A polypeptide and its use in improving pigmentation
Patent Information
- Application Number
- CN202610853453.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-12
- Publication Date
- 2026-08-21
AI Technical Summary
（1）本发明所述多肽SFPYHY能够显著抑制细胞中酪氨酸酶活性，且抑制能力随着浓度的升高而增加，对细胞中酪氨酸酶的抑制作用与熊果苷相差不大，且经过细胞活力测定证明了多肽SFPYHY对细胞无毒性。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of polypeptide technology, specifically to a polypeptide and its application in improving pigmentation. Background Technology
[0002] Besides the skin that is frequently exposed to sunlight, which is prone to darkening, localized pigmentation abnormalities can also cause problems, especially for women. Women pay great attention to their appearance and skin tone, and melasma has the highest incidence rate among women. It is a very typical skin pigmentation disorder caused by ultraviolet radiation. Generally, hormonal imbalances in women and the use of birth control pills can lead to melasma. Melasma on women's skin usually appears as yellowish-brown or brown patches, which affects a woman's visual appearance in the eyes of others.
[0003] Hirudo medicinalis ( Poecilobdella manillensis Lesson The golden-edged leech (Hirudo medicinalis), also known as the Mani medicinal leech, belongs to the phylum Annelida, class Hirudinea, order Hirudoformes, family Hirudinidae, and genus Hirudo. It is one of the larger species among common blood-sucking leeches. my country has abundant resources of Hirudo medicinalis, mainly distributed in Guangxi, Guangdong, Fujian, Hainan, and Hong Kong in southern my country. Abroad, it is mostly distributed in Southeast Asian countries such as the Philippines, Thailand, and Vietnam. It has the effects of breaking up blood stasis and promoting menstruation, and can be used to treat amenorrhea due to blood stasis, sprains, and other ailments. The pharmacological effects of Hirudo medicinalis are basically the same as those of leeches, and it also contains various active ingredients such as hirudin and hirudin. Hirudo medicinalis has anticoagulant effects, and its antithrombotic and thrombolytic effects are very significant, even better than those of leeches. Summary of the Invention
[0004] To address or partially address the problems existing in the related technologies, one of the objectives of this invention is to provide a polypeptide whose amino acid sequence is shown in SEQ ID NO:1, specifically the amino acid sequence Ser-Phe-Pro-Tyr-His-Tyr (SFPYHY).
[0005] A second objective of this invention is to provide a method for preparing the aforementioned polypeptide, wherein the polypeptide is obtained by hydrolyzing leeches with an alkaline protease, and the specific steps are as follows: (1) The freeze-dried leech powder was reconstituted in a phosphate buffer (PBS) system with a pH of 8.6, filtered, enzymatically digested with alkaline protease and heat-inactivated, centrifuged and ultrafiltered to obtain the target component.
[0006] (2) The target component obtained in step (1) is placed in a low temperature environment for freeze drying to obtain a dry powder.
[0007] (3) The dry powder obtained in step (2) was analyzed in depth using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to identify its peptide sequence characteristics and resolve its molecular structure information.
[0008] A third objective of this invention is to provide applications of the polypeptide in the following two aspects: (1) The application of the polypeptide in the preparation of daily chemical products that help improve pigmentation.
[0009] (2) Application of the polypeptide in the preparation of health products that help improve melasma.
[0010] The beneficial effects of this invention are: (1) The polypeptide SFPYHY described in this invention can significantly inhibit the activity of tyrosinase in cells, and the inhibitory ability increases with increasing concentration. Its inhibitory effect on tyrosinase in cells is not much different from that of arbutin. Furthermore, cell viability assays have proven that the polypeptide SFPYHY is non-toxic to cells.
[0011] (2) The polypeptide SFPYHY described in this invention can significantly inhibit the activation of melanocyte-stimulating hormone melanocorticoids on melanin synthesis-related factor proteins TYR, MITF, TRP-1 and DCT, and reduce the melanin content in cells. This discovery not only provides a highly promising innovative raw material for the development of new whitening agents, but also significantly enhances the economic added value of the entire Hirudo medicinalis industry chain. Attached Figure Description
[0012] Figure 1 This is a graph showing the cell proliferation activity assay results of the polypeptide SFPYHY in Example 5 of this invention.
