Walnut protein source angiotensin converting enzyme selenium-enriched peptide as well as preparation method and application thereof in lowering blood pressure

Abstract

The invention discloses a selenium-rich walnut protein angiotensin converting enzyme inhibitory active peptide as well as a preparation method and application thereof. The peptide SeMYHAN (682.1934 Da) provided by the invention forms 10 hydrogen bonds with 8 amino acid residues on ACE (Angiotensin Converting Enzyme), and can be used as a non-competitive inhibitor to be combined with the ACE to play a role (IC50 is equal to 0.123 mg / mL). In addition, the SeMYHAN can increase the NO release amount of EA.hy926 cells and reduce the expression amount of ET-1 in the cells, and the blood pressure lowering effect of the SeMYHAN is verified on the cellular level. In a word, the selenium-rich walnut ACE inhibitory peptide SeMYHAN is expected to serve as a good antihypertensive drug, and meanwhile, a new theoretical basis is provided for high-value utilization of natural organic selenium.

Description

Technical Field

[0001] The invention relates to the technical field of active peptides, and in particular to a walnut protein-derived angiotensin converting enzyme selenium-enriched peptide, a preparation method thereof, and an application thereof in lowering blood pressure. Background Art

[0002] Walnut belongs to the family of Carya, and is a food and medicine. It is rich in fatty acids, proteins, vitamins and mineral elements, and has high nutritional and economic value. However, the current development and utilization of walnut resources is still mainly based on primary agricultural products and oil extraction. As a by-product of oil extraction, the protein in walnut meal has not been efficiently utilized, which greatly limits the economic benefits of the walnut industry and the added value of products.

[0003] Selenium (Se) is an essential trace element for the human body and has multiple physiological functions, such as anticancer activity, immunomodulatory effects, prevention of cardiovascular and cerebrovascular diseases, and antioxidant effects. Insufficient selenium intake is closely related to human diseases and can easily cause diseases such as cancer, Keshan disease, immune response, and thyroid hormone dysfunction. Epidemiological studies have shown that selenium deficiency affects up to 1 billion people worldwide. Selenium exists in inorganic and organic forms, but natural organic selenium has the characteristics of low toxicity, multiple biological activities, and high bioavailability, and generally has more advantages than inorganic selenium. Therefore, how to supplement natural selenium through food resources has become a hot topic of research in recent years.

[0004] Hypertension, as one of the most common chronic cardiovascular and cerebrovascular diseases, seriously affects human life and health. It is estimated that by 2025, the number of hypertensive patients in the world will exceed 1.5 billion. Angiotensin-I can be converted into angiotensin II, which causes vasoconstriction, by angiotensin-converting enzyme (ACE). At the same time, it can also cause the inactivation of bradykinin, which has the effect of dilating blood vessels, and ultimately lead to increased blood pressure. Therefore, in order to prevent and treat hypertension, ACE inhibitors can be used to reduce the activity of ACE so that it cannot act on angiotensin-I. However, some patients who take chemically synthesized ACE inhibitors will have side effects to varying degrees.

[0005] Therefore, food-borne ACE inhibitory peptides with high safety and few side effects have become a research hotspot. Summary of the invention

[0006] In order to overcome the problems existing in the prior art, one of the purposes of the present invention is to provide a selenium-enriched walnut protein angiotensin converting enzyme inhibitory active peptide.

[0007] The second object of the present invention is to provide the application of the above-mentioned selenium-enriched walnut protein angiotensin converting enzyme inhibitory active peptide.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A selenium-enriched walnut protein angiotensin converting enzyme inhibitory active peptide has the following amino acid sequence: Se-Met-Tyr-His-Ala-Asn (SeMYHAN); wherein Se represents selenocysteine.

[0010] Furthermore, the active peptide is prepared by enzymatic hydrolysis of selenium-enriched walnuts, solid phase synthesis or genetic engineering technology.

[0011] A pharmaceutical composition comprises the above active peptide and a pharmaceutically acceptable excipient.

[0012] A salt is a pharmaceutically acceptable salt of the above active peptide.

[0013] A nucleic acid encoding the above active peptide.

[0014] A biological material, comprising the above-mentioned nucleic acid, is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

[0015] Application of the above active peptide, pharmaceutical composition, salt, nucleic acid or biological material in the preparation of angiotensin converting enzyme inhibitors.

