KR102085679B1 - New compounds and cosmetic composition including the same - Google Patents

Abstract

The present invention relates to a novel compound and a cosmetic composition containing the same, wherein the compound has no cytotoxicity even at high concentrations, inhibits melanin production, reduces the expression of various enzymes (tyrosinase, TRP-1, TRP-2) involved in melanin synthesis, and reduces a microphthalmia-associated transcription factor (MITF) regulating the expression of melanin-producing enzymes as a transcription factor specific for melanocytes, thereby having excellent whitening activity.

Description

Novel compounds and cosmetic compositions comprising the same {NEW COMPOUNDS AND COSMETIC COMPOSITION INCLUDING THE SAME}

The present invention relates to a novel compound and a cosmetic composition comprising the same.

Skin is a very important tissue that protects the human body while being in direct contact with the external environment and has biochemical and physical functions. This tissue is largely divided into three parts: epidermis, dermis and subcutaneous tissue. Human skin color depends mainly on the number, size, type and distribution of melanocytes containing melanin contained in skin cells.

The chemical action on melanin production, which determines the color of the skin, has been reported in many papers until now. The melanocyte-producing melanocytes (melanocytes) function to protect cells from ultraviolet rays in the intracellular organelles called melanosomes. After synthesizing melanin, the melanin is known to migrate from melanocytes to keratinocytes, and it has been reported that melanin transferred to keratinocytes determines skin color.

The precursor required in the melanin biosynthesis process is an amino acid called tyrosine, which is converted to dihydroxyphenylalanine (DOPA), and then through a substance called dopaquinone to finally convert to melanin. It is known. In connection with such melanin biosynthesis, tyrosine-related protein 1 or dopachrome tautomerase (TRP-2) is known to affect melanin biosynthesis and can regulate these. Microphthalmia-associated transcription factor (MITF), a major transcription factor, is also known to play a role in the regulation of melanin biosynthesis. In particular, an enzyme that plays a pivotal role in melanin biosynthesis is tyrosinase (TYR), which is required for the oxidation of tyrosine to DOPA and DOPA to dopaquinone. Inhibition of the enzyme activity is known to inhibit melanin synthesis.

In the conventional cosmetic field, as a whitening component, for example, substances that inhibit tyrosinase enzyme activity such as kojic acid, arbutin, hydroquinone, vitamin C (L-ascorbic acid) ) And their derivatives and various plant extracts have been used. However, the substance that inhibits the tyrosinase enzyme activity is deteriorated due to its low stability in the prescription, or colored, or its use is limited due to the occurrence of off-flavor, efficacy at the biological level, unclear effect and safety issues. It is true.

On the other hand, quercetin (quercetin) is a kind of flavonoids (flavonoids) is one of the highest physiological activity among the flavonoids. Such quercetin is known to have antioxidant activity, and thus may be a functional material. However, the safety and effectiveness of the active ingredient is important in the cosmetic industry, quercetin is not only a highly toxic material, but also has a low skin absorption rate, so it is difficult to use as a cosmetic preparation.

Under these circumstances, the present inventors have diligently searched for an active ingredient having a skin whitening effect. As a result, a novel quercetin compound prepared by chemically changing quercetin is not cytotoxic even at high concentrations and does not irritate the skin. The present invention was completed by inhibiting melanin production, reducing the expression of various enzymes (tyrosinase, TRP-1, TRP-2) involved in melanin synthesis, and reducing the MITF, thereby completing the present invention. .

Korean Patent No. 1570123

An object of the present invention is to provide a novel compound.

An object of the present invention is to provide a cosmetic composition comprising the compound.

1. A compound represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof:

[Formula 1]

Wherein R ₁ , R ₂ , R _3, R ₄ , R ₅ independently of one another are hydrogen, a saturated or unsaturated chained hydrocarbon group of 5 to 20 carbon atoms, at least one of which is not hydrogen.

2. according to the above 1, wherein the compound of Formula 1 is a compound represented by the following Formula 2, a compound, a pharmaceutically acceptable salt thereof or solvate thereof:

[Formula 2]

Wherein R ₁ , R ₂ , R ₃ are independently of each other hydrogen, a saturated or unsaturated chain hydrocarbon group having 5 to 20 carbon atoms, at least one of which is not hydrogen.

3. according to the above 1, wherein the compound is represented by any one of the following Formulas 3 to 5, the compound, a pharmaceutically acceptable salt thereof or solvate thereof:

[Formula 3]

[Formula 4]

[Formula 5]

.

.

4. A method for preparing a compound represented by the formula (1), a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, comprising reacting a compound represented by the following formula (6) with a compound represented by the following formula (7) :

[Formula 1]

Wherein R ₁ , R ₂ , R _3, R ₄ , R ₅ independently of one another are hydrogen, a saturated or unsaturated chained hydrocarbon group of 5 to 20 carbon atoms, at least one of which is not hydrogen.

[Formula 6]

[Formula 7]

(Wherein R is a saturated or unsaturated chain hydrocarbon group having 5 to 20 carbon atoms and X is F, Cl, Br or I).

5. In the above 4, wherein in Formula 7 X is Br, the compound of Formula 6 and the compound of Formula 7 1: 1: 1 to 2 equivalents, the method.

6. Cosmetic composition comprising a compound of the above 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

7. In the above 6, wherein the cosmetic composition is for skin whitening, cosmetic composition.

8. according to the above 6, wherein the compound is represented by the following Formula 3, cosmetic composition:

[Formula 3]

.

