KR102615923B1 - Skin-lightening Composition Using Xanthatin - Google Patents

Abstract

The present invention provides a composition for skin whitening using zansatin, which has the activity of inhibiting melanin production in a concentration-dependent manner and suppressing the expression of tyrosinase, TRP-1, and TRP-2 proteins in B16F10, a mouse-derived melanoma cell line. Begin.

Description

Composition for skin whitening using xanthatin {Skin-lightening Composition Using Xanthatin}

The present invention relates to a composition for skin whitening using xanthatin (CAS No. 26791-73-1).

As living standards improve in modern society, interest in not only health but also beauty is gradually increasing. Among these, product development is active to improve pigmentation-type skin diseases such as melasma and freckles caused by skin damage and aging caused by ultraviolet rays, air pollution, etc., and to improve overall skin tone.

Melanin, which exists in the basal layer of the epidermis, is a major pigment that determines skin color and protects the skin by preventing damage from ultraviolet rays and removing free radicals. However, when melanin is locally overproduced, it causes skin diseases such as freckles, freckles, dark spots, and skin cancer. In addition, if continuously overproduced melanin stays in the dermis, it turns the skin color black or makes the complexion dull, thereby inducing the desire for whitening (Pigment Cell Res. 2007 Feb;20(1):2-13.; Curr Biol. 2011 Nov 22;21(22):1906-11.;Ann Dermatol Venereol. 2012 Nov;139 Suppl 3:S73-7).

Melanin is a phenolic polymer natural pigment that is widely present in living organisms. In the human body, it is synthesized in melanosomes within melanocytes in the basal layer of the epidermis and is transferred to surrounding keratinocytes, giving the human skin color. If melanin is abnormally low, it causes skin lesions such as vitiligo. Conversely, if it is produced in excess, it causes acquired hyperpigmentation such as melasma, postinflammatory melanoderma, and solar lentigo. It can cause various skin diseases, including.

The synthesis of melanin is accomplished by creating melanosomes in melanocytes present in the basal layer of the skin, and the series of processes by which melanin is synthesized are collectively called melanogenesis. Melanin production uses tyrosine, one of the amino acids, as a substrate, and DOPA (3, After being converted to DOPA quinone through 4-dihydroxyphenylalanine), melanin is synthesized through a polymerization reaction with amino acids or proteins after going through a non-enzymatic reaction and spontaneous oxidation process (Korean J. FOOD SCI. TECHNOL., 2000. 32(3):736; Journal of Life Science, 2013. 23(12):1445; Pigment Cell Res.1999. 12(1):4-12.). Recently, research has been conducted on the inhibition of melanin synthesis using proteins such as tyrosinase, TRP-1, and TRP-2, as well as MITF (microphtalmia associated transcription factor). The main intracellular signaling pathway is the cAMP/PKA (cyclic monophosphate/protein kinase A) pathway. cAMP promotes the expression of MITF via PKA and CREB1 (cAMP responsive element binding protein 1), and MITF is important in the melanin synthesis process. As a transcriptional regulator, it promotes the transcription of tyrosinase, TRP-1, and TRP-2 (Pharmazie. 2015 Oct;70(10):646-9.; Int J Mol Sci. 2015 Oct 13;16(10): 24219-42; Curr Pharm Biotechnol. 2015;16(12):1111-9.).

Since tyrosinase plays the most important role in the first step of producing melanin, research on whitening agents mainly focuses on substances that inhibit tyrosinase activity or antagonists of tyrosinase substrates in order to control tyrosinase activity. Research has been conducted in the direction of developing (Kor. J. Pharmacogn., 44(3):220, 2013).

Arbutin, currently the most widely used whitening agent, is an antagonist of tyrosine, and kojic acid chelates the active site of tyrosinase and inhibits the process from tyrosine to dopa and dopaquinone (Korean J. Plant Res., 26(5):526, 2013; J. Korean Soc. Food Sci. Nutr., 41(2):156, 2012). Arbutin and kojic acid are known to have a strong whitening effect, but due to some side effects such as skin irritation, research is being actively conducted to find substances with whitening activity from natural substances with relatively few side effects.

The present invention discloses the whitening activity of zansatin isolated and identified from natural products.

The purpose of the present invention is to provide a composition for skin whitening using xanthine.

Other or specific purposes of the present invention will be presented below.

As confirmed in the examples and experimental examples below, the present invention inhibits melanin production in a concentration-dependent manner when xanthin of the formula (1) is treated with B16F10, a mouse-derived melanoma cell line, and also inhibits tyrosinase, TRP- This was accomplished by suppressing the expression of 1 and TRP-2 proteins.

<Formula 1>

Considering the above, the composition for skin whitening of the present invention is characterized by containing xanthine as an active ingredient.

