KR100889460B1 - Composition for enhancing melanogenesis in hair containing gdnf, method for enhancing melanogenesis in hair using gdnf, and method for screening hair-melanogenesis enhancer using gdnf expression level - Google Patents

Abstract

A white hair suppressing composition and a kit for melanogenesis promotion of hair are provided to promote melanogenesis of hair using GDNF protein or gene ciphering protein. A white hair suppressing composition comprises a neurotrophic factor receptor genesis growth factor as an active ingredient. The white hair suppressing composition promotes melanogenesis of hair by promoting expression of microphthalmia-associated transcription factor. The white hair suppressing composition is coated scalp or hair. A kit for melanogenesis promotion of hair comprises gene ciphering the neurotrophic factor receptor genesis growth factor or protein.

Description

Composition for promoting melanin production of hair comprising WIND, method for promoting melanin production of hair using WNDF and method for screening hair melanogenesis promoter using expression level of WNDF {Composition for enhancing melanogenesis in hair containing GDNF, method for enhancing melanogenesis in hair using GDNF, and method for screening hair-melanogenesis enhancer using GDNF expression level}

The present invention provides a composition for promoting melanin production of hair comprising a GDNF protein, a method for promoting melanin production of hair comprising administering a GDNF protein or a gene encoding the protein, and a hair melanogenesis promoter using a degree of expression of GDNF. It relates to a method of screening. Specifically, on the basis of the fact that the expression of GDNF is significantly reduced in white hair compared to the black hair and the fact that the expression of GDNF is related to melanin synthesis, a composition for inhibiting white hair, a method for inhibiting white hair and white hair, It relates to inhibitor screening methods.

Hair is composed of three parts: cuticle, fur, and medulla. The cortex that surrounds the medulla is the thickest part of the hair (80-90%), with melanin and lipid membranes, tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2) and other proteins. It consists of granules called melanosomes. Melanin pigment is synthesized in melanocytes, which are organelles in melanocytes, and migrates to the surrounding keratinocytes through dendritic processes of melanocytes. In the synthesis route of melanin, tyrosinase is involved in oxidizing the amino acid tyrosine to produce dopa and oxidizing dopa to produce dopaquinone, and the subsequent reaction proceeds automatically. Enzymes such as TRP-2 are known to accelerate the reaction.

The color of the hair is determined by factors such as the type and distribution ratio of eumelanin and peomelanin formed from the amino acid tyrosine as a raw material. In other words, it can be said that it determines various hair colors such as black, red, blonde according to the type of melanin and the size of granules. White hair is a hair color that appears without pigment granules.

Melanin is a protein that does not dissolve in water, giving color to the hair. The amino acid tyrosine is a substrate of melanin produced in breast tea. In the case of white hair, it does not make tyrosinase enzyme, so it cannot be oxidized and stops its action in dopaquinone. Therefore, hair is formed by keratinizing (raking up). That is, white hair is a kind of aging phenomenon that occurs because melanin production stops in melanocytes.

Dopaquinone is polymerized in two ways. Dopaquinone passes through dopachrome to produce dark brown eumelanin, and dopaquinone combines with cysteine present in keratin protein to produce reddish brown pheomelanin. Highly cysteine-rich hair contains a lot of pheomelanin. In this reaction, aromatic amines and phenolmonomers are oxidatively polymerized and further bind to amino acid residues in the hair.

The resulting melanin is delivered to keratinocytes and then moves upwards with hair growth. White hair, which is recognized as a phenomenon of physiological aging, is caused by a decrease in the number of hair melanocytes and a decrease in melanin production due to a decrease in melanocyte function.

 Since white hair generally occurs as aging progresses, it gives a strong impression of old age, so that a dye that blackens white hair in order to hide the generated white hair has been studied and widely marketed. However, dyeing must be dyed again by the growth of white hair in a temporary manner, and oxidative black hair dyeing, which is widely used now, damages the skin by the use of an oxidizing agent or the like.

