KR20230039105A - Skin-lightening Composition Using Xanthatin - Google Patents

Abstract

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

Description

Skin whitening composition using Xanthatin {Skin-lightening Composition Using Xanthatin}

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

As living standards improve in modern society, interest in beauty as well as health is gradually increasing. Among them, development of products for improvement of pigmentation-type skin diseases such as melasma and freckles caused by skin damage and aging caused by ultraviolet rays, air pollution, etc., and improvement of overall skin tone is active.

Melanin present in the basal layer of the epidermis is a major pigment that determines skin color and plays a role in protecting the skin by preventing damage caused by ultraviolet rays and removing active oxygen. However, when melanin is locally overproduced, it causes skin diseases such as melasma, freckles, dark spots, and skin cancer. In addition, when continuously overproduced melanin stays in the dermis, it turns the skin color black or dull, leading to a desire for whitening (Pigment Cell Res. 2007 Feb;20(1):2-13.; Curr Biol. 2011 Nov. 22;21(22):1906-11.; Ann Dermatol Venereol.

Melanin is a phenolic polymeric natural pigment widely present in living organisms. In the human body, it is synthesized in melanosomes in melanocytes in the basal layer of the epidermis, and is transferred to surrounding keratinocytes to show human skin color. When melanin is abnormally low, skin lesions such as vitiligo are induced, and on the contrary, when it is produced in excess, acquired hyperpigmentation such as melasma, postinflammatory melanoderma, and solar lentigo A variety of skin diseases, including

The synthesis of melanin is achieved by making melanosomes in melanocytes present in the basal layer of the skin, and a series of processes in which melanin is synthesized are collectively referred to as melanogenesis. Melanin production uses tyrosine, one of the amino acids, as a substrate, and DOPA (3, After conversion to DOPA quinone through 4-dihydroxyphenylalanine), melanin is synthesized by polymerization with amino acids or proteins after undergoing 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, studies on the inhibition of melanin synthesis have been conducted using proteins such as tyrosinase, TRP-1 and TRP-2 as well as microphtalmia associated transcription factor (MITF). The major intracellular signal transduction 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 plays an important role in melanin synthesis. 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 melanin production, studies on whitening agents are mainly focused on tyrosinase activity inhibitors or antagonists for tyrosinase substrates to regulate tyrosinase activity. Research has been conducted in the direction of developing (Kor. J. Pharmacogn., 44(3):220, 2013).

Arbutin, the most commonly used whitening agent, is a tyrosine antagonist, and kojic acid chelates the active site of tyrosinase to inhibit the process of tyrosine to dopa and dopaquinone (Korean J. Plant Res., 26(5):526, 2013; J. Korean Soc. Food Sci. Nutr., 41(2):156, 2012). Although arbutin and kojic acid are known to have strong whitening effects, due to some side effects such as skin irritation, studies are being actively conducted to find substances having 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.

An object of the present invention is to provide a composition for skin whitening using zansatin.

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

As confirmed in the following examples and experimental examples, the present invention inhibits melanin production in a concentration-dependent manner and also inhibits tyrosinase, TRP- 1 and TRP-2 proteins were inhibited.

<Formula 1>

Considering the foregoing, the skin whitening composition of the present invention is characterized in that it contains zansatin as an active ingredient.

In the present specification, "active ingredient" means a component that exhibits the desired activity alone or can exhibit activity in combination with a carrier having no activity itself.

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. to 15% by weight. Here, "effective amount" means that when the composition of the present invention is administered to a mammal, preferably a human, during the administration period according to the advice of medical experts, skin experts, etc., the intended medical and dermatological effects such as skin whitening effect It refers to the amount of the active ingredient contained in the composition of the present invention that can be expressed. Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

In addition to the active ingredients, the composition of the present invention has similar activities such as skin whitening activity, skin wrinkle improvement activity, skin hypersensitivity reaction inhibition activity, skin protection activity (inhibition of skin damage by ultraviolet rays, skin moisturizing, etc.) In order to enhance the convenience of intake, intake, and use through addition, any compound or natural extract known to have a corresponding activity and whose safety has already been verified in the art may be further included.

