Polypeptides and their application in preparing products with whitening function

Abstract

The present invention relates to the field of polypeptide technology, and in particular to polypeptides and their use in preparing products with whitening functions. The polypeptides provided by the present invention include carnosine and nonapeptide-1, which have the activity of inhibiting melanin synthesis and anti-oxidation, two mechanisms of action strongly related to whitening. Moreover, the effect of the polypeptide is superior to that of a physical mixture of carnosine and nonapeptide-1.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, in particular to polypeptides and applications thereof in preparing products with whitening function. Background Art

[0002] With rising consumer demand for whitening skincare products, the whitening peptide market presents significant growth potential. Whitening products currently available primarily utilize active ingredients such as peptides, vitamin C, niacinamide, tranexamic acid, and glabridin. These products achieve whitening effects by reducing melanocyte stimulation, blocking melanin-promoting signals, and reducing melanin synthesis. Whitening peptides, in particular, have been widely adopted in various whitening products due to their broad range of physiological activities, diverse skincare benefits, well-defined mechanisms, high safety profile, and ease of absorption. Furthermore, since peptides are amino acid compounds, their metabolites are amino acids, which generally have minimal or no side effects on the human body. For example, most whitening peptide skincare products currently available utilize peptides such as nonapeptide-1, hexapeptide-2, and tetrapeptide-30 as their whitening active ingredients. These peptides share a similar structure to fragments of melanocyte-stimulating hormone (α-MSH). The signaling pathway between α-MSH and the melanocortin 1 receptor (MC1-R) is a major driver of melanin formation. Nonapeptide-1, as a biomimetic peptide specific for α-MSH, has a high affinity for the MC1-R receptor. It can competitively prevent the binding of α-MSH to the MC1-R receptor, thereby preventing the activation of the melanin synthesis pathway in melanocytes and the further activation of tyrosinase from the source, thereby blocking the synthesis of melanin and achieving the effect of whitening and lightening spots.

[0003] However, existing whitening products generally suffer from a single mechanism of action, resulting in limited whitening effectiveness, increased safety risks, and reduced market competitiveness. Melanin production is regulated by multiple pathways, including tyrosinase activity, oxidative stress, and melanin transport. Whitening products with a single mechanism of action are unlikely to fully block melanin production. For example, when antioxidant levels are insufficient, the skin continues to be damaged by free radicals, indirectly promoting melanin production. Long-term, high-concentration use of active ingredients with a single mechanism of action, whose metabolism relies on the liver and kidneys, can lead to active ingredient accumulation, increasing the burden on the liver and kidneys and raising safety risks. Furthermore, modern consumers prefer "all-in-one" skincare products. Whitening products with a single mechanism of action, unable to meet the comprehensive needs of antioxidant, repair, and moisturizing, are gradually losing ground in the market.

[0004] Prior art reports have reported using a combination of plant extracts and excipients to inhibit tyrosinase activity. While this approach offers both whitening and antioxidant benefits while removing freckles, it still fails to address the multi-step synergistic regulation of melanin production. Therefore, developing peptides with multiple whitening mechanisms to achieve synergistic regulation of the multi-step pathways of melanin production is key to improving the stability of whitening effects, enhancing product efficacy, expanding the market reach, and increasing market competitiveness. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a polypeptide and its application in preparing products with whitening function.

[0006] The present invention couples two whitening polypeptides, nonapeptide-1 and carnosine, to form a peptide with the following sequence: β-Ala-L-His-L-Gly-L-Gly-L-Gly-L-Gly-L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2. The amino acid sequence of nonapeptide-1 is L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2, while the amino acid sequence of carnosine is β-Ala-L-His-NH2. Five glycine residues (Gly) are used to connect the two peptide sequences to form a new peptide with the following sequence: β-Ala-L-His-L-Gly-L-Gly-L-Gly-L-Gly-L-Gly-L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2. The polypeptides provided by the present invention include carnosine and nonapeptide-1.

