Donkey-hide gelatin peptide and application thereof in brightening and whitening skin color

Abstract

The invention discloses a colla corii asini peptide. The amino acid sequence of the colla corii asini peptide is GPIGPVG. Meanwhile, the invention discloses application of the donkey-hide gelatin peptide in preparation of a whitening composition. The colla corii asini peptide disclosed by the invention has good tyrosinase inhibitory activity and an effect of inhibiting melanin cells from synthesizing melanin, can be used as a whitening functional component, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and more specifically to a donkey-hide gelatin peptide and the use of compositions containing the peptide. Background Technology

[0002] Human skin color depends on the epidermis, determined by the melanin produced by melanocytes located in the epidermis. Melanin is produced from melanosomes within melanocytes. After maturation, melanin migrates from the dendritic ends of melanocytes to the stratum corneum of the skin, where it is transferred to surrounding keratinocytes, completing skin pigmentation. Therefore, effectively controlling pigmentation and reducing melanin production is a primary approach to skin whitening and blemish removal.

[0003] Traditional skin-whitening active ingredients have drawbacks such as significant cytotoxicity, strong irritation, high sensitization, and numerous adverse reactions, making it difficult to meet the needs of a wide range of consumers. For example, phenylethyl resorcinol (377), a cosmetic raw material with significant skin-whitening and spot-removing effects, has a regulated upper limit of 0.5% for its addition; concentrations exceeding this level pose a risk of skin irritation. Therefore, the search for safe and effective melanin inhibitors derived from natural products and traditional Chinese medicine has become one of the hot topics in domestic and international research in recent years.

[0004] Donkey-hide gelatin (Ejiao) is sweet and neutral in nature, possessing the effects of nourishing blood and yin, moistening dryness, and stopping bleeding. Therefore, it is often used for blood deficiency and chlorosis, dizziness and palpitations, muscle weakness, restlessness and insomnia, internal wind due to deficiency, dry cough, hemoptysis due to overwork, hematemesis, hematuria, hematochezia, metrorrhagia, and threatened abortion. As early as the Eastern Han Dynasty, the famous medical work *Shennong Bencao Jing* listed Ejiao as a "superior" medicine, recording that "long-term use benefits qi and lightens the body." There is considerable research on Ejiao in the health food industry, and its safety has been fully proven. However, there are currently few reports on obtaining whitening active peptides from Ejiao. Summary of the Invention

[0005] To address the above problems, the present invention provides a donkey-hide gelatin peptide, which contains the amino acid sequence shown in SEQ ID NO:1.

[0006] In some embodiments, the amino acid sequence of the donkey-hide gelatin peptide is as shown in SEQ ID NO:1 or has at least 90% sequence identity with it.

[0007] In some embodiments, the amino acid sequence of the donkey-hide gelatin peptide is GPIGPVG.

[0008] This invention provides a method for screening the aforementioned donkey-hide gelatin peptides, comprising the following steps:

[0009] (1) Obtaining donkey-hide gelatin oligopeptides: Add donkey-hide gelatin to water, heat to boiling, dissolve, remove gelatin foam and impurities, and filter to obtain a uniform gelatin solution; when the gelatin solution is cooled to 50-55℃, add papain, pepsin and bromelain for enzymatic hydrolysis for 2 hours, stirring continuously during the process; inactivate the enzymes by steam heating; concentrate the enzyme-inactivated gelatin solution using a vacuum concentrator; filter the concentrated gelatin solution, and then dry it into powder using a vacuum continuous dryer to obtain donkey-hide gelatin oligopeptide powder.

[0010] (2) Identification by liquid chromatography-tandem mass spectrometry: The oligopeptides of donkey-hide gelatin were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). UPLCMS was used for detection, and Agilent Q-TOF high-resolution mass spectrometry combined with the pFind software for proteomics data identification was applied to identify the peptides derived from donkey-hide gelatin in the oligopeptide samples. The small molecule donkey-hide gelatin peptide sequences were obtained, and the identified peptides were further confirmed using Agilent Bioconfirm software and Agilent Qualitative Analysis software.

[0011] (3) Efficacy Verification: The peptide sequence obtained by molecular docking screening was chemically synthesized using a solid-phase method. Its whitening activity was verified using an A375 cell in vitro model and tyrosinase inhibition experiments. The donkey-hide gelatin peptide described in this invention can inhibit melanin synthesis in A375 cells and inhibit tyrosinase activity.