.

9. The composition for external application for skin comprising the compound of the above 1, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

10. In the above 9, wherein the external composition for skin is for skin whitening, the external composition for skin.

11. In the above 9, wherein the compound is represented by the following formula 3, the external composition for skin:

[Formula 3]

.

.

The novel compounds of the present invention are not cytotoxic even at high concentrations, inhibit melanogenesis and reduce the expression of various enzymes (tyrosinase, TRP-1, TRP-2) involved in melanin synthesis, As a transcription factor specific for melanocytes, it has an effect of reducing MITF (Microphthalmia-associated transcription factor) that regulates the expression of melanogenesis enzymes.

The novel compounds of the present invention can promote transdermal permeation through enhancing skin affinity and improving skin absorption, and thus can be usefully used as functional cosmetics and external skin preparations for imparting whitening activity.

1 shows the results of TLC analysis of a compound according to an embodiment of the present invention.
2a and 2b show the results of ¹³ C-NMR and ¹ H-NMR measurement of the compound according to an embodiment of the present invention.
Figure 3 is the result of measuring the cell survival rate according to the treatment according to the concentration of the compound according to an embodiment of the present invention. OQ is an abbreviation for the compound of the present invention.
Figure 4 is a result of measuring the melanin content in the melanoma cells treated with α-MSH (1 ㎍ / ml) and the concentration according to the embodiment of the present invention (50 ㎍ / ml, 100 ㎍ / ml) . OQ is an abbreviation for the compound of the present invention.
FIG. 5 shows tyrosinase and TRP- according to the treatment of melanoma cells with α-MSH (1 μg / ml) and concentrations of compounds according to an embodiment of the present invention (50 μg / ml, 100 μg / ml). 1, TRP-2 protein expression was measured by Western blot analysis (A) and quantified it is shown in the graph (B). TYR stands for tyrosinase and OQ stands for compound of the present invention.
6 is a protein expression level of MIITF according to the treatment of a-MSH (1 μg / ml) and the concentration of the compound according to an embodiment of the present invention (50 μg / ml, 100 μg / ml) to melanoma cells. The result measured by blot analysis (A) and it is quantified and graphically represented (B). OQ is an abbreviation for the compound of the present invention.
7 shows p-CREB and p-PKA according to α-MSH (1 μg / ml) and the concentration-specific treatment (50 μg / ml, 100 μg / ml) of melanoma cells according to an embodiment of the present invention. The expression level of the protein was measured by Western blot analysis (A) and it is quantified and shown in the graph (B). OQ is an abbreviation for the compound of the present invention.
FIG. 8 shows tyrosinase and TRP- according to the treatment of melanoma cells with concentrations of α-MSH (1 μg / ml) and compounds according to an embodiment of the present invention (50 μg / ml, 100 μg / ml). 1, mRNA expression level of TRP-2 was measured by RT-PCR analysis. OQ is an abbreviation for the compound of the present invention.
9 is a result of measuring the melanin content change after the treatment according to the concentration of the compound according to an embodiment of the present invention in zebrafish (50 ㎍ / ml, 100 ㎍ / ml). OQ is an abbreviation for the compound of the present invention.
Figure 10 is the result of measuring the cell survival rate according to the treatment of the concentration of quercetin compound (5, 10, 30 ㎍ / ml).
Figure 11 shows protein expression of tyrosinase, TRP-1, TRP-2 according to the treatment of the concentration of α-MSH (1 μg / ml) and quercetin compound (5 μg / mL 10 μg / mL) in melanoma cells The amount was measured by Western blot analysis.
12 is a result of measuring the melanin content change and tyrosinase activity after treatment of the quercetin compound in zebrafish by concentration (2.5 μg / ml, 5 μg / ml). A: Image of zebrafish observed under microscope, B: Mortality rate (%), C: Pigmentation area (%), D: Tyrosinase activity (%)
Figure 13 shows the results of ¹³ C-NMR and ¹ H-NMR of the compound according to an embodiment of the present invention.
Figure 14 shows the results of ¹³ C-NMR and ¹ H-NMR measurement of the compound according to an embodiment of the present invention.
Figure 15 shows the tyrosinase inhibitory activity of the compound according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof.

[Formula 1]

Wherein R ₁ , R ₂ , R _3, R ₄ , R ₅ independently of one another are hydrogen, a saturated or unsaturated chained hydrocarbon group of 5 to 20 carbon atoms, at least one of which is not hydrogen.

The saturated or unsaturated chain hydrocarbon group having 5 to 20 carbon atoms may have 5 to 10 carbon atoms, specifically 6 to 18 carbon atoms, an alkyl group or at least some of the chains may be unsaturated, and in the case of an unsaturated chain, a double bond May be 1 to 3, specifically 1 to 2, but is not limited thereto.

As used herein, the term “pharmaceutically acceptable salts” refers to salts or complexes that retain the desired biological activity of the compound of Formula 1 above and exhibit minimal or no undesirable toxic effects. Specifically, the pharmaceutically acceptable salt is formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; Or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert Acid addition salts formed with organic acids such as butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid;

As used herein, the term “solvate” refers to a higher-order compound formed between molecules or ions of a solute and molecules or ions of a solvent, and hydrates of water are also included therein.

The compound of Formula 1 of the present invention may specifically be a compound of Formula 2, more specifically, may be a compound of Formulas 3 to 5, and most specifically, a compound of Formula 3.