In this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

The composition of the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit skin whitening activity, etc. depending on the product, formulation, etc., and a typical effective amount is 0.001% by weight based on the total weight of the composition. It will be determined within the range of 15% by weight. Here, “effective amount” means that when the composition of the present invention is administered to mammals, preferably humans, to which it is applied during the administration period recommended by medical experts, skin experts, etc., the intended medical and dermatological effects, such as skin whitening effects, are achieved. It refers to the amount of active ingredient contained in the composition of the present invention that can be expressed. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In addition to the active ingredients, the composition of the present invention contains similar activities such as skin wrinkle improvement activity, skin hypersensitivity reaction inhibition activity, and skin protection activity (inhibition of skin damage caused by ultraviolet rays, skin moisturization, etc.) for the purpose of increasing and reinforcing skin whitening activity, etc. In order to improve the convenience of taking, ingesting, or using by adding, any compound or natural extract that has already been proven safe in the art and is known to have the corresponding activity may be additionally included.

These compounds or extracts include the Pharmacopoeia of each country (in Korea, “Korean Pharmacopoeia”), each country’s Code of Health Functional Foods (in Korea, “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety), and the Code of Functional Cosmetics of each country (in Korea, “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety). Compounds or extracts listed in compendia such as "Functional Cosmetics Standards and Test Methods"), compounds or extracts approved as items in accordance with the laws of each country governing the manufacture and sale of pharmaceuticals (in Korea, this is the "Pharmaceutical Affairs Act"), health Compounds or extracts recognized as functional in accordance with the laws of each country that govern the manufacture and sale of functional foods (in Korea, this is the “Health Functional Foods Act”), and the laws of each country that govern the manufacture and sale of functional cosmetics (in Korea, it is the “Cosmetics Act”) 」) includes compounds or extracts whose functionality has been recognized. For example, skin whitening ingredients include arbutin, niacinamide, ascorbyl glucoside, alpha-bisabolol, and Oil Soluble Licorice (Glycyrrhiza) Extract, and skin wrinkle improvement ingredients include retinol and retinyl palmitate. , adenosine, polyethoxylated amide, etc., and examples of ultraviolet protection ingredients include drometrizole, drometrizole trisiloxane, digalloyl trioleate, dimethylcodiethylbenzalmalonate, and diethylhexylbutamido. Examples include complexes such as triazone and pine bark extract, phosphatidylserine, finger root extract powder, and complex extracts such as red ginseng and Cornus officinalis. In addition, moisturizing ingredients include AP collagen enzyme decomposition peptide, collactive collagen peptide, N-acetylglucosamine, konjac potato extract, complex extracts such as dandelion, rice bran extract, corn germ extract, low molecular weight collagen peptide, agar extract powder, phosphatidylserine, hyaluronic acid, etc. Functional ingredients for 'improving sensitive skin conditions' include L. sakei Probio 65, oil containing gamma-linolenic acid, L. plantarum CJLP133, a lactic acid bacteria derived from fruit and vegetables, and probiotic ATP. One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

In a specific aspect, the composition for skin whitening of the present invention can be considered a cosmetic composition.

When the composition of the present invention is recognized as a cosmetic composition, the cosmetic composition may be classified as an arbitrary product in terms of its use and law, and may specifically be a functional cosmetic with a use such as skin whitening, a non-functional general cosmetic, etc. The product form can take any product form, specifically solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax. It can be formulated as foundation, spray, etc. Specific product forms may include softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

In addition to the active ingredient, the cosmetic composition of the present invention may contain ingredients commonly used in cosmetic compositions, such as stabilizers, solubilizers, surfactants, vitamins, conventional auxiliaries such as pigments and flavorings, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. can be used.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention can be prepared according to a cosmetic composition manufacturing method commonly used in the art, except that it contains an active ingredient that exhibits skin whitening activity.

In a specific aspect, the composition of the present invention can be viewed as a food composition.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc.

In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to the Korean 「Act on Health Functional Foods」, or confectionery, beans, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (Ministry of Food and Drug Safety Notification 「Food Standards and Specifications」) , special purpose food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the Food Additives Code of Laws of each country that regulates the manufacturing and distribution of food (in Korea, it is the Food Sanitation Act), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification “Food Additive Standards and Specifications”), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified into sweeteners and flavors in terms of function. It is classified into preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and either natural or synthetic ones can be used in the composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and the synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc.

Preservatives include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, Examples include pectin, and acidulants include acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms.

As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that can be included in the food composition of the present invention, each country's food code or food additive code can be referred to.

In a specific aspect, the composition for skin whitening of the present invention can be considered a pharmaceutical composition.

When the composition for skin whitening of the present invention is understood as a pharmaceutical composition, the target disease can be understood as skin hyperpigmentation, and specific examples of such hyperpigmentation include freckles, senile spots, liver spots, freckles, brown or black spots, and solar pigments. Spots, blue melanosis, hyperpigmentation after use of drugs such as calcium antagonists, sequelae of hyperpigmentation following tissue sclerotherapy, brown spots of pregnancy, melasma in women taking oral contraceptives, wounds or dermatitis including scratches and burns. This may include, but is not limited to, post-inflammatory hyperpigmentation, phototoxic reaction, or other similar small fixed pigmented lesions.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art. Here, the route of administration may be any suitable route, including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case where two or more dosage forms of drugs according to the administration route are combined, for example, when one drug is administered firstly through an intravenous route and the other drug is administered secondarily through a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and for specifics, reference can be made to each country's pharmacopoeia, including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, and wafers using a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease. Serol, etc. can be mentioned. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. can be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, and wax, and as a lubricant, oleic acid. Examples include sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, and polydethylene glycol. Disintegrants include starch and methyl cellulose. , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, etc. Additionally, diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier. Specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the carrier is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

As described above, according to the present invention, a composition for skin whitening using xanthine can be provided.

