Polypeptide capable of combining and dyeing hair keratin as well as preparation method and application of polypeptide

Abstract

The invention relates to a polypeptide capable of combining and dyeing hair keratin as well as a preparation method and application of the polypeptide capable of combining and dyeing the hair keratin. Polypeptide molecules provided by the invention are stably combined with the keratin, so that the hair can be dyed without damaging a cuticle, and the completeness of the hair is protected; the chemical bonding property ensures lasting dyeing and reduces color fading; the polypeptide molecule characteristics guarantee uniform coverage, and the problem of non-uniform traditional dyeing is solved; the polypeptide with high biocompatibility is used, so that irritant chemical substances are avoided, and the allergy risk is reduced; accurate color customization is realized by regulating and controlling the amino acid sequence and proportion of the polypeptide; the specific polypeptide can have dyeing and hair care functions at the same time; the polypeptide material is easy to biodegrade and high in environmental protection property; the hair conditioner is suitable for various hair qualities, including chemically treated hair; the natural hair color can be better simulated, and the artificial feeling is avoided. The advantages are derived from the molecular characteristics of the polypeptide and the interaction mechanism of the polypeptide and hair protein, and a new direction is provided for the hair dyeing technology.

Description

Technical Field

[0001] The present invention relates to the field of medical aesthetics technology, and specifically to a polypeptide that binds to and dyes hair keratin, a preparation method and an application thereof. Background Art

[0002] Hair dyeing is one of the most common services in the beauty industry, and chemical dyeing has always been a relatively mainstream choice due to its significant effects and relatively simple operation. However, as people's awareness of health and environmental protection increases, many shortcomings of traditional chemical dyeing technology have gradually emerged.

[0003] First, safety is the most prominent issue. Traditional chemical dyes often contain irritating and potentially harmful substances such as ammonia, hydrogen peroxide, and paraphenylenediamine. These ingredients may cause scalp irritation, allergic reactions, and even more serious health problems in some cases. Long-term exposure to these chemicals may increase the risk of skin inflammation and certain cancers, although research results in this area have not yet been completely conclusive.

[0004] Secondly, the damage that chemical dyeing causes to the hair structure cannot be ignored. During the dyeing process, in order to allow the dye to better penetrate the hair, it is usually necessary to open the hair scale layer. This process will inevitably damage the hair's natural protective layer, causing the hair to become dry, brittle, and more prone to breakage and split ends. Repeated dyeing will aggravate this damage, causing the hair to lose its luster and elasticity, affecting the overall hair quality.

[0005] The durability and uniformity of color are also challenges faced by chemical dyeing. Many dyeing products fade quickly after repeated washing, especially bright colors are more difficult to maintain. This not only requires users to frequently dye, increasing the cost of use, but also aggravates the cumulative damage to the hair. In terms of dyeing uniformity, traditional technology is difficult to guarantee completely consistent results, especially for hair that has been dyed or bleached, which may have spots or uneven tones.

[0006] Environmental impact is another issue that cannot be ignored. The wastewater generated during the chemical dyeing process contains a variety of harmful substances. If not properly handled, it will pollute water and soil. In addition, the production and packaging of hair dyes also consume a lot of resources and create an environmental burden.

[0007] In addition, chemical dyeing also has some restrictions on the use of special groups. For example, people who are allergic to certain chemical ingredients may not be able to use traditional hair dye products at all.

[0008] Faced with these challenges, the industry is actively exploring other dyeing technologies. For example, peptide combined with hair keratin dyeing technology is a promising direction, which is expected to provide a safer, more durable and less damaging dyeing effect on hair. Plant-based dyes are also receiving more and more attention. Although the effect may not be as significant as chemical dyes, they have advantages in safety and environmental protection.

[0009] In general, although chemical dyeing technology has made significant progress in the past few decades, its inherent limitations still exist. Future research should focus on developing dyeing technologies that are safer, more environmentally friendly, more natural, and do not damage hair. Summary of the invention

[0010] In order to solve or partially solve the problems existing in the related art, the present invention provides a polypeptide for binding to and dyeing hair keratin, a preparation method and application.

[0011] The first aspect of the present application provides a polypeptide having a general formula (I): CKRKLFRLFKRKCPPGYGRGDSYC (I); and its cosmetically or pharmaceutically acceptable salts, hydrates or solvates.

