A bovine collagen active peptide, and a preparation method and application thereof

Abstract

The application discloses a bovine collagen active peptide as well as a preparation method and application thereof. The amino acid sequence of the bovine collagen active peptide is selected from SEQ ID NO. 1 to SEQ ID NO. 5, and the bovine collagen active peptide can be used for screening and / or preparation of biomedical materials or medicines for resisting inflammation, oxidation or atherosclerosis. The bovine collagen active peptide disclosed by the application is not only non-toxic and non-sensitizing, but also has good free radical scavenging capacity, Fe 2+ chelating capacity and total antioxidant capacity, and can inhibit inflammation of Raw264.7 macrophages induced by ox-LDL.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioactive peptides, and particularly relates to a bovine collagen active peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Atherosclerosis (AS) is caused by endothelial cell damage caused by high blood lipids, high blood sugar, high blood pressure, etc., and is the cause of cardiovascular diseases, acute myocardial infarction, coronary heart disease, stroke, peripheral necrosis and other diseases. The number of people who die of atherosclerotic cardiovascular diseases accounts for a large part of the number of deaths in the world. The drugs for preventing and treating atherosclerosis are widely used, and common ones are statins or fibrates. Such drugs have side effects in the use process and have a bad impact on the body. For example, statins can cause muscle pain, liver damage and other symptoms, and fibrates can cause vomiting, indigestion and other symptoms.

[0003] Therefore, a safer and more effective drug or natural dietary supplement needs to be developed to prevent atherosclerosis for a long time. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a bovine collagen active peptide as well as a preparation method and application thereof.

[0005] The present application specifically adopts the following technical solutions:

[0006] I. A bovine collagen active peptide

[0007] The amino acid sequence of the bovine collagen active peptide is selected from SEQ ID NO. 1 to SEQ ID NO. 5.

[0008] II. A polynucleotide

[0009] The polynucleotide can encode the bovine collagen active peptide.

[0010] III. An expression vector

[0011] The expression vector comprises the polynucleotide.

[0012] IV. An anti-inflammatory drug

[0013] The anti-inflammatory drug comprises the bovine collagen active peptide, the polynucleotide or the expression vector.

[0014] V. An antioxidant drug

[0015] The antioxidant drug comprises the bovine collagen active peptide, the polynucleotide or the expression vector.

[0016] Six, an anti-atherosclerosis drug

[0017] The drug comprises the above-mentioned active peptide of bovine collagen, the above-mentioned polynucleotide or the above-mentioned expression vector.

[0018] Seven, application of the above-mentioned active peptide of bovine collagen, the above-mentioned polynucleotide or the above-mentioned expression vector in preparation of an anti-inflammatory, anti-oxidation or anti-atherosclerosis biomedical material.

[0019] Eight, application of the above-mentioned active peptide of bovine collagen, the above-mentioned polynucleotide or the above-mentioned expression vector in screening and / or preparation of an anti-inflammatory drug, an anti-oxidation drug or an anti-atherosclerosis drug.

[0020] Nine, a preparation method of the above-mentioned active peptide of bovine collagen

[0021] The preparation method comprises the following steps:

[0022] S1, preparing a bovine dry skin powder solution with a pH of 9.0-10.0;

[0023] S2, adding alkaline protease to the bovine dry skin powder solution, and performing enzymolysis reaction at 45-50 DEG C for 4-6 h, then stopping the enzymolysis reaction to obtain an enzymolysis solution;

[0024] S3, centrifuging the enzymolysis solution to obtain supernatant, and freeze-drying the supernatant to obtain an enzymolysis product;

[0025] S4, separating small-molecule enzymolysis products with a molecular weight of <1 kDa by high-performance liquid chromatography, and separating purified components by using dextran gel chromatography with ultrapure water as an eluent;

[0026] S5, performing desalination treatment on the purified components by using a C18 desalination column, and separating bovine collagen active peptides by using a C18 chromatographic column with pure water containing 0.1% volume percentage trifluoroacetic acid as eluent A and acetonitrile containing 0.1% volume percentage trifluoroacetic acid as eluent B.

[0027] The present application has the following advantages:

[0028] 1. The bovine collagen peptides prepared by the present application have good free radical scavenging capacity, hydroxyl radical scavenging capacity and ferrous ion chelating capacity, and can effectively play an anti-oxidation role.

