A bioactive peptide and its application

Abstract

The present invention belongs to the field of biotechnology and relates to a bioactive peptide and its application. The amino acid sequence of the bioactive peptide is shown as any one of SEQ ID NO: 1-4. The bioactive peptide of the present invention has the characteristics of high permeability, strong antioxidant property, and strong repair ability, and has great application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a bioactive peptide and its application. Background Art

[0002] Bioactive peptides are a general term for different peptides composed of natural amino acids with different compositions and arrangements, ranging from dipeptides to complex linear and cyclic structures, and are multifunctional compounds derived from proteins. Bioactive peptides are involved in human growth and development, metabolism, and aging, and have functions such as enhancing immunity, antibacterial, antiviral, antioxidant, anti-aging, and blood pressure lowering. Therefore, they have great potential application prospects in the fields of medicine, cosmetics, functional foods, etc. Currently, they are mainly classified by function into antioxidant peptides, hormone regulatory peptides, antibacterial active peptides, neuroactive peptides, immunomodulatory peptides, and antihypertensive active peptides.

[0003] Antioxidant active peptides are a class of natural active peptides that have been widely studied recently. They can effectively scavenge excess reactive oxygen free radicals in the body, protect the normal structure of cell membranes and mitochondria, and prevent lipid peroxidation. Oxidation is closely related to natural aging in humans and the occurrence and development of many diseases such as cancer, diabetes, arteriosclerosis, and Alzheimer's disease. The most studied antioxidant active peptides are carnosine and glutathione. Glutathione (GSH) is a tripeptide compound formed by the condensation of L-cysteine, L-glutamic acid, and glycine through peptide bonds. It is the most important small molecule active peptide for maintaining the redox balance in the body and has the effects of scavenging free radicals, antioxidant, whitening the skin, fading spots, and anti-aging. It has currently been applied in many fields such as food, medicine, health products, and cosmetics. Another widely used one is oligopeptide. Human oligopeptide-1, namely epidermal growth factor, is an active peptide existing in the human body. It can promote cell proliferation and differentiation, replace aging and dead cells with new cells, and is widely used in the field of regenerative medicine for the treatment of burns, wounds, and the healing of traumatic wounds. However, the use of human oligopeptide-1 has uncontrollability and difficult production processes, so there are strict requirements for application scenarios.

[0004] Another type is traditional bioactive peptides extracted from animals and plants, including snake venom peptides, marine bioactive peptides, soybean peptides, etc., which all belong to exogenous active peptides. The extraction of these active peptides is difficult and requires huge costs. Moreover, they are easily degraded into amino acids when entering the human body, greatly reducing their activity. There are also exogenous bioactive peptides produced by specific proteolysis, but the enzymatic hydrolysis products of this method have complex components, containing hundreds to thousands of peptide segments, and it is difficult to separate and purify peptide segments with similar molecular weights or equal charges, which brings great difficulties to subsequent structure identification. Not only does it take a lot of time for separation and purification, but it is also difficult to obtain high-purity and single substances. In addition, the protein source of the polypeptides obtained through semi-rational design is not clear, and subsequent applications need to solve their safety problems.

[0005] The appropriate addition of bioactive peptides in the diet can not only regulate physiological functions, but also help individuals prevent diseases, reduce the dependence on drug treatment, and thus lower the global healthcare costs. Therefore, it has received increasing attention in the field of application of medical foods for special dietary use. For example, when applied in medical foods for special dietary use for cancer patients, it can play an anti-tumor role through mechanisms such as regulating the release of cancer cells, oxidative stress response, cancer-related signaling pathways and the expression of their transcription factors, regulating the release of inflammatory factors, and increasing the total protein level in the body. For example, the marine peptide preparation studied by Yu Fengmei et al. can increase the visceral protein indexes such as TP, Alb, and globulin in patients (The effect of marine peptides on the nutritional status and immune function of patients with malignant tumor chemotherapy); the marine collagen peptide studied by Liang Jiang et al. can increase the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the serum of mice (Inhibitory effect of long-term feeding of marine collagen peptides on spontaneous tumors in SD rats); the goat milk whey protein peptide product studied by CAKIR et al. can regulate the energy metabolism mechanism and oxidative stress signal transduction mechanism of cancer cells (Potential Anticarcinogenic Effect of Goat Milk-Derived Bioactive Peptides on HCT-116 Human Colorectal Carcinoma Cell Line). With the continuous development of theories and technologies in medicine, pharmacy, medical immunology, and molecular pharmacology, people have a deeper understanding of the biological activities of polypeptide substances. The research and development of bioactive peptides will become the focus in various fields and have very broad development prospects.

