A walnut protein-derived angiotensin-converting enzyme selenium-enriched peptide, its preparation method, and its application in lowering blood pressure.

Abstract

This invention discloses a selenium-enriched walnut protein angiotensin-converting enzyme (ACE) inhibitory peptide, its preparation method, and its applications. The peptide SeMYHAN (682.1934 Da) provided by this invention forms 10 hydrogen bonds with 8 amino acid residues on ACE and may act as a non-competitive inhibitor binding to ACE (IC50). 50 =0.123 mg / mL). Furthermore, SeMYHAN increased NO release and decreased intracellular ET-1 expression in EA.hy926 cells, validating its antihypertensive effect at the cellular level. In conclusion, the selenium-enriched walnut ACE inhibitory peptide SeMYHAN shows promise as a good antihypertensive drug, and also provides a new theoretical basis for the high-value utilization of natural organic selenium.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to a walnut protein-derived angiotensin-converting enzyme selenium-enriched peptide, its preparation method, and its application in lowering blood pressure. Background Technology

[0002] Walnuts, belonging to the walnut family, are a food with both medicinal and culinary uses. They are rich in fatty acids, proteins, vitamins, and minerals, possessing high nutritional and economic value. However, current walnut resource development and utilization primarily focus on primary agricultural products and oil extraction. As a byproduct of oil extraction, the protein in walnut meal is not efficiently utilized, significantly limiting the economic benefits and added value of the walnut industry.

[0003] Selenium (Se) is an essential trace element for the human body, possessing various physiological functions such as anti-cancer activity, immunomodulatory effects, prevention of cardiovascular and cerebrovascular diseases, and antioxidant effects. Insufficient selenium intake is closely related to human diseases, easily leading to cancer, Keshan disease, immune system disorders, and thyroid hormone dysfunction. Epidemiological studies indicate that selenium deficiency affects up to one billion people worldwide. Selenium exists in both inorganic and organic forms, but natural organic selenium is characterized by low toxicity, diverse biological activities, and high bioavailability, generally offering more advantages than inorganic selenium. Therefore, how to supplement natural selenium through food resources has become a research hotspot in recent years.

[0004] Hypertension, as one of the most common chronic cardiovascular and cerebrovascular diseases, seriously affects human health. It is estimated that by 2025, the number of people with hypertension worldwide will exceed 1.5 billion. Angiotensin I can be converted into angiotensin II, which causes vasoconstriction, by angiotensin-converting enzyme (ACE). ACE also inactivates bradykinin, which has a vasodilatory effect, ultimately leading to elevated blood pressure. Therefore, to prevent and treat hypertension, ACE inhibitors can be used to reduce the activity of ACE, preventing it from acting on angiotensin I. However, some patients taking chemically synthesized ACE inhibitors experience varying degrees of side effects.

[0005] Therefore, food-derived 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 objectives of the present invention is to provide a selenium-enriched walnut protein angiotensin-converting enzyme inhibitory active peptide.

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

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

[0010] Furthermore, the active peptides are prepared by enzymatic hydrolysis of selenium-enriched walnuts, solid-phase synthesis, or genetic engineering techniques.

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

[0012] A salt that is pharmaceutically acceptable for the aforementioned active peptide.

[0013] A nucleic acid that encodes the aforementioned active peptide.

[0014] A biomaterial comprising the aforementioned nucleic acid, wherein the biomaterial is recombinant DNA, an expression cassette, a transposon, a vector, or a host cell.

[0015] The application of the above-mentioned active peptides, pharmaceutical compositions, salts, nucleic acids or biological materials in the preparation of angiotensin-converting enzyme inhibitors.

[0016] The use of the above-mentioned active peptides, pharmaceutical compositions, salts, nucleic acids or biological materials in the preparation of drugs for the prevention and / or treatment of cardiovascular and cerebrovascular diseases.

[0017] The use of the above-mentioned active peptides, pharmaceutical compositions, salts, nucleic acids or biological materials in the preparation of drugs for the prevention and / or treatment of hypertension.

[0018] The above-mentioned preparations or drugs can be made into various formulations, and can be administered orally or non-orally. Non-oral administration can be done by subcutaneous injection, intravenous injection, or rectal administration. The preparation of the injection solution can use any of the following: physiological saline, glucose, stabilizers, preservatives, suspending agents, or emulsifiers.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] The peptide SeMYHAN (682.1934 Da) provided by this invention forms 10 hydrogen bonds with 8 amino acid residues on ACE, and may act as a non-competitive inhibitor binding to ACE (IC50). 50=0.123 mg / mL). Furthermore, SeMYHAN increased NO release and decreased intracellular ET-1 expression in EA.hy926 cells, validating its antihypertensive effect at the cellular level. In conclusion, the selenium-enriched walnut ACE inhibitory peptide SeMYHAN shows promise as a good antihypertensive drug, and also provides a new theoretical basis for the high-value utilization of natural organic selenium. Attached Figure Description

[0021] Figure 1 The figure shows the results of the study on the inhibitory effect of peptide SeMYHAN on ACE.

