Lactoferrin-derived bioactive polypeptide and preparation and application thereof

Abstract

The invention discloses a lactoferrin-derived bioactive polypeptide as well as preparation and application thereof. The amino acid sequence of the bioactive polypeptide conforms to the general formula TKCX1QWQRX2MX3K, wherein X1 is phenylalanine (F), arginine (R) or alanine (A); x2 is alanine (A), asparagine (N), tryptophan (W) or ornithine (O), and X3 is arginine (R) or lysine (K). In-vitro experiment results prove that the bioactive polypeptide disclosed by the invention has good effects of resisting oxidation, relieving and brightening skin color; when the composition is applied to cosmetics, damage to skin caused by environmental stimulation and endogenous pressure can be relieved, and the skin color can be improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bioactive polypeptide derived from lactoferrin and its preparation and application. Background Technology

[0002] Lactoferrin is an iron-binding glycoprotein found in milk and other exocrine fluids, and it has attracted much attention in cosmetics and personal care products due to its various biological activities. It possesses broad-spectrum antibacterial activity, inhibiting the growth of a variety of bacteria, including Gram-positive and Gram-negative bacteria. Its antibacterial mechanism is mainly through iron chelation, depriving bacteria of the iron they need for growth, thereby inhibiting bacterial reproduction. Simultaneously, it enhances the host's immune response, helping to resist pathogen invasion. Lactoferrin also exhibits significant antioxidant properties, reducing the production of reactive oxygen species (ROS), protecting cells from oxidative damage. Furthermore, it can activate intracellular antioxidant defense mechanisms, enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx). In terms of brightening skin tone, lactoferrin's effectiveness is mainly attributed to its inhibition of tyrosinase activity and its soothing effect. Tyrosinase is a key enzyme in melanin synthesis, and lactoferrin reduces melanin production by inhibiting its activity. External stimuli-induced inflammation leads to increased melanin synthesis, and lactoferrin can reduce melanin production signals at the source by inhibiting the expression of inflammatory factors. Furthermore, skin radiance depends on a plump structure; collagen loss leads to dull skin. Reducing collagen degradation caused by environmental stimuli and promoting collagen production also play important roles in improving skin tone. Summary of the Invention

[0003] To address the aforementioned technical problems in the prior art, this invention provides a bioactive polypeptide derived from lactoferrin, its preparation, and its application.

[0004] Specifically, the present invention solves the above-mentioned technical problems through the following technical solutions.

[0005] A first aspect of the present invention provides a bioactive polypeptide having an amino acid sequence as shown in SEQ ID NO: 11, but not SEQ ID NO: 1;

[0006] SEQ ID NO: 11 is shown below:

[0007] TKCX1QWQRX2MX3K, where X1 is phenylalanine (F), arginine (R) or alanine (A); X2 is alanine (A), asparagine (N), tryptophan (W) or ornithine (O); and X3 is arginine (R) or lysine (K).

[0008] The bioactive peptides have improved antioxidant activity, improved tyrosinase inhibition activity, and / or improved melanin production inhibition activity.

[0009] In some embodiments, the amino acid sequence of the bioactive polypeptide is as shown in SEQ ID NO: 11, and is not SEQ ID NO: 1;

[0010] SEQ ID NO: 11 is shown below:

[0011] TKCX1QWQRX2MX3K, where X1 is phenylalanine (F), arginine (R) or alanine (A); X2 is alanine (A), asparagine (N), tryptophan (W) or ornithine (O); and X3 is arginine (R) or lysine (K).

[0012] In some embodiments, the bioactive peptide has improved antioxidant activity, improved tyrosinase inhibition activity, and / or improved melanin production inhibition activity.

[0013] In some embodiments, the bioactive peptide has improved antioxidant activity, improved tyrosinase inhibition activity, and / or improved melanin production inhibition activity relative to the peptide with the amino acid sequence shown in SEQ ID NO: 1.

