A polypeptide for inhibiting melanin production, and its preparation method and application

Abstract

The present invention discloses a polypeptide for inhibiting melanin production, a preparation method thereof, and an application thereof. The polypeptide of the present invention is obtained from the hydrolyzed protein of Babylonia square areolata, has an amino acid sequence of leucine-glycine-alanine-glycine-phenylalanine-glycine-glycine-glycine-leucine, can significantly inhibit melanin production in cells, and has the characteristics of low toxicity, low irritation, good stability, safety and high efficiency, and can be used in whitening cosmetics.

Description

Technical Field

[0001] The present invention relates to a polypeptide, in particular to a polypeptide for inhibiting melanin production, and a preparation method and application thereof. Background Art

[0002] In daily life, skin is affected by external stimuli such as ultraviolet rays, medications, and stress, leading to the increasing appearance of skin pigmentation. Skin color is primarily determined by the amount and distribution of melanin. When melanin is excessive, skin color darkens, ultimately causing spots. To address this problem, new whitening products are constantly being developed. Currently, some commonly used whitening ingredients include hydroquinone, kojic acid, arbutin, and L-ascorbic acid. However, these whitening ingredients are highly toxic, ineffective, and unstable. Therefore, there is an urgent need to develop safer and more effective whitening products.

[0003] The development of peptide-based whitening products, which offer high safety and stability, has been a research focus in recent years. Peptides are intermediate products of protein hydrolysis, composed of two or more amino acids linked by peptide bonds. They are widely present in organisms and possess diverse functions. In recent years, researchers have discovered a variety of peptides with tyrosinase inhibitory activity through natural source screening and synthetic design. These peptides achieve whitening effects by inhibiting tyrosinase activity and reducing melanin production. Their targeted, mild, and bioactive properties have led to their increasing application in whitening cosmetics.

[0004] However, there is currently no documented evidence of polypeptides extracted from Babylonia square-shaped conchs for inhibiting intracellular melanin. Summary of the Invention

[0005] The present invention aims to provide a polypeptide for inhibiting melanin production, and its preparation method and application. The polypeptide of the present invention can significantly inhibit melanin production in cells and has the characteristics of low toxicity, low irritation, good stability, safety and high efficiency.

[0006] The technical solution of the present invention is a polypeptide for inhibiting melanin production, wherein the amino acid sequence of the polypeptide is: leucine-glycine-alanine-glycine-phenylalanine-glycine-glycine-glycine-leucine.

[0007] The aforementioned polypeptide for inhibiting melanin production is obtained by hydrolyzing and filtering Babylonia squarellae.

[0008] In the aforementioned polypeptide for inhibiting melanin production, the molecular weight of the polypeptide is less than 3 kDa.

[0009] In the aforementioned polypeptide for inhibiting melanin production, the polypeptide is used at a concentration of less than 0.05 mg / mL.

[0010] In the aforementioned polypeptide for inhibiting melanin production, the polypeptide is used at a concentration of 0.01 mg / mL.

[0011] The method for preparing the above-mentioned polypeptide comprises the following steps:

[0012] (1) Preparation of Babylonia areolata powder: mince and homogenize the meat of Babylonia areolata to obtain a homogenate, then mix and stir the homogenate with isopropyl alcohol in a volume ratio of 1:(3-6), remove the isopropyl alcohol, dry and grind to obtain Babylonia areolata powder;

[0013] (2) Preparation of hydrolysate: Mix the powder of Babylonia areolata with the protease solution, perform the hydrolysis reaction in a buffer solution, terminate the hydrolysis reaction by inactivating the enzyme, and then centrifuge to obtain the supernatant, which is the hydrolysate;

[0014] (3) Preparation of polypeptide: The hydrolyzate is filtered through a membrane to obtain a polypeptide with a molecular weight of less than 3 kDa and an amino acid sequence of LGAGFGGGL.

[0015] In the aforementioned method for preparing the polypeptide, in step (2), the hydrolysis ratio of Babylonia areolata powder to protease is (0.1-1):10, and the buffer solution is 15-25 mM Tris-HCl buffer containing 100-200 mM NaCl.

