A polypeptide for binding and dyeing hair keratin, preparation method and application

Abstract

This invention application is about a polypeptide that combines with and dyes hair keratin, its preparation method and applications. The polypeptide molecule provided by this invention forms a stable bond with keratin, enabling dyeing without damaging the cuticle, thus protecting the integrity of hair quality; the chemical bonding property ensures long-lasting dyeing and reduces fading; the characteristics of the polypeptide molecule guarantee uniform coverage and solve the problem of uneven dyeing in traditional dyeing; using polypeptides with high biocompatibility avoids irritating chemical substances and reduces the risk of allergies; by regulating the amino acid sequence and proportion of the polypeptide, precise color customization can be achieved; specific polypeptides can simultaneously have the functions of dyeing and hair care; the polypeptide material is easily biodegradable and has strong environmental protection; it is applicable to various hair qualities, including chemically treated hair; it can better simulate natural hair color and avoid an artificial feeling. These advantages stem from the characteristics of the polypeptide molecule and its interaction mechanism with hair proteins, providing a new direction for 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 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] Facing these challenges, the industry is actively exploring other dyeing techniques. For example, the polypeptide binding to hair keratin dyeing technique is a promising direction, which is expected to provide a safer, more durable and less damaging dyeing effect on hair quality. Plant-based dyes have also received increasing attention. Although their effects may not be as significant as chemical dyes, they have advantages in terms of safety and environmental protection.

[0009] Generally speaking, although significant progress has been made in chemical dyeing techniques in the past few decades, their inherent limitations still exist. Future research directions should focus on developing dyeing techniques that are safer, more environmentally friendly, have a more natural effect and do not damage hair quality. Summary of the Invention

[0010] To solve or partially solve the problems existing in the related technologies, the present invention application provides a polypeptide for binding and dyeing hair keratin, a preparation method and an application.

[0011] The first aspect of the present invention application provides a polypeptide having the structure of general formula (I):

[0012] CKRKLFRLFKRKCPPGYGRGDSYC (I); and its pharmaceutically or cosmetically acceptable salts, hydrates or solvates.

[0013] The second aspect of the present invention application provides the application of the above polypeptide in the preparation of hair dyes.

[0014] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present invention application.

[0015] Advantageous Technical Effects of the Present Invention:

[0016] The polypeptide hair dyeing technology of the present invention is based on the binding mechanism between a specifically designed polypeptide and hair keratin, and has the following core advantages: The polypeptide molecule forms a stable bond with keratin, and can be dyed without damaging the cutin layer, protecting the integrity of hair quality; The chemical binding property ensures long-lasting dyeing and reduces fading; The characteristics of the polypeptide molecule ensure uniform coverage and solve the problem of uneven traditional dyeing; Using polypeptides with high biocompatibility, avoiding irritating chemical substances and reducing the risk of allergy; By regulating the amino acid sequence and ratio of the polypeptide, precise color customization can be achieved; Specific polypeptides can have both dyeing and hair care functions at the same time; The polypeptide material is easily biodegradable and has strong environmental protection; It is suitable for various hair qualities, including chemically treated hair; It can better simulate natural hair color and avoid an artificial feeling. These advantages stem from the characteristics of the polypeptide molecule and its interaction mechanism with hair proteins, providing a new direction for hair dyeing technology. Description of the Drawings

[0017] Figure 1 Test Example 2 of the present invention is a comparison chart of experimental results. Detailed Embodiments

[0018] The optional embodiments of the present invention application will be described in more detail below with reference to the accompanying drawings. Although the optional embodiments of the present invention application are shown in the drawings, it should be understood that the present invention application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention application more thorough and complete, and to fully convey the scope of the present invention application to those skilled in the art.

[0019] The terms used in the present invention application are for the purpose of describing specific embodiments only and are not intended to limit the present invention application. The singular forms "a", "the" and "said" used in the present invention application and the appended claims are also intended to include the plural forms unless the context clearly dictates otherwise. It should also be understood that the term "and / or" used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0020] The following details a polypeptide, preparation method and application for combining and dyeing hair keratin in the present invention application

[0021] as follows:

[0022] The present invention application provides a polypeptide having the structure of general formula (I):

[0023] CKRKLFRLFKRKCPPGYGRGDSYC (I); and its pharmaceutically or cosmetically acceptable salts, hydrates or solvates.

