Active peptide with whitening effect as well as preparation method and application thereof

Abstract

The invention discloses an active peptide with a whitening effect as well as a preparation method and application thereof, and belongs to the technical field of biological medicines and cosmetics. The amino acid sequence of the active peptide is shown as SEQ ID No.1-26, the active peptide has a good inhibition effect on melanin synthesis and tyrosinase activity, and the active peptide has whitening activity and application potential as a whitening product, has the advantages of being safe, effective, free of toxic and side effects, good in biocompatibility and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and cosmetics, and particularly relates to an active peptide with whitening effect and a preparation method and application thereof. Background Art

[0002] Melanin is a biological pigment commonly found in plants and animals. Melanin can protect the skin from harm caused by ultraviolet radiation, oxidation and other factors. However, the skin is affected by ultraviolet rays, endocrine, inflammatory mediators, improper diet and other factors, which can cause abnormal melanin metabolism, excessive growth and uneven distribution of skin melanin, and then cause local skin darkening and pigmentation, manifested as chloasma, freckles and post-inflammatory pigmentation. The actual color of human skin mainly depends on the content and distribution of melanin. Most of the whitening agents on the market are regulated based on the two aspects of melanin production and transport, with the aim of reducing melanin production or transfer.

[0003] The synthesis of melanin is a series of complex enzymatic oxidation reactions, with tyrosine or dopa as substrate, oxidized to produce dopaquinone under the catalysis of tyrosinase. This oxidation reaction process is the key rate-limiting reaction in melanin synthesis, and the overexpression of tyrosinase is the main cause of excessive pigmentation. Therefore, inhibiting the oxidation reaction or the activity of tyrosinase can block the biosynthesis reaction chain of melanin, reduce the production of melanin, and achieve a whitening effect.

[0004] Reducing the production of melanin can be achieved by inhibiting the activity of tyrosinase. In recent years, more and more compounds have been proven to effectively inhibit the activity of tyrosinase, which are called tyrosinase inhibitors, such as kojic acid, arbutin, vitamin C derivatives and some natural extracts. However, vitamin C is unstable and not easily absorbed by the skin, making it difficult to be widely used; arbutin is a phenolic compound, which is safe but has a slow inhibitory effect on tyrosinase and good light absorption, making it unsuitable for daytime use; kojic acid has a significant whitening and freckle removal effect but has certain toxicity and cannot be used for a long time.

[0005] The defects of chemical whitening ingredients will have a great impact on the effect of whitening products and the experience of consumers. Therefore, it is of great significance to find natural, non-toxic and stable new whitening ingredients. The polypeptide has a small molecular weight, is easy to be absorbed through the skin, and has good stability in the formula. In recent years, polypeptides have gradually come into the public eye as new whitening agents, which have a very high market prospect, and related polypeptide products have been reported. Decapeptide-12 is a tyrosinase inhibitor that can interact with the C-terminal residue of tyrosinase. He et al. found that scytonemin separated and purified from seaweed and cyanobacteria extracts has a good in vitro mushroom tyrosinase inhibitory effect, and the inhibitory activity is greater than that of the commercially available kojic acid standard product. It can be used as a skin whitening agent or an auxiliary drug for the treatment of melanoma, and has a high cosmeceutical value. CN113980098A discloses a nonapeptide-1 derivative, which can inhibit tyrosinase activity and the synthesis of melanin. Therefore, finding a safe and effective melanin production inhibitory peptide has broad application prospects. Virtual screening refers to the pre-screening of compound molecules on a computer according to pre-set conditions before biological activity screening to identify small molecules that are most likely to bind to the target, thereby greatly reducing the number of compounds actually screened and improving the efficiency of lead compound discovery. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a group of active peptides that inhibit melanin production and have whitening effects, preparation methods and applications thereof. The present invention obtains active peptides that may inhibit melanin production and have whitening effects through a virtual screening method, and then further optimizes and designs the active peptides obtained through virtual screening. And through experimental verification, the active peptides of the present invention have the potential to be used as melanin production inhibitors. The application of the active peptides of the present invention in inhibiting melanin production, whitening, etc. is of great significance.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention uses computer-aided design and computer virtual screening technology to screen out active peptides containing 1 to 2 tyrosines. Based on the above screening results, further optimization design is performed, and the results show that sequences containing cysteine ​​have higher activity.

