A chicken gizzard peptide with ACE inhibitory activity, its preparation method and application

Abstract

This invention discloses a chicken gizzard membrane peptide with ACE inhibitory activity, its preparation method, and its application, belonging to the field of bioactive peptide technology. This chicken gizzard membrane peptide is a tetrapeptide with the amino acid sequence SRVW and a molecular weight of 546.29143 Da, exhibiting significant ACE inhibitory activity. The advantages of this invention are: (1) it is derived from chicken gizzard membrane, a traditional Chinese medicine, and is a natural ACE inhibitory peptide, making it safer for the human body; (2) its small molecular weight makes it easier for the human body to absorb; and (3) it inhibits ACE at IC50 levels. 50 With a value of 0.022 mM (0.012 mg / mL), it exhibits significant ACE inhibitory activity and has a higher antihypertensive activity than most existing ACE inhibitory peptides. It can be used to prepare antihypertensive drugs or compositions (for treating hypertension) and antihypertensive foods (for preventing hypertension), and has important application value and market prospects in the development of antihypertensive functional products.

Description

Technical Field

[0001] This invention relates to small molecule bioactive peptides, their preparation methods and applications, specifically to a chicken gizzard peptide with ACE inhibitory activity, its preparation method and applications, belonging to the field of bioactive peptide technology. Background Technology

[0002] Hypertension, a common cardiovascular disease, is a significant risk factor for various serious complications such as coronary heart disease, heart failure, stroke, and atherosclerosis. The renin-angiotensin-aldosterone system plays a crucial role in the molecular mechanisms of blood pressure regulation. Angiotensin-converting enzyme (ACE), a core component of this system, catalyzes the conversion of angiotensin I into angiotensin II, which has a potent vasoconstrictive effect. Therefore, inhibiting ACE activity is one of the effective strategies for treating hypertension. While commonly used ACE inhibitors (such as captopril and enalapril) have significant antihypertensive effects, their use may cause serious side effects, such as cough, inflammation, or kidney damage. Therefore, developing novel, highly effective, and low-toxicity ACE inhibitors is of great importance. In recent years, food-derived bioactive peptides have attracted widespread attention due to their wide availability, high safety, and multiple physiological regulatory functions, showing broad prospects for development and application.

[0003] Chicken gizzard lining, also known as chicken yellow skin, is the dried inner lining of the gizzard of the domestic chicken (Pheasantidae family) and is one of my country's traditional medicinal materials. Studies have shown that chicken gizzard lining is rich in various bioactive substances and has high medicinal and economic value. However, research on bioactive peptides derived from chicken gizzard lining in the treatment of hypertension is currently lacking, and its potential ACE inhibitory activity urgently needs to be developed. Summary of the Invention

[0004] The purpose of this invention is to provide a small molecule bioactive peptide (chicken gizzard peptide) derived from chicken gizzard membrane with ACE inhibitory activity, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A chicken gizzard peptide with ACE inhibitory activity, wherein the chicken gizzard peptide is a tetrapeptide with an amino acid sequence of SRVW and a molecular weight of 546.29143 Da.

[0007] A method for preparing the aforementioned chicken gizzard peptide with ACE inhibitory activity, employing an enzymatic hydrolysis method, includes the following steps:

[0008] (1) Add chicken gizzard powder to a 1% sodium bicarbonate solution at a ratio of 1g:10mL, mix thoroughly, and then sonicate at 37℃ to obtain chicken gizzard homogenate.

[0009] (2) Adjust the pH of the chicken gizzard homogenate to 2.0, add simulated gastric juice at a volume ratio of 2:3, place the mixture in a shaker at 37°C and shake to digest for 2 hours, then use sodium hydroxide solution to adjust the pH of the mixture to 7.6 to obtain chicken gizzard gastric digestion solution;

[0010] (3) Add simulated intestinal fluid to the chicken gizzard digestion solution at a volume ratio of 2:3. Place the mixture in a shaker at 37°C for 2 hours to digest. Then place the mixture in a boiling water bath to inactivate the enzyme. Centrifuge the enzyme-inactivated mixture and collect the supernatant to obtain the chicken gizzard digestion solution.