[0012] The present invention also provides a method for preparing the said donkey-hide gelatin peptide, comprising the following steps:

[0013] (1) Place 2 mmol CTC resin in a 100 mL solid-phase synthesis reactor, add 4 mmol Fmoc-Gly-OH amino acid, 20 mL DCM, and 2 mL DIEA, and react at 25 °C for 3 hours. Add 3 mL methanol and react for 5 minutes. Filter and wash the resin twice each with 20 mL DCM, methanol, and DMF. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 minutes, filter to remove the deprotection solution, and then wash 6 times with 20 mL DMF solution. Dry and set aside for later use.

[0014] (2) Take 4 mmol Fmoc-Val-OH and 4 mmol HOBt in a 50 mL beaker, cool to 2-8℃, add 2 mL DMF and 4 mmol DIC, let stand for 10-20 minutes, add the solution in the beaker to the solid-phase synthesis reactor, stir and react for 1.5 hours until the reaction is complete. Wash the resin three times with 20 mL of DMF solution each time. After washing, add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter to remove the deprotection solution, then wash 6 times with 5 mL of DMF solution, and dry for later use. According to the above operation, couple Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, and Fmoc-Gly-OH sequentially.

[0015] (3) Then, wash twice with 5 mL of methanol, twice with DCM, and twice with methanol. Dry under vacuum to obtain peptide resin H-Gly-Pro-Ile-Gly-Pro-Val-Gly-CTC.

[0016] (4) The obtained peptide resin was cut with 40 mL of cutting solution TFA / anisin thioether / PhOH / H2O / EDT=87.5 / 5 / 2.5 / 2.5 / 2.5 at 30℃ for 2.5 hours. The cutting solution was added to 800 mL of diethyl ether (2-8℃) solution, and a white solid was precipitated. After centrifugation, a white solid crude peptide was obtained. After vacuum drying, a crude peptide powder H-Gly-Pro-Ile-Gly-Pro-Val-Gly-OH was obtained.

[0017] The present invention also provides a whitening composition comprising the aforementioned donkey-hide gelatin peptide.

[0018] In some embodiments, the weight range of the donkey-hide gelatin peptide in the whitening composition is 0.0000001% to 10% based on the total weight of the composition. In some specific embodiments, the weight range of the donkey-hide gelatin peptide is 0.0000001% to 1%.

[0019] In some embodiments, the whitening composition further comprises an active agent for enhancing the whitening effect of the donkey-hide gelatin peptide. In some specific embodiments, the active agent is selected from one or more of the following: peptides, vitamin C and its derivatives, B vitamins, dehydroepiandrosterone (DHEA), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, amino sugars, azoles, and metal salts.

[0020] In some embodiments, the whitening composition further comprises cosmetic excipients. In one specific embodiment, the cosmetic excipients are selected from one or more of the following: thickeners, surfactants, co-surfactants, skin conditioning agents, preservatives, fragrances, lubricants, disintegrants, humectants, binders, and fillers.

[0021] The present invention also provides the use of the said donkey-hide gelatin peptide or the said whitening composition in the preparation of products with whitening or freckle-removing effects.

[0022] The present invention also provides the use of the said donkey-hide gelatin peptide or the said whitening composition in skin whitening or freckle removal for non-therapeutic purposes.

[0023] In some implementations, the whitening or spot-removing effect includes inhibiting tyrosinase activity and / or inhibiting the synthesis of melanin by human melanocytes.

[0024] In some embodiments, the donkey-hide gelatin peptide or the whitening composition of the present invention can be used on all parts of the body, and more specifically, according to the corresponding indication, on the face, body, collar or scalp, in any form or medium known to those skilled in the art, particularly in the form of a solution, dispersion, emulsion, paste or powder, alone or in a premixed form, or transported alone or premixed with a carrier (e.g., macrocapsules, microcapsules or nanocapsules, macrospheres, microspheres or nanospheres, liposomes, oil bodies or chylomicrons, large particles, microparticles or nanoparticles, macrosponges, microsponges or nanosponges, microemulsions or nanoemulsions, or adsorbed on powdered organic polymers, talc, bentonite, spores or outer walls and other mineral or organic carriers).

[0025] In some embodiments, the donkey-hide gelatin peptide or the whitening composition of the present invention is used in cosmetics, particularly in the areas of skin care for the face, body, hair and scalp, and in the area of ​​cosmetic care.