These compounds or extracts include each country's Pharmacopoeia ("Korean Pharmacopoeia" in Korea), each country's Health Functional Food Codex (in Korea, it is "Health Functional Food Standards and Specifications", a notification by the Ministry of Food and Drug Safety), and each country's Functional Cosmetics Codex (in Korea, a notification by the Ministry of Food and Drug Safety Compounds or extracts listed in compendial documents such as "Functional Cosmetics Standards and Test Methods"), compounds or extracts approved as items under the laws of each country governing the manufacture and sale of pharmaceuticals ("Pharmaceutical Affairs Act" in Korea), health In accordance with the laws of each country regulating the manufacture and sale of functional foods (in Korea, it is the 「Health Functional Food Act」), the laws of each country regulating the manufacture and sale of compounds or extracts with recognized functionality, and the manufacture and sale of functional cosmetics (in Korea, the 「Cosmetics Act」 ”), compounds or extracts whose functionality has been recognized are included. For example, skin whitening ingredients include arbutin, niacinamide, ascorbyl glucoside, alpha-bisabolol, oil soluble licorice (Glycyrrhiza) Extract, etc., and skin wrinkle improvement ingredients include retinol and retinyl palmitate. , adenosine, polyethoxylated amide, and the like, and examples of ultraviolet protection components include drometrizole, drometrizoletrisiloxane, digaloyltrioleate, dimethicodyethylbenzalmalonate, and diethylhexylbutamido. 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, as moisturizing ingredients, 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, weed extract powder, phosphatidylserine, hyaluronic acid, etc. Functional ingredients for 'improving sensitive skin conditions' include L. sakei Probio 65, gamma-linolenic acid-containing oil, L.plantarum CJLP133, a lactic acid bacterium derived from fruits and vegetables, and probiotics 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 embodiment, the composition for skin whitening of the present invention can be regarded as 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 specifically, may be a functional cosmetic having a purpose such as skin whitening, a non-functional general cosmetic, and the like. In product form, it may 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 a foundation, spray, and the like. In a specific product form, it may be a softening lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder formulation.

The cosmetic composition of the present invention may include, in addition to the active ingredient, ingredients commonly used in cosmetic compositions, for example, stabilizers, solubilizers, surfactants, vitamins, conventional adjuvants such as pigments and fragrances, and carriers.

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

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant 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 esters, polyethylene glycol, fatty acid esters of sorbitan, and the like can be used.

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

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention may be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except for including an active ingredient exhibiting skin whitening activity.

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

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

In addition, the food composition of the present invention may take any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, health functional food according to the Korean 「Health Functional Food Act」, or snacks, legumes, teas, beverages according to each food type according to the Food Code (MFDS notification 「Food Standards and Specifications」) of the Korea 「Food Sanitation Act」 , special purpose foods, etc.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives may generally be understood as substances that are added to, mixed with, or infiltrated into food in manufacturing, processing, or preserving food. Since food is consumed daily and for a long period of time, its safety must be guaranteed. According to the Food Additives Code under the laws of each country governing the manufacture and distribution of food (in Korea, it is the 「Food Sanitation Act」), safety-guaranteed food additives are limitedly regulated in terms of ingredients or functions. In the Korean Food Additives Codex (MFDS notification 「Standards and Specifications for Food Additives」), food additives are classified into chemical synthetic products, natural additives and mixed preparations in terms of ingredients and are regulated. It is divided into additives, preservatives, emulsifiers, acidulants, and thickeners.

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

Flavors may be used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and synthetic flavors may include esters, alcohols, aldehydes, terpenes, and the like.

As preservatives, sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used, and as emulsifiers, acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like, and examples of the acidulant include acid salt, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Acidulants may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to improving taste.

As the thickening agent, a suspending agent, a sedimentation agent, a gel forming agent, a swelling agent, and the like may be used.