[0006] Most bioactive peptides must penetrate the skin to exert their effects, but most current bioactive peptides have poor skin permeability and are difficult to enter the dermis to play their roles. This is mainly because bioactive peptides have the characteristics of high molecular weight and hydrophilicity, and contain ionizable groups in their structures, making them easily affected by proteolytic enzymes in the skin. Therefore, the transdermal absorption problem of bioactive peptides is also one of the difficult problems in their wide application.

[0007] References:

[0008] Yu Fengmei, Rao Zhiyong, Liu Yuan, et al. The effect of marine peptides on the nutritional status and immune function of patients with malignant tumor chemotherapy [J]. West China Medical Journal, 2011, 26(8): 1203-1207.

[0009] Liang Jiang, Pei Xinrong, Wang Nan, et al. Inhibitory effect of long-term feeding of marine collagen peptides on spontaneous tumors in SD rats [J]. Food and Fermentation Industries, 2011, 37(5): 1-6.

[0010] Cakir, B. and Tunali-Akbay, T. (2021) Potential Anticarcinogenic Effect of Goat Milk-Derived Bioactive Peptides on HCT-116 Human Colorectal Carcinoma Cell Line. Analytical Biochemistry, 622, Article ID: 114166.

[0011] Guo Y, Zhang T, Jiang B, et al. The effects of an antioxidative pentapeptide derived from chickpea protein hydrolysates on oxidative stress in Caco-2 and HT-29 cell lines[J]. Journal of Functional Foods, 2014, 7: 719 - 726.

[0012] García-Nebot M J, Recio I, Hernández-Ledesma B. Antioxidant activity and protective effects of peptide lunasin against oxidative stress in intestinal Caco-2 cells[J]. Food and Chemical Toxicology, 2014, 65: 155 - 161.

[0013] He R, Ju X, Yuan J, et al. Antioxidant activities of rapeseed peptides produced by solid state fermentation[J]. Food Research International, 2012, 49(1): 432 - 438.

[0014] Liu C, Ren D, Li J, et al. Cytoprotective effect and purification of novel antioxidant peptides from hazelnut (C. heterophylla Fisch) protein hydrolysates[J]. Journal of Functional Foods, 2018, 42: 203 - 215.

[0015] Mahgoub S, Alagawany M, Nader M, et al. Recent development in bioactive peptides from plant and animal products and their impact on the human health[J]. Food Reviews International, 2021: 1 - 26.

[0016] Mayeur S, Spahis S, Pouliot Y, et al. Lactoferrin, a pleiotropic protein in health and disease[J]. Antioxidants & redox signaling, 2016, 24(14): 813 - 836.

[0017] Zhang Q, Li W H. Research status of antioxidant peptides[J]. Food and Fermentation Industries, 2021, 47(2): 298 - 304.

[0018] Jiang H, Tong T, Sun J, et al. Purification and characterization of antioxidative peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate[J]. Food Chemistry, 2014, 154: 158 - 163.

[0019] Aliaga C, Lissi E A. Reactions of the radical cation derived from 2,2'-azinobis

[0020] (3-Ethylbenzothiazoline-6-sulfonic acid)(ABTS·+) with aminoacids. Kinetics and mechanism[J]. Canadian Journal of Chemistry, 2000, 78(8): 1052 - 1059.

