Endothelium corneum gigeriae galli peptide with ACE inhibitory activity as well as preparation method and application thereof

Abstract

The invention discloses an endothelium corneum gigeriae galli peptide with ACE inhibitory activity and a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The endothelium corneum gigeriae galli peptide is tetrapeptide, the amino acid sequence is SRVW, the molecular weight is 546.29143 Da, and obvious ACE inhibitory activity is achieved. The ACE inhibitory peptide has the beneficial effects that (1) the ACE inhibitory peptide is derived from the traditional Chinese medicinal material endothelium corneum gigeriae galli in China, is a natural ACE inhibitory peptide and is safer to a human body; (2) the molecular weight is small, and absorption by the human body is easier; (3) the IC50 value to ACE is 0.022 mM (0.012 mg / mL), the ACE inhibitory activity is remarkable, the antihypertensive activity of the ACE inhibitory peptide is higher than that of most existing ACE inhibitory peptides, the ACE inhibitory peptide can be applied to preparation of antihypertensive drugs or compositions (for treating hypertension) and antihypertensive food (for preventing hypertension), and the ACE inhibitory peptide has important application value and market prospect in development of antihypertensive functional products.

Description

Technical Field

[0001] The present invention relates to small molecule bioactive peptides and preparation methods and applications thereof, and in particular to a chicken gizzard peptide with ACE inhibitory activity and a preparation method and application thereof, belonging to the technical field of bioactive peptides. Background Art

[0002] Hypertension, a common cardiovascular disease, is a significant risk factor for numerous serious complications, including 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 to angiotensin II, a potent vasoconstrictor. Therefore, inhibiting ACE activity is an effective strategy for treating hypertension. While commonly used ACE inhibitors (such as captopril and enalapril) offer significant antihypertensive effects, they can also cause serious side effects, such as cough, inflammation, and renal impairment. Therefore, the development of novel, highly effective, and low-toxic ACE inhibitors is of vital importance. In recent years, food-derived bioactive peptides have attracted widespread attention due to their wide availability, safety, and multiple physiological regulatory functions, presenting promising prospects for development and application.

[0003] Chicken gizzard lining, also known as chicken yellow skin, is the dried inner wall of the gizzard of the domestic chicken (Phasianidae) and is a traditional Chinese medicinal ingredient. Research has shown that chicken gizzard lining is rich in various bioactive substances and possesses high medicinal and economic value. However, research on bioactive peptides derived from chicken gizzard lining for the treatment of hypertension remains limited, and their potential ACE inhibitory activity needs to be explored urgently. Summary of the Invention

[0004] The purpose of the present invention is to provide a small molecule bioactive peptide (chicken gizzard peptide) derived from chicken gizzard lining and having ACE inhibitory activity, as well as a preparation method and application thereof.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions: The invention discloses 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.

[0006] A method for preparing the aforementioned chicken gizzard peptide having ACE inhibitory activity, using an enzymatic hydrolysis method, comprises the following steps: (1) Add chicken gizzard powder to 1% sodium bicarbonate solution at a ratio of 1 g:10 mL, mix thoroughly, and then ultrasonicate at 37°C to obtain a homogenate of chicken gizzard; (2) The pH value of the homogenate of Gallus gallus was adjusted to 2.0, and simulated gastric fluid was added at a volume ratio of 2:3. The mixture was placed in a shaker at 37°C for 2 h, and then the pH value of the mixture was adjusted to 7.6 with sodium hydroxide solution to obtain Gallus gallus gallus gastric digestion fluid; (3) Add simulated intestinal fluid to the chicken gizzard lining stomach digestive fluid at a volume ratio of 2:3, place the mixture in a shaker at 37°C for 2 h, then place the mixture in a boiling water bath to inactivate the enzyme, centrifuge the inactivated mixture, and collect the supernatant to obtain the chicken gizzard lining intestinal digestive fluid; (4) Ultrafiltration was performed on the chicken gizzard intestinal digestive fluid using an ultrafiltration tube with a molecular weight cutoff of 5 kDa, and the components with a molecular weight <5 kDa were collected and spray-dried to obtain chicken gizzard peptide powder, which contained a tetrapeptide with an amino acid sequence of SRVW.

[0007] The application of the chicken gizzard peptide powder prepared by the above enzymatic hydrolysis method in the preparation of anti-hypertensive food.