[0016] In the aforementioned method for preparing the polypeptide, in step (2), the hydrolysis conditions are: pH 8.0, rotation speed 100-150 rpm, temperature 45-55°C, and hydrolysis time 40-80 min; the enzyme inactivation operation is: heating at 85-95°C for 8-12 minutes to terminate the reaction.

[0017] The present invention also provides the use of the polypeptide in preparing whitening cosmetics that inhibit melanin production.

[0018] In the aforementioned application, the whitening cosmetics include any one of facial cleanser, facial cream, facial mask, essence, toner, lotion, body lotion, and shower gel.

[0019] The present invention also provides a whitening composition comprising the above polypeptide.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a new small molecule polypeptide with excellent melanin production inhibition function, which can be applied in the fields of cosmetics, medical beauty, health care, etc. and has great application value.

[0022] Moreover, the polypeptide of the present invention can inhibit melanin production at a concentration of 0.01 mg / mL (i.e., 10 ppm), effectively reducing the amount of raw materials used, lowering production costs, and helping to improve product safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of the polypeptide of the present invention on the intracellular melanin content, where "*" indicates the significance level compared with the blank control group, and "****" indicates the significance level P <0.0001; “#” indicates the significance level compared with the positive control group, and “####” indicates the significance level P <0.0001. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0025] Comparison table of amino acid names used in this invention:

[0026]

[0027] Example:

[0028] A polypeptide that inhibits melanin production, the amino acid sequence of the polypeptide is: LGAGFGGGL, i.e., leucine-glycine-alanine-glycine-phenylalanine-glycine-glycine-glycine-leucine, composed of 9 amino acids, and belongs to type I cytokeratin.

[0029] The above polypeptide is obtained from the hydrolyzed protein of Babylonia areolata.

[0030] The preparation method of the above polypeptide comprises the following steps:

[0031] (1) The Babylonia areolata was placed under running water for thorough washing, and then anesthetized on ice for 15 minutes; thereafter, the meat of the Babylonia areolata was separated from the shell and chopped; the chopped meat was placed in a blender and fully homogenized to obtain a homogenate.

[0032] The homogenate of Babylonia square spot was mixed with isopropyl alcohol at a volume ratio of 4:1 and stirred continuously at room temperature for 60 minutes. The isopropyl alcohol was then effectively removed from the mixture using vacuum filtration technology, and the mixture was then dried in an oven at 60°C to obtain a homogeneous dry product.

[0033] (2) The homogenized dried product was placed in a blender and further ground into a fine powder to obtain the Babylonia areolata powder. To ensure its quality, the Babylonia areolata powder was properly stored in a desiccator for subsequent research or application.

[0034] (3) The amino acid composition of the hydrolysate was analyzed by liquid chromatography using an Agilent Poroshell 120 HILIC-Z column (3.0 × 100 mm, 2.7 µm particle size) at a column temperature of 40 °C and a flow rate of 1 mL / min. A Sciex liquid chromatography coupled with a triple quadrupole instrument was used simultaneously to detect the types and contents of amino acids in the hydrolysate in the ESI positive ion mode.

[0035] (3.1) Preparation of total amino acids in the hydrolyzate: Weigh approximately 0.25 g of Babylonia areolata powder into a hydrolysis tube, add 10 mL of 6.0 mol / L hydrochloric acid, add 2–3 drops of phenol, mix thoroughly, seal the tube with nitrogen, and hydrolyze in a 110°C oven for 20 h. Remove the hydrolysis tube, cool to room temperature, and filter. The filtrate is concentrated on a rotary evaporator to remove the hydrochloric acid. Wash the tube several times with water (2 mL each time) until the hydrochloric acid is completely evaporated. Add water to make the volume up to 10 mL. Mix thoroughly, then take an appropriate amount of the sample solution and centrifuge at 12,000 rpm for 5 min to obtain the total amino acid supernatant. Because some amino acids in the total amino acid supernatant exceed the measurement range, precisely pipette 1 mL of the total amino acid supernatant into 10, 50, and 250 mL volumetric flasks. Add water to the mark and mix thoroughly to obtain the total amino acid sample.

