WO2024000088A1 - Pearl-derived whitening polypeptide and use thereof - Google Patents

Abstract

Disclosed in the present invention are a pearl-derived whitening polypeptide and a use thereof. The polypeptide is a polypeptide having an amino acid sequence according to any one of SEQ ID NOs. 1-5. The molecular docking result shows that the polypeptide has relatively strong affinity with tyrosinase, indicating that the polypeptide has a potential tyrosinase inhibition capability, so that melanin generation is inhibited. By means of cell experiments, it is further verified that the polypeptide can remarkably inhibit melanin generation of B16 melanoma cells, so that the pearl-derived polypeptide of the present invention has a potential whitening effect. Moreover, the pearl-derived polypeptide is a natural active peptide, has the advantages of good stability, weak cytotoxicity, and being easy to absorb by the human body, etc., and has large application prospects in preparation of a skin care product having a whitening effect.

Description

A pearl-derived whitening polypeptide and its application Technical field

The invention belongs to the field of biomedicine and relates to a pearl-derived whitening polypeptide and its application.

Background technique

With the improvement of living standards, people pay more attention to personal image and skin care. Whitening is a hot topic in modern life. Many people invest a lot of time and energy, but the results are often unsatisfactory. Tyrosinase (EC 1.14.18.1) is a copper enzyme and a key enzyme in the synthesis of melanin. It catalyzes the hydroxylation of monophenols to o-diphenols and then the oxidation of o-diphenols to o-quinones, ultimately leading to the formation of melanin.

Some whitening ingredients achieve skin whitening effects by inhibiting tyrosinase, such as ascorbic acid, kojic acid, hydroquinone and arbutin, but they also have certain side effects. Ascorbic acid is widely used as a whitening agent, but it loses its efficacy after a rapid redox reaction. Kojic acid, hydroquinone, and arbutin are widely used in cosmetics to achieve skin whitening effects, however, previous studies have reported genotoxic and carcinogenic effects of these compounds. Therefore, it is of great significance to find natural, safe and efficient whitening agents. Pearl is a precious organic gemstone whose main component is CaCO ₃ with a small amount of organic matter and water. At present, there are many documents proving that pearls have whitening effect. At the same time, we have previously proved through experiments that pearl extract can inhibit the activity of tyrosinase, thereby reducing the production of melanin. Protein derivatives, peptides and protein hydrolysates of natural origin are reported to have whitening effects and are used in the treatment of skin diseases. Therefore, peptides isolated from pearl extracts may have potential whitening effects. We identified 1,437 pearl peptides through mass spectrometry. After determining the amino acid sequences of these peptides, we used molecular docking technology to predict peptides with potential affinity activity for tyrosinase, and synthesized them through solid-phase synthesis. The experiment screened out peptides with melanin inhibitory activity, which greatly broadened the application prospects of pearls in whitening.

Contents of the invention

In a first aspect, the present invention provides a whitening polypeptide, which is selected from one or more polypeptides with amino acid sequences such as SEQ ID NO.1-5; the amino terminal sequence of SEQ ID NO.1 is EGGGGFG ; The amino terminal sequence of SEQ ID NO.2 is MHFHEVN; the amino terminal sequence of SEQ ID NO.3 is GGFDGGF; the amino terminal sequence of SEQ ID NO.4 is ASAGGFG; the amino terminal sequence of SEQ ID NO.5 is GSEHRIN.

Taking the pearl-derived whitening polypeptide as the core, any corresponding adjustments or modifications thereto fall within the protection scope of the present invention.

The invention provides a pearl-derived whitening polypeptide, whose amino acid sequence is shown as SEQ ID NO: 1-SEQ ID NO: 5. The molecular docking results show that the polypeptide SEQ ID NO: 1-SEQ ID NO: 5 and Tyrosinase (PDB: 2Y9X) has a strong affinity, indicating that the pearl-derived whitening peptide has potential tyrosinase inhibitory activity.

In a second aspect, the present invention provides a method for preparing the aforementioned whitening polypeptide, which method includes using a polypeptide synthesizer to synthesize the target polypeptide according to the amino acid sequence; in some specific embodiments, using a polypeptide solid-phase synthesis reactor to synthesize the target polypeptide.

In the third aspect, the present invention uses the aforementioned whitening polypeptides to prepare cosmetics or pharmaceuticals with melanin inhibitory activity; in some embodiments, the aforementioned polypeptides are added to the cell culture medium, and the cells after the polypeptide treatment are measured. The melanin content of B16 melanoma cells showed that the polypeptides showed significant ability to inhibit melanin production, indicating that the polypeptides SEQ ID NO. 1-5 provided by the invention have potential whitening effects.

Existing polypeptides on the market have all undergone melanin-promoting pretreatment with B16 cells in the melanin-inhibiting activity test. After being tested using the method of this embodiment, they have almost no melanin-inhibiting activity; in the embodiment of the present invention, the B16 provided The cells are cells that are in a natural state and have not been subjected to melanin-promoting tests. They can better reflect the true melanin inhibitory activity of the active peptide composition and more truly reflect the melanin inhibitory activity of the highly active polypeptide mixture.

