An antioxidant peptide derived from cytochrome c oxidase subunit of red jungle fowl and application thereof

Abstract

The application discloses an antioxidant peptide derived from a cytochrome C oxidase subunit of red jungle fowl and application thereof, and the amino acid sequence of the antioxidant peptide is LRFPIQRNL, and the molecular weight is 1155 Da. The polypeptide is identified from the red jungle fowl by a high performance liquid chromatography technology for the first time, and an antioxidant peptide with an antioxidant activity and not reported is screened out. The antioxidant peptide is synthesized by a solid phase method, has the characteristics of safety, non-toxicity, good water solubility and stability, and the antioxidant property is obviously improved after being combined with vitamin B5, and 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 in particular to an antioxidant peptide derived from the cytochrome C oxidase subunit of red junglefowl and its applications. Background Technology

[0002] The Red Junglefowl is a high-quality local chicken breed from the Hongyuan Plateau region of Sichuan Province, China. Growing in alpine grassland environments, it possesses excellent cold resistance, disease resistance, and strong adaptability to grazing. This breed is primarily bred using natural feed and semi-free-range methods. Its meat is tender, has a unique flavor, low fat deposition, and is rich in various functional proteins and bioactive peptides, making it an important resource for developing functional livestock and poultry products. Recent studies have discovered that the muscle tissue of the Red Junglefowl can release a variety of bioactive antioxidant peptides. These antioxidant peptides exhibit good DPPH and ABTS free radical scavenging abilities in vitro and can alleviate cellular oxidative stress responses through mechanisms such as chelating metal ions and inhibiting lipid peroxidation. Some peptide fragments are rich in electron-donating amino acid residues, such as His, Tyr, Cys, and Trp, endowing them with strong free radical scavenging and antioxidant capabilities. Therefore, the Red Junglefowl is not only a high-quality meat source poultry, but its protein resources also have the potential for developing natural antioxidant peptides and applying them in functional foods and biomedicine.

[0003] Antioxidant peptides are a class of low-molecular-weight functional peptides released from animal, plant, or microbial sources. Due to their rich structure containing amino acid residues that can participate in free radical reactions (such as Trp, Tyr, His, and Cys), they possess excellent free radical scavenging, peroxidation inhibition, and metal chelation capabilities. These peptides have small molecular weights and are easily absorbed. In vivo, they can effectively alleviate oxidative stress-induced damage to cells and tissues through multiple pathways, including regulating redox-related signaling pathways and enhancing the activity of endogenous antioxidant enzymes. In recent years, antioxidant peptides have been widely used in food preservation, nutritional intervention, anti-aging, and adjuvant treatment of chronic diseases. Their high safety, good biocompatibility, and lack of toxic side effects make them ideal substitutes for synthetic antioxidants (such as BHT and BHA). With the continuous deepening of peptide screening, structure-function relationship analysis, and in vivo mechanism research, antioxidant peptides are gradually moving from experimental research to industrial transformation, showing broad application prospects. However, there are currently no reports of antioxidant peptides derived from the cytochrome C oxidase subunit of red junglefowl. 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 the cytochrome C oxidase subunit of red junglefowl 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 the cytochrome C oxidase subunit of red junglefowl is provided, with the amino acid sequence LRFPIQRNL, as shown in SEQ ID NO: 1.

[0008] The antioxidant peptide has a molecular weight of 1155 Da, a positive charge of +2, an isoelectric point (pI) of 12, 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 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 B5.

[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 B5 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 B5. This composite antioxidant agent possesses high antioxidant activity and has broad application potential in antioxidant 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 3 The results show the DPPH free radical scavenging capacity of vitamin B5, the antioxidant peptide of the present invention, and the composite antioxidant of the antioxidant peptide of the present invention and vitamin B5.

[0021] In this invention, the antioxidant peptide and vitamin B5 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 B5, the antioxidant peptide of the present invention, and the compound antioxidant agent of the antioxidant peptide of the present invention and vitamin B5.

[0023] Figure 5 The results show the hydroxyl radical scavenging capacity of vitamin B5, the antioxidant peptide of the present invention, and the compound antioxidant agent of the antioxidant peptide of the present invention and vitamin B5.

[0024] Figure 6 The determination of the iron ion reducing power of vitamin B5, the antioxidant peptide of the present invention, and the compound antioxidant agent of the antioxidant peptide of the present invention and vitamin B5.

[0025] Figure 7 This is a diagram showing the docking results of vitamin B5 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 B5 composite antioxidant of the present invention with DPPH free radical molecules.