[0014] In some implementations, SEQ ID NO: 11:

[0015] X1 is R, X2 is N, W, A or O, and X3 is K or R;

[0016] Alternatively, X1 is A, X2 is N, W, A or O, and X3 is K or R;

[0017] Alternatively, X1 is F, X2 is N, and X3 is K.

[0018] In some implementations, in SEQ ID NO: 11: X1 is A, X2 is W, A or O, and X3 is R.

[0019] In some specific embodiments, the amino acid sequence of the bioactive polypeptide is as shown in any of SEQ ID NO: 2-10.

[0020] A second aspect of the present invention provides an isolated nucleic acid that encodes a bioactive polypeptide as described in the first aspect of the present invention.

[0021] A third aspect of the present invention provides a recombinant vector comprising the nucleic acid as described in the second aspect of the present invention.

[0022] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention.

[0023] A fifth aspect of the invention provides a composition comprising a bioactive polypeptide as described in the first aspect of the invention, and at least one of a pharmaceutically acceptable carrier, excipient, and diluent.

[0024] In some embodiments, the composition is a cosmetic composition.

[0025] In some embodiments, the cosmetic composition is in the form of a foam, liniment, gel, patch, spray, cream, emulsion, solution, tincture, or ointment.

[0026] In some embodiments, the effective content of the bioactive polypeptide in the composition is 1-10000 ppm.

[0027] A sixth aspect of the present invention provides a method for preparing bioactive polypeptides, the method comprising culturing a transformant as described in the fourth aspect of the present invention under suitable conditions to obtain a culture product containing the bioactive polypeptide; or directly synthesizing the bioactive polypeptide as described in the first aspect of the present invention using a chemical synthesis method.

[0028] In some embodiments, the chemical synthesis method is a liquid-phase synthesis method or a solid-phase synthesis method.

[0029] The seventh aspect of the present invention provides the use of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, a bioactive polypeptide as described in the first aspect of the present invention, or a composition as described in the fifth aspect of the present invention in the preparation of products for antioxidant, soothing, skin repair, or skin brightening purposes.

[0030] In some implementations, the product is a cosmetic or a pharmaceutical.

[0031] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0032] The reagents and raw materials used in this invention are all commercially available.

[0033] The positive and progressive effects of this invention are as follows:

[0034] Based on the structure-activity relationship study of the active site of lactoferrin, this invention optimizes and screens a series of lead peptides that show good effects in anti-oxidation, soothing and brightening of skin tone. When applied in cosmetics, they can protect the skin from environmental stimuli and endogenous damage and improve skin tone. Attached Figure Description

[0035] Figure 1 The effect of bioactive peptides on the content of ROS free radicals in cells (relative fluorescence intensity).

[0036] Figure 2 The effect of bioactive peptides on the content of ROS free radicals in cells (cell fluorescence intensity and distribution).

[0037] Figure 3 The effect of bioactive peptides on the expression level of the POMC gene in cells.

[0038] Figure 4 The effect of bioactive peptides on the expression level of the IL6 gene in cells.

[0039] Figure 5 The effect of bioactive peptides on the expression level of the CXCL8 gene in cells.

[0040] Figure 6 The effect of bioactive peptides on the expression level of the IL33 gene in cells.

[0041] Figure 7 The effect of bioactive peptides on the expression level of the ET-1 (END1) gene in cells.

[0042] Figure 8 The effect of bioactive peptides on the expression level of the HSP70 gene in cells. Detailed Implementation

[0043] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0044] The term "antioxidant" refers to mitigating the damage to the skin caused by oxidative stress by reducing reactive oxygen species (ROS).

[0045] The term "soothing" refers to relieving skin irritation or discomfort through anti-inflammatory and sedative methods.

[0046] The term "skin repair" refers to repairing the damaged skin barrier structure and restoring its function of resisting external damage and maintaining internal environmental stability.

[0047] The term "skin brightening" refers to improving dull skin tone by inhibiting melanin production and other methods.