[0007] The polypeptide described in this invention combines the tyrosinase-inhibiting activity of nonapeptide-1 with the free radical-reducing activity of carnosine, and its efficacy is superior to that of a physical mixture of the two polypeptides. Furthermore, through its multi-pathway action, the polypeptide may reduce the concentration requirement of a single component, thereby minimizing irritation or side effects.

[0008] The structure of the polypeptide of the present invention is:

[0009] [(Carnosine)a-(Nonapeptide-1)b]c or [(Nonapeptide-1)b-(Carnosine)a]c.

[0010] Wherein: a and b are independently selected from integers greater than or equal to 0, and are not both 0;

[0011] c is an integer greater than or equal to 1.

[0012] In the structure of the polypeptide described herein, the letter a represents the number of repeats of carnosine, the letter b represents the number of repeats of nonapeptide-1, and the letter c represents the number of repeats of the unit composed of carnosine and nonapeptide-1 in the polypeptide. Within the polypeptide, a, b, and c may or may not be equal, and this is not a limitation of the present invention.

[0013] As a feasible example, in the polypeptide, a is an integer from 0 to 10, b is an integer from 0 to 10, and c is an integer from 1 to 10.

[0014] Taking a=1, b=1 and c=1 as an example, the structure of the fusion protein of the present invention is carnosine-nonapeptide-1 or nonapeptide-1-carnosine.

[0015] Taking a=2, b=1 and c=1 as an example, the structure of the fusion protein of the present invention is carnosine-carnosine-nonapeptide-1 or nonapeptide-1-carnosine-carnosine.

[0016] When c is not 1, the composition of the polypeptides in the multiple units may be the same or different, and the present invention does not limit this.

[0017] Taking a=0 or 1, b=1 and c=2 as an example, the structure of the polypeptide of the present invention is: carnosine-nonapeptide-1-carnosine, or nonapeptide-1-carnosine-nonapeptide-1.

[0018] The polypeptide structure described in the present invention also includes a linker fragment. In the present invention, the linker is located between two adjacent fragments, for example, between carnosine and nonapeptide-1, or between two carnosines, or between two nonapeptides-1, and the present invention does not limit this. Alternatively, the length of the linker is 1 to 10 amino acids, or 1 to 5 amino acids, and the present invention does not limit this. Preferably, the amino acids used in the linker described in the present invention do not contain side chain groups. For example, the linker is G, GGG, or GGGGG.

[0019] In a specific embodiment, the structure of the polypeptide is shown in Formula I:

[0020]

[0021] Formula I.

[0022] Furthermore, the present invention also provides a method for preparing the above polypeptide, which comprises obtaining the polypeptide by coupling according to the peptide sequence.

[0023] In the present invention, the coupling includes sequential coupling or segmented coupling, and the preparation adopts solid phase synthesis and / or liquid phase synthesis. As a feasibility example, the preparation of the polypeptide in the present invention adopts a fully automatic continuous microwave peptide instrument for synthesis or manual solid phase synthesis.

[0024] Furthermore, the present invention also provides the use of the aforementioned polypeptide in the preparation of skin care products.

[0025] In the present invention, the skin care product has a whitening effect, and the whitening effect includes inhibiting tyrosinase activity, anti-oxidation and / or reducing melanin levels. More specifically, the anti-oxidation effect includes scavenging free radicals. The free radical is DPPH.

[0026] Furthermore, the present invention also provides a skin care product comprising the polypeptide as described above.

[0027] In the skin care product of the present invention, the concentration of the aforementioned polypeptide is 0.1 to 10 mM. In a specific embodiment, the concentration of the aforementioned polypeptide in the skin care product is 0.5 mM.

[0028] In the present invention, the skin care product is a whitening skin care product. The skin care product of the present invention also includes auxiliary materials acceptable in cosmetics.