[0026] In some embodiments, the donkey-hide gelatin peptides of the present invention can be used in any form, in combination, incorporated or adsorbed onto large particles, micro particles and nanoparticles or large capsules, micro capsules and nanocapsules, for treating textiles, natural or synthetic fibers, wool and any material intended to come into contact with the skin and to be used in clothing, underwear (day or night), handkerchiefs or tissues, to exert their cosmetic or therapeutic effects through such skin / textile contact and to allow continuous local delivery.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention provides the first identification of a donkey-hide gelatin peptide with the amino acid sequence Gly-Pro-Ile-Gly-Pro-Val-Gly (GPIGPVG). Compared to 377, this donkey-hide gelatin peptide exhibits better stability and lower cytotoxicity. It also inhibits tyrosinase activity and melanin synthesis by melanocytes, thus demonstrating excellent whitening activity. Therefore, it can be used to prepare products with whitening effects or for non-therapeutic whitening treatments of the skin, and has wide applications in the cosmetics field.

[0029] Terminology Definition

[0030] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0031] Unless the context clearly indicates, as used herein, the singular form of a word includes the plural, and vice versa. Therefore, “a,” “an,” and “the” generally include the plural of the corresponding term. For example, “a type of donkey-hide gelatin peptide” includes the plural of the stated “donkey-hide gelatin peptide.” Similarly, the term “for example,” particularly when preceding a series of terms, is merely exemplary and explanatory and should not be considered exclusionary or comprehensive.

[0032] In this invention, "donkey-hide gelatin peptide or its salt" includes metal salts of donkey-hide gelatin peptide, salts formed by donkey-hide gelatin peptide and inorganic or organic acids, and salts formed by donkey-hide gelatin peptide and organic bases.

[0033] Unless otherwise specified, the practice of this invention will take place using conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. Unless otherwise defined, 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 pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0034] To a certain extent, the terms “comprising,” “including,” and “having, has, with,” or variations thereof, are used in the specific implementation and / or claims, and these terms are intended to include a manner similar to the term “comprising.”

[0035] The "effective" dose depends on a variety of factors, such as age, the severity of the condition, and the method of application. An effective dose is a non-toxic amount sufficient to achieve the desired effect. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0037] Figure 1 The images show the total ion chromatogram (TIC) and base peak chromatogram (BPC) of donkey-hide gelatin oligopeptides in the LC-MS / MS analysis process.

[0038] Figure 2 The primary mass spectrum (A) and secondary mass spectrum (B) of the peptide obtained using pFind software for the identification of donkey-hide gelatin peptide GPIGPVG.

[0039] Figure 3 This diagram illustrates the results of donkey-hide gelatin peptide inhibiting tyrosinase activity. Kojic acid was used as the positive control. * indicates P < 0.05, ** indicates P < 0.01. *** This means P < 0.001.

[0040] Figure 4 This diagram illustrates the results of donkey-hide gelatin peptides inhibiting melanin synthesis. BC: blank control (cell control well A375 cells), positive control: kojic acid, * indicates P < 0.05. ** This means P < 0.01. *** This means P < 0.001. Detailed Implementation

[0041] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0042] Example 1: Identification of active peptide sequences from donkey-hide gelatin

[0043] (1) Preparation of donkey-hide gelatin oligopeptides, the preparation method includes the following steps:

[0044] S1 gelatinization: Take donkey-hide gelatin, crush it into powder, weigh it, put it in a reaction vessel, add drinking water according to a solid-liquid ratio of 1:2-1:5 v / w, heat and melt it according to a temperature gradient program of 60℃±2℃ for 5 min, 70℃±2℃ for 10 min, 85℃±2℃ for 15 min and 100℃±5℃ for 30 min, and continue stirring to obtain gelatin solution.

[0045] S2 enzymatic hydrolysis: Adjust the temperature of the homogeneous gel solution of S1 to 50-55℃, adjust the pH to neutral, add papain, pepsin and bromelain preparations at a ratio of 4000-6000U / g, and enzymatically hydrolyze at 50℃ for 2 hours.

[0046] S3 Inactivation: The gel solution after enzymatic hydrolysis in S2 is heated to 95℃ for 30 minutes to inactivate the enzyme, and then cooled to obtain the enzymatic hydrolysis mixture of gel solution;

[0047] S4 Concentration: After the enzymatic hydrolysis mixture in S3 is sieved through a 100-120 mesh sieve, the mixture is concentrated to a relative density of 1.17-1.28 by alternating continuous boiling and reduced pressure concentration. Then, it is filtered through a 120-200 mesh filter to obtain donkey-hide gelatin oligopeptides for later use.