[0021] IMURA Y, CHODA N, MATSUZAKI K, et al. Magainin 2 in action: distinct modes of membrane permeabilization in living bacterial and mammalian cells[J]. Biophys J, 2008, 95(12): 5757 - 5765. Summary of the Invention

[0022] First, based on existing machine learning and artificial intelligence tools, the present invention designs a polypeptide library through an overlapping peptide library, screens bioactive peptides with higher permeability scores with the help of MLCPP 2.0 and BChemRF-CPPred prediction tools, and further screens out bioactive peptides with antioxidant activity and functionality through cell experiments for wide application in the fields of biomedicine and cosmetics. To achieve the above object, the technical solution provided by the present invention is as follows:

[0023] The present invention provides a bioactive peptide, the amino acid sequence of which is shown as any one of SEQ ID NO: 1 - 4.

[0024] More preferably, its amino acid sequence is as shown in SEQ ID NO: 1.

[0025] The present invention also provides a coding polynucleotide of the bioactive peptide.

[0026] The present invention provides the application of the bioactive peptide in the preparation of a preparation with antioxidant effect.

[0027] Specifically, the application is in food, cosmetics, medicine or health products.

[0028] Among them, the bioactive peptide plays an antioxidant role.

[0029] Specifically, the skin product with antioxidant effect is a skin medicine, such as a topical skin medicine, for example, a hand cream, an eye cream, a skin moisturizing product, a skin anti-aging product or a skin sunscreen product.

[0030] More specifically, the bioactive peptide has a wound healing or repair effect.

[0031] The polypeptide of the present invention is a functional active polypeptide based on food sources, with good human compatibility. It is derived from natural food proteins and human growth-related factors, has a safe source, is non-toxic to cells, non-irritating to the skin, has good biocompatibility, and can be obtained by chemical synthesis, biosynthesis, purification and extraction, etc. The bioactive peptide of the present invention has the characteristics of strong antioxidant property and strong repair ability, and at the same time has high skin permeability, which is beneficial to penetrate into the dermis layer of the skin to play a role. The polypeptide of the present invention can be used in the fields of food, cosmetics, medicine or health products, and has great application value. Description of the Drawings

[0032] Figure 1 Effect of glutathione on oxidatively damaged cells.

[0033] Figure 2 Effect of SA-LFP1 on oxidatively damaged cells.

[0034] Figure 3 Effect of SA-LFP3 on oxidatively damaged cells.

[0035] Figure 4 Effect of SA-LFP4 on oxidatively damaged cells.

[0036] Figure 5 Effect of SA-LFP13 on oxidatively damaged cells.

[0037] Figure 6 Scavenging effect of SA-LFP1 on intracellular ROS.

[0038] Figure 7 Quantitative analysis chart of the effect of SA-LFP1 on intracellular ROS.

[0039] Figure 8 Effect of SA-LFP1 on cell migration.

[0040] Figure 9 Promoting healing ability of SA-LFP1 on cell scratch. Detailed Embodiments

[0041] The present invention will be further described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation to the present invention.

[0042] Example 1, Peptide Library Establishment and Screening

[0043] 1) Determine the protein source

[0044] Lactotransferrin is a multifunctional globular protein that widely exists in various secretions of animals and humans. Lactotransferrin has various biological activities such as antibacterial, antioxidant, and anticancer effects, and is applied in products such as infant formula foods and cosmetics, and has high safety. It can not only be used as a source of human nutrition to supplement iron and amino acids, but also be used as a drug for preventing and treating various human diseases, for maintaining the balance of intestinal flora, preventing infections and resisting viruses, inhibiting the occurrence and metastasis of tumors, and preventing the generation of free radicals in the body from damaging the body, etc.

