KR102502972B1 - A composition for promoting melanin synthesis comprising syringetin - Google Patents

Abstract

The present invention relates to a composition for promoting melanin production comprising syringentin or a physiologically acceptable salt thereof, wherein syringentin promotes tyrosinase activity, melanin-related enzyme (TRP-1) It was confirmed that it increases the expression of protein and promotes melanin biosynthesis, and it can be used for improvement, prevention, or treatment of vitiligo, gray hair, or hypopigmentation by promoting melanin production.

Description

A composition for promoting melanin production comprising syringetin {A composition for promoting melanin synthesis comprising syringetin}

The present invention relates to a composition for generating melanin comprising syringetin or a physiologically acceptable salt thereof.

Melanin, which is produced by melanocytes present in the basal layer of the epidermis of human skin, refers to a phenolic polymer material having a complex form of black pigment and protein. The melanin determines the skin color of a person according to its concentration and distribution, and also serves to protect the human skin from ultraviolet rays.

Excessive production of melanin causes pigmentation such as spots and freckles. However, on the contrary, if the production of melanin is excessively suppressed, skin lesions of vitiligo may appear, the number and function of melanocytes in the hair root may decrease, resulting in gray hair, or hypopigmentation, which may become a risk factor for skin cancer.

In many patients with vitiligo, white spots are known to have a great impact on the quality of life, and recent newspaper articles reported that vitiligo patients have serious problems with employment and interpersonal relationships. This means that while vitiligo may seem like a minor disorder, it can affect psychological self-esteem and social functioning in people with severe hypopigmentation disease.

White hair, a disease similar to vitiligo, is also attracting attention as interest in appearance increases, and it is becoming a research subject in terms of health and beauty and psychology. As with vitiligo, the more white the hair, the more depressed or stressed, and feel uncomfortable in interpersonal relationships or social activities.

Currently, in the treatment of vitiligo, topical corticosteroids, calcineurin inhibitors, vitamin D derivatives, phototherapy (UVA, narrow band UVB, photochemotherapy), surgical treatment to transplant epidermis with normal melanocytes, and local treatment Various treatments, including a combination of light therapy and phototherapy, are used, but have side effects such as increasing the incidence of non-melanoma skin cancer and melanoma.

In addition, various hair dyes are currently being used as a solution to gray hair. However, since these permanent hair dyes are made of vegetable hair dyes, metallic hair dyes, synthetic hair dyes, etc., they have side effects such as damage and irritation to hair and scalp due to chemical action.

However, since these methods are not fundamental prevention, improvement, or treatment methods, research on inducing blackening by promoting melanin production is required for a more fundamental solution.

Republic of Korea Patent No. 10-1669359

An object to be solved by the present invention is to provide a composition for promoting melanin production comprising syringentin or a physiologically acceptable salt thereof as an active ingredient.

In addition, to provide a composition for preventing, treating, or improving vitiligo, gray hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient, and the composition includes a cosmetic composition, a pharmaceutical composition, and a food composition.

In order to solve the above problems, the present invention provides a composition for promoting melanin production comprising syringentin or a physiologically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a cosmetic composition for preventing or improving vitiligo, gray hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating vitiligo, gray hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving vitiligo, gray hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

In the present invention, it was confirmed that syringentin promotes melanin biosynthesis, tyrosinase activity, and TRP-1 expression. Syringentin promotes melanin production in the skin or hair root to induce blackening, and thus It can be used for improvement, prevention or treatment of melanin deficiency diseases (Vitiligo, gray hair or hypopigmentation).

1 is a result of analyzing B16F10 cell viability according to the treatment concentration of syringetin. Data are expressed as mean ± standard deviation (SD) for at least 4 independent experiments (n = 4). *p <0.05, **p <0.01, ***p <0.001
Figure 2 is a result of analyzing the change in melanin content according to the treatment concentration of syringetin (syringetin) in B16F10 cells. B16F10 cells were treated with syringetin at a concentration of 0.75 to 3 μg/ml for 72 hours, and α-MSH was used as a positive control. Melanin content was expressed as a percentage compared to the measured values of control cells (untreated). Data are expressed as mean ± standard deviation (SD) for at least 4 independent experiments (n = 4). *p <0.05, **p <0.01, ***p <0.001
Figure 3 is a result of analyzing the change in tyrosinase activity according to the treatment concentration of syringetin (syringetin) in B16F10 cells. B16F10 cells were treated with syringetin at a concentration of 0.75 to 6 μg/ml for 72 hours, and α-MSH was used as a positive control. Melanin content was expressed as a percentage compared to the measured values of control cells (untreated). Data are expressed as mean ± standard deviation (SD) for at least 4 independent experiments (n = 4). ***p < 0.001
Figure 4 is a result of analyzing TRP-1 expression changes according to syringetin (syringetin) in B16F10 cells. Protein expression levels were analyzed by Western blot. Western analysis of TRP-1 expression in cells treated with syringetin at 1.5 to 6 μg/ml (a) was performed using beta actin as a standard, and the percentage of expression level compared to that of the control group was graphed (b). Data are expressed as mean ± standard deviation (SD) for at least 4 independent experiments (n = 3). ***p < 0.001