[0029] The excipients include but are not limited to solvents (water, ethanol, propylene glycol, butylene glycol), emulsifiers (glyceryl stearate, polysorbate, sodium lauryl sulfate), thickeners (carbomer, xanthan gum, hydroxyethyl cellulose), moisturizers (glycerin, hyaluronic acid, panthenol), preservatives (phenoxyethanol, paraben, chlorphenesin), antioxidants (vitamin E, tocopheryl acetate, tea polyphenols), flavors (synthetic flavors, natural plant essential oils), pigments (iron oxides, CI series dyes, pearlescent pigments), pH Conditioners (triethanolamine, citric acid, sodium hydroxide), film formers (polyvinyl alcohol, acrylate copolymers), soothing agents (allantoin, bisabolol, dipotassium glycyrrhizate), sunscreens (titanium dioxide, zinc oxide, ethylhexyl methoxycinnamate), antistatic agents (quaternary ammonium salts, polyquaternium salts), propellants (propane, butane, dimethyl ether, for aerosols), adsorbents (talc, kaolin, silica), defoaming agents (silicone, polyether), excipients (petroleum jelly, paraffin, beeswax).

[0030] The skin care products are lotions, emulsions, creams, powders, gels, aerosols, soaps, facial masks, essential oils, essences, facial cleansing products, makeup removers or hand soaps.

[0031] The lotion includes but is not limited to toner, softening water, astringent water, conditioning water, essence water, moisturizing water, balancing water, spray water, mineral spray, activating water, tenderizing water, secondary cleansing water or soothing water.

[0032] The lotion includes but is not limited to moisturizing lotion, oil-control lotion, soothing lotion, sunscreen lotion, primer, repair lotion, refreshing lotion or repair lotion.

[0033] The creams include but are not limited to facial cream, night cream, day cream, moisturizing cream, repair cream, essence cream, anti-aging cream, sunscreen, isolation cream, natural cream, BB cream, CC cream, foundation cream, concealer, contour cream, highlighter cream, blush cream, lipstick, lip balm, eye cream, neck cream, hand cream, body cream, body lotion, massage cream, acne cream, anti-allergic cream, and aftershave cream.

[0034] The polypeptide provided by the present invention includes carnosine and nonapeptide-1. The polypeptide has the activity of inhibiting melanin synthesis and anti-oxidation, two mechanisms of action strongly related to whitening. Moreover, the effect of the polypeptide is better than that of a physical mixture of carnosine and nonapeptide-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The MALDI-TOF MS spectra of the dual-action whitening polypeptides prepared in Example 1 and Comparative Examples 1-2 are shown, wherein: a is the MS spectrum of the polypeptide prepared in Example 1, b is the MS spectrum of the polypeptide prepared in Comparative Example 1, and c is the MS spectrum of the polypeptide prepared in Comparative Example 2;

[0036] Figure 2 The chromatogram of the sample prepared in Example 1 detected by HPLC is shown;

[0037] Figure 3 The NMR spectrum of the sample prepared in Example 1 is shown;

[0038] Figure 4 1 shows the Fourier transform infrared spectrum of the sample prepared in Example 1;

[0039] Figure 5 Cytotoxicity test results of each group of samples;

[0040] Figure 6 Shows the inhibitory effect of each group of samples on melanin;

[0041] Figure 7 Statistical analysis of the inhibitory effect of each group of samples on melanin;

[0042] Figure 8 Shows the inhibitory effect of each group of samples on tyrosinase activity;

[0043] Figure 9 Shown are the statistical analysis of the inhibitory effects of each group of samples on tyrosinase activity;

[0044] Figure 10 Comparison of DPPH scavenging effects of the polypeptide prepared in Example 1 and 9-peptide-1;

[0045] Figure 11 Shows the DPPH clearance experimental effect. DETAILED DESCRIPTION

[0046] The present invention provides polypeptides and their use in the preparation of products with whitening functions. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0048] The terms "include," "comprising," and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0049] The term "and / or" describes the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0050] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0051] The numerical ranges and parameters involved in this disclosure are presented as precisely as possible in the specific examples. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure are subject to reasonable deviation within a certain range, for example, within ±10%, ±5%, ±1%, or ±0.5%.