[0045] Epidermal growth factor (EGF) is a heat-resistant single-chain low-molecular polypeptide without a glycosyl part, which is very stable, heat-resistant and acid-resistant, and widely exists in the body fluids of animals and humans and in various glands. EGF can accelerate the generation of new cells, increase the number of new cells, promote the metabolism of skin cells, the dead skin layer gradually falls off, and keep the skin texture smooth, can enhance skin elasticity and reduce skin wrinkles. Therefore, EGF is often used to help tissue regeneration, repair surface wounds and ulcers, and is applied in drugs and cosmetics.

[0046] Transforming growth factor-β (TGF-β) belongs to the multifunctional cytokines of the transforming growth factor family and is a secreted protein. It can be regulated by immune cells for cell growth and differentiation, and its key role is to regulate the inflammatory process. It is currently mainly used as a cytokine to promote the differentiation of fibroblasts. Some studies also suggest that TGF-β has potential application prospects in treating wound healing, promoting cartilage and bone repair, and treating autoimmune diseases and transplant rejection through immunosuppression, etc.

[0047] The above-mentioned proteins are mostly distributed in the body fluids of humans and animals, and have a reparative effect, and lactotransferrin has been applied in foods. Therefore, a polypeptide library is designed based on the above-mentioned green and safe proteins derived from animals and humans.

[0048] 2) AI high-throughput screening peptide library

[0049] (1) Screen the above-mentioned peptide library through the MLCPP 2.0 and BChemRF-CPPred servers, set the polypeptide length range to 6 - 30 amino acids, and obtain the transmembrane strength and score of the above-mentioned peptides through prediction and comparison.

[0050] (2) Based on the polypeptide scoring threshold predicted in step (1) > 0.7, 40 peptide sequences were screened. Although the mechanisms of polypeptide transdermal promotion and cell membrane penetration are not exactly the same, there are commonalities. Currently, there is still a lack of a direct transdermal effect prediction tool. In the present invention, transdermal polypeptides were screened by predicting cell membrane penetration. It was found that among the 40 polypeptides studied, some polypeptides (with Cys amino acid residues) have good antioxidant potential. The relevant polypeptide sequences are shown in Table 1, and the transmembrane scores of these 4 polypeptides are all ≥ 0.95. Therefore, the antioxidant properties and other biological activities of the relevant polypeptides were subsequently studied.

[0051] (3) Polypeptide synthesis

[0052] The 4 polypeptide sequences screened according to step (2) were synthesized by solid-phase chemical synthesis for subsequent experiments.

[0053] Table 1. Sequence information of relevant polypeptides

[0054] SEQ ID NO Name Peptide sequence Length SPP score 1 SA-LFP1 CLAGRRRRSV 10 0.96 2 SA-LFP3 CLAGRRRRS 9 0.97 3 SA-LFP4 KLCKLRKGNC 10 0.95 4 SA-LFP13 RCQHRDLKWW 10 0.97

[0055] Example 2. Detection of the in vitro antioxidant capacity of bioactive peptides

[0056] (1) DPPH radical scavenging ability

[0057] Preparation of relevant reagents:

[0058] 1) DPPH solution: Prepare a 0.2 mM DPPH solution with absolute ethanol.

[0059] 2) Polypeptide solution: The polypeptides are selected from SA-LFP1 (SEQ ID NO: 1), SA-LFP3 (SEQ ID NO: 2), SA-LFP4 (SEQ ID NO: 3), SA-LFP13 (SEQ ID NO: 4) with Cys amino acid residues, customized and synthesized from GenScript Biotech Corporation, and dissolved in pure water to a stock solution of 2 mg / mL for storage.

[0060] Experimental protocol:

[0061] Sample#1

[0062] Respectively pipette 100 μL of polypeptide solutions with different concentrations, add 100 μL of 0.2 mM DPPH ethanol solution, shake well, and place at room temperature in the dark for 30 min. Measure the absorbance At at 517 nm. At the same time, measure the absorbance Ar of 100 μL of polypeptide solution + 100 μL of ethanol solution at 517 nm, and then measure the absorbance Ao of 100 μL of DPPH solution + 100 μL of ethanol at 517 nm. Three parallels were designed for the same measurement. The results are shown in Tables 2 and 3.