[0009] In a first aspect, the present application provides an active peptide, the amino acid sequence of the active peptide being shown in any one of SEQ ID No. 1 to 26.

[0010] SEQ ID No. Amino acid sequence Remark 1 CWRRR / 2 R / 3 HRWGC / 4 RRRGC / 5 CRRWW / 6 CRRR / 7 CRGDR / 8 HWCH / 9 YCW / 10 YWCH / 11 WY / 12 WCH / 13 HCWH / 14 HYCH / 15 RCWRSD / 16 YW / 17 DSRWC / 18 WRRC / 19 CWRSD / 20 CWRSD The head and tail are cyclically bonded by amide bonds 21 Wlq / 22 RYF / 23 CRRWW The head and tail are cyclically bonded by amide bonds 24 CRYWYWC / 25 KKWYW / 26 QUR /

[0011] In a second aspect, the present invention provides an active peptide, wherein the amino acid sequence of the active peptide is greater than or equal to 75% identical to the amino acid sequence shown in any one of SEQ ID No. 1 to 26.

[0012] Preferably, the amino acid sequence of the active peptide has an identity of greater than or equal to 80% with the amino acid sequence shown in any one of SEQ ID No. 1 to 26.

[0013] Preferably, the amino acid sequence of the active peptide has an identity of greater than or equal to 85% with the amino acid sequence shown in any one of SEQ ID No. 1 to 26.

[0014] Preferably, the amino acid sequence of the active peptide has an identity of greater than or equal to 90% with the amino acid sequence shown in any one of SEQ ID No. 1 to 26.

[0015] Preferably, the active peptide has 4 to 10 amino acids.

[0016] Preferably, the active peptide has 4, 5, 6 or 7 amino acids.

[0017] On the other hand, the present invention provides an active peptide mutant, wherein at least one amino acid in any of the amino acid sequences shown in SEQ ID No. 1 to 26 is modified. Chemical modification of key amino acids of active peptides according to different requirements can improve their biological activity while making the peptide segment have more optimized functional properties.

[0018] Preferably, the active peptide mutant satisfies: one or two amino acids in the amino acid sequence shown in any one of SEQ ID No. 1 to 26 are modified.

[0019] Preferably, the modification includes at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification, C-amidation modification, N-palmitoylation modification, N-myristoylation modification and lipidation modification.

[0020] More preferably, the modification is N-acetylation modification and / or C-amidation modification.

[0021] In another aspect, the present invention provides a polynucleotide encoding the above-mentioned active peptide.

[0022] Furthermore, the polynucleotide encodes an active peptide shown in any one of SEQ ID No. 1-26.

[0023] On the other hand, the present invention provides a composition comprising an effective dose of an active ingredient and excipients, wherein the active ingredient is at least one of the above-mentioned active peptides, active peptide mutants or polynucleotides.

[0024] On the other hand, the present invention provides a use of the above-mentioned active peptide, active peptide mutant, polynucleotide or composition in at least one of the following (a) to (c):

[0025] (a) preparing a whitening product;

[0026] (b) preparing a product for inhibiting melanocyte progenitor cells;

[0027] (c) preparing a product for inhibiting melanin production.

[0028] Preferably, the product is a medicine, cosmetic or skin care product having the efficacy of whitening or treating or preventing melanin deposition diseases.

