KR100883357B1 - Composition for the Improvement of Leukoderma - Google Patents

Abstract

The present invention discloses melanin hypopigmentation disease improving composition. Specifically, the present invention discloses a melanin hypopigmentation disease composition comprising a polymethoxyflavone-based compound as an active ingredient.

Melanin, hypopigmentation, polymethoxyflavones

Description

Composition for the Improvement of Leukoderma

The present invention relates to a melanin hypopigmentation disease improving composition, and relates to a melanin hypopigmentation disease improving composition comprising a polymethoxyflavone-based compound as an active ingredient.

Human hair or skin color is generally determined by the amount of melanin present in the hair or skin.

Melanin is produced by tyrosinase through waveguide from tyrosine in melanocytes, which are pigment cells in the basal layer of the epidermis, and in addition to tyrosinase, Trp-1 / 2 ( Tyrosinase associated protein-1 / 2) (Korner and Pawelek, Science (1982) 217: 1163-1165; Kobayashi et al., EMBO J (1994) 13: 5818-5825; Yokoyama et al., Biochim Biophys Acta ( 1994) 1217: 317-321), as well as a higher signal for tyrosinase expression, is known to be involved in the cyclic AMP (cAMP) and the sustained activation of ERK1 / 2 (Extracellular signal-activated kinases-1 / 2). (Marshall, Cell (1995) 80: 179-185; Busca and Ballotti, Pigment Cell (2000) 13: 60-69).

Melanocytes are affected by various factors such as external conditions such as ultraviolet rays and inflammation, hormones such as melanocyte stimulating hormone (MSH) and thyroid stimulating hormone (TSH).

Melanin absorbs the light energy of ultraviolet rays from sunlight to protect cells deep in the skin from damage caused by ultraviolet rays.However, abnormal production of melanin causes abnormal skin pigmentation such as blemishes, freckles, and pigmentation. , On the contrary, if less produced, vitiligo, depigmental nevus, white pseudoleucoderma atopicum, Tinea versicolor, ecchymosis, allergy, post-inflammatory bleaching, idiopathic hypopigmentation, depigmentation nevus, partial leukemia Melanin hypopigmentation disease (Leukoderma) such as (Bolognia and Pawelek, J Am Acad Dermatol (1988) 19: 217-255; Pinto and Bolognia, Pediatr Clin North Am (1991) 38: 991-1017).

Among the melanin hypopigmentation diseases, vitiligo is a disease caused by the destruction of melanocytes, and the remaining diseases are caused by a decrease in the amount of melanin produced in the melanocytes.

As a treatment for the disease, photochemical methods using photosensitizers such as soralene, surgical methods such as surgery, and ingredients of ginger, pipally and black pepper (Ancient Science of Life, (1990) Vol. IV , No. 4, 202-206), and the like, and methods of inducing recoloration of the skin by administration of a drug having proliferative activity or melanin production promoting activity.

On the other hand, Korean Patent Laid-Open Publication No. 2005-0019956 discloses a photochemical method using a photosensitizer such as Hypericin and chemical light, and International Publication No. 2004-112815 (WO 2004/112815) discloses melanin. Disclosed is a drug that can induce recoloring of the skin by using the extract of King Danggae or its active ingredient, lupeol fatty acid ester, which has a production promoting activity.

The present invention also discloses a drug that can improve melanin hypopigmentation disease using a polymethoxyflavone-based compound having melanin production promoting activity.

An object of the present invention to provide a melanin hypopigmentation disease improving composition using a polymethoxy flavone-based compound.

Other objects, specific embodiments, and the like of the present invention will be presented below.