[0008] Another method for preparing the aforementioned chicken gizzard peptide having ACE inhibitory activity adopts a solid phase synthesis method, comprising the following steps: The tetrapeptide powder was obtained by solid phase synthesis using Fmoc-protected amino acid as raw material and polystyrene resin as solid phase carrier and adopting Fmoc solid phase synthesis strategy.

[0009] Application of the tetrapeptide powder prepared by the solid phase synthesis method in the preparation of antihypertensive drugs or compositions.

[0010] The present invention is beneficial in that: (1) The chicken gizzard peptide SRVW provided by the present invention is derived from the traditional Chinese medicinal material chicken gizzard, and is a natural ACE inhibitory peptide that is safer for the human body; (2) The chicken gizzard peptide SRVW provided by the present invention is composed of 4 amino acids and has a small molecular weight (546.29143Da), making it more easily absorbed by the human body; (3) IC of the chicken endogenous peptide SRVW provided by the present invention 50 The value is 0.022mM (0.012mg / mL), which has significant ACE inhibitory activity and higher antihypertensive activity than most existing ACE inhibitory peptides. It can be used to prepare antihypertensive drugs or compositions (for the treatment of hypertension) and antihypertensive foods (for the prevention of hypertension). It has important application value and market prospects in the development of antihypertensive functional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a three-dimensional structural diagram of the molecular docking of a peptide with the amino acid sequence RSW and an ACE receptor; Figure 2This is a three-dimensional structural diagram of the molecular docking of a peptide with the amino acid sequence NNPKRW and the ACE receptor; Figure 3 This is a three-dimensional structural diagram of the molecular docking of a peptide with the amino acid sequence SRVW and an ACE receptor; Figure 4 This is a three-dimensional structural diagram of the molecular docking of a peptide with the amino acid sequence RQKW and the ACE receptor; Figure 5 This is a graph showing the inhibition of ACE by a peptide having the amino acid sequence SRVW. DETAILED DESCRIPTION

[0012] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0013] 1. Preparation, separation and purification of chicken gizzard lining digestion products 1. Preparation of Chicken Gizzard Homogenate The dried chicken gizzard slices were placed in a grinder and crushed at high speed to obtain uniform fine powder. The fine powder was sieved using a 100-mesh cell sieve, and the chicken gizzard powder was collected for later use.

[0014] Accurately weigh 1.0 g of chicken gizzard lining powder, add 10 mL of 1% (w / v) sodium bicarbonate solution, mix thoroughly, and ultrasonically treat at 37°C, 3000 W, 40 kHz for 35 min to obtain a chicken gizzard lining homogenate for use.

[0015] 2. Simplified in vitro digestion simulation based on the INFOGEST in vitro digestion model Adjust the pH value of the chicken gizzard lining homogenate to 2.0, add simulated gastric fluid at a volume ratio of 2:3 (take 2.0g sodium chloride and 10.0g pepsin (containing 3-3.5 activity units per mg), add 7mL hydrochloric acid, and add water to make up to 1000mL, so that the pH value of the solution is 2.0). Place the mixture in a 37°C constant temperature shaker and oscillate at 180rpm for 2h (simulating gastric digestion process). Monitor the pH value of the mixture in real time during the experiment and keep it at 2.0. Subsequently, use a sodium hydroxide solution with a concentration of 1mol / L to adjust the pH value of the mixture to 7.6 (to terminate the gastric digestion reaction) to obtain chicken gizzard lining gastric digestion fluid.

[0016] Simulated intestinal fluid (6.8 g potassium dihydrogen phosphate, dissolved in 500 mL of water, and adjusted to pH 7.6 with 0.1 mol / L sodium hydroxide solution) was added to the chicken gizzard lining digestive fluid in a 2:3 volume ratio. Separately, 10.0 g pancreatic enzyme (4.041 activity units per mg) and 60.0 g porcine bile salt were dissolved in appropriate amounts of water. The two solutions were mixed and the volume was adjusted to 1000 mL with water. The mixture was then placed in a 37°C incubator and shaken at 180 rpm for another 2 hours to simulate intestinal digestion. The pH of the mixture was monitored throughout the experiment and maintained at 7.6. The mixture was then placed in a boiling water bath to inactivate the enzymes for 15 minutes (terminating the intestinal digestion reaction). The inactivated mixture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected to obtain the chicken gizzard lining intestinal digestive fluid.

[0017] 3. Separation and purification The chicken gizzard lining intestinal digestive fluid was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 5 kDa, and the components with a molecular weight <5 kDa were collected and spray-dried to obtain chicken gizzard lining peptide powder for later use.