[0036] Among them, the total amino acid sample is diluted 10 times to obtain the sample solutions to be tested of cystine and histidine; the total amino acid sample is diluted 50 times to obtain the sample solutions to be tested of isoleucine, alanine, aspartic acid, glycine, methionine, serine, threonine, tyrosine and lysine; the total amino acid sample is diluted 250 times to obtain the sample solutions to be tested of leucine, proline, valine, phenylalanine, arginine and glutamic acid.

[0037] (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 mark, and sonicate at 40 kHz, 300 W for 30 min. Remove, cool, and mix thoroughly. Take an appropriate amount of sample solution and centrifuge at 12,000 rpm for 5 min to obtain a free amino acid supernatant. The concentrations of leucine, proline, alanine, valine, phenylalanine, tyrosine, threonine, and glutamic acid in the free amino acid supernatant exceed the measurement range. Therefore, precisely pipette 1 mL of the free amino acid supernatant into a 10 mL volumetric flask, add water to the mark, and mix thoroughly to obtain the free amino acid sample. Then, plot a standard curve to determine the molecular weight of each amino acid component. This provides the raw material basis and theoretical basis for the subsequent preparation of peptides with specific sequences.

[0038] (4) The hydrolyzate of Babylonia areolata powder was prepared using the protease subtilisin A (EC3.4.21.62, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0039] In a 20 mM Tris-HCl buffer containing 150 mM NaCl, pH 8.0, the prepared enzyme solution (2.5 AU / g) and Babylonia square-shaped snail powder were mixed together at a substrate to enzyme hydrolysis ratio of 0.5:10. The hydrolysis reaction was carried out on a shaker at a speed of 125 rpm and a temperature of 50°C for 60 minutes, and the hydrolysis reaction was terminated by heating at 90°C for 10 minutes to obtain a hydrolyzate. The hydrolyzate was centrifuged at 10,000×g for 15 minutes, the supernatant was collected to obtain the hydrolyzate, and stored at -4°C for subsequent use.

[0040] (5) The hydrolyzate of Babylonia areata was fractionated using ultrafiltration centrifuge tubes (Changde Beekman Biotechnology Co., Ltd.): the hydrolyzate obtained in step (4) was filtered through 10 kDa, 5 kDa and 3 kDa molecular weight cutoff membranes with decreasing pore sizes. During this process, all fractions obtained by membrane filtration were collected, including: fractions with a molecular weight greater than 10 kDa that were retained by the 10 kDa membrane (i.e., the retentate of the 10 kDa membrane), fractions with a molecular weight between 5 kDa and 10 kDa that were retained by the 5 kDa membrane but failed to pass through the 10 kDa membrane (i.e., the retentate of the 5 kDa membrane), fractions with a molecular weight less than 5 kDa that permeated through the 5 kDa membrane (i.e., the permeate of the 5 kDa membrane), fractions with a molecular weight between 3 kDa and 5 kDa that were retained by the 3 kDa membrane but failed to pass through the 5 kDa membrane (i.e., the retentate of the 3 kDa membrane), and fractions with a molecular weight less than 3 kDa that permeated through the 3 kDa membrane (i.e., the permeate of the 3 kDa membrane), ultimately obtaining polypeptides with a molecular weight of approximately less than 3 kDa.

[0041] (6) Peptides were identified using liquid chromatography-mass spectrometry (MS) using an LC-MS / MS system. The LC system used was a Vanquish series, manufactured by ThermoFisher. The Q_Exactive mass spectrometer is a high-performance mass spectrometer launched by ThermoFisher. The chromatographic column was manufactured by Waters, using a 1.7 μm particle size and C18 reversed-phase packing. The column length was 2.1 cm, the inner diameter was 100 mm, and the pore size was 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 UV absorption of the peptide. By using the 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 belonged, to identify the protein in the sample and the digested peptide segment. Finally, the target peptide was identified, with the amino acid sequence of LGAGFGGGL, defined as P08779.

[0042] Experimental example:

[0043] Test materials:

[0044] Biological safety cabinet (Likang Biomedical Technology Holdings Co., Ltd., NU-437-400S), CO2 incubator (Panasonic Corporation, MCO-18AIC), water bath (Qun'an Scientific Instrument (Zhejiang) Co., Ltd., WB100-1F), centrifuge (Sigma-Aldrich, USA, 2-16p), analytical balance (Sartorius Group, Germany, QUINTIX224-1CN), microscope (Nippon Kogaku Kogyo Co., Ltd., Ts2R-FL), and microplate reader (Tecan (Shanghai) Trading Co., Ltd., SPARK).