[0013] Figure 2 This is a graph showing the experimental results of cellular tyrosinase activity of the polypeptide SFPYHY in Example 6 of this invention.
[0014] Figure 3 This is a diagram showing the experimental results of cellular melanin synthesis of polypeptide SFPYHY in Example 7 of this invention.
[0015] Figure 4 This refers to the determination of the effect of polypeptide SFPYHY on the expression of melanin synthesis-related factor proteins in Example 8 of the present invention. Figure 5 This is a quantitative diagram of the expression of melanin synthesis-related factor proteins by the polypeptide SFPYHY in Example 8 of the present invention. Detailed Implementation
[0016] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described therein; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and all raw materials used can be purchased through conventional commercial channels.
[0017] Example 1 Preparation of polypeptides from Hirudo medicinalis The leech sample used in this embodiment was provided by Huazhi Pharmaceutical Co., Ltd., with product batch number HZFYZ-20240315. The leech sample is a lyophilized powder (300 mesh). The specific steps are as follows: (1) Using the degree of hydrolysis (DH) as the core evaluation index, the kinetic efficiency of the enzymatic hydrolysis reaction was systematically evaluated: the enzymatic hydrolysis effects of alkaline protease, flavor protease, pepsin, lipase, trypsin, neutral protease and bromelain on the freeze-dried powder of Hirudo medicinalis were compared. The results showed that the enzymatic hydrolysis effect of alkaline protease was the best. The solid-liquid ratio of alkaline protease to freeze-dried Hirudo medicinalis powder was then optimized to 1:5, the hydrolysis time was 4h, the temperature was 55℃ and the enzyme amount was 3000U / g.
[0018] (2) The lyophilized leech powder was reconstituted in a phosphate buffer (PBS) system with a pH of 8.6 and a solid-liquid ratio of 1:1. After homogenization, the mixture was initially filtered through a dialysis membrane to collect the clear filtrate. 8% alkaline protease was added to the filtrate and enzymatically hydrolyzed at 55°C for 4 h. After the hydrolysate was heat-inactivated at 55°C for 30 min, impurities were removed by centrifugation. The supernatant obtained was then passed through 10 kDa and 3 kDa ultrafiltration membranes in sequence using an organic membrane separator to obtain three polypeptide fractions: FNZ-8 (>10 kDa), FNZ-7 (3-10 kDa) and FNZ-6 (0-3 kDa).
[0019] (3) The obtained components were freeze-dried and the activity of the three polypeptide components obtained in step (2) was determined by MTT colorimetric method. The results showed that the FNZ-6 (0-3kDa) component had good proliferative activity. Therefore, the FNZ-6 (0-3kDa) component was selected for the next experiment.
[0020] (4) The components of FNZ-6 (0-3kDa) were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS): The chromatographic separation conditions are as follows: ①Chromatographic column system: Analytical column: RP-C18, 0.15mm × 150mm (column Technology Inc., Fremont, CA, USA), equilibrated with 95% A phase.
[0021] Peptide trap column: Zorbax 300SB-C18 (Agilent Technologie, Wilmington, DE, USA).
[0022] ② Column temperature: 30℃.
[0023] ③Mobile phase: Phase A is an aqueous solution of formic acid (0.1% formic acid), and Phase B is an aqueous solution of formic acid and acetonitrile (0.1% formic acid, containing 84% acetonitrile).
[0024] ④ The sample was injected into the Zorbax 300SB-C18 peptide trap using an autosampler and separated on a liquid chromatography column. The elution gradient and flow rate are shown in Table 1. Table 1 Elution gradient and flow rate of mobile phase in liquid chromatography Mass spectrometry conditions are: Instrument model: Q-Exactive mass spectrometer (Thermo Fisher, Boston, MA, USA).