[0018] 2. Sequence Identification and Analysis of Chicken Gynecological Peptides The peptide sequence of the obtained chicken gizzard peptide powder was identified and analyzed using liquid chromatography-tandem Q Exactive HF-X mass spectrometry system (Thermo Fisher).

[0019] A 0.1% (v / v) formic acid aqueous solution was used as solution A, and a 0.1% (v / v) formic acid acetonitrile aqueous solution (the concentration of acetonitrile was 84%, v / v) was used as solution B.

[0020] A reverse phase C18 column (0.15 mm × 150 mm, RP-C18, Column Technology Inc.) was used as the liquid chromatography column.

[0021] The specific mass spectrometry analysis conditions are as follows: detection mode is positive ion, mass spectrometry scanning 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.

[0022] The mass-to-charge ratios of peptides and their fragments were collected as follows: 10 fragmentation spectra (MS2 scans) were collected after each full scan, the MS2 activation type was HCD, the separation window was 2 m / z, and the secondary mass spectrometry resolution was 17500.

[0023] The amino acid sequence of the peptide was determined by comparison with the UniProt database.

[0024] A total of 768 peptides were obtained by mass spectrometry identification.

[0025] 3. Preliminary screening of peptides with potential ACE inhibitory activity through bioinformatics The PeptideRanker tool was used to predict the biological activities of 768 peptides identified by mass spectrometry. The screening threshold was set at 0.5, and peptides with scores > 0.5 were retained as they were considered to have potential ACE inhibitory activity. Peptides with scores < 0.5 were not further screened.

[0026] The amino acid sequences of the peptides were queried for novelty using the UNIPORT and BIOPEP databases to exclude amino acid sequences with existing activity reports and ensure the innovation of the screened peptides.

[0027] The ToxinPred tool was used to predict the potential toxicity and physicochemical properties of peptides and screen non-toxic peptides.

[0028] The AlgPred 2.0 tool was used to assess the allergenic risk of peptides and screen out peptides that were not allergenic.

[0029] The PLifePred tool was used to evaluate the stability of peptides in the blood, and peptides with a half-life value >800s were screened, which were considered to have good stability in the blood.

[0030] The PeptideCutter tool was used to predict the digestion resistance 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 (Chymotrypsin, EC 3.4.21.1) and trypsin (Trypsin, EC 3.4.21.4), the peptide is considered to have the potential to resist gastrointestinal digestion.

[0031] The CPPpred tool was used to predict the cell membrane penetration ability of peptides, and candidate peptides with scores > 0.5 were screened for subsequent experimental verification.

[0032] Of the 768 peptides identified by mass spectrometry, 266 had PeptideRanker scores >0.5 (showing potential ACE inhibitory activity), and 502 had scores <0.5 (not reaching the activity threshold). Further novelty screening of these 266 peptides with potential ACE inhibitory activity revealed 16 previously reported sequences and 250 novel sequences. Based on multi-dimensional screening based on toxicity, allergenicity, digestibility, cell membrane permeability, and stability, four candidate peptides were selected for subsequent molecular docking analysis. Sequence information for these four candidate peptides is shown in Table 1.

[0033] Table 1 Sequence information of 4 candidate peptides obtained from preliminary screening

[0034] 4. Virtual screening of peptides with the strongest ACE inhibitory activity through molecular docking The crystal structure of ACE protein (PDB ID: 1O8A) was obtained from the PDB database (http: / / www.rcsb.org / ).

[0035] The crystal structure of ACE protein was preprocessed using Pymol software, specifically including removing water molecules, zinc ions and chloride ions on the protein.

[0036] The pretreated ACE protein was used as the receptor for molecular docking.

[0037] 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.

[0038] Blind docking analysis was performed using AutoDock Vina software, and molecular visualization technology was used to analyze the binding sites and intermolecular interaction forces between the peptide and ACE protein.

[0039] Binding energy (ΔG) was used as the evaluation index for molecular docking, and the peptide with the lowest binding energy was selected as the optimal candidate peptide for subsequent in vitro activity determination experiments.

[0040] The molecular docking results (binding energy) of the four candidate peptides with ACE are shown in Table 2.