For this purpose, the inventors identified five polymethoxyflavone-based compounds isolated and identified from citrus fruits, namely 5,6,7,8,3 ', 4'-hexamethoxy, as identified in the following examples. Flavones (5,6,7,8,3 ', 4'-hexamethoxyflavone) (nobiletin, nobililet), 5,6,7,3'4'-pentamethoxyflavones (5,6,7,3'4 '-pentamethoxyflavone) (sinensetin), 5,6,7,8,4'-pentamethoxyflavone (5,6,7,8,4'-pentamethoxyflavone) (tangeretin), 3,5 , 6,7,8,3 ', 4'-heptamethoxyflavone (3,5,6,7,8,3', 4'-heptamethoxyflavone) and 5-hydroxy-6,7,8,3 ' , 4'-pentamethoxyflavone (5-hydoxy-6,7,8,3 ', 4'-pentamethoxyflavone) (These compounds are all known compounds and their separation or preparation methods are described in "<Wang, X. , Li, F., Zhang, H., Geng, Y., Yuna, J., Jiang, T. Preparative isolation and purification of polymethoxylated flavones from Tangeretine peel using high-speed counter-current chromatography, Journal od Chromatography A, 1090 : 188-192 , 2005; Roman, G., Jayaprakasha, GK, Cho, MH, Brodbelt, J., Patil, BS, Rapid adsorptive separation of citrus polymethoxylated flavones in non-aqueous conditions, Separation and purification Technology 45: 147-152, 2005> It is confirmed through melanoma B16 / F10 cells whether or not (these documents are regarded as part of the present specification) has activity to promote melanin production.

As a result, it was confirmed that all five compounds have the activity of promoting the production of melanin in the melanoma B16 / F10 cells, and furthermore, only in the nobilete, the inventors showed that the melanin production promoting activity is most excellent The molecular mechanism of melanin-promoting activity of melanin was investigated by increasing the concentration of cAMP, activating EPK1 / 2, and increasing melanin-producing tyrosinase. It was also confirmed that it promotes production.

In view of the experimental results described above, in one aspect, the present invention can be grasped as a melanin hypopigmentation disease improver composition comprising the compound of the formula (1) as an active ingredient.

<Formula 1>

Wherein R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ is H or OMe, R ₇ is OMe and R ₈ points to H.

The compound of Formula 1 preferably refers to each of the five compounds.

Melanin hypopigmentation disease improving composition of the present invention can be used in any disease caused by melanin production, whether the disease caused by the loss of melanin cells or the disease caused by inhibition of melanogenesis, does not reach the normal level, For example, it can be used for alleviating vitiligo, albinism, depigmentation nevus, white nasal ganglia, stings, post-inflammatory bleaching, ecchymosis, partial leukemia, idiopathic red hypopigmentation, pointy leukemia.

Preferably, the melanin hypopigmentation disease improving composition of the present invention is a disease caused by a decrease in melanogenesis, namely whiteness, depigmentation nevus, white nasal mucus, stinging, post-inflammatory bleaching, ecchymoses, partial leukemia, idiopathic red hypopigmentation It is used when it is used for symptom, mumps leukemia, and the like.

As used herein, "improved" is meant to include the prevention and treatment of diseases, including alleviation of symptoms.

The hypopigmentation disorder improving agent composition of the present invention may include the compound of <Formula 1> as an active ingredient in any amount according to the use, formulation, formulation purpose, etc., usually 0.001 based on the total weight of the composition Weight to 15% by weight.

The hypopigmentation disorder improving agent composition of the present invention may further include a known compound or composition known to have a melanin production promoting effect in order to increase and enhance the melanin production promoting effect of the compound of Formula 1 as an active ingredient in the composition. Such compounds or compositions may include the substances exemplified above for treating melanin hypopigmentation disease, extracts of Gopipi (Korean Patent Application No. 2003-0056823), extracts of Taraxacum officinale and its active ingredient. Phosphorus lufeol and its analogues (Biological and Pharmaceutical Bulletin by Keishi Hata et al., 2000, Vol. 23, No. 8, p.962 to 967; Journal of Natural Products, Keishi Hata et al., 2002, Vol. 65, p.645 to 648 , Biological and Pharmaceutical Bulletin (Sonchus asper) and Sonchus oleraceus (Keishi Hata et al., 2000), Vol. 23, No. 8, p.962 ~ 96 7), extract of King Ddung, and its active ingredient, rufeol fatty acid ester and its analogues (WO 2004/112825), melanocyte stimulating hormone (α-MSH, β-glutamyl, β-arginyl MSH), and the like. Can be mentioned.

