Polypeptide with whitening effect as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide with a whitening effect and a preparation method and application thereof.The polypeptide is obtained from hydrolyzed protein of babylonia areolata, the amino acid sequence of the polypeptide is cysteine-arginine-proline-threonine-cysteine-serine-arginine-leucine-alanine-cysteine, and the amino acid sequence of the polypeptide is cysteine-arginine-proline-threonine-cysteine-serine-arginine-leucine-alanine. The compound can obviously inhibit the generation of melanin in cells and the activity of tyrosinase in vitro, has the characteristics of low toxicity, low irritation, good stability, safety and high efficiency, and can be applied to whitening cosmetics.

Description

Technical Field

[0001] The present invention relates to a polypeptide, in particular to a polypeptide with whitening efficacy, and its preparation method and application. Background Art

[0002] In daily life, the skin is affected by external stimuli such as ultraviolet rays, drugs, and stress, resulting in an increasingly obvious problem of skin pigmentation. And the skin color is mainly determined by the quantity and distribution of melanin. When there is an excessive amount of melanin, the skin color will darken, ultimately leading to skin pigmentation. During the melanin generation process, many enzyme-catalyzed reactions are involved. Among them, tyrosinase is the key enzyme for melanin generation. An effective way to block melanin generation is to inhibit the activity of tyrosinase. Tyrosinase, also known as tyrosine hydroxylase, tyrosine 3-monooxygenase, polyphenol oxidase, and catechol oxidase, is a copper-containing oxidoreductase that widely exists in animals, plants, and microorganisms. The active center of tyrosinase consists of two copper ion sites. Tyrosine is hydroxylated by tyrosinase to form dopa, and further undergoes oxidation, decarboxylation and other reactions to become indolequinone. Finally, indolequinone is further synthesized into melanin.

[0003] To solve the problem of skin pigmentation, people have continuously developed new whitening products. Currently, some conventional whitening raw materials are: hydroquinone, kojic acid, arbutin, L-ascorbic acid, etc. However, these whitening raw materials have characteristics such as high toxicity, poor efficacy, and instability. Therefore, there is an urgent need to develop some safer and more efficient whitening products.

[0004] Developing whitening products based on peptides, with high safety and good stability, is a research direction in recent years. A peptide is an intermediate product of protein hydrolysis, formed by the connection of two or more amino acids through peptide bonds, and is a bioactive substance widely existing in organisms with diverse functions. In recent years, researchers have discovered a variety of polypeptides with tyrosinase inhibitory activity through natural source screening and synthetic design. These polypeptides inhibit the activity of tyrosinase, reduce melanin generation, achieve a whitening effect, and have characteristics such as targeting, mildness, and biological activity, thus gradually being applied in whitening cosmetics.

[0005] However, there is currently no document recording a polypeptide substance extracted from Babylonia areolata that can inhibit intracellular melanin generation and in vitro tyrosinase activity. Summary of the Invention

[0006] The purpose of the present invention is to provide a polypeptide with whitening efficacy, and its preparation method and application. The polypeptide of the present invention can significantly inhibit intracellular melanin generation and in vitro tyrosinase activity, and has the characteristics of low toxicity, low irritation, good stability, safety and high efficiency.

[0007] Technical solution of the present invention: A polypeptide with whitening effect, the amino acid sequence of the polypeptide is: Cysteine - Arginine - Proline - Threonine - Cysteine - Serine - Arginine - Leucine - Alanine - Cysteine.

[0008] In the polypeptide with whitening effect described above, the polypeptide is obtained by hydrolyzing and filtering Babylonia areolata.

[0009] In the polypeptide with whitening effect described above, the molecular weight of the polypeptide is less than 3 kDa.

[0010] In the polypeptide with whitening effect described above, the usage concentration of the polypeptide is less than 0.1 mg / mL.

[0011] In the polypeptide with whitening effect described above, the usage concentration of the polypeptide is 0.05 mg / mL.

