An antioxidant peptide derived from mouse hemoglobin and application thereof

Abstract

The application discloses an antioxidant peptide derived from mouse hemoglobin and application thereof, and the amino acid sequence of the antioxidant peptide is LAKADKRAVGAEALAR, and the molecular weight is 1652.9686 g / mol. The polypeptide is identified from mouse hemoglobin by high performance liquid chromatography for the first time, and an unreported antioxidant peptide with antioxidant activity is screened out. The antioxidant peptide derived from mouse hemoglobin is synthesized by a solid phase method, and has the characteristics of safety, non-toxicity, good water solubility and stability. After being combined with vitamin B1, the antioxidant property of the antioxidant peptide is significantly improved, and the antioxidant peptide can be widely applied to preparation of food, cosmetics, medicines or health products, and has important economic value and social significance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to an antioxidant peptide derived from mouse hemoglobin and its applications. Background Technology

[0002] Mouse hemoglobin is the most abundant iron-containing protein in mouse erythrocytes, responsible for transporting oxygen and carbon dioxide in the circulatory system. Its basic structure is similar to human hemoglobin, consisting of two pairs of globin subunits (commonly α and β chains), each containing a heme group that can reversibly bind an oxygen molecule. Gene expression of mouse hemoglobin is tissue-specific and developmentally stage-dependent, with its synthesis primarily occurring in bone marrow erythroid cells. As a classic model organism, mouse hemoglobin is not only of significant reference value in the study of anemia and hemoglobinopathies (such as sickle cell disease and thalassemia), but is also widely used to evaluate the effects of drugs, nutritional interventions, or functional factors on the hematopoietic system and oxidative stress. Furthermore, the structural stability, oxygen affinity, and antioxidant properties of mouse hemoglobin molecules are important parameters in physiological regulation research and disease model construction. Therefore, systematically analyzing the structural and functional characteristics of mouse hemoglobin is helpful for a deeper understanding of the oxygen transport mechanisms in mammals and the pathogenesis of related diseases.

[0003] Antioxidant peptides are a class of small peptides derived from the degradation of natural proteins, typically composed of 2 to 20 amino acids. They possess multiple biological functions, including scavenging free radicals, inhibiting lipid peroxidation, chelating metal ions, and regulating intracellular antioxidant enzyme systems (such as SOD, CAT, and GSH-Px). Their antioxidant activity mainly derives from the electron-donating ability and free radical stability conferred by specific amino acid residues (such as His, Tyr, Cys, and Trp), effectively alleviating cell damage caused by oxidative stress both in vitro and in vivo. In recent years, the development of functional peptides using hemoglobin as a raw material has attracted attention, especially mouse hemoglobin, a typical iron porphyrin protein in model animals, which has a well-defined structure and stable source, possessing potential advantages for developing antioxidant peptides. However, no antioxidant peptides based on mouse hemoglobin have been reported to date. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antioxidant peptide derived from mouse hemoglobin and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] One of the technical solutions adopted by this invention to solve its technical problem is:

[0007] An antioxidant peptide derived from mouse hemoglobin is provided, with the amino acid sequence LAKADKRAVGAEALAR, as shown in SEQ ID NO: 1.

[0008] The antioxidant peptide has a molecular weight of 1652.9686 g / mol, a positive charge of +2, an isoelectric point (pI) of 10.67, and good water solubility.

[0009] The present invention also provides a nucleic acid encoding the above-mentioned antioxidant peptide, an expression vector containing the nucleic acid, and a recombinant cell containing the expression vector.

[0010] The present invention also provides the application of the above-mentioned antioxidant peptides or their encoded nucleic acids in the preparation of antioxidant products.

[0011] Preferably, the product is one or more of cosmetics, health products, or pharmaceuticals.

[0012] Preferably, the above-mentioned antioxidant peptides are used as active ingredients or additives.

[0013] The second technical solution adopted by this invention to solve its technical problem is:

[0014] An antioxidant is provided, the antioxidant comprising the aforementioned antioxidant peptide and vitamin B1.

[0015] The antioxidants of the present invention can be synthesized using methods known to those skilled in the art, such as solid-phase synthesis, and purified using methods known to those skilled in the art, such as high-performance liquid chromatography.

