discovery of peptides with specific ACE-inhibiting activity can realize the high-value utilization of this marine resource. 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 was identified from the protein hydrolysate of red algae agar residue, 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:

[0008] A pentapeptide ES5 with ACE inhibitory activity, wherein the amino acid sequence of the pentapeptide ES5 is ERQFS.

[0009] The aforementioned method for preparing the pentapeptide ES5 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 ES5 in a solid phase.

[0010] The preparation method of the aforementioned pentapeptide ES5 with ACE inhibitory activity adopts an enzymatic hydrolysis method, as follows: (1) Red algae agar waste residue is placed in water, heated to 40°C, and yeast is added. Enzymatic hydrolysis is carried out at this temperature for 2 hours; (2) The temperature is further increased to 48°C, and alkaline protease and neutral protease are added. Enzymatic hydrolysis is carried out at this temperature for 2 hours; (3) The temperature is further increased to 58°C, and papain is added. Enzymatic hydrolysis is carried out at this temperature for 3 hours; (4) The temperature is further increased to 85°C and held for 30 minutes; (5) The enzymatic hydrolysate is allowed to stand and precipitate, the supernatant is centrifuged, and the supernatant after centrifugation is spray-dried to obtain red algae agar waste residue protein peptide, which contains pentapeptide ES5; wherein, by mass, the ratio of red algae agar waste residue, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2.

[0011] Preferably, the aforementioned red algae is Gracilaria zeylanica.

[0012] The aforementioned pentapeptide ES5 with ACE inhibitory activity is used in the preparation of antihypertensive drugs or functional foods that help lower blood pressure.

[0013] The advantages of this invention are as follows: The pentapeptide ES5 provided by this invention was identified from the protein hydrolysate of red algae agar waste. Molecular docking revealed that pentapeptide ES5 has a potential interaction with ACE. In vitro ACE inhibition activity tests showed that the ACE inhibition rate of pentapeptide ES5 was 90.84%, which is significantly increased 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) (p<0.001). Pentapeptide ES5 can be used to prepare antihypertensive drugs or functional foods that help lower blood pressure. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the binding mode between pentapeptide ES5 and ACE;

[0015] Figure 2 The graph shows the ACE inhibition rate of pentapeptides ES5, VPP, and IPP. *** indicates p < 0.001. Detailed Implementation

[0016] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0017] 1. Collect red algae agar waste

[0018] Agar was extracted from Gracilaria verrucosa, a red algae, according to the method described in the local standard DB35T2159-2023, and the waste residue of the red algae agar was collected.

[0019] II. Preparation of protein peptides from red algae agar residue

[0020] The method for preparing protein peptides from red algae agar residue includes the following steps:

[0021] (1) Put 100g of red algae agar waste into 1000mL of water, heat to 40℃ and add 1g of yeast, and enzymatically hydrolyze at this temperature for 2h;

[0022] (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.

[0023] (3) Continue to heat up to 58°C and add 2g of papain. At this temperature, enzymatically hydrolyze for 3 hours.

[0024] (4) Continue to heat up to 85℃ and hold for 30 minutes;

[0025] (5) Let the enzymatic 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 red algae agar waste protein peptide.

[0026] III. Obtaining the polypeptide sequence from the protein peptides in the waste residue of red algae agar

[0027] The red algae agar waste protein peptides obtained above were analyzed by LC-MS / MS mass spectrometry. The mass spectrometry results were analyzed using mass spectrometry analysis software to obtain several polypeptide sequences.

[0028] The LC-MS / MS determination conditions are as follows:

[0029] (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%.

[0030] (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.

[0031] IV. Screening peak area ≥ 5.00 × 10 6 Active peptides with ≤6 amino acids

[0032] From the several polypeptide sequences obtained above, 62 peak areas ≥ 5.00 × 10⁻⁶ were finally selected. 6 The screening results for bioactive peptides with ≤6 amino acid counts are shown in Tables 1-1, 1-2, and 1-3.

[0033] Table 1-1 High-abundance bioactive peptides in red algae agar waste residue (Part 1)

[0034]

[0035] Table 1-2 High-abundance bioactive peptides in red algae agar waste residue (II)

[0036]

[0037] Table 1-3 High-abundance bioactive peptides in red algae agar waste residue (Part III)

[0038]

[0039] V. Screening for bioactive peptides with strong ACE binding ability

[0040] Using Discovery Studio software, the active peptide sequences in Tables 1-1, 1-2, and 1-3 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.