[0024] Design Principle:

[0025] CK and YC: Cysteine (C) is at both ends, which can form disulfide bonds to increase stability.

[0026] RKLFRL: This part contains basic amino acids (R and K) that can bind to keratin, as well as hydrophobic amino acids (L and F), which helps to bind to hair keratin.

[0027] FKRK: Another group of basic and hydrophobic amino acids that enhance the binding to keratin.

[0028] CPP: This is a cell-penetrating peptide sequence that can help the polypeptide better penetrate into the hair.

[0029] GYGRGDS: This is a cell adhesion sequence that helps the polypeptide bind to the hair surface. At the same time, glycine (G) provides flexibility.

[0030] Dyeing Part:

[0031] In the polypeptides designed by the present invention, a dye molecule can be linked to the N-terminus or C-terminus.

[0032] In one embodiment of the present invention application, the use of the above polypeptide in the preparation of a hair dye is provided.

[0033] For the sake of clarity, the following will be described in detail through the following examples.

[0034] Example 1

[0035] Materials and equipment:

[0036] 1. Peptide synthesizer (such as the 433A of Applied Biosystems or equivalent equipment);

[0037] 2. Fmoc-protected amino acids;

[0038] 3. Resin: Rink Amide MBHA resin;

[0039] 4. Solvents: N,N-dimethylformamide (DMF), dichloromethane (DCM);

[0040] 5. Activator: HBTU (O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate);

[0041] 6. Base: DIEA (N,N-diisopropylethylamine);

[0042] 7. Deprotection reagent: piperidine;

[0043] 8. Cleavage reagent: TFA (trifluoroacetic acid), TIS (triisopropylsilane), water, EDT (1,2-ethanedithiol).

[0044] Specific steps:

[0045] 1. Resin preparation:

[0046] Weigh an appropriate amount of Rink Amide MBHA resin (about 0.1 mmol scale);

[0047] Swell the resin with DMF for 30 minutes.

[0048] 2. Removal of the Fmoc protecting group:

[0049] Treat the resin with a 20% piperidine / DMF solution for 10 minutes and repeat twice;

[0050] Wash the resin with DMF 5 times.

[0051] 3. Amino acid coupling:

[0052] Perform amino acid coupling in sequence from the C-terminus to the N-terminus;

[0053] Each coupling uses 4 equivalents of Fmoc - amino acid, 4 equivalents of HBTU and 8 equivalents of DIEA;

[0054] React in DMF for 45 - 60 minutes;

[0055] After coupling, wash the resin 5 times with DMF.

[0056] 4. Repeat steps 2 and 3 until the synthesis of the entire sequence is completed.

[0057] 5. Finally, remove the N - terminal Fmoc protecting group.

[0058] 6. Cleavage and side - chain deprotection:

[0059] Prepare the cleavage mixture: TFA / TIS / water / EDT (94:2.5:2.5:1, v / v);

[0060] Transfer the resin to a reaction flask and add the cleavage mixture;

[0061] React at room temperature for 2 - 3 hours;

[0062] Filter to collect the solution, and precipitate the peptide with ice - cold ether;

[0063] Centrifuge to collect the precipitate and wash it 2 - 3 times with ether.

[0064] 7. Purification:

[0065] Purify the polypeptide using reverse - phase high - performance liquid chromatography (RP - HPLC);

[0066] Use a C18 column with water / acetonitrile (containing 0.1% TFA) as the mobile phase;

[0067] Collect the main peak and lyophilize to obtain the pure product, namely the polypeptide with the structural formula CKRKLFRLFKRKCPPGYGRGDSYC.

[0068] Experimental Example 1

[0069] Experimental purpose:

[0070] Verify whether the designed polypeptide can specifically bind to hair keratin and evaluate its binding efficiency.