Therefore, various studies have been conducted on the white hair inhibitor which prevents generation of white hair. Use black sesame seeds, rosacea, Hannyeoncho and honey as a supplementary ingredient, or mix it with Sukjihwang, Cheonmundong, and Macmundong, or put them in a container with black beans along with red sesame seeds, cypress leaves, sperm, Hannyeoncho, Seoksaeng, Sukjihwang, Cheonmundong, and Macmundong. After boiling at a temperature of 50 ℃, the chemical solution is to maintain the black soybeans, black sesame seeds, black pine leaves, using the kelp, the extract of the extract using the extract of the organol as an active ingredient. In addition, those using pituitary hormones, using C-AMP (adenosine-3,5 'cyclic phosphate), using vitamin D3, and the like are disclosed. However, the active ingredients of recent white hair inhibitors are mainly extracts of plants, animals, or components that are involved in the biological activities of animals. The good phenomenological side is the majority, and the mechanistic research is still insufficient. Although research on the mechanism of white hair is being conducted worldwide, the research on hair follicle melanocytes is different from that of the epidermis, so the research is slow.

In general, GDNF protein is a glial cell line derived neurotrophic factor, which is a neurotropic factor associated with the TGF-b family. NCBI accession ID is NP_000505, and its molecular weight is 15KDa. GDNF, as its name suggests, is involved in the survival of neurons, and their primary role is to promote the growth of neurons and axons in neurons. Therefore, much research has been done on the neurological side of GDNF.

The present inventors have found that the expression of GDNF is significantly reduced in white hair compared to the black hair and that the expression of GDNF is related to melanin synthesis. Therefore, an object of the present invention is to provide a composition for promoting melanin production of hair comprising a GDNF protein based on the above facts. Another object of the present invention is to provide a method for promoting melanin production of hair by administering a GDNF protein or a gene encoding the protein. In addition, an object of the present invention is to provide a method for screening a hair melanogenesis promoter using the expression level of GDNF.

In order to solve the above problems, the present invention provides a composition for promoting melanin production and a composition for inhibiting white hair of hair containing a glial cell line derived growth factor (Glial cell line derived neurotrophic factor, GDNF) as an active ingredient.

The present invention provides a method for promoting melanin production of hair, comprising administering GDNF or a gene encoding the protein.

In another aspect, the present invention is a method for screening whether or not to promote the hair melanin production for the test substance, hair melanin production promoter screening comprising the step of confirming whether the test substance promotes the expression of GDNF by ELISA or Western blot (immuno blot) method Transfecting the cell with a method or a plasmid comprising a promoter and reporter gene of GDNF; Treating the cell with a test substance; And it provides a hair melanin production promoter screening method comprising the step of confirming whether the test substance promotes the expression of the reporter gene.

According to one embodiment of the present invention, based on the fact that the expression of GDNF is significantly reduced in white hair compared to the black hair and the fact that the expression of GDNF is related to melanin synthesis, using a GDNF protein or a gene encoding the protein It can promote the melanin production of hair. In addition, the GDNF may be used as a target for developing a hair melanin production promoter, and may be used as a method for screening the developed hair melanin production promoter.

The present invention reveals that the expression of GDNF is significantly reduced in white hair compared to the black hair and that the expression of GDNF is related to melanin synthesis, and based on this, a composition for promoting melanin production or inhibiting white hair of GDNF. And a method for promoting melanin production or suppressing white hair using the gene encoding GDNF or GDNF. The present invention also provides a method for screening a hair melanin production promoter or a white hair inhibitor using the expression level of GDNF. In one embodiment of the present invention, GDNF may inhibit the white hair by promoting the melanin production of hair by promoting the expression of MITF, and promotes the melanin production of hair.

When applied to cosmetics it contains a cosmetically acceptable medium or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, and / or It may be provided in the form of a nonionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. These compositions can be prepared according to conventional methods in the art. In particular, it may be prepared in the form of a shampoo, rinse, conditioning, tonic or scalp essence applied to the scalp or hair.

Cosmetic composition according to an embodiment of the present invention is a fatty material, an organic solvent, a dissolving agent, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, ionic or nonionic Such as type emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in cosmetics It may contain adjuvants commonly used in the cosmetic field. These adjuvants are introduced in amounts generally used in the cosmetic field.