[0029] Preferably, the melanin hyperpigmentation disease is selected from chloasma, freckles, age spots, spots, café au lait spots, Baker's nevus, chloasma, Mongolian spots, nevus of Ota, acquired bilateral nevus of Ota-like spots, nevus of Ito, blue nevus, junctional nevus, mixed nevus, intradermal nevus, halo nevus, congenital melanocytic nevus, Spitz nevus, dysplastic nevus, and sun spots.

[0030] Preferably, the dosage form of the cosmetic includes ointment, cream, emulsion, water, oil, gel, powder, tablet, mud, patch, film, aerosol, spray, freeze-dried preparation or nano preparation.

[0031] Preferably, the cosmetics or skin care products are selected from one or more of facial cleansers, facial creams, facial masks, essences, toners, lotions, sunscreens, and eye creams.

[0032] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0033] In the present invention, the terms "peptide", "short peptide", "polypeptide", "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), which may be recombinant peptides, natural peptides, or synthetic peptides. The peptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with a labeling component).

[0034] In the present invention, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs, and all their D and L stereoisomers.

[0035] The production method of the peptide of the present invention may include the following steps: (1) construction of genetically engineered bacteria, such as Escherichia coli; (2) fermentation culture and induction expression of the genetically engineered bacteria; (3) purification and enzyme cleavage of the polypeptide.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] The active peptide with whitening effect of the present invention and its preparation method and application have both high-efficiency tyrosinase inhibitory activity and the functions of promoting fibroblast proliferation and resisting oxidation and aging to protect normal cells. At the same time, the short peptide sequence can be controlled at a relatively low production cost and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The HPLC test results of the active peptide shown in SEQ ID No.6;

[0039] Figure 2 The mass spectrometry results of the active peptide shown in SEQ ID No.6;

[0040] Figure 3 The HPLC test result of the active peptide shown in SEQ ID No.19;

[0041] Figure 4 The mass spectrometry results of the active peptide shown in SEQ ID No.19;

[0042] Figure 5 To evaluate the inhibitory activity of active peptides on melanin production at the cellular level, Figure A is the detection result of the active peptide shown in SEQ ID No.19, Figure B is the detection result of the active peptide shown in SEQ ID No.6, and Figure C is the detection result of the active peptide shown in control group 1. DETAILED DESCRIPTION

[0043] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0044] Example 1 Synthesis of active peptides

[0045] The active peptide compound and its derivative provided by the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and DMSO is used for oxidation to form two pairs of disulfide bonds in the molecule. The synthetic carrier is Fmoc-Gly-Wang Resin resin. In the synthesis process, the Fmoc-Gly-Wang Resin resin is first fully swollen in N,N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The cut linear precursor crude product is subjected to disulfide bond oxidation in a weak alkaline solution to obtain the target polypeptide crude product. The crude polypeptide was purified and separated by C-18 reverse phase preparative chromatography in 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure polypeptide and its derivatives.

[0046] Experimental reagents

[0047]

[0048]

[0049] 1. Preparation of the active peptide shown in SEQ ID No.6

[0050] Step 1: Coupling of the first amino acid Fmoc-Arg(Pbf)-OH

[0051] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Arg(Pbf)-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin to react at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0052] Step 2: Linear peptide chain synthesis

[0053] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized in the order from the second amino acid at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0054] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0055] • Rinse the resin 6-8 times with DMF until neutral pH.

[0056] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0057] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0058] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0059] Step 3: Cleavage of the linear precursor peptide chain

[0060] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0061] Step 4: Peptide purification

[0062] After filtering through a 0.45um membrane, the mixture was separated using a reverse phase high performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The product-related fractions were collected, and after HPLC purity identification, the fractions with >95% were combined and freeze-dried to obtain the pure polypeptide.

[0063] Step 5: Detection and characterization methods

[0064] The peptide product from step 4 was subjected to analytical HPLC and LC / MS to determine purity and compound. The results of HPLC and LC-MS tests are as follows: Figure 1 , Figure 2 shown.