[0048] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0049] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0050] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0051] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0052] Example 1: Preparation of Bioactive Peptides

[0053] I. Polypeptide Synthesis

[0054] The solid-phase synthesis method for polypeptides is described using SEQ ID NO: 1 as an example. Other sequences can be prepared using a similar method by replacing the Fmoc-protected amino acids at specific sites.

[0055] 1. Weigh 3g of RINK resin (degree of substitution 0.3mmol / g) into a 150ml reactor and soak it in 50ml of dichloromethane (DCM). After 2 hours, wash the resin with 3 times its volume of DMF, then dry it. Repeat this process four times. After drying the resin, set it aside for later use.

[0056] 2. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4, v / v) to the reactor and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting groups from the resin. After deprotection, wash four times with 3 times the volume of DMF. Take a small amount of resin and test the reaction using the ninhydrin method; color development of the resin indicates successful deprotection.

[0057] 3. Weigh appropriate amounts of Fmoc-protected amino acid Lys and 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, add 20ml of DMF to dissolve them, then add 3ml of N,N-diisopropylcarbodiimide (DIC) and shake well for 1min. After the solution becomes clear, add it to the reactor and then place the reactor in a shaker at 30℃ to react.

[0058] 4. After 2 hours, cap the head with a certain amount of acetic anhydride (acetic anhydride:DIEA:DCM=1:1:2, v / v / v) for half an hour, then wash four times with 3 times the volume of DMF, and dry for later use.

[0059] 5. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4, v:v) to the reactor, place it on a decolorizing shaker and shake for 20 min to remove the Fmoc protecting group. Wash the resin four times with DMF and dry it. Detect the reaction using the ninhydrin method.

[0060] 6. Repeat steps 3-5, sequentially adding the other amino acids from the sequence.

[0061] 7. After the reaction was complete, the Fmoc protection was removed, the resin was washed four times with DMF, and then the resin was dried with methanol. The peptide was cleaved from the resin using a cleavage buffer (trifluoroacetic acid:triisopropylsilane:water = 95:2.5:2.5, v / v / v) (10 mL of cleavage buffer was added per gram of resin), and the peptide was centrifuged four times with ice-cold diethyl ether (cleavage buffer:diethyl ether = 1:9, v / v). The solvent was then removed under vacuum to obtain the crude peptide.

[0062] 8. Dissolve the crude peptide, purify it by high performance liquid chromatography, collect the main product, select acetic acid as the mobile phase, and freeze-dry to obtain the corresponding polypeptide product.

[0063] Table 1. Sequence Information:

[0064]

[0065] Wherein, X1 is phenylalanine F, arginine R or alanine A, X2 is alanine A, asparagine N, tryptophan W or ornithine O, and X3 is arginine R or lysine K.

[0066] II. Confirmation of peptide purity and structure

[0067] Purity was determined using HPLC. Analytical method:

[0068] Instrument Model: SHIMADZU Prominence LC-20A High Performance Liquid Chromatograph

[0069] Column: ACE Excel 5 C18 amide, 250 × 4.6 mm

[0070] Elution system: 0.1% TFA in MeCN / 0.1% TFA in H2O

[0071] Table 2. HPLC elution procedure

[0072]

[0073] Test sample solvent: H2O

[0074] Sample concentration: 1 mg / mL

[0075] Injection volume: 30 μL

[0076] Flow rate: 1 mL / min

[0077] Detection wavelength: 210 nm

[0078] The purity of the target molecule was determined by the peak area integral method.

[0079] The structure was determined using LCMS:

[0080] High-resolution mass spectrometry instrument model: Thermo Fisher Q Exactive

[0081] Ion source: ESI source

[0082] Test sample solvent: 100% water

[0083] Mobile phase: 50% MeOH / H2O

[0084] Injection volume: 1 μL

[0085] The target molecular weight is determined by comparing characteristic ion peaks.

[0086] Table 3. LCMS Analysis Results

[0087]

[0088] HPLC and MS tests confirmed that the synthesized active peptide had high purity and was consistent with the theoretical sequence.