[0025] Ion source: positive ion mode.
[0026] Analysis time: 60 minutes.
[0027] Mass spectrometry data were analyzed using MaxQuant 1.5.5.1. The UniProt database search settings were as follows: MS / MS tolerance was 0.2 Da, with a maximum of two missing fragments, and methionine oxidation was modified as a variable. The confidence level for positive protein identification was determined based on the high protein and peptide portions in the search results.
[0028] The Byonic database was used for searching the target protein in the database uniprotkb_taxonomy_Poecilobdellamanillensis. De novo sequencing was performed to analyze the components of the identified peptide fragments. Peptide fragments meeting the "Score>60" criterion were screened from the identified peptides. The bioinformatics tool BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) was then used for bioactivity prediction, retaining peptides with tyrosinase-inhibiting biological activity. The bioactivity prediction was combined with peptide confidence scores, water solubility, and low molecular weight analysis to select a peptide with a high score, good water solubility, and activity related to improving pigmentation. The results are shown in Table 2.
[0029] Table 2 shows peptides with potential biological activity after screening for "Score > 60". The polypeptide SFPYHY has multiple activities related to improving pigmentation, mainly antioxidant and tyrosinase inhibitory activities. Its key active sites are "PY" and "FPY", and it has the potential to promote wound healing.
[0030] Example 2 The specific steps for predicting the absorption, distribution, metabolism, excretion, and toxicity of the peptide SFPYHY (ADMET) are as follows: First, the amino acid sequence of peptide SFPYHY shown in Table 2 was converted into a simplified molecular input line input canonical system (SMILES). AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar2) was used to predict the ADMET properties of the screened peptides. Peptides with excellent water solubility, extremely low risk of drug interactions, long half-life, low blood-brain barrier penetration, and low (or non-toxic) toxicity were further analyzed for molecular docking. The screening results are shown in Table 3. Peptide SFPYHY has good solubility, extremely low risk of drug interactions, high local safety, and no central toxicity.
[0031] Table 3 shows the ADME characteristic prediction of the screened peptides using SMILES codes. Example 3 The peptide SFPYHY was molecularly docked with TYR (tyrosinase PPO3) and MITF (melanocyte-inducible transcription factor). The specific steps are as follows: The 3D structure of the peptide was constructed using Chem Draw 20.0 and energy minimization was performed, and the structure was converted to "mol2" format. The structures of TYR and MITF were obtained from the RCSB protein database (http: / / www.rcsb.org), with PDBIDs of 2Y9X and 4ATI, respectively. The receptor proteins (TYR and MITF) were dehydrated, impurity removed, and hydrogenated using PyMol 3.0.4 and AutoDockTools 1.5.7, and then exported as files as receptors. Molecular docking was performed using AutoDock Vina. All results were visualized and analyzed using PyMol and LigPlot. The optimal binding conformation of the peptide with TYR and MITF was predicted based on the binding energy score, and the interaction between the peptide and the active sites of TYR and MITF was constructed. The results are shown in Table 4. SFPYHY established 6 hydrogen bond interactions with TYR, namely LYS-5 (2.2Å), GLN-74 (2.5Å), LEU-75 (2.0Å), HIS-76 (2.8Å), GLYS-5 (2.2Å), GLN-74 (2.5Å), LEU-75 (2.0Å), HIS-76 (2.8Å), GLYS-74 (2.5Å), GLN-75 (2.0 ... GLN-75 (2.8 -326 (2.8 Å), ASN-57 (2.2 Å), the binding energy of SFPYHY with TYR is -9.4 kcal / mol; SFPYHY establishes four hydrogen bond interactions with MITF, namely DA-12 (2.5 Å), DG-4 (2.5 Å), DC-14 (2.6 Å), DT-16 (2.2 Å) hydrogen bonds, the binding energy of SFPYHY with TYR is -8.4 kcal / mol, which indicates that the two molecules are relatively stable and have good binding activity.