[0065] 1. Preparation of the active peptide shown in SEQ ID No.19

[0066] Step 1: Coupling of the first amino acid Fmoc-Asp(OtBu)-OH

[0067] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Arg(Pbf)-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin to react at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0068] Step 2: Linear peptide chain synthesis

[0069] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized in the order from the second amino acid at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0070] · Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0071] • Rinse the resin 6-8 times with DMF until neutral pH.

[0072] Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0073] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0074] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0075] Step 3: Cleavage of the linear precursor peptide chain

[0076] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 1 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0077] Step 4: Peptide purification

[0078] After filtering through a 0.45um membrane, the mixture was separated using a reverse phase high performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The product-related fractions were collected, and after HPLC purity identification, the fractions with >95% were combined and freeze-dried to obtain the pure polypeptide.

[0079] Step 5: Detection and characterization methods

[0080] The peptide product from step 4 was subjected to analytical HPLC and LC / MS to determine purity and compound. The results of HPLC and LC-MS tests are as follows: Figure 3 , Figure 4 shown.

[0081] The remaining active peptides of the present invention can be prepared by referring to the above-mentioned Fmoc solid phase synthesis method.

[0082] Example 2 Inhibitory activity of active peptides on mushroom tyrosinase

[0083] 1. Experimental materials:

[0084]

[0085] 2. Detection of the inhibitory activity of active peptides on mushroom tyrosinase catalysis

[0086] First, prepare 0.2KU / mL (5X) mushroom tyrosinase, 10mM (1.6X) substrate DOPA, 10mM (5X) kojic acid inhibitor control, and 1mM (5X) active peptide to be tested for single-concentration screening. At the same time, prepare 100mM (5X) kojic acid inhibitor control and 5mM (5X) active peptide to be tested, and dilute them three-fold with PBS to eight concentrations for testing IC 50 test.

[0087] 10 mM (5X) kojic acid is used as the inhibitor control for the active peptide tested at a single concentration of 1 mM (5X). 100 mM (5X) kojic acid is used as the inhibitor control for the active peptide tested at three-fold serial dilution from 5 mM (5X).

[0088] Add 10 μL active peptide or 10 μL kojic acid or 10 μL PBS blank control to a black transparent bottom 384-well plate. Then add 10 μL PBS to each blank control well, and add 10 μL mushroom tyrosinase to other experimental wells, and incubate at 37°C for 20 min. Finally, add 30 μL substrate DOPA to all wells and incubate at 37°C. Detect OD at 0 min, 30 min, and 60 min of incubation. 475 .

[0089] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 475 Experimental group-blank control) / (OD 475Positive control-blank control)(%)

[0090] Use Graphpad to plot the inhibition rate and calculate the IC 50 .

[0091] 3. Detection of the inhibitory activity of active peptides on mushroom tyrosinase catalysis

[0092] First, prepare 0.2KU / mL (5X) mushroom tyrosinase, 5mM (1.6X) substrate L-tyrosine, 0.3mM (5X) kojic acid inhibitor control, 3mM (5X) positive peptide Decapeptide-12 (sequence: YRSRKYSSWY) inhibitor control, and 1mM (5X) active peptide to be tested for single concentration. At the same time, prepare 10mM (5X) kojic acid inhibitor control, 10mM (5X) positive peptide Decapeptide-12 inhibitor control, and 5mM (5X) active peptide to be tested, and dilute them three times with PBS to eight concentrations for testing IC 50 test.

[0093] 0.3mM (5X) Kojic acid and 3mM (5X) Decapeptide-12 were used as inhibitor controls when the inhibition rate of the active peptide was tested at a single concentration of 1mM (5X). 10mM (5X) Kojic acid and 10mM (5X) Decapeptide-12 were used as inhibitor controls when the active peptide was diluted three times from 5mM (5X).