[0052] The polypeptide provided by the present invention is the preferred solution after multiple attempts. Preliminary experiments attempted to link different types of peptides, but the results were not as good as those obtained by fusing carnosine with nonapeptide-1. Furthermore, different linking methods and numbers of linkings were also tried, and all achieved a certain degree of whitening or antioxidant effect. However, the polypeptide prepared in Example 1 showed the best results.

[0053] The test materials used in the present invention are all common commercial products and can be purchased on the market. It should be understood that in the various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution. Some or all steps can be executed in parallel or in sequence. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The present invention is further described below in conjunction with the embodiments:

[0054] Example 1: Preparation of dual-action whitening peptide (5 Gly linkages)

[0055] The sequence of the dual-action whitening peptide is: β-Ala-L-His-L-Gly-L-Gly-L-Gly-L-Gly-L-Gly-L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2, with a theoretical molecular weight of 1699.97Da.

[0056] The peptide was synthesized using a CEM Liberty Blue fully automatic continuous microwave peptide analyzer in the United States with a synthesis concentration of 0.20 M. The amount of various amino acids was calculated according to the preset sequence, and 0.47 g of L-phenylalanine was dissolved in 6 mL of N'N dimethylformamide (DMF), 2.86 g of L-glycine was dissolved in 48 mL of DMF, 1.36 g of L-histidine was dissolved in 11 mL of DMF, 0.57 g of L-lysine was dissolved in 6 mL of DMF, 0.45 g of L-methionine was dissolved in 6 mL of DMF, 0.75 g of L-proline was dissolved in 11 mL of DMF, 1.43 g of L-arginine was dissolved in 11 mL of DMF, 0.41 g of L-valine was dissolved in 6 mL of DMF, 0.46 g of D-phenylalanine was dissolved in 6 mL of DMF, and 0.63 g of D-tryptophan was dissolved in 6 mL Weigh 0.68 g of β-alanine and dissolve it in 11 mL of DMF. Dissolve the peptide under ultrasound. After dissolution, install the reagent tubes on the instrument. Weigh 14.21 g of solid Oxime and dissolve it in 100 mL of DMF to prepare an Oxime solution. Mix 7.8 mL of DIC solution in 92.2 mL of DMF to prepare a DIC solution. Oxime and DIC are used as coupling agents. Finally, weigh 90 mL of 20% piperidine solution as the deprotection solvent. Finally, weigh 0.596 mg of Rink amide AM resin (degree of substitution: 0.338) and place it in the reactor. CEM Liberty The Blue automated continuous microwave peptide analyzer performs the following steps for peptide synthesis: swelling the resin, adding the deprotection solvent, microwave heating (heating to 90°C), removing the deprotection solvent, washing three times, adding the corresponding amino acid, adding the coupling agent, microwave heating (heating to 90°C for 110 seconds or 230 seconds), and washing with DMF three times. This process is repeated 16 times for a total of 16 amino acids in the sequence, completing the solid-phase synthesis.

[0057] Resin Cleavage: After solid-phase synthesis, add 10 mL of cleavage buffer containing 9.25 mL of trifluoroacetic acid, 0.25 mL of water, 0.25 mL of 1,2-ethanedithiol, and 0.25 mL of triisopropylsilane to the resin. Place the resin on a rotator and allow to cleave for 2 hours. After cleavage, add 30 mL of anhydrous ether, mix thoroughly, and centrifuge at 8000 rpm for 5 minutes. Repeat this process three times to obtain a crude white peptide. Cleavage should be performed at the junction of the amino group and the bis-benzyl group on the branch.

[0058] Purification of crude product: The crude product was dissolved in a 10 mg / mL mixed solvent with a 6:4 ratio of water to acetonitrile. Purification was then performed using a preparative liquid phase (Shimadzu, Japan) with acetonitrile as mobile phase A and water containing 0.1% TFA as mobile phase B. The elution gradient was 80%-20% water, 20%-80% acetonitrile over 30 minutes. Pure product was collected based on an absorption peak around 20 minutes.

[0059] The obtained pure product solution was subjected to rotary evaporation to remove acetonitrile and most of the water, retaining 20 mL of water, placed in liquid nitrogen for cooling, and then placed in a freeze dryer with a cold trap temperature of -80°C and a vacuum degree of 1.0 Pa. The pure product powder was obtained by freeze drying for 72 hours.