In another aspect, the present invention can be understood as a composition for promoting melanin production comprising the compound of Formula 1 as an active ingredient.

<Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ points to H.

The composition for promoting melanin production of the present invention may be used for the treatment or prevention of melanin hypopigmentation disease, but may also be used for blackening or browning of hair, prevention of white hair, and blackening of skin for cosmetic purposes. Therefore, the composition for promoting melanin production of the present invention should be understood as a meaning including all of the composition for blackening or browning of the hair, the composition for preventing gray hair, and the composition for skin blackening.

In the melanin composition of the present invention, the melanin hypopigmentation disorder improving agent composition of the present invention in relation to a preferred embodiment of the compound of <Formula 1>, which is an active ingredient, its content, and a known compound or composition for increasing and enhancing the effect The foregoing is valid as is.

Melanin hypopigmentation disease improving composition and the composition for promoting melanin production of the present invention can be used as a cosmetic composition in a specific embodiment.

When the composition of the present invention is used as a cosmetic composition, the cosmetic composition of the present invention does not attenuate the melanin production promoting effect of the compound of <Formula 1> as an active ingredient, in addition to including the compound of <Formula 1> as an active ingredient. As long as the human body is not toxic as long as the human body is suitable for contact with the skin, it may be prepared including components conventionally used for preparing cosmetic compositions.

Such conventional ingredients include, for example, oils, surfactants, humectants, polyalcohols, thickeners, water soluble polymers, film formers, water-insoluble polymers, powders, pigments, dyes, lakes, lower alcohols, ultraviolet absorbers, metal ion chelating agents, organics Amines, pH adjusters, drug active ingredients, sugars, preservatives, antioxidants, fragrances, water and the like.

Since the components commonly used in the preparation of the cosmetic composition are already known in the art, those skilled in the art can select and use appropriate components. For example, as the oil, olive oil, avocado oil, castor oil, palm oil, etc. As the surfactant, polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, glycerin fatty acid ester, polyoxyethylene hardened castor oil and the like can be used, and as a moisturizing agent, glycerin, 1,3-butylene glycol, polyethylene glycol Carboxyvinyl polymer, carboxymethyl cellulose, etc. may be used as the thickener, and paraamino benzoic acid, octylmethoxycinnamate, 2-ethoxyethyl-p-methoxycinnamate, etc. may be used as the sunscreen. As the amines, monoethanolamine, Triethanolamine etc. can be used, Tocopherols, dibutylhydroxytoluene, etc. can be used as antioxidant, Ethyl paraben, butyl paraben, sodium benzoate, etc. can be used as an antiseptic.

Components commonly used in the manufacture of cosmetics may be included in the range of 85% to about 99.99% by weight based on the total weight of the composition of the present invention.

Cosmetic compositions of the present invention can be prepared in a variety of forms, including emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and water-based), anhydrous products (oils and glycols) ), Gels, masks, packs, powders and the like.

In another specific embodiment, the compositions of the present invention can be used as pharmaceutical compositions.

The pharmaceutical composition of the present invention may include known pharmaceutically acceptable ingredients in addition to the compound of <Formula 1> as an active ingredient. These pharmaceutically acceptable ingredients are commonly used in pharmaceutical formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

The pharmaceutical compositions of the present invention may further comprise lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives and the like. Components that can be used as such lubricants, wetting agents, and the like are known in the art and those skilled in the art can select and use appropriate components as needed.

Such ingredients may be included in the pharmaceutical composition of the present invention in an amount of about 85% to about 99.99% by weight relative to its total weight.

On the other hand, the pharmaceutical composition of the present invention can be administered by various routes, for example, orally or rectally, or by injection into veins, muscles, subcutaneous, intrauterine dura, and the like. It is preferably the case where it is applied directly to the skin or hair.

The pharmaceutical compositions of the present invention may be prepared in unit dose form or formulated into pharmaceutically acceptable carriers and / or excipients or incorporated into multi-dose containers. In this case, the formulation may be in the form of a solution, suspension, or emulsion, or may be in the form of elexirs, extracts, powders, granules, tablets, ointments, and the like.