[0011] (4) Use an ultrafiltration tube with a molecular weight cutoff of 5 kDa to ultrafilter the chicken gizzard intestinal digestive fluid, collect the components with a molecular weight <5 kDa, spray dry them to obtain chicken gizzard peptide powder, which contains a tetrapeptide with the amino acid sequence SRVW.

[0012] The application of chicken gizzard peptide powder prepared by the aforementioned enzymatic hydrolysis method in the preparation of antihypertensive food.

[0013] Another method for preparing the aforementioned chicken gizzard peptide with ACE inhibitory activity employs a solid-phase synthesis method, comprising the following steps:

[0014] Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, a solid-phase synthesis strategy based on Fmoc was adopted to obtain tetrapeptide powder.

[0015] The application of the tetrapeptide powder prepared by the aforementioned solid-phase synthesis method in the preparation of antihypertensive drugs or compositions.

[0016] The advantages of this invention are:

[0017] (1) The chicken gizzard peptide SRVW provided by this invention is derived from chicken gizzard, a traditional Chinese medicine. It is a natural ACE inhibitory peptide and is safer for the human body.

[0018] (2) The chicken gizzard peptide SRVW provided by the present invention is composed of 4 amino acids and has a small molecular weight (546.29143 Da), making it easier for the human body to absorb.

[0019] (3) The IC of chicken gizzard peptide SRVW provided by the present invention 50 With a value of 0.022 mM (0.012 mg / mL), it exhibits significant ACE inhibitory activity and has a higher antihypertensive activity than most existing ACE inhibitory peptides. It can be used to prepare antihypertensive drugs or compositions (for treating hypertension) and antihypertensive foods (for preventing hypertension), and has important application value and market prospects in the development of antihypertensive functional products. Attached Figure Description

[0020] Figure 1 This is a three-dimensional structural diagram of the molecular docking of a polypeptide with the amino acid sequence RSW with an ACE receptor.

[0021] Figure 2 This is a three-dimensional structural diagram of the molecular docking of a polypeptide with the amino acid sequence NNPKRW with the ACE receptor.

[0022] Figure 3 This is a three-dimensional structural diagram of the molecular docking of a polypeptide with the amino acid sequence SRVW with the ACE receptor.

[0023] Figure 4 This is a three-dimensional structural diagram of the molecular docking of a polypeptide with the amino acid sequence RQKW with the ACE receptor.

[0024] Figure 5 This is a curve showing the inhibition of ACE by a polypeptide with the amino acid sequence SRVW. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0026] I. Preparation, Separation and Purification of Chicken Gizzard Membrane Digestion Products

[0027] 1. Preparation of chicken gizzard homogenate slurry

[0028] Place the dried chicken gizzard lining slices in a pulverizer and crush them at high speed to obtain a uniform fine powder. Use a 100-mesh cell sieve to sieve the fine powder and collect the chicken gizzard lining powder for later use.

[0029] Accurately weigh 1.0g of chicken gizzard powder, add 10mL of 1% (w / v) sodium bicarbonate solution, mix thoroughly, and then sonicate at 37℃, 3000W, and 40kHz for 35min to obtain a chicken gizzard homogenate for later use.

[0030] 2. Simplified in vitro digestion simulation based on the INFOOGSEST in vitro digestion model

[0031] The pH of the chicken gizzard homogenate was adjusted to 2.0. Simulated gastric juice (prepared by adding 2.0 g sodium chloride and 10.0 g pepsin (containing 3-3.5 activity units per mg) at a volume ratio of 2:3, along with 7 mL hydrochloric acid and water to a final volume of 1000 mL, with a pH of 2.0) was then placed in a 37°C constant-temperature shaker and digested at 180 rpm for 2 hours (simulating gastric digestion). The pH of the mixture was monitored in real-time during the experiment and maintained at 2.0. Subsequently, the pH of the mixture was adjusted to 7.6 using a 1 mol / L sodium hydroxide solution (to terminate the gastric digestion reaction), yielding the chicken gizzard homogenate gastric digestion solution.