[0029] 2. The bovine collagen peptides prepared by the present application are obtained by enzymolysis, are non-toxic and non-sensitizing, and have good total antioxidant capacity and an effect of inhibiting the generation of inflammatory factors.

[0030] 3. The enzymatic method adopted by the present application is a process of decomposing biological macromolecular substances into small molecular substances by using the catalytic action of enzymes, which is carried out under mild conditions without high temperature and high pressure. The enzymatic product obtained by the method has high quality and activity, and can improve production efficiency and reduce environmental pollution. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 are mass spectra of the active peptides of bovine dermal collagen in the present application; wherein A~E are bovine dermal collagen active peptide A, bovine dermal collagen active peptide B, bovine dermal collagen active peptide C, bovine dermal collagen active peptide D, and bovine dermal collagen active peptide E, respectively;

[0032] Figure 2 are structural formulas of the active peptides of bovine dermal collagen in the present application; wherein A~E are bovine dermal collagen active peptide A, bovine dermal collagen active peptide B, bovine dermal collagen active peptide C, bovine dermal collagen active peptide D, and bovine dermal collagen active peptide E, respectively;

[0033] Figure 3 are antioxidant capacities of the active peptides of bovine dermal collagen in the present application; wherein A is DPPH free radical scavenging capacity; B is hydroxyl radical scavenging capacity; C is Fe 2+ chelating capacity;

[0034] Figure 4 are total gastrointestinal digestion antioxidant capacities of the active peptides of bovine dermal collagen in the present application;

[0035] Figure 5 are anti-inflammatory capacities of the active peptides of bovine dermal collagen in the present application; wherein A is IL-1β secretion amount; B is TNF-α secretion amount; C is IL-6 secretion amount. DETAILED DESCRIPTION

[0036] The specific implementation of the present application is further described below in combination with the drawings and examples, but the implementation and protection of the present application are not limited thereto. It should be noted that if the following processes are not specifically described in detail, they can be implemented or understood by those skilled in the art with reference to the prior art. If the reagents or instruments used are not specifically described, they are considered to be conventional products that can be purchased on the market.

[0037] The first aspect of the present application provides a bovine dermal collagen active peptide. The amino acid sequence of the bovine dermal collagen active peptide is selected from SEQ ID NO. 1~SEQ ID NO. 5. Specifically:

[0038] The bovine dermal collagen active peptide is one of the following polypeptides:

[0039] A: GPAWR, the amino acid sequence of which is Gly-Pro-Ala-Trp-Arg;

[0040] B; GPWR, whose amino acid sequence is Gly-Pro-Trp-Arg;

[0041] C: KGPWR, whose amino acid sequence is Lys-Gly-Pro-Trp-Arg;

[0042] D: KWCAGPR, whose amino acid sequence is Lys-Trp-Cys-Ala-Gly-Pro-Arg;

[0043] E:LGPRW, with the amino acid sequence Leu-Gly-Pro-Arg-Trp.

[0044] The present invention also provides a polynucleotide capable of encoding the above-mentioned bovine collagen active peptides A to E.

[0045] The present invention also provides an expression vector comprising the above-mentioned polynucleotide. The expression vector enables the overexpression of the above-mentioned polynucleotide.

[0046] Preferably, the expression vector is a plasmid, virus, or bacteriophage.

[0047] The present invention also provides an anti-inflammatory drug comprising the above-mentioned bovine collagen active peptides A-E, the above-mentioned polynucleotides, or the above-mentioned expression vector.

[0048] The present invention also provides an antioxidant drug comprising the above-mentioned bovine collagen active peptides A to E, the above-mentioned polynucleotides, or the above-mentioned expression vector.

[0049] The present invention also provides an anti-atherosclerotic drug comprising the above-mentioned bovine collagen active peptides A-E, the above-mentioned polynucleotides, or the above-mentioned expression vector.

[0050] The present invention also provides the application of the above-mentioned bovine collagen active peptides A~E, the above-mentioned polynucleotides, or the above-mentioned expression vectors in the preparation of anti-inflammatory, antioxidant, or anti-atherosclerotic biomedical materials.

[0051] The present invention also provides the application of the above-mentioned bovine collagen active peptides A~E, the above-mentioned polynucleotides, or the above-mentioned expression vectors in screening and / or preparing anti-inflammatory drugs, antioxidant drugs, or anti-atherosclerotic drugs.