[0022] Figure 2 Figure shows the results of a study on the effect of peptide SeMYHAN on the activity of EA.hy926 cells.

[0023] Figure 3 Figure 1 shows the results of a study on the effect of peptide SeMYHAN on NO secretion in EA.hy926 cells.

[0024] Figure 4 Figure 1 shows the results of a study on the effect of peptide SeMYHAN on ET-1 secretion in EA.hy926 cells.

[0025] Figure 5 The image shows the results of the visualization analysis of the peptide SeMYHAN-ACE complex; where A is the 3D structure of the complex and B is the interaction between the peptide and ACE amino acid residues. Detailed Implementation

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

[0027] The angiotensin-converting enzyme (ACE, derived from rabbit lung), hydroxyethylpiperazine ethanesulfonic acid (HEPES), furanylacryl 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), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco, USA; and all other chemicals and reagents were of analytical grade.

[0028] Example 1: Synthesis of ACE-inhibiting peptides

[0029] The selenium-enriched walnut ACE inhibitory peptide SeMYHAN was synthesized by Nanjing Synpeptide Co., Ltd. The purity of the synthesized peptide was determined to be 98% by high-performance liquid chromatography (HPLC), and its molecular weight was determined by LC / ESI-MS. Its ACE inhibitory activity was verified through in vitro and cellular 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 40 μL of 80 mmol / L HEPES buffer was mixed with 50 μL of FAPPG for the blank group. 10 μL of 0.1 U / mL ACE was added to both groups simultaneously. Immediately after adding ACE, the absorbance of both groups was measured at a wavelength of 340 nm. The blank group was recorded as A1, and the experimental group as B1. After incubation at 37℃ in the dark, the absorbance of both groups was measured again at 340 nm and recorded as A2 and B2, respectively. The obtained data 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 cells EA.hy926 were cultured in complete medium (DMEM containing 20% ​​fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin solution). The culture medium was stored at 37°C in a 5% carbon dioxide environment. Cytotoxicity was determined by the MTT assay. 100 μL of cell suspension was seeded into each well of a white 96-well plate to obtain a seeding density of 1 × 10⁶ cells per well. 4Cells were cultured at 37°C for 24 hours. To avoid changes in the surrounding environment, PBS was added to the outermost well of the plate. After 24 hours of cell culture, the medium was changed. 100 μL of complete culture 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 captopril of the same concentration was added to the control group for 24 hours of further culture. 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. After discarding the MTT solution, the cells were washed with PBS and 100 μL of DMSO was added. The cells were shaken thoroughly for 10 minutes and the OD value was measured at 490 nm using a microplate reader. The cell viability 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] 100 μL of cell suspension was seeded into each well of a white 96-well plate to obtain a seeding density of 1 × 10⁶ cells per well. 4 Cells were collected and incubated at 37°C for 24 hours. After 24 hours, the medium was changed. The normal group received 100 μL of complete culture medium, the experimental groups received 100 μL of peptides at different concentrations (0.1, 0.25, 0.5 mg / mL), and the control group received the same concentration of captopril. Incubation was continued for 24 hours. Cell supernatant was collected and stored for later analysis. NO content in the cells was determined using a NO assay kit.

[0039] 2.4 Determination of Endothelin (ET-1) Content

[0040] 100 μL of cell suspension was seeded into each well of a white 96-well plate to obtain a seeding density of 1 × 10⁶ cells per well. 4 Cells were collected and incubated at 37°C for 24 hours. After 24 hours, the medium was changed. The normal group received 100 μL of complete culture medium, the experimental groups received 100 μL of peptides at different concentrations (0.1, 0.25, 0.5 mg / mL), and the control group received the same concentration of captopril. Incubation was continued for 24 hours. After 24 hours, the cell culture medium was centrifuged at 1000 x g, and the supernatant was collected for analysis. The ET-1 content in the cells was determined using the ET-1 kit.

[0041] 2.5 Statistical Analysis

[0042] Statistical analysis was performed using SPSS 20.0 software. All experiments were repeated three times, and results are expressed as mean ± standard deviation. One-way ANOVA was then performed. p A value <0.05 indicates that the difference is significant.

[0043] 2.6 Results and Analysis

[0044] (1) Assay for inhibitory peptide activity: such as Figure 1 As shown, the ACE inhibitory activity IC of peptide SeMYHAN 50 With a concentration of 0.102 mg / mL, it exhibits strong ACE inhibitory activity and can be used as an ACE inhibitor in conjunction with antihypertensive drugs. Its activity is significantly higher than that of the previously reported selenium-enriched tea protein ACE inhibitory peptide SeTAPep (IC50). 50 =13.39 mg / mL). Therefore, we will continue to investigate the effects of SeMYHAN on NO and ET-1 secretion in EA.hy926 cells.