[0016] Implementing this invention has the following beneficial effects:

[0017] The antioxidant peptides of this invention are safe, non-toxic, have good water solubility, stability, and high in vitro antioxidant activity, making them suitable for preparing antioxidant products. Furthermore, the binding energy of the composite antioxidant agent of the antioxidant peptides and vitamin B1 to DPPH and ABTS free radicals is lower than that of either peptide alone, indicating that the antioxidant capacity is significantly enhanced after mixing with vitamin B1. This composite antioxidant agent possesses high antioxidant activity and has broad application potential in antioxidant health products, pharmaceuticals, and cosmetics. Attached Figure Description

[0018] Figure 1 This is a mass spectrometry analysis diagram of the antioxidant peptide of this invention.

[0019] Figure 2 This is a schematic diagram of the 3D structure of the antioxidant peptide of the present invention.

[0020] Figure 3The results show the DPPH free radical scavenging capacity of vitamin B1, the antioxidant peptide of the present invention, and the composite antioxidant of the antioxidant peptide of the present invention and vitamin B1.

[0021] In this invention, the antioxidant peptides and vitamin B1 are mixed in a 1:1 ratio, with concentration gradients of 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL.

[0022] Figure 4 The results show the ABTS free radical scavenging capacity of vitamin B1, the antioxidant peptide of the present invention, and the compound antioxidant of the antioxidant peptide of the present invention and vitamin B1.

[0023] Figure 5 The results show the hydroxyl radical scavenging capacity of vitamin B1, the antioxidant peptide of the present invention, and the compound antioxidant agent of the antioxidant peptide of the present invention and vitamin B1.

[0024] Figure 6 The present invention relates to the determination of the iron ion reducing power of vitamin B1, antioxidant peptides, and the complex antioxidant agent of antioxidant peptides and vitamin B1.

[0025] Figure 7 This is a diagram showing the docking results of vitamin B1 with DPPH free radical molecules.

[0026] Figure 8 This is a diagram showing the docking results of the antioxidant peptides of this invention with DPPH free radical molecules.

[0027] Figure 9 This is a diagram showing the docking results of the antioxidant peptide and vitamin B1 composite antioxidant of the present invention with DPPH free radical molecules.

[0028] Figure 10 This is a diagram showing the docking results of vitamin B1 with ABTS free radical molecules.

[0029] Figure 11 This is a diagram showing the docking results of the antioxidant peptide of this invention with ABTS free radical molecules.

[0030] Figure 12 This is a diagram showing the docking results of the antioxidant peptide and vitamin B1 composite antioxidant of the present invention with ABTS free radical molecules. Detailed Implementation

[0031] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Structural identification of antioxidant peptides derived from mouse hemoglobin

[0035] 1. Sample pretreatment: Weigh 1 mg of mouse hemoglobin lyophilized powder, add 200 μL of 0.1% TFA (trifluoroacetic acid) aqueous solution, sonicate homogenize, centrifuge at 14000 g for 10 min, collect the supernatant, ultrafilter the supernatant using a 10 kDa ultrafiltration tube (PALL, OD010C35), centrifuge at 13500 g for 10 min, and use a nanodrop 2000C UV-Vis spectrophotometer (thermo) for peptide quantification. Desalt the filtrate using a C18 solid-phase extraction column (66871, Sigma). The specific steps are as follows: first wash the extraction column with acetonitrile, then equilibrate the extraction column with 0.1% TFA, load the ultrafiltered sample solution, and elute the peptides with 70% acetonitrile solution.

[0036] 2. LC-MS / MS detection: Solution A used in the liquid chromatography was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile content was 84%).

[0037] The liquid chromatography column (0.15 mm * 150 mm, RP-C18, Column Technology Inc.) was equilibrated with 95% solution A. The sample was loaded into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) via an autosampler and then separated by the liquid chromatography column. The relevant liquid gradient settings are as follows:

[0038] From 0 minutes to 50 minutes, the linear gradient of solution B increased from 4% to 50%.

[0039] From 50 to 54 minutes, the linear gradient of solution B increased from 50% to 100%.

