KR20220078295A - A composition for promoting melanin synthesis comprising linarin - Google Patents

Abstract

The present invention relates to a composition for promoting melanin production comprising linarin or a physiologically acceptable salt thereof, and it has been confirmed that linarin promotes tyrosinase activity to promote melanin biosynthesis. , it can be used for improving, preventing or treating vitiligo, white hair or hypopigmentation by promoting melanin production.

Description

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

The present invention relates to a composition for promoting melanin production comprising linarin or a physiologically acceptable salt thereof.

Melanin produced in melanocytes existing in the epidermal basal layer of human skin refers to a phenolic polymer having a complex form of black pigment and protein. The melanin also determines the color of a person's skin according to the concentration and distribution, and also serves to protect the skin of the human body from ultraviolet rays.

Excessive production of melanin causes pigmentation such as spots and freckles. On the other hand, if melanin production is excessively suppressed, vitiligo skin lesions may appear, the number and function of melanocytes in the hair root may decrease, resulting in white hair, and hypopigmentation, which may be a risk factor for skin cancer.

In many patients with vitiligo, white spots are known to have a significant impact on the quality of life, and recent newspaper articles reported that vitiligo patients' employment and interpersonal problems were serious. This means that while vitiligo may appear to be a minor disorder, it can affect psychological self-esteem and social activity in people with severe pigment deficiency disease.

White hair, a disease similar to vitiligo, is also attracting attention with increasing interest in appearance, and is being studied in terms of health, beauty, and psychology. As with vitiligo, the more white hair there is, the more psychologically depressed or stressed, the more uncomfortable they are in interpersonal relationships and social activities.

Currently, in the treatment of vitiligo, local corticosteroids, calcineurin inhibitors, vitamin D derivatives, phototherapy (UVA, narrow band UVB, photochemotherapy), surgical treatment by transplanting the epidermis with normal melanocytes, and local treatment Various treatments, including a combination of phototherapy and phototherapy, have been used, but they 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 for graying 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 the hair and scalp due to chemical action.

However, since these methods are not their fundamental prevention, improvement or treatment methods, studies are needed to promote melanin production to induce blackening for a more fundamental solution.

Republic of Korea Patent No. 10-1669359

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

In addition, to provide a composition for preventing, treating or improving vitiligo, white hair or hypopigmentation comprising the composition for promoting melanin production as an active ingredient, the composition comprising 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 linarin or a physiologically acceptable salt thereof as an active ingredient.

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

In the present invention, it was confirmed that linarin promotes melanin biosynthesis and tyrosinase activity, linarin promotes melanin production in the skin or hair root to induce blackening, and thus melanin deficiency disease (Vitiligo) , white hair or hypopigmentation) can be used for improvement, prevention or treatment.

1 is a result of analyzing B16F10 cell viability according to linarin treatment concentration (6.25 to 50 μg/ml).
Each cell was treated with linarin at the indicated concentration and treated for 72 hours. Cell viability was expressed as a percentage relative to untreated controls. Data are expressed as mean ± standard deviation (SD) for at least 3 independent experiments (n = 3). *p <0.05, **p <0.01, ***p <0.001
Figure 2 is a result of analyzing the change in melanin content according to the concentration of linarin (linarin) treatment in B16F10 cells.
B16F10 cells were treated with linarin at a concentration of 3.125 to 25 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 value 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
3 is a result of analyzing the change in tyrosinase activity according to the concentration of linarin treatment in B16F10 cells.
B16F10 cells were treated with linarin at a concentration of 3.125 to 25 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 value of control cells (untreated). Data are expressed as mean ± standard deviation (SD) for at least 4 independent experiments (n = 4). ***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 melanin production comprising linarin or a physiologically acceptable salt thereof as an active ingredient.

In the present invention, linarin is a kind of flavonoid and is represented by the following Chemical Formula 1, and the compound name is called acacetin-7-O-b-D-rutinoside.

In the present invention, “physiologically acceptable salt” refers to any salt that possesses the desired biological or physiological activity of the compound and exhibits minimal undesirable toxicological effects. It refers to a salt prepared according to a method conventional in the art, and such preparation method is known to those skilled in the art. Specifically, the physiologically acceptable salts include, but are not limited to, salts derived from cosmetically, pharmacologically, or physiologically acceptable inorganic acids, organic acids, and bases. Examples of suitable acids include 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 linarin represented by Formula 1 or a physiologically acceptable salt thereof, as well as an isomer thereof or a possible solvate or hydrate prepared therefrom.

In an embodiment of the present invention, it was confirmed that linarin maintains 90% or more of cell viability in B16F10 cells at a concentration of 25 μg/ml or less, and promotes melanin production and tyrosinase activity. As it was confirmed, the composition comprising linarin is characterized in that it promotes melanin production.