The daily dosage of the pharmaceutical composition of the present invention is usually 0.001 ~ 150 mg / kg body weight range, it can be administered once or divided into several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.

The composition of the present invention may be regarded as a food composition in specific embodiments.

The food composition of the present invention may be prepared from gums, vitamin complexes, dietary supplements, special nutritional supplements, functional drinks, and the like.

The food composition of the present invention, in addition to the compound of the formula (1), glucose, monosaccharides such as fructose, disaccharides such as maltose, sucrose, or polysaccharides such as dextrin, cyclodextrin can be added, xylitol, sorbitol Sugar alcohols, such as erythritol, may also be added, sweeteners such as taurine, stevia extract, and the like may also be added, and other food additives may also be added.

Meanwhile, the composition for promoting melanin production in the composition of the present invention, specifically, the composition for skin blackening, the composition for blackening or browning hair, and the composition for preventing white hair can also be used as a soap composition.

When the composition for promoting melanin production of the present invention is used as a soap composition, the soap composition of the present invention may be prepared including components commonly used in soap formulations in addition to the compound of <Formula 1> as an active ingredient.

Such components include soap bases, skin moisturizers, emulsifiers, water softeners, antibacterial agents, dispersants, antifoam agents, solvents, scale inhibitors, corrosion inhibitors, flavoring agents, pigments, metal ion sequestrants, antioxidants, preservatives and the like.

The components may be those known in the art, for example, as a soap base, vegetable oils such as palm oil, palm oil, soybean oil, palm oil, olive oil, palm kernels or animal fats and oils such as tallow, pork, sunny, fish oil, etc. may be used. As the skin moisturizing agent, materials exemplified in connection with the cosmetic composition of the present invention, such as glycerin, may be used. As the emulsifier, natural oil, wax, fatty alcohol, hydrocarbons, and the like may be used. Sodium ID, etc. can be used.

In the soap composition of the present invention, the components may be included in any amount as long as there is no toxicity above the adaptable to the human body without inhibiting the melanin production promoting effect of the compound of the general formula (1) as an active ingredient, soap typically 85% to 99.99% by weight based on the total weight of the composition.

In the composition for promoting melanin production of the present invention, the composition for blackening or browning hair and the composition for preventing gray hair can be used as a shampoo composition.

When the composition for promoting melanin production of the present invention is used as a shampoo composition, the shampoo composition of the present invention may be prepared including components commonly used in shampoo formulations in addition to the compound of <Formula 1> as an active ingredient, The components include surfactants, foam enhancers, emulsifiers, thickeners, perfumes, preservatives, pH adjusters, fungicides, anti-dandruff agents, tonic agents, moisturizers and the like.

The surfactants, bubble enhancers, emulsifiers, thickeners, perfumes (hydrotrope), preservatives, pH adjusters, fungicides, anti-dandruff agents, tonic agents and the like sugars such as those exemplified in relation to the cosmetic composition of the present invention Any known in the art can be used.

The above components may also be included in any amount so long as they do not poison the human body and do not inhibit the melanin production promoting effect of the compound of Formula 1, which is typically based on the total weight of the sampu composition. When it will contain from 85% to 99.99% by weight.

As described above, the present invention provides a melanin hypopigmentation disease improving composition. Also provided in accordance with the present invention is a melanin production promoter composition.

The compositions may be used in the form of cosmetic compositions, pharmaceutical compositions, food compositions or soap compositions and shampoo composition.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Melanin production promoting activity measurement experiment