[0032] Table 4. Interactions between peptide SFPYHY and TYR, MITF Example 4 The peptide SFPYHY was synthesized via solid-phase synthesis, and the specific steps are as follows: (1) Resin swelling: 2-Chlorotrityl Chloride Resin (2-chlorotriphenylmethyl chloride resin) was placed in a reaction tube, and DMF (N,N-dimethylformamide, CAS: 7087-68-5) was added at a rate of 15 mL / g. The mixture was shaken for 60 min.
[0033] (2) Add the first amino acid: filter the solvent through a sand core, add 3 times the molar excess of Fmoc-protected serine (the first amino acid at the C-terminus), then add 10 times the molar equivalent of DIEA (N,N-diisopropylethylamine, CAS: 7087-68-5), and finally add DMF to dissolve and shake for 30 min; add methanol to carry out the end-capping reaction and shake for 30 min.
[0034] (3) Deprotection: DMF was removed by filtration through a sand core, and a DMF solution containing 20% piperidine was added at a rate of 15 mL / g. The reaction was carried out for 5 min. After filtration again, a DMF solution containing 20% piperidine was added at a rate of 15 mL / g. The reaction was carried out for 15 min.
[0035] (4) Deprotection detection: Filter out the DMF solution containing 20% piperidine, take 15 resin grains, wash three times with ethanol, add 2 drops of Kaiser reagent (ninhydrin colorimetric reagent), heat at 108℃ for 5 min, and the dark blue color indicates a positive reaction, indicating that deprotection is complete.
[0036] (5) After the test is completed, wash twice with DMF (10mL / g), twice with methanol (10mL / g), and twice with DMF (10mL / g).
[0037] (6) Condensation: Add 3 times molar excess of Fmoc to protect amino acids, 3 times molar excess of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate, CAS: 94790-37-1), then add 10 times molar excess of DIEA, and finally add DMF to dissolve and shake for 45 min.
[0038] (7) Detection of condensation reaction: Take 15 resin grains, wash three times with ethanol, add 2 drops of Kaiser reagent, heat at 108℃ for 5 min, and the colorless reaction is a negative reaction, indicating that all free amino groups have been successfully condensed with amino acids.
[0039] (8) After the test is completed, wash once with DMF (10mL / g), twice with methanol (10mL / g), and twice with DMF (10mL / g).
[0040] (9) Repeat steps (3)-(9) and connect them sequentially from left to right according to the sequence phenylalanine-proline-tyrosine-histidine-tyrosine until the last amino acid Fmoc protecting group is removed.
[0041] (10) Wash the resin as follows: After the solvent is removed, wash it with DMF (10 mL / g) twice, DCM (10 mL / g) three times, and methanol (10 mL / g) four times in sequence, and then remove it for 10 min.
[0042] (11) Cutting: Prepare the cutting solution, in which the volume percentage of TFA is 95%; the volume percentage of water is 2%; the volume percentage of EDT (1,2-ethylenedithiol, CAS: 540-63-6) is 2%; the volume percentage of TIS (triisopropylsilane, CAS: 6485-79-6) is 1%. The amount of cutting solution used is 10 mL / g. Cut for 180 min to cut the complete peptide chain from the resin.
[0043] (12) Drying and washing: Filter to remove resin, blow the filtrate dry with nitrogen as much as possible, pour the filtrate containing the product into ether to precipitate, centrifuge to remove supernatant, wash the precipitate with ether six times, and then evaporate to dryness at room temperature to obtain crude product.
[0044] (13) Purification: A new process was developed to purify the crude product to 98% using high performance liquid chromatography.
[0045] (14) The purified liquid sample was placed in a freeze-drying system for concentration treatment, and finally a white powder product was obtained. Then, the prepared peptide was reconstituted with sterile distilled water to prepare a synthetic peptide SFPYHY solution with a concentration of 10 mg / mL.