[0016] Use of the above active peptides, pharmaceutical compositions, salts, nucleic acids or biological materials in the preparation of drugs for preventing and / or treating cardiovascular and cerebrovascular diseases.

[0017] Use of the above active peptide, pharmaceutical composition, salt, nucleic acid or biological material in the preparation of drugs for preventing and / or treating hypertension.

[0018] The above-mentioned preparations or drugs can be made into various preparations, which can be administered orally or parenterally; parenterally, they can be administered subcutaneously, intravenously, or anorectally; and the preparation of injections can arbitrarily use physiological saline, glucose, tranquilizers, preservatives, suspending agents, or emulsifiers.

[0019] Compared with the prior art, the present invention has the following advantages and effects:

[0020] The peptide SeMYHAN (682.1934Da) provided by the present invention forms 10 hydrogen bonds with 8 amino acid residues on ACE and may act as a non-competitive inhibitor binding to ACE (IC 50=0.123mg / mL). In addition, SeMYHAN can increase the NO release of EA.hy926 cells and reduce the expression of ET-1 in cells, verifying its antihypertensive effect at the cellular level. In conclusion, the selenium-rich walnut ACE inhibitory peptide SeMYHAN is expected to be a good antihypertensive drug, and also provides a new theoretical basis for the high-value utilization of natural organic selenium. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the results of a study on the inhibitory effect of peptide MSeYHAN on ACE.

[0022] Figure 2 This is a graph showing the results of a study on the effect of peptide MSeYHAN on the activity of EA.hy926 cells.

[0023] Figure 3 This is a graph showing the results of a study on the effect of peptide MSeYHAN on NO secretion by EA.hy926 cells.

[0024] Figure 4 This is a graph showing the results of a study on the effect of peptide MSeYHAN on ET-1 secretion by EA.hy926 cells.

[0025] Figure 5 This is a visualization analysis result diagram of the peptide MSeYHAN-ACE complex; wherein A is the 3D structure diagram of the complex, and B is the interaction between the peptide and the amino acid residues of ACE. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below through specific examples, but the embodiments of the present invention are not limited thereto.

[0027] Angiotensin converting enzyme (ACE, from rabbit lung), hydroxyethylpiperazineethanesulfonic acid (HEPES), furan acryloyl tripeptide (FAPGG), phosphate buffer saline (PBS), and dimethyl sulfoxide (DMSO) used in the following examples 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.

[0028] Example 1: Synthesis of ACE inhibitory peptides

[0029] Selenium-enriched walnut ACE inhibitory peptide MSeYHAN 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.

[0030] Example 2: Effects of ACE inhibitory peptides on EA.hy 926 cells

[0031] 2.1 Determination of ACE inhibitory activity

[0032] The ACE inhibitory activity was determined according to the method of Memarpooryazdi M, with some improvements: 40 μL of sample solution was mixed with 50 μL of 1.0 mmol / L FAPPG, and the blank group was mixed with 40 μL of 80 mmol / L HEPES buffer and 50 μL of FAPPG. 10 μL of 0.1 U / mL ACE was added to both groups at the same time. After adding ACE, the absorbance values ​​of the two groups were immediately measured at a wavelength of 340 nm. The blank group was recorded as A1 and the experimental group was recorded as B1. After incubation at 37°C in the dark, the samples were taken out and the absorbance values ​​of the two groups were measured again at 340 nm and recorded as A2 and B2, respectively. The data obtained were calculated according to the following formula (1):

[0033] ACE inhibition rate (%) = [(A1-A2) - (B1-B2)] / (A1- A2) × 100 (1)

[0034] 2.2 Cytotoxicity assay

[0035] Human umbilical vein cell fusion cell EA.hy926 cells were cultured in complete culture medium (DMEM with 20% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin solution). The culture medium was stored at 37°C and 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 1×10 per well. 4cells and incubated at 37°C for 24 hours. To avoid changes in the surrounding environment, PBS was added to the outermost wells of the plate. After 24 hours of cell culture, the medium was changed. The normal group added 100 μL complete medium, the experimental group added 100 μL of peptides of different concentrations (0.025, 0.05, 0.1, 0.25, 0.5, 1, 2 mg / mL), and the control group added the same concentration of captopril and continued to culture for 24 hours. After 24 hours of cell culture, the medium was changed. 100 μL of 0.5 mg / mL MTT solution was added to each well, and the cells were cultured in the dark for 4 hours. After the MTT solution was discarded, the cells were 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 a microplate reader. The cell survival rate was calculated according to formula (2):