Melanoma B16 / F10 cells were dispensed in 6-well plates at 5 × 10 ⁴ cell counts / mL and 37 ° C., 5% CO ₂ for 24 h. After incubation under the conditions, five samples, such as nobiliretin, anthrax retinin, and sinencetin, were treated at a concentration of 10 μM and incubated for 36 hours. The medium was exchanged, and the above-described samples of nobilithin and the like were treated again at the same concentration and further incubated for 36 hours. Meanwhile, in another experiment, in order to compare the melanin production promoting effect of α-MSH, which is a melanin biosynthesis inducing substance, with the melanin production promoting effect of the sample such as nobilithin, α-MSH was treated with 0.1 μM and cultured under the same conditions. After culturing for a total of 72 hours, cells were harvested using trypsin-EDTA. 500 μl of 67 mM phosphate buffer solution (pH 6.8) containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 1% Triton X-100 was added, and the cells were crushed using an ultrasonic grinder and stored on ice for 1 hour, followed by centrifugation. To separate the precipitate. 1 N NaOH solution containing 10% DMSO was added to the precipitate and dissolved in a 65 ° C. constant temperature water bath for 3 hours, after which the melanin content was analyzed. Melanin content was evaluated by measuring the absorbance at 405 nm, expressed as a relative melanin content compared to the control (untreated group of samples such as nobiletein).

As a result, as shown in Figure 1, all samples such as nobiliretin, anthletin, sinencetin significantly promoted melanin biosynthesis in B16 / F10 melanoma cells (Fig. 1).

In Figure 1, A, B, C, D and E are nobiliretin, tangeretine, sinencetin, 3,5,6,7,8,3 ', 4'-heptamethoxyflavone (3,5,6, 7,8,3 ', 4'-heptamethoxyflavone) and 5-hydroxy-6,7,8,3', 4'-pentamethoxyflavone (5-hydoxy-6,7,8,3 ', 4' -pentamethoxyflavone).

Example 2 Experiment for Molecular Mechanism of Melanin Promoting Activity

In order to confirm whether the melanin production is promoted by the molecular mechanism only for the nobilete shown to be the highest activity in the <Example 1>, the following experiment was carried out.

<Example 2-1> Tyrosinase activity and production promoting activity measurement experiment of nobilete

In this experiment, it was confirmed whether the melanogenesis-promoting activity of nobilithin is due to the increased expression of tyrosinase. Melanoma B16 / F10 cells were dispensed in 6-well plates at 5 × 10 ⁴ cell counts / mL and 37 ° C., 5% CO ₂ for 24 h. After incubation in the conditions, the nobiletin was treated at concentrations of 0 (untreated), 2.5, 5, 10, 20 μM and incubated for 36 hours. The medium was exchanged and treated again with the same concentration of nobililet and then incubated for another 36 hours.

After culturing for a total of 72 hours, cells were harvested using trypsin-EDTA. 500 μl of 67 mM phosphate buffer solution (pH 6.8) containing 0.2 mM PMSF and 1% Triton X-100 was added and the cells were crushed using an ultrasonic grinder and stored on ice for 1 hour, followed by centrifugation to obtain supernatant. It was. Using the isolated supernatant as an enzyme source, the tyrosinase activity contained in the cells was analyzed. Tyrosinase activity was analyzed by measuring the color of DOPA chrome produced by the enzymatic reaction of tyrosinase by colorimetric method. To 120 µl of 67 mM phosphate buffer (pH 6.8), 5 ml of 1.5 µM L-tyrosine and 40 µl of 25 µM L-DOPA were added as substrates, and 100 µl of the supernatant as an enzyme source. The reaction was carried out for a minute. The resulting waveguide (L-DOPA) and dopachrome (DOPA chrome) were measured at 475 nm. The enzyme activity was measured by comparing the standard curve of isolated mushroom tyrosinase (0.39-25 Units), and the enzyme activity was corrected by the amount of protein.

In another experiment, we examined the tyrosinase expression by Western blot after culturing the cells under the same conditions to observe whether the inhibition of tyrosinase activity of nobiletein is due to the inhibition of tyrosinase expression. After cell culture, the medium was removed, the cells were washed with phosphate buffer solution, and the whole protein was extracted with lysis buffer. The extract was centrifuged and the supernatant was taken to quantify the protein with protein quantitative reagent (Bio-Rad). 30 μg of protein was electrophoresed on a 10% SDS-polyacrylamide gel and the protein was transferred to PVDF membrane. After blocking for 1 hour with 5% whole milk powder, an antibody against tyrosinase diluted in an appropriate ratio (Santa-Cruz, USA) was added and reacted at 4 ° C. overnight. Thereafter, the mixture was washed 5 times with TTBS (Tween 20 / Tris-buffered saline) solution for 5 minutes. Next, a goat secondary antibody diluted in an appropriate ratio was added and reacted at room temperature for 1 hour.

