Preparation method and application of whitening peptide with tyrosinase inhibition function

Abstract

The invention discloses a preparation method and application of a whitening peptide with a tyrosinase inhibition function, and relates to the field of active peptide materials. The non-natural whitening peptide disclosed by the invention has the beneficial effects that the non-natural whitening peptide has good light stability and can play a whitening role through a barrier effect of skin, so that excessive generation of melanin caused by ultraviolet radiation is prevented; the whitening peptide is low in toxicity and good in biocompatibility, so that the skin irritation reaction and other side effects cannot be caused, and the whitening peptide can be widely applied to skin protection scenes such as spot treatment and skin color uniformization and has wide application in the aspects of preparation of whitening drugs, tyrosinase inhibitors, cosmetics and the like.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a whitening peptide with tyrosinase inhibition function, belonging to the technical field of active peptide materials. Background Art

[0002] Prolonged exposure to ultraviolet (UV) radiation can cause abnormal tyrosinase activity in melanocytes, leading to skin tanning. Traditional whitening products (such as arbutin, hydroquinone, and kojic acid) may cause side effects such as skin sensitivity and skin cancer. Currently, many natural active peptides extracted from organisms have been shown to inhibit tyrosinase activity and have potential skin whitening effects, but research results on non-natural whitening peptides in skin whitening are still lacking. Peptide chains with excessively large molecular weights will be blocked by the barrier function of the skin and will not be able to penetrate deeply into the skin layer where melanocytes are located to better exert their whitening effects. Therefore, peptide chains that use the inhibition of tyrosinase as a whitening principle need to have a small molecular weight or use a transmembrane effect to achieve skin penetration.

[0003] In light of the limitations of existing technologies and materials, the present invention designed and screened a whitening peptide (P1: CASYIG, sequence from segments C to N) capable of inhibiting tyrosinase through solid-phase synthesis. The P1 peptide was tested for its potential as a skin-whitening agent, including its physicochemical properties and whitening characteristics. The biological whitening performance was tested using a mouse pigmentation model. The experimental results demonstrated that the P1 peptide described herein can be used for localized skin whitening under illumination, with improved patient compliance and no adverse reactions. Summary of the Invention

[0004] The present invention aims to overcome the problems of arbutin being unable to be used during the day and the difficulty of natural tyrosinase inhibitory peptides in penetrating the skin, thereby providing a non-natural whitening peptide with skin-whitening efficacy. The whitening peptide has a low binding energy with tyrosinase, can tightly bind to tyrosinase, and inhibit tyrosinase activity. The whitening peptide can also inhibit the formation of melanin, thereby exhibiting good whitening activity. The peptide has a wide range of uses in the preparation of whitening drugs, tyrosinase inhibitors, and cosmetics.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] 1. Arrangement by using tyrosine and amino acids with antioxidant capacity;

[0007] 2. Screen whitening peptide chains through solid phase synthesis.

[0008] The preparation method of the present invention comprises the following steps:

[0009] A peptide library with TYR inhibitory ability was synthesized by solid-phase synthesis and the peptide chain P1 was screened from it: NH2-Cys-Ala-Ser-Tyr-Ile-Gly-COOH, with a molecular weight of 612.69Da.

[0010] The peptide chain sequence synthesis followed a classic solid-phase synthesis method with some modifications. 2-ChlorotritylChloride Resin beads served as the synthesis support, Pybop / HoBt was used as the amide reaction coupling agent, piperidine was used to remove the Fmoc group, and the reaction endpoint was determined by the colorimetric reaction of ninhydrin with amino acids. Click synthesis was performed step by step according to the sequence. Trifluoroacetic acid was used as a cleavage agent to cleave the peptide from the beads, precipitated with glacial ether, and freeze-dried. Finally, the product was purified by HPLC. The final product was characterized by MALDI-TOF and transmission electron microscopy (TEM).