[0032] Simulated intestinal fluid (6.8 g potassium dihydrogen phosphate dissolved in 500 mL of water, pH adjusted to 7.6 with 0.1 mol / L sodium hydroxide solution; 10.0 g trypsin (containing 4.041 activity units per mg) and 60.0 g porcine bile salts dissolved in water; the two solutions were mixed and diluted to 1000 mL with water) was placed in a 37°C constant temperature shaker and shaken at 180 rpm for 2 hours (simulating intestinal digestion). The pH of the mixture was monitored in real time during the experiment and maintained at 7.6. The mixture was then placed in a boiling water bath for 15 minutes to inactivate the enzyme (terminating the intestinal digestion reaction). The enzyme-inactivated mixture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected to obtain the chicken gizzard intestinal digestion fluid.

[0033] 3. Separation and purification

[0034] The chicken gizzard lining intestinal digestive fluid was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 5 kDa. Components with a molecular weight <5 kDa were collected, spray-dried, and then chicken gizzard lining peptide powder was obtained for later use.

[0035] II. Identification and Analysis of Chicken Gizzard Peptide Sequences

[0036] The obtained chicken gizzard peptide powder was analyzed for peptide sequence identification using a liquid chromatography-tandem Q Exactive HF-X mass spectrometry system (Thermo Fisher).

[0037] Solution A is a 0.1% (v / v) formic acid aqueous solution, and solution B is a 0.1% (v / v) formic acid acetonitrile aqueous solution (acetonitrile concentration is 84%, v / v).

[0038] The liquid chromatography column used is a reverse-phase C18 column (0.15 mm × 150 mm, RP-C18, Column Technology Inc.).

[0039] The specific mass spectrometry analysis conditions are as follows: detection mode is positive ion, mass spectrometry scan range is 300-1800 m / z, primary mass spectrometry resolution is 70000, and AGC (Automatic Gain Control) target is 3e. 6 The maximum injection time is 10ms, and the dynamic exclusion time is 40.0s.

[0040] The peptide and fragment mass-charge ratio of the peptide were acquired as follows: 10 fragment spectra were acquired after each full scan (MS2 scan), with HCD activation type, separation window of 2 m / z, and secondary mass spectrometry resolution of 17500.

[0041] The amino acid sequence of the peptide was determined by comparison with the database (UniProt).

[0042] A total of 768 polypeptides were obtained by mass spectrometry identification.

[0043] III. Preliminary screening of peptides with potential ACE inhibitory activity using bioinformatics.

[0044] The bioactivity of 768 peptides obtained by mass spectrometry was predicted using the PeptideRanker tool. A screening threshold of 0.5 was set, and peptides with a score >0.5 were retained as having potential ACE inhibitory activity. Peptides with a score <0.5 were not further screened.

[0045] The novelty of the amino acid sequences of peptides was searched using the UNIPORT and BIOPEP databases to exclude amino acid sequences with existing activity reports, ensuring the novelty of the peptides screened.

[0046] The ToxinPred tool was used to predict the potential toxicity and physicochemical properties of peptides, and non-toxic peptides were screened.

[0047] The AlgPred 2.0 tool was used to assess the sensitization risk of peptides and screen for peptides that do not have sensitizing properties.

[0048] The stability of peptides in blood was assessed using the PLifePred tool. Peptides with a half-life > 800 s were screened and considered to have good stability in blood.

[0049] The PeptideCutter tool was used to predict the digestibility of peptides. If the amino acid sequence of a peptide does not contain sites that can be cleaved by pepsin (Pepsin PH 1.3 and PH>2.0, EC 3.4.23.1), chymotrypsin (EC 3.4.21.1), and trypsin (EC 3.4.21.4), then the peptide is considered to have the potential to resist gastrointestinal digestion.

[0050] The cell membrane penetration ability of peptides was predicted using the CPPpred tool, and candidate peptides with a score >0.5 were screened for subsequent experimental validation.