[0052] Bovine collagen active peptides A-E clear the core driving factors of oxidative stress and inhibit ox-LDL production through antioxidant effects, thus blocking endothelial damage initiating AS. They also reduce inflammatory cell infiltration and foam cell formation by inhibiting pro-inflammatory factors such as IL-1β, TNF-α, and IL-6 and their downstream signaling pathways. At the same time, they stabilize plaque structure and improve vascular function, thus intervening in the pathological process of AS driven by oxidative stress and inflammation in multiple dimensions, providing potential mechanistic support for the prevention and adjuvant intervention of AS.

[0053] Preferably, the atherosclerosis is early-stage atherosclerosis.

[0054] This invention also provides a method for preparing the above-mentioned bovine collagen active peptides. The preparation method of this invention includes the following steps:

[0055] S1. Prepare a solution of dried cowhide powder with a pH of 9.0~10.0;

[0056] S2. Add alkaline protease to the cowhide powder solution, and after enzymatic hydrolysis at 45~50℃ for 4~6 h, terminate the enzymatic hydrolysis to obtain the enzymatic hydrolysate.

[0057] S3. After centrifuging the enzyme hydrolysate, take the supernatant and freeze-dry it to obtain the enzyme hydrolysate;

[0058] S4. Small molecule enzymatic hydrolysates with a molecular weight <1 kDa were separated by high performance liquid chromatography, and purified components were obtained by dextran gel chromatography with ultrapure water as the eluent.

[0059] S5. The purified components were desalted using a C18 desalting column, and then the bovine collagen active peptides were separated using a C18 chromatographic column with pure water containing 0.1% (v / v) trifluoroacetic acid as eluent phase A and acetonitrile containing 0.1% (v / v) trifluoroacetic acid as eluent phase B.

[0060] The bovine collagen protein hydrolysate and bovine collagen active peptides provided by this invention both have good DPPH free radical scavenging ability, hydroxyl free radical scavenging ability, ferrous ion chelating ability and total antioxidant capacity.

[0061] Furthermore, the bovine collagen protein hydrolysate and bovine collagen active peptides provided by this invention can both reduce the production of inflammatory factors in cells.

[0062] Furthermore, the inflammatory factors are one or more of the pro-inflammatory factors IL-1β, IL-6, and TNF-α.

[0063] Specific embodiments of the present invention are as follows:

[0064] (I) Preparation of bovine collagen protein hydrolysate

[0065] Take an appropriate amount of dried bovine hide powder and add distilled water at a material-to-liquid ratio of 1:20 (m / v). After thorough mixing, adjust the pH of the solution to the optimal pH of alkaline protease (9.0) using 1 mol / L NaOH. Add alkaline protease at a concentration equivalent to 1% (w / w) of the bovine hide powder and perform enzymatic hydrolysis at 50℃ for 4 h. Measure the degree of hydrolysis during this period. After the reaction is complete, heat in a boiling water bath for 15 min to terminate the enzymatic hydrolysis. After the hydrolysate cools, centrifuge at 11000 rpm for 15 min, collect the supernatant, freeze-dry it to obtain bovine hide collagen protease hydrolysate powder, and store it at -20℃ for later use.

[0066] Calculate the degree of hydrolysis (%) of each enzymatic hydrolysis product using the following formula:

[0067] Degree of hydrolysis (%) = [(B·N)] B ) / (M·h tot ·α)]×100%

[0068] Where B is the volume (mL) of NaOH consumed; N B The concentration of NaOH is (mol / L); M is the total protein mass (g); h tot The total number of peptide bonds in the substrate (mmol / g); α is the average degree of dissociation of α-NH2 released during hydrolysis, and the value of α can be calculated according to the following formula:

[0069] α=10 pH-pK / (1+10 pH-pK )

[0070] In the formula, pH is the actual pH value of the enzymatic hydrolysate, and pK is the average pK value of the α-NH2 groups released during hydrolysis. The pK value is temperature-dependent and can be calculated using the following formula:

[0071] pK = 7.8 + [(298-T) / (298T)] × 2400

[0072] In the formula, T represents temperature.

[0073] Calculations show that the degree of hydrolysis of the hydrolysate obtained by alkaline protease hydrolysis is 8.72.