[0063] DPPH radical scavenging rate (%) = [1 - (At - Ar) / Ao] × 100

[0064] Ao: Absorbance value of 100 μL absolute ethanol + 100 μL DPPH solution;

[0065] At: Absorbance value of 100 μL sample solution + 100 μL DPPH solution;

[0066] Ar: Absorbance value of 100 μL sample solution + 100 μL absolute ethanol.

[0067] Experimental results:

[0068] DPPH free radical is a stable nitrogen-centered purple free radical, and antioxidants can reduce DPPH free radical to a yellow compound. The reason for this phenomenon is that DPPH free radical can accept an electron or a hydrogen atom to form a stable diamagnetic molecule. Four polypeptides and GSH were uniformly determined for DPPH free radical scavenging activity to comprehensively evaluate their antioxidant activities. The results are shown in Table 2. For the four bioactive peptides, when the concentration was 0.5 mg / mL, their DPPH free radical scavenging rates were 80.18% - 95.18%. The IC50 values of DPPH free radical scavenging activities of SA-LFP1, SA-LFP3, SA-LFP 4, and SA-LFP 13 were 0.024, 0.031, 0.042, and 0.030 mg / mL, respectively (see Table 3). In addition, the DPPH free radical scavenging activity of glutathione was 0.032 mg / mL. Obviously, in terms of DPPH free radical scavenging activity, glutathione has very high activity. Compared with glutathione, the DPPH free radical scavenging activities of the four peptides were comparable to that of glutathione, and even the DPPH free radical scavenging activities of SA-LFP1, SA-LFP3, and SA-LFP13 were better than that of glutathione. It can be clearly seen that the four polypeptides and glutathione all have a common Cys amino acid residue. Due to the presence of the sulfhydryl group of the Cys amino acid residue, it can directly react with free radicals (Jiang H, Tong T, Sun J, et al. Purification and characterization of antioxidative peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate [J]. Food Chemistry, 2014, 154: 158 - 163.), so this type of polypeptide has strong DPPH free radical scavenging activity.

[0069] (2) ABTS free radical scavenging ability

[0070] Preparation of related reagents:

[0071] 1) ABTS solution: Weigh 19.2 mg of ABTS and add 5 mL of pure water to dissolve it into a 7 mM stock solution.

[0072] 2) Potassium persulfate solution: Weigh 189 mg of potassium persulfate and add 5 mL of pure water to dissolve it into a 140 mM stock solution.

[0073] 3) ABTS stock solution: Mix 5 mL of ABTS solution and 5 mL of potassium persulfate solution, and let it stand overnight for 16 h at room temperature in the dark to form the ABTS stock solution.

[0074] 4) Polypeptide solution: Custom synthesized from GenScript Biotech Corporation and stored as a 2 mg / mL stock solution dissolved in pure water.

[0075] Experimental protocol:

[0076] Sample#1

[0077] 1) Dilute the above ABTS stock solution with pure water to a working solution, and the absorbance of the working solution at a wavelength of 734 nm is 0.7 ± 0.02.

[0078] 2) Pipette 100 μL of polypeptide solutions with different concentrations respectively, add 100 μL of ABTS working solution, shake for 1 - 2 min, place at 37 °C for 10 min, then measure the absorbance As at 734 nm. Use 100 μL of ABTS working solution + 100 μL of distilled water as the blank absorbance Ac. Use the absorbance of the mixture of 100 μL of polypeptide solution + 100 μL of distilled water as Ab. Design 3 parallels for the same determination. The results are shown in Table 2 and Table 3.

[0079] ABTS radical scavenging rate (%) = [1 - (As - Ab) / Ac] × 100

[0080] As: Absorbance of 100 μL of sample solution added to 100 μL of ABTS solution;

[0081] Ab: Absorbance of 100 μL of sample solution added to 100 μL of distilled water;

[0082] Ac: Absorbance of 100 μL of ABTS solution added to 100 μL of distilled water.