[Formula 2]

Wherein R ₁ , R ₂ , R ₃ are independently of each other hydrogen, a saturated or unsaturated chain hydrocarbon group having 5 to 20 carbon atoms, at least one of which is not hydrogen)

[Formula 3]

[Formula 4]

[Formula 5]

The compound of formula 1 of the present invention may be synthesized or obtained from natural products.

In addition, the present invention relates to a method of preparing a compound of Formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

The compound of Formula 1 may be obtained by nucleophilic substitution reaction of the compound represented by the following formula (6), more specifically, may be obtained by reacting the compound represented by the formula (6) with a compound represented by the formula (7) .

[Formula 6]

[Formula 7]

(Wherein R is a saturated or unsaturated chain hydrocarbon group having 5 to 20 carbon atoms and X is F, Cl, Br or I).

Specifically, the compound of Formula 7 may be a compound of Formula 8 to 10. The compound of formula 3 is obtained by reacting the compound of formula 6 with the compound of formula 8, the compound of formula 4 with the compound of formula 6, the compound of formula 9, and the compound of formula 5 with the compound of formula 10 It may be.

[Formula 8]

[Formula 9]

[Formula 10]

Wherein X is F, Cl, Br or I.

The nucleophilic substitution reaction can be carried out using a solvent, a catalyst known in the art.

Specifically, a polar solvent such as DMSO (dimethyl sulfoxide), DMF (N, N'dimethylformamide), acetone, DMA (dimethylacetamide) or HMPA (hexamethylphosphoramide) may be used. More specifically, DMSO may be used. It is not limited to this.

Specifically, Na ₂ CO ₃ may be used as the catalyst, but is not limited thereto.

The equivalent ratio of the compound of Formula 6 to the compound of Formula 7 may be, for example, 1: 1 to 2, specifically 1: 1 to 1.8, but is not limited thereto.

In addition, when using a catalyst, for example, the equivalent ratio of the compound of Formula 6, the compound of Formula 7 and the catalyst may be 1: 1 to 2: 1 to 2, specifically 1: 1 to 1.8: 1 to 1.8 days May be, but is not limited thereto.

The present invention also relates to a composition comprising the compound of Formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

The composition of the present invention may be a cosmetic composition, a topical skin composition, a pharmaceutical composition.

The compositions of the present invention may have skin whitening uses.

The compound of formula 1, a pharmaceutically acceptable salt thereof, or solvate thereof may be included in the composition of the present invention at a concentration of 0.001 to 10,000,000 μg / ml.

The cosmetic composition is not particularly limited in formulation, and may be appropriately selected as desired. As a specific example, the formulation may be selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, powders, soaps, oils, sprays, and the like, but is not limited thereto.

Specific formulations of cosmetic compositions include softening lotions, gels, water soluble liquids, milk lotions, nourishing creams, massage creams, essences, oil-in-water emulsions, water-in-oil emulsions, flake anhydrous products, solid anhydrous products, globules in aqueous phase Oil dispersions, ionic lipid vesicles, nonionic lipid vesicles, ointments, cleansing foams, cleansing water, packs, body oils, oil-in-water makeup bases, water-in-oil makeup bases, foundations, skin covers, lipsticks, lip gloss, One powder selected from the group consisting of face powder, two-way cake, eye shadow, mascara, cheek color and eyebrow pencils.

Cosmetic compositions include purified water, stabilizers, emulsifiers, thickeners, moisturizers, liquid crystal film strengthening agents, pH adjusting agents, antibacterial agents, water soluble polymers, coating agents, metal ion sequestrants, amino acids, organic amines, polymer emulsions, pH adjusters, skin nutrients, antioxidants At least one aqueous additive selected from antioxidants, preservatives, fragrances and the like; And at least one oily additive selected from fats and oils, waxes, hydrocarbon oils, higher fatty acid oils, higher alcohols, synthetic ester oils and silicone oils.

At this time, the aqueous additive is not limited as long as it is a raw material generally used in the art, specific examples include glycerin, dipropylene glycol, butylene glycol, pentylene glycol, methyl propanediol, sorbitol, diglycerol, Erythritol, pentaerythritol, polybutylene glycol-10, polyglycerol-3, polyglycerol-4, polyglycerol-6, polyglycerol-10, polyglycerol-20, polyglycerol-40, sorbeth-5, sorbeth -6, sorbeth-20, sorbeth-30, sorbeth-40, inositol, maltitol, maltose, met, mannitol, mannose, lactitol, lactose, dihydroxypropylPG-glucoside, dithiaoctanediol, fructose Lactose, Glucamine, Methylglucamine, Glucose, 1,2,6-hexanechiol, Methylgluses-10, Methylgluses-20, Ozoneized glycerin, Pytantriol, Thioglycerin, Traceol, Trimethyl Olpropane, Xylitol, IDTI EDTA), guar gum, quinsseed, carrageenan, galactan, gum arabic, pectin, metnan, starch, xanthan gum, curdlan, methylcellulose, hydroxyethylcellulose, carboxymethyl cellulose, methyl hydroxypropyl cellulose, chondroitin sulfate , Dermatan sulfate, glycogen, heparan sulfate, hyaluronic acid, sodium hyaluronate, tragant gum, keratan sulfate, chondroitin, mucotin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, kerat sulfate, Locust bean gum, succinoglucan, caronic acid, chitin, chitosan, carboxymethyl chitin, agar, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium polyacrylate, polyethylene glycol, bentonite, methylparaben, propylparaben, It may be at least one selected from phenoxyethanol, 1,2-hexanediol, ethylhexylglycerine and the like. In addition, the oil-based additive is not limited as long as it is a raw material commonly used in the art, and liquid oils such as olive oil, camellia oil, jojoba oil, triglycerides, glycerin trioctanoate and glycerin triisopalmitate, palm oil, hardened palm oil, palm oil And solid fats and oils such as hardened oil and hardened castor oil, beeswax, candelilla wax, carnauba wax, lanolin, jojoba wax and the like. Hydrocarbon oils include liquid paraffin, squalene, petrolatum, microcrystalline wax and the like. Examples of higher fatty acids include waxes such as lauric acid, myristic acid, palmitic acid, stearic acid, and behenic acid, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, cetostearyl alcohol, and the like. Synthetic ester oil may be isopropyl myristate, cetyloctanoate, octyldodecyl myristate, isopropyl palmitate, hexyl laurate, myristyl myristate, cetyl lactate, isocetyl isostearate, neopentyl Higher alcohols such as glycol dicaprate, ethylhexylglycerin, cetylethylhexanoate, ethylhexyl palmitate and cetostearyl alcohol, chain silicone oils such as dimethylpolysiloxane, methylphenylpolysiloxane and methylhydrogenpolysiloxane, dodecamethylcyclohexa Selected from cyclic silicone oils such as siloxane, octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane It is, but is not limited thereto.