[0089] Example 2: Antioxidant Test

[0090] I. DPPH Free Radical Scavenging Rate Test

[0091] The antioxidant properties of bioactive peptides were tested in accordance with the national standard GB / T 39100-2020 "Determination of Antioxidant Properties of Peptides by DPPH and ABTS Methods".

[0092] Test method:

[0093] Accurately weigh DPPH standard, glutathione standard, and the bioactive polypeptide sample obtained in Example 1, and prepare a test solution with anhydrous ethanol to a concentration of 100 mg / L. Glutathione is used as a positive control.

[0094] Add 3.5 mL of DPPH anhydrous ethanol solution to a small test tube, add 0.5 mL of anhydrous ethanol, mix thoroughly, and zero the microscope at 517 nm using anhydrous ethanol as a reference. Measure the absorbance value A0. Add 3.5 mL of DPPH anhydrous ethanol solution to a small test tube, add 0.5 mL of sample solution, mix thoroughly, let stand in the dark for 30 min, then centrifuge at 8000 r / min for 5 min. Take the supernatant and zero the microscope at 517 nm using anhydrous ethanol as a reference. Measure the absorbance value Ai. Add 3.5 mL of anhydrous ethanol to a small test tube, add 0.5 mL of sample solution, mix thoroughly, let stand in the dark for 30 min, then centrifuge at 8000 r / min for 5 min. Take the supernatant and zero the microscope at 517 nm using anhydrous ethanol as a reference. Measure the absorbance value Aj. Vitamin C is used as a positive control. Three parallel samples are set up for each experimental group.

[0095] Calculation of clearance rate SR / %: SR / % = [1 - (Ai - Aj) / Ao] * 100%. See the table below for the corresponding results:

[0096] Table 4. Results of DPPH free radical scavenging experiments

[0097]

[0098] II. ABTS Free Radical Scavenging Experiment

[0099] The antioxidant properties of bioactive peptides were tested in accordance with the national standard GB / T 39100-2020 "Determination of Antioxidant Properties of Peptides by DPPH and ABTS Methods".

[0100] Test method:

[0101] Weigh 200 mg of ABTS and 34.4 mg of potassium persulfate, dissolve them in 50.0 mL of distilled water, shake well, and let stand at room temperature in the dark for 24 hours to obtain the ABTS stock solution. Weigh an appropriate amount of the ABTS stock solution and dilute it with 95% ethanol until the absorbance at 734 nm is within 0.70 ± 0.02 to obtain the ABTS assay solution. This solution should be prepared fresh before use. Accurately weigh glutathione standard and the bioactive polypeptide sample obtained in Example 1, and prepare a 100 mg / L test solution with anhydrous ethanol. Glutathione serves as a positive control.

[0102] Add 3.6 mL of ABTS solution and 0.4 mL of glutathione solution to a test tube as a control group, and measure their absorbance data as A. b Add 3.6 mL of ABTS solution and 0.4 mL of sample solvent solution to a test tube as a blank group, and measure its absorbance data as A. cAdd 3.6 mL of ABTS solution and 0.4 mL of sample solution to a test tube as the experimental group, and measure its absorbance data as A. b Each experimental group had 3 parallel samples. The SR% was calculated using the formula SR%=(A b -A c / A c )*100 calculates the clearance rate.

[0103] Table 5. Results of ABTS free radical scavenging experiments

[0104]

[0105] It was found that bioactive peptides have good scavenging effects on both DPPH and ABTS free radicals, exhibiting antioxidant efficacy. They can reduce the impact of external environmental stress on the skin by scavenging free radicals.

[0106] Example 3: Tyrosinase Activity Inhibition Test

[0107] The inhibitory effect of bioactive peptides on tyrosinase activity was detected according to T / SHRH 015-2018 "Cosmetics - Experimental Method for Inhibition of Tyrosinase Activity".