[0046] Example 5 The viability assay of the synthetic peptide SFPYHY on B16F10 cells (mouse skin melanoma cells) was performed using the following steps: (1) When the coverage of B16F10 cells in the culture dish reaches about 70%, wash the cells with 2 mL of phosphate-buffered saline (PBS) to remove serum and dead cell residue; then, add 1 mL of trypsin for digestion and dissociation, remove the trypsin after digestion for 30 seconds, and repeatedly pipette with 2 mL of complete culture medium to ensure that the cells detach from the bottle wall and form a uniform single-cell suspension. Prepare a uniform cell suspension and count the cells.
[0047] (2) Cell plating and culture: based on 5 × 10⁵ cells per well 3 The seeding density of cells was determined, the total volume was accurately calculated, and 90 μL of cell suspension was evenly dispensed into each well of a 96-well plate and cultured in a cell culture incubator for 24 hours.
[0048] (3) After culturing the cells for 24 hours, SFPYHY solution with concentrations of 125, 250 and 500 µg / mL was added to different cell wells. Three replicates were set for each concentration. The cells were cultured for another 24 hours to allow the synthetic peptide SFPYHY to act. The wells without SFPYHY solution were used as the control group (Ctrl).
[0049] (4) Cell fixation: After 24 hours of treatment, 5 mg / mL of freshly prepared MTT solution (thiazolyl blue solution, CAS: 298-93-1) was added to each well, and the reaction was carried out in a constant temperature incubator in the dark for 4 hours. After the reaction was terminated, the supernatant was removed, and 100 μL of dimethyl sulfoxide (DMSO) was introduced to dissolve the formed formazan crystals.
[0050] (5) Mix well by shaking at 37℃ for 10 min, and use an ELISA reader to detect the OD value of each well at a wavelength of 570 nm and record it in detail; calculate the average OD value for each concentration. To ensure accurate and stable experimental results, each cell proliferation experiment should be repeated at least 3 times, and 3 replicate wells should be set for each concentration each time; the calculation results are as follows. Figure 1As shown, the synthetic peptide SFPYHY at different concentrations was not toxic to B16F10 cells.
[0051] Example 6 The effects of the synthetic peptide SFPYHY on tyrosinase activity in B16F10 cells were investigated using the following steps: (1) Culture melanoma cells (B16F10 cells) in a culture dish. When the coverage of B16F10 cells in the culture dish reaches about 70%, wash the cells with 2 mL of phosphate-buffered saline (PBS) to remove serum and dead cell residue. Then, add 1 mL of trypsin for digestion and dissociation. After digestion for 30 seconds, remove the trypsin and add 2 mL of complete culture medium to repeatedly pipette to ensure that the cells detach from the bottle wall and form a uniform single-cell suspension. Prepare a uniform cell suspension and count the cells.
[0052] (2) Cell plating and culture: based on 5 × 10⁵ cells per well 3 The seeding density of cells was determined, the total volume was accurately calculated, and 90 μL of cell suspension was evenly dispensed into each well of a 96-well plate and cultured in a cell culture incubator for 24 hours.
[0053] (3) Add 200 nM of alpha-corticosteroids to 15 wells, add 500 μg / mL of arbutin to 3 wells, and add 125, 250 and 500 µg / mL of SFPYHY solution to 9 wells respectively. Perform 3 replicates for each group. Use the wells without alpha-corticosteroids, arbutin and SFPYHY solution as blank controls and continue culturing.
[0054] (4) Then add 100 μL of Triton X-100 solution (polyethylene glycol tert-octylphenyl ether) with a volume concentration of 10% to each well, place the 96-well plate in a -80℃ freezer for 30 min and then take it out.
[0055] (5) After thawing, add 10 μL of levodopa solution (CAS: 59-92-7) to each well, incubate at 37℃ in the dark for 2 h, and finally detect the absorbance at 475 nm and calculate the inhibitory effect of the sample on tyrosinase in B16F10 cells. The above experiment should be repeated at least 3 times; the results are as follows. Figure 2 The synthetic peptide SFPYHY shown inhibited tyrosinase activity in B16F10 cells.