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

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

The food composition of the present invention may include the above-mentioned food additives in an appropriate amount to achieve the purpose of addition according to the type of product.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to national food codes or food additive codes.

In a specific aspect, the composition for skin whitening of the present invention can be understood as 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, melasma, brown or dark spots, solar pigment Melasma blue, hyperpigmentation after the use of drugs such as calcium antagonists, hyperpigmentation sequelae following tissue sclerotherapy, brown spots of pregnancy, melasma in women taking oral contraceptives, wounds or dermatitis including scratches and burns may include, but are not limited to, post-inflammatory hyperpigmentation, phototoxic reaction, or other similar small fixed pigmented lesions.

The pharmaceutical composition of the present invention may be formulated into an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The route of administration herein may be any appropriate 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 in which two or more formulations of drugs are combined according to the route of administration, for example, one drug is firstly administered intravenously and the other drug is secondly administered through a topical route.

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

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer according to a method known in the art together with a suitable carrier. It can be prepared in formulations 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, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, Serol etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may 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, wax, and the like, and oleic acid as a lubricant Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. may be mentioned. As disintegrants, starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like can be used.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. These documents are considered as 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, sex, age, severity of the patient, and route of administration. /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 present 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 as food, cosmetics, medicine, and the like.

1 is a result showing that zansatin 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 zansatin 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 > Xanxatin of whitening active

1. Materials and test methods

1.1 Preparation of Xanthine

Xanthine was independently obtained by isolation and identification from natural products.

1.2 Cell culture

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

1.3 Measurement of melanin synthesis (Melanin contents)

B16F10 cell culture was performed using DMEM medium containing 10% fetal bovine serum and 100 nM α-MSH (melanocyte stimulating hormone) (Sigma-Aldrich Co.) at 37°C and 5% CO ₂ by culturing the cell line. used

B16F10 cells cultured in DMEM medium were dispensed in a 24-well plate at 1 × 10 ⁵ cells/well and cultured for one day to be used in experiments. Afterwards, the samples were treated by concentration and cultured for 3 days. After culturing, the cells were washed with phosphate buffer (pH 7.4) and treated with 200 μl of 1N NaOH in each well to dissolve the cells. The lysed cells were transferred to a 96-well plate and 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 is a sample untreated group (α-MSH only treated group).

1.4 Analysis of protein expression by Western blot

In order to confirm the expression level of tyrosinase and TRP-1 among genes involved in melanin production, B16F10 cells were cultured and samples were treated for 72 hours at each concentration, and then the cells were treated with protease inhibitor cocktail (Sigma, St. Louis, MO USA) was added to the RIPA buffer (10 mM Tris-HCl (pH 7.5), 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA). . Thereafter, 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). SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) was performed after adding a buffer solution for electrophoresis (sample buffer) to the protein extracted from the cells and heating at 100 ° C. for 5 minutes. After electrophoresis, the 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, after reacting in a buffer solution (phosphate buffered saline; PBS (pH 7.4), 0.1% Tween 20, 5% skimmed milk) at room temperature for 1 hour, the primary antibody was diluted as follows used The antibody was 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 the reaction, wash 3 to 4 times for 5 minutes with washing buffer (0.1% Tween-20, PBS) and then use a 1:5000 diluted secondary antibody (HRP-conjugated anti-goat IgG antibody) solution for 1 hour at room temperature. reacted After the secondary antibody reaction, the protein band to be analyzed was colored 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 FIG. 1, xanthine inhibited melanin production in a concentration-dependent manner when compared to the α-MSH treated group, which is a positive control group. 1 also shows the results of the cytotoxicity test, and xanthine did not show any particular cytotoxicity even at 5 μM.

2.2 Western blot results

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

Claims (4)

A composition for skin whitening comprising xanthine as an active ingredient.
According to claim 1,
The composition is a composition for skin whitening, characterized in that the cosmetic composition.
According to claim 1,
The composition is a composition for skin whitening, characterized in that the food composition.
According to claim 1,
The composition is a composition for skin whitening, characterized in that the pharmaceutical composition.

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