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

In one aspect, the present invention relates to a composition for promoting melanogenesis comprising syringentin or a physiologically acceptable salt thereof as an active ingredient.

Syringetin in the present invention is a compound having the structure of Formula 1.

The syringentin is 3',5'-O-dimethylmyricetin (3',5'-O-Dimethylmyricetin), 3',5'-dimethoxy-3,5,7,4'-tetrahydride Roxiflavone (3',5'-Dimethoxy-3,5,7,4'-tetrahydroxyflavone) or 3,5,7,4'-tetrahydroxy-3',5'dimethoxyflavone (3,5,7 ,4'-Tetrahydroxy-3',5'dimethoxyflavone).

In the present invention, "physiologically acceptable salt" means any salt that has the desired biological or physiological activity of the compound and exhibits minimal undesirable toxicological effects. It means a salt prepared according to a conventional method in the art, and such a preparation method is known to those skilled in the art. Specifically, the physiologically acceptable salt includes, but is not limited to, cosmetically, pharmacologically, or physiologically acceptable salts derived from inorganic and organic acids and bases. Examples of suitable acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium or potassium, alkaline earth metals such as magnesium.

In addition, the composition of the present invention may include not only syringentin represented by Formula 1 or a physiologically acceptable salt thereof, but also an isomer thereof or a solvate or hydrate that may be prepared therefrom.

In one embodiment of the present invention, it was confirmed that syringentin maintains a cell viability of 90% or more at a concentration of 6.25 μg/ml or less in B16F10 cells, promotes melanin production, promotes tyrosinase activity, As confirmed by increasing the protein expression of an enzyme involved in melanin production (TRP-1), a composition containing syringentin or a physiologically acceptable salt thereof is characterized in that it promotes melanin production.

The composition for promoting melanin production of the present invention can be used to alleviate, improve, prevent, improve, or treat vitiligo, gray hair, or hypopigmentation caused by melanin deficiency by increasing melanin biosynthesis in skin or hair follicle cells.

In another aspect, the present invention relates to a cosmetic composition for preventing or improving vitiligo, gray hair, or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

The cosmetic composition of the present invention contains a composition for promoting melanin production containing syringentin or a physiologically acceptable salt thereof as an active ingredient to promote melanin production in the skin or hair root, resulting in vitiligo, gray hair or Shows the effect of preventing or improving hypopigmentation.

The "vitiligo" refers to an acquired depigmentation disease in which white spots of various sizes and shapes appear on the skin due to destruction of melanocytes due to an abnormality in autoimmune function.

The “gray hair” refers to a phenomenon in which the hair turns white as the number and function of melanocytes in the hair root decrease.

The "hypochromatism" refers to a disease in which the skin at the lesion site temporarily turns white due to functional disorders in melanocytes locally due to skin diseases such as eczema, atopic dermatitis, and inflammatory dermatitis.

In the present invention, "prevention, improvement, or treatment of vitiligo, gray hair, or hypopigmentation" is to prevent, improve, or treat symptoms of vitiligo, gray hair, or hypopigmentation by producing melanin in melanocytes present in skin or hair follicles. .

In addition to syringentin, the cosmetic composition of the present invention may further contain a compound or natural extract known to have a melanin production promoting effect so as to increase or reinforce the effect of preventing or improving vitiligo, gray hair or hypopigmentation.

In the cosmetic composition for preventing or improving vitiligo, gray hair, or hypopigmentation of the present invention, the active ingredient promotes melanin production to exhibit an effect of preventing or improving vitiligo, gray hair, or hypopigmentation, use, dosage form, It may be included in an arbitrary amount (effective amount) depending on the purpose of blending, etc., and a typical effective amount may be included within the range of 0.001% by weight to 99.99% by weight based on the total weight of the composition. Here, "effective amount" refers to the amount of an active ingredient capable of inducing antioxidant, anti-inflammatory or antibacterial effects. Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

The cosmetic composition of the present invention may be prepared in various forms, for example, the cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, a solution, suspension, paste, gel, cream , Lotion, powder, cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray may be formulated, but is not limited thereto. In addition, specifically, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, essence, nutrient essence, pack, soap, shampoo, It may have a formulation selected from the group consisting of cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder, and loose powder, but is not limited thereto.