The cosmetic composition may be formulated by containing a compound of Formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof in a nanoliposome to stabilize it. When the ingredient is contained in the nanoliposomes, the active ingredient is stabilized to solve problems such as precipitation formation, discoloration, and odor when formulated, and can increase the solubility and transdermal absorption of the component to maximize the efficacy expected from the compound. Can be expressed.

Nanoliposomes are liposomes having an average particle diameter of 10 to 500 nm as having a conventional liposome form. Specifically, the average particle diameter of the nanoliposomes is 50-300 nm, more preferably 100-200 nm. When the average particle diameter of the nanoliposomes exceeds 500 nm, the improvement of skin penetration and the improvement of formulation stability are very weak among the technical effects to be achieved in the present invention.

Nanoliposomes can be prepared by a mixture comprising polyols, oily ingredients, surfactants, phospholipids, fatty acids and water.

The polyols used for the nanoliposomes are not particularly limited, and preferably, propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methyl propanediol, isopropylene glycol, pentylene glycol, erythritol, xylitol, sorbitol and At least one selected from the group consisting of mixtures thereof. The amount used is 10 to 80% by weight, preferably 30 to 70% by weight based on the total weight of nanoliposomes.

The oil component used in the preparation of the nanoliposomes may be various oils known in the art, preferably hydrocarbon oils such as hexadecane and paraffin oil, synthetic oils of esters, dimethicone and Silicone oils such as cyclomethicone, sunflower oil, corn oil, soybean oil, avocado oil, animal and vegetable oils such as sesame oil and fish oil, ethoxylated alkyl ether oil, propoxylated alkyl ether oil, phytos Sphingoidoid lipids such as pingosine, sphingosine and sphinginine, cerebroside cholesterol, cytosterol cholesterylsulfate, cytosterylsulfate, C10-40 fatty alcohols and mixtures thereof. The amount may be 1.0 to 30.0% by weight based on the total weight of the nanoliposomes, preferably 3.0 to 20.0% by weight.

Surfactants used in the preparation of nanoliposomes can be used in any known in the art. For example, anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants can be used. Preferred are anionic surfactants and nonionic surfactants. Specific examples of anionic surfactants include alkylacylglutamate, alkylphosphates, alkyllactylates, dialkylphosphates and trialkylphosphates. Specific examples of nonionic surfactants include alkoxylated alkylethers, alkoxylated alkylesters, alkylpolyglycosides, polyglyceryl esters and sugar esters. Particularly preferred surfactants are polysorbates belonging to nonionic surfactants. The amount used may be 0.1 to 10% by weight, and preferably 0.5 to 5.0% by weight based on the total weight of the nanoliposomes.

Phospholipids, another component used in the preparation of nanoliposomes, include amphoteric lipids, natural phospholipids (eg egg yolk lecithin or soy lecithin, sphingomyelin) and synthetic phospholipids (eg dipalmitoyl). Phosphatidylcholine or hydrogenated lecithin), preferably lecithin. In particular, naturally-occurring unsaturated lecithin or saturated lecithin extracted from soybean or egg yolk are preferred. Typically, lecithin derived from nature has an amount of phosphatidylcholine of 23-95%, and an amount of phosphadidylethanolamine of 20% or less. In the preparation of the nanoliposomes of the present invention, the amount of phospholipid used is 0.5 to 20.0% by weight, preferably 2.0 to 8.0% by weight, based on the total weight of the nanoliposomes.

Fatty acids used for the preparation of nanoliposomes include higher fatty acids, preferably saturated or unsaturated fatty acids of the C12-22 alkyl chain, such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid. The amount used may be 0.05 to 3.0% by weight relative to the total weight of the nanoliposomes, preferably 0.1 to 1.0% by weight.

Water used for the preparation of nanoliposomes is generally deionized distilled water, the amount of which may be 5.0 to 40% by weight relative to the total weight of nanoliposomes.