[0074] Molecular weight distribution was determined by high-performance liquid chromatography (HPLC) using a TSKgel G2000 SWXL 300 mm × 7.8 mm column. Sample concentration: 1 mg / mL; injection volume: 10 μL; mobile phase: 45% acetonitrile + 55% trifluoroacetic acid solution (containing 0.1% TFA); flow rate: 0.5 mL / min; temperature: 25℃; detection wavelength: 214 nm; detection time: 25 min; standard: cytochrome C (M... w=12400 Da), aprotinin (M) w =6511.44 Da), bacitracin (M w =1422.69 Da), Arg-Pro-Pro-Leu-Pro (M w =578.69 Da), Gly-Gly-Gly (M w =189.17 Da).

[0075] Calculations showed that the molecular weight of the enzymatic hydrolysates obtained by alkaline protease hydrolysis was mainly distributed between 100 and 4000 Da. The molecular weight components with a molecular weight of less than 100 Da, 100-1000 Da, 1000-4000 Da, and greater than 4000 Da accounted for 3.04±0.02%, 72.43±0.09%, 23.44±0.03%, and 1.10±0.05% of all peptide components, respectively.

[0076] (II) Screening of bovine collagen peptides

[0077] The obtained alkaline protease hydrolysate solution with a molecular weight <1 kDa was separated and purified by dextran gel chromatography using Sephadex G-15 as the dextran gel. 100 g of Sephadex G-15 was weighed and dissolved in 500 mL of water, and swollen at room temperature for 48 h. After swelling, the supernatant was removed. The swollen Sephadex G-15 was packed into a 1.6 cm × 100 cm column, allowing the gel to settle freely until the liquid level in the packing material no longer decreased. A 3 mL sample with a concentration of 100 mg / mL was prepared, filtered, and loaded onto the column. The eluent was ultrapure water, the flow rate was 0.6 mL / min, and the elution wavelength was 280 nm. The separated fractions were collected, freeze-dried, and stored at -20°C for later use.

[0078] A suitable amount of purified bovine collagen fraction was desalted using a C18 desalting column, and then analyzed by LC-MS / MS equipped with an online nanospray ionization source. The entire system was an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, USA) with EASY-nanoLC 1200 in series. A total of 3 μL of sample was loaded (C18 column: 20 cm × 75 μm id, 1.9 μm particle size). The bovine collagen active peptides were separated using pure water (containing 0.1% v / v trifluoroacetic acid) in phase A and acetonitrile (containing 0.1% v / v trifluoroacetic acid) in phase B as eluents. A gradient separation was performed over 60 min, with the column flow rate controlled at 350 nL / min, the column temperature at 40°C, and the electrospray voltage at 2 kV. The gradient started with 2.2% phase B, increased non-linearly to 90% in 54.5 min, increased to 99% within 0.5 min, and maintained for 5 min.

[0079] The mass spectrometer operates in data-dependent acquisition mode and automatically switches between MS and MS / MS acquisition. The mass spectrometry parameters are set as follows: (1) MS: Scan range (m / z): 200~1500; resolution: 60000; Normalized AGC target: 300%; maximum injection time: 25 ms; (2) HCD-MS / MS: resolution: 15000; Normalized AGC target: 50%; maximum injection time: 22 ms; collision energy 30%; dynamic exclusion time: 30 s.

[0080] The retention time, peak area, peak area percentage, and peak height of the bovine collagen active peptides A-E of this invention are shown in Table 1 below:

[0081] Table 1. Retention time, peak area, peak area percentage, and peak height of peptides

[0082]

[0083] For peptides obtained from mass spectrometry analysis, novel anti-AS peptides were screened using an activity virtual screening tool, including the following steps: PeptideRanker was used to predict the probability of peptide bioactivity, and peptides with a score > 0.85 were screened for further analysis. CPPpred was used to assess the probability of peptide penetration into cells, and peptides with a score > 0.5 were screened for further analysis. AllergenFP v1.1 was used to predict the potential sensitization of the screened peptides, and peptides predicted to be non-sensitizing were selected for further analysis. ToxinPred was used to predict the potential toxicity of the screened peptides, and peptides predicted to be non-toxic were selected for further analysis. AdmetSAR was used to predict and analyze the hydrophobicity, amphiphilicity, and isoelectric point of key peptides.

[0084] The amino acid sequences of bovine collagen active peptides A to E of the present invention are shown in SEQ ID NO.1 to SEQ ID NO.5, respectively.