[0036] Survival rate (%) = OD value of experimental group / OD value of normal group × 100 (2)

[0037] 2.3 Determination of nitric oxide (NO) content

[0038] 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. The cell supernatant was collected and stored for testing. The NO content of the cells was determined by referring to the NO kit.

[0039] 2.4 Determination of endothelin (ET-1) content

[0040] 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.

[0041] 2.5 Statistical analysis

[0042] 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.

[0043] 2.6 Results and Analysis

[0044] (1) Determination of inhibitory peptide activity: Figure 1 As shown, the ACE inhibitory activity IC of peptide SeMYHAN 50 The value was 0.102 mg / mL, showing strong ACE inhibitory activity, and can be used as an ACE inhibitor for synergistic antihypertensive drugs. Its activity is significantly higher than that of the reported selenium-enriched tea protein ACE inhibitory peptide SeTAPep (IC 50 =13.39 mg / mL). Therefore, we will continue to explore the effect of SeMYHAN on the secretion of NO and ET-1 by EA.hy926 cells.

[0045] (2) Cytotoxicity test of selenium-enriched walnut ACE inhibitory peptide: The MTT method was used to conduct cytotoxicity experiments to explore the effect of peptide SeMYHAN on EA.hy926 cells. The results are as follows Figure 2 As shown. Within the concentration range of 0.025-0.5 mg / mL, both the active peptide and the positive control captopril can maintain cell viability above 95%, indicating that both are not toxic to cells. According to the experimental results, the low-dose group, medium-dose group, and high-dose group were selected with concentrations of 0.1, 0.25, and 0.5 mg / mL, respectively, for subsequent experiments.

[0046] (3) Determination of nitric oxide (NO) content in cells: NO can participate in the peripheral and central regulation of cardiovascular function and is an important factor in regulating endothelial cell function. It can relax vascular smooth muscle cells, dilate blood vessels, and balance the body's blood pressure. The cells were treated with ACE inhibitory peptides and captopril at low, medium, and high concentrations, and the amount of NO released in the cell culture medium was measured. Figure 3 As shown. Compared with the blank group, low concentrations of peptide and captopril have shown significant differences (p < 0.05). With the increase of concentration, the amount of NO released increased significantly in a concentration-dependent manner. At the maximum concentration of 0.5 mg / mL, the amount of NO released by ACE inhibitory peptide was 3.21 μmol / L, which was 5.4 times that of the blank group. These results indicate that peptide SeMYHAN can stimulate EA.hy926 cells to release NO and lower blood pressure.

[0047] (4) Determination of endothelin (ET-1) content in cells: ET-1 is a factor secreted by endothelial cells that has vasoconstriction and pressor properties. Excessive ET-1 content can lead to cardiovascular diseases such as atherosclerosis and hypertension. The ET-1 content of cells treated with peptide SeMYHAN and captopril is shown in Figure 2. Figure 4As shown. Compared with the blank group, low, medium and high concentrations of peptides can significantly reduce the ET-1 content of EA.hy926 cells, and its inhibitory ability increases with the increase of concentration. At 0.5 mg / mL, the peptide SeMYHAN can reduce the ET-1 content in cells to 14.95 pg / mL, which is 36.9% lower than the blank group, which is comparable to the antihypertensive effect of the chemical synthetic drug captopril (13.64 pg / mL).

[0048] The above results of in vitro ACE inhibitory activity evaluation show that SeMYHAN exhibits good ACE inhibitory activity. In cell experiments, it was found that SeMYHAN can play a role in lowering blood pressure by increasing the release of NO in cells and reducing the expression of ET-1, and its effect is comparable to the currently commonly used antihypertensive drug captopril. In general, selenium-rich walnut ACE inhibitory peptides can be developed into a dietary supplement that synergizes with chemical drugs to exert antihypertensive activity, which can not only reduce the side effects of drugs on the human body, but also improve the bioavailability of walnut processing by-products.