[0040] For 54 to 60 minutes, solution B remained at 100%.

[0041] 3. The enzymatic hydrolysis products were separated by capillary high-performance liquid chromatography (HPLC) and then analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (ThermoFisher). The analysis time was 60 min. The detection method was positive ion. The mass-charge ratio of the peptide and peptide fragments was collected using the following method: 10 fragment spectra were collected after each full scan (MS2 scan).

[0042] 4. Database Search: The raw mass spectrometry test file was searched in the relevant database using MaxQuant 1.5.5.1 software to obtain the protein identification and quantitative analysis results. The mass spectrum of the antioxidant peptide LAKADKRAVGAEALAR (as shown in SEQ ID NO: 1) is shown below. Figure 1 As shown.

[0043] Example 2: Bioinformatics of antioxidant peptides derived from mouse hemoglobin

[0044] The test steps are as follows:

[0045] 1. Prediction of Potential Biological Activity of Peptides: The potential biological activity of obtained peptide sequences was analyzed using the Peptide Ranker online platform (http: / / distilldeep.ucd). Peptides were ranked according to their predicted biological activity probability. The preset threshold of the Peptide Ranker prediction model was 0.5; a threshold greater than 0.5 was considered to indicate biological activity.

[0046] 2. Using Expasy The pI / Mw tool (https: / / web.expasy.org / computepi / ) and the PepDraw server (https: / / pepcalc.com) analyzed the isoelectric point (pI) and water solubility of peptides.

[0047] 3. Using the online tool Toxinpred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html), the potential toxicity of the new peptide was predicted based on the SVM (Swiss-Port) algorithm. After potential toxicity analysis, the antioxidant peptide LAKADKRAVGAEALAR was identified as non-Toxin (non-toxic). Its physicochemical properties are shown in Table 1. The sequence was then used for subsequent synthesis and verification.

[0048] Table 1 Physicochemical properties of antioxidant peptides

[0049]

[0050] 4. The three-dimensional structure of the antioxidant peptide LAKADKRAVGAEALAR was predicted using Pymol 2.0 and Swiss-Model. A schematic diagram of its 3D structure is shown below. Figure 2 As shown.

[0051] Example 3: In vitro antioxidant activity of vitamin B1, the antioxidant peptide of the present invention, and a compound antioxidant of the antioxidant peptide of the present invention and vitamin B1.

[0052] 1. Determination of DPPH free radical scavenging ability

[0053] The antioxidant peptides and vitamin B1 of this invention were prepared into solutions with different concentration gradients (31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL) using distilled water, with a mixing ratio of 1:1. 100 μL of each of the different concentrations of the antioxidant peptide solution, vitamin B1 solution, and mixed solution were added, along with 100 μL of DPPH ethanol solution (the DPPH stock solution was diluted with anhydrous ethanol to 0.1 mol / L, resulting in an absorbance range of 0.6-1 at 517 nm). After mixing, the solutions were incubated in the dark at room temperature for 30 min, and the absorbance at 517 nm was measured using a microplate reader. The experimental results are as follows: Figure 3 As shown in the figure, vitamin B1 has a weak ability to scavenge DPPH free radicals. The antioxidant peptide of this invention has a DPPH free radical scavenging ability of 30% to 60%. When the two are mixed, the antioxidant capacity is significantly improved, reaching more than 80%.

[0054] DPPH free radical scavenging capacity (%) = [1 - (X1 - X2) / X0] × 100

[0055] X1: Absorbance of the sample solution / mixed solution and DPPH ethanol solution mixture;

[0056] X2: Absorbance of the sample solution / mixed solution and distilled water mixture;

[0057] X0: Absorbance of a mixture of DPPH ethanol solution and distilled water.