[0094] Add 10μL active peptide / 10μL kojic acid / 10μL / Decapeptide-12 / 10μL PBS blank control to a black transparent bottom 384-well plate, then add 10μL PBS to each well of the blank control, add 10μL mushroom tyrosinase to the wells of the other experimental groups, and incubate at 37℃ for 20min. Finally, add 30μL substrate L-tyrosine to all wells and incubate at 37℃. Detect OD at 0min, 30min, and 60min of incubation. 475 .

[0095] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 475 Experimental group-blank control) / (OD 475 Positive control-blank control)(%)

[0096] Use Graphpad to plot the inhibition rate and calculate the IC 50 .

[0097] 4. Detection of DOPA-inhibitory activity of active peptides inhibiting human aminotransferase

[0098] First, prepare 13 μg / mL (5x) of in-house synthesized human tyrosinase, 10 mM (1.6X) substrate DOPA, 100 mM (5X) kojic acid inhibitor control, and 5 mM (5X) active peptide to be tested, and dilute them three-fold with PBS to eight concentrations to test IC 50 .

[0099] First, add 10 μL active peptide / 10 μL kojic acid / 10 μL PBS blank control, then add 10 μL PBS to each well of the blank control, and add 10 μL human tyrosinase to the wells of the other experimental groups, and incubate at 37°C for 20 min. Finally, add 30 μL substrate dopa to all wells and incubate at 37°C. Detect OD at 0 min, 30 min, and 60 min of incubation. 475 . Use Graphpad to draw graphs and calculate IC 50 .

[0100] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 475 Experimental group-blank control) / (OD 475 Positive control-blank control)(%)

[0101] 5. Experimental Results

[0102] In vitro inhibitory activity of active peptides on tyrosinase IC 50 As shown in Tables 1 to 3, when DOPA or tyrosine is used as a substrate, the whitening active peptides of the present invention can effectively inhibit the activity of tyrosinase.

[0103] Table 1 IC of the active peptides of the present invention inhibiting mushroom tyrosinase activity using DOPA as substrate 50

[0104]

[0105] Table 2 IC of the active peptides of the present invention inhibiting mushroom tyrosinase activity using tyrosine as substrate 50

[0106]

[0107]

[0108] Table 3 IC of the active peptides of the present invention inhibiting human tyrosinase activity using DOPA as substrate 50

[0109]

[0110] Example 3 Detection of inhibitory activity of active peptides in inhibiting melanin production at the cellular level

[0111] 1. Experimental Procedure

[0112] B16-F10 mouse melanoma cells from the previous 17 generations were plated in 6-well plates at 60,000 cells / well and placed at 37°C with 5% CO 2 After 24 hours in the incubator, discard the original medium and add new medium or 3 μM α-MSH prepared active peptides to be tested and inhibitor controls. The volume of medium or drug in each well is 3 mL. After adding drugs, place the cells at 37°C and 5% CO 2 Incubate in the incubator for 48 hours. 48 hours after drug addition, discard the medium and drug, rinse the cells once with PBS, collect the cells with trypsin, centrifuge at 1600rpm / 3min to obtain the cell pellet, discard the supernatant medium, add 110μL 1mM NaOH to each tube, seal with sealing film, and place in a 65℃ water bath for 1 hour. Take 100μL to a 96-well plate and detect OD 495 value.

[0113] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 495 Experimental group-blank control) / (OD 495 Positive control-blank control) (%).

[0114] According to the experimental results of the above experiments, after determining the better active peptide, further optimize the drug concentration and control drug group to repeat the above experimental steps.

[0115] 2. Experimental results

[0116] The experimental results are as follows Figure 5 , as shown in Table 4. Among them, the polypeptide amino acid sequence used in the control group 1 is CRVI (DOI: 10.1124 / mol.114.094185).

[0117] Table 4 Results of evaluating the inhibitory activity of active peptides on melanin production at the cellular level

[0118]

[0119] According to the above experimental results, the active peptides of the present invention have good melanin production inhibition activity at the cellular level, and the active peptides of the present invention have whitening effects. The active peptides shown in SEQ ID No. 6 and SEQ ID No. 19 have the strongest melanin production inhibition activity.

