Angiotensin I converting enzyme inhibitory peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses an angiotensin I converting enzyme inhibitory peptide. According to the present invention, the action mechanisms of two ACE inhibitory peptides H5 (LIIPQH, 719.85 Da) and H6 (LIIPEH, 720.85 Da) extracted, separated and purified from rice wine residue are researched, and the in vitro activity of the ACE inhibitory peptides is researched through an EA.hy926 cell model, such that the two ACE inhibitory peptides can provide the activity effect by promoting the release of NO, the expression of eNOS and SOD, and inhibiting the release secretion of ET-1 and the release of ROS; meanwhile, the cells are subjected to transcriptomics analysis, and the pathway mechanism of polypeptide action is defined. The ACE inhibitory peptide is expected to serve as a potential antihypertensive drug or a functional food ingredient, the high-value utilization of the rice wine residues is improved, and a new application way is provided for the food-borne ACE inhibitory peptide in the aspect of treating hypertension.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and discloses an angiotensin I converting enzyme inhibitory peptide and application thereof. Background Art

[0002] Hypertension is the main cause of cardiovascular disease, which is also the disease with the highest number of deaths worldwide. Therefore, controlling and lowering blood pressure can effectively prevent cardiovascular disease. According to research, many systems in the human body are closely related to the regulation of blood pressure. Antihypertensive drugs can act on any link that affects blood pressure regulation to reduce blood pressure. The mechanism of action of hypertension is complex, and the pathways affect each other. Among them, the most important hypertension regulation system in the human body is the renin-angiotensin system (RAS). ACE inhibitors can reduce the mortality rate of patients with hypertension. Chemical synthetic drugs such as captopril and lisinopril are used clinically to treat hypertension, but they may be accompanied by some side effects. Compared with the side effects of commonly used antihypertensive drugs, food-derived antihypertensive peptides are generally accepted by the public because of their natural sources and no toxic side effects. In addition to daily healthy diet, increasing the intake of fruits, vegetables and high-quality protein can lower blood pressure, and the active peptide fragments contained in protein are also very helpful to human health.

[0003] Rice is one of the most important crops in the world, with high nutritional value. The functional properties of the protein in it are good, and it has biological activities such as antioxidant and blood pressure lowering. However, the current level of development and comprehensive utilization of rice resources is low. Rice wine is one of the ways to process and utilize rice. During the production process, a large amount of fermentation base material, namely rice wine residue, will be produced. Due to its large output and difficult storage characteristics, the main treatment method is currently drying and selling it as feed, and the price is low. Some are even directly discharged as waste, which causes a large amount of protein resources. Waste is not conducive to environmental protection. It is known that part of the protein in rice after fermentation can be converted into polypeptides, which is a good raw material for the development of new polypeptides. Rice wine residue is also rich in various amino acids, vitamins and various trace elements, and is rich in nutrients. Many studies have also proved that high-activity antihypertensive peptides can be extracted, separated and purified from rice wine residue. If rice wine residue can be used at a high value, it can not only improve the economic benefits of by-products, but also reduce environmental pollution, forming a win-win situation. Summary of the invention

[0004] One of the purposes of the present invention is to provide an ACE inhibitory peptide derived from rice wine dregs.

[0005] Another object of the present invention is to provide the use of the above inhibitory peptide.

[0006] The above technical objectives are achieved through the following solutions:

[0007] An angiotensin I converting enzyme inhibitory peptide, whose amino acid sequence is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0008] The present invention also claims to protect the use of the angiotensin I converting enzyme inhibitory peptide in the preparation of therapeutic and preventive hypotensive drugs.

[0009] Furthermore, the drug also includes a pharmaceutically acceptable salt.

[0010] Furthermore, the dosage form of the drug is granules, tablets, capsules, aqueous solutions, suspensions, emulsions, sprays or powders.

[0011] Furthermore, the drug can be added into various foods as health food for suppressing blood pressure.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention studies the mechanism of action of two ACE inhibitory peptides H5 (LIIPQH, 719.85Da) and H6 (LIIPEH, 720.85Da) extracted, separated and purified from rice wine dregs, and explores the in vitro ACE inhibitory activity through the EA.hy926 cell model. It is found that ACE inhibitory peptides H5 and H6 can play an active role by promoting the release of NO, the expression of eNOS and SOD, and inhibiting the secretion of ET-1 and the release of ROS. At the same time, transcriptomics analysis was also performed on the cells to clarify the pathway mechanism of the polypeptide action. The ACE inhibitory peptide is expected to be used as a potential antihypertensive drug or functional food ingredient. The present invention improves the high-value utilization of rice wine dregs and also provides a new application path for food-borne ACE inhibitory peptides in the treatment of hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The structural identification results of antihypertensive peptides H5 and H6 are shown in Figure 1. Figure 1 a is the liquid chromatogram of H5 and H6 and the result of ACE inhibition rate determination. Figure 1 b is the PPSQ sequencing result diagram of H5 and H6;