[0083] Experimental results:

[0084] In the reaction system, ABTS will be oxidized to generate ABTS free radicals, which are stable and water-soluble free radicals with a blue-green color and a maximum absorbance value at 734nm. The reaction of ABTS free radicals with antioxidants can make the characteristic color of the former fade, thereby reducing the absorbance value. Under the same conditions, the antioxidant activity of biological samples is proportional to the degree of decrease in the absorbance value of the reaction solution. The ABTS free radical scavenging rates of the four peptides are shown in Table 2. At a concentration of 0.5 mg / mL, their ABTS free radical scavenging rates are 90.01% to 97.79%. Their IC50s are 0.012, 0.036, 0.028 and 0.048 mg / mL, respectively (Table 3).

[0085] Table 2. Scavenging ability of 0.5 mg / mL peptide on DPPH free radical and ABTS free radical

[0086] Number DPPH radical scavenging rate % ABTS radical scavenging rate % SA-LFP1 90.02±0.56 97.79±0.32 SA-LFP3 84.48±0.49 96.22±0.71 SA-LFP4 95.18±0.77 95.41±0.41 SA-LFP13 80.18±0.68 90.01±0.29

[0087] Table 3. IC values of scavenging ability of 4 peptides and glutathione on DPPH free radical and ABTS free radical 50 value

[0088]

[0089]

[0090] Studies have shown that the reaction rate of ABTS free radicals with amino acids is mainly determined by whether the amino acid side chain has unstable hydrogen atoms, and is related to the pH of the solution and the concentration of the sample. Under the same reaction conditions, the activity of several amino acids in scavenging free radicals is ranked as follows: Cys>Trp>Tyr>His (Aliaga C, Lissi E A. Reactions of the radical cation derived from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)(ABTS·+)with amino acids. Kinetics and mechanism[J]. Canadian Journal of Chemistry, 2000, 78(8):1052-1059.). Consistent with the above research conclusions, peptides with strong ABTS free radical scavenging ability all contain Cys amino acids that are prone to lose hydrogen atoms in the reaction. It can be seen that the ABTS free radical scavenging activity of the polypeptide is related to the type of its amino acid residues, and different types of amino acid residues have different activities in free radical reactions.

[0091] Example 3. Verification of Antioxidant Activity of Bioactive Peptides (Cell Model)

[0092] Preparation of related reagents:

[0093] 1) AAPH

[0094] Weigh 2.034 g of AAPH, dissolve it in 15 mL of PBS to prepare a 500 mM stock solution, filter it and dispense it into EP tubes, and store it at -20°C in the dark.

[0095] 2) Preparation of polypeptide stock solution

[0096] Use sterilized distilled water as the solvent to prepare a stock solution with a concentration of 2 mg / mL, and dispense it for storage at -20°C.

[0097] 3) DCFH mixture

[0098] Purchased from Beyotime Biotechnology Co., Ltd., and the specific operation can refer to the instruction manual.

[0099] Experimental protocol

[0100] Protective effect of Sample#1 bioactive peptide on oxidatively damaged cells

[0101] Seed HaCaT cells in a 96-well plate at a density of 1.0×10^5. After 24 h of stable cell culture, add 85 mM AAPH to the model group and experimental groups to act on the cells for 2 h. Then discard the culture medium. Add culture medium containing different concentrations of polypeptide (1, 10, 100 μg / mL) to the experimental groups, and add complete culture medium to the blank control group and model group. Culture at 37°C and 5% CO2 for 24 h. Then discard the culture medium. Add 90 μL of basic culture medium and 10 μL of CCK8 to each well, mix well, and incubate at 37°C for 40 min. After incubation, measure the absorbance at 450 nm. Cell viability (%) = (OD of experimental group - OD of blank well) / (OD of blank control group - OD of blank well) * 100%.