[0051] Of the 768 peptides identified by mass spectrometry, 266 had a PeptideRanker score >0.5 (possessing potential ACE inhibitory activity), while 502 had a score <0.5 (not reaching the activity threshold). Further novelty screening of the 266 peptides with potential ACE inhibitory activity revealed 16 previously reported sequences and 250 novel sequences. Based on multi-dimensional screening considering toxicity, allergenicity, digestibility, cell membrane permeability, and stability, four candidate peptides were ultimately selected for subsequent molecular docking analysis. The sequence information of the four candidate peptides is shown in Table 1.

[0052] Table 1. Sequence information of the four candidate peptides obtained from the initial screening.

[0053]

[0054] IV. Virtual screening of peptides with the strongest ACE inhibitory activity via molecular docking

[0055] The crystal structure of the ACE protein (PDB ID: 1O8A) was obtained from the PDB database (http: / / www.rcsb.org / ).

[0056] The crystal structure of the ACE protein was pretreated using Pymol software, specifically by removing water molecules, zinc ions, and chloride ions from the protein.

[0057] The pretreated ACE protein was used as the acceptor for molecular docking.

[0058] The three-dimensional structures of the four candidate peptides in Table 1 were constructed using Chem 3D software, and the optimal molecular conformation after energy minimization optimization was used as the ligand for molecular docking.

[0059] Blind docking analysis was performed using AutoDock Vina software, and molecular visualization techniques were used to analyze the binding sites and intermolecular interactions between peptides and ACE proteins.

[0060] Using binding energy (ΔG) as an evaluation index for molecular docking, the peptide with the lowest binding energy was selected as the optimal candidate peptide for subsequent in vitro activity assays.

[0061] The molecular docking results (binding energies) of the four candidate peptides with ACE are shown in Table 2.

[0062] Table 2. Molecular docking results of four candidate peptides with ACE.

[0063]

[0064] The three-dimensional structures of the molecular docking of the four candidate peptides with ACE are shown in the figures below. Figure 1 , Figure 2 , Figure 3 and Figure 4 The visualization results show:

[0065] The peptide RSW forms eight hydrogen bonds with GLU-162, ASN-277, THR-282, CYS-370, ASP-377, ASP-415, ASP-453, and LYS-454. Figure 1 );

[0066] The peptide NNPKRW forms seven hydrogen bonds with ASN-70, ALA-356, ASP-358, TYR-360, ARG-402, and GLY-404. Figure 2 );

[0067] The peptide SRVW forms 11 hydrogen bonds with GLU-162, HIS-353, THR-372, GLU-376, ASP-377, ASP-415, ASP-453, and HIS-513. Figure 3 );

[0068] The peptide RQKW forms 11 hydrogen bonds with ASN-70, SER-355, ALA-356, TYR-360, TYR-394, ARG-402, and TYR-523. Figure 4 ).

[0069] As shown in Table 2, the peptide SRVW has the lowest binding energy to ACE protein, indicating that it has the strongest binding affinity to ACE protein.

[0070] Therefore, peptide SRVW was selected as the optimal candidate peptide for subsequent in vitro activity assays.

[0071] V. Verification of the in vitro ACE inhibitory activity of peptide SRVW

[0072] The optimal candidate peptide SRVW was synthesized in a solid-phase manner. Specifically, Fmoc-protected amino acids were used as raw materials, polystyrene resin was used as a solid-phase support, and the Fmoc solid-phase synthesis strategy was adopted to obtain tetrapeptide powder.

[0073] The tetrapeptide powder was prepared into sample solutions with concentrations of 1 mM, 0.25 mM, 0.1 mM, 0.05 mM, 0.025 mM, and 0.0125 mM, respectively.