[0048] S5 Vacuum Continuous Drying and Powdering: The donkey-hide gelatin oligopeptides obtained in S4 are dried and powdered using a vacuum continuous dryer.

[0049] S6 is prepared and shaped.

[0050] (2) Identification of amino acid sequences of active peptides derived from donkey-hide gelatin

[0051] Accurately weigh the donkey-hide gelatin oligopeptide powder and dissolve it in ultrapure water to prepare a solution of approximately 5 mg / mL. Transfer the prepared solution to a 10K ultrafiltration centrifuge tube and centrifuge at 10,000 rpm for 10 minutes. Collect the filtrate for LC-MS / MS analysis. Using UPLC-MS, three batches of donkey-hide gelatin oligopeptides were analyzed, with each batch analyzed three times to ensure that the obtained peptide sequences were derived from donkey-hide gelatin oligopeptides.

[0052] The total flow rate of the liquid chromatography was 0.3 mL / min. Mobile phase A was deionized water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.2% formic acid. The elution conditions were as follows: 0-5 min, 3% B; 5-60 min, 35% B; 60-61 min, 90% B. The chromatographic column was an Agilent AdvanceBio Peptide Plus, 2.1 x 150 mm, 2.7 μm, and the column temperature was 40 °C.

[0053] Agilent Q-TOF high-resolution mass spectrometry (6545XT LC / Q-TOF) was used with an electrospray ionization source in positive ion scanning mode. The mass spectrometry range was 50–3200 m / z, capillary voltage was 175 V, nebulizer pressure was 40 psig, ESI gas temperature was 325 °C, and flow rate was 10 L / min. The pFind software was used for proteomics data identification. The donkey collagen library downloaded from Uniprot was selected for data processing. This allowed for the identification of donkey-hide gelatin-derived peptides in oligopeptide samples, obtaining small molecule donkey-hide gelatin peptide sequences. Agilent Bioconfirm and Agilent Qualitative Analysis software were used for further confirmation of the identified peptides. After analysis, several stable detectable peptides were obtained, with the strongest ionic strength being the donkey-hide gelatin peptide GPIPVG (SEQ ID NO:1). Figure 1 As shown, the LC-MS / MS analysis procedure displays the total ion chromatogram (TIC) and base peak chromatogram (BPC) of a specific batch of donkey-hide gelatin oligopeptides from three batches. Figure 2 As shown, the primary mass spectrum (A) and secondary mass spectrum (B) of the GPIGPVG peptide obtained by pFind software are presented respectively.

[0054] Example 2: Synthesis and preparation of peptide H-Gly-Pro-Ile-Gly-Pro-Val-Gly-OH

[0055] CTC resin (1.78 g, 2 mmol) was placed in a 100 mL solid-phase synthesis reactor. Amino acid Fmoc-Gly-OH (1.20 g, 4 mmol) was added, followed by 20 mL of dichloromethane (DCM). Then, DIEA (2.0 mL) was added, and the reaction was carried out at 25 °C for 3 hours. Finally, 3 mL of methanol was added, and the reaction was continued for 5 minutes. The mixture was filtered, and the resin was washed twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF.

[0056] Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter to remove the deprotection solution, then wash 6 times with 20 mL of DMF solution, and dry for later use.

[0057] Take Fmoc-Val-OH (1.36 g, 4 mmol) and HOBt (0.56 g, 4 mmol) in a 50 mL beaker, cool to 2-8 °C, add 2 mL of DMF solution and DIC (0.63 mL, 4 mmol), and let stand for 10-20 minutes. Then, add the solution from the 50 mL beaker to a 100 mL solid-phase synthesis reactor and stir for 1.5 hours until the reaction is complete. Wash the resin three times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction.

[0058] Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter to remove the deprotection solution, then wash 6 times with 5 mL of DMF solution, and dry for later use.

[0059] According to the described protocol, each coupling contained 0.56 g HOBt (4 mmol), 2 mL DMF solution, and 0.64 mL LDI C (4 mmol). After each coupling, the resin was washed as described in the general procedure, and the deprotection treatment of the Fmoc group was repeated. In order to couple the following amino acids, Fmoc-Pro-OH (1.35 g, 4 mmol), Fmoc-Gly-OH (1.20 g, 4 mmol), Fmoc-Ile-OH (1.40 g, 4 mmol), Fmoc-Pro-OH (1.35 g, 4 mmol), and Fmoc-Gly-OH (1.20 g, 4 mmol) were coupled sequentially.