[0049] Example 3: Molecular docking and visualization analysis

[0050] In order to explore the mechanism of action of peptide SeMYHAN in inhibiting ACE, AutoDock Vina was used to perform molecular docking between peptide SeMYHAN and ACE. The theoretical binding mode of SeMYHAN and ACE is as follows Figure 5As shown. Studies have shown that most ACE inhibitors can bind to the amino acid residues of ACE through hydrogen bonds, hydrophobic interactions, electrostatic interactions, van der Waals forces, etc., among which hydrogen bonds play a very important role in the stability of the complex (Production of antioxidant and ACE-inhibitory peptides from Kluyveromycesmarxianus protein hydrolysates: Purification and molecular docking, 2018). Selenium-enriched walnut peptide SeMYHAN forms 10 hydrogen bonds with the eight amino acid residues of ACE, Thr171, Asn285, Met299, Thr301, Thr302, Asn374, Leu375, Glu376 and Lys449, respectively, forms carbon-hydrogen bonds with Asn285 and Thr301, forms electrostatic forces with Leu375, and forms hydrophobic interactions with Asp453. This result shows that SeMYHAN can form a stable complex with ACE. 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, His353, Lys 511, His 513, and Tyr 520, and S1' contains Glu162 (Shih YH, Discovery and study of novel antihypertensive peptides derived from cassia obtusifolia seeds, 2019). The ACE inhibitor captopril is a competitive inhibitor. Its mechanism of action is to bind to the active pocket of ACE, prevent it from binding to other substrates, and play a role in raising blood pressure. SeMYHAN does not bind to the amino acid residues in the active pocket, and it may be a non-competitive inhibitor. The reason why it exerts ACE inhibitory activity may be that it binds to the inactive pocket site of ACE, making ACE inactive, and even if the substrate binds to ACE, it cannot raise the body's blood pressure. At present, most natural ACE inhibitory peptides are competitive inhibitors. Therefore, selenium-rich walnut peptides can be used as a non-competitive inhibitor in conjunction with competitive antihypertensive drugs such as captopril.

[0051] 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. A selenium-enriched walnut protein angiotensin converting enzyme inhibitory active peptide, characterized in that: It has the following amino acid sequence: Se-Met-Tyr-His-Ala-Asn; wherein Se represents selenocysteine.

2. The selenium walnut protein angiotensin converting enzyme inhibitory active peptide according to claim 1, characterized in that: The active peptide is prepared by enzymatic hydrolysis of selenium-enriched walnuts, solid phase synthesis or genetic engineering technology.

3. A pharmaceutical composition, characterized in that: The composition comprises the active peptide as claimed in claim 1 and a pharmaceutically acceptable excipient.

4. A salt, characterized in that: It is a pharmaceutically acceptable salt of the active peptide described in claim 1.

5. A nucleic acid, characterized in that: The nucleic acid encodes the active peptide described in claim 1.

6. A biomaterial, characterized in that: The biological material comprises the nucleic acid described in claim 5 and is a recombinant DNA, an expression cassette, a transposon, a vector or a host cell.

7. Use of the active peptide according to claim 1, the pharmaceutical composition according to claim 3, the salt according to claim 4, the nucleic acid according to claim 5 or the biomaterial according to claim 6 in the preparation of angiotensin converting enzyme inhibitors.

8. Use of the active peptide according to claim 1, the pharmaceutical composition according to claim 3, the salt according to claim 4, the nucleic acid according to claim 5 or the biomaterial according to claim 6 in the preparation of drugs for preventing and / or treating cardiovascular and cerebrovascular diseases.

9. Use of the active peptide according to claim 1, the pharmaceutical composition according to claim 3, the salt according to claim 4, the nucleic acid according to claim 5 or the biomaterial according to claim 6 in the preparation of a drug for preventing and / or treating hypertension.

10. The use according to any one of claims 7 to 9, characterized in that: When used, the drug is administered orally or parenterally; parenterally, the drug may be administered subcutaneously, intravenously, or anorectally; the injection solution may be prepared using any of physiological saline, glucose, a tranquilizer, a preservative, a suspending agent, or an emulsifier.

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