[0120] Example 4 Detection of the cytotoxicity of active peptides to B16-F10 cells

[0121] 1. Experimental materials and reagents:

[0122] Material Name factory Part Number KOJICacid Aladdin K105452-25g B16-F10 Punosa CL-0319 Cell Counting Kit-8 (CCK-8) GLPBIO GK10001 Transparent 96-well plate Guangzhou Jiete CAP011096 1640 Punosa PM150110P FBS ExCellBio FSP500 α-MSH MCE 581-05-5

[0123] 2. Experimental Procedure

[0124] B16-F10 mouse melanoma cells of the first 17 generations (cultured and passaged in our laboratory) were selected and plated in a transparent 96-well plate at 2000 cells / well and placed at 37°C with 5% CO 2 In the incubator, after culturing for 24 hours, the original medium was discarded and 1640 complete medium or 3 μM α-MSH prepared active peptides to be tested and inhibitor controls (prepared in 1640 complete medium) were added. The volume of medium or drug in each well was 100 μL. After adding drugs, the cells were placed at 37°C and 5% CO 2 Incubate the cells in an incubator for 48 h. Add 10 μL of CCK8 reagent to each well and incubate at 37°C, 5% CO 2 Incubate in the incubator for 1 h and measure OD with an enzyme-labeled instrument 450 The control is the compound MMAE (Monomethyl auristatin E), which is an analog of Dolastatin 10. It effectively inhibits mitosis by inhibiting tubulin polymerization and has extremely strong cytotoxicity.

[0125] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 450 EXP-OD 450 BLANK) / (OD 450 Negative control - OD 450 BLANK

[0126] 3. Experimental results

[0127] Multiple tests on B16-F10 cells showed that the active peptide of the present invention had no killing effect.

[0128] Example 5 Detection of cytotoxicity of active peptides to NHDF cells

[0129] 1. Experimental materials and reagents:

[0130] Material Name factory Part Number KOJICacid Aladdin K105452-25g NHDF Beina Biotechnology BNCC358600 Cell Counting Kit-8 (CCK-8) GLPBIO GK10001 Transparent 96-well plate Guangzhou Jiete CAP011096 DMEM Punosa PM150210P FBS ExCellBio FSP500 α-MSH MCE 581-05-5

[0131] 2. Experimental Procedure

[0132] Normal human dermal fibroblasts (NHDF) were selected and plated in a transparent 96-well plate at 2000 cells / well and placed at 37°C with 5% CO. 2 In the incubator, after culturing for 24 hours, the original medium was discarded and DMEM complete medium or 3 μM α-MSH prepared active peptides to be tested and inhibitor controls (prepared in DMEM complete medium) were added. The volume of medium or drug in each well was 100 μL. After adding drugs, the cells were placed at 37°C and 5% CO 2 Incubate the cells in an incubator for 48 h. Add 10 μL of CCK-8 reagent to each well and incubate at 37°C, 5% CO 2Incubate in the incubator for 2 h and measure OD with an enzyme-labeled instrument 450 Absorbance value.

[0133] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 450EXP -OD 450BLANK ) / (OD 450阴性对照 -OD 450BLANK )

[0134] 3. Experimental results

[0135] Multiple tests on NHDF cells showed that the active peptide of the present invention had no killing effect, and had a growth-promoting effect on NHDF cells. The active peptide of the present application has obvious cell proliferation-promoting activity and has obvious anti-wrinkle effect.