The preparation of the nanoliposomes can be accomplished through various methods known in the art, but is preferably prepared by applying a mixture comprising the above components to a high pressure homogenizer. The preparation of nanoliposomes by high pressure homogenizer can be carried out under various conditions (eg, pressure, frequency, etc.) according to the desired particle size, preferably 1-5 times high pressure homogenizer under pressure of 600-1200 bar. Nanoliposomes can be prepared by passing through.

The cosmetic composition may be used alone or in duplicate, or may be used in combination with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention may be used according to a conventional method of use, and the number of times of use may vary depending on the skin condition or taste of the user.

"Skin external preparation" is a general concept encompassing all materials used for external application of the skin. Non-limiting examples of the formulation include PLASTERS, lotion (LPTIONS), LINIMENTS, liquid formulations. LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, Ointments, FLUIDEXTRACTS, Emulsions, Suspensions, Capsules, Creams, Creams ), Soft, hard gelatin capsules, patches, or sustained release agents.

The skin external preparation may be a parenteral dosage form formulated in solid, semisolid or liquid form with the addition of a compatible inorganic or organic carrier, excipient and diluent. For parenteral administration, the preparation may be a transdermal dosage form selected from the group consisting of drops, ointments, lotions, gels, creams, patches, sprays, suspensions and emulsions, but is not limited thereto.

Carriers, excipients and diluents that may be included in the external preparation include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the external preparation composition for skin by each formulation, other ingredients other than the composition of the present invention described above can be appropriately selected and blended by those skilled in the art according to the formulation or use purpose of the external preparation for skin, and in this case, simultaneously applied with other raw materials. If you do, synergistic effects may occur.

The pharmaceutical composition may further contain pharmaceutical aids such as preservatives, stabilizers, hydrating or emulsifying accelerators, salts or buffers for the control of osmotic pressure and other therapeutically useful substances, and parenteral dosage forms according to conventional methods. It can be formulated as.

The composition of the present invention may be applied to the skin of an individual to prevent or ameliorate pigmentation in the skin or to correct or repair symptoms due to pigmentation. Symptoms caused by the pigmentation include blemishes, freckles, seborrheic keratosis (aging black surplus, blotch) and hyperpigmentation after inflammation or stimulation.

In the present invention, the term "individual" means all animals, including humans, which may or may have developed pigmentation on the skin as described above, or whose symptoms may or may be expressed due to the pigmentation, and the composition of the present invention. By applying to the subject can effectively prevent or improve pigmentation in the skin or correct or recover the symptoms caused by pigmentation. The composition of the present invention can be used in combination with a conventional skin whitening pharmaceutical composition.

In the present invention, the term "application" means any method of contacting the composition of the present invention with the skin of an individual in any suitable way, through which the purpose is to absorb the composition into the skin.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example

1. Compound Synthesis

(1) a compound of formula 3

Substitution of quercetin was carried out using oleyl bromide. Na ₂ CO ₃ (SigmaAldrich, USA) was used as a catalyst and DMSO (dimethyl sulfoxide, Sigma Aldrich, USA) was used as a solvent. Quercetin per one equivalent of the nucleophile material and Na ₂ CO ₃ gave put by 1.2 equivalents or 1.5 equivalents, respectively. Into a 100 mL two-neck round-bottom flask, quercetin, nucleophile, Na ₂ CO ₃ , and DMSO were added, followed by magnetic stirring by controlling the RPM and temperature of the hot plate. The synthesized sample was placed in a separatory funnel, 100 mL of EtOAc and 40 mL of NH ₄ Cl aqueous solution were added to the separatory funnel, and then shaken vigorously and the supernatant was discarded. 40 mL of aqueous NH ₄ Cl solution was added to the remaining supernatant, followed by vigorous shaking. The lower supernatant was discarded to remove Na ₂ CO ₃ . 40 mL of NaCl aqueous solution was repeated twice in the same manner as above, and finally, the remaining solution was added to remove anhydrous (anhydrous) MgSO ₄ . After filtering using a glass filter, concentrated to complete the sample pretreatment. About half of the amount of pretreated sample was separated using an open column. The column was lowered and received in glass tubes for each fraction, followed by TLC to identify materials, and then the same materials were combined and concentrated. After concentration it was finally concentrated using Speed vac (MaxiVac, Neutec, USA). TLC analysis results are shown in FIG. 1.

(2) a compound of formula 4

Compounds were synthesized in the same manner as in (1), except that hexyl bromide was used instead of oleyl bromide.

(3) a compound of formula 5

Compounds were synthesized in the same manner as in (1), except that dodecyl bromide was used instead of oleyl bromide.

2. Structure Analysis

(1) a compound of formula 3

In order to confirm the structure of the compound prepared in Example 1, ¹ H-, ¹³ C-NMR analysis was performed. ¹ H-NMR analysis confirmed the H of the oleyl carbon chain (-CH ₃ , -CH ₂ ) at 0.7-1.92 ppm and the H of the double bond of oleyl carbon at 5.23 ppm. At 3.90 ppm, quercetin and oleyl bond were confirmed. The H of the quercetin aromatic ring was found at 6.20-7.50 ppm. ¹³ C-NMR analysis confirmed the oleyl carbon chain at 13.05-72.55 ppm and the double bond of oleyl carbon at 129.41-129.44 ppm. A quercetin aromatic ring was identified at 92.12-178.83 ppm. ¹ H- and ¹³ C-NMR analysis confirmed that oleyl and quercetin were synthesized, and ¹ H-NMR analysis confirmed that two oleyls instead of one were bound to quercetin according to the integral ratio of H. That is, the oleyl-quercetin compound prepared in Example 1 was confirmed to be '3,7-dioleyl quercetin' including two oleyl groups at 3 and 7 carbon positions of quercetin.