[0015] Figure 2 The results of the cytotoxicity CCK-8 experiment of the antihypertensive peptides H5 and H6;

[0016] Figure 3 This is the result diagram of the effect of antihypertensive peptides H5 and H6 on cellular NO secretion;

[0017] Figure 4 This is the result diagram of the effect of antihypertensive peptides H5 and H6 on the expression of cellular eNOS;

[0018] Figure 5 This is the result diagram of the effect of antihypertensive peptides H5 and H6 on ET-1 secretion in cells;

[0019] Figure 6 This is the result diagram of the effect of antihypertensive peptides H5 and H6 on the expression of cellular SOD;

[0020] Figure 7 This is the result diagram of the effect of antihypertensive peptides H5 and H6 on cellular ROS release;

[0021] Figure 8 Figure 8 is the result of cell transcriptomics analysis of antihypertensive peptides H5 and H6, where 8a is the differential gene analysis diagram, 8b is the GO pathway analysis result diagram, and 8c is the KEGG pathway analysis result diagram. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0024] 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), penicillin-streptomycin solution, and 0.25% trypsin were provided by Gibco; phosphate buffer saline (PBS) was provided by Hangzhou Jinuo Biomedical Technology Co., Ltd.; dimethyl sulfoxide (DMSO) was provided by Sigma; CCK-8 kit, nitric oxide (NO) kit, BCA kit, SOD kit, and ROS kit were provided by Beyotime; endothelin (ET-1) kit and eNOS kit were provided by Nanjing Jiancheng; other chemicals and reagents were of analytical grade.

[0025] Example 1 Molecular docking simulation and activity evaluation of ACE inhibitory peptides

[0026] 1. Preparation and structural identification of ACE inhibitory peptides

[0027] The samples were collected according to the peak shape of the liquid phase spectrum, and it was found that the ACE inhibition rate of H5 and H6 was significantly higher than that of other components. Figure 1 As shown in a. Then, H5 and H6 components were collected several times, and their N-terminal absolute sequences were detected using PPSQ. The results are shown in Figure 1 As shown in Figure b, the sequences of ACE inhibitory peptides H5 and H6 can be determined to be Leu-Ile-Ile-Pro-Gln-His (LIIPQH) and Leu-Ile-Ile-Pro-Glu-His (LIIPEH), respectively. This is also the first time that peptides LIIPQH and LIIPEH with high ACE inhibitory activity have been isolated from natural products derived from food.

[0028] 2. Evaluation of the antihypertensive activity of antihypertensive peptides H5 and H6 on EA.hy926

[0029] 2.1 Toxicity test

[0030] The cytotoxic effects of antihypertensive peptides at concentrations of 0.01 to 2 mg / mL were evaluated based on the CCK-8 method. Figure 2 As shown. Most concentrations of H5 have the effect of promoting cell proliferation, especially at 0.25 mg / ml. H6 has no significant inhibitory effect on proliferation except at 2 mg / mL. In summary, in order to reduce the impact of cell proliferation on index determination, the two groups of samples were selected in the concentration gradient of 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL for subsequent experiments.

[0031] 2.2 Effects of peptides H5 and H6 on NO release from cells

[0032] The NO content in the cell supernatant was determined using a kit. Figure 3 As shown. All concentration groups of H5 and H6 can promote the release of NO from endothelial cells compared with the Control group, and there is a dependence between the amount of NO released and the sample concentration. The higher the concentration, the more NO is released. Among them, the effects of the high-concentration groups of H5 and H6 on NO content were significantly different from those of the Control group, but not higher than the positive control group. NO release is one of the important indicators for evaluating anti-hypertension. It can act as a vasodilator, prevent the uncontrolled growth and migration of vascular smooth muscle cells, and avoid excessive contraction of blood vessels causing hypertension. The results show that ACE inhibitory peptides can lower blood pressure by affecting NO release.

[0033] 2.3 Effects of peptides H5 and H6 on cellular eNOS expression

[0034] The expression of eNOS in the cell supernatant was determined using an Elisa kit. Figure 4As shown. The low and medium concentration groups of H5 and H6 had no effect on eNOS expression, while the high concentration group increased eNOS expression compared with the control group, and was higher than the positive control group. eNOS is a key regulatory enzyme for vascular homeostasis and intracellular pathways in human endothelial cells. It can catalyze the precursor L-arginine to produce NO, thereby regulating blood pressure, vascular remodeling and angiogenesis. The experimental results of eNOS and NO were similar, confirming that increased eNOS expression would promote the release of NO.