In some specific embodiments, the cosmetics or pharmaceuticals include cosmetics or pharmaceuticals that have the effect of whitening or treating or preventing melanin pigmentation diseases; specifically, the hypermelanin pigmentation diseases include but are not limited to chloasma, freckles, and age spots. , spots, café-au-lait spots, Baker’s nevus, nevus, Mongolian spot, nevus of Ohta, acquired bilateral nevus of Ohta, Ito nevus, blue nevus, junctional nevus, mixed nevus, intradermal nevus, halo nevus, congenital melanocytic nevus Moles, Spitz nevus, dysplastic nevus, sun spots.

In the fourth aspect, the present invention provides a method for preparing a whitening polypeptide. The whitening polypeptide is one of the polypeptides with an amino acid sequence as shown in SEQ ID NO. 1-5. The preparation method includes using polypeptide synthesis according to the amino acid sequence. instrument synthesis.

In some specific embodiments of the present invention, a polypeptide mixture is obtained by extracting pearls, and molecular docking is performed through Discovery Sdudio 2019 software to screen out pearl polypeptides with potential affinity for tyrosinase (PDB: 2Y9X). On this basis, the inventors took the polypeptide with strong affinity to tyrosinase in the molecular docking results as the target polypeptide, and chemically synthesized it through solid-phase synthesis. Finally, the synthesized peptide was added to the cell culture medium, the melanin content of B16 melanoma cells was measured, and highly active whitening peptides SEQ ID NO. 1-5 were screened out.

The polypeptide provided by the invention is a pearl polypeptide. As a natural active peptide, it has the advantages of good stability, weak cytotoxicity, and easy absorption by the human body. It has great application prospects in the preparation of skin care products with whitening effect.

Description of drawings

Figure 1 3D structure diagram of tyrosinase (PDB: 2Y9X).

Figure 2 shows the melanin content of B16 melanoma cells after treatment with pearl-derived whitening peptide SEQ ID NO.1-5.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific implementation examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

Table 1 is a comparison table of amino acid name definitions cited in the present invention.

Glutamic acid E(Glu) Glycine G(Gly) Phenylalanine F(Phe) Methionine M(Met) Histidine H(His) valine V(Val) asparagine N(Asn) asparagine D(Asp) Alanine A(Ala) Serine S(Ser) Arginine R(Arg) Isoleucine I(Ile)

Pearl-derived whitening peptides EGGGGFG, MHFHEVN, GGFDGGF, ASAGGFG and GSEHRIN were obtained through solid-phase synthesis with a purity greater than 95%.

Example 1 Molecular docking

Preprocessing of target protein: Download the PDB format file of tyrosinase protein (PDB: 2Y9X) in the PDB database (https://www.pdbus.org/), then open the file with Discovery Sdudio 2019 software, and click on " Click "Prepare Protein" in Macromolecules" to perform protein preprocessing. The new protein obtained after preprocessing has a total of 66 sites.

Preparation of peptide ligands: Use Chemdraw 19.0 software to draw the molecular structure of the peptide, save it in "sdf" format, then use Discovery Sdudio 2019 software to open the peptide file, click "Prepare Ligands" in "Small Molecules", and proceed with it preprocessing.

Molecular docking: According to experience, the polypeptide has a higher possibility of docking with sites 1-10 of the tyrosinase protein. Therefore, sites 1-10 of the tyrosinase were selected as docking sites, respectively with the polypeptide. Perform rigid docking (LibDock), and record the number of Pose docked at each site and the highest rigid docking score (LibDockScore). The results are shown in Table 1.

Table 1 Molecular docking results of pearl-derived whitening peptide SEQ ID NO.1-5 and tyrosinase

As can be seen from Table 1, the pearl-derived whitening peptide SEQ ID NO.1-5 can be successfully docked with multiple sites of tyrosinase, and has a high pose number, especially EGGGGFG and ASAGGFG, positions 1-10 The cumulative number of poses reached 751 and 835 respectively. In addition, MHFHEVN, GGFDGGF and GSEHRIN have higher docking scores, with the highest values being 230.206, 216.46 and 224.105 respectively. The above results show that the pearl polypeptide has a strong affinity with tyrosinase, suggesting that it has potential whitening effect.

Example 2 Solid-phase synthesis of polypeptides (taking SEQ ID NO. 2MHFHEVN as an example)

Step 1: Weigh 0.3mmol MBHA amino resin into the reaction tube, swell the resin with dichloromethane (DCM) for 40 minutes, clean the resin with N,N-dimethylformamide (DMF) and drain it, and clean it 5 times in total.

Step 2: Use 20% piperidine (dissolved in DMF) to remove the Fmoc protecting group for 3 minutes, drain it, wash with DMF and drain it, use 20% piperidine again to remove the Fmoc protecting group for 7 minutes, drain it, and clean the resin with DMF Then drain it and wash it 5 times in total.