[0085] The mass spectra of bovine collagen active peptides A-E of this invention are as follows: Figure 1 As shown in A~E, the structural formulas are respectively as follows Figure 2 As shown in Figures A through E, the basic physicochemical properties are shown in Table 2 below:

[0086] Table 2 Basic physicochemical properties of polypeptides

[0087] SEQ ID NO Peptide sequence Length (n) Molecular weight (Da) Allergenicity Toxicity BBB Caco-2 permeability Hydrophobicity Amphiphilicity pI A GPAWR 5 585.64 No No BBB-(0.9009) 0.8647 -0.21 0.49 10.11 B GPWR 4 514.57 No No BBB-(0.9593) 0.7844 -0.33 0.61 10.11 C KGPWR 5 642.74 No No BBB-(0.6917) 0.8381 -0.48 1.22 11.01 D KWCAGPR 7 816.95 No No BBB+(0.5824) 0.8405 -0.30 0.87 9.55 E LGPRW 5 627.72 No No BBB+(0.5895) 0.7995 -0.15 0.49 10.11

[0088] In the table, BBB represents the likelihood of a drug crossing the blood-brain barrier. BBB+ indicates a stronger ability to cross the blood-brain barrier, while BBB- indicates a weaker ability. The larger the value in parentheses, the greater the likelihood of crossing. Caco-2 permeability represents the ability of a drug to penetrate Caco-2 cells; the higher the value, the stronger the intestinal absorption.

[0089] (III) Antioxidant activity of bovine skin collagen peptides

[0090] 1. Determination of DPPH free radical scavenging capacity, including the following steps: Weigh 0.0079 g of DPPH (2,2-Diphenyl-1-picrylhydrazyl, 1,1-diphenyl-2-trinitrophenylhydrazine) and dissolve it in anhydrous ethanol, then dilute to 100 mL to obtain a 0.2 mmol / L DPPH-anhydrous ethanol solution, which is stored in the dark. Mix 1.5 mL of the sample (1 mg / mL) with 1.5 mL of the DPPH-anhydrous ethanol (0.2 mmol / L) solution and store in the dark for 30 min. Measure the absorbance at 517 nm using a UV spectrophotometer. Calculate the scavenging capacity according to the following formula:

[0091] DPPH free radical scavenging capacity (%) = {[1-(A1-A2)] / A0} × 100%

[0092] Wherein, A1 is the absorbance of a mixture of 1.5 mL sample solution and 1.5 mL DPPH-anhydrous ethanol solution; A2 is the absorbance of a mixture of 1.5 mL sample solution and 1.5 mL anhydrous ethanol solution; and A0 is the absorbance of a mixture of 1.5 mL DPPH-anhydrous ethanol solution and 1.5 mL anhydrous ethanol solution.

[0093] 2. Determination of hydroxyl radical scavenging ability, including the following steps: Weigh 0.25 g FeSO4, and dilute to 100 mL with distilled water to obtain a 9 mmol / L FeSO4 solution. Weigh 0.12 g salicylic acid, and dilute to 100 mL with anhydrous ethanol to obtain a 9 mmol / L salicylic acid-anhydrous ethanol solution. Measure 1 mL of 30% hydrogen peroxide, dilute to 100 mL with distilled water, and dilute tenfold before use to obtain an 8.8 mmol / L hydrogen peroxide solution. Mix 2 mL of sample (1 mg / mL) with 2 mL FeSO4 (9 mmol / L), 2 mL H2O2 (8.8 mmol / L), and 2 mL salicylic acid-anhydrous ethanol solution. After incubation at 37°C for 30 min, measure the absorbance at 510 nm using a UV spectrophotometer. Calculate the scavenging activity using the following formula:

[0094] Hydroxyl radical scavenging capacity (%) = {[A0] ’ -(A1 ’ -A2 ’ )] / A0 ’}×100%

[0095] Among them, A1 ’ This represents the absorbance of a mixture of 2 mL FeSO4 solution, 2 mL salicylic acid-anhydrous ethanol solution, 2 mL sample solution, and 2 mL H2O2 solution; A2 ’ Indicates the absorbance of the solution in the absence of H2O2; A0 ’ This indicates the absorbance of the solution when no sample is present.