[0012] The preparation method of the above-mentioned polypeptide includes the following steps: (1) Prepare Babylonia areolata powder: Chop and homogenize the meat of Babylonia areolata to obtain a homogenate, then mix the homogenate and isopropanol in a volume ratio of 1:(3 - 6), stir, then remove isopropanol, dry and grind to obtain Babylonia areolata powder; (2) Prepare a hydrolyzate: Mix the Babylonia areolata powder with a protease solution, carry out a hydrolysis reaction in a buffer solution, and terminate the hydrolysis reaction through an enzyme inactivation operation, then perform a centrifugation treatment, and take the supernatant, which is the hydrolyzate; (3) Prepare a polypeptide: Filter the hydrolyzate through a membrane to obtain a polypeptide with a molecular weight less than 3 kDa and an amino acid sequence of CRPTCSRLAC.

[0013] In the preparation method of the above-mentioned polypeptide, in step (2), the hydrolysis ratio of Babylonia areolata powder to protease is (0.1 - 1):10, and the buffer solution uses a 15 - 25 mM Tris - HCl buffer solution containing 100 - 200 mM of NaCl.

[0014] In the preparation method of the above-mentioned polypeptide, in step (2), the hydrolysis conditions are: pH is 8.0, the rotation speed is 100 - 150 revolutions per minute, the temperature is 45 - 55 °C, and the hydrolysis time is 40 - 80 min; the enzyme inactivation operation is: heating at 85 - 95 °C for 8 - 12 minutes to terminate the reaction.

[0015] The present invention also provides the application of the above-mentioned polypeptide in the preparation of a whitening cosmetic for inhibiting melanin production and / or inhibiting the activity of tyrosinase in vitro.

[0016] In the above-mentioned application, the whitening cosmetic includes any one of facial cleanser, cream, facial mask, essence, toner, emollient lotion, body lotion, body wash.

[0017] The present invention also discloses a whitening composition comprising the above-mentioned polypeptide.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a new small molecule polypeptide with excellent functions of inhibiting melanin production and inhibiting tyrosinase, which can be applied to fields such as cosmetics, medical aesthetics, and health care, and has great application value.

[0019] Moreover, the polypeptide of the present invention can play a role in inhibiting melanin production when the concentration is 0.05 mg / mL (i.e., 50 ppm), and can play a role in inhibiting tyrosinase when the concentration is 0.03 mg / mL, effectively reducing the usage amount of raw materials, lowering the production cost, and at the same time contributing to improving the safety of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a diagram showing the effect of the polypeptide of the present invention on the intracellular melanin content, where the "*" represents the significance level label compared with the blank control group, and "****" represents the significance level P <0.0001; where the "#" represents the significance level label compared with the positive control group, and "#" represents the significance level P <0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The following examples are used to further illustrate the present invention, but are not used as a basis for limiting the present invention.

[0022] The comparison table of the amino acid name definitions cited in the present invention:

[0023] Example: A polypeptide with whitening efficacy, the amino acid sequence of the polypeptide is: CRPTCSRLAC, that is, cysteine - arginine - proline - threonine - cysteine - serine - arginine - leucine - alanine - cysteine, consisting of 10 amino acids.

[0024] The above-mentioned polypeptide is obtained from the hydrolyzed protein of Babylonia areolata.

[0025] The preparation method of the above-mentioned polypeptide includes the following steps: (1) Thoroughly wash Babylonia areolata under tap water, and then perform anesthetizing treatment on ice for 15 minutes; afterwards, separate the snail meat of Babylonia areolata from the shell and chop it; put the chopped snail meat into a blender and perform sufficient homogenization treatment to obtain a homogenate; The homogenate of Babylonia areolata was mixed with isopropanol at a volume ratio of 4:1 and continuously stirred at room temperature for 60 minutes. Then, the isopropanol was removed using vacuum filtration technology, and the mixture was placed in an oven at 60 °C for drying to obtain a homogeneous dry product.