[0012] The second aspect of the present application provides the use of the above polypeptide in the preparation of hair dyes.

[0013] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention.

[0014] Beneficial technical effects of the present invention: The polypeptide hair dyeing technology of the present invention is based on the binding mechanism of a specifically designed polypeptide and hair keratin, and has the following core advantages: the polypeptide molecule forms a stable bond with keratin, and dyeing can be done without destroying the stratum corneum, thereby protecting the integrity of the hair; the chemical binding property ensures that the dyeing lasts and reduces fading; the characteristics of the polypeptide molecule ensure uniform coverage, solving the problem of uneven traditional dyeing; the use of highly biocompatible polypeptides avoids irritating chemicals and reduces the risk of allergies; precise color customization is achieved by regulating the polypeptide amino acid sequence and ratio; specific polypeptides can have both dyeing and hair care functions; polypeptide materials are easily biodegradable and highly environmentally friendly; they are suitable for all kinds of hair, including chemically treated hair; and they can better simulate natural hair color and avoid artificial feeling. These advantages stem from the characteristics of polypeptide molecules and their interaction mechanism with hair proteins, providing a new direction for hair dyeing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Test Example 2 of the present invention is a comparison chart of experimental results. DETAILED DESCRIPTION

[0016] The optional embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the optional embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0017] The terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0018] The present invention is applied for a polypeptide that binds and dyes hair keratin, a preparation method and an application thereof. The detailed description is as follows: The present invention provides a polypeptide having a general formula (I): CKRKLFRLFKRKCPPGYGRGDSYC (I); and its cosmetically or pharmaceutically acceptable salts, hydrates or solvates.

[0019] Design principle: CK and YC: Cysteine ​​(C) at both ends can form disulfide bonds to increase stability.

[0020] RKLFRL: This part contains basic amino acids (R and K) that can bind to keratin, and hydrophobic amino acids (L and F) that help bind to hair keratin.

[0021] FKRK: Another group of basic and hydrophobic amino acids that enhances binding to keratin.

[0022] CPP: This is the cell penetrating peptide sequence that helps the peptide penetrate into the hair better.

[0023] GYGRGDS: This is the cell adhesion sequence that helps the peptide bind to the hair surface. At the same time, the glycine (G) provides flexibility.

[0024] Dyeing part: In the polypeptide designed by the present invention, the dye molecule can be connected to the N-terminus or the C-terminus.

[0025] In one embodiment of the present application, there is provided the use of the above polypeptide in the preparation of a hair dye.

[0026] For the purpose of greater clarity, the invention is described in detail through the following examples.

[0027] Example 1 Materials and Equipment: 1. Peptide synthesizer (such as Applied Biosystems 433A or equivalent); 2. Fmoc-protected amino acids; 3. Resin: Rink Amide MBHA resin; 4. Solvent: N,N-dimethylformamide (DMF), dichloromethane (DCM); 5. Activator: HBTU (O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate); 6. Base: DIEA (N,N-diisopropylethylamine); 7. Deprotection reagent: piperidine; 8. Cutting reagents: TFA (trifluoroacetic acid), TIS (triisopropylsilane), water, EDT (1,2-ethanedithiol).

[0028] Specific steps: 1. Resin preparation: Weigh an appropriate amount of Rink Amide MBHA resin (about 0.1 mmol scale); The resin was swelled with DMF for 30 minutes.

[0029] 2. Removal of Fmoc protecting group: Treat the resin with 20% piperidine / DMF solution for 10 min, repeated twice; The resin was washed 5 times with DMF.

[0030] 3. Amino acid coupling: The amino acids were coupled sequentially from the C-terminus to the N-terminus according to the sequence; Four equivalents of Fmoc-amino acid, four equivalents of HBTU, and eight equivalents of DIEA were used for each coupling; React in DMF for 45-60 minutes; After coupling the resin was washed 5 times with DMF.

[0031] 4. Repeat steps 2 and 3 until the synthesis of the entire sequence is completed.

[0032] 5. Finally remove the N-terminal Fmoc protecting group.