When applied to pharmaceuticals, parenteral dosage forms (injection, transdermal or external application) in the form of solid, semi-solid or liquid by adding a commercially available inorganic or organic carrier using the GDNF protein according to an embodiment of the present invention as an active ingredient ) Can be formulated. Formulations for parenteral administration include topical injections, ointments, lotions, sprays, suspensions and the like.

In order to formulate the active ingredient according to an embodiment of the present invention, it can be easily formulated according to the conventional method, and a surfactant, an excipient, a coloring agent, a spice, a preservative, a stabilizer, a buffer, a suspending agent, and other commonly used auxiliaries can be suitably used. Can be.

GDNF dosage according to an embodiment of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and can be applied at the level of those skilled in the art by external application to the site where the white hair occurs for the preparation except the injection, and in the case of injection, dermis of the scalp with advanced white hair. It can be injected topically into the subcutaneous and subcutaneous fat. Generally dosages range from 0.001 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosages are 1 μg / kg / day to 100 μg / kg / day.

 GDNF protein according to an embodiment of the present invention may be in various modified forms by replacing, deleting or / and adding the amino acid residue of the natural amino acid sequence to another residue as long as it can suppress the white hair or promote hair melanin production It may also be in a form in which modifications such as phosphorylation, saccharification, methylation, and pansylation have occurred.

In one embodiment of the invention, various methods can be used to administer genes encoding GDNF or pseudoproteins. Viral vectors or non-viral vectors may be used to administer genes. Viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and the like, and non-viral vectors include micelles or liposomes. The same structure and the like, DNA can also be administered by electroporation or gene gun without a vector. In particular, liposomes may be administered by carrying a gene encoding GDNF or a similar protein.

Screening of white hair inhibitor or hair melanogenesis promoter using the expression level of GDNF confirms whether the test substance promotes the expression of GDNF protein by immunoassay (e.g., ELISA or Immunoblot), or includes a reporter gene comprising a promoter of GDNF. The plasmid may be transfected into cells and the cells treated with a test substance to determine whether the test substance promotes expression of the reporter gene.

The reporter gene assay for screening inhibitors uses a reporter gene vector prepared by common molecular biology techniques. As the reporter gene, a firefly luciferase gene or a Gaussian luciferase gene (GLuc) may be used. Promoters of GDNF can be readily designed and obtained by those skilled in the art using Gene ID 2668 from the US NCBI database. After transfection of the prepared plasmid into primary cultured melanocytes or immortalized melanocytes or melanoma cells, treatment with a candidate for a white hair suppressor and measurement of luciferase activity by a luminometer (Luminometer) The effect of a white hair inhibitor drug on the expression of GDNF can be identified.

Hereinafter, the present invention will be described in more detail with reference to the following test examples, but the present invention is not limited only to these examples.

Test Example 1 Comparative Analysis of Cytokine Expression in White Hair and Black Hair

A total of three scalp biopsies were obtained from the white hair and the black hair, respectively, and then the melanocytes were separated from each other and cultured. For each cultured melanosite, 50000 cells per ml are placed in plate medium and grown for 24 hours. This culture is then walked and named primary culture. After adding fresh medium to it and incubating for 72 hours, the culture solution is called a secondary culture solution. Mix the primary and secondary cultures in a 1: 1 ratio to form a suitable medium (CM). Using prepared white and black hair CM, experiments are carried out by using a cytokine assay kit (product number: AAH-GF1) purchased from Raybiotech by standard test methods provided in the kit. The results are shown in FIG. As a result of the comparative analysis, it was confirmed that the expression level of GDNF was lower in culture of white hair than in culture of black hair.

In the cytokine assay results, the expression of GDNF was reduced in white hair, and the expression of mRNA was examined to determine the expression level of GDNF in human hair and white hair.

Test Example 2 Expression of GDNF mRNA in Black Hair and White Hair

A total of three scalp and biopsies were biopsied from each of the scalp, and RNA was extracted from the tissue to perform RT-PCR (2-a). In addition, the melanosite was cultured from the tissue and RNA was extracted from the cells to perform RT-PCR (2-b). The results are shown in FIG. RT-PCR showed that the black hair expresses more GDNF than the white hair in both tissues and cells.