[0026] (2)The homogeneous dry product was put into a blender and further ground into a fine powder to obtain Babylonia areolata powder. To ensure its quality, the Babylonia areolata powder was properly stored in a desiccator for subsequent research or application.

[0027] (3)The amino acid composition of the hydrolysate was analyzed by liquid chromatography using an Agilent Poroshell 120 HILIC-Z chromatographic column (with a specification of 3.0×100 mm and a particle size of 2.7 µm) at a column temperature of 40 °C and a flow rate of 1 mL / min. Meanwhile, a Sciex liquid chromatography tandem triple quadrupole instrument was used to detect the types and contents of amino acids in the hydrolysate in the ESI positive ion mode.

[0028] (3.1)Preparation of total amino acids in the hydrolysate: Weighed approximately 0.25 g of Babylonia areolata powder into a hydrolysis tube, added 10 mL of 6.0 mol / L hydrochloric acid, added 2 - 3 drops of phenol, mixed well, filled with nitrogen and sealed, placed in an oven at 110 °C for hydrolysis for 20 h. The hydrolysis tube was taken out, cooled to room temperature, filtered by suction, the filtrate was taken and concentrated to dryness of hydrochloric acid on a rotary evaporator, washed with water (2 mL each time) several times until hydrochloric acid was completely evaporated, added water to a volume of 10 mL, mixed well, and an appropriate amount of the sample solution was taken and centrifuged at a high speed of 12000 rpm for 5 min to obtain the total amino acid supernatant. Since the contents of some amino acids in the total amino acid supernatant exceeded the range, 1 mL of the total amino acid supernatant was accurately transferred to 10, 50, and 250 mL volumetric flasks, filled with water to the scale, and mixed well to obtain the total amino acid samples.

[0029] Among them, the total amino acid sample diluted 10 times was the sample solution to be measured for cystine and histidine; the total amino acid sample diluted 50 times was the sample solution to be measured for isoleucine, alanine, aspartic acid, glycine, methionine, serine, threonine, tyrosine, and lysine; the total amino acid sample diluted 250 times was the sample solution to be measured for leucine, proline, valine, phenylalanine, arginine, and glutamic acid.

[0030] (3.2)Preparation of free amino acids: Weigh approximately 0.5 g of Babylonia areolata powder into a 10 mL volumetric flask, add pure water to the scale, sonicate at 40 kHz and 300 W for 30 min, take out, let it cool, mix well, take an appropriate amount of the sample solution, centrifuge at a high speed of 12000 rpm for 5 min to obtain the free amino acid supernatant. Since the contents of leucine, proline, alanine, valine, phenylalanine, tyrosine, threonine and glutamic acid in the free amino acid supernatant are out of range, accurately pipette 1 mL of the above free amino acid supernatant into a 10 mL volumetric flask, add water to the scale, and mix well to obtain the free amino acid sample. Then, draw a standard curve and measure the molecular weights of the components of each amino acid, providing a raw material basis and theoretical basis for the subsequent preparation of polypeptides with specific sequences.

[0031] (4)Prepare the hydrolysate of Babylonia areolata powder using protease subtilisin A (EC 3.4.21.62, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0032] In a 20 mM Tris-HCl buffer containing 150 mM of NaCl, under the condition of pH 8.0, mix the enzyme solution (2.5 AU / g) and Babylonia areolata powder according to the hydrolysis ratio of substrate to enzyme of 0.5:10, and carry out the hydrolysis reaction on a shaker at a speed of 125 revolutions per minute and a temperature of 50 °C for 60 min, and heat at 90 °C for 10 minutes to terminate the reaction to obtain the hydrolysate; centrifuge the hydrolysate at 10,000×g for 15 minutes, collect the supernatant to obtain the hydrolysate, and store it at -4 °C for subsequent use.

