An antioxidant peptide derived from bullfrog mucus and application thereof

Abstract

The application discloses an antioxidant peptide derived from bullfrog mucus and application thereof, and the amino acid sequence of the antioxidant peptide is KFLNVVSGLLTSKYR, and the molecular weight is 1724 g / mol. The polypeptide is identified from the bullfrog mucus 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 the bullfrog mucus 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 B3, the antioxidant property of the antioxidant peptide is significantly improved, and the antioxidant peptide can be widely applied to preparation of cosmetics, medicines or health products, and has important economic value and social significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to an antioxidant peptide derived from bullfrog mucus and application thereof. BACKGROUND

[0002] Bullfrog mucus is a natural substance secreted from the skin of bullfrog, which has multiple biological activities and protective effects. The mucus contains various proteins, peptides, enzymes, and antimicrobial and antioxidant substances, which can effectively resist external pathogens and oxidative stress. In recent years, it has been found that the bullfrog mucus contains rich antioxidant components, which have the potential to scavenge free radicals and slow down cell oxidative damage. These antioxidant substances such as phenolic compounds and peptides have good antioxidant activity and are widely used in medicine, health products and cosmetics. The natural antioxidant components of bullfrog mucus not only can enhance the antioxidant capacity of the human body, but also have multiple biological activities such as anti-inflammatory and antibacterial, and have important application prospects.

[0003] Antioxidant peptides are a class of polypeptide molecules with antioxidant activity, which can protect cells from oxidative damage by scavenging free radicals, and can directly react with free radicals to convert them into harmless molecules, thereby reducing cell damage; some antioxidant peptides can also chelate transition metal ions such as iron and copper to prevent them from catalyzing harmful oxidation reactions. However, there is still no report on antioxidant peptides derived from bullfrog mucus. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art, and provides an antioxidant peptide derived from bullfrog mucus and application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] One of the technical solutions adopted by the present application to solve its technical problems is:

[0007] An antioxidant peptide derived from bullfrog mucus is provided, and the amino acid sequence thereof is KFLNVVSGLLTSKYR, as shown in SEQ ID NO: 1.

[0008] The molecular weight of the antioxidant peptide is 1724 g / mol, the positive charge carried is +3, the isoelectric point (pI) is 10.29, and the water solubility is good.

[0009] The present application 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 application also provides the use of the above-mentioned antioxidant peptide or its encoding nucleic acid in the preparation of antioxidant products.

[0011] Preferably, the product is one or more of a cosmetic product, a health product or a pharmaceutical product.

[0012] Preferably, the antioxidant peptide is used as an active ingredient or an additive.

[0013] The second technical solution adopted by the present application to solve its technical problems is:

[0014] An antioxidant is provided, which comprises the antioxidant peptide and vitamin B3.

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

[0016] The present application has the following beneficial effects:

[0017] The antioxidant peptide of the present application is safe, non-toxic, has good water solubility, stability and good in-vitro antioxidant activity, and can be used to prepare antioxidant products. At the same time, the docking energy of the complex antioxidant of the antioxidant peptide and vitamin B3 with the receptors DPPH and ABTS free radicals is lower than that of the two alone with DPPH free radicals and ABTS free radicals, indicating that the antioxidant capacity of the antioxidant peptide and vitamin B3 after mixing is significantly improved, and the complex antioxidant has high antioxidant activity, and has a wide application space in the fields of antioxidant health products, pharmaceuticals and cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The figure is a mass spectrum analysis chart of the antioxidant peptide of the present application.

[0019] Figure 2 The figure is a 3D structure schematic diagram of the antioxidant peptide of the present application.

[0020] Figure 3 The figure is the determination result of the high-efficiency antioxidant DPPH free radical scavenging capacity of vitamin B3, the antioxidant peptide of the present application and the antioxidant peptide of the present application combined with vitamin B3.

[0021] The antioxidant peptide of the present application and vitamin B3 are mixed in a 1:1 ratio, and the concentration gradient is 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, respectively.

[0022] Figure 4 The figure is the determination result of the high-efficiency antioxidant ABTS free radical scavenging capacity of vitamin B3, the antioxidant peptide of the present application and the antioxidant peptide of the present application combined with vitamin B3.

[0023] Figure 5 Results of determination of hydroxyl radical scavenging capacity of vitamin B3, the antioxidant peptide of the present application and the high-efficiency antioxidant of the antioxidant peptide of the present application combined with vitamin B3.