The composition for skin whitening of the present invention can be commercialized into foods, cosmetics, drugs, etc.

Figure 1 shows results showing that xanthine inhibits melanin production in a concentration-dependent manner in B16F10, a mouse-derived melanoma cell line.
Figure 2 is a Western blot result showing that xanthine inhibits the expression of tyrosinase, TRP-1, and TRP-2 proteins in B16F10, a mouse-derived melanoma cell line.

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

< Example > Xanthine of whitening activity

1. Materials and experimental methods

1.1 Preparation of xanthine

Xanthin was obtained by self-isolation and identification from natural products and was used.

1.2 Cell culture

B16F10 cells, a mouse-derived melanoma cell line, were purchased from the Korean Cell Line Bank and incubated with 1% antibiotics (Gibco, USA) and 10% (100 mg/mL) fetal bovine serum (Gibco, USA). The cells were cultured in a 5% CO ₂ incubator at 37°C using DMEM medium (Dulbecco's modified Eagle's medium) containing (Grand Island, USA), and subculture was performed once every 4 days.

1.3 Measurement of melanin synthesis (Melanin contents)

B16F10 cells were cultured using DMEM medium containing 10% fetal bovine serum and 100 nM α-MSH (melanocyte stimulating hormone) (Sigma-Aldrich Co.) in an environment of 37°C and 5% CO _2. used.

B16F10 cells cultured in DMEM medium were dispensed at 1 × 10 ⁵ cells/well in a 24-well plate, cultured for one day, and used in experiments. Afterwards, the samples were treated at different concentrations and cultured for 3 days. After incubation, the cells were washed with phosphate buffer (pH 7.4) and 200 ㎕ of 1N NaOH was added to each well to lyse the cells. The dissolved cells were transferred to a 96-well plate and the absorbance was measured at 405 nm using a microplate reader (Bioteck, cytation3). The melanin production inhibition rate was calculated as a percentage compared to the control group, which was an untreated group (a group treated only with α-MSH).

1.4 Protein expression analysis through Western blot

To determine the level of expression of tyrosinase and TRP-1 among the genes involved in melanin production, B16F10 cells were cultured and samples were treated at different concentrations for 72 hours, and then the cells were treated with a protease inhibitor cocktail (Sigma, St. Louis, MO USA) was dissolved in RIPA buffer (10mM Tris-HCl (pH 7.5), 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150mM NaCl, 1mM EDTA). . Afterwards, the obtained cell lysate was centrifuged at 12,000 × g for 5 minutes at 4°C, and the protein concentration was measured using a BCA protein kit (Thermo Scientific). Sample buffer for electrophoresis was added to the protein extracted from cells, heated at 100°C for 5 minutes, and then SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) was performed. After electrophoresis, proteins separated by molecular weight were transferred from the gel to a nitrocellulose membrane and quantified on the nitrocellulose membrane. At this time, in order to minimize non-specific reactions, a buffer solution (phosphate buffered saline; PBS (pH 7.4), 0.1 % Tween 20, 5 % skimmed milk) was reacted at room temperature for 1 hour, and then the primary antibody was diluted as follows. used. Antibodies were diluted by reacting at room temperature for 1 hour using a 5% non-fat dry milk solution, and the dilution ratio was 1:1000 for anti-tyrosinase antibody and 1:1000 for anti-TRP-1 antibody. After reaction, wash 3 to 4 times for 5 minutes with washing buffer (0.1% Tween-20, PBS) and then wash with secondary antibody (HRP-conjugated anti-goat IgG antibody) solution diluted 1:5000 for 1 hour at room temperature. reacted. After the secondary antibody reaction, the protein band to be analyzed was developed and quantified using an enhanced chemiluminescence (ECL) reagent.

2. Experimental results

2.1 Measurement of melanin synthesis (Melanin contents)

The results are shown in Figure 1. Referring to Figure 1, xanthine inhibited melanin production in a concentration-dependent manner compared to the α-MSH treated group, which is a positive control group. Figure 1 also shows the results of the cytotoxicity test, where zansatin did not show any particular cytotoxicity even at 5 μM.

2.2 Western blot results

The results are shown in Figure 2. As seen in Figure 2, it was confirmed that as the sample treatment concentration increased, the expression levels of tyrosinase, TRP-1, and TRP-2 proteins decreased compared to the α-MSH treatment group, which is a positive control group. Therefore, it is believed that zansatin inhibits melanin synthesis by regulating the expression of tyrosinase, TRP-1, and TRP-2 proteins involved in melanin synthesis.

Claims (4)

A cosmetic composition for skin whitening containing xanthine as an active ingredient.
delete A food composition for skin whitening containing zansatin as an active ingredient.
A pharmaceutical composition for the treatment or prevention of skin hyperpigmentation comprising zansatin as an active ingredient.