[0045] (2) Cytotoxicity detection of selenium-enriched walnut ACE inhibitory peptide: The MTT assay was used to investigate the effect of peptide SeMYHAN on EA.hy926 cells. The results are as follows: Figure 2 As shown, within a concentration range of 0.025-0.5 mg / mL, both the active peptide and the positive control captopril maintained cell viability above 95%, indicating that neither was cytotoxic. Based on the experimental results, low-dose, medium-dose, and high-dose groups 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 blood pressure. Cells were treated with low, medium, and high concentrations of ACE inhibitory peptide and captopril, and the NO release in the cell culture medium was measured as follows: Figure 3 As shown in the figure, low concentrations of peptides and captopril already showed significant differences compared to the control group. p <0.05). With increasing concentration, NO release significantly increased in a concentration-dependent manner. At the maximum concentration of 0.5 mg / mL, the NO release from the ACE inhibitory peptide was 3.21 μmol / L, which was 5.4 times that of the control group. These results indicate that the peptide SeMYHAN can stimulate EA.hy926 cells to release NO and lower blood pressure.

[0047] (4) Determination of endothelin-1 (ET-1) content in cells: ET-1 is a factor secreted by vascular endothelial cells with vasoconstrictive and pressor properties. Excessive ET-1 levels can lead to cardiovascular diseases such as atherosclerosis and hypertension. The ET-1 content in cells treated with peptide SeMYHAN and captopril is as follows: Figure 4As shown in the figure, compared with the control group, low, medium, and high concentrations of peptides all significantly reduced the ET-1 content in EA.hy926 cells, and their inhibitory ability increased with increasing concentration. At 0.5 mg / mL, the peptide SeMYHAN reduced the ET-1 content in cells to 14.95 pg / mL, a reduction of 36.9% compared with the control group, which is comparable to the antihypertensive effect of the chemically synthesized drug captopril (13.64 pg / mL).

[0048] The results of the in vitro ACE inhibitory activity evaluation above indicate that SeMYHAN exhibits good ACE inhibitory activity. In cell experiments, it was found that SeMYHAN exerts its antihypertensive effect by increasing NO release and decreasing ET-1 expression, with effects comparable to the commonly used antihypertensive drug captopril. In summary, selenium-enriched walnut ACE inhibitory peptides can be developed into a dietary supplement that synergistically enhances the antihypertensive activity of chemical drugs. This would not only reduce the side effects of drugs on the human body but also improve the bioavailability of walnut processing byproducts.

[0049] Example 3: Molecular docking and visualization analysis

[0050] To investigate the mechanism of action of the peptide SeMYHAN in inhibiting ACE, AutoDock Vina was used to perform molecular docking between 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 ACE amino acid residues through hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. Among these, hydrogen bonds play a crucial role in the stability of the complex (Production of antioxidant and ACE-inhibitory peptides from Kluyveromycesmarxianus protein hydrolysates: Purification and molecular docking, 2018). The selenium-enriched walnut peptide SeMYHAN forms 10 hydrogen bonds with eight amino acid residues of ACE: Thr171, Asn285, Met299, Thr301, Thr302, Asn374, Leu375, Glu376, and Lys449. It also forms carbon-hydrogen bonds with Asn285 and Thr301, electrostatic interactions with Leu375, and hydrophobic interactions with Asp453. This result indicates that SeMYHAN can form a stable complex with ACE. Studies have shown that ACE has three active pockets: S1, S2, and S1'. 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 obtusifoliaseeds, 2019). Captopril, an ACE inhibitor, is a competitive inhibitor. Its mechanism of action is to bind to the active pockets of ACE, preventing it from binding to other substrates and thus increasing blood pressure. SeMYHAN, however, does not bind to amino acid residues in the active pockets, suggesting it may be a non-competitive inhibitor. Its ACE-inhibiting activity may stem from binding to inactive pocket sites on ACE, rendering it inactive and preventing blood pressure increases even when substrates bind to ACE. Currently, most naturally occurring ACE-inhibiting peptides are competitive inhibitors. Therefore, selenium-enriched walnut peptides can be used as a non-competitive inhibitor in synergy with competitive antihypertensive drugs such as captopril.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A selenium-enriched walnut protein angiotensin-converting enzyme inhibitory peptide, characterized in that: The amino acid sequence is as follows: Se-Met-Tyr-His-Ala-Asn; where Se represents selenocysteine.

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

3. A pharmaceutical composition, characterized in that: It comprises the active peptide as described 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 biomaterial comprises the nucleic acid described in claim 5, which is recombinant DNA, expression cassette, transposon, vector, or host cell.

7. The use of the active peptide of claim 1, the pharmaceutical composition of claim 3, the salt of claim 4, the nucleic acid of claim 5, or the biomaterial of claim 6 in the preparation of a drug for the prevention and / or treatment of hypertension.

8. The application according to claim 7, characterized in that: When using this product, it can be administered orally or non-orally. Non-oral administration includes subcutaneous injection, intravenous injection, or rectal administration. The injection solution can be prepared using physiological saline, glucose, stabilizers, preservatives, suspending agents, or emulsifiers.

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