[0024] Figure 6 Results of determination of iron ion reducing power of vitamin B3, the antioxidant peptide of the present application and the high-efficiency antioxidant of the antioxidant peptide of the present application combined with vitamin B3.

[0025] Figure 7 Docking result figure of vitamin B3 and DPPH radical molecule.

[0026] Figure 8 Docking result figure of the antioxidant peptide of the present application and DPPH radical molecule.

[0027] Figure 9 Docking result figure of the high-efficiency antioxidant of the antioxidant peptide of the present application combined with vitamin B3 and DPPH radical molecule.

[0028] Figure 10 Docking result figure of vitamin B3 and ABTS radical molecule.

[0029] Figure 11 Docking result figure of the antioxidant peptide of the present application and ABTS radical molecule.

[0030] Figure 12 Docking result figure of the high-efficiency antioxidant of the antioxidant peptide of the present application combined with vitamin B3 and ABTS radical molecule. DETAILED DESCRIPTION

[0031] In order to better understand the present application, the present application will be further described in detail below in combination with examples and drawings, but those skilled in the art should understand that the following examples are not a limitation to the protection scope of the present application, and any changes and variations made on the basis of the present application are within the protection scope of the present application.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0033] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0034] Example 1 Structure identification of antioxidant peptide derived from bullfrog mucus

[0035] 1. Sample pretreatment: 1 mg of freeze-dried powder of bullfrog mucus was weighed, 200 μΐ of 0.1% TFA (trifluoroacetic acid) aqueous solution was added, and the sample was homogenized by ultrasonic treatment, centrifuged at 14000 g for 10 minutes, and the supernatant was taken. The supernatant was ultrafiltered using a 10 kD ultrafiltration tube (PALL, OD010C35), centrifuged at 13500 g for 10 min, and the filtrate was subjected to polypeptide quantification using a nanodrop 2000C UV-visible spectrophotometer (thermo). The filtrate was desalted using a C18 solid phase extraction column (66871, Sigma). The specific steps were as follows: first, the extraction column was washed with acetonitrile, then the extraction column was equilibrated with 0.1% TFA, the ultrafiltered sample solution was loaded, and the peptide fragments were eluted using a 70% acetonitrile solution.

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

[0037] The liquid chromatography column (0.15 mm*150 mm, RP-C18, Column Technology Inc.) was equilibrated with 95% A liquid, the sample was loaded onto the Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by automatic sampler, and then separated by liquid chromatography column. The relevant liquid phase gradient is set as follows:

[0038] 0 min---50 min, B liquid linear gradient from 4% to 50%;

[0039] 50 min---54 min, B liquid linear gradient from 50% to 100%;

[0040] 54 min---60 min, B liquid maintained at 100%.

[0041] 3. After the enzymatic product was separated by capillary high performance liquid chromatography, mass spectrometry was performed using a Q Exactive HF-X mass spectrometer (ThermoFisher). The analysis time was 60 min. The detection mode was positive ion. The mass-to-charge ratio of the polypeptide and the fragments of the polypeptide was collected according to the following method: 10 fragment maps (MS2 scan) were collected after each full scan.

[0042] 4. Database search: The mass spectrometry test raw file (Raw File) was searched using the software MaxQuant 1.5.5.1 to search the corresponding database, and finally the protein identification and quantitative analysis results were obtained. The mass spectrum of the antioxidant peptide KFLNVVSGLLTSKYR is shown in Figure 1 .

[0043] Example 2 Bioinformatics of antioxidant peptides derived from bullfrog mucus

[0044] 1. The isoelectric point (pI) and water solubility of the peptide were analyzed using the Expasy-pI / Mw tool (https: / / web.expasy.org / computepi / ) and the PepDraw server (https: / / pepcalc.com).

[0045] 2. The online tool Toxinpred, (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) platform based on SVM (Swiss-Port) algorithm was used to predict the potential toxicity of the new peptide. After potential toxicity analysis, the antioxidant peptide KFLNVVSGLLTSKYR was Non-Toxin (non-toxic), and its physicochemical properties are shown in Table 1. The sequence was followed by synthesis and verification.

[0046] Table 1 Physicochemical properties of antioxidant peptides

[0047]

[0048] 3. The three-dimensional structure of the antioxidant peptide KFLNVVSGLLTSKYR was predicted using Pymol2.0 and Swiss-Model, and the 3D structure diagram is shown in Figure 2 .