[0033] (5)Fractionate the Babylonia areolata hydrolysate using an ultrafiltration centrifugal tube (Changde Bicman Biotechnology Co., Ltd.): Filter the hydrolysate obtained in step (4) through molecular weight cut-off membranes with pore sizes decreasing in turn of 10 kDa, 5 kDa and 3 kDa. During this process, collect all the components obtained by membrane filtration, including: the fraction with a molecular weight greater than 10 kDa retained by the 10 kDa membrane (i.e., the retentate of the 10 kDa membrane), the fraction with a molecular weight between 5 kDa and 10 kDa retained by the 5 kDa membrane but not retained by the 10 kDa membrane (i.e., the retentate of the 5 kDa membrane), the fraction with a molecular weight less than 5 kDa permeated through the 5 kDa membrane (i.e., the permeate of the 5 kDa membrane), the fraction with a molecular weight between 3 kDa and 5 kDa retained by the 3 kDa membrane but not retained by the 5 kDa membrane (i.e., the retentate of the 3 kDa membrane), and the fraction with a molecular weight less than 3 kDa permeated through the 3 kDa membrane (i.e., the permeate of the 3 kDa membrane), finally obtaining polypeptides with a molecular weight of approximately less than 3 kDa.

[0034] (6) The polypeptide was identified by liquid chromatography - mass spectrometry (MS) using an LC - MS / MS system, where the LC system was of the Vanquish series produced by ThermoFisher. The Q_Exactive mass spectrometer is a high - performance mass spectrometer launched by ThermoFisher. The chromatographic column was produced by Waters, with a particle size of 1.7 microns and C18 reverse - phase packing material, a column length of 2.1 cm, an inner diameter of 100 mm, and a pore size of 130 Å. The column temperature was stabilized at 60 °C, the flow rate was controlled at 0.1 mL / min, and 220 nm was selected as the detection wavelength to accurately capture the ultraviolet absorption of the polypeptide. By using the database search function of the data analysis software, the experimental data was compared with the theoretical data in the whole - proteome sequence database of the species to which the sample to be tested belongs to identify the proteins and digested peptide fragments in the sample. Finally, the target polypeptide with the amino acid sequence CRPTCSRLAC was identified and defined as P60413.

[0035] Experimental example: Test materials: Biological safety cabinet (Likon Biomedical Co., Ltd., NU - 437 - 400S), CO2 incubator (Panasonic Corporation, MCO - 18AIC), water bath (Qunan Scientific Instruments (Zhejiang) Co., Ltd., WB100 - 1F), centrifuge (Sigma - Aldrich, USA, 2 - 16p), analytical balance (Sartorius Group, Germany, QUINTIX224 - 1CN), microscope (Nikon Corporation, Japan, Ts2R - FL), microplate reader (Tecan Trading AG (Shanghai), SPARK).

[0036] Phosphate buffer (Lanjieke Technology Co., Ltd., BL302A), high-glucose DMEM medium (Gibco Life Technologies, 3062439), non-essential amino acids (Guangzhou Saiku Biotechnology Co., Ltd., CM1008L), 0.25% trypsin solution (Gibco Life Technologies, 27250018), secondary antibody (Gibco Life Technologies, 15240062), fetal bovine serum (Shanghai Beyotime Biotechnology Co., Ltd., C0234), cck8 reagent (Yakeyin Biotechnology Co., Ltd., BMU106), kojic acid (Shanghai Macklin Biochemical Co., Ltd., K3125), α-arbutin (Shanghai Macklin Biochemical Co., Ltd., A821853), dimethyl sulfoxide DMSO (Shanghai Macklin Biochemical Co., Ltd., D6258), mushroom tyrosinase (Sigma-Aldrich, USA, T3824), L-levodopa (Shanghai Aladdin Biochemical Technology Co., Ltd., D111048), NaOH (Shanghai Macklin Biochemical Co., Ltd., S817977).

[0037] Reagent preparation: Preparation of polypeptide P60413 solution: Reagent grade, a water-soluble raw material. Dissolve the polypeptide powder directly in an EP tube containing PBS buffer, shake well, and wait until completely dissolved. This is used as the stock solution, i.e., the mother liquor concentration. Dilute it to the corresponding concentration with PBS buffer during the experiment.