[0060] Example 2: Preparation method of dual-action whitening polypeptide

[0061] The sequence of the dual-action whitening peptide is: β-Ala-L-His-L-Gly-L-Gly-L-Gly-L-Gly-L-Gly-L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2.

[0062] It was prepared by manual solid phase synthesis:

[0063] Using Rink AM resin (loading capacity 0.338 mmol / g), amino acids were coupled sequentially from the C-terminus to the N-terminus using an orthogonal protection strategy. The main-chain amino groups were all protected with 9-fluorenylmethyloxycarbonyl (Fmoc) groups, and the side chains were all acid-sensitive protecting groups. After synthesis, the peptide was cleaved from the resin using a newly configured cleavage reagent, simultaneously removing the side chain protecting groups. The specific steps are as follows:

[0064] 1. Resin swelling and Fmoc protection group removal: Weigh 1 g of resin into a 25 mL glass solid phase tube and add 10 mL of DMF to swell for 30 minutes. After removing the DMF, add another 10 mL of DMF and purge the resin with air for 1 minute before draining the liquid. Repeat this process three times. Then, add 10 mL of a DMF solution containing 20% ​​piperidine to remove the Fmoc protection group. Shake on an oscillator for 30 minutes, then remove the solution and repeat this process twice. After removing the Fmoc protection group, wash the resin three times with DMF and then with dichloromethane (DCM).

[0065] 2. Amino Acid Coupling: Add 10 mL of DMF to the 25 mL glass solid phase tube containing the resin to swell the resin for 10 minutes. Then, remove the solvent under reduced pressure and add 10 mL of DMF to wash the resin. Repeat the washing process three times. Add Fmoc-Val-OH (475.2 mg, 4 eq) and HBTU (530.9 mg, 4 eq) to the solid phase tube and stir thoroughly in 10 mL of DMF to dissolve. Then, add DIPEA (310 μL, 10 eq) and shake continuously for 4 hours. Then, drain the reaction solution and wash the resin three times with DMF and then DCM. After washing, remove a small amount of resin and monitor the completion of the coupling reaction using the Kaiser test. The reaction is complete when the resin turns colorless or light yellow. Repeat the Fmoc protecting group removal and amino acid coupling process to couple the next amino acid. Amino acid coupling is carried out sequentially from the C-terminus to the N-terminus.

[0066] 3. Resin Cleavage and Side Chain Protecting Group Removal: After all couplings are complete, cleave the resin using 20 mL of freshly prepared cleavage reagent. The cleavage solution consists of trifluoroacetic acid (TFA), deionized water, and triisopropylsilane (TIPS) in a ratio of 95:2.5:2.5 (v / v / v). Cleave the resin at room temperature for 2 h. Filter the resin and rinse twice with 4 mL of TFA. Combine the filtrate and washes, and evaporate the cleavage solution to less than 15 mL using flowing nitrogen. Precipitate the peptide with 40 mL of icy ether. Centrifuge at 8000 rpm for 5 min, discard the supernatant, and repeat this process three times to obtain a crude white peptide.

[0067] 4. Purification of crude product: The crude product was dissolved in a 10 mg / mL mixed solvent with a water to acetonitrile ratio of 6:4. It was then purified using a preparative liquid phase (Shimadzu, Japan) with acetonitrile as the mobile phase A and 0.1% TFA in water as the mobile phase B. The elution gradient was 80%-20% water, 20%-80% acetonitrile over a 30-minute period. The pure product was collected based on the absorption peak after about 20 minutes. The resulting pure product solution was subjected to rotary evaporation to remove acetonitrile and most of the water, retaining 20 mL of water. The solution was placed in liquid nitrogen to cool, then placed in a freeze dryer with a cold trap temperature of -80°C and a vacuum of 1.0 Pa. The pure product powder was obtained by freeze drying for 72 hours.

