A blood pressure lowering active peptide and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a blood pressure lowering active peptide and its application. The present invention uses molecular docking technology to explore the mechanism of action of Guangdong glutinous rice wine peptide MGYTSY (720.2789Da), and finds that it can interact with the active site of ACE to form a tight complex. The in vitro ACE inhibitory activity of the active peptide was explored, and it was found that MGYTSY (IC 50 =0.123mg / mL) has a non-competitive inhibition mode on ACE. In addition, the active peptide MGYTSY can significantly increase the release of NO in EA.hy926 cells and reduce the release of ET-1 in cells, making it an ideal raw material source for antihypertensive functional food ingredients and / or antihypertensive drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a blood pressure lowering active peptide and application thereof. Background Art

[0002] The bioactive peptides in Guangdong glutinous rice wine are produced by the secretion of microbial enzymes to degrade proteins during the fermentation process, and are generally composed of several amino acids. At present, the specific composition and efficacy of the active peptides in Guangdong glutinous rice wine are still understudied.

[0003] In recent years, cardiovascular disease has become the main cause of death, and hypertension is closely related to cardiovascular disease. Hypertension is a common chronic disease that can easily lead to major diseases such as myocardial infarction and heart failure. Therefore, antihypertensive treatment can effectively prevent cardiovascular disease.

[0004] Human blood pressure is mainly regulated by the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), in which angiotensin converting enzyme (ACE) plays an important role. In the RAS, ACE can convert angiotensin I into angiotensin II with strong vasoconstrictor activity, causing vasoconstriction. In the KKS, ACE can inactivate the vasodilator bradykinin, ultimately leading to increased blood pressure. Therefore, inhibiting the activity of ACE is an effective way to treat patients with hypertension.

[0005] At present, studies have shown that chemically synthesized antihypertensive drugs have many side effects and are not suitable for patients to take for a long time. Therefore, food-derived ACE inhibitors with few side effects and easy to obtain have attracted widespread attention and become an ideal substitute or supplement for antihypertensive drugs. In recent years, many blood pressure-lowering peptides have been extracted and identified from a variety of food-derived proteins, and it has been found that they have ACE inhibitory activity that is not inferior to chemical drugs. For example, NMAINPSKENLCSTFCK from casein and LSGYGP in tilapia protein. Summary of the invention

[0006] To solve the related problems, the primary purpose of the present invention is to provide a blood pressure lowering active peptide.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned blood pressure lowering active peptide.

[0008] Another object of the present invention is to provide the application of the above-mentioned blood pressure lowering active peptide.

[0009] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0010] A blood pressure lowering active peptide, the amino acid sequence of which is MGYTSY (Met-Gly-Tyr-Thr-Ser-Tyr).

[0011] The preparation method of the active peptide is to use solid phase peptide synthesis technology to continuously add, react and synthesize amino acids from the carboxyl end to the amino end according to the amino acid sequence of the active peptide to obtain the active peptide.

[0012] Application of the active peptide in the preparation of ACE inhibitors, blood pressure lowering functional food ingredients and / or blood pressure lowering drugs.

[0013] Furthermore, in the application, the ACE inhibitor is a non-competitive ACE inhibitor.

[0014] Furthermore, in the application, the blood pressure lowering functional food ingredient and / or blood pressure lowering drug has the effect of promoting the release of NO and reducing the release of ET-1.

[0015] Furthermore, in the application, the ACE inhibitor, blood pressure lowering functional food ingredient and / or blood pressure lowering drug uses the active peptide as an active ingredient, and can be added with food or pharmaceutically acceptable carriers or excipients.

[0016] An ACE inhibitor comprises the active peptide as an active ingredient.

[0017] Furthermore, the ACE inhibitor also contains a pharmaceutically acceptable carrier or excipient.

[0018] A functional food ingredient for lowering blood pressure comprises the active peptide as an active ingredient.

[0019] Furthermore, the blood pressure lowering functional food ingredients also include food-acceptable carriers or auxiliary materials.

[0020] A blood pressure lowering drug comprises the active peptide as an active ingredient.

[0021] Furthermore, the antihypertensive drug also contains a pharmaceutically acceptable carrier or excipient.