The composition for promoting melanin production of the present invention increases melanin biosynthesis in skin or hair follicle cells, thereby alleviating, improving, preventing, improving or treating vitiligo, albinism or hypopigmentation caused by melanin deficiency.

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

The cosmetic composition of the present invention contains a composition for promoting melanin production including linarin as an active ingredient to promote melanin production in the skin or hair root, thereby preventing or improving vitiligo, white hair or hypopigmentation due to melanin deficiency. indicates

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

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

The "hypochromatism" refers to a disease in which the skin at the lesion site temporarily appears white due to a functional disorder 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, white hair, or hypopigmentation" means to produce melanin in melanocytes present in the skin or hair follicles to prevent, improve or treat the symptoms of vitiligo, white hair or hypopigmentation. .

In addition to linarin, the cosmetic composition of the present invention may further include a compound or natural extract known to have a melanin production promoting effect so as to enhance or reinforce the prevention or improvement effect of vitiligo, white hair or hypopigmentation.

In the cosmetic composition for preventing or improving vitiligo, white hair or hypopigmentation of the present invention, the active ingredient promotes the production of melanin so as to exhibit the prevention or improvement effect of vitiligo, white hair or hypopigmentation, use, formulation, It may be included in any amount (effective amount) depending on the purpose of blending, etc., and a typical effective amount may be included within the range of 0.001 wt % to 99.99 wt % 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 ability of one of ordinary skill in the art.

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 conventionally prepared in the art, for example, a solution, suspension, paste, gel, cream , lotion, powder, cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. In addition, specifically, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, essence, nourishing 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 an acceptable carrier in the cosmetic preparation in addition to the active ingredient. Here, "acceptable carrier in cosmetic formulations" means a known and used compound or composition that can be included in a cosmetic formulation, or a compound or composition to be developed in the future, which has no toxicity, instability, or irritation beyond what the human body can adapt 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 from about 1% to about 99.99% by weight, preferably from about 5% to about 99% by weight of 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 site (face or hand) or its desired application amount, the ratio is intended to limit the scope of the present invention in any aspect. should not be understood

On the other hand, as the carrier, alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, sunscreen, color developer, fragrance, and the like may be exemplified. Alcohols, oils, surfactants, fatty acids, silicone oils, humectants, humectants, viscosity modifiers, emulsifiers, stabilizers, sunscreens, coloring agents, perfumes, 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, white hair, or hypopigmentation comprising the composition for promoting melanin production as an active ingredient.

In the present invention, the composition for promoting melanin production, vitiligo, white hair, hypopigmentation, prevention or treatment are the same as described above.

The pharmaceutical composition of the present invention includes a composition for promoting melanin production including linarin as an active ingredient to promote melanin production in the skin or hair root to prevent or treat vitiligo, white hair or hypopigmentation due to melanin deficiency. show the effect.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable diluent, excipient or carrier. As used herein, the term “pharmaceutically acceptable” means exhibiting non-toxic properties 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, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents and suppositories It may have a dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like 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, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can 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 subject's type and severity, age, sex, and disease. It may be determined according to the type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, 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 may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, 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 an individual by any suitable method, and the route of administration of the composition is oral or parenteral, as long as it can reach the target tissue. It may be administered through, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation or intradermal routes may be administered in a conventional manner.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of treating vitiligo, white hair or hypopigmentation, and any one is applicable. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs, goats, etc., humans, birds, and fish can be used, and the pharmaceutical composition is parenteral, It may be administered subcutaneously, intraperitoneally, intrapulmonary and intranasally, and for local treatment, if necessary, it may be administered by a suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may 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 in an amount of 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 administered in divided doses from once to several times a day.

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

In the present invention, the composition for promoting melanogenesis, vitiligo, white hair, hypopigmentation, prevention or improvement is the same as described above.

The food composition of the present invention contains a composition for promoting melanin production including linarin as an active ingredient to promote melanin production in the skin or hair root, thereby preventing or improving vitiligo, white hair or hypopigmentation due to melanin deficiency. indicates

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, and there is no particular limitation on the type of food to which the linarin of the present invention can be added, For example, there are various beverages, chewing gum, tea, vitamin complexes, and health supplements.

In addition to linarin, other ingredients may be added to the food composition, and the type thereof is not particularly limited. For example, it may contain, as an additional component, various herbal extracts, food-logically acceptable food supplement additives or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

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. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.