[0068] Comparative Example 1 Preparation Method of Dual-Action Mechanism Whitening Peptide (3 Gly Linked)

[0069] The sequence of the dual-action whitening peptide is: β-Ala-L-His-L-Gly-L-Gly-L-Gly-L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2, with a theoretical molecular weight of 1586 Da.

[0070] The preparation parameters are the same as in Example 1.

[0071] Comparative Example 2 Preparation Method of Dual-Action Mechanism Whitening Peptide (1 Gly Linked)

[0072] The sequence of the dual-action whitening peptide is: β-Ala-L-His-L-Gly-L-Met-L-Pro-D-Phe-L-Arg-D-Trp-L-Phe-L-Lys-L-Pro-L-Val-NH2, with a theoretical molecular weight of 1472 Da.

[0073] The preparation parameters are the same as in Example 1.

[0074] Example 3: Identification and analysis of dual-action whitening peptides

[0075] The dual-action whitening polypeptide obtained by the method described in Example 1 was subjected to mass spectrometry, 1 Its chemical structure was confirmed by H-NMR nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy.

[0076] The physical and chemical properties are as follows:

[0077] 1. White amorphous powder.

[0078] 2.Molecular weight: 1699.97 Da.

[0079] 3. Molecular formula: C 80 H 113 N 23 O 17 S, the structural formula is shown below:

[0080]

[0081] 4. The peptides prepared by the methods of Example 1 and Comparative Examples 1-2 were detected by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS): The molecular weight of the peptide prepared in Example 1 was 1699.7 Da ( Figure 1 In a), the molecular weight of the polypeptide obtained in Comparative Example 1 was measured to be 1586.3 Da ( Figure 1 b), the molecular weight of the polypeptide obtained in Comparative Example 2 was measured to be 1472.4 Da ( Figure 1 (c) Both are consistent with the theoretical molecular weight.

[0082] 5. Purity Analysis: The sample prepared in Example 1 was dissolved in deionized water at a concentration of 1 mg / mL. Purity analysis was then performed using a high performance liquid chromatography (HPLC) analytical liquid phase (Shimadzu, Japan). Mobile phase A was acetonitrile, mobile phase B was water containing 0.1% TFA, and the elution gradient was 80%-20% water, 20%-80% acetonitrile, over 30 minutes. The sample purity was over 95% according to the peak analysis of the liquid phase. The results are as follows: Figure 2 .

[0083] 6. 1 H-NMR nuclear magnetic resonance spectrum: The sample prepared in Example 1 was dissolved in deuterium oxide (D2O) and measured at 400 MHz for more than 128 times. The results are shown in Figure 3 :

[0084] 7. Fourier transform infrared spectroscopy (FT-IR) (Bruker, Germany) was used to characterize the structure of the samples, and the results are shown in Figure 4 :

[0085] The dual-action whitening polypeptide obtained by the method described in Example 1 was subjected to mass spectrometry, high performance liquid chromatography, 1 H-NMR nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy confirmed the successful preparation of the dual-action mechanism whitening peptide, and its purity met the usage requirements (over 95%).

[0086] Example 4: Cytotoxicity experiment of dual-action whitening peptide on mouse melanoma cells (B16F10 cells)

[0087] Test method:

[0088] 1. The mouse melanoma cells (B16F10 cells) involved in the experiment were cultured in RPMI 1640 medium, which is a complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone, USA). B16F10 cells were cultured in a 37°C, 5% CO2 incubator.

[0089] 2. After B16F10 cells grew to an appropriate density, 2×10 4 The cells were transferred to a 96-well plate at a density of 100 μL / well, and 100 μL of cell suspension was added to each well. The cells were cultured in a 37°C and 5% CO2 incubator for 18 h to allow them to adhere to the plate.

[0090] 3. The dual-action whitening peptide prepared in Example 1 was dissolved in RPMI 1640 complete medium at a concentration of 0.5 mM. The following three control groups were set up:

[0091] A physical mixture of 0.5 mM 9-peptide-1 and 0.5 mM carnosine, a 0.5 mM 9-peptide-1 group, and a blank control group were used. After cells were completely attached, the supernatant was aspirated and discarded. The above samples were added to the wells of a 96-well plate, with six replicates per group and a sample volume of 100 μL per well.