[0022] Compared with the prior art, the present invention has the following advantages and effects:

[0023] The present invention uses molecular docking technology to explore the antihypertensive mechanism of Guangdong glutinous rice wine peptide MGYTSY (720.2789Da), and finds that it can interact with the active site of ACE to form a tight complex. The in vitro ACE inhibitory activity of the active peptide was explored, and it was found that MGYTSY (IC 50=0.123mg / mL) has a non-competitive inhibition mode on ACE. In addition, the active peptide MGYTSY can significantly increase the release of NO in EA.hy926 cells and reduce the release of ET-1 in cells, and is an ideal raw material source for antihypertensive functional food ingredients and / or antihypertensive drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the secondary mass spectrum of peptide MGYTSY;

[0025] Figure 2 Figure 1 is a diagram showing the molecular docking results of the peptide MGYTSY and the ACE inhibitory peptide; A is a schematic diagram showing the structure of the active peptide MGYTSY; B is a schematic diagram showing the molecular docking results of the blood pressure lowering active peptide;

[0026] Figure 3 This is the result diagram of the inhibitory effect of peptide MGYTSY on ACE;

[0027] Figure 4 The Lineweaver-Burk plot of the ACE inhibition by peptide MGYTSY;

[0028] Figure 5 The figure is the result of the cytotoxicity study of the peptide MGYSTY;

[0029] Figure 6 This is the result diagram of the effect of peptide MGYSTY on cellular NO secretion;

[0030] Figure 7 This is a graph showing the effect of peptide MGYSTY on intracellular ET-1 secretion. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0032] The sources of the experimental materials in the following examples are as follows: Guangdong glutinous rice wine was provided by Guangdong Heyuan Green Pure Food Co., Ltd., angiotensin converting enzyme (ACE, from rabbit lung), hydroxyethylpiperazineethanesulfonic acid (HEPES), furan acryloyl tripeptide (FAPGG), phosphate buffer saline (PBS), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA); human umbilical vein cell fusion cells (EA.hy926) were purchased from the cell bank of the Chinese Academy of Sciences; DMEM (dulbecco's modified eagle medium) culture medium, fetal bovine serum (FBS) and 0.25% trypsin were purchased from Gibco, USA; other chemicals and reagents were of analytical grade.

[0033] Example 1: Synthesis and determination of angiotensin converting enzyme inhibitory peptides

[0034] 1. Molecular docking

[0035] MarvinSketch software was used to draw the structure of the active peptide, and the energy minimization structure was optimized and exported as a mol2 format file for subsequent docking. The crystal structure of the receptor molecule ACE protein (1O8A) was downloaded from the PDB database (RCSB PDB: Homepage), and the ligands and water molecules contained in the receptor were removed using Pymol software, and finally saved as a pdb format file. The receptor molecule and the synthetic peptide were hydrogenated and charged using AutodockTools 1.5.6 software and exported as a pdbqt format file, and then docked using Vina. The Vina docking binding energy was used to sort them, and the peptides with stronger affinity were selected for visualization analysis using Pymol and Discovery Studio 4.5 software.

[0036] 2. Synthesis of blood pressure lowering peptides

[0037] The antihypertensive active peptide MGYTSY was synthesized by Nanjing Synpeptide Co., Ltd. The purity of the synthetic peptide was determined to be 98% by high performance liquid chromatography, and the molecular weight of the synthetic peptide was determined by LC / ESI-MS. Its ACE inhibitory activity was verified by in vitro experiments and cell experiments.

[0038] 3. Analysis of the physicochemical properties of antihypertensive active peptides

[0039] The physicochemical properties of the antihypertensive peptide MGYTSY, including toxicity, hydrophobicity, and stability, were evaluated. The toxicity of the peptide was predicted using the ToxinPred tool (ToxinPred (iiitd.edu.in)). The hydrophobicity of the peptide was predicted using the PepDraw tool (PepDraw (tulane.edu)). The stability of the peptide was predicted using the ProtParam tool (http: / / web.expasy.org / protparam / ).