[0056] Example 7 The effect of the synthetic peptide SFPYHY on melanin content in B16F10 cells was investigated through the following steps: (1) Culture melanoma cells in a culture dish. When the coverage of B16F10 cells in the culture dish reaches about 70%, wash the cells with 2 mL of phosphate-buffered saline (PBS) to remove serum and dead cell residue. Then, add 1 mL of trypsin for digestion and dissociation. After digestion for 30 seconds, remove the trypsin and add 2 mL of complete culture medium to repeatedly pipette to ensure that the cells detach from the bottle wall and form a uniform single-cell suspension. Prepare a uniform cell suspension and count the cells.
[0057] (2) Cell plating and culture: based on 5 × 10⁵ cells per well 3 Based on the seeding density of individual cells, the total volume was calculated, and 90 μL of cell suspension was evenly dispensed into each well of a 96-well plate and cultured in a cell culture incubator for 24 h.
[0058] (3) Add 200 nM of alpha-corticosteroids to 15 wells, add 500 μg / mL of arbutin to 3 wells, and add 125, 250 and 500 µg / mL of SFPYHY solution to 9 wells respectively. Perform 3 replicates for each group. Use the wells without alpha-corticosteroids, arbutin and SFPYHY solution as blank controls and continue culturing.
[0059] (4) Then add 1 mL of 1 mol / L NaOH solution to each well (the NaOH solution contains 10% DMSO by volume) and incubate the 96-well plate in an 80°C water bath for 60 min.
[0060] (5) After the cells are completely dissolved and broken, the absorbance is measured at a wavelength of 405 nm and the effect of the sample on the melanin content in the cells is calculated. The above experiment is repeated at least 3 times; the results are as follows. Figure 3 The synthetic peptide SFPYHY shown reduces melanin content in B16F10 cells.
[0061] Example 8 The effects of the synthetic peptide SFPYHY on melanin synthesis-related factor proteins were investigated through the following steps: (1) Culture B16F10 cells in culture dishes. When the coverage of B16F10 cells in the culture dish reaches about 70%, wash the cells with 2 mL of phosphate-buffered saline (PBS) to remove serum and dead cell residue. Then, add 1 mL of trypsin for digestion and dissociation. After digestion for 30 seconds, remove the trypsin and repeatedly pipette with 2 mL of complete culture medium to ensure that the cells detach from the flask wall and form a homogeneous single-cell suspension. Prepare a homogeneous cell suspension and count the cells. After counting, adjust the cell concentration to 3 × 10⁻⁶ cells / mL. 6Cells were seeded at 1.8 mL / well in a 6-well plate and cultured for 24 h. Then, 200 nmol / L of arbutin was added to three wells, 500 μg / mL of arbutin was added to one well containing arbutin, 500 µg / mL of SFPYHY solution was added to another well containing arbutin, and the remaining well containing arbutin served as a negative control. Cells without arbutin, arbutin, and SFPYHY solution served as a blank control. The cells were cultured for another 24 h.
[0062] (2) After culturing for 24 hours, the 6-well plate was transferred to the operating table, the culture medium was discarded, and the plate was washed 3 times with PBS. The plate was placed in an ice box, and 350 µL of protein lysis buffer containing mercaptoethanol was added to each well. The plate was repeatedly pipetted with a 1 mL pipette to remove the protein from the wall. The plate was then placed on ice for 10 min to lyse. The lysis buffer was transferred to a 1.5 mL centrifuge tube and sonicated until the liquid was no longer viscous (3 min each time). The protein was then heated in a 98 °C metal bath for 10 min to denature it. The plate was centrifuged at 14000 rpm and 4 °C for 10 min. The supernatant was aliquoted and stored in a refrigerator.
[0063] (3) Prepare an 8% concentration separating gel. Quickly pipette the prepared gel solution into the gap of the installed glass plate. Then add 1 mL of isopropanol to remove the air bubbles on the top of the separating gel and keep the separating gel in a straight line. Wait 2 hours until the separating gel solidifies. Pour out the layered isopropanol. Then pipette the pre-prepared 4% concentration stacking gel into the gap of the glass plate and quickly insert the comb into the stacking gel. Wait for the stacking gel to solidify.