[0058] 2. Determination of ABTS free radical scavenging ability

[0059] The total antioxidant capacity assay kit (ABTS method) from Beyotime Biotechnology Co., Ltd. was used for determination. Sample mixtures with different concentration gradients were prepared according to the above method. First, the ABTS working stock solution was prepared: ABTS solution and potassium persulfate solution were mixed at a ratio of 1:1 (v:v) and left to stand overnight at room temperature to obtain the ABTS working stock solution. 10 μL of the above sample was added to 200 μL of ABTS working solution (the working stock solution was diluted with PBS until the absorbance at 734 nm reached approximately 0.7), shaken to mix, and left to stand at room temperature for 2-6 min. The absorbance at 734 nm was then measured. The experimental results are as follows. Figure 4As shown in the figure, vitamin B1 has a weak ability to scavenge ABTS free radicals. The antioxidant peptide of this invention has an ABTS free radical scavenging ability of 10% to 50%. The antioxidant capacity is significantly improved after the two are mixed, reaching more than 50%.

[0060] ABTS free radical scavenging capacity (%) = [1 - (X1 - X2) / X0] × 100

[0061] X1: Absorbance of the sample solution / mixed solution and ABTS solution mixture;

[0062] X2: Absorbance of the sample solution / mixed solution and distilled water mixture;

[0063] X0: Absorbance of the mixture of ABTS solution and distilled water.

[0064] 3. Determination of hydroxyl radical scavenging ability

[0065] Sample solutions and mixed solutions of different concentration gradients were prepared according to the above method. 250 μL of the sample to be tested was placed in a test tube, and 250 μL of 9 mmol / L ferrous sulfate solution and 250 μL of 9 mmol / L salicylic acid ethanol solution were added. Then, 250 μL of 8.8 mmol / L hydrogen peroxide solution was quickly added, and the mixture was thoroughly mixed. The reaction was carried out at 37℃ for 30 min, and after cooling to room temperature, the absorbance was measured at 510 nm. The experimental results are as follows: Figure 5 As shown in the figure, vitamin B1 has a weak ability to scavenge hydroxyl radicals. The antioxidant peptide of this invention has a scavenging ability of 20% to 60% against hydroxyl radicals. The antioxidant capacity is significantly improved after the two are mixed, and can reach more than 80% even under the lowest concentration conditions.

[0066] Hydroxyl radical scavenging capacity (%) = [1 - (X1 - X2) / X0] × 100

[0067] X1: Absorbance of the sample solution / mixed solution and reaction system mixture;

[0068] X2: Absorbance of the mixture of sample solution / mixed solution and reaction system (using distilled water instead of hydrogen peroxide solution);

[0069] X0: Absorbance of the mixture of distilled water and reaction system.

[0070] 4. Determination of the reducing power of iron ions (FRAP)

[0071] Sample solutions and mixed solutions with different concentration gradients were prepared according to the above method. 250 μL of the sample solution to be tested was added to 250 μL of PBS buffer (0.2 mol / L, pH=6.8) and 250 μL of potassium ferricyanide solution (1%, w / v), mixed, and incubated in a water bath at 50℃ for 20 min. After removal, it was rapidly cooled, and the reaction was terminated by adding 1 mL of trichloroacetic acid solution (10%, w / v). The mixture was centrifuged at 5000 r / min for 5 min, and 1 mL of the supernatant was collected. 1 mL of distilled water and 200 μL of ferric chloride solution (0.1%, w / v) were added. Vitamin C solution was used as a positive control, and the iron atom reducing ability was characterized by the absorbance at 700 nm. The experimental results are as follows: Figure 6 As shown in the figure, it can be seen that vitamin B1 has a weak reducing power for iron ions. The antioxidant peptide of this invention has a reducing power for iron ions of 20% to 40%. The reducing power for iron ions is significantly improved after the two are mixed. As the concentration increases, the reducing power for iron ions increases from 30% to more than 60%.

[0072] Iron ion reducing power = X1 - X2

[0073] X1: Absorbance of the sample solution / mixed solution and the reaction system;

[0074] X2: Absorbance of the sample solution / mixed solution and the reaction system (using distilled water instead of potassium ferricyanide solution).

[0075] Example 4: Molecular docking analysis of vitamin B1 with DPPH and ABTS free radicals, the antioxidant peptide of the present invention with DPPH and ABTS free radicals, and the antioxidant peptide of the present invention combined with vitamin B1 as a complex antioxidant with DPPH and ABTS free radicals.