[0040] 4. ACE inhibitory activity assay

[0041] Take 40μL of sample solution and mix it with 50μL of 1.0mmol / L FAPPG. For the blank group, use 40μL of 80mmol / L HEPES buffer and mix it with 50μL FAPPG. Add 10μL of 0.1U / mL ACE to both groups at the same time. After adding ACE, immediately measure the absorbance of both groups at a wavelength of 340nm. The blank group is recorded as A1 and the experimental group is recorded as B1. After incubation at 37℃ in the dark, take out and measure the absorbance of both groups again at 340nm, and record them as A2 and B2 respectively. The data obtained are calculated according to the following formula (1):

[0042] ACE inhibition rate (%) = [(A1-A2)-(B1-B2)] / (A1-A2)×100 (1).

[0043] 5. ACE inhibition kinetics assay

[0044] 0.33, 0.67, 1.33, 3.08 mmol / L of substrate were mixed with 0.5 and 1.0 mg / mL of peptide respectively, and then ACE enzyme was added to react. A Lineweaver-Burk plot was made with 1 / V as the ordinate and 1 / S as the abscissa.

[0045] 6. Effects of ACE inhibitory peptides on EA.hy 926 cells

[0046] 6.1 Cytotoxicity assay

[0047] EA.hy926 cells were cultured in complete medium (DMEM supplemented with 20% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin solution). The culture medium was stored at 37°C in an environment of 5% carbon dioxide. Cytotoxicity was determined by MTT assay. 100 μL of cell suspension was inoculated per well in a white 96-well plate to obtain a seeding density of 1×104 cells per well and incubated at 37°C for 24 h. To avoid changes in the surrounding environment, PBS was added to the outermost wells of the plate. After 24 h of cell culture, the medium was changed. 100 μL of complete medium was added to the normal group, 100 μL of peptides of different concentrations (0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 mg / mL) were added to the experimental group, and the control group was added with the same concentration of captopril and continued to culture for 24 h. After 24 hours of cell culture, the medium was changed and 100 μL of 0.5 mg / mL MTT solution was added to each well. The cells were cultured in the dark for 4 hours. The MTT solution was discarded and washed with PBS and 100 μL of DMSO was added. The cells were fully shaken for 10 minutes and the OD value was measured at 490 nm using an enzyme reader. The cell survival rate was calculated according to formula (2):

[0048] Survival rate (%) = OD value of experimental group / OD value of normal group × 100 (2).

[0049] 6.2 Determination of nitric oxide (NO) content

[0050] Inoculate 100 μL of cell suspension per well in a white 96-well plate to obtain a seeding density of 1×10 4 Cells were added and incubated at 37°C for 24 hours. After 24 hours, the medium was changed, 100 μL of complete medium was added to the normal group, 100 μL of peptides of different concentrations (0.1, 0.25, 0.5 mg / mL) were added to the experimental group, and the same concentration of captopril was added to the control group, and incubated for 24 hours. The cell supernatant was collected and stored for testing. The NO content of the cells was determined by referring to the NO kit.

[0051] 6.3 Determination of endothelin (ET-1) content

[0052] Inoculate 100 μL of cell suspension per well in a white 96-well plate to obtain a seeding density of 1×10 4 cells and incubated at 37°C for 24 hours. After 24 hours, the medium was changed, 100 μL of complete medium was added to the normal group, 100 μL of peptides of different concentrations (0.1, 0.25, 0.5 mg / mL) were added to the experimental group, and the same concentration of captopril was added to the control group, and incubated for 24 hours. After 24 hours, the cell culture medium was centrifuged at 1000xg and the supernatant was taken for testing. The ET-1 content of the cells was determined by referring to the ET-1 kit.

[0053] 7. Statistical Analysis

[0054] Statistical analysis was performed using SPSS 20.0 statistical analysis software. All experiments were repeated three times, and the results were expressed as mean ± standard deviation. After one-way analysis of variance, p < 0.05 indicated a significant difference.

[0055] 7.1 Molecular docking visualization analysis

[0056] Studies have shown that the inhibitory activity of ACE inhibitory peptides is related to the C-terminal peptide segment in their sequence. The binding effect of peptides to ACE is also significantly associated with aromatic amino acids (Phe, Trp and Tyr) or proline (Pro) in the sequence, as well as branched side chain amino acids (Ala, Gly, Ile, Leu, Met and Val). Therefore, we screened MGYTSY for molecular docking with ACE receptor protein to explore its ACE inhibitory mechanism.