[0096] 3. Metal ions Fe 2+The chelating ability was determined using the following steps: 0.04 g of FeCl2 was weighed and diluted to 100 mL with distilled water to obtain a 2 mmol / L FeCl2 solution. 0.025 g of phenanthroline was weighed and diluted to 10 mL with distilled water to obtain a 5 mmol / L phenanthroline solution. 60 μL of the sample (1 mg / mL) was mixed with 18 μL of FeCl2 solution (2 mmol / L) and 505 μL of distilled water. After incubation at 37°C for 15 min, 61 μL of phenanthroline solution (5 mmol / L) was added, and the mixture was incubated at room temperature for 10 min. The absorbance at 562 nm was measured using a microplate reader. The chelating ability was calculated using the following formula:

[0097] Fe 2+ Chelation capacity (%) = [(A0) ’’ -A1 ’’ ) / A0 ’’ ]×100%

[0098] Among them, A1 ’’ Indicates the absorbance of the solution in the presence of the sample; A0 ’’ This indicates the absorbance of the solution when no sample is present.

[0099] Experimental results are as follows Figure 3 As shown, all five peptides exhibit good DPPH free radical scavenging ability. Figure 3 A) Hydroxyl radical scavenging ability ( Figure 3 B), Fe 2+ Chelating ability ( Figure 3 (C). Among them, KWCAGPR and LGPRW peptides have the best antioxidant effects.

[0100] 4. Simulated gastrointestinal digestion experiment, including the following steps: Prepare simulated gastric juice SGF and simulated intestinal juice SIF according to the Chinese Pharmacopoeia. SGF: Mix 16.4 mL of 9.8% (w / w) hydrochloric acid with 800 mL of water, add 10 g of pepsin, and dilute the solution to 1000 mL (pH 1.5) with water; SIF: Dissolve 6.8 g of KH₂PO₄ in 500 mL of water, adjust the pH of the solution to 6.8 with 0.1 mol / L NaOH, add 10 g of trypsin, and dilute to 1000 mL with water. Mix 1 mg / mL peptide with SGF at a ratio of 1:4 (v / v), incubate at 37℃ for 2 h, and collect 1 mL of gastric digestion fluid every 0.5 h. Add 0.1 mol / L NaOH to the remaining digested liquid to adjust the pH of the reaction mixture to 7.0. Mix the above reaction solution with SIF at a ratio of 1:3 (v / v) and incubate at 37°C for 2 h. Take 1 mL of gastrointestinal digested liquid every 0.5 h. Terminate digestion by boiling in a water bath for 10 min. Centrifuge the reaction solution at 5000 g for 5 min and collect the supernatant.

[0101] 5. Total antioxidant capacity determination, including the following steps: Adjust the concentration of the supernatant sample to 0.25 mg / mL. Add 10 μL of supernatant sample to each well of a 96-well plate, add 10 μL of distilled water to each blank control well, and add 10 μL of Trolox standard solutions of various concentrations to each standard curve well. Add 200 μL of ABTS working solution to each detection well. Mix gently and incubate at room temperature for 2–6 min. Measure the absorbance at 734 nm using a microplate reader.

[0102] Total antioxidant capacity (mmol / g) = Trolox concentration corresponding to A734 / sample concentration

[0103] The total antioxidant capacity of the five peptides is as follows Figure 4 As shown, all five peptides exhibited good total antioxidant capacity after simulating gastrointestinal digestion, indicating that their antioxidant capacity was not weakened in vivo. Among them, KWCAGPR and LGPRW peptides had the highest total antioxidant capacity.

[0104] (iv) Bovine skin collagen peptides improve ox-LDL-induced lipid oxidation model in RAW264.7 macrophages

[0105] Cell culture was performed using DMEM high-glucose medium containing DMEM basal medium, 10% fetal bovine serum, and 1% penicillin-dextrose antibody, and incubated at 37°C in a 5% CO2 incubator. When the cell density reached 80%–90%, the cells were passaged. The old medium was discarded, and the cells were gently rinsed with 3 mL of PBS. Then, 4 mL of basal medium was added and the cells were thoroughly resuspended by pipetting. The cell suspension was centrifuged at 1200 rpm for 3 min, the medium was discarded, and 2 mL of complete medium was added for resuspending. The cells were then transferred to a new T25 culture flask, and 3 mL of complete medium was added before incubation.