[0108] Test method:

[0109] 14.33 g of disodium hydrogen phosphate dodecahydrate was dissolved in 200 mL of water and stirred with a glass rod until dissolved, yielding solution a; 2.1 g of citric acid monohydrate was dissolved in 100 mL of water and stirred with a glass plate until dissolved, yielding solution b; 154.5 mL of solution a and 45.5 mL of solution b were mixed to obtain a disodium hydrogen phosphate-citric acid buffer solution. Tyrosinase (activity ≥1000 units / mg solid) was dissolved in the buffer solution to 100 u / mL and prepared fresh before use; levodopa (purity ≥98%) was dissolved in the buffer solution to 1 mg / mL and stored protected from light. Kojic acid, a positive control, was prepared into gradient solutions with concentrations of 1000, 200, 40, 8, and 1.6 ppm, and active peptide samples were prepared into gradient solutions with concentrations of 1000, 500, 250, 125, and 62.5 ppm.

[0110] Set up sample group T, sample background group T0, blank group C, and blank background group C0. Add 1 mL of sample solution and 0.5 mL of tyrosinase solution to the sample group; add 1 mL of sample solution and 0.5 mL of buffer solution to the sample background group; add 1 mL of buffer solution and 0.5 mL of tyrosinase solution to the blank group; and add 1.5 mL of buffer solution to the blank background group. Mix thoroughly and incubate in a 37℃ water bath for 10 minutes. Add 2 mL of levodopa solution to each tube, ensuring a consistent reaction time of 5 minutes per tube, and immediately measure the absorbance at 475 nm. Set up three parallel samples for each sample. Calculate the tyrosinase activity inhibition rate using the following formula: SR / % = [1 - (T - T0) / (C - C0)] * 100%. Calculate the IC50 concentration at which the inhibition rate reaches 50% based on the inhibition rate fitting curve of the gradient concentration. 50 value.

[0111] Table 6. Results of Tyrosinase Activity Inhibition Test

[0112]

[0113] It was found that bioactive peptides have a good inhibitory effect on tyrosinase activity. By inhibiting tyrosinase activity, they can reduce melanin production, alleviate environmentally induced skin pigmentation, and achieve a brightening effect on skin tone.

[0114] Example 4: Cell viability test

[0115] The effects of bioactive peptides on the activity of HaCaT keratinocytes and B16 melanocytes were detected using the MTT assay.

[0116] Test method:

[0117] HaCaT cells and B16 cells were seeded into 96-well plates and incubated overnight in a CO2 incubator. A solvent control group and a sample group (the bioactive peptide prepared in Example 1) were set up. Drug administration was performed when the cell deposition rate in the 96-well plates reached 50-60%, with three replicates per group. 200 μL of DMEM culture medium was added to each well of the solvent control group; 200 μL of culture medium containing the corresponding concentration of the test sample was added to each well of the sample group, with concentration gradients of 1000, 500, 250, 125, 62.5, and 31.25 ppm. After drug administration, the 96-well plates were incubated in a CO2 incubator for 24 h. After 24 h of cell incubation, the supernatant was discarded, and 0.5 mg / mL MTT working solution was added, followed by incubation at 37°C in the dark for 4 h. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well, with OD values ​​read at 490 nm.

[0118] Calculate cell viability: After measuring the OD values ​​of each group, calculate the average value of each group, and then calculate the cell proliferation rate of each group using the following formula: Cell viability (%) = OD value of drug-treated group / OD value of control group × 100%. Calculate the concentration of bioactive peptides when the cell viability is greater than 90%, and record it as the CV90 value.