[0057] Studies have shown that ACE has three active pockets, namely S1, S2 and S1', of which S1 contains Ala354, Glu384, and Tyr 523; S2 contains Gln 281, His 353, Lys 511, His 513, and Tyr 520, and S1' contains Glu162. ACE inhibitory peptides bind to key sites in these active pockets with different forces to form a stable complex structure, thereby achieving the purpose of inhibiting ACE from playing a role in the blood pressure regulation system. The theoretical binding mode of the antihypertensive active peptide MGYTSY and ACE receptor protein is shown in Figure 2. Figure 2 As shown in B. MGYTSY forms hydrogen bonds with key sites Ala354 and Tyr523, as well as hydrogen bonds with other sites Asn70 and Asn66, carbon-hydrogen bonds with Ser355, Pi-Pi bonds with Phe512, hydrophobic interactions with Ala63, and interaction forces with Zn701.

[0058] The stability of the peptide-ACE complex is maintained by different forces, including hydrogen bonds, carbon-hydrogen bonds, salt bridges, hydrophobic interactions, van der Waals forces, Metal-Acceptor, etc., among which the most important force is hydrogen bonds. As shown in Table 1, the docking binding energy of MGYTSY is -9.1, the number of hydrogen bonds is 8, and it can bind to the key sites of the three active pockets in the ACE protein to varying degrees. This shows that MGYTSY can have a good binding effect with the ACE receptor protein.

[0059] In addition, ACE inhibitors can also inhibit the activity of ACE when they interact with the zinc ions in ACE. ACE inhibitors such as captopril, enalapril, and fosinopril can all form chemical bonds with the zinc ions of ACE. This result has been confirmed in recent years: GPAGPAGL extracted from lemon basil seeds can form Metal-Acceptor bonds with zinc ions in molecular docking, and MEVFVP extracted from flounder also forms Metal-Acceptor bonds with zinc ions in molecular docking. In this study, MGYTSY can also form Metal-Acceptor bonds with Zn701, from which it can be inferred that this active peptide has a clear action site with ACE and forms a stable complex.

[0060] Table 1. Docking energy and interaction sites of active peptides and ACE protein

[0061]

[0062] 7.2 Analysis of the physicochemical properties of antihypertensive active peptides

[0063] Since the in vivo and in vitro evaluation systems have the characteristics of high cost and complex operation, analytical tools that can preliminarily predict the physicochemical properties of peptide sequences have been widely used. The toxicity, hydrophobicity and stability of the peptide MGYTSY were evaluated by analytical tools, and the results are shown in Table 2. Although bioactive peptides have the characteristics of strong activity, low production cost and easy absorption, their potential toxicity is still a problem worthy of attention in food processing and production. The results show that MGYTSY is non-toxic and can be used as an active substance in the development of functional foods. Since the C-terminal domain of ACE is a hydrophobic environment, high hydrophobic antihypertensive active peptides are more likely to bind to it and exert an inhibitory effect. MGYTSY has good hydrophobicity of 7.67kcal / mol. In terms of stability, the instability index of MGYTSY is -19.97, which is judged to be stable by the analytical tool.

[0064] Table 2. Prediction of physicochemical properties of antihypertensive active peptides

[0065]

[0066] 7.3 Determination of ACE inhibitory activity of antihypertensive peptides

[0067] IC of ACE inhibitory activity of peptide MGYTSY and positive control captopril 50 Value Figure 3 MGYTSY exhibits strong ACE inhibitory activity and is a new type of effective ACE inhibitor, with significantly higher activity than the reported yeast protein peptide YGKPVAVPAR (IC 50=0.42mg / mL) and bonito muscle protein peptide VDRYF (IC 50 =0.28 mg / mL), and the inventor's previous patent CN202210484022.4-Guangdong glutinous rice wine antioxidant and ACE inhibitory active peptides and peptide VLSGA (IC 50 =0.50 mg / mL) and VISGA (IC 50 =0.81 mg / mL). Therefore, the ACE inhibition mode of the active peptide MGYTSY and its antihypertensive activity at the cellular level will be further explored.