[0041] Table 2 Molecular docking results of 4 candidate peptides with ACE

[0042] The three-dimensional structures of the molecular docking of the four candidate peptides with ACE are shown in Figure 1 、 Figure 2 、 Figure 3 and Figure 4 The visualization results show: The peptide RSW forms 8 hydrogen bonds with GLU-162, ASN-277, THR-282, CYS-370, ASP-377, ASP-415, ASP-453 and LYS-454 ( Figure 1 ); The peptide NNPKRW forms 7 hydrogen bonds with ASN-70, ALA-356, ASP-358, TYR-360, ARG-402 and GLY-404 ( Figure 2 ); 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 ); 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 ).

[0043] It can be seen from Table 2 that the binding energy of polypeptide SRVW to ACE protein is the lowest, indicating that it has the strongest binding affinity to ACE protein.

[0044] Therefore, the peptide SRVW was selected as the optimal candidate peptide for subsequent in vitro activity determination experiments.

[0045] 5. Verification of the in vitro ACE inhibitory activity of the SRVW peptide The optimal candidate peptide SRVW was subjected to solid phase synthesis. Specifically, Fmoc-protected amino acids were used as raw materials, polystyrene resin was used as a solid phase carrier, and Fmoc solid phase synthesis strategy was used for solid phase synthesis to obtain tetrapeptide powder.

[0046] 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.

[0047] 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 FAPGG (N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine) solution was added. The initial absorbance of the control group (a1) and the initial absorbance of the sample group (b1) at a wavelength of 340 nm were immediately measured. The plates were then incubated at 37°C for another 30 min, and the absorbance of the control group (a2) and the absorbance of the sample group (b2) at a wavelength of 340 nm were measured again.

[0048] The calculation formula of ACE inhibitory activity (ACE inhibition rate) is as follows:

[0049] The results of the calculation of the inhibition rate of ACE by peptide SRVW at different concentrations are shown in Figure 5 .

[0050] Depend on Figure 5 It can be seen that the IC of peptide SRVW 50 The value is 0.022mM (0.012mg / mL).

[0051] The above results indicate that the polypeptide SRVW screened in the present invention has good ACE inhibitory activity and can be applied to the field of antihypertensive drugs or compositions, and antihypertensive foods, and can be used to prepare antihypertensive drugs or compositions (for the treatment of hypertension) and antihypertensive foods (for the prevention of hypertension).

[0052] It should be noted that the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A chicken glucoside peptide having ACE inhibitory activity, characterized in that: The chicken gizzard peptide is a tetrapeptide with an amino acid sequence of SRVW and a molecular weight of 546.29143 Da.

2. The method for preparing the chicken glucoside peptide having ACE inhibitory activity according to claim 1, wherein The enzymatic hydrolysis method includes the following steps: (1) Add chicken gizzard powder to 1% sodium bicarbonate solution at a ratio of 1 g:10 mL, mix thoroughly, and then ultrasonicate at 37°C to obtain a homogenate of chicken gizzard; (2) The pH value of the homogenate of Gallus gallus was adjusted to 2.0, and simulated gastric fluid was added at a volume ratio of 2:

3. The mixture was placed in a shaker at 37°C for 2 h, and then the pH value of the mixture was adjusted to 7.6 with sodium hydroxide solution to obtain Gallus gallus gallus gastric digestion fluid; (3) Add simulated intestinal fluid to the chicken gizzard lining stomach digestive fluid at a volume ratio of 2:3, place the mixture in a shaker at 37°C for 2 h, then place the mixture in a boiling water bath to inactivate the enzyme, centrifuge the inactivated mixture, and collect the supernatant to obtain the chicken gizzard lining intestinal digestive fluid; (4) Ultrafiltration was performed on the chicken gizzard intestinal digestive fluid using an ultrafiltration tube with a molecular weight cutoff of 5 kDa, and the components with a molecular weight <5 kDa were collected and spray-dried to obtain chicken gizzard peptide powder, which contained a tetrapeptide with an amino acid sequence of SRVW.

3. Application of the chicken gizzard peptide powder prepared by the method according to claim 2 in the preparation of antihypertensive foods.

4. The method for preparing the chicken glucoside peptide having ACE inhibitory activity according to claim 1, wherein The solid phase synthesis method includes the following steps: The tetrapeptide powder was obtained by solid phase synthesis using Fmoc-protected amino acid as raw material and polystyrene resin as solid phase carrier and adopting Fmoc solid phase synthesis strategy.

5. Use of the tetrapeptide powder prepared by the method of claim 4 in the preparation of antihypertensive drugs or compositions.

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