[0041] 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.

[0042] The molecular docking results of the above 62 active peptides with ACE are shown in Tables 2-1, 2-2 and 2-3.

[0043] Table 2-1 Predicted results of interactions between bioactive peptides and ACE (Part 1)

[0044]

[0045] Table 2-2 Predicted results of interactions between bioactive peptides and ACE (Part II)

[0046]

[0047] Table 2-3 Predicted results of interactions between bioactive peptides and ACE (Part III)

[0048]

[0049] VI. Molecular docking analysis

[0050] Among the 62 bioactive peptides mentioned above, EREFK (denoted as pentapeptide EK5, SEQ ID NO: 29) and ERQFS (denoted as pentapeptide ES5, SEQ ID NO: 14) had the highest docking scores, at 99.0072 kcal / mol and 92.8689 kcal / mol, respectively. Considering that the peak area of ​​pentapeptide EK5 is 6.98 × 10⁻⁶... 6 The peak area of ​​pentapeptide ES5 is 37.00 × 10⁻⁶. 6 The peak area of ​​the latter is 5 times that of the former. Therefore, ERQFS (pentapeptide ES5) was selected for further molecular docking analysis.

[0051] Analysis revealed that the binding mode of pentapeptide ES5 to ACE is as follows: Figure 1 As shown, the molecular docking is as follows:

[0052] The pentapeptide ES5 forms one salt bridge interaction, 14 HH bond interactions, 3 CH bond interactions and 5 electrostatic interactions with ACE. Thirteen amino acid residues are involved in the interaction between the pentapeptide ES5 and ACE.

[0053] VII. Evaluation of the ACE inhibitory activity of pentapeptide ES5

[0054] 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 pentapeptide ES5 (purity >90%).

[0055] The pentapeptide ES5, VPP (positive control), or IPP (positive control) obtained by solid-phase synthesis was dissolved in ultrapure water to prepare ES5 solution, VPP solution, and IPP solution with a concentration of 0.1 mg / mL, respectively.

[0056] Sample group: Take 10 μL of ES5 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.

[0057] 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.

[0058] 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.

[0059] The formula for calculating the ACE inhibition rate is as follows:

[0060]

[0061] Where A represents the hippuric acid content in the control group and B represents the hippuric acid content in the sample group.

[0062] Calculations showed that at a concentration of 0.1 mg / mL, the ACE inhibition rates of pentapeptides ES5, VPP, and IPP were 90.84±1.8%, 82.32±1.2%, and 78.00±1.4%, respectively. (See the comparison below.) Figure 2 .

[0063] Depend on Figure 2 It can be seen that, at the same concentration, the ACE inhibition rate of pentapeptide ES5 was significantly increased compared with the positive controls VPP and IPP (p<0.001).

[0064] In summary, pentapeptide ES5 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.

[0065] This discovery not only provides core raw materials for the development of a new generation of antihypertensive drugs and functional foods that help lower blood pressure, but also enables the high-value utilization of red algae processing by-products.

[0066] 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 ES5 with ACE inhibitory activity, characterized in that, The amino acid sequence of the pentapeptide ES5 is ERQFS.

2. The method for preparing the pentapeptide ES5 with ACE inhibitory activity as described in claim 1, characterized in that, The pentapeptide ES5 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 ES5 with ACE inhibitory activity as described in claim 1, characterized in that, The enzymatic hydrolysis method is used, as detailed below: (1) Put the red algae agar waste into water, heat it to 40°C and add yeast. At this temperature, enzymatic hydrolysis is carried out for 2 hours. Among them, the red algae is Gracilaria fusiforme. (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 enzyme hydrolysate stand to precipitate, take the supernatant and centrifuge, and spray dry the supernatant after centrifugation to obtain red algae agar waste protein peptide, which contains pentapeptide ES5; The ratio of red algae agar waste, yeast, alkaline protease, neutral protease and papain by mass is 100:1:3:2:

2.

4. The use of the pentapeptide ES5 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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