After the two tests confirmed that the expression of GDNF in the white hair is lower than that of normal black hair, it was examined whether GNDF can actually promote melanogensis (melanogensis). First, we investigated whether the activity of the gene is increased in relation to the microphthalmia-associated transcription factor (MITF), which is known to be important in the early stage of melanin synthesis, and also measured the amount of melanin, the end product of melanin synthesis.

Test Example 3 Effect of GDNF on MITF Promoter Activity

The melan-a cell system with the MITF promoter was used to measure MITF activity against GDNF using GLuc assay. The amount of melanin was also quantified using the cells used at this time.

Specifically, a small amount of medium was removed from the cell culture dish and transferred to the measurement plate, and 1 × GLuc assay working solution (NEB) was added to the medium in a ratio of 4: 1, and the amount of light generated at 470 nM using a luminometer. Was measured. After 500,000 cells were plated using a 24-hole plate, GDNF (R & D system, cat no. 212-GD) was treated at 50 ng / ml and IBMX was treated at 100 uM. IBMX was used as a positive control. GLuc analysis is performed 24 hours and 48 hours after incubation. The analysis results are shown in Figure 3a.

And on day 3, the medium was changed to fresh medium containing the substance, and then cells were collected on day 5. After adding 1N NaOH to dissolve the cells, the absorbance at OD405 nm was measured to measure the amount of melanin. The melanin amount measurement result is shown in FIG. 3B.

As can be seen from the results of Figures 3a and 3b, GDNF shows that GDNF has the effect of promoting melanin synthesis by increasing the MITF promoter activity by about 60% and melanin production by about 30% or more.

Test Example 4 Effect on Normal Human Melanosite

In order to examine the effects of GDNF on human melanocytes, we examined the effects of melanin synthesis on normal human melanocytes. To assess this, we measured tyrosinase activity, which plays an important role in the last step in the melanin synthesis process. This was done for tyrosinase activity using DOPA staining in a way to find out on the man-hour weight (in situ). Put 100,000 normal human melanocytes on the 6-hole medium, and add 50ng / ml of GDNF and incubate for 5 days. At this time, change to a new badge on the third day. After 5 days, the cells were washed with PBS and sonicated using 0.05M sodium phosphate buffer (pH6.8) containing PMSF and 1.0% Triton X-100. Then, it is left for 30 minutes at 4 ° C to separate tyrosinase from the melanosomal membrane. After centrifugation at 40000g for 20 minutes, only supernatant is collected to obtain protein. 10 ug of protein is mixed with SDS sample buffer and subjected to electrophoresis using Tris-Glycine gel. After electrophoresis, the gel is incubated for 15 minutes in 0.5 M sodium phosphate buffer (pH 6.5). Thereafter, the gel was added to 0.1 M sodium phosphate buffer (pH6.8) containing 0.2% L-DOPA and reacted at 37 ° C. Stop the reaction when the band appears. The results are shown in Figure 4a. And it was quantified and shown in Figure 4b as a graph. As shown in Figure 4 GDNF was confirmed to exhibit a tyrosinase activity promoting effect.

The composition according to an embodiment of the present invention may be prepared by the following preparation example, but is not limited thereto.

Production Example 1 Hair Shampoo

Ingredient Name weight % Sodium Lauryl Sulfate Solution (30%) 20.0 Palm oil fatty acid diethanolamide 5.0 Polyquaternium-10 0.3 Propylene glycol 2.0 GDNF 0.01 Pyrotonolamine 0.5 Yellow 203 Quantity Paraoxybenzoic Acid Ester 0.2 Combination Quantity Citric acid Quantity Purified water Remaining amount

Preparation Example 2 Hair Conditioning

Ingredient Name weight % Cetyltrimethylammonium chloride (29%) 7.0 Distearyl dimethylammonium chloride (75%) 4.0 Cetostearyl alcohol 3.5 Polyoxyethylene stearyl ether 1.0 Liquid paraffin 2.0 Propylene glycol 1.5 GDNF 0.01 Combination Quantity Citric acid Quantity Purified water Remaining amount