[0060] After each reaction and washing, 5 mL of 20% Pip / DMF solution was added, and the mixture was stirred for 30 min. The mixture was then filtered to remove the deprotection solution, followed by washing six times with 5 mL of DMF solution and drying under vacuum. The final washings included two times with 5 mL of methanol, two times with 5 mL of DCM solution, and two times with 5 mL of methanol. The mixture was then dried under vacuum to obtain the peptide resin of H-Gly-Pro-Ile-Gly-Pro-Val-Gly-CTC.

[0061] The peptide resin was cleaved at 30°C for 2.5 hours using 40 mL of TFA / anisole / PhOH / H2O / EDT = 87.5 / 5 / 2.5 / 2.5 / 2.5. The cleavage solution was then added to 800 mL of diethyl ether (2-8°C), precipitating a white solid. This solid was centrifuged to obtain a white, crude peptide. The white, crude peptide was dried under vacuum to obtain crude peptide powder H-Gly-Pro-Ile-Gly-Pro-Val-Gly-OH (0.94 g). The theoretical yield was 2 mmol H-Gly-Pro-Ile-Gly-Pro-Val-Gly-OH, with a yield of 78.6%.

[0062] Example 3: Inhibition of Tyrosinase Activity – Determination of Inhibition Rate of Tyrosinase Activity in In Vitro

[0063] (1) Experimental instruments and reagents

[0064] UV-Vis spectrophotometer, centrifuge, constant temperature water bath, adjustable pipette, 96-well plate, tyrosinase, L-tyrosine, DMSO, kojic acid, phosphate buffer solution, PBS.

[0065] (2) Experimental methods

[0066] In the same 96-well plate, a sample group (donkey-hide gelatin peptide prepared in Example 2) and a positive control group (kojic acid) were set up.

[0067] In the sample (donkey-hide gelatin peptide) group, 10 μL of donkey-hide gelatin peptide solution of different concentrations (0.63, 1.25, 2.5, and 5 mg / mL) was added to the administration wells, followed by 130 μL of PBS and 10 μL of tyrosinase (666.67 U / mL) via a pipette. In the background wells, 10 μL of donkey-hide gelatin peptide solution of different concentrations was added, followed by 140 μL of PBS via a pipette. In the control wells, 140 μL of PBS and 10 μL of tyrosinase (666.67 U / mL) were added via a pipette. In the blank wells, 150 μL of PBS was added.

[0068] In the positive control (kojic acid) group, a kojic acid solution of the same concentration was used instead of a donkey-hide gelatin peptide solution.

[0069] After incubating the 96-well plate at room temperature for 5 min, add 50 μL of L-tyrosine solution (2 mM) to each well using a pipette. Incubate the reaction mixture at 37 °C for 20 min, and then measure the absorbance (OD) at 475 nm. Calculate the relative inhibition rate of melanin synthesis using the following formula:

[0070] Tyrosinase inhibition rate (%) = [1 - (OD of drug delivery well - OD of background well) / (OD of control well - OD of blank well)] × 100%

[0071] Experimental results are as follows Figure 3 As shown, compared with the positive control (kojic acid) group, the tyrosinase inhibition rate of the sample (donkey-hide gelatin peptide) group at concentrations of 0.63 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL was better than that of kojic acid at the same concentration. This indicates that the donkey-hide gelatin peptide has excellent inhibitory effect on tyrosinase, can reduce the amount of melanin produced, and has a whitening effect.

[0072] Example 4: Inhibition of Melanin Synthesis in A375 Cells – Determination of Melanin Synthesis Inhibition Rate

[0073] (1) Experimental instruments and reagents

[0074] Biosafety cabinet, inverted fluorescence microscope, vortex shaker, CO2 incubator, microplate reader, 24-well plate, 96-well plate, 6cm culture dish, DMSO, NaOH, cell lysis buffer, DMEM medium, pen Strep antibiotic, fetal bovine serum, 0.05% Trypsin-EDTA, PBS, MTT.