[0038] Preparation of kojic acid solution: Reagent grade, a water-soluble raw material. Weigh 10 mg of kojic acid powder accurately, dissolve it in 1 mL of PBS buffer, shake well, and wait until completely dissolved. This is used as the stock solution. Dilute it to the corresponding concentration with PBS buffer during the experiment. Kojic acid is used as a positive control.

[0039] Preparation of α-arbutin solution: Reagent grade, a water-soluble raw material. Weigh 10 mg of arbutin powder accurately, dissolve it in 1 mL of PBS buffer, shake well, and wait until completely dissolved. This is used as the stock solution. Dilute it to the corresponding concentration with PBS buffer during the experiment. α-Arbutin is used as a positive control.

[0040] Preparation of L-levodopa (L-DOPA) solution: Weigh 0.01479 g of L-DOPA powder accurately, dissolve it completely with 1 mL of 1 mol / L HCl solution, and then dilute it to 50 mL with PBS buffer to make the molar concentration of L-DOPA 1.5 mmol / L. Since the L-DOPA solution is easily oxidized and turns black, it should be prepared freshly and used within 4 hours after fresh preparation.

[0041] 1. Safety test of polypeptide: 1.1 Test method: Experimental grouping: Set up a blank control group (Control), a sample group, and a zero-adjustment group, with 3 - 6 replicate wells in each group. Blank control group: Contains only cells and culture medium; Sample group: Contains cells and a certain concentration of the sample; Zero-adjustment group: Does not contain cells and only adds culture medium. Among them, 7 concentration gradients are set for each sample in the sample group, and 6 replicate wells are set under each concentration gradient.

[0042] Cell seeding: After resuscitating the cells and passing them once, when the confluence of the passaged cells reaches 70% - 80%, plate them. Uniformly seed the cells into a 96-well plate, 100 μL per well; The zero-adjustment group and the outer wells of the 96-well plate do not contain cells, add 100 μL of PBS to prevent the evaporation of water in the cell culture medium. Place the 96-well plate in an incubator (37 °C, 5% CO2) and incubate overnight.

[0043] Washing and drug administration: When observing under a microscope that the cell confluence rate in the 96-well plate reaches 40% - 60%, discard the old culture medium, change the liquid and administer drugs in groups. Add 100 μL of culture medium containing the corresponding concentration of the test substance to each well in the sample group; Add 100 μL of culture medium to each well in the blank control group; If the PBS in the zero-adjustment group and the outer wells of the 96-well plate has evaporated, it can be appropriately supplemented. After loading the samples, place the 96-well plate in an incubator (37 °C, 5% CO2) and incubate for 48 h.

[0044] ELISA reader detection: After culturing for 48 h, discard the supernatant, add cck-8 reagent, incubate at 37 °C in the dark for 1 - 4 h. After the incubation ends, read the OD value at 450 nm.

[0045] Calculation of relative cell viability: Calculate according to the formula, relative cell viability (%) = (OD of sample well - OD of zero-adjustment well) / (OD of blank control well - OD of zero-adjustment well) × 100%.

[0046] Data processing: Use software for statistical analysis and drawing. All values are expressed as Mean ± SD (mean ± standard deviation). Adopt t-test or one-way ANOVA for statistical comparison, and P < 0.05 is considered a significant difference and has statistical significance.

[0047] 1.2 Test results: Table 1 Effects of polypeptide P60413 on the toxicity of melanocytes

[0048] Note: Mean ± SD represents mean ± standard deviation.

[0049] According to the above test results, when the polypeptide concentration is between 0.001 and 0.1 mg / mL, the cell viability reaches over 90%, which is non-toxic to melanocytes and has high safety.

[0050] 2. Melanin content test of the polypeptide: 2.1 Test method: Set up a blank control group, a positive control group, a sample group and a zero-adjustment group, with at least 3 replicate wells in each group. Blank control group (with cells, only add 2 mL of complete medium); positive control group (with cells, containing a certain concentration of kojic acid and α-arbutin); sample group (with cells, containing a certain concentration of polypeptide sample).