Preparation Example 3 Scalp Hair Tonic

Ingredient Name weight % menthol 0.1 D-panthenol 0.6 Salicylic acid 0.05 glycerin 1.0 Polyoxyethylene Cured Castor Oil 0.8 Tocopherol Acetate 0.03 Combination Quantity GDNF 0.02 ethanol 30.0 Purified water Remaining amount

Preparation Example 4 Scalp Essence

Ingredient Name weight % ethanol 30.0 Polysorbate 60 1.5 glycerin 3.0 Carboxy Vinyl Polymer 0.1 Triethanolamine 0.2 GDNF 0.03 antiseptic A reasonable amount Spices and Colors A reasonable amount Purified water Remaining amount

Production Example 5 Injection

Ingredient Name Weight ratio (per 1ml) Sodium chloride 9mg Sodium carboxymethylcellulose 30.0mg Tween 20 1.0mg GDNF 100ug Distilled water for injection Remaining amount

[Manufacture example 6] Ointment

Ingredient Name weight % Caprine / Capryl Triglycerides 10 Liquid paraffin 10 Solbitan Sesquioleate 6 Octyldodeceth-25 9 3,7 hexanoate 10 Squalane One Salicylic acid One glycerin 15 Sorbitol One GDNF 0.03 Preservative, coloring, flavoring Quantity Purified water Remaining amount

[Manufacture example 7] Lotion

Ingredient Name weight % GDNF 0.01 Beeswax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Caprylic / Capric Triglycerides 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxy Vinyl Polymer 0.1 Triethanolamine 0.2 Preservatives, Colors and Flavors Quantity Purified water Remaining amount

[Manufacture example 8] Spray

Ingredient Name weight % GDNF 0.01 ethanol 80 Tween 80 5 glycerin 5 Tocopherol Acetate One Preservatives, Colors and Flavors Quantity Purified water Remaining amount

Figure 1 is a photograph showing the results of performing a cytokine assay to compare cytokine (cytokine) expression in white hair and black hair.

Figure 2 shows the results of RT-PCR extraction of RNA from tissue (2a) and melanocytes (2b) to compare the mRNA expression of GDNF in black hair and white hair.

Figure 3 is a graph showing the MITF activity (3a), and the amount of melanin quantified from the cells (3b) for GDNF measured by GLuc assay using a melan-a cell system containing a MITF promoter.

Figure 4 is the result of measuring the tyrosinase activity in situ ( in situ ) to confirm the melanin synthesis effect of GDNF in human melanocytes. Figure 4a is a photograph showing the gel DOPA stained, 4b is a graph quantifying the tyrosinase activity.

Claims (14)

Composition for inhibiting white hair containing glial cell line derived growth factor (Glial cell line derived neurotrophic factor, GDNF) as an active ingredient. The composition of claim 1, wherein the composition promotes the melanin production of hair by promoting the expression of a microphthalmia-associated transcription factor (MITF). The composition for inhibiting white hair according to claim 1 or 2, wherein the composition is applied to the scalp or hair. 4. The composition for inhibiting white hair according to claim 3, wherein the composition has a formulation of shampoo, conditioning, tonic, scalp essence, lotion, ointment, spray or topical injection. delete The composition for inhibiting white hair according to claim 1, wherein the composition suppresses white hair by promoting melanin production of hair. delete delete Kit for promoting melanin production of hair comprising a glial cell line derived growth factor (Glial cell line derived neurotrophic factor, GDNF) or the gene encoding the protein. delete The kit for promoting melanin production of hair according to claim 9, wherein the gene encoding the GDNF or the protein is composed of a formulation for injection, transdermal administration or external application. A method for screening whether or not to promote hair melanin production for a test substance, A method for screening a hair melanogenesis promoter comprising confirming whether the test substance promotes expression of GDNF. The method for screening hair melanogenesis promoter of claim 12, wherein the expression promotion is confirmed by ELISA or immunoblot. Transfecting the cells with a plasmid comprising a promoter and reporter gene of GDNF; Treating the cell with a test substance; And A method for screening a hair melanogenesis promoter comprising checking whether the test substance promotes expression of the reporter gene.

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