Polypeptide with whitening effect, and preparation method and application thereof

Abstract

The present application provides a kind of polypeptide, wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO.1 or the amino acid sequence as shown in SEQ ID NO.2;The inventors of the present application prepare a kind of high activity peptide mixture by the process of pearl powder enzymolysis, ultrafiltration, freeze-drying etc., the high activity peptide mixture has the efficacy of whitening, anti-aging etc.On this basis, the present application obtains two polypeptides SEQ ID NO.1, SEQ ID NO.2, the amino acid sequence of the SEQ ID NO.1 is TKLDHAVT, the amino acid sequence of the SEQ ID NO.2 is VPGF;In another embodiment of the present application, the inventors of the present application obtain polypeptide SEQ ID NO.1, SEQ ID NO.2 by self-synthesis method.In some specific embodiments of the present application, the inventors of the present application find that the aforementioned two polypeptides SEQ ID NO.1, SEQ ID NO.2 have significant melanin inhibitory activity.

Description

Technical Field

[0001] This invention belongs to the fields of cosmetic technology and medical aesthetic functional peptides, and relates to the preparation method and application of one or more pearl polypeptides with tyrosinase inhibitory activity. Background Technology

[0002] As we all know, melanin protects the skin from environmental pollution factors such as ultraviolet radiation and oxidative stress. However, excessive accumulation of melanin can also lead to many skin conditions, such as freckles, age spots, and melanoma. Therefore, effectively controlling pigmentation and reducing melanin production is a major approach to skin whitening and blemish removal.

[0003] In recent years, peptides have gradually gained popularity as novel skin-whitening agents, showing great market potential, and related peptide products have been reported. Peptides are compounds formed by α-amino acids linked together by peptide bonds. They are intermediate products of protein hydrolysis, have small molecular weights, are easily absorbed through the skin, and exhibit good stability in formulations. Pearls have long been considered to have excellent skin-whitening effects, containing a large amount of pearl protein. Through extraction, enzymatic hydrolysis, and ultrafiltration processes, highly active peptide mixtures are produced. Screening for specific functional peptides from these mixtures enhances product efficacy, representing a significant leap forward in the deep processing of freshwater products. Summary of the Invention

[0004] In a first aspect, the present invention provides a polypeptide, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO.1 or as shown in SEQ ID NO.2; any corresponding adjustments or modifications made to the whitening polypeptide as the core are within the scope of protection of the present invention.

[0005] In some specific embodiments of the present invention, the inventors prepared a highly active peptide mixture by enzymatic hydrolysis, ultrafiltration, and freeze-drying of pearl powder. This highly active peptide mixture possesses whitening and anti-aging effects. Based on this, the present invention obtained two polypeptides, SEQ ID NO.1 and SEQ ID NO.2, where the amino acid sequence of SEQ ID NO.1 is TKLDHAVT and the amino acid sequence of SEQ ID NO.2 is VPGF. In other embodiments of the present invention, the inventors obtained polypeptides SEQ ID NO.1 and SEQ ID NO.2 through a self-synthesis method. In some specific embodiments of the present invention, the inventors discovered that the aforementioned two polypeptides, SEQ ID NO.1 and SEQ ID NO.2, possess significant melanin-inhibiting activity.

[0006] Since the polypeptide provided by this invention is derived from pearl, as a natural active peptide, it has the characteristics of good stability, low cytotoxicity, and easy absorption. Its application in cosmetics or medical aesthetics fields has high market value.

[0007] In some specific embodiments of the present invention, a polypeptide mixture is obtained by extracting, enzymatically hydrolyzing, separating, and purifying pearl. The small molecule peptides are then screened for whitening activity using the molecular docking software Discovery Studio 2019. The receptor protein (2Y9X) is downloaded from the PDB database, opened in Discovery Studio 2019, and pretreated. This pretreatment includes removing water molecules and adding hydrogen to the protein, resulting in a new protein with 66 sites. The polypeptide structure is drawn using Chemdraw 20.0, then opened in Discovery Studio 2019 to prepare ligands. Rigid docking (LibDock) is selected for docking, and polypeptides TKLDHAVT and VPGF, which exhibit strong inhibitory activity against tyrosinase, are screened out.