[0119] Table 7. Results of cell viability test

[0120]

[0121] Example 5: Cellular level antioxidant activity assay – ROS fluorescent probe method

[0122] Test method:

[0123] HaCaT cells were seeded at a density of 2 × 10⁵ cells / well in 24-well plates and incubated overnight in a CO₂ incubator. The experiment consisted of a blank control group, a negative control group, a positive control group, and an experimental group, with three replicates in each group. The positive control group received 30 ppm glutathione, the experimental group received 30 ppm of the active peptide, and the negative control group received no treatment. When the cell deposition rate reached 30-50%, the cells were treated in groups, with 2 mL of sample added to each well, in triplicate. After treatment, the cells were incubated for 24 hours. Subsequently, except for the blank control group, appropriate concentrations of hydrogen peroxide solution were added to the other groups to induce ROS generation. After incubation, the cells were washed with PBS, and then 1 mL of 10 μM DCFH-DA probe was added to each well. The cells were incubated for 30 minutes. After incubation, the culture medium containing the probe was discarded, and the cells were washed with DPBS. The washed cells were then digested with 0.25% trypsin and washed again with DPBS. Cell culture dishes were placed under a fluorescence microscope, and appropriate excitation and emission wavelengths were selected (DCFH-DA excitation wavelength approximately 488 nm, emission wavelength approximately 525 nm). Cell fluorescence intensity and distribution were observed and photographed. 96-well plates were placed in a microplate reader, and the same wavelength parameters were set to detect the fluorescence intensity values ​​of each well. Intracellular ROS levels were expressed as relative fluorescence intensity. Test results are shown below. Figure 1 and Figure 2 In the attached figure, * indicates p≤0.05, and ** indicates p≤0.01.

[0124] It can be found that bioactive peptides have a good scavenging effect on intracellular ROS, exhibiting antioxidant effects, and can reduce the impact of external environmental stress on the skin by scavenging free radicals.

[0125] Example 6: Detection of gene levels of factors related to melanin synthesis

[0126] Melanin synthesis is regulated by multiple signaling pathways, and inflammation induced by external environmental stimuli increases melanin synthesis. By influencing the expression of melanin synthesis-related factors, the amount of melanin produced in the skin can be controlled, thereby improving skin tone. The following experimental protocol was used to detect the effect of bioactive peptides on the gene expression levels of melanin synthesis-related factors.

[0127] Experimental Groups:

[0128] Blank group (without UVB modeling)

[0129] Model group (UVB modeling)

[0130] Positive control group (UVB modeling + 30 ppm tranexamic acid)

[0131] Experimental group (UVB modeling + 30 ppm bioactive peptides)

[0132] Irradiation conditions: UVB irradiation intensity of 950 μW / cm 2 The total irradiation dose was 30 mJ / cm. 2

[0133] Experimental methods:

[0134] 1. Cell preparation: Resuscitate cells and culture them in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin. When the cell confluence reaches 80-90%, digest with 0.25% Trypsin-EDTA and passage at a ratio of 1:3.

[0135] 2. Plating: After digestion and counting of cells in the logarithmic growth phase, the cells were evenly seeded into culture plates. After a suitable incubation period, the cells were confirmed to be well adherent and growing. The culture medium was changed (the culture medium of the control group and the experimental group contained the drug), and the cells were placed back into the incubator for 24 h. When the confluence reached 40-60%, the supernatant was gently discarded, and the cells were washed twice with PBS to completely remove the culture medium. Subsequently, the model group and the control group were irradiated to create the model.

[0136] 3. UVB molding

[0137] ① After the light source is preheated and stabilized, the irradiation intensity should be measured using an irradiation meter to be 950 μW / cm². 2 Calculate the irradiation time using the following formulas (total irradiation dose is 30 mJ / cm). 2 ).

[0138] Irradiation dose (energy density) = Radiation intensity × Irradiation time

[0139] Note: The unit for irradiation intensity is generally μW / cm². 2 The unit conversion relationships are as follows:

[0140] 1 mW / cm 2 = 1000 μW / cm 2

[0141] ② Add PBS pre-cooled to 4°C to each well. The volume of PBS buffer should be adjusted according to the size of the culture substrate, approximately 100 μl per square centimeter. Then, irradiate with UVB without a cover (Note: the irradiation time varies for different irradiation distance groups).