¹ H NMR (CD3OD): δ = 0.77 (m, 3H), 1.22 (m, 16H), 1.30 (m, 2H), 1.39 (m, 4H), 1.65 (m, 2H) 1.92 (m, 4H), 3.90 (dt, 2H, J = 48.00 Hz, J = 6.40 Hz), 5.23 (m, 2H), 6.20 (d, 1H, J = 2.00 Hz, H-8), 6.43 (d, 1H, J = 2.40 Hz , H-6), 6.79 (d, 1H, J = 8.40 Hz, H-5 '), 7.42 (dd, 1H, J = 8.40 Hz, J = 2.00 Hz, H-6') 7.50 (d, 1H, J = 2.40 Hz, H-2 '). ¹³ C NMR (CD3OD): δ = 13.05, 22.33, 25.60, 26.69, 26.73, 28.67, 28.75, 28.80, 28.95, 29.05, 29.21, 29.36, 29.45, 29.54, 31.67, 72.552, 92.12 (C-8), 97.80 ( C-6), 105.31 (C-4a), 114.81 (C-2 '), 115.40 (C-5'), 121.18 (C-6 '), 121.63 (C-3), 129.41, 129.49, 137.44 (C -1 '), 145.02 (C-2), 148.59 (C-3'), 156.91 (C-4 '), 157.25 (C-5), 161.50 (C-8a), 165.22 (C-7), 178.83 (C-4).

¹ H- and ¹³ C-NMR analysis results are shown in FIGS. 2A and 2B.

(2) the compound of Formulas 4 and 5

In order to confirm the structure of hexyl quercetin (Formula 4) and dodecyl quercetin (Formula 5), ¹ H-, ¹³ C-NMR analysis was performed. Hexyl quercetin and dodecyl quercetin ¹ H-NMR analysis confirmed the H of the alkyl chains (-CH ₃ , -CH ₂ ) at 0.7-1.92 ppm, and the quercetin and alkyl chain bonds were identified at 3.90-4.08 ppm. The H of the quercetin aromatic ring was found at 6.20-7.50 ppm. ¹³ C-NMR analysis confirmed an alkyl chain at 14-30 ppm and quercetin and an alkyl chain bond at 68-72 ppm. Quercetin aromatic rings were identified at 92.12-178.83 ppm. ¹ H- and ¹³ C-NMR analysis confirmed that hexyl and dodecyl-coupled quercetin were synthesized, and ¹ H-NMR analysis showed that two alkyl chains instead of one were bound to quercetin according to the integral ratio of H.

The results are shown in Figure 13 (Formula 4), Figure 14 (Formula 5).

3. Cytotoxicity Assessment

The cytotoxicity of the 3,7-dioleyl quercetin compound of the present invention prepared in Example 1 was evaluated. In detail, B16F10 melanoma cells were inoculated at 1 × 10 ⁵ cells / well in 24 well plates and incubated for 24 hours. Treatment of 3,7-dioleyl quercetin compound of the present invention by concentration (25, 50, 100 ㎍ / mL), and after 24 hours incubation treated with 500 μL of MTT stock solution (2 mg / mL) to the cultured cells After 1 hour, the medium was removed, 200 μl of DMSO was added to dissolve the MTT-formazan crystals produced in the cells, and 100 μl of the 96 wells was measured. The absorbance was measured at 550 nm with an ELISA reader.

As a result, as shown in Figure 3, the 3,7-dioleyl quercetin compound of the present invention prepared in Example 1 was found to be cytotoxic to a concentration of 100 μg / mL.

4. Evaluation of whitening activity

In order to evaluate the whitening activity of the compound of the present invention prepared in Example 1, first, the melanin content according to the concentration-specific treatment of the 3,7-dioleyl quercetin compound was analyzed; Expression levels of proteins involved in melanin formation were analyzed by Western blot analysis; Gene expression levels of enzymes involved in melanin synthesis were analyzed by RT-PCR; Finally, the whitening activity using zebrafish was confirmed.

For reference, tyrosinase plays an important role in the synthesis of melanin, which has tyrosine hydroxylase activity that converts tyrosine to DOPA and DOPA oxidase activity that oxidizes DOPA to DOPAquinone. It is an enzyme. TRP-1 (tyrosinase-related protein l (DHICA oxidase) is involved in melanin synthesis, oxidizes DHICA to indole-5, & quinone-2-carboxylate and finally proceeds to the synthesis of eumelanin DHICA oxidase. In addition to its function as DHICA oxidase, TRP-1 has also been reported to contribute to the stabilization of the enzyme group in melanosomes. Tyrosinase-related protein 2: DOPA chrome tautomerase (TRP-2) converts DOPA chromium produced by tyrosinase-induced synthesis into TPR-2 with 5,6-dihydroxyindole-2-carboxylic acid and indole It is known to form a skeleton.