[0011] Application of whitening peptides

[0012] The whitening peptides of the present invention can be used in various applications requiring skin whitening, such as preventing tanning and even skin tone. Specifically, the active peptides of the present invention can be applied to exposed skin or injected into the skin. Application or administration of the whitening peptides prevents skin darkening caused by prolonged UV exposure without causing side effects.

[0013] 1. In vitro tyrosinase inhibition test

[0014] An indirect method was used to evaluate the inhibition of whitening peptide on tyrosinase, measuring the extent to which the substrate was catalyzed by tyrosinase within a fixed time, and parallel tests were performed using PBS solution as a control.

[0015] 2. Cell melanin production inhibition test

[0016] The melanin production inhibition test was modified based on previous reports. The inhibition level of whitening peptides was evaluated by the amount of melanin produced by cells. The less melanin produced, the stronger the whitening ability.

[0017] 3. Animal Experiments

[0018] The actual whitening effect of whitening peptides was evaluated using a mouse pigmentation model.

[0019] In general, the technical solution described in the present invention has the following beneficial results compared with the existing technology:

[0020] The whitening peptide with whitening effect can overcome the limitations of arbutin usage conditions and inhibit the formation of melanin by binding to tyrosinase, thereby having good whitening activity and having a wide range of uses in the preparation of whitening drugs, tyrosinase inhibitors, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the effect of inhibiting mushroom tyrosinase activity provided in Example 1 of the present invention.

[0022] Figure 2 3D and 2D visualization diagrams of the docking of the whitening peptide with whitening efficacy provided in Example 1 of the present invention with tyrosinase (2Y9X), wherein Figure 2 a is a 3D visualization diagram of the docking of whitening peptide with whitening effect and tyrosinase (2Y9X), Figure 2 b is a 2D visualization diagram of the docking of the whitening peptide with whitening effect and tyrosinase (2Y9X).

[0023] Figure 3 This is a diagram showing the relative active substrate catalytic effect of intracellular tyrosinase provided in Example 2 of the present invention.

[0024] Figure 4 This is a diagram showing the inhibitory effect of cell melanin production provided in Example 2 of the present invention.

[0025] Figure 5 This is a diagram showing the skin penetration effect of the whitening peptide with whitening efficacy provided in Example 3 of the present invention.

[0026] Figure 6 This is an experimental effect diagram of the effect of the whitening peptide with whitening effect on the melanin content of mouse skin provided in Example 3 of the present invention, wherein Figure 6 a is a schematic diagram of the skin changes of mice. Figure 6 b is the melanin distribution diagram of mouse skin sections after the experiment. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to specific embodiments. Unless otherwise specified, the raw materials and equipment used in the present invention are commercially available. The methods described in the following embodiments are conventional methods in the art unless otherwise specified. The details and forms of the technical solutions of the present invention may be modified and replaced without departing from the spirit and scope of the present invention. Such modifications and replacements fall within the scope of protection of the present invention.

[0028] Example 1 Evaluation of Mushroom Tyrosinase Inhibitory Activity of Whitening Peptides with Whitening Efficacy

[0029] 1) Peptide sequence synthesis:

[0030] The peptide sequence was synthesized using the solid-phase Fmoc method. First, 2-ChlorotritylChloride Resin was swollen in DCM for 1-2 hours, followed by the addition of amino acids and DIEA for 1-2 hours. Unreacted sites were then protected by the addition of methanol and DIEA. The Fmoc protecting group was removed using 20% ​​piperidine, and the reaction was verified by a colorimetric reaction between ninhydrin and the amino acid. Subsequently, the amino acid, PyBoP, HoBT, and DIEA were added and reacted for 1-2 hours to remove the Fmoc protecting group. This step was repeated until the last amino acid was attached. Finally, the polypeptide chain was cleaved from the resin beads using a prepared trifluoroacetic acid cleavage agent. The peptide was precipitated in icy ether and washed and purified at least three times. After purification, the solid product was lyophilized. The molecular weight of the obtained product was P1: 612.69 Da.