[0136] Example 6 Detection of the toxicity of active peptides to HaCaT cells

[0137] 1. Experimental materials and reagents:

[0138] Material Name factory Part Number KOJICacid Aladdin K105452-25g HaC Oricell H5-1001 Cell Counting Kit-8 (CCK-8) GLPBIO GK10001 Transparent 96-well plate Guangzhou Jiete CAP011096 DMEM Punosa PM150210P FBS ExCellBio FSP500 α-MSH MCE 581-05-5

[0139] 2. Experimental Procedure

[0140] Human immortalized epidermal cells HaCaT were plated at 2000 cells / well in a transparent 96-well plate and placed at 37°C with 5% CO 2 In the incubator, after culturing for 24 hours, the original medium was discarded and DMEM complete medium or 3 μM α-MSH prepared active peptides to be tested and inhibitor controls (prepared in DMEM complete medium) were added. The volume of medium or drug in each well was 100 μL. After adding drugs, the cells were placed at 37°C and 5% CO 2 Incubate the cells in an incubator for 48 h. Add 10 μL of CCK-8 reagent to each well and incubate at 37°C, 5% CO 2 Incubate in the incubator for 2 h and measure OD with an enzyme-labeled instrument 450 Absorbance value.

[0141] Inhibition rate calculation: Inhibition rate (%) = 1-(OD 450EXP -OD 450BLANK ) / (OD 450阴性对照 -OD 450BLANK )

[0142] 3. Experimental results

[0143] Multiple tests on HaCaT cells showed that the active peptide of the present invention had no killing effect.

[0144] The above cytotoxicity experiments show that the active peptides of the present invention are safe for use on the skin.

[0145] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure may be implemented in different specific forms without changing its technical spirit and essential features. Therefore, it should be understood that the above-mentioned embodiments are not restrictive in all aspects, but illustrative. The scope of the present disclosure is limited by the appended claims, rather than by the preceding description thereof, and therefore all changes and modifications fall within the boundaries and scope of the claims, or the equivalents of such boundaries and scope are therefore intended to be covered by the claims.

Claims

1. An active peptide, characterized in that The amino acid sequence of the active peptide is shown in any one of SEQ ID No. 1-26.

2. An active peptide, characterized in that: The amino acid sequence of the active peptide has an identity of greater than or equal to 75% with the amino acid sequence of claim 1, and the active peptide has 4 to 10 amino acids.

3. An active peptide mutant, characterized in that: The active peptide mutant satisfies the requirement that at least one amino acid in the amino acid sequence of claim 1 is modified.

4. The active peptide mutant according to claim 3, characterized in that The modification includes at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification, C-amidation modification, N-palmitoylation modification, N-myristoylation modification and lipidation modification.

5. A polynucleotide, characterized in that The polynucleotide encodes the active peptide according to claim 1 or 2.

6. A composition comprising an effective dose of an active ingredient and an adjuvant, characterized in that: The active ingredient is at least one of the active peptide according to claim 1 or 2, the active peptide mutant according to claim 3 or 4, or the polynucleotide according to claim 5.

7. Use of the active peptide according to claim 1 or 2, the active peptide mutant according to claim 3 or 4, the polynucleotide according to claim 5 or the composition according to claim 6 in at least one of the following (a) to (c): (a) preparing a whitening product; (b) preparing a product for inhibiting melanocyte progenitor cells; (c) preparing a product for inhibiting melanin production.

8. The use according to claim 7, characterized in that The product is a medicine, cosmetic or skin care product with the function of whitening or treating or preventing melanin deposition diseases.

9. The use according to claim 7 or 8, characterized in that The melanin hyperpigmentation disease is selected from chloasma, freckles, age spots, spots, café au lait spots, Baker's nevus, lentigo, Mongolian spots, nevus of Ota, acquired bilateral nevus of Ota-like spots, nevus of Ito, blue nevus, junctional nevus, mixed nevus, intradermal nevus, halo nevus, congenital melanocytic nevus, Spitz nevus, dysplastic nevus, and sun spots.

10. The use according to claim 8, characterized in that The cosmetics or skin care products are selected from one or more of facial cleansers, facial creams, facial masks, essences, toners, lotions, sunscreens, and eye creams.

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