On the other hand, MITF (Microphthalmia-associated transcription factor) is known to regulate the expression of melanogenesis enzyme as a transcription factor specific to melanocytes. Melanocite receptors increase intracellular cAMP levels in the presence of stimuli such as ultraviolet light, and an increase in cAMP leads to activation of protein kinase A (PKA) followed by phosphorylation of cAMP response element-binding protein (CREB). . Phosphorylated CREB causes the expression of MITF, a specific transcription factor of melanocytes. Expression of MITF eventually promotes the binding of tyrosinase, TRP-1, and TRP-2 promoters, which are the major enzymes of melanogenesis, to increase melanin biosynthesis.

(1) melanin content analysis

To quantify the resulting melanin, B16 / F10 melanoma cells were dispensed in 6-well plates at a concentration of 1 × 10 ⁵ cells / well and incubated for one day at 37 ° C. and 5% CO ₂ conditions. Subsequently, the stimulant α-MSH (1 μg / ml) and the 3,7-dioleyl quercetin compound of the present invention were treated by concentration (50 μg / mL 100 μg / mL) and incubated for 3 days. The cell precipitate obtained by centrifuging the cultured cells for 5 minutes was lysed with 1 mL of PBS. 200 nL of a 1 N NaOH (+ 10% DMSO) solution was added to the pellets, and the absorbance was measured at 405 nm. The untreated group was used as a control, and the arbutin treated group (arbutin 100 µg / ml) was used as a positive control.

As a result, as shown in Figure 4, when treated with 1 ㎍ / ml α-MSH the amount of melanin increased by 300% compared to the untreated group (100%), 3,7-diol of the present invention with α-MSH In the experimental group treated with the rail quercetin compound by concentration (50 μg / mL, 100 μg / mL), the amount of melanin was reduced depending on the treatment concentration. In particular, the experimental group treated with the 3,7-dioleyl quercetin compound of the present invention at a concentration of 100 μg / mL showed a better melanin reduction effect than the arbutin treated group as a positive control group.

(2) Western blot analysis

B16F10 melanoma cells were inoculated at 1 × 10 ⁵ cells / well, incubated for 24 hours, the samples were treated by concentration, and after 1 hour, lysis buffer was added to dissolve the cells, followed by centrifugation at 3000 × g for 10 minutes. After incubation for 12 hours after α-MSH treatment, 100 μl of the incubated cell culture supernatant was taken and transferred to a 96-well plate. Protein quantitation was measured using the Bradford method. 20 μg / mL of protein was denatured with 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and transferred to a polyvinylpolyvinylidene fluoride (PVDF) membrane. After blocking the membrane, the primary antibody was added to Tris-buffered saline containing 0.2% PBST containing Tris buffer saline containing 2.5% nonfat dry milk and at room temperature for 3 hours or 4 ° C. The reaction was overnight (10 hours). Membranes reacted with the primary antibody were washed three times with PBST, reacted with the secondary antibody for 1 hour at room temperature, and then washed three times with PBST to confirm the pattern of each band using an image quant detection system (LAS 4000 mini). The protein pattern was confirmed by treating the 3,7-dioleyl quercetin compound of the present invention by concentration (1-50 μg / mL), and the arbutin treated group (arbutin 100 μg / ml) was used as a positive control.

Primary antibodies of TYR (tyrosinase), TRP-1, TRP-2, MITF, p-CREB, CREB, p-PKA, PKA and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

20 μg / mL of protein was denatured with 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and transferred to PVDF membrane. After blocking the membrane, the primary antibody was added to Tris-buffered saline with 0.2% PBST containing 2.5% nonfat dry milk and reacted at room temperature for 3 hours or over night (10 hours). The membranes reacted with the primary antibody were washed three times with PBST, and then reacted with the secondary antibody for 1 hour at room temperature, and then washed three times with PBST to confirm the pattern of each band with an ImageQuant detection system (LAS 4000 mini).

As a result, as shown in Figures 5 to 7, the treatment of α-MSH protein expression of tyrosinase, TRP-1, TRP-2, MITE, p-CREB, p-PKA markedly increased compared to the control group On the other hand, in the experimental group treated with the 3,7-dioleyl quercetin compound of the present invention, the expression level of the protein associated with enhanced melanin formation was significantly reduced by α-MSH treatment.

(3) RT-PCR analysis

In order to examine the effects of the 3,7-dioleyl quercetin compound of the present invention on the expression of melanogenic enzymes in B16F10 stimulated with α-MSH, total mRNA in the cells was first extracted, followed by tyrosinase. MRNA levels of Aze, TRP-1 and TRP-2 were analyzed via qRT-PCR.

Total RNA was extracted using TRIZOL (Invitrogen, USA). CDNA for RT-PCR was synthesized using a high-capacity cDNA synthesis kit (Bioneer, Daejon, Korea), and synthesized cDNA using SYBR green premix (Applied Biosystems, USA) and STEP ONE (Applied Biosystems, USA) The specificity of the amplified PCR product was confirmed by analyzing the melting curve of the amplified product with a Realtime RT-PCR machine.

gene primer order SEQ ID NO: Tyrosinase Forward (F) 5'-ATAACAGCTCCCACCAGTGC-3 ' One Reverse (R) 5'-CCCAGAAGCCAATGCACCTA-3 ' 2 TRP-1 Forward (F) 5'-GCTGCAGGAGCCTTCTTTCTC-3 ' 3 Reverse (R) 5'-GCTGCAGGAGCCTTCTTTCT-3 ' 4 TRP-2 Forward (F) 5'-GGATGACCGTGAGCAATGGCC-3 ' 5 Reverse (R) 5'-CGGTTGTGACCAATGGGTGCC-3 ' 6

As a result, as shown in Figure 8, the mRNA levels of the melanogenesis enzymes tyrosinase, TRP-1 and TRP-2 significantly increased by α-MSH treatment, this increase is 3,7 of the present invention -Significantly reduced by treatment with dioleyl quercetin compound. In particular, it was confirmed that the 3,7-dioleyl quercetin compound of the present invention more effectively inhibits the mRNA expression level of melanin-producing enzyme compared to the positive control.