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

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the linarin 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 when preparing food. However, in the case of long-term intake for health and hygiene purposes or for health control, the above amount may be used below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in the ordinary 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

B16F10 cells were cultured in DMEM containing 10% FBS and 1% penicillin as a medium at 37° C., 5% CO ₂ in a humidified incubator.

2. Cytotoxicity Assessment

The MTT method was performed to measure cytotoxicity according to the concentration of linarin treatment. B16F10 cells were seeded in a 24-well plate at 8×10 ³ cells/well per well, cultured for 24 hours, and samples were treated by concentration and cultured for 72 hours. Then, all of the supernatant was removed, and 500 μl of MTT reagent (0.2 mg/ml in DMEM) was added per well and reacted for 4 hours. After the reaction time was over, all the MTT reagent was removed and DMSO was added to dissolve the formed formazan crystals. Absorbance was measured at 560 nm using a spectrophotometric microplate reader.

3. Measurement of melanin content

In a 6-well plate (well-plate), 5×10 ⁴ cells/well were aliquoted and cultured for 24 hours, and then samples were treated by concentration and cultured for 72 hours. The cultured cells were washed with 1×PBS and harvested by centrifugation. Then, 500 μl of 1 N NaOH supplemented with 10% DMSO was added and melanin was dissolved at 80° C. for 20 minutes. Absorbance was measured at 405 nm using a spectrophotometric microplate reader.

4. Tyrosinase Activity Assessment

In a 6-well plate (well-plate), 5×10 ⁴ cells/well were aliquoted and cultured for 24 hours, then samples were treated by concentration and cultured for 72 hours. After washing the cultured cells with 1× PBS, RIPA buffer (NaCl: 150mM Tritonx-100: 1% sodium deoxycholate: 1% SDS: 0.1% Tris-HCI, pH7.5: 50mM with Protease inhibitor cocktail added at 1%) The cells were lysed with EDTA, pH 8.0: 2 mM). The supernatant was separated by centrifugation. The protein concentration of the supernatant was quantified with a BCA protein assay kit. Then, 20 μl of the quantified protein and 80 μl of 2 mg/ml L-DOPA were added to a 96-well plate and reacted at 37° C. for 2 hours. Then, the absorbance was measured at 490 nm using a spectrophotometric microplate reader.

<Experimental Example 1> Cytotoxicity evaluation of linarin

In order to evaluate the effect of linarin on cell survival in B16F10 melanoma cells, an MTT experiment was performed. B16F10 melanoma cells were treated with linarin at 6.25 to 50 μg/ml for 72 hours. Cell viability was expressed as a percentage of untreated cells.

As a result of the analysis, when treated at a concentration of 50 μg/ml, the cell viability was 83%, confirming that it exhibited some cytotoxicity. On the other hand, it was confirmed that the survival rate of 90% or more was maintained when treated with a concentration of 25 μg/ml or less ( FIG. 1 ).

Therefore, in subsequent experiments, the activity was evaluated at a concentration of 25 μg/ml or less.

<Experimental Example 2> Analysis of melanin synthesis promoting effect of linarin

In order to confirm the effect of linarin on melanin synthesis, B16F10 cells were treated at a concentration of 3.125, 6.25, 12.5, or 25 μg/ml, which did not affect B16F10 cell viability, for 72 hours. As a positive control, α-MSH (100 nM) was used.

As a result of the analysis, it was confirmed that the melanin content increased as the linarin treatment concentration increased from 3.125 µg/ml to 25 µg/ml, and it was confirmed that the increase in melanin content was higher than that of the positive control ( FIG. 2 ).

<Experimental Example 3> Effect of linarin to promote tyrosinase activity

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

B16F10 cells were treated with α-MSH (100 nM) as a positive control, linarin was treated at a concentration of 3.125, 6.25, 12.5 or 25 μg/ml, and changes in tyrosinase activity were analyzed.

As a result of the analysis, it was confirmed that the tyrosinase activity was increased in a concentration-dependent manner in the group treated with linarin compared to the untreated control group. Specifically, the group treated with high concentration (25 μg/ml) of linarin showed the greatest increase in tyrosinase activity, and tyrosinase (tyrosinase) compared to the positive control group (α-MSH, 100 nM). ) it was confirmed that the activity was high (Fig. 3).

Claims (4)

A composition for promoting melanin production comprising linarin or a physiologically acceptable salt thereof as an active ingredient. A cosmetic composition for preventing or improving vitiligo, white hair or hypopigmentation comprising the composition for promoting melanin production of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating vitiligo, white hair or hypopigmentation comprising the composition for promoting melanin production of claim 1 as an active ingredient. A food composition for preventing or improving vitiligo, white hair or hypopigmentation comprising the composition for promoting melanin production of claim 1 as an active ingredient.

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