[0142] ③ Recovery culture: After irradiation, discard the PBS and immediately add fresh complete culture medium (containing the drug in the culture medium of the control and experimental groups) to each well. Carefully observe the condition of the model group cells. If too many cells die (more than 40%), it may indicate that the irradiation is too strong, and the experimental protocol needs to be readjusted. If the cells are normal, return them to the incubator and continue culturing for 24 hours.

[0143] 4. RNA extraction and reverse transcription;

[0144] 5. qPCR detection: internal reference gene and POMC, IL6, CXCL8, IL33 and ET-1 (EDN1).

[0145] The experimental results are shown in Figure 3-7 In the attached figure, * indicates p≤0.05, and ** indicates p≤0.01.

[0146] It has been found that bioactive peptides can regulate the gene expression levels of factors related to melanin synthesis, thereby achieving the effect of brightening skin tone.

[0147] Example 7: HSP70 gene level test for maintaining skin protein homeostasis

[0148] The arrangement and density of collagen affect how light is reflected and refracted on the skin surface. Skin with high collagen content typically exhibits an even, smooth, and plump appearance, and usually has a brighter color. Conversely, when collagen content decreases, changes in skin structure lead to light scattering and refraction, making the skin appear dull. Heat shock protein HSP70 can repair skin damage caused by external stimuli and increase collagen synthesis.

[0149] The effect of the bioactive peptide on the expression level of the HSP70 gene in HaCaT cells was detected using a method similar to that in Example 6. Results are shown below. Figure 8 In the attached figure, * indicates p≤0.05, and ** indicates p≤0.01.

[0150] It was found that bioactive peptides can increase the expression level of the HSP70 gene, thereby mitigating the impact of environmental stimuli on skin protein homeostasis.

Claims

1. A bioactive polypeptide, characterized in that, The amino acid sequence of the bioactive polypeptide is shown in SEQ ID NO: 11, and is not SEQ ID NO: 1; SEQ ID NO: 11 is shown below: TKCX1QWQRX2MX3K, where X1 is phenylalanine (F), arginine (R) or alanine (A); X2 is alanine (A), asparagine (N), tryptophan (W) or ornithine (O); and X3 is arginine (R) or lysine (K). The bioactive peptides have improved antioxidant activity, improved tyrosinase inhibition activity, and / or improved melanin production inhibition activity.

2. The bioactive polypeptide according to claim 1, characterized in that, In SEQ ID NO: 11: X1 is R, X2 is N, W, A or O, and X3 is K or R; Alternatively, X1 is A, X2 is N, W, A or O, and X3 is K or R; Alternatively, X1 is F, X2 is N, and X3 is K; Preferably, the amino acid sequence of the bioactive polypeptide is as shown in any one of SEQ ID NO: 2-10.

3. An isolated nucleic acid, characterized in that, The nucleic acid encodes the bioactive polypeptide as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid as described in claim 3.

5. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 3 or the recombinant expression vector as described in claim 4.

6. A composition, characterized in that, The composition comprises the bioactive polypeptide as described in claim 1 or 2, and at least one of a pharmaceutically acceptable carrier, excipient, and diluent.

7. The composition according to claim 6, characterized in that, The composition is a cosmetic composition; Preferably, the cosmetic composition is in the form of a foam, liniment, gel, patch, spray, cream, emulsion, solution, tincture, or ointment.

8. The composition according to claim 6 or 7, characterized in that, The effective content of the bioactive polypeptide is 1-10000 ppm.

9. A method for preparing bioactive polypeptides, characterized in that, The method includes culturing the transformant as described in claim 5 under suitable conditions to obtain a culture product containing the bioactive polypeptide; or directly synthesizing it using chemical synthesis methods. The bioactive polypeptide as described in claim 1 or 2 is preferably synthesized by liquid-phase synthesis or solid-phase synthesis.

10. The use of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, a bioactive polypeptide as described in claim 1 or 2, or a composition as described in any one of claims 6-8 in the preparation of products for antioxidant, soothing, skin repair, or skin brightening purposes; Preferably, the product is a cosmetic or a pharmaceutical.

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