[0068] 7.4 Determination of ACE inhibition kinetics of antihypertensive active peptides

[0069] The Lineweaver-Burk plot was used to analyze the inhibition mode of peptide MGYTSY on ACE. Figure 4 As shown in the figure, MGYTSY mainly exhibits non-competitive inhibition on ACE, which indicates that MGYTSY can not only bind to free ACE, but also does not occupy the active area where the substrate binds to ACE, and has the ability to bind to the substrate-ACE complex. When the peptide binds to free ACE, the peptide reduces the binding ability of ACE to the substrate; while the peptide binds to the substrate-ACE complex, it can prevent the complex from generating reaction products, and can also achieve the purpose of lowering blood pressure. Similarly, VSQLTR extracted from soleus muscle protein is also a non-competitive ACE inhibitor. These results also further verify the feasibility of molecular docking technology. From the visual analysis of MGYTSY, it can be concluded that it mainly binds to the non-active site of ACE and does not need to compete with the substrate for the active area of ​​ACE. Therefore, we believe that the antihypertensive active peptide derived from glutinous rice wine is expected to be used as a functional food ingredient in conjunction with chemical drugs such as captopril, while ensuring the antihypertensive effect while reducing the patient's dependence on chemical drugs.

[0070] 7.5 Effects of antihypertensive peptides on EA.hy926 cells

[0071] 7.5.1 Cytotoxicity assay

[0072] To further verify the safety of the antihypertensive active peptide, we evaluated the cytotoxicity of MGYSTY at 0.025-2 mg / mL based on the MTT method. Figure 5 As shown. The cell activity of captopril at 0.025-2 mg / mL was above 90%, but the cell survival rate of MGYSTY at 2 mg / mL was less than 90%. In summary, the active peptide MGYSTY had no cytotoxicity to EA.hy926 cells within the concentration range of 1 mg / mL.

[0073] 7.5.2 Effect of active peptides on cellular nitric oxide (NO) content

[0074] Studies have shown that the use of ACE inhibitors to lower blood pressure is mainly achieved through endothelium-dependent vasodilation, which is due to the production of vasodilator factor NO by endothelial nitric oxide synthase in the renin-angiotensin system (RAS), which maintains normal blood pressure levels. Figure 6 As shown in the figure, after treatment with different concentrations of peptide MGYTSY, the amount of NO released from cells was significantly increased compared with the blank group. At 0.5 mg / mL, MGYTSY made the NO release of cells reach the highest level, 2.79 μmol / L, which is 4.7 times that of the blank group. It has been reported that AHIII from sea squirt and YRK from Antarctic krill have the ability to inhibit ACE activity and increase cellular NO production. Therefore, the antihypertensive active peptides derived from glutinous rice wine can inhibit the decomposition of vasodilators and directly stimulate endothelial cells to release NO.

[0075] 7.5.3 Effect of active peptides on endothelin-1 (ET-1) content

[0076] In addition to the renin angiotensin system (RAS), the role of the endothelin system in blood pressure regulation is increasingly valued, among which the overexpression of ET-1 can lead to cardiovascular diseases such as arteriosclerosis and hypertension. Therefore, peptides that can reduce the level of ET-1 in the body also have potential antihypertensive activity. Figure 7 As shown in the figure, compared with the blank group, the peptide MGYSTY can significantly reduce the level of intracellular ET-1, and there is a significant dose-dependent relationship between the low, medium and high dose groups, indicating that it has potential antihypertensive activity. At 0.5 mg / mL, MGYSTY made the intracellular ET-1 content reach the lowest level, which was 47.77 pg / mL, which was 27.1% lower than the blank group. This result is comparable to the effect of captopril.

[0077] 7.6 Conclusion

[0078] In this study, we found that the glutinous rice wine-derived active peptide MGYTSY has potential antihypertensive activity. It can bind to the key sites of ACE with different forces and form a Metal-Acceptor bond with Zn701. The active peptide MGYTSY has a significant inhibitory effect on ACE, and it is a non-competitive inhibition of ACE. The results of cell experiments showed that both MGYTSY and captopril can play a role in lowering blood pressure by inducing cells to increase NO release and reduce ET-1 release. We believe that the peptide MGYTSY is expected to be a dietary supplement or functional food ingredient that can lower blood pressure.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. The use of active peptides in the preparation of antihypertensive drugs, characterized in that: The amino acid sequence of the active peptide is Met-Gly-Tyr-Thr-Ser-Tyr; the IC 50 It is 0.123mg / mL.

2. The use according to claim 1, characterized in that: The blood pressure lowering drug further comprises a pharmaceutically acceptable carrier or excipient.

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