[0064] (4) Sample loading and electrophoresis: After the gel has completely solidified, fix the SDS-PAGE gel and glass clamp, put them into the electrophoresis tank, pour in 1× electrophoresis solution, remove the comb, and add the protein and marker to the corresponding wells in the order of sample loading. Then concentrate the protein into a straight line at a fixed voltage of 60V. When the protein enters the separation gel, change the voltage to a constant voltage of 130V to separate the protein in the separation gel. Stop electrophoresis when the sample is 1cm away from the bottom of the glass plate.
[0065] (5) Gel cutting and transfer: Activate the 0.45µm PVDF membrane by soaking it in anhydrous methanol for 5 min; then attach the membrane tightly to the SDS-PAGE gel and place it in the transfer device; set a constant current of 250mA and transfer the membrane for 1.5 h to transfer the protein from the gel to the membrane.
[0066] (6) Blocking and antibody incubation: After the transfer was completed, the electrotransfer PVDF membrane was placed in the prepared 5% skim milk powder and blocked at room temperature for 2 hours. After blocking, the band was cut according to the molecular weight of the target band and placed in the prepared primary antibody dilution solution and incubated overnight at 4°C. The primary antibodies were: MIT (1:3000; Wuhan Sanying Biotechnology Co., Ltd.), TYR (1:3000; Wuhan Sanying Biotechnology Co., Ltd.), TRP-1 (1:3000; Wuhan Sanying Biotechnology Co., Ltd.), DCT (1:3000; Wuhan Sanying Biotechnology Co., Ltd.), and GAPDH (1:10000; Wuhan Sanying Biotechnology Co., Ltd.).
[0067] (7) Washing and secondary antibody incubation: After primary antibody incubation, the bands were placed in 1×TBST washing buffer (Tris buffered saline containing Tween 20) and washed 5 times, 5 min each time; after incubation with dilution containing secondary antibody at room temperature for 1.5 h, the bands were washed 5 times with 1×TBST, 5 min each time. The secondary antibodies were HRP-labeled anti-rabbit (1:10000; Wuhan Sanying Biotechnology Co., Ltd.) or anti-mouse (1:10000; Wuhan Sanying Biotechnology Co., Ltd.).
[0068] (8) Development: Prepare the developer solution using Factek ultrasensitive ECL chemiluminescence solution (Meilun Biotechnology, MA0186). The ratio of Factek ultrasensitive ECL solution A to Factek ultrasensitive ECL solution B is 1:1. Mix well. Lay the cleaned membrane strip flat on plastic wrap, add the developer solution, and let it stand in the dark for 2 minutes. Immediately develop the membrane using a chemiluminescence analyzer and take a picture for storage. The results are as follows: Figure 4 As shown, when both alphacorticin and the synthetic peptide SFPYHY are present, the synthetic peptide SFPYHY can inhibit the activation of melanin synthesis-related factors proteins TYR (tyrosinase), MITF (melanocyte-inducible transcription factor), TRP-1 (tyrosinase key protein 1), and DCT (dopachrome tautomerase) by alphacorticin, and the inhibitory effect is not much different from that of the melanin inhibitor arbutin.
[0069] (9) Quantitative analysis of the expression levels of the above key proteins was performed, and the results are as follows: Figure 5 As shown, compared with the group containing arbutin, the synthetic peptide SFPYHY significantly inhibited the expression of TYR, MITF, TRP-1 and DCT, and the inhibitory effect was not much different from that of the arbutin group.
[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A polypeptide, characterized in that, Its amino acid sequence is: Ser-Phe-Pro-Tyr-His-Tyr.
2. The use of the polypeptide of claim 1 in the preparation of daily chemical products that help improve pigmentation.
3. The use of the polypeptide of claim 1 in the preparation of health products that help improve melasma.