[0071] Experimental materials:

[0072] The polypeptide designed in the present invention (labeled as MP - 1);

[0073] Hair keratin (extracted from human hair);

[0074] Control polypeptide (a polypeptide with a length similar to MP-1 but a random sequence, labeled as CP-1);

[0075] Fluorescent labeling reagent (FITC);

[0076] Dialysis bag;

[0077] Fluorescence spectrophotometer;

[0078] Gel filtration chromatography column.

[0079] Experimental procedure:

[0080] 1. Polypeptide labeling:

[0081] Fluorescently label MP-1 and CP-1 with FITC respectively.

[0082] Use a dialysis bag to remove unbound FITC.

[0083] 2. Binding experiment:

[0084] Prepare a series of concentrations of hair keratin solutions (0, 1, 2, 5, 10, 20 μg / mL).

[0085] Mix the labeled MP-1 and CP-1 (at a fixed concentration, such as 5 μg / mL) with keratin solutions at different concentrations respectively.

[0086] Incubate at room temperature for 2 hours.

[0087] 3. Gel filtration separation:

[0088] Use a gel filtration chromatography column to separate the bound and unbound polypeptides.

[0089] Collect the eluate, 0.5 mL per fraction.

[0090] Fluorescence detection:

[0091] Measure the fluorescence intensity of each eluate fraction using a fluorescence spectrophotometer (excitation wavelength 495 nm, emission wavelength 519 nm).

[0092] Experimental results:

[0093]

[0094] Result analysis:

[0095] MP-1 showed a high degree of specific binding to hair keratin, and the binding rate increased significantly with the increase in keratin concentration.

[0096] The control polypeptide CP-1 showed extremely low non-specific binding and remained at a low level even at high concentrations of keratin.

[0097] Test Example 2

[0098] Experimental purpose:

[0099] Verify whether the designed and synthesized polypeptide-black dye complex can produce a visible and long-lasting black dyeing effect on hair.

[0100] Experimental materials:

[0101] 1. The polypeptide (CKRKLFRLFKRKCPPGYGRGDSYC) designed and synthesized in the present invention;

[0102] 2. Maleimide-modified black dye molecule;

[0103] 3. Untreated human hair samples (white hair);

[0104] 4. PBS buffer (pH 7.4);

[0105] 5. Reducing agent (TCEP);

[0106] 6. Dyeing tray;

[0107] 7. Constant temperature water bath;

[0108] 8. Digital camera;

[0109] 9. Black standard color card (including different shades of black).

[0110] Experimental steps:

[0111] 1. Prepare the polypeptide-black dye complex:

[0112] Dissolve the polypeptide in PBS buffer.

[0113] Add a reducing agent to activate the cysteine thiol group at the N-terminus of the polypeptide.

[0114] Add the maleimide-modified black dye molecule and react at room temperature for 2 hours to form a polypeptide-dye complex.

[0115] Remove the unreacted dye molecules by gel filtration or dialysis.

[0116] 2. Prepare the hair samples:

[0117] Select white human hair, with each strand about 5 cm in length.

[0118] Gently wash the hair with PBS buffer to remove surface impurities.

[0119] 3. Dyeing process:

[0120] Immerse the prepared hair samples in the polypeptide-black dye complex solution.

[0121] Place the dyeing tray into a thermostatic water bath preheated to 37°C.

[0122] Set the dyeing time to 30 minutes (adjustable according to actual circumstances).

[0123] 4. Rinsing and drying:

[0124] After the dyeing is completed, gently rinse the hair samples with PBS buffer solution, and then rinse with distilled water until the rinsing solution is clear.

[0125] Air-dry the hair samples naturally at room temperature.

[0126] 5. Immediately conduct an initial evaluation after the initial dyeing:

[0127] Observe the hair color with the naked eye and record in detail.

[0128] Measure the Lab* values using a spectrophotometer.

[0129] Take high-definition digital photos as a reference.

[0130] 6. Conduct simulated shampooing and regular evaluations:

[0131] Prepare a mild colorless shampoo aqueous solution.