[0074] For the sample group, 10 μL of ACE solution (0.1 U / mL, dissolved in 0.1 mol / L sodium borate buffer (pH=8.3, containing 0.3 M sodium chloride)) and 20 μL of sample solutions of different concentrations were added to a 96-well microplate. For the control group, 10 μL of ACE solution and 20 μL of ultrapure water were added to a 96-well microplate. After mixing, the plates were incubated at 37 °C for 10 min. Then, 50 μL of 0.8 mmol / L FAPPGG (N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine) solution was added. The initial absorbance of the control group (a1) and the sample group (b1) at a wavelength of 340 nm were measured immediately. After incubation at 37 °C for another 30 min, the absorbance of the control group (a2) and the sample group (b2) at a wavelength of 340 nm were measured again.

[0075] The formula for calculating ACE inhibitory activity (ACE inhibition rate) is as follows:

[0076]

[0077] The calculated inhibition rates of different concentrations of the peptide SRVW on ACE are shown in the figure. Figure 5 .

[0078] Depend on Figure 5 It can be seen that the IC50 of the peptide SRVW 50 The value was 0.022 mM (0.012 mg / mL).

[0079] The above results indicate that the peptide SRVW obtained by screening in this invention has good ACE inhibitory activity and can be applied to the fields of antihypertensive drugs or compositions and antihypertensive foods, for the preparation of antihypertensive drugs or compositions (for the treatment of hypertension) and antihypertensive foods (for the prevention of hypertension).

[0080] It should be noted that the above embodiments of the present invention 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 embodiments here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing chicken gizzard peptides with ACE inhibitory activity, wherein the amino acid sequence of the chicken gizzard peptides is SRVW, characterized in that, The enzymatic hydrolysis method includes the following steps: (1) Add chicken gizzard powder to a 1% sodium bicarbonate solution at a ratio of 1g:10mL, mix thoroughly, and then sonicate at 37℃ to obtain chicken gizzard homogenate. (2) Adjust the pH of the chicken gizzard homogenate to 2.0, add simulated gastric juice at a volume ratio of 2:3, place the mixture in a shaker at 37°C and shake at 180 rpm for 2 hours to simulate the gastric digestion process. Monitor the pH of the mixture in real time during the digestion process and keep it at 2.

0. Then use sodium hydroxide solution to adjust the pH of the mixture to 7.6 to terminate the gastric digestion reaction and obtain chicken gizzard gastric digestion solution. The simulated gastric juice is prepared by taking 2.0 g sodium chloride and 10.0 g pepsin, adding 7 mL hydrochloric acid, and adding water to make up to 1000 mL. Each 1 mg of pepsin contains 3-3.5 activity units. (3) Add simulated intestinal fluid to the chicken gizzard digestion solution at a volume ratio of 2:

3. Place the mixture in a shaker at 37°C and shake at 180 rpm for 2 hours to simulate intestinal digestion. Monitor the pH of the mixture in real time during digestion and keep it at 7.

6. Then place the mixture in a boiling water bath to inactivate enzymes for 15 minutes to terminate the intestinal digestion reaction. Centrifuge the enzyme-inactivated mixture at 5000 rpm for 10 minutes and collect the supernatant to obtain the chicken gizzard digestion solution. The simulated intestinal fluid is prepared as follows: take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve, adjust the pH to 7.6 with 0.1 mol / L sodium hydroxide solution, take 10.0 g of pancreatic enzyme and 60.0 g of pig bile salt, add an appropriate amount of water to dissolve, the pancreatic enzyme contains 4.041 activity units per 1 mg, mix the two solutions, and add water to make up to 1000 mL. (4) Use an ultrafiltration tube with a molecular weight cutoff of 5 kDa to ultrafilter the chicken gizzard intestinal digestive fluid, collect the components with a molecular weight <5 kDa, spray dry them to obtain chicken gizzard peptide powder, which contains a tetrapeptide with the amino acid sequence SRVW.

2. A method for preparing chicken gizzard peptide with ACE inhibitory activity, wherein the amino acid sequence of the chicken gizzard peptide is SRVW, characterized in that, The solid-phase synthesis method includes the following steps: Using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid-phase carrier, a solid-phase synthesis strategy based on Fmoc was adopted to obtain tetrapeptide powder.

3. The use of the tetrapeptide powder prepared by the method of claim 2 in the preparation of antihypertensive drugs or compositions.

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