The cosmetic composition of the present invention may include a carrier acceptable in cosmetic formulations in addition to the active ingredient. Here, "acceptable carrier in cosmetic formulations" is a compound or composition that is already known and used that can be included in cosmetic formulations, or a compound or composition to be developed in the future, which does not have toxicity, instability, or irritation more than the human body can adapt to when in contact with the skin. say that The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% to about 99.99% by weight, preferably about 5% to about 99% by weight, based on the total weight of the composition. However, since the ratio varies depending on the formulation of the cosmetic composition of the present invention and its specific application area (face or hand) or its preferred application amount, the ratio is intended to limit the scope of the present invention in any aspect. should not be understood

Meanwhile, as the carrier, alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, sunscreen, coloring agent, fragrance, and the like may be exemplified. Alcohols, oils, surfactants, fatty acids, silicone oils, humectants, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, coloring agents, compounds/compositions that can be used as fragrances, etc. that can be used as the carrier are already known in the art. Therefore, those skilled in the art can select and use an appropriate material/composition.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating vitiligo, gray hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

Description of the composition for promoting melanin production, vitiligo, white hair, hypopigmentation, prevention or treatment in the present invention is as described above.

The pharmaceutical composition of the present invention contains a composition for promoting melanin production containing syringentin or a physiologically acceptable salt thereof as an active ingredient to promote melanin production in the skin or hair root, thereby preventing vitiligo and gray hair due to melanin deficiency. or exhibits a preventive or therapeutic effect on hypopigmentation.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable diluent, excipient or carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits characteristics that are not toxic to cells or humans exposed to the composition.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried agents, and suppositories. It may have a dosage form, and may be various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, and disease. It may be determined according to factors including type, activity of drug, sensitivity to drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject by any suitable method, and the route of administration of the composition can be various oral or parenteral routes as long as it can reach the target tissue. It can be administered through, specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalational or intradermal routes.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of treating vitiligo, gray hair, or hypopigmentation, and any one can be applied. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, humans, birds and fish may be used, and the pharmaceutical composition may be used for parenteral, It can be administered subcutaneously, intraperitoneally, intrapulmonaryly and intranasally, and for local treatment, it can be administered by any suitable method including, if necessary, intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the subject, the severity of the disease, the drug type, the route and duration of administration, but can be appropriately selected by those skilled in the art.

A suitable total daily amount can be determined by a treating physician within the scope of sound medical judgment, and is generally 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/kg. The amount of kg can be divided and administered once a day to several times.

In another aspect, the present invention relates to a food composition for preventing or improving vitiligo, gray hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

Description of the composition for promoting melanin production, vitiligo, white spots, hypopigmentation, prevention or improvement in the present invention is as described above.

The food composition of the present invention contains a composition for promoting melanin production containing syringentin or a physiologically acceptable salt thereof as an active ingredient to promote melanin production in the skin or hair root, resulting in vitiligo, gray hair or Shows the effect of preventing or improving hypopigmentation.

The food composition of the present invention may include pills, powders, granules, infusions, tablets, capsules, liquids, etc., and foods to which syringentin or a physiologically acceptable salt thereof of the present invention may be added. There is no particular limitation on the type of , and there are, for example, various beverages, chewing gum, tea, vitamin complexes, health supplements, and the like.

In addition to syringentin, other ingredients may be added to the food composition, and the type is not particularly limited. For example, it may contain various herbal extracts, food-acceptable food additives, or natural carbohydrates as additional ingredients, like conventional foods, but is not limited thereto.

In the present invention, the term "food supplement additive" refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, etc.), stevia extract (rebaudioside A, glycir hijin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can advantageously be used.

The food composition of the present invention may include health functional food. The term "health functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. Here, functional means obtaining useful effects for health purposes such as adjusting nutrients for the structure and function of the human body or physiological functions.

The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a drug for a long time by using food as a raw material, and it can be excellent in portability.

