A hexapeptide NS6 with ACE inhibitory activity, its preparation method and application

Abstract

This invention discloses a hexapeptide NS6 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 hexapeptide NS6 is NLLELS, and it can be prepared by solid-phase synthesis and enzymatic hydrolysis. The hexapeptide NS6 was identified from the hydrolysate of *Polygonum multiflorum* and exhibits potential interaction with ACE. At a concentration of 0.1 mg / mL, its ACE inhibition rate is 82.74%. Compared with VPP and IPP (whose ACE inhibition rates at the same concentration are 82.32% and 78.00%, respectively), although the ACE inhibitory activity does not surpass that of VPP, it is significantly superior to that of IPP (p<0.05). This provides a wider range of options for the development of adjunctive antihypertensive functional foods that require precise formulation design within a specific activity range. Hexapeptide NS6 can be used to prepare antihypertensive drugs or adjunctive antihypertensive functional foods.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide technology, specifically to a hexapeptide NS6 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. Angiotensin-converting enzyme (ACE) plays a crucial role in the renin-angiotensin system, which regulates blood pressure. Precise inhibition of ACE activity has become a key strategy for clinical intervention in hypertension. Currently, chemically synthesized ACE inhibitors such as captopril are commonly used clinically, but long-term use carries potential adverse reaction risks, limiting long-term medication adherence for some patients. Therefore, developing ACE inhibitory components derived from natural foods that are mild in action and have high safety profiles can meet the practical needs of blood pressure management in sub-healthy populations.

[0003] Food-derived ACE inhibitory peptides are considered ideal functional factors due to their high safety, easy absorption, and minimal side effects. Among them, 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.

[0004] Marine organisms, due to their unique living environment, often possess special amino acid sequences in their proteins that are not found in terrestrial organisms, making them an excellent source for discovering novel bioactive peptides. The protein resources of *Polysiphonia urceolata* (mainly distributed along the coast of the Yellow and Bohai Seas in China) have not yet been fully recognized and utilized. Systematic exploration of *Polysiphonia urceolata* proteins and the discovery of peptides with specific ACE inhibitory activities is an effective way to achieve high-value utilization of this marine resource. Summary of the Invention

[0005] 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 *Polygonum multiflorum*, as well as the preparation method and application of the small molecule peptide.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A hexapeptide NS6 with ACE inhibitory activity, wherein the amino acid sequence of the hexapeptide NS6 is NLLELS.

[0008] The aforementioned method for preparing the hexapeptide NS6 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 support to synthesize the hexapeptide NS6 in a solid phase.

[0009] The preparation method of the aforementioned hexapeptide NS6 with ACE inhibitory activity adopts an enzymatic hydrolysis method, as follows: (1) Place *Polystomia spp.* in water, heat to 40°C, add yeast, and hydrolyze at this temperature for 2 hours; (2) Continue heating, heat to 48°C, add alkaline protease and neutral protease, and hydrolyze at this temperature for 2 hours; (3) Continue heating, heat to 58°C, add papain, and hydrolyze at this temperature for 3 hours; (4) Continue heating, heat to 85°C, and maintain for 30 minutes; (5) Allow the hydrolysate to settle, take the supernatant, centrifuge, and spray dry the supernatant after centrifugation to obtain *Polystomia spp.* protein peptide, which contains hexapeptide NS6; wherein, by mass, the ratio of *Polystomia spp.*, yeast, alkaline protease, neutral protease and papain is 100:1:3:2:2.

[0010] The aforementioned application of the hexapeptide NS6 with ACE inhibitory activity in the preparation of antihypertensive drugs or functional foods that help lower blood pressure.