[0075] (2) Experimental methods

[0076] Cell seeding: 1×10 5 A375 cells (Chinese Academy of Sciences Stem Cell Bank) were seeded into 24-well plates at a density of cells / well and incubated overnight in an incubator (37℃, 5% CO2). After 24 hours of culture, according to the experimental design, the cells were divided into groups and treated accordingly. 1 mL of cell culture medium was added to each well of the blank control group; 1 mL of culture medium containing the corresponding concentration of kojic acid was added to each well of the positive control group; and 1 mL of culture medium containing the corresponding concentration of glycyrrhizin / donkey-hide gelatin peptide was added to each well of the sample group. After treatment, the 24-well plates were incubated in an incubator (37℃, 5% CO2) for 24 hours.

[0077] Detection: 72 hours after the third medium change, the melanin content of the cells was measured. Cells were washed twice with 1×PBS, the PBS was removed, and 200 μL of 0.25% trypsin was added to each well to digest the cells. The cells were then incubated in a CO2 incubator for 5 minutes. 2 mL of PBS was added twice, and the cells were collected by pipetting. The cells were then centrifuged at 4000 rpm for 5 minutes. The remaining PBS in the centrifuge tubes was carefully removed. 200 μL of melanin extraction solution was added to each tube, and the mixture was shaken well. The tubes were then heated in an 80°C water bath for 1 hour. After cooling, the droplets on the tube wall were centrifuged moderately, and the mixture was pipetted well. 150 μL of the solution from each centrifuge tube was transferred to a 96-well plate, and the absorbance (OD) at 405 nm was measured using a microplate reader. The relative inhibition rate of melanin synthesis was calculated using the following formula:

[0078] Cellular melanin synthesis inhibition rate (%) = [1 - (OD of drug administration well - OD of blank well) / (OD of cell control well - OD of blank well)] × 100%

[0079] (3) Experimental Results

[0080] like Figure 4As shown, compared with the blank control group (BC), the positive control group showed a significantly increased inhibition rate of melanin synthesis, indicating the effectiveness of the experimental system. Compared with the BC group, the GPIPVG peptide increased the inhibition rate of melanin synthesis at a concentration of 0.1 mg / mL and significantly increased it at a concentration of 0.5 mg / mL, indicating that this GPIPVG peptide has a whitening effect by inhibiting melanin synthesis in A375 cells. Furthermore, at concentrations of 0.1 mg / mL and 0.5 mg / mL, the inhibition rate of GPIPVG peptide on melanin synthesis was superior to that of traditional whitening active ingredients kojic acid and glycyrrhizin at the same concentration.

[0081] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A whitening composition, characterized in that, The whitening composition contains donkey-hide gelatin peptides; the donkey-hide gelatin peptides contain an amino acid sequence as shown in SEQ ID NO:1 (GPIGPVG).

2. The whitening composition according to claim 1, characterized in that, The amino acid sequence of the donkey-hide gelatin peptide is as shown in SEQ ID NO:1 or an amino acid sequence having at least 90% sequence identity with it.

3. The whitening composition according to any one of claims 1-2, characterized in that, In the whitening composition, the weight range of the donkey-hide gelatin peptide is 0.0000001% to 10% based on the total weight of the composition, more preferably 0.0000001% to 1%.

4. The whitening composition according to any one of claims 1-3, characterized in that, The whitening composition also contains an active agent for enhancing the whitening effect of the donkey-hide gelatin peptide.

5. The whitening composition according to claim 4, characterized in that, The active agent is selected from one or more of the following: peptides, vitamin C and its derivatives, vitamins derived from the B group, dehydroepiandrosterone (DHEA), phytosterols, salicylic acid and its derivatives, retinoids, flavonoids, amino sugars, azoles and metal salts.

6. The whitening composition according to any one of claims 1-5, characterized in that, The whitening composition also includes cosmetic excipients.

7. The whitening composition according to claim 6, characterized in that, The cosmetic excipients are selected from one or more of the following: thickeners, surfactants, co-surfactants, skin conditioning agents, preservatives, fragrances, lubricants, disintegrants, humectants, adhesives, and fillers.

8. Use of the whitening composition according to any one of claims 1-7 in the preparation of products having whitening or freckle-removing effects.

9. Use of the whitening composition according to any one of claims 1-7 in skin whitening or freckle removal for non-therapeutic purposes.

10. The use according to claim 8 or 9, characterized in that, The whitening or freckle-removing effects include inhibiting tyrosinase activity and / or inhibiting the synthesis of melanin by human melanocytes.

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