[0051] Cell seeding: After resuscitating the cells and passing them on for one generation, when the confluence of the passaged cells reaches 70% - 80%, plate them. Uniformly seed the cells into a 6-well plate, 2 mL per well, and incubate overnight in an incubator (37°C, 5% CO2).

[0052] Medium replacement and drug administration: When the cell confluence rate in the well plate reaches 40% - 60% under the microscope, discard the old culture medium and replace the medium and administer drugs in groups. Add 2 mL of culture medium to each well in the blank control group; add 2 mL of culture medium containing the corresponding concentration of kojic acid to each well in the positive control group; add 2 mL of culture medium containing the corresponding concentration of the sample to each well in the sample group. After loading the samples, place the 6-well plate in an incubator (37°C, 5% CO2) and incubate for 48 h.

[0053] Collect cells: Prepare in advance by setting the constant temperature water bath to 80°C and pre-cooling the PBS buffer. Then digest the cells in the well plate with trypsin, centrifuge to discard the supernatant and collect the cells into a new EP tube. Subsequently, wash twice with pre-cooled PBS to terminate the drug reaction.

[0054] Lyse cells: After washing, add 200 μL of 1 M NaOH to each tube, and then place the EP tube on a foam buoy for water bath heating for about 5 - 10 min (the incubation time can be appropriately extended, and the incubation time is based on the complete dissolution of melanin), until the melanin is completely dissolved.

[0055] Determination of melanin absorbance: Take 100 μL of the lysate from each tube into a 96-well plate and read the OD value at 405 nm.

[0056] Result calculation: Melanin content (%) = (OD value of sample melanin content / OD value of blank melanin content), melanin inhibition rate (%) = (blank control group - sample group) / blank control group.

[0057] Data processing: The software performs statistical analysis and draws graphs. All numerical values are expressed as Mean±SD (mean ± standard deviation). T-test or one-way ANOVA is used for statistical comparison, and P<0.05 indicates significant difference and statistical significance.

[0058] 2.2 Test results: Table 2 Effects of polypeptide P60413 on melanin content

[0059] Note: Statistical analysis was performed among groups in the table. Control represents the blank control, and α-Arbutin represents α-arbutin, which is the positive control.

[0060] The test results are shown in Table 2 and Figure 1 as follows. According to the above research results, compared with the blank control group, polypeptide P60413 has the ability to reduce melanin content at a concentration of 0.05 mg / mL, and the inhibition rate of melanin content in melanocytes is 17.83%, showing a whitening effect; and the polypeptide of the present invention has a higher melanin inhibition rate at 0.05 mg / mL than α-Arbutin at the same concentration, indicating that the polypeptide of the present invention has a better whitening effect.

[0061] 3. In vitro tyrosinase activity test of polypeptide: 3.1 Test method: Experimental grouping: This experiment was carried out in a 96-well plate, and the grouping is shown in Table 3. The activities of mushroom tyrosinase and human tyrosinase were detected respectively.

[0062] Table 3 Grouping table for in vitro tyrosinase activity test

[0063] Note: A means that the test sample is not included in the reaction system; B means that the reaction system is the solvent control; C means that the test sample is included in the reaction system; D means that tyrosinase is not included in the reaction system, that is, the test sample itself is the blank control to exclude the color interference of the test sample and the solvent medium; the test samples include kojic acid and P60413, kojic acid sample is used as the positive control, and phosphate buffer PBS solvent is used as the negative control, and each sample is repeated three times.

[0064] Reaction system: Add L-DOPA, the test sample and PBS solution to the 96-well plate and mix well. Then, incubate the 96-well plate with tyrosinase solution in a constant temperature environment at 37°C for 10 min. Then add tyrosinase solution to each well and continue to react at 37°C. After 10 min, immediately put it into the microplate reader.