[0035] 2.4 Effects of peptides H5 and H6 on ET-1 release from cells

[0036] The ET-1 release in the cell supernatant was measured using an Elisa kit. Figure 5 As shown. All concentration groups of H5 significantly inhibited the release of ET-1 compared with the Control group, and the medium and high concentration groups of H6 could significantly inhibit the release of ET-1. Among them, the effects of all concentrations of H5 and the high concentration group of H6 were stronger than those of the positive control group. ET-1 is a vasoconstrictor factor, and its overexpression is one of the important causes of hypertension. The results show that ACE inhibitory peptides can treat hypertension by reducing the release of ET-1.

[0037] 2.5 Effects of peptides H5 and H6 on cell SOD expression

[0038] The expression level of SOD in the cell supernatant was measured using a kit. Figure 6 As shown. High concentration of H5 and medium and high concentrations of H6 all significantly promoted the activity of SOD enzyme, among which the promoting effect of H5 high concentration group was higher than that of the positive control group. Increased expression of SOD enzyme can effectively reduce ROS. SOD is an antioxidant enzyme, and its activity is essential for reducing oxidative stress during Ang II activation. At the same time, increased SOD expression can also reduce ROS content. The role of redox signaling in vascular function and hypertension has been confirmed, and antihypertensive drugs can achieve the purpose of lowering blood pressure by promoting the expression of SOD.

[0039] 2.6 Effects of peptides H5 and H6 on cell ROS release

[0040] The cell samples treated with H5 and H6 and installed with fluorescent probes were placed in an incubator and incubated in the dark. The differences in fluorescence intensity were observed under a fluorescence microscope. The results are as follows: Figure 7As shown. It can be roughly judged from the figure that with the increase of H5 and H6 concentrations, the ROS content in endothelial cells shows a downward trend, and the fluorescence intensity is significantly weakened compared with the Control group. The weakening of fluorescence intensity in the positive control group is the most significant. Activating redox signals can affect the regulatory function of endothelial cells. Reduced oxidative capacity may cause vascular dysfunction and induce hypertension. The vascular system has a variety of antioxidant systems, such as the superoxide dismutase (SOD) family, catalase, glutathione, etc. Angiotensin Ang II can stimulate the production of ROS in endothelial cells, leading to endothelial dysfunction and hypertension. In summary, ACE inhibitory peptides can achieve the purpose of lowering blood pressure by increasing SOD expression and reducing ROS levels.

[0041] 2.7 Cellular transcriptomic analysis

[0042] This analysis will be conducted from three aspects: expression level clustering of differentially expressed genes, GO enrichment, and KEGG enrichment. Figure 8 As shown. Figure 8 As can be seen in a, there are differences in genes between the sample treatment group and the Control group. Compared with Control, H5 has 377 differentially expressed genes, of which 198 are downregulated and 179 are upregulated; compared with Control, H6 has 580 differentially expressed genes, of which 322 are downregulated and 258 are upregulated. Figure 8 b analyzed the GO enrichment results. H5 was mainly related to protein peptidyl prolyl isomerization, peptidyl proline modification, ribonucleoprotein complex, mitochondrial part, transmembrane transporter complex, etc., and H6 was mainly related to DNA metabolic process, microtubule movement, cell nucleus, ion channel complex, actin cytoskeleton, etc. Figure 8 c KEGG pathway analysis revealed that among the top 20 enriched pathways, H5 and H6 shared three common pathways, namely, cell cycle, AMPK signaling pathway, and cell senescence; the unique pathway of H5 was the FoxO signaling pathway, and the unique pathway of H6 was programmed cell death. The pathways will be further analyzed in the future to clarify the gene pathway mechanisms involved in the regulation of blood pressure by ACE inhibitory peptides H5 and H6.

[0043] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. An angiotensin I converting enzyme inhibitory peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. Use of the angiotensin I converting enzyme inhibitor peptide according to claim 1 in the preparation of a drug for treating and preventing hypotensive disorders.

3. The use according to claim 2, characterized in that: The medicament also includes pharmaceutically acceptable salts.

4. The use according to claim 2, characterized in that: The dosage form of the drug is granules, tablets, capsules, aqueous solutions, suspensions, emulsions, sprays or powders.

5. The use according to claim 2, characterized in that: The medicine can be added into various foods as health food for suppressing blood pressure.

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