[0008] In some embodiments of the present invention, peptides TKLDHAVT and VPGF were synthesized using a solid-phase synthesis method. Melanin content was detected using B16 melanocytes, and the melanin inhibition rate was calculated. The results showed that TKLDHAVT and VPGF exhibited significant melanin-inhibiting activity. In some specific embodiments, the melanin inhibition rates of TKLDHAVT and VPGF at a concentration of 100 ppm were 246.71% and 154.27% of those at a high concentration of 1000 ppm arbutin, respectively. Secondly, the present invention uses the aforementioned peptides to prepare cosmetics or pharmaceuticals with melanin-inhibiting activity. In some specific embodiments, the inventors of the present invention found that the two peptides SEQ ID NO.1 and SEQ ID NO.2 provided by the present invention are significantly superior to arbutin in melanin inhibition. In some specific embodiments, under the same test conditions, at a concentration of 100 ppm, the melanin production inhibition rate of SEQ ID NO.1 was 36.07%, and the melanin production inhibition rate of SEQ ID NO.2 was 39.75%. Regarding the inhibition of melanin activity, the two polypeptides SEQ ID NO.1 and SEQ ID NO.2 provided by this invention have achieved a multiple improvement compared to arbutin.

[0009] In some specific embodiments, the effects of two peptides, SEQ ID NO.1 and SEQ ID NO.2, on the viability of B16 melanocytes were tested. The results showed that neither peptide was cytotoxic at concentrations of 1000 ppm, 500 ppm, 250 ppm, 100 ppm, 50 ppm, and 10 ppm, and peptide SEQ ID NO.1 also had the effect of promoting cell viability.

[0010] Thirdly, the present invention provides a polypeptide composition comprising the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] Fourthly, the present invention uses the aforementioned polypeptides to prepare cosmetics or pharmaceuticals with whitening or anti-aging effects.

[0012] Fifthly, the present invention uses the aforementioned polypeptides to prepare cosmetics or pharmaceuticals that prevent and / or treat melanin hyperpigmentation disorders; in some embodiments, the melanin hyperpigmentation disorders are selected from melasma, freckles, age spots, freckles, café-au-lait spots, Becker's nevus, lentigines, Mongolian spots, nevus of Ota, acquired bilateral nevus of Ota-like spots, nevus of Ito, blue nevus, junctional nevus, compound nevus, intradermal nevus, halo nevus, congenital melanocytic nevus, Spitz's nevus, dysplastic nevus, and sunspots.

[0013] Sixthly, the present invention uses the aforementioned polypeptides as tyrosinase inhibitors. In some specific embodiments, experiments have demonstrated that the two polypeptides SEQ ID NO.1 and SEQ ID NO.2 can significantly inhibit tyrosinase activity at concentrations of 1000 ppm, 100 ppm, and 10 ppm.

[0014] In a seventh aspect, the present invention provides a method for preparing the aforementioned polypeptide, the method comprising synthesizing the polypeptide using a polypeptide synthesizer according to the amino acid sequence.

[0015] The polypeptide provided by this invention, as a natural active peptide, has the characteristics of good stability, low cytotoxicity, and easy absorption. Its application in cosmetics or medical aesthetics fields has high market value. Attached Figure Description

[0016] Figure 1 The melanin inhibition rate of the two polypeptides provided by this invention;

[0017] Figure 2 The effects of the two polypeptides provided in this invention on the viability of B16 melanocytes;

[0018] Figure 3 The tyrosinase inhibition rate of the two polypeptides provided in this invention. Detailed implementation method:

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Reference table of amino acid names cited in this invention

[0021] Phenylalanine F(Phe) Histidine H(His) Valine V(Val) Asparagine D(Asp) alanine A(Ala) proline P(Pro) threonine T(Thr) Lysine K(Lys) Leucine L(Leu)

[0022] Example 1: Molecular docking of peptide with mushroom tyrosinase (2Y9X)

[0023] The skin-whitening activity of small peptides was screened using the molecular docking software Discovery Studio 2019. The receptor protein (2Y9X) was downloaded from the PDB database, opened in Discovery Studio 2019, and pretreated. The pretreatment process included removing water molecules and adding hydrogen to the protein, resulting in a new protein with 66 sites. The peptide structure was drawn using Chemdraw 20.0, then opened in Discovery Studio 2019, and ligands were prepared. Rigid docking (LibDock) was performed; successful docking indicated that the peptide possessed tyrosinase inhibitory activity.