[0033] 6. Cleavage and side chain deprotection: Prepare cutting mixture: TFA / TIS / water / EDT (94:2.5:2.5:1, v / v); Transfer the resin to the reaction bottle and add the cutting mixture; React at room temperature for 2-3 hours; The solution was collected by filtration, and the peptide was precipitated with ice-cold ether; The precipitate was collected by centrifugation and washed 2-3 times with ether.

[0034] 7. Purification: The peptides were purified using reverse phase high performance liquid chromatography (RP-HPLC); A C18 column was used with water / acetonitrile (containing 0.1% TFA) as the mobile phase; The main peak is collected and freeze-dried to obtain a pure product, which is the polypeptide contained in claim 1 of the present invention.

[0035] Test Example 1 Purpose: Verify whether the designed peptide can specifically bind to hair keratin and evaluate its binding efficiency.

[0036] Experimental Materials: The polypeptide designed by the present invention (labeled as MP-1); Hair keratin (extracted from human hair); control peptide (a peptide of similar length to MP-1 but with random sequence, labeled as CP-1); Fluorescent labeling reagent (FITC); Dialysis bags; Fluorescence spectrophotometer; Gel filtration chromatography columns.

[0037] Experimental steps: 1. Peptide labeling: MP-1 and CP-1 were fluorescently labeled with FITC, respectively.

[0038] Unbound FITC was removed using a dialysis bag.

[0039] 2. Combined experiment: A series of hair keratin solutions were prepared (0, 1, 2, 5, 10, 20 μg / mL).

[0040] The labeled MP-1 and CP-1 (fixed concentration, such as 5 μg / mL) were mixed with keratin solutions of different concentrations.

[0041] Incubate at room temperature for 2 hours.

[0042] 3. Gel filtration separation: Bound and unbound peptides were separated using a gel filtration chromatography column.

[0043] Collect the eluate in 0.5 mL portions.

[0044] Fluorescence detection: The fluorescence intensity of each eluate was measured using a fluorescence spectrophotometer (excitation wavelength 495 nm, emission wavelength 519 nm).

[0045] Experimental results:

[0046] Result analysis: MP-1 showed highly specific binding to hair keratin, with the binding rate increasing significantly with increasing keratin concentration.

[0047] The control peptide CP-1 showed very low nonspecific binding, which remained low even at high keratin concentrations.

[0048] Test Example 2 Purpose: To verify whether the designed and synthesized peptide-black dye complex can produce a visible and lasting black dyeing effect on hair.

[0049] Experimental Materials: 1. The polypeptide designed and synthesized by the present invention (CKRKLFRLFKRKCPPGYGRGDSYC); 2. Maleimide-modified black dye molecules; 3. Untreated human hair samples (white hair); 4. PBS buffer (pH 7.4); 5. Reducing agent (TCEP); 6. Staining tray; 7. Constant temperature water bath; 8. Digital camera; 9. Black standard color card (including different shades of black).

[0050] Experimental steps: 1. Prepare the peptide-black dye complex: The peptides were dissolved in PBS buffer.

[0051] Add a reducing agent to activate the cysteine ​​thiol group at the N-terminus of the polypeptide.

[0052] Maleimide-modified black dye molecules were added and reacted at room temperature for 2 hours to form a peptide-dye complex.

[0053] Unreacted dye molecules are removed by gel filtration or dialysis.

[0054] 2. Prepare hair samples: Select white human hair, each strand is about 5 cm long.

[0055] Gently wash the hair with PBS buffer to remove surface impurities.

[0056] 3. Dyeing process: The prepared hair sample was immersed in the peptide-black dye complex solution.

[0057] Place the staining dish in a thermostatic water bath preheated to 37 °C.

[0058] The dyeing time is set to 30 minutes (can be adjusted according to actual conditions).

[0059] 4. Rinse and dry: After staining, the hair samples were gently rinsed with PBS buffer and then with distilled water until the rinse solution was clear.

[0060] The hair samples were air-dried at room temperature.

[0061] 5. After initial staining, immediately perform an initial assessment: Observe the hair color with your naked eyes and record it in detail.

[0062] The Lab* value was measured using a spectrophotometer.

[0063] Take high-resolution digital photos as a baseline reference.

[0064] 6. Perform simulated shampooing and regular assessment: Prepare a mild, colorless shampoo solution.