Again rinsed with TTBS solution 3 times at 5 minute intervals and then developed.

The results are shown in FIG. As shown in FIG. 2, nobiliretin increased the enzyme activity of intracellular tyrosinase concentration-dependently and increased the protein expression of intracellular tyrosinase.

Example 2-2 Experimental Observation of Protein Expression Patterns of Signaling System Related to Melanin Biosynthesis by Nobilete

In this experiment, we observed the expression patterns of Trp-1 / 2, ERK 1/2 and tyrosinase, which are known to be the enzyme related to melanogenesis.

Nobilete was treated under the same conditions as in <Example 2-1>, and melanoma B16 / F10 cells were cultured.

After culturing for a total of 72 hours, the medium was removed, the cells were washed with phosphate buffer solution, and the whole cell protein was extracted with lysis buffer. The extract was centrifuged and the supernatant was taken to quantify the protein with protein quantitative reagent (Bio-Rad). 30 μg of protein was electrophoresed on a 10% SDS-polyacrylamide gel and the protein was transferred to PVDF membrane. After blocking for 1 hour with 5% whole milk powder, a solution diluted with an appropriate ratio of the antibody to the protein to be observed was added and reacted at 4 ° C. overnight. The mouse monoclonal antibody anti-phospho-ERK1 / 2 (Santa-Cruz, USA) was used to observe the phosphorylated ERK1 / 2 expression. Tyrosinase was a goat polyclonal antibody anti-Tyrosinase (Santa-Cruz, USA). , Trp-1 is goat polyclonal antibody anti-Trp-1 (Santa-Cruz, USA), Trp-2 is rabbit polyclonal antibody anti-Trp-2 (Santa-Cruz, USA), β-actin is mouse monoclonal Antibody anti-β-actin (Sigma, USA) was used. Thereafter, the mixture was washed 5 times with TTBS (Tween 20 / Tris-buffered saline) solution for 5 minutes. Next, a mouse, rabbit or goat secondary antibody diluted in an appropriate ratio was added and reacted at room temperature for 1 hour. Again rinsed with TTBS solution 3 times at 5 minute intervals and developed.

The results are shown in FIG. As shown in FIG. 3, nobilithin increased the protein expression of tyrosinase, Trp-1, which is involved in melanin biosynthesis, and continuously activated ERK1 / 2 until 72 hours. However, expression of the Trp-2 protein was not affected by nobilithin.

<Example 2-3> Nobile retinoic cAMP concentration increasing activity measurement and cell morphology observed in the experiments

Previously, it was confirmed that nobilithin continuously activates ERK1 / 2, and in this experiment, the effect of nobilithin on the concentration of intracellular cyclic AMP (cAMP) was observed.

B16 / F10 melanoma cells were dispensed in 6-well plates at 5 × 10 ⁴ cell count / ml and maintained at 37 ° C., 5% CO ₂ for 24 h. After culturing under conditions, nobilithin was treated with a concentration of O, 20 μM and further incubated for 24 hours. After incubation, the cell morphology was observed under an optical microscope (Olympus, Japan). In order to measure the concentration of intracellular cyclic AMP, cells were incubated for 48 hours after treatment with nobililet under the same conditions. After incubation, the medium was removed, the cells were washed with phosphate buffer solution, lysis buffer was added, lysed for 30 minutes and centrifuged to remove cell membrane components and the like. Intracellular cyclic AMP content was measured using a cyclic AMP assay kit (R & D systems, USA).

The results are shown in FIG. As shown in FIG. 4, the nobilithin (20 μM) of the present invention increased the concentration of intracellular cyclic AMP in B16 / F10 melanoma cells by about two times as compared to the control (nobilitin untreated group). These results indicate that nobiletein continuously activates ERK1 / 2 by increasing the concentration of cyclic AMP, the intracellular secondary signaler, increasing the expression of tyrosinase and subsequently promoting melanin biosynthesis.