[0024] Simulation results revealed that two polypeptides with amino acid sequences TKLDHAVT and VPGF exhibited high tyrosinase inhibitory activity.

[0025] Example 2: Peptide Synthesis

[0026] (1) Weigh 0.3 mmol of resin into a reaction tube, swell the resin with dichloromethane for 40 min, clean the resin with N,N-dimethylformamide (DMF) and dry it, and clean it a total of 5 times.

[0027] (2) Remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group with 20% piperidine (dissolved in DMF) for 3 min, dry under vacuum, wash with DMF and dry under vacuum, remove Fmoc again with 20% piperidine for 7 min, dry under vacuum, wash the resin with DMF and dry under vacuum, and wash a total of 5 times.

[0028] (3) Add 0.9 mmol Fmoc-Phe-OH, 0.85 mmol O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), 0.85 mmol 1-hydroxybenzotriazole (HOBT) and 1.8 mmol N,N-diisopropylethylamine (Dipea) to the reaction tube, react at room temperature for 45 min, dry under vacuum, wash the resin with DMF and dry under vacuum, and wash a total of 5 times.

[0029] (4) Take a small amount of resin in a test tube, wash it twice with DMF and methanol, add about 0.3 mL of 2% ninhydrin indicator, heat for 5 minutes and observe the color change of the resin. If no blue color is observed, it indicates that the reaction is complete. If a blue color is observed, it indicates that the reaction is incomplete and needs to be continued.

[0030] (5) Replace Fmoc-Phe-OH in (3) with Fmoc-Gly-OH, Fmoc-Pro-OH and Fmoc-Val-OH in sequence, and repeat steps 2-4 until the amino acids on the polypeptide sequence are completely linked.

[0031] (6) After removing the Fmoc protection from the last amino acid Fmoc-Val-OH, the resin was first washed with DMF 5 times, and then washed with dichloromethane 5 times.

[0032] (7) Add 5 mL of trifluoroacetic acid (TFA):H2O:TIPS (95:2.5:2.5) to the reaction tube, react at room temperature for 2 h, filter, rinse with a small amount of TFA and collect the liquid in a centrifuge tube. Slowly add diethyl ether to the liquid, during which a pale yellow solid will be formed. Place the centrifuge tube in a -20℃ refrigerator for 2 h, centrifuge and discard the supernatant, add diethyl ether again, centrifuge and discard the supernatant.

[0033] (8) The obtained solid was dried in a fume hood, then dissolved in water and filtered through a 0.22 μm polysulfone ether (PES) membrane. Purification was performed using preparative grade reversed-phase high-performance liquid chromatography (SemiPrep RP-HPLC) with a mobile phase of water (Buffer A) containing 0.1% TFA and acetonitrile (Buffer B) containing 0.1% TFA. The detection wavelength was set to 214 nm. The collected peptide was characterized by analytical grade reversed-phase high-performance liquid chromatography (Analytical RP-HPLC) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and then lyophilized to obtain the target peptide VPGF. Another peptide was synthesized using the same method, differing only in the replacement of the corresponding amino acid with the Fmoc protecting group.

[0034] Example 3: Assay for melanin inhibition activity

[0035] (1) B16 melanocytes grown in high-glucose DMEM medium containing 10% FBS and in the logarithmic growth phase were subjected to (10-15)×10 4 Cells were seeded at a rate of 100 cells / well in 100 mm cell culture dishes, with 10 mL of culture medium added to each well. The dishes were then incubated at 37 °C in a 5% CO2 incubator for 24 h.