[0076] Molecular docking simulations were performed on vitamin B1, the antioxidant peptide of this invention, and a composite antioxidant formed by mixing the two to identify the antioxidant activity of the composite antioxidant peptide and vitamin B1, and to predict the binding mode of the composite antioxidant peptide and vitamin B1 to free radicals. The 3D structures of vitamin B1 (CID: 124951) free radicals ABTS (CID: 9570474) and DPPH (CID: 74358) were downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The 2D structure of the antioxidant peptide was drawn using the PepDraw website and converted to a 3D structure with energy minimization and stability optimization using Chem3D 2021. Before docking, the molecules were imported into AutoDockTools software (v. 1.5.7) for water molecule removal, addition of polar hydrogen, charge calculation, and spin bond setting. Using AutoDock Vina software (v. 1.2.5), simulations were performed to demonstrate the formation of complex antioxidants by vitamin B1 with DPPH and ABTS free radicals, antioxidant peptides with DPPH and ABTS free radicals, and the antioxidant peptides of this invention with vitamin B1, followed by their binding to DPPH and ABTS free radicals. The molecular docking binding energies are shown in Table 2. Lower molecular docking binding energies indicate more stable ligand-receptor binding. Based on the docking results, the complex antioxidants of this invention, composed of antioxidant peptides and vitamin B1, exhibit low docking binding energies with DPPH and ABTS free radicals, indicating higher antioxidant activity. Finally, the docking model with the lowest binding free energy was selected, and visualization and analysis were performed using PyMOL 2.6. Figure 7 - Figure 12 As shown.

[0077] from Figure 7 and Figure 10 From this, it can be concluded that the binding energies of vitamin B1 with DPPH free radicals and ABTS free radicals are -3.0 kcal / mol (DPPH... + ) and -2.6 kcal / mol (ABTS) + The values ​​were all above -4 kcal / mol, indicating that the hydrogen bond interaction between vitamin B1 and the two free radicals was weak, and its own free radical scavenging activity was extremely weak. Meanwhile, the values ​​of vitamin B1 and free radicals were both above -4 kcal / mol, indicating that the hydrogen bond interaction between vitamin B1 and the two free radicals was weak. Figure 8 and Figure 11 It can be seen that the docking energy of the antioxidant peptide of this invention with the two free radicals is reduced to -4.3 kcal / mol (DPPH). + ) and -4.9 kcal / mol (ABTS) +Both of these values ​​are above the activity threshold of -4.0 kcal / mol, indicating moderate binding strength, suggesting that the hydrogen bonding between the antioxidant peptides of this invention and the two free radicals is stronger than that of vitamin B1. When vitamin B1 forms a complex with the antioxidant peptides, the binding energy further decreases significantly to -6.719 kcal / mol (DPPH). + ) and -7.801 kcal / mol (ABTS) + ), Figure 9 , Figure 12 This indicates that both have a synergistic effect, jointly stabilizing free radicals through hydrogen bond networks and hydrophobic interactions, thus greatly improving the efficiency of free radical scavenging. This result corroborates the aforementioned in vitro antioxidant experiment results, indicating that the composite antioxidant formed by vitamin B1 and the antioxidant peptide of this invention also has greatly enhanced antioxidant properties. This synergistic system not only clarifies the molecular interaction mechanism from a theoretical perspective, but also provides a scientific experimental basis for the development of highly efficient and low-toxic cosmetics, pharmaceuticals, or health products, and points the way for subsequent improvements in formulation and enhancement of antioxidant effects.

[0078] Table 2 Molecular docking binding energy

[0079]

[0080] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An antioxidant peptide derived from mouse hemoglobin, characterized in that, The amino acid sequence of the antioxidant peptide is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the antioxidant peptide as described in claim 1.

3. An expression vector containing the nucleic acid as described in claim 2.

4. Recombinant cells containing the expression vector as described in claim 3.

5. The use of the antioxidant peptide or its encoded nucleic acid as described in claim 1 in the preparation of antioxidant products.

6. The application as described in claim 5, characterized in that, The product is one or more of the following: cosmetics, health products, or pharmaceuticals.

7. The application as described in claim 6, characterized in that, The antioxidant peptides are used as active ingredients or additives.

8. An antioxidant, characterized in that, The antioxidants include the antioxidant peptides of claim 1 and vitamin B1.

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