[0102] The experimental results are as Figures 1 to 5 shown. It can be seen from the figure that compared with the blank control group, the viability of oxidatively damaged cells treated with AAPH was significantly reduced, and the cell viability was only 60% (P < 0.05). After treatment with SA-LFP1, the cell viability was significantly increased and showed a concentration-dependent manner. When the concentration was 1 μg / mL, the cell viability reached 77% (P < 0.05), indicating that SA-LFP1 had exerted a repair effect on oxidatively damaged cells at a low concentration. While glutathione had no significant repair effect at a low concentration of 1 μg / mL. SA-LFP4 only had a significant repair effect at a high concentration of 100 μg / mL. It shows that compared with glutathione and other polypeptides, SA-LFP1 has the best repair effect on oxidatively damaged cells.

[0103] Scavenging effect of Sample#2 bioactive peptide on intracellular ROS in oxidatively damaged cells

[0104] HaCaT cells were seeded in 6-well plates at a density of 1.0×10^5. After 24 h of stable cell culture, 2 mM AAPH was added to the model group and the experimental group for 2 h. Then, the culture medium was discarded. The experimental group was added with the culture medium containing different concentrations of SA-LFP1 (1, 10, 100 μg / mL), and the blank control group and the model group were added with complete culture medium. Incubate at 37°C and 5% CO2 for 24 h. DCFH was diluted 1:1000 with serum-free medium to make the probe concentration 10 μM. Then, 1 mL of the mixed culture medium was added to the wells and incubated for 20 - 30 min. Then, wash 2 - 3 times with serum-free medium to wash away the DCFH that did not enter the cells, and then observe under a fluorescence microscope.

[0105] Experimental results:

[0106] It can be seen from Figure 5 that compared with the blank control group, the intracellular ROS in oxidatively damaged cells treated with AAPH increased significantly, and the fluorescence intensity increased significantly, indicating that the cells were severely oxidatively damaged. As Figure 6 shown in the results, after treatment with different concentrations of SA-LFP1 (1 μg / mL, 10 μg / mL, 100 μg / mL), the fluorescence intensity in the experimental group cells decreased significantly and showed a concentration-dependent manner. The higher the polypeptide concentration, the lower the intracellular ROS content. It shows that SA-LFP1 can scavenge intracellular ROS free radicals.

[0107] Example 4. Detection of the wound healing promoting ability of bioactive peptide

[0108] Cell scratch assay

[0109] Preparation of related reagents:

[0110] 1) Preparation of SA-LFP1 polypeptide stock solution

[0111] Using sterilized distilled water as the solvent, prepare a stock solution with a concentration of 2 mg / mL, and aliquot and store at -20°C.

[0112] 2) bFGF stock solution

[0113] Add 100 μL of sterile water to 20 μg of bFGF. After standing at room temperature for 20 min to fully dissolve, add 10% BSA for further dilution and aliquot, and then store at -80°C. The mother liquor concentration is 200 μg / mL.

[0114] Experimental protocol

[0115] Sample#1:

[0116] In a biosafety cabinet, draw lines at intervals of 0.6 cm on the bottom of a 6-well plate. Then, seed HaCaT cells in the 6-well plate at a density of 12×10^5, and culture them at 37°C and 5% CO2 for 24 h. After the cells are stable, use a 200 μL pipette tip to make scratches perpendicular to the drawn lines, making 3 scratches in each well. Slowly wash the cells with PBS to remove floating cells, and then add different concentrations of SA-LFP1 polypeptide (1 μg / mL, 10 μg / mL, 100 μg / mL) and bFGF (0.1 μg / mL, 1 μg / mL) prepared with basic medium DMEM to act on the cells. The blank control group is added with DMEM. Then, fix 3 sites in each well, and at 0 h, 4 h, 6 h, 24 h, and 48 h after the sample acts, record the scratch conditions by taking pictures under a microscope.