[0049] Example 3 In vitro antioxidant activity of vitamin B3, the antioxidant peptide of the present application, and the complex antioxidant of the antioxidant peptide of the present application and vitamin B3

[0050] 1. Determination of DPPH free radical scavenging ability

[0051] The antioxidant peptide and vitamin B3 were respectively prepared into solutions with different concentration gradients (31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) using distilled water, and the mixing ratio of the antioxidant peptide and vitamin B3 was 1:1. 100 μL of the antioxidant peptide solution, the vitamin B3 solution and the mixed solution with different concentrations were taken, 100 μL of DPPH ethanol solution (the DPPH mother liquor was diluted with anhydrous ethanol to 0.1 mol / L, and the final absorbance range at 517 nm was 0.6-1), was added, mixed well, and then placed in the dark at room temperature for 30 min. The absorbance value of the solution at 517 nm was measured using an enzyme marker. The experimental results are shown in Figure 3As shown in the figure, it can be seen that vitamin B3 has weak DPPH free radical scavenging ability, the DPPH free radical scavenging ability of the antioxidant peptide of the application is 15% to 40%, and the antioxidant ability is significantly improved after mixing, and can reach more than 75%.

[0052] DPPH free radical scavenging ability (%) = [1- (X1-X2) / X0] x 100

[0053] X1: sample solution / mixed solution and DPPH ethanol solution mixture absorbance;

[0054] X2: sample solution / mixed solution and distilled water mixture absorbance;

[0055] X0: DPPH ethanol solution and distilled water mixture absorbance.

[0056] 2. Determination of ABTS free radical scavenging ability

[0057] Using the total antioxidant capacity detection kit (ABTS method) of Biyun Tian Biotechnology Co., Ltd. to determine. The sample mixed solution with different concentration gradients is prepared according to the above method. First, the ABTS working mother liquor is prepared: ABTS solution and potassium persulfate solution are mixed at 1:1 (v:v), and are placed at room temperature overnight to obtain the ABTS working mother liquor. 10 μL of the above sample to be tested is added to 200 μL of ABTS working solution (the working mother liquor is diluted with PBS, and the final absorbance at 734 nm is about 0.7), and is shaken and mixed, and then is placed at room temperature for 2-6 min, and the absorbance at 734 nm is determined. The experimental results are shown in Figure 4 As shown in the figure, it can be seen that vitamin B3 has weak ABTS free radical scavenging ability, the ABTS free radical scavenging ability of the antioxidant peptide of the application is 20% to 40%, and the antioxidant ability is significantly improved after mixing, and can reach more than 80%.

[0058] ABTS free radical scavenging ability (%) = [1- (X1-X2) / X0] x 100

[0059] X1: sample solution / mixed solution and ABTS solution mixture absorbance;

[0060] X2: sample solution / mixed solution and distilled water mixture absorbance;

[0061] X0: ABTS solution and distilled water mixture absorbance.

[0062] 3. Determination of hydroxyl radical scavenging ability

[0063] The sample solution and mixed solution with different concentration gradient were prepared according to the above method. 250 μL of the sample to be tested was taken in a test tube, 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 fully mixed. The reaction was carried out at 37°C for 30 min, and then the absorbance was measured at 510 nm after cooling to room temperature. The experimental results are shown in Figure 5 As can be seen from the figure, the vitamin B3 has weak hydroxyl radical scavenging ability, the antioxidant peptide of the application has hydroxyl radical scavenging ability of 20% to 40%, and the antioxidant ability is significantly improved after mixing, and can reach more than 75% under the condition of the lowest concentration.

[0064] Hydroxyl radical scavenging ability (%) = [1-(X1-X2) / X0]x100

[0065] X1: absorbance of sample solution / mixed solution and reaction system mixture;

[0066] X2: absorbance of sample solution / mixed solution and reaction system (distilled water instead of hydrogen peroxide solution) mixture;

[0067] X0: absorbance of distilled water and reaction system mixture.

[0068] 4. Determination of iron ion reducing power (FRAP)

[0069] The sample solution and mixed solution with different concentration gradient were prepared according to the above method. 250 μL of the sample to be tested was taken in a test tube, 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 fully mixed. The reaction was carried out at 37°C for 30 min, and then the absorbance was measured at 510 nm after cooling to room temperature. The experimental results are shown in Figure 6 As can be seen from the figure, the vitamin B3 has weak hydroxyl radical scavenging ability, the antioxidant peptide of the application has hydroxyl radical scavenging ability of 20% to 40%, and the antioxidant ability is significantly improved after mixing, and can reach more than 75% under the condition of the lowest concentration.