[0106] The levels of inflammatory factors IL-1β, IL-6, and TNF-α in cell supernatant were determined using a double-antibody sandwich assay. Serum was centrifuged at 3000 rpm for 15 min, and the supernatant was collected. Different concentrations of standard wells, blank wells, and sample wells were prepared on an ELISA plate. In the sample wells, 40 μL of sample dilution was added to the ELISA plate, followed by 10 μL of the sample to be tested, and the mixture was gently shaken to mix. The plate was sealed with a sealing film and incubated at 37°C for 30 min. After incubation, the sealing film was carefully removed, the liquid was discarded, and the plate was shaken dry. Each well was filled with washing buffer, allowed to stand for 30 s, and then discarded. This process was repeated 5 times, and the plate was then patted dry. 50 μL of enzyme-labeled reagent was added to each well, except for the blank wells. The incubation and washing steps were repeated. After incubation, 50 μL of chromogenic reagent A was added to each well, followed by 50 μL of chromogenic reagent B, and the mixture was gently shaken to mix. The plate was then incubated at 37°C in the dark for 15 min. The reaction was stopped by adding 50 μL of stop solution to each well, at which point the liquid in the well changed from blue to yellow. The absorbance at 450 nm was measured using a blank well as the zeroing setting. The contents of IL-1β, IL-6, and TNF-α in the sample were calculated based on the standard curve.

[0107] The results of the ox-LDL-induced Raw 264.7 mouse monocyte-macrophage experiment are as follows: Figure 5 As shown in the figure. K, M, GA, GP, KG, KW, and LG represent the blank control group, ox-LDL induction model group, peptide GPAWR treatment group, peptide GPWR treatment group, peptide KGPRW treatment group, peptide KWCAGPR treatment group, and peptide LGPRW treatment group, respectively. Different letters indicate significant differences between groups (p<0.05), while the same letter indicates no significant difference (p≥0.05).

[0108] It is evident that all five peptides can effectively reduce the pro-inflammatory factor IL-1β (IL-1β). Figure 5 A), TNF-α ( Figure 5 B), IL-6 ( Figure 5 The generation of C). Among them, KWCAGPR and LGPRW have significant anti-inflammatory effects.

[0109] Therefore, the mechanism by which peptides improve early atherosclerosis is as follows: bovine collagen peptides clear the core driving factors of oxidative stress and inhibit ox-LDL production through antioxidant effects, thus blocking endothelial damage that initiates atherosclerosis (AS); and reduce inflammatory cell infiltration and foam cell formation by inhibiting pro-inflammatory factors such as IL-1β, TNF-α, and IL-6 and their downstream signaling pathways; at the same time, they stabilize plaque structure and improve vascular function, thus intervening in the pathological process of AS driven by oxidative stress and inflammation in multiple dimensions, providing potential mechanistic support for the prevention and adjuvant intervention of AS.

[0110] The amino acid sequence of the antibody involved in this invention is as follows:

[0111] SEQ ID NO.1:

[0112] Name: Bovine Skin Collagen Active Peptide A

[0113] Sequence type: AA

[0114] Biological origin: Bos taurus

[0115] GPAWR

[0116] SEQ ID NO.2:

[0117] Name: Bovine Skin Collagen Active Peptide B

[0118] Sequence type: AA

[0119] Biological origin: Bos taurus

[0120] GPWR

[0121] SEQ ID NO.3:

[0122] Name: Bovine Skin Collagen Active Peptide C

[0123] Sequence type: AA

[0124] Biological origin: Bos taurus

[0125] KGPWR

[0126] SEQ ID NO.4:

[0127] Name: Bovine Skin Collagen Active Peptide D

[0128] Sequence type: AA

[0129] Biological origin: Bos taurus

[0130] KWCAGPR

[0131] SEQ ID NO.5:

[0132] Name: Bovine Skin Collagen Active Peptide E

[0133] Sequence type: AA

[0134] Biological origin: Bos taurus

[0135] LGPRW.

Claims

1. An active peptide of bovine collagen, characterized in that: The amino acid sequence of the bovine collagen active peptide is SEQ ID NO.

5.

2. A polynucleotide, characterized in that: It can encode the bovine collagen active peptide as described in claim 1.

3. An expression vector, characterized in that: It contains the polynucleotide as described in claim 2.

4. An anti-inflammatory drug, characterized in that: It comprises the bovine collagen active peptide as described in claim 1, the polynucleotide as described in claim 2, or the expression vector as described in claim 3.

5. An antioxidant drug, characterized in that: It comprises the bovine collagen active peptide as described in claim 1, the polynucleotide as described in claim 2, or the expression vector as described in claim 3.

6. The use of the bovine collagen active peptide as described in claim 1, the polynucleotide as described in claim 2, or the expression vector as described in claim 3 in the preparation of an anti-atherosclerotic drug.

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