[0132] Conduct evaluations after the 1st, 10th, and 20th shampoos. Each evaluation is carried out according to the following steps: a. Immerse the dyed hair samples in the shampoo aqueous solution and gently scrub for 30 seconds. b. Rinse the hair samples with warm water. c. Air-dry naturally. d. Observe the hair color with the naked eye and record any changes. e. Measure the Lab* values again using a spectrophotometer and compare with the initial values and the previous measurement values. f. Observe the dyeing situation on the hair surface with an optical microscope, paying attention to the dye distribution and possible loss. g. Take digital photos and compare with the initial photo and the previous photos. h. Use a black standard color card for comparison to evaluate the color retention degree.

[0133] 7. Data recording and analysis:

[0134] Create a data table to record the Lab* values and the results of visual observations for each evaluation.

[0135] Calculate the percentage of color change relative to the initial value after each shampoo.

[0136] Show the changes in L*, a*, and b* values with the number of shampooings.

[0137] Compare the performance differences between the polypeptide-black dye complex and commercially available permanent black hair dyes.

[0138] Experimental results:

[0139] Initial dyeing effect:

[0140]

[0141] Note: The smaller the L* value, the darker the color. The closer the a* value is to 0, the less tendency to red or green. The b* value is positive but close to 0, indicating a slight warm tone.

[0142] Results of the persistence test

[0143]

[0144] The L* value still remains low, indicating a dark color.

[0145] The a* value is slightly greater than 0, indicating a very slight tendency to red, which is common in black hair dyeing.

[0146] The b* value is positive but close to 0, indicating a slight yellow / warm tone, which also conforms to the characteristics of most black hair dye products.

[0147] The difference between the experimental group and the positive control is very small, indicating that the dyeing performance of the polypeptide-black dye complex is comparable to that of commercially available products (see the specific comparison chart in the appendix of the specification Figure 1 )

[0148] Test Example 3

[0149] 1. In vitro skin irritation test:

[0150] Specific steps: a. Prepare the EpiSki human skin model and equilibrate it in a 6-well plate for 2 hours. b. Prepare the test substances:

[0151] Experimental group: 5% solution of the polypeptide-black dye complex prepared in Test Example 2

[0152] Positive control: 5% SDS solution

[0153] Negative control: PBS buffer c. Apply 30 μL of each test substance to the surface of the skin model, covering an area of approximately 0.38 cm². d. Incubate at 37°C under 5% CO2 for 15 minutes. e. Rinse the skin model 10 times with PBS buffer. f. Transfer the skin model to a new culture medium and continue culturing for 42 hours. g. Use the MTT method to determine cell viability. h. Collect the culture medium and use an ELISA kit to determine the concentration of IL-1α.

[0154] 2. Cytotoxicity test:

[0155] Specific steps: a. Culture HaCaT cells in a 96-well plate at 5000 cells per well for 24 hours. b. Prepare polypeptide-black dye complexes at different concentrations: 0.1%, 0.5%, 1%, 5%. c. Expose the cells to the test substances at different concentrations, with 6 replicates for each concentration. d. Incubate at 37°C and 5% CO2 for 24 hours and 48 hours. e. Add MTT solution (5 mg / mL) and continue to incubate for 4 hours. f. Aspirate the culture medium and add DMSO to dissolve formazan. g. Measure the absorbance at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. h. Calculate the cell viability.

[0156] Experimental results:

[0157] In vitro skin irritation test:

[0158]

[0159] Cytotoxicity test:

[0160]

[0161] Result analysis:

[0162] In vitro skin irritation test:

[0163] The cell viability of the polypeptide-black dye complex was 95.8%, close to the negative control, indicating that it was almost non-irritating to the skin.

[0164] The release amount of IL-1α (6.7 pg / mL) was closer to the negative control (5.1 pg / mL), further confirming its extremely low irritation.

[0165] Cytotoxicity test:

[0166] At all test concentrations, the polypeptide-black dye complex showed higher cell viability (>93%).

[0167] Even at the highest concentration (5%) and the longest exposure time (48 hours), the cell viability remained at 93.2%.

[0168] As the concentration increased and the exposure time extended, the decrease in cell viability was slower.

[0169] The embodiments of the present invention application have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice 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 ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide structure is as follows: CKRKLFRLFKRKCPPGYGRGDSYC.

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

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