The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food, the delon of the present invention may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount below the above range may be used.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, etc., and includes all health functional foods in a conventional sense.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Experiment method>

1. Cell culture

Mouse melanoma cells (B16F10, mouse melanoma cells) were purchased from ATCCⓡ (The Global Bioresource Center). Cells were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO ₂ conditions. The cells were subcultured at 3 × 10 ⁵ cells/100π dish in a 3-day cycle.

2. Cytotoxicity assessment

Cytotoxicity according to the sample concentration was evaluated using the MTT assay. The MTT experiment is an experiment to evaluate the metabolic activity of colorimetric assay cells. When cells are treated with yellow water-soluble MTT reagent by NAD (P) H- dependent cellular oxidoreductase enzyme in the mitochondria of living cells, purple-violet insoluble formazan is produced. is returned to At this time, the degree of cell survival or death can be evaluated by measuring the amount of reduced formazan. The higher the formazan concentration, the deeper the purple color and the higher the absorbance.

B16F10 cells were placed in a 24-well plate at a concentration of 0.8 × 10 ⁴ cells/well at a concentration of 500 μl/well, cultured for 24 hours in DMEM medium containing 10% FBS and 1% penicillin/streptomycin, and Syringetin was treated at concentrations of 0, 0.75, 1.5, 3, 6, and 12.5 μg/ml. After 72 hours, 500 μl/well of 0.2 mg/ml MTT diluted with a medium of 5 mg/ml was added and reacted for 4 hours. Then, remove the supernatant, add 500 μl/well of DMSO, dissolve formazan for 30 minutes, transfer 100 μl of the solution to a 96-well plate, and use a microplate reader (Tecan, Mannedorf, Switzerland). The absorbance was measured at a wavelength of 560 nm.

3. Measurement of melanin content

B16F10 cells were placed in a 6-well dish at a concentration of 0.5 × 10 ⁵ cells/well (cells/well) at a concentration of 3000 μL each, cultured for 24 hours in DMEM medium containing 10% FBS and 1% penicillin/streptomycin, and then syringetin (syringetin ) was treated for 72 hours at concentrations of 0, 0.75, 1.5, and 3 μg/ml. At this time, α-MSH (200 nM) was used as a positive control.

After 72 hours of sample treatment, the supernatant was removed, washed twice with 1 × PBS, and 200 μl of RIPA buffer (1% inhibitor cocktail) was added, followed by reaction at 4 ° C for 30 minutes, and then in a 1.5 mL e-tube. Cells were recovered. The recovered cells were centrifuged at 15,000 rpm, -8 ° C for 20 minutes using a deep freezer centrifuge, and the supernatant was skimmed off, only the pellet was obtained, and 1 N NaOH (10% DMSO ) into 500 μl, and melanin was dissolved at 70° C. for 1 hour. Thereafter, 100 μl of each dissolved pellet was placed in a 96-well plate, and absorbance was measured at 405 nm using a microplate reader (Tecan, Mannedorf, Switzerland).

4. Assessment of tyrosinase activity

B16F10 cells were put into a 60π dish plate at a concentration of 0.8 × 10 ⁵ cells/well (cells/well) at a concentration of 3000 μl, cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin for 24 hours, and then syringetin (syringetin ) was treated for 72 hours at concentrations of 0, 0.75, 1.5, 3, and 6 μg/ml. At this time, α-MSH (200 nM) was used as a positive control. 72 hours after the sample, the cells were recovered, washed twice with 1 × PBS, 200 μl of RIPA buffer (1% inhibitor cocktail) was added, reacted at 4 ° C for 30 minutes, and then cells were placed in a 1.5 ml e-tube. recovered. The recovered cells were centrifuged in a deep freezer centrifuge at 15,000 rpm, -8 ° C for 20 minutes to skim off the protein supernatant, and then protein assay was performed using the BCA protein assay kit. Concentration was quantified. Each supernatant was diluted to 1 μg/ml, and 20 μl of protein solution and 80 μl of L-Dopa_(phenyl-d3) (2 mg/ml) were added to a 96-well plate, followed by 1 hour and 30 minutes at 37°C. reacted during After that, it was measured at 490 nm using a microplate reader (Tecan, Mannedorf, Switzerland).

5. Western blot: analysis of TRP-1 expression

B16F10 cells were placed in a 60π dish plate at a concentration of 0.8 × 10 ⁵ cells/well, cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin for 24 hours, and then syringetin was added at 0, 0.75, 1.5 , 3, and 6 μg/ml for 48 hours. At this time, α-MSH (200 nM) was used as a positive control.