[0045] Phosphate buffer (Lanjieke Technology Co., Ltd., BL302A), 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 (Yacoin Biotechnology Co., Ltd., BMU106), α-arbutin (Shanghai MacLean Biochemical Technology Co., Ltd., A821853), and dimethyl sulfoxide (DMSO) (Shanghai MacLean Biochemical Technology Co., Ltd., D6258).

[0046] Reagent preparation:

[0047] Preparation of polypeptide P08779 solution: Reagent grade, water-soluble raw material. Dissolve the polypeptide powder directly in an EP tube containing PBS buffer, shake and mix until completely dissolved. This is used as the stock solution or mother liquor concentration. Dilute with PBS buffer to the corresponding concentration during the experiment.

[0048] Preparation of α-Arbutin Solution: Precisely weigh 10 mg of reagent-grade, water-soluble arbutin powder in 1 mL of PBS buffer and shake thoroughly until completely dissolved. This serves as the stock solution. Dilute to the desired concentration in PBS buffer during the experiment.

[0049] 1. Safety test of peptides:

[0050] 1.1 Test method:

[0051] 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. The blank control group contains only cells and culture medium; the sample group contains cells and a certain concentration of sample; the zero-adjustment group contains only culture medium without cells. Within each sample group, set up seven concentration gradients for each sample, with six replicate wells in each concentration gradient.

[0052] Cell seeding: After thawing, subculture cells for one generation. When the cell confluency reaches 70%-80%, plate the cells evenly into a 96-well plate, using 100 μL per well. Add 100 μL of PBS to the zero-adjustment group and the outer wells of the 96-well plate to prevent evaporation of the cell culture medium. Incubate the 96-well plate in an incubator (37°C, 5% CO2) overnight.

[0053] Washing and Dosing: When the cell confluence in the 96-well plate reaches 40%-60% by microscopic observation, discard the old culture medium and replace the medium for dosing in each group. For the sample group, add 100 μL of culture medium containing the corresponding test substance concentration to each well; for the blank control group, add 100 μL of culture medium to each well. If PBS evaporates in the zero-adjustment group and the outer wells of the 96-well plate, replenish it as needed. After sample loading, incubate the 96-well plate in an incubator (37°C, 5% CO2) for 48 hours.

[0054] Detection by microplate reader: After 48 hours of incubation, discard the supernatant, add CCK-8 reagent, and incubate at 37°C in the dark for 1-4 hours. After incubation, read the OD value at 450 nm.

[0055] Calculation of relative cell viability: Calculated according to the formula, relative cell viability (%) = (OD value of sample well - OD value of zero adjustment well) / (OD value of blank control well - OD value of zero adjustment well) × 100%.

[0056] Data processing: Statistical analysis and plotting were performed using software. All values ​​are expressed as mean ± SD. Statistical comparisons were performed using the t-test or one-way analysis of variance. P < 0.05 was considered statistically significant.

[0057] 1.2 Test results:

[0058] Table 1 Effects of peptide P08779 on melanocyte toxicity

[0059]

[0060] According to the above test results, when the polypeptide concentration is ≤0.05 mg / mL, the cell viability reaches more than 90%, is non-toxic to melanocytes, and is highly safe.

[0061] 2. Peptide whitening test:

[0062] 2.1 Test method:

[0063] Set up a blank control group (with cells, only 2 mL of complete culture medium added); a positive control group (with cells and a certain concentration of α-arbutin); and a sample group (with cells and a certain concentration of peptide sample). Each group should have at least three replicates.

[0064] Cell seeding: After thawing, subculture the cells for one generation. When the cell confluency reaches 70%-80%, plate the cells evenly. Inoculate the cells into 6-well plates with 2 mL per well and incubate in an incubator (37°C, 5% CO2) overnight.