[0036] (2) Dilute and dissolve peptides TKLDHAVT and VPGF with PBS to a final concentration of 100 ppm. Set up 3 replicates for each peptide. Add arbutin to the positive control to a final concentration of 1000 ppm. Add an equal volume of PBS to the blank control. Continue culturing for 24 h.

[0037] (3) Collect cells by digestion with trypsin, discard the supernatant, wash twice with PBS, add 300 μL of 1N sodium hydroxide containing 10% dimethyl sulfoxide (DMSO) to each tube to dissolve melanin granules, and incubate in a water bath at 80℃ for 40 min. Take 100 μL and add it to a 96-well plate, and measure the absorbance at 405 nm using an ELISA reader.

[0038] Specific experimental results are shown in Table 1 and... Figure 1 Both peptides showed excellent inhibitory effects on melanin production. The melanin inhibition rates of 100 ppm TKLDHAVT and VPGF were 246.71% and 154.27% of those of 1000 ppm high-concentration arbutin, respectively.

[0039] Table 1. Melanin suppression rate of peptides

[0040]

[0041] Example 4 Cytotoxicity Assay

[0042] (1) B16 cells grown in high-glucose DMEM medium containing 10% FBS and in logarithmic growth phase were subjected to (5-10)×10 4 Cells were seeded per well in 96-well cell culture plates, with 100 μL of culture medium added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0043] (2) Add 10 μL of peptide sample to each well, with 5 replicates for each concentration, and continue incubation. After 24 h of incubation, discard the supernatant, wash once with PBS, prepare 10% CCK8 solution with incomplete culture medium, add 100 μL to each well, and incubate for 1-4 h. Observe the color frequently; when it darkens, measure the absorbance with a microplate reader.

[0044] Specific experimental results are shown in Table 2 and... Figure 2 Both peptides showed no cytotoxicity at concentrations of 1000ppm, 500ppm, 250ppm, 100ppm, 50ppm, and 10ppm, and TKLDHAVT also promoted cell viability.

[0045] Table 2. Effects of peptides on B16 melanocyte viability

[0046]

[0047] Example 5: Tyrosinase Activity Assay

[0048] (1) B16 cells grown in high-glucose DMEM medium containing 10% FBS and in logarithmic growth phase were subjected to (5-10)×10 4 Cells were seeded per well in 96-well cell culture plates, with 100 μL of culture medium added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0049] (2) Add 10 μL of peptide sample to each well, with 5 replicates for each concentration, and continue incubation. After 24 h of culture, discard the supernatant, wash three times with PBS, add 80 μL of 1% octylphenol polyoxyethylene ether (Triton X-100) solution to each well, and quickly freeze at -81℃ for 30 min. Then thaw at room temperature to completely lyse the cells, preheat at 37℃ for 5 min, and add 20 μL of 1% levodopa solution. React at 37℃ for 2 h, and measure the absorbance at 490 nm using a microplate reader.

[0050] Specific experimental results are shown in Table 3 and... Figure 3 Both peptides inhibited tyrosinase activity at concentrations of 1000 ppm, 100 ppm, and 10 ppm.

[0051] Table 3 Tyrosinase inhibition rate of peptides

[0052]

[0053] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

[0054] sequence list

[0055] <120> Peptides with whitening effects, their preparation methods and applications

[0056] <160> 2

[0057] <170> SIPOSequenceListing 1.0

[0058] <210> 1

[0059] <211> 8

[0060] <212> PRT

[0061] <213> Artificial Sequence

[0062] <400> 1

[0063] Thr Lys Leu Asp His Ala Val Thr 1 5

[0065] <210> 2

[0066] <211> 4

[0067] <212> PRT

[0068] <213> Artificial Sequence

[0069] <400> 2

[0070] Val Pro Gly Phe 1

Claims

1. A polypeptide, wherein, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The application of the polypeptide as described in claim 1 in the preparation of cosmetics with whitening effects.

3. The method for preparing the polypeptide according to claim 1, wherein the method comprises synthesizing the polypeptide using a polypeptide synthesizer according to the amino acid sequence.

4. A polypeptide composition, wherein, The polypeptide composition comprises the polypeptide as described in claim 1.

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