[0028] Figure 10 This is a diagram showing the docking results of vitamin B5 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 B5 composite antioxidant of this 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 the cytochrome C oxidase subunit of red junglefowl

[0035] 1. Sample pretreatment: Weigh 1 mg of lyophilized red junglefowl powder, add 200 μL of 0.1% TFA (trifluoroacetic acid) aqueous solution, sonicate to homogenize, centrifuge at 14000 g for 10 minutes, collect the supernatant, ultrafilter the supernatant using a 10 kD ultrafiltration tube (PALL, OD010C35), centrifuge at 13500 g for 10 min, and use a nanodrop 2000C UV-Vis spectrophotometer (thermo) to quantify peptides. 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 (0.15mm*150mm, RP-C18, Column Technology Inc.) was equilibrated with 95% solution A. The sample was loaded into a 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). Analysis time: 60 min. Detection mode: positive ion. The mass-charge ratio of the peptide and peptide fragments was collected using the following method: 10 fragment spectra were acquired 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 LRFPIQRNL (as shown in SEQ ID NO: 1) is as follows. Figure 1 As shown.

[0043] Example 2: Bioinformatics of antioxidant peptides derived from the cytochrome C oxidase subunit of red junglefowl

[0044] The test steps are as follows:

[0045] 1. Prediction of Potential Biological Activity of Peptides: The potential biological activity of the 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 for the Peptide Ranker prediction model was 0.5; peptides with a threshold greater than 0.5 were considered to possess biological activity. The predicted value for the antioxidant peptides screened in this application was 0.54.

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

[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 LRFPIQRNL was determined to be 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 LRFPIQRNL 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 B5, the antioxidant peptide of the present invention, and a compound antioxidant of the antioxidant peptide of the present invention and vitamin B5.

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

[0053] The antioxidant peptides and vitamin B5 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 B5 solution, and their mixture were added, along with 100 μL of DPPH ethanol solution (DPPH stock solution diluted to 0.1 mol / L with anhydrous ethanol, 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 B5 has a weak ability to scavenge DPPH free radicals. The antioxidant peptide of this invention has a DPPH free radical scavenging ability of 20% to 50%. When the two are mixed, the antioxidant capacity is significantly improved, reaching more than 90%.

[0054] DPPH 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 final absorbance at 734 nm reached approximately 0.7), and the mixture was shaken to mix thoroughly. After standing at room temperature for 2-6 min, the absorbance at 734 nm was measured. The experimental results are as follows: Figure 4 As shown in the figure, vitamin B5 has a weak ability to scavenge ABTS free radicals. The antioxidant peptide of this invention has a scavenging ability of 30% to 50% against ABTS free radicals. When the two are mixed, the antioxidant capacity is significantly improved, reaching more than 90%.

[0060] ABTS 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 B5 has a weak ability to scavenge hydroxyl radicals. The antioxidant peptide of this invention has a scavenging ability of 50% to 60% against hydroxyl radicals. The antioxidant capacity is significantly improved after the two are mixed, and can reach more than 70% 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, vitamin B5 has a weak reducing power for iron ions. The antioxidant peptide of this invention has a reducing power for iron ions of 10% 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 20% 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 B5 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 B5 as a complex antioxidant with DPPH and ABTS free radicals.

[0076] Molecular docking simulations were performed on vitamin B5, 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 B5, and to predict the binding mode of the composite antioxidant peptide and vitamin B5 to free radicals. The 3D structures of vitamin B5 (CID: 786784), 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 a complex antioxidant by vitamin B5 with DPPH and ABTS free radicals, the antioxidant peptide of this invention with DPPH and ABTS free radicals, and the antioxidant peptide of this invention with vitamin B5, followed by their binding to DPPH and ABTS free radicals. The molecular docking binding energies are shown in Table 2. A lower molecular docking binding energy indicates a more stable ligand-receptor binding. Based on the docking results, the complex antioxidant of this invention, consisting of the antioxidant peptide and vitamin B5, exhibits 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. Figures 7-12 As shown.

[0077] from Figure 7 and Figure 10 It can be concluded that single vitamin B1 and DPPH + and ABTS + The binding energies are -2.6 kcal / mol (DPPH). + ) and -2.0 kcal / mol (ABTS) + The concentrations of these elements were all above -4 kcal / mol, indicating weak hydrogen bonding and hydrophobic interactions, and negligible free radical scavenging activity. Meanwhile, the concentrations of these elements were also higher than -4 kcal / mol, revealing weak hydrogen bonding and hydrophobic interactions, and negligible free radical scavenging activity. 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 -4.6 kcal / mol (DPPH). + ) and -4.0 kcal / mol (ABTS) +The binding energy was close to the activity threshold of -4.0 kcal / mol, indicating a moderate binding capacity, suggesting that the hydrogen bonding between the antioxidant peptide of this invention and the two free radicals is stronger than that of vitamin B1 alone. When vitamin B1 forms a complex with the antioxidant peptide, the binding energy further decreases to -5.894 kcal / mol (DPPH). + ) and -5.778 kcal / mol (ABTS) + () Figure 9 , Figure 12 The results confirmed that the two significantly enhanced free radical stability through hydrogen bond network and hydrophobic synergy, with a scavenging efficiency approximately 1.3-1.8 times higher than that of the monomer. This result is highly consistent with in vitro antioxidant experiments, indicating that the vitamin B5-peptide complex system has practical application potential in cosmetics or pharmaceuticals, and provides a direct theoretical basis for subsequent formulation optimization.

[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 the cytochrome C oxidase subunit of red junglefowl, 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 cosmetics 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 B5.

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