[0031] 2) Mushroom tyrosinase inhibition test

[0032] L-DOPA was used as the substrate and PBS was selected as the solvent. 80 μL of L-DOPA solution (0.3 mg / ml) was added to 40 μL of peptide solution and 40 μL of tyrosinase solution (50 U / mL). The mixture was incubated at 37°C for 10 min, and then 80 μL of L-DOPA solution (0.3 mg / ml) was added to initiate the reaction. The absorbance at 0 min and 30 min was measured using a microplate reader at a wavelength of 475 nm. The tyrosinase inhibition rate was calculated according to formula (1).

[0033]

[0034] Wherein: A0 is the absorbance of the substrate and PBS system; A1 is the absorbance of the substrate, tyrosinase and PBS system; A is the absorbance of the substrate, tyrosinase and whitening peptide system.

[0035] 3) Molecular docking

[0036] The whitening peptide was molecularly docked with tyrosinase (PDB ID: 2Y9X). The molecular docking process was as follows: the synthetic P1 peptide was constructed using molecular docking software (MOE 2019), and the three-dimensional structure of P1 was energy minimized. Then, the RCSB PDB database (http: / / www.rcsb.org / 2Y9X) was used to screen the protein target and the crystal structure with high resolution was used as the molecular docking receptor. The protein was dehydrated, dephosphorylated, and saved as a PDB file using PyMOL software. The compound was energy minimized using Molecular Operating Environment 2019 software, the target protein was pretreated, and the active pocket was found. Finally, MOE 2019 was run for molecular docking. The binding activity of the two was evaluated based on the binding energy, and the results were visualized using MOE and PyMOL software.

[0037] After molecular docking, lower binding energies indicate more stable binding between the ligand and the tyrosinase active center. The docking results for a whitening peptide (sequence: CASYIG) and tyrosinase (2Y9X) are shown in Table 1. The binding energy for this whitening peptide is -7.3772 kcal / mol, indicating excellent binding to tyrosinase (2Y9X). This theoretically demonstrates that this pearl shell active peptide has the ability to tightly bind to the tyrosinase active center.

[0038] Example 2 Evaluation of the inhibitory effect of whitening peptides on melanin production in cells

[0039] 1) Intracellular tyrosinase activity inhibition test

[0040] B16F10 cells (1×10 5 Cells (100 cells / well) were seeded into 6-well plates and incubated with 1640 medium for 24 hours. Cells were then treated with 1 mL of the peptide solution for 72 hours. After removing the peptide solution, cells were treated with fixative for 15 minutes. After removing the fixative, 1 mL of L-DOPA solution (0.3 mg / mL) was added to initiate the reaction. After incubation at 37°C for 3.5 hours, the L-DOPA solution was removed, and melanin production was observed using a fluorescence microscope.

[0041] B16F10 cell lysate was centrifuged at 12,000 rpm for 10 minutes at 4°C to obtain a supernatant containing tyrosinase. 10 μL of the supernatant was mixed with 10 μL of the peptide solution and incubated at 37°C for 10 minutes. 80 mM L-DOPA solution (0.3 mg / ml) was added to initiate the reaction. The absorbance at 0 and 30 minutes was measured using a microplate reader at a wavelength of 475 nm. The relative activity of intracellular tyrosinase was calculated according to formula (2).

[0042]

[0043] Wherein: A0 is the absorbance of the substrate and PBS system; A1 is the absorbance of the substrate, supernatant and PBS system; A is the absorbance of the substrate, supernatant and whitening peptide.

[0044] 2) Test of the inhibitory effect on cell melanin production

[0045] B16F10 cells (1×10 5 Cells / well were seeded in 6-well plates and incubated with DMEM medium for 24 h. After treatment with 1 mL of peptide solution for 48 h, the peptide solution was removed and the cells were treated with a melanin staining kit and observed under an inverted fluorescence microscope.