[0070] Iron ion reducing ability = X1-X2

[0071] X1: absorbance of sample solution / mixed solution and reaction system mixture;

[0072] X2: Absorbance of sample solution / mixed solution and reaction system (distilled water instead of potassium ferricyanide solution).

[0073] Example 4 Molecular docking analysis of vitamin B3 and DPPH radical and ABTS radical, antioxidant peptide of the present application and DPPH radical and ABTS radical, and complex antioxidant of antioxidant peptide of the present application and vitamin B3 and DPPH radical and ABTS radical

[0074] Molecular docking simulation was performed on the complex antioxidant of vitamin B3 and antioxidant peptide of the present application mixed together to identify the antioxidant activity of the complex antioxidant of antioxidant peptide of the present application and vitamin B3 and predict the binding mode of the complex antioxidant of antioxidant peptide of the present application and vitamin B3 and free radicals. The 3D structures of vitamin B3 (CID: 936), 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 into a 3D structure optimized for energy minimization and stability using Chem3D 2021. Before docking, the molecules were imported into the AutoDockTools software (v. 1.5.7) for removal of water molecules, addition of polar hydrogens, charge calculation, and rotation key setting. Using AutoDock Vina software (v. 1.2.5), vitamin B3 and DPPH radical and ABTS radical, antioxidant peptide of the present application and DPPH radical and ABTS radical, and the complex antioxidant of antioxidant peptide of the present application and vitamin B3 were simulated to bind to DPPH radical and ABTS radical, respectively, and the molecular docking binding energy is shown in Table 2. The lower the molecular docking binding energy, the more stable the binding of the ligand and the receptor. According to the docking results, the complex antioxidant of antioxidant peptide of the present application and vitamin B3 has a low docking binding energy with DPPH radical and ABTS radical, and has high antioxidant activity. Finally, the docking model with the lowest binding free energy was selected and visualized and analyzed using PyMOL 2.6.

[0075] As shown in Figure 7 - Figure 12 , the binding energy of vitamin B3 alone with DPPH + and ABTS + is only -2.4 kcal / mol and -2.4 kcal / mol, respectively, both of which are higher than -4 kcal / mol, indicating that the hydrogen bond and hydrophobic interaction between them and the free radicals are weak, and the radical scavenging activity is negligible. The binding energy of antioxidant peptide of the present application with DPPH + , ABTS +The binding energies of the combination of the two are reduced to -5.2 kcal / mol and -5.7 kcal / mol, respectively, which is significantly lower than the activity threshold of -4 kcal / mol, indicating that it has moderate binding ability to the two free radicals, and the force is stronger than vitamin B3. After vitamin B3 forms a complex with the antioxidant peptide of the present application, the binding energy is further reduced to -7.79 kcal / mol (DPPH + ) and -8.125 kcal / mol (ABTS⁺), which confirms that the two significantly enhance the stability of free radicals through hydrogen bond network and hydrophobic synergistic effect, and the removal efficiency is about 1.5 times higher than that of the monomer.

[0076] In summary, the complex system composed of vitamin B3 and the antioxidant peptide of the present application has a significant synergistic antioxidant effect, and has high efficiency and good biocompatibility, which lays a solid theoretical and experimental foundation for subsequent formulation upgrading and industrial application in cosmetics, drugs and health products.

[0077] Table 2 Binding energy of molecular docking

[0078]

[0079] Although the specific embodiments of the present application are described above, those skilled in the art should understand that the specific examples described are only illustrative, and are not intended to limit the scope of the present application, and equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present application should be covered within the scope of the claims of the present application.

Claims

1. An antioxidant peptide derived from the mucus of bullfrog, 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 of claim 1.

3. An expression vector containing the nucleic acid of claim 2.

4. A recombinant cell containing the expression vector of claim 3.

5. Use of the antioxidant peptide of claim 1 or a nucleic acid encoding the same in the preparation of an antioxidant cosmetic, health care or pharmaceutical product.

6. Use according to claim 5, wherein The antioxidant peptide is used as an active ingredient or additive.

7. An antioxidant characterized by, The antioxidant agent comprises the antioxidant peptide of claim 1 and vitamin B3.

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