1 is an experimental result showing that a polymethoxyflavone-based compound such as nobilithin has an activity of promoting melanin production in melanoma B16 / F10 cells.

2 is an experimental result showing that nobilitin promotes tyrosinase activity and increases the expression of tyrosinase.

Figure 3 is an experimental result showing that nobiletein increases the protein expression of tyrosinase, Trp-1 involved in melanin biosynthesis, and continuously activates ERK1 / 2.

Figure 4 shows that nobilithin increases the concentration of intracellular cyclic AMP in B16 / F10 melanoma cells.

Claims (19)

Melanin hypopigmentation disease improving agent pharmaceutical composition comprising a compound of the formula (1) as an active ingredient. <Formula 1>

Wherein R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ is H or OMe, R ₇ is OMe and R ₈ is H. The method of claim 1, The compound of <Formula 1> is nobiliretin, sinencetin, tangeretine, 3,5,6,7,8,3'4'-heptamethoxyflavone, or 5-hydroxy-6,7,8,3 Melanin hypopigmentation disease improver pharmaceutical composition, characterized in that the ', 4'-pentamethoxy flavone. The method of claim 1, The melanin hypopigmentation disease is melanin hypopigmentation disease, characterized by vitiligo, albinism, depigmentation nevus, white nasal cavity, rash, post-inflammatory depigmentation, ecchymoses, partial leukemia, idiopathic hypopigmentation or pointy leukemia Enhancer drug composition. A pharmaceutical composition for promoting melanin production comprising the compound of Formula 1 as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H. The method of claim 4, wherein The compound of <Formula 1> is nobiliretin, sinencetin, tangeretine, 3,5,6,7,8,3'4'-heptamethoxyflavone, or 5-hydroxy-6,7,8,3 A pharmaceutical composition for promoting melanin production, which is', 4'-pentamethoxyflavone. delete Melanin hypopigmentation disease improving agent food composition comprising a compound of the formula (1) as an active ingredient. <Formula 1>

Wherein R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ is H or OMe, R ₇ is OMe and R ₈ is H. delete Food composition for promoting melanin production comprising the compound of the formula (1) as an active ingredient. <Formula 1>

Wherein R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ is H or OMe, R ₇ is OMe and R ₈ is H. Soap composition for promoting melanin production comprising the compound of the formula (1) as an active ingredient. <Formula 1>

Wherein R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ is H or OMe, R ₇ is OMe and R ₈ is H. Sampo composition for promoting melanin production comprising the compound of the formula (1) as an active ingredient. <Formula 1>

Wherein R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ is H or OMe, R ₇ is OMe and R ₈ is H. A pharmaceutical composition for preventing white hair, comprising the compound of Formula 1 as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H. The method of claim 12, The compound of <Formula 1> is nobiliretin, sinencetin, tangeretine, 3,5,6,7,8,3'4'-heptamethoxyflavone, or 5-hydroxy-6,7,8,3 Anti-white hair pharmaceutical composition, characterized in that the ', 4'-pentamethoxy flavone. A pharmaceutical composition for skin blackening comprising the compound of the following <Formula 1> as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H. The method of claim 14, The compound of <Formula 1> is nobiliretin, sinencetin, tangeretine, 3,5,6,7,8,3'4'-heptamethoxyflavone, or 5-hydroxy-6,7,8,3 A pharmaceutical composition for skin blackening, characterized in that the ', 4'-pentamethoxy flavone. Cosmetic composition for preventing white hair comprising the compound of the formula (1) as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H. White hair anti-steam composition comprising the compound of the formula (1) as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H. Cosmetic composition for skin blackening comprising the compound of the formula <1> as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H. Soap composition for skin blackening comprising the compound of the formula (1) as an active ingredient. <Formula 1>

Likewise, R ₁ is H or OMe (methoxy group), R ₂ is OH (hydroxy group) or OMe, R ₃ is OMe, R ₄ is OMe, R ₅ is H or OMe, and R ₆ Is H or OMe, R ₇ is OMe and R ₈ is H.

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