[0011] The advantages of this invention are as follows: The hexapeptide NS6 provided by this invention was identified from the protein hydrolysate of *Polygonum multiflorum*. Molecular docking revealed that hexapeptide NS6 has a potential interaction with ACE. In vitro ACE inhibitory activity tests showed that the ACE inhibition rate of hexapeptide NS6 was 82.74%. 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), the ACE inhibitory activity of hexapeptide NS6, although not exceeding that of VPP, is significantly better than that of IPP (p<0.05). This provides a wider range of options for the development of auxiliary antihypertensive functional foods that require precise formulation design within a specific activity range. Hexapeptide NS6 can be used to prepare antihypertensive drugs or auxiliary antihypertensive functional foods. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the binding mode of hexapeptide NS6 and ACE;

[0013] Figure 2 The graph shows the ACE inhibition rate of hexapeptides NS6, VPP, and IPP. * indicates p < 0.05, and ns indicates no statistical difference. 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 Polytubularia protein peptides

[0016] The method for preparing polytubular algal protein peptides specifically includes the following steps:

[0017] (1) Put 100g of Polytubularia 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 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 multitube algae protein peptide.

[0022] II. Obtaining the polypeptide sequence from the polytubular algae protein peptide

[0023] The obtained polychaete algae protein peptides were analyzed by LC-MS / MS, and the results were analyzed using mass spectrometry analysis software to obtain several polypeptide 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, 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%.

[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 > 5.00 × 10 7 Active peptides with ≤6 amino acids

[0028] From the several polypeptide sequences obtained above, 38 peak areas > 5.00 × 10⁻⁶ were finally selected. 7 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 Polychaete algae protein peptides (Part 1)

[0030]

[0031] Table 1-2 High-Abundance Bioactive Peptides in Polychaete Algae Protein Peptides (Part 2)

[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 38 active peptides with ACE are shown in Tables 2-1 and 2-2.

[0037] Table 2-1 Predicted Interactions Between 38 Bioactive Peptides and ACE (Part 1)

[0038]

[0039] Table 2-2 Predicted Interactions Between 38 Bioactive Peptides and ACE (Part 2)

[0040]

[0041] V. Molecular docking analysis

[0042] Among the 38 bioactive peptides mentioned above, NLLELS (denoted as hexapeptide NS6, SEQ ID NO: 11) had the highest docking score, at 101.0760 kcal / mol. Therefore, NLLELS (hexapeptide NS6) was selected for further molecular docking analysis.

[0043] Analysis revealed that the binding mode of hexapeptide NS6 to ACE is as follows: Figure 1 As shown, the molecular docking is as follows:

[0044] The hexapeptide NS6 forms one salt bridge interaction, eight HH bond interactions, two CH bond interactions, and two electrostatic interactions with ACE. Ten amino acid residues are involved in the interaction between the hexapeptide NS6 and ACE.

[0045] VI. Evaluation of the ACE inhibitory activity of hexapeptide NS6

[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 NS6 (purity >90%).

[0047] The hexapeptide NS6, VPP (positive control), or IPP (positive control) obtained by solid-phase synthesis was dissolved in ultrapure water to prepare NS6 solution, VPP solution, and IPP solution with a concentration of 0.1 mg / mL, respectively.

[0048] Sample group: Take 10 μL of NS6 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 hexapeptides NS6, VPP, and IPP were 82.74±5.9%, 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, compared with the positive controls VPP and IPP, the ACE inhibitory activity of hexapeptide NS6 did not exceed that of VPP (p>0.05), but was significantly better than that of IPP (p<0.05).

[0056] In summary, hexapeptide NS6 provides a wider range of options for the development of functional foods that require precise formulation design within a specific activity range to assist in lowering blood pressure. Hexapeptide NS6 can be used to prepare antihypertensive drugs or functional foods that assist in lowering 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 NS6 with ACE inhibitory activity, characterized in that, The amino acid sequence of the hexapeptide NS6 is NLLELS.

2. The method for preparing the hexapeptide NS6 with ACE inhibitory activity as described in claim 1, characterized in that, 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 NS6.

3. The method for preparing the hexapeptide NS6 with ACE inhibitory activity as described in claim 1, characterized in that, The enzymatic hydrolysis method is used, as detailed below: (1) Place the Polytubularia in 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 multi-tube algal protein peptide, which contains hexapeptide NS6; The ratio of the amounts of Polysaccharidea, yeast, alkaline protease, neutral protease and papain by mass is 100:1:3:2:

2.

4. The use of the hexapeptide NS6 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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