[0117] Experimental results: The results are as Figure 7 and Figure 8 shown. It can be seen from Figure 7 that after SA-LFP1 polypeptide acts on cells for 6 h, the cell migration rate is comparable to that of bFGF, indicating that SA-LFP1 has a strong repair effect similar to bFGF; Figure 8 it can be seen that compared with the blank control group, after SA-LFP1 polypeptide is incubated for 6 h, the cells migrate to the gap, and the scratch wound becomes flat and narrow; after further incubation for 24 h, SA-LFP1 polypeptide promotes further cell migration, and the cell migration rate reaches 30%, which is much higher than that of the blank control group and is comparable to the effect of the positive control bFGF, indicating that SA-LFP1 polypeptide can significantly promote wound healing and has a good repair effect.

[0118] Example 5: Verification of the non-toxic and non-irritating properties of bioactive peptides based on a cell model

[0119] Preparation of related reagents:

[0120] 1) Cells

[0121] Human keratinocytes (HaCaT)

[0122] 2) Preparation of bioactive peptide stock solution

[0123] Using sterilized distilled water as a solvent, prepare a stock solution of transdermal enhancing peptide with a concentration of 3 mM, and then aliquot and store it at -20°C for later use. The polypeptides selected in the present invention are those shown in the following amino acid sequences: SA-LFP1, SA-LFP3, SA-LFP4, and SA-LFP13.

[0124] 3) Cell lysate

[0125] 1M NaOH: Weigh 4.0 g of sodium hydroxide solid and make it up to 100 mL of sodium hydroxide solution.

[0126] Experimental protocol:

[0127] Cytotoxicity Detection of Sample#1

[0128] HaCaT cells were seeded in 96-well plates at a density of 1.0×10^5. After 24 h of culture and cell stabilization, different concentrations of the above polypeptides were added to the cells. The cells were cultured at 37 °C and 5% CO2 for 24 h. Then, the culture medium was discarded. 90 μL of basal medium and 10 μL of CCK8 were added to each well and mixed evenly. The mixture was incubated at 37 °C for 40 min. After the incubation, the absorbance was measured at 450 nm. Cell viability (%) = (OD of experimental group - OD of blank well) / (OD of blank control group - OD of blank well) * 100%. When the cell viability < 70%, it indicates that the bioactive peptide has cytotoxicity.

[0129] Experimental Results:

[0130] The cytotoxicity detection results of the bioactive peptides are shown in Table 4. The results indicate that these polypeptides are non-toxic to HaCaT cells within the tested concentration range (5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 200 μM).

[0131] Table 4 Cytotoxicity Results of Bioactive Peptides

[0132] Polypeptide 5 μM 10 μM 20 μM 50 μM 100 μM 200 μM SA-LFP1 － － － － － － SA-LFP3 － － － － － － SA-LFP4 － － － － － － SA-LFP13 － － － － － －

[0133] +: Cytotoxic; -: Non-cytotoxic.

Claims

1. A bioactive peptide, characterized in that, Its amino acid sequence is as shown in SEQ ID NO:

1.

2. The coding polynucleotide of the bioactive peptide according to claim 1.

3. The use of the bioactive peptide according to claim 1 in the preparation of a preparation having an antioxidant effect.

4. The application according to claim 3, wherein The preparation is a food, a cosmetic or a pharmaceutical.

5. The application according to claim 4, characterized in that The bioactive peptide has an antioxidant effect.

6. The application according to claim 5, characterized in that The preparation having an antioxidant effect is a skin product.

7. The application according to claim 6, wherein The preparation having an antioxidant effect is a topical skin medicine.

8. The application according to claim 7, wherein The bioactive peptide has a wound healing or repair effect.

9. The application according to claim 7, wherein The skin product having an antioxidant effect is a skin moisturizing product, a skin anti-aging product or a skin sunscreen product.

10. The application according to claim 9, characterized in that, The skin product having an antioxidant effect is a hand cream or an eye cream.

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