[0065] Absorbance measurement: Use a microplate reader to measure the absorbance value of the 96-well plate at 475 nm.

[0066] The settings of each group are designed to comprehensively evaluate the effect of the sample on tyrosinase activity. The blank control group is used to determine the experimental background absorbance value, the solvent control group is used to correct the potential effect of the solvent on tyrosinase activity. The positive control group (i.e., kojic acid) is used to verify the accuracy of the experimental system. The test sample polypeptide P60413 is used to verify the tyrosinase inhibition effect of the test sample.

[0067] Finally, the group data after the reaction are exported and analyzed. By excluding the interference of some external factors through the blank control group and the solvent control group, the test results of the inhibition of tyrosinase activity by the test sample are obtained through the following formula, . The test results are shown in Table 4.

[0068] 3.2 Test results: Table 4 Effects of polypeptide P60413 on tyrosinase activity in vitro

[0069] According to the above test results, it shows that polypeptides P60413 at different concentrations have inhibitory effects on both mushroom and human tyrosinase, and at the same concentration of 0.1 mg / mL, the inhibitory effect of polypeptide P60413 on mushroom tyrosinase is higher than that of kojic acid.

[0070] The usage concentration of the polypeptide in the whitening cosmetic or whitening composition is 0.01 - 5%.

[0071] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. For those skilled in the art, the technical solutions described in the above embodiments can be modified, or some of the technical features can be equivalently replaced; and all such modifications and replacements should fall within the protection scope of the appended claims of the present invention.

Claims

1. A polypeptide with whitening effect, characterized in that: The amino acid sequence of the polypeptide is: cysteine - arginine - proline - threonine - cysteine - serine - arginine - leucine - alanine - cysteine.

2. The polypeptide with whitening effect according to claim 1, characterized in that: The polypeptide is obtained by hydrolyzing and filtering Babylonia areolata.

3. The polypeptide with whitening effect according to claim 1, wherein: The molecular weight of the polypeptide is less than 3 kDa.

4. The polypeptide with whitening effect according to claim 1, characterized in that: The usage concentration of the polypeptide is less than 0.1 mg / mL.

5. The polypeptide with whitening effect according to claim 1, wherein: The usage concentration of the polypeptide is 0.05 mg / mL.

6. The preparation method of the polypeptide according to any one of claims 1-5, characterized in that: It includes the following steps: (1) Prepare Babylonia areolata powder: Chop and homogenize the muscle of Babylonia areolata to obtain a homogenate, then mix the homogenate with isopropanol and stir, and then remove the isopropanol, dry and grind to obtain Babylonia areolata powder; (2) Prepare the hydrolyzate: Mix the Babylonia areolata powder with a protease solution, carry out a hydrolysis reaction in a buffer solution, and terminate the hydrolysis reaction by inactivating the enzyme, then carry out centrifugation and take the supernatant, which is the hydrolyzate; (3) Prepare the polypeptide: Filter the hydrolyzate through a membrane to obtain a polypeptide with a molecular weight less than 3 kDa and an amino acid sequence of CRPTCSRLAC.

7. The preparation method according to claim 6, wherein: In step (2), the hydrolysis ratio of Babylonia areolata powder to protease is (0.1 - 1):10, and the buffer solution is a 15 - 25 mM Tris - HCl buffer solution containing 100 - 200 mM of NaCl.

8. The preparation method according to claim 6, characterized in that: In step (2), the hydrolysis conditions are: pH is 8.0, the rotation speed is 100 - 150 revolutions per minute, the temperature is 45 - 55 °C, and the hydrolysis time is 40 - 80 min; the enzyme inactivation operation is: heat at 85 - 95 °C for 8 - 12 minutes to terminate the reaction.

9. Use of the polypeptide according to any one of claims 1 - 5 in the preparation of a whitening cosmetic for inhibiting melanin production and / or inhibiting the activity of tyrosinase in vitro.

10. A whitening composition, characterized in that: It contains the polypeptide according to any one of claims 1 - 5.

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