[0065] Medium exchange and drug administration: When the cell confluence in the well plate reaches 40%-60% as observed under a microscope, discard the old medium and exchange the medium for drug administration in each group. For the blank control group, add 2 mL of medium per well; for the positive control group, add 2 mL of medium containing the corresponding concentration of α-arbutin per well; for the sample group, add 2 mL of medium containing the corresponding concentration of sample per well. After sample loading, incubate the 6-well plate in an incubator (37°C, 5% CO2) for 48 hours.

[0066] Cell harvesting: Set a thermostatic water bath to 80°C and pre-chill the PBS buffer. Then, use trypsin to digest the cells in the well plate, centrifuge, discard the supernatant, and collect the cells into a new EP tube. Wash twice with pre-chilled PBS to terminate the drug reaction.

[0067] Lyse cells: After washing, add 200 μL of 1 M NaOH to each tube. Then place the EP tube on a foam float and heat it in a water bath for about 5 to 10 minutes (the time can be extended appropriately, and the incubation time should be based on the complete dissolution of the melanin) until the melanin is completely dissolved.

[0068] Melanin absorbance determination: 100 μL of lysate was transferred from each tube to a 96-well plate and the OD value was read at 405 nm.

[0069] Result calculation: melanin content (%) = (OD value of melanin content in sample / OD value of melanin content in blank), melanin inhibition rate (%) = (blank control group - sample group) / blank control group.

[0070] Data processing: Statistical analysis and plotting were performed using Statistical Analysis Software. All values ​​are expressed as mean ± SD. Statistical comparisons were performed using the t-test or one-way analysis of variance. P < 0.05 was considered statistically significant.

[0071] The specific experimental group design is shown in Table 2.

[0072] Table 2 Whitening test design

[0073]

[0074] 2.2 Test results:

[0075] Table 3 Effect of peptide P08779 on melanin content

[0076]

[0077] Note: Statistical analysis was performed between the groups in the table. Control refers to the blank control, and α-Arbutin refers to α-arbutin, which is the positive control.

[0078] The test results are shown in Table 3 and Figure 1 The above research results show that the polypeptide P08779 has the ability to reduce melanin content at a concentration of 0.01 mg / mL, with an inhibition rate of 19.80% for melanin content in melanocytes, demonstrating a whitening effect. Furthermore, the melanin inhibition rate of the polypeptide of the present invention is higher than that of α-Arbutin at the same concentration, indicating that the polypeptide of the present invention has a better whitening effect.

[0079] The concentration of peptides used in whitening cosmetics or whitening compositions is 0.01~5%.

[0080] 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. Those skilled in the art may modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein; and all these modifications and replacements should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A polypeptide for inhibiting melanin production, characterized in that: The amino acid sequence of the polypeptide is: leucine-glycine-alanine-glycine-phenylalanine-glycine-glycine-glycine-leucine.

2. The polypeptide for inhibiting melanin production according to claim 1, characterized in that: The polypeptide is obtained by hydrolyzing and filtering Babylonia squarellae.

3. The polypeptide for inhibiting melanin production according to claim 1, wherein: The molecular weight of the polypeptide is less than 3 kDa.

4. The polypeptide for inhibiting melanin production according to claim 1, wherein: The polypeptide is used at a concentration of less than 0.05 mg / mL.

5. The polypeptide for inhibiting melanin production according to claim 1, wherein: The polypeptide was used at a concentration of 0.01 mg / mL.

6. The method for preparing a polypeptide according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Preparation of Babylonia areolata powder: mince and homogenize the meat of Babylonia areolata to obtain a homogenate, then mix and stir the homogenate with isopropyl alcohol, remove the isopropyl alcohol, dry and grind to obtain Babylonia areolata powder; (2) Preparation of hydrolysate: Mix the powder of Babylonia areolata with the protease solution, perform the hydrolysis reaction in a buffer solution, terminate the hydrolysis reaction by inactivating the enzyme, and then centrifuge to obtain the supernatant, which is the hydrolysate; (3) Preparation of polypeptide: The hydrolyzate is filtered through a membrane to obtain a polypeptide with a molecular weight of less than 3 kDa and an amino acid sequence of LGAGFGGGL.

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

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

9. Use of the polypeptide according to any one of claims 1 to 5 in the preparation of whitening cosmetics that inhibit melanin production.

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

Images