After 48 hours of sample treatment, the supernatant was removed, washed twice with 1 × PBS, and 200 μl of RIPA buffer (1% inhibitor cocktail) was added and reacted at 4 ° C for 30 minutes. Then, in a 1.5 mL e-tube Cells were recovered. The collected cells were centrifuged at 15,000 rpm, -8 ° C for 20 minutes using a deep freezer centrifuge to skim off the protein supernatant, and then use the BCA protein assay kit The protein concentration was quantified. Equal amounts of protein (10 μg) were loaded on each lane and separated via electrophoresis on a 10% polyacrylamide gel. It was transferred to a nitrile cellulose membrane and incubated with anti-TRP-1 for 12 hours. After that, it was washed with TBST (Tris-Buffered Saline containing Tween 20) and incubated with a secondary antibody (anti-mouse IgG HRP-linked antibody).

Bands showing immunoreactivity were detected using an enhanced chemiluminescence (ECL, Amersham, Bucks, UK) system. The intensity of the band was measured using the Image J program (NIH, Bethesda, MD, USA), and the intensity of the band was evaluated using β-actin as an internal control.

<result>

1. Cytotoxicity evaluation of Syringetin

MTT experiments were conducted to evaluate the effect of Syringetin on cell survival in B16F10 melanoma cells. Syringetin was treated for 72 hours at various concentrations (1.56 to 12.5 μg/ml). Cell viability was evaluated based on 100% of untreated cells.

As a result of the analysis, when treated at a concentration of 12.5 μg/ml, the viability of cells was less than 90%, confirming that some cytotoxicity was exhibited. On the other hand, when treated with a concentration of 6.25 μg / ml or less, it was confirmed that the survival rate was maintained at 90% or more (FIG. 1).

Therefore, in subsequent experiments, the activity was evaluated at concentrations of 6.25 μg/ml or less.

2. Analysis of the melanin synthesis promotion effect of Syringetin

To confirm the effect of Syringetin on melanin synthesis, B16F10 cells were treated with Syringetin at concentrations of 0, 0.75, 1.5, and 3 μg/ml, which do not affect B16F10 cell survival, for 72 hours. processed. As a positive control group, α-MSH (200 nM) was used. As a result of the analysis, it was confirmed that the melanin content tended to increase as the concentration of Syringetin increased from 0.75 μg/ml to 3 μg/ml. Specifically, it was confirmed that the melanin content was highest at a concentration of 3 μg/ml among the treatment concentrations and higher than that of the positive group (FIG. 2).

3. Syringetin's tyrosinase activity promoting effect

From the above experimental results, it was confirmed that Syringetin increases the melanin content, and the effect of Syringetin on the activity of tyrosinase, an important enzyme for melanin synthesis in B16F10 cells, was analyzed. .

B16F10 cells were treated with α-MSH (200 nM) as a positive control, and treated with 1.5, 3, or 6 μg/ml of Syringetin, and changes in tyrosinase activity were analyzed.

As a result of the analysis, it was confirmed that the tyrosinase activity increased in a concentration-dependent manner in the group treated with Syringetin compared to the untreated control group. Specifically, it was confirmed that the tyrosinase activity of the group treated with high concentration (6 μg/ml) of Syringetin increased the most when compared to the control group and the positive control group (FIG. 3).

4. Syringetin promotes TRP-1 expression

In the process of synthesizing melanin, tyrosinase, TRP-1 and TRP-2 play an important role in the process of synthesizing melanin. In the above experiment, it was confirmed that Syringetin increases melanin synthesis and tyrosinase activity. Therefore, in order to prove this more effectively, changes in melanin-related enzyme (TRP-1) protein expression according to the treatment concentration of Syringetin were analyzed. TRP-1 protein expression level was analyzed by performing a western blot.

As a result of the analysis, it was confirmed that Syringetin significantly increased the protein expression of TRP-1 in a concentration-dependent manner (FIG. 4).

Summarizing the above results, it was confirmed that Syringetin promotes TRP-1 protein expression in B16F10 cells, increases tyrosinase activity, and promotes melanin biosynthesis.

Claims (4)

delete A cosmetic composition for preventing or improving vitiligo, white hair, or hypopigmentation, comprising a compound represented by Formula 1 below as an active ingredient.
[Formula 1]

A pharmaceutical composition for preventing or treating vitiligo, white hair, or hypopigmentation, comprising a compound represented by Formula 1 below as an active ingredient.
[Formula 1]

A food composition for preventing or improving vitiligo, white hair, or hypopigmentation, comprising a compound represented by Formula 1 below as an active ingredient.
[Formula 1]

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