[0092] 4. After incubating the 96-well plate in a CO2 cell culture incubator (37°C, 5% CO2) for 24 hours, use CCK-8 to measure cell viability. The specific method is: after aspirating the supernatant, add 100 μL of incomplete culture medium containing 10 μL of CCK-8 to each well, and then measure the absorbance using a microplate reader. Cell viability = C t / C0×100%, where C t is the absorbance of each experimental group at 450 nm, and C0 is the absorbance of the blank control group.

[0093] The cytotoxicity test results obtained by the above method are as follows Figure 5 The results showed that all samples in the tested concentrations had no cytotoxicity to mouse melanoma cells.

[0094] Example 5: Effects of dual-action whitening peptides on melanin levels in mouse melanoma cells (B16F10 cells)

[0095] Test method:

[0096] 1. The mouse melanoma cells (B16F10 cells) involved in the experiment were cultured in RPMI 1640 medium, which is a complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone, USA). B16F10 cells were cultured in a 37°C, 5% CO2 incubator.

[0097] 2. When B16F10 cells grow to 70% to 80% confluence, trypsinize the cells and replace the culture medium with high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone, USA). The high-glucose DMEM cell culture medium is used to induce B16F10 cells to produce melanin. After digestion, the cells were collected and plated at 3×10 5 Cells were transferred to a 6-well plate at a density of 100 cells / well, 3 mL of cell suspension was added to each well, and cultured at 37°C, 5% CO2. After the cells were completely attached, the dual-action whitening peptide prepared in Example 1 was dissolved in high-glucose DEME complete medium at a concentration of 0.5 mM. The following five control groups were set, and 3 replicates were set for each sample concentration:

[0098] 0.5 mM of the dual-action whitening peptide prepared in Comparative Example 1;

[0099] 0.5 mM of the dual-action whitening peptide prepared in Comparative Example 2;

[0100] 0.5 mM 9-peptide-1 and 0.5 mM carnosine physical mixture group;

[0101] 0.5 mM of 9-peptide-1 group;

[0102] Blank control group.

[0103] 3. After culturing B16F10 cells for another 48 hours, harvest the co-cultured cells and aspirate the supernatant medium from the wells into a centrifuge tube. Add 800 µL of trypsin to each well for digestion. Add 2 mL of complete culture medium to create a cell suspension, which is then transferred to the aforementioned centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash with 2 mL of PBS. Centrifuge at 1000 rpm for 5 minutes, then wash twice with PBS. Collect the cell pellet for later use.

[0104] 4. Process the collected cells: add 200 μL of 1 mol / L NaOH containing 10% DMSO to lyse the cells, incubate at 80°C for 2 h, measure the absorbance of the cell lysate and supernatant at 405 nm using a microplate reader, and calculate the melanin synthesis rate. Melanin synthesis rate = B t / B0×100%, where B t is the absorbance of each experimental group at 405 nm, and B0 is the absorbance of the blank control group.

[0105] Melanin synthesis rate is used to express the relative content and relative activity of melanin. The melanin synthesis rate of each group of samples measured by the above method is as follows: Figures 6 and 7 The results showed that the dual-action whitening peptide prepared in Example 1 can more effectively reduce the melanin content, and has a significant advantage over the use of 9-peptide-1 alone or the combination of 9-peptide-1 and carnosine, with p < 0.01.

[0106] Example 6: Effects of dual-action whitening peptides on tyrosinase activity in mouse melanoma cells (B16F10 cells)

[0107] Test method:

[0108] 1. The mouse melanoma cells (B16F10 cells) involved in the experiment were cultured in RPMI 1640 medium, which is a complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone, USA). B16F10 cells were cultured in a 37°C, 5% CO2 incubator.