Step 3: Add 0.9mmol Fmoc-Asn-OH, 0.85mmol HBTU, 0.85mmol HOBT and 1.8mmol Dipea to the reaction tube, react at room temperature for 45 minutes, drain, wash the resin with DMF and drain, wash 5 times in total.

Step 4: Put a small amount of resin into a test tube, wash it twice with DMF and MeOH, add about 0.3 mL of 2% ninhydrin indicator, and observe the color change of the resin after heating for 5 minutes. If no blue color appears, the reaction is complete. Blue indicates that the reaction is incomplete and needs to continue.

Step 5: Replace Fmoc-Asn-OH in step 3 with Fmoc-Val-OH, Fmoc-Glu-OH, Fmoc-His-OH, Fmoc-Phe-OH, Fmoc-His-OH, Fmoc-Met- OH, and repeat steps 2-4 until the amino acids on the polypeptide sequence are completely connected.

Step 6: After deprotecting the last connected amino acid Fmoc-Met-OH, wash the resin 5 times with DMF and then 5 times with DCM.

Step 7: Add 5 mL of TFA:H ₂ O:TIPS (95:2.5:2.5) to the reaction tube, react at room temperature for 2 hours, filter, rinse with a small amount of TFA and collect the liquid into a centrifuge tube. Slowly add diethyl ether dropwise to the liquid. During this period, a light yellow solid will be generated. Place the centrifuge tube in a -20°C refrigerator for 2 hours, centrifuge and discard the supernatant. Add diethyl ether again, centrifuge and discard the supernatant.

Step 8: Dry the obtained solid in a fume hood, then dissolve it in water and pass it through a 0.22μm PES filter membrane, and purify it with SemiPrep RP-HPLC. The mobile phase is water containing 0.1% TFA (Buffer A) and water containing 0.1% TFA. TFA's acetonitrile (Buffer B), the detection wavelength is set to 214nm. The collected peptides were characterized and confirmed using Analytical RP-HPLC and MALDI-TOF-MS, and then freeze-dried with a freeze dryer to obtain the target peptide SEQ ID NO. 2 MHFHEVN.

SEQ ID NO.1: EGGGGFG; SEQ ID NO.3: GGFDGGF; SEQ ID NO.4: ASAGGFG;

SEQ ID NO.5: GSEHRIN is also synthesized using a similar method.

Example 3 Melanin Inhibition Experiment

B16 melanoma cells grown in DMEM complete medium and in the logarithmic growth phase were seeded into 100mm cell culture dishes at a density of (10-15) × 10 ⁴ cells/mL, and 10 mL of complete culture medium was added to each culture dish. , placed in a carbon dioxide incubator at 37°C and 5% CO ₂ concentration. After 24 hours, add polypeptide solution (dissolved in PBS) to the culture dish. The final concentration of the polypeptide is 100mg/L (100ppm). Add an equal amount of PBS solution to the control group, and then return it to the incubator to continue culturing. After 24 hours, the cells were digested with trypsin and centrifuged to collect the cells. The supernatant was discarded and washed twice with PBS. 300 μL of 1 M NaOH containing 10% DMSO was added and placed in an 80°C water bath to lyse the cells. After 30 minutes, take 100 μL of cell lysate into a 96-well plate, read the absorbance at 405 nm with a microplate reader, and finally convert the absorbance into melanin content.

As shown in Figure 2, at a concentration of 100mg/L, pearl-derived whitening peptide SEQ ID NO.1-5 can significantly reduce melanin content (P<0.01), reducing it by 27.03%, 32.43%, and 31.23% respectively. , 26.72% and 22.02%, indicating that the polypeptides have strong whitening effects.

Claims (7)

A whitening polypeptide, characterized in that the polypeptide is selected from one or more polypeptides with amino acid sequences such as SEQ ID NO. 1-5. The preparation method of whitening polypeptide according to claim 1, characterized in that the preparation method includes synthesizing using a polypeptide synthesizer according to the amino acid sequence. The method for preparing a whitening polypeptide according to claim 1, characterized in that a polypeptide solid-phase synthesis reactor is used to synthesize the target polypeptide. Application of the whitening polypeptide according to claim 1 in the preparation of cosmetics or pharmaceuticals with melanin inhibitory activity. Application of the whitening polypeptide as claimed in claim 1 in the preparation of cosmetics or pharmaceuticals with the effect of whitening or treating or preventing melanin pigmentation diseases. Use as claimed in claim 4, characterized in that the hypermelanosis disease is selected from the group consisting of chloasma, freckles, age spots, freckles, café-au-lait spots, Baker’s nevus, nevus, Mongolian spots, nevus of Ohta, acquired bilateral Ota nevus, Ito nevus, blue nevus, junctional nevus, mixed nevus, intradermal nevus, halo nevus, congenital melanocytic nevus, Spitz nevus, dysplastic nevus, sun spots. A method for preparing a whitening polypeptide, characterized in that the whitening polypeptide is one of the polypeptides with an amino acid sequence as shown in SEQ ID NO. 1-5. The preparation method includes synthesizing using a polypeptide synthesizer according to the amino acid sequence.

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