[0065] Evaluations were performed after the 1st, 10th, and 20th shampooings, each performed as follows: a. Dip the dyed hair sample into the shampoo solution and gently scrub for 30 seconds. b. Rinse the hair sample with warm water. c. Air dry. d. Visually observe the hair color and record any changes. e. Measure the Lab* value again using a spectrophotometer and compare it to the initial value and the previous measurement. f. Observe the dyeing of the hair surface using an optical microscope, paying attention to dye distribution and possible loss. g. Take a digital photo and compare it to the initial and previous photos. h. Compare using a black standard color chart to evaluate the degree of color retention.

[0066] 7. Data recording and analysis: Create a data sheet to record the Lab* values ​​and visual observations for each evaluation.

[0067] The percentage color change after each shampooing relative to the initial value was calculated.

[0068] Shows the changes in L*, a*, and b* values ​​with the number of shampooing times.

[0069] Compare the performance differences between the peptide-black dye complex and commercially available permanent black hair dye.

[0070] Experimental results: Initial dyeing effect:

[0071] Note: The smaller the L* value, the darker the color; an a* value close to 0 indicates no red or green tendency; a b* value that is positive but close to 0 indicates a slightly warm tone.

[0072] Durability Test Results

[0073] The L* value still remains low, indicating a dark color.

[0074] An a* value slightly greater than 0 indicates a very slight red tendency, which is common in black hair dyeing.

[0075] The b* value is positive but close to 0, indicating a slight yellow / warm tint, which is consistent with the characteristics of most black hair dye products.

[0076] The difference between the experimental group and the positive control was small, indicating that the dyeing performance of the peptide-black dye complex is comparable to that of the commercially available product (see the attached manual for specific comparison chart). Figure 1 ).

[0077] Test Example 3 1. In vitro skin irritation test: Specific steps: a. Prepare EpiSki human skin model and equilibrate in a 6-well plate for 2 hours. b. Prepare test substances: Experimental group: 5% Example 2 Preparation of polypeptide-black dye complex solution Positive control: 5% SDS solution Negative control: PBS buffer c. Apply 30μL of the test substance to the surface of the skin model, covering an area of ​​approximately 0.38cm². d. Incubate at 37°C, 5% CO2 for 15 minutes. e. Rinse the skin model 10 times with PBS buffer. f. Transfer the skin model to new culture medium and continue to culture for 42 hours. g. Determine cell activity using the MTT method. h. Collect the culture medium and determine the IL-1α concentration using an ELISA kit.

[0078] 2. Cytotoxicity test: Specific steps: a. Culture HaCaT cells in 96-well plates, 5000 cells per well, for 24 hours. b. Prepare different concentrations of peptide-black dye complex: 0.1%, 0.5%, 1%, 5%. c. Expose cells to different concentrations of test substances, with 6 replicates for each concentration. d. Incubate at 37°C, 5% CO2 for 24 hours and 48 hours. e. Add MTT solution (5mg / mL) and continue incubation for 4 hours. f. Aspirate the culture medium and add DMSO to dissolve the formazan. g. Measure the absorbance at a wavelength of 570nm using a microplate reader. h. Calculate cell viability.

[0079] Experimental results: In vitro skin irritation test:

[0080] Cytotoxicity test:

[0081] Result analysis: In vitro skin irritation test: The cell viability of the peptide-black dye complex was 95.8%, close to that of the negative control, indicating that it was almost non-irritating to the skin.

[0082] The amount of IL-1α released (6.7 pg / mL) was closer to that of the negative control (5.1 pg / mL), further confirming its extremely low irritation.

[0083] Cytotoxicity test: The peptide-black dye complex showed higher cell viability (>93%) at all tested concentrations.

[0084] Even at the highest concentration (5%) and longest exposure time (48 h), cell viability remained at 93.2%.

[0085] As the concentration increased and the exposure time prolonged, the cell viability decreased more slowly.

[0086] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A polypeptide and its cosmetically or pharmaceutically acceptable salt, hydrate or solvate, characterized in that: The polypeptide has a structure of formula (I): CKRKLFFRLFKRKCPPGYGRGDSYC(I).

2. Use of the polypeptide according to claim 1 in the preparation of a hair dye.

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