[0109] 2. When B16F10 cells grow to 70% to 80% confluence, trypsinize the cells and replace the culture medium with high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone, USA). The high-glucose DMEM cell culture medium is used to induce B16F10 cells to produce melanin. After digestion, the cells were collected and plated at 2×10 4 Cells were transferred to a 96-well plate at a density of 100 μL per well. 100 μL of cell suspension was added to each well and cultured in a 37°C, 5% CO2 incubator for 18 h to allow attachment. After the cells were fully attached, the dual-action whitening peptide prepared in Example 1 was dissolved in high-glucose DEME complete medium at a concentration of 0.5 mM. The following five control groups were set up, with 6 replicates per group:

[0110] 0.5 mM of the dual-action whitening peptide prepared in Comparative Example 1;

[0111] 0.5 mM of the dual-action whitening peptide prepared in Comparative Example 2;

[0112] 0.5 mM 9-peptide-1 and 0.5 mM carnosine physical mixture group;

[0113] 0.5 mM of 9-peptide-1 group;

[0114] Blank control group.

[0115] 3. After 48 hours of co-culture, wash cells twice with 0.01 mol / L PBS (pH 7.2) and resuspend in 100 μL of PBS containing 1% Triton X-100. Lyse cells at -20°C for 1 hour. Preheat the lysate to 37°C for 5 minutes and then react with 100 μL of 1% L-DOPA at 37°C for 2 hours. Measure absorbance (A) at 470 nm using a microplate reader. Relative tyrosinase activity = At / A0 × 100%, where At is the absorbance at 470 nm for each experimental group and A0 is the absorbance of the blank control.

[0116] The tyrosinase activity results of each group of samples measured according to the above method are as follows: Figures 8 and 9The results showed that the dual-action whitening peptide prepared in Example 1 could more effectively inhibit tyrosinase activity, and had a significant advantage over the use of 9-peptide-1 alone or the combination of 9-peptide-1 and carnosine (p<0.01).

[0117] Example 7: Antioxidant effect experiment of dual-action whitening peptide:

[0118] The antioxidant effect of the dual-action whitening peptide prepared in Example 1 was evaluated using a DPPH free radical scavenging experiment. Test method:

[0119] 1. Dissolve 1 mg of solid 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) in 24 mL of anhydrous ethanol. Ultrasonicate for 10 minutes to homogenize the solution. Protect from light during the experiment. Dilute 1 mL of the DPPH solution with anhydrous ethanol to an absorbance between 0.6 and 1.0.

[0120] 2. Use anhydrous ethanol as the solvent to prepare 0.5 mM dual-action whitening peptide and 0.5 mM 9-peptide-1, respectively. Take 1 mL of the prepared DPPH solution and then add 1 mL of the prepared dual-action whitening peptide and 9-peptide-1 samples. Add the same volume of anhydrous ethanol to the blank control group. Set up 3 parallel samples for each group. After mixing, react at room temperature in the dark for 30 minutes, and then take a photo to record the absorbance at a wavelength of 517 nm. The scavenging rate of DPPH free radicals = (D0-D t ) / D0×100%, where D t is the absorbance of each experimental group at 517 nm, and D0 is the absorbance of the blank control group.

[0121] The above results show that the dual-action whitening polypeptide prepared in Example 1 has the effects of inhibiting melanin production and anti-oxidation ( Figures 10-11 ).

[0122] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide having the structure shown in Formula I: Formula I.

2. A method for preparing the polypeptide according to claim 1, comprising obtaining the polypeptide by coupling according to the peptide sequence.

3. The preparation method according to claim 2, characterized in that The coupling includes sequential coupling or segmented coupling, and the preparation adopts solid phase synthesis and / or liquid phase synthesis.

4. Use of the polypeptide according to claim 1 in preparing a skin care product with whitening effect. A skin care product comprising the polypeptide according to claim 1.

6. The skin care product according to claim 5, characterized in that The concentration of the polypeptide according to claim 1 is 0.5 mM.

7. The skin care product according to claim 5 or 6, characterized in that It can be lotion, emulsion, cream, gel, aerosol, soap, facial mask, essential oil, serum, cleanser, makeup remover or hand soap.

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