[0046] B16F10 cells (1×10 5 Cells (100 cells / well) were seeded in a 6-well plate and incubated with DMEM medium for 24 hours. After treatment with 1 mL of the peptide solution for 48 hours, the peptide solution was removed and 500 μL of 1 M NaOH solution containing 1% DMSO was added for dissolution at 80°C for 1 hour. The absorbance at 0 and 30 minutes was measured using a microplate reader at a wavelength of 475 nm. The relative content of melanin production was calculated according to formula (3).

[0047]

[0048] Where: A0 is the absorbance value of the cell lysate in the blank group; A is the absorbance value of the cell lysate in the whitening peptide-treated group.

[0049] Example 3 Study on the whitening efficacy of whitening peptides based on mouse models

[0050] 4) Penetration model:

[0051] During this study, the mouse ear skin was used as a penetration model to evaluate the penetration ability of P1. C57 mice were randomly divided into an experimental group (30min, 60min, 90min) and a control group. Before the evaluation, the mice were anesthetized, and mouse PI-FITC was applied to the surface of the mouse skin and placed in a dark environment. After a period of time, the mice were killed, and the mouse ear skin was collected and slices were made to observe the penetration effect of P1. We found that as time went on, the fluorescence intensity deep in the skin gradually increased, and after 90 minutes, the mouse ear skin was filled with this FITC-labeled P1. This means that the whitening peptide described in the present invention can penetrate the skin well.

[0052] 5) Pigment deposition model:

[0053] In this study, mice were used as an animal model for pigmentation to evaluate the melanin-inhibiting ability of P1. C57 mice were randomly divided into an experimental group, a positive control group (arbutin), and a blank group (propylene glycol). The backs of the C57 mice were depilated before the experiment. Except for the control group, the ears of the mice were exposed to UVB light (dose of 100mJ / cm 2 UVB irradiation was performed 5 times per week for 2 weeks. The sample prepared with propylene glycol was topically applied 2 hours before irradiation to ensure complete absorption. The distribution of melanin on the back of the mice was observed. After the experiment, the mice were killed and melanin-stained back skin sections were prepared. The mouse ear skin tissue samples were sliced ​​and the tissue sections were observed and photographed using a fluorescence microscope.

Claims

1. A novel active short peptide, characterized by: It has the ability to penetrate the skin and inhibit melanin production. Its sequence is: NH2-Cys-Ala-Ser-Tyr-Ile-Gly-COOH, named P1, with a molecular weight of 612.69 Da.

2. The novel active short peptide according to claim 1, characterized in that Modification, extension, shortening of peptide chains and replacement of amino acids.

3. The novel active short peptide according to claim 1, characterized in that: By competitively binding to tyrosinase, it reduces melanin production and achieves skin whitening.

4. The novel active short peptide according to claim 1, characterized in that It has certain skin penetration ability, low toxicity and good biocompatibility.

5. The novel active short peptide according to claim 1, characterized in that: The tyrosinase inhibition rate was 71% at a concentration of 2 mM.

6. The novel active short peptide according to claim 1, characterized in that: Apply active short peptides to cosmetics with whitening effects.

7. The novel active short peptide according to claim 1, characterized in that: The active short peptide with whitening effect is used in the preparation of a tyrosinase inhibitor that combines with the amino acid residues LYS28, ASP144, ILE148 and LYS218 of tyrosinase through hydrogen bonds.

8. The novel active short peptide according to claim 1, characterized in that: The active short peptide with whitening effect is used in the preparation of a tyrosinase inhibitor that binds to the amino acid residues ARG156 and LYS218 of tyrosinase through electrostatic interaction.

9. The application method of the novel active short peptide according to claim 1, characterized in that: Including whitening spray, facial cream application, and loading in other skin care products and other methods of use.

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