(4) Evaluation of whitening activity using zebrafish

Zebrafish embryos were cultured in 24 wells and 10 per well. The 3,7-dioleyl quercetin compound of the present invention was treated by dissolving in 0.1% DMSO at each concentration (50, 100 µg / ml) and left for 72 hours to evaluate the whitening activity. In this study, 0.2 mmol / L of PTU (1-phenyl2-thiourea, tyrosinase dependent step block), an anti-cell deposition drug, was used as a positive control. Pigmentation of zebrafish was observed with a stereomicroscope and images were analyzed at 55 hpf by calculating the pigmentation area of each zebrafish with Image Pro Plus Analysis Software. For reference, the survival rate was 100% in zebrafish embryo cultures treated with the 3,7-dioleyl quercetin compound of the present invention (results not shown).

As a result, as shown in Figure 9, the experimental group treated with the 3,7-dioleyl quercetin compound of the present invention was found that the number of pigment cells is reduced and the pigment of some individuals is light, especially 3, Treatment of the 7-dioleil quercetin compound at a concentration of 100 μg / ml showed melanin content similar to that of the positive control.

(5) Furthermore, the tyrosinase inhibitory activity of the compounds of the formulas (4) and (5) was further evaluated.

Kojic acid was used as a positive control.

Mushroom tyrosinase activity was measured to confirm the whitening effect of the synthesized new material. The experimental method is based on the principle that L-DOPA is converted to DOPA chrome by tyrosinase activity. 20 μl of synthesized novel substance prepared with 220 μl of 0.1 M sodium phosphate buffer (pH 6.5) and 50 to 1000 μg / mL, L-tyrosine (Sigma, USA) 1.5 mM 40 μl, mushroom prepared with 1700 unit 20 μl of tyrosinase (Sigma, USA) was added and reacted at 37 ° C. for 15 minutes. After the reaction, the amount of DOPA chrome formed from DOPA by tyrosinase was measured at 490 nm. The tyrosinase inhibitory activity was expressed as a decrease in absorbance of the added and non-added samples.

As a result, it was confirmed that the compounds of Formulas 4 and 5 had excellent tyrosinase inhibitory activity (FIG. 15).

Comparative Example 1. Evaluation of Cytotoxicity of Quercetin Compounds

The cytotoxicity of the quercetin compound was evaluated. B16F10 melanoma cells were inoculated at 1 × 10 ⁵ cells / well in 24 well plates and incubated for 24 hours. After treatment with quercetin compounds by concentration (5, 10, 30 ㎍ / ml), the cultured cells were treated with 500 μl of MTT stock solution (2 mg / mL) for 24 hours, and after 1 hour, the medium was removed and DMSO 200 μl was added to dissolve the MTT-formazan crystals produced in the cells, and then 100 μl were taken into 96 wells and the absorbance was measured at 550 nm with an ELISA reader.

As a result, as shown in FIG. 10, the quercetin compound showed high cytotoxicity even at a concentration of 10 μg / ml. This low concentration of cytotoxicity is considered to be unsuitable for cosmetics due to human safety reasons.

The above results are in complete contrast with the results of the 3,7-dioleyl quercetin compound of the present invention having no cytotoxicity up to a concentration of 100 μg / ml. As described in the above examples, the 3,7-dioleyl quercetin compound of the present invention did not show cytotoxicity to a concentration of 100 ㎍ / ml was confirmed to be a material having a human (skin) safety.

Comparative Example 2. Evaluation of the whitening activity of the quercetin compound

In order to evaluate the whitening activity of the quercetin compound, the expression levels of the proteins related to melanin formation following the treatment with the quercetin compound in the melanoma cells were analyzed by Western blot analysis; Whitening activity was confirmed using zebrafish; Tyrosinase activity was analyzed.

(1) Western blot analysis

The quercetin compound was treated in B16F10 melanoma cells at different concentrations (5, 10 μg / mL) to measure the expression of melanin-related enzymes (tyrosinase, TRP1, TRP2) by Westin blot analysis. Western blot analysis was performed in the same manner as in the above example except for the type and concentration of the compound to be treated in the cells.

As a result, as shown in Figure 11, when the quercetin compound is treated it was confirmed that the protein expression amount of tyrosinase, TRP1, TRP2 is further increased. These results indicate that the quercetin compound itself has no whitening activity and, conversely, inhibits whitening activity.

(2) Evaluation of whitening activity using zebrafish

Zebrafish embryos were cultured in 24 wells and 10 per well. The quercetin compound was treated by dissolving in 0.1% DMSO at each concentration (2.5, 5 μg / ml) and left for 72 hours to evaluate the whitening activity. In this study, 30 μg / mL of PTU (1-phenyl2-thiourea, tyrosinase dependent step block) was used as a positive control. Pigmentation of zebrafish was observed with a stereomicroscope and images were analyzed at 55 hpf by calculating the pigmentation area of each zebrafish with Image Pro Plus Analysis Software.

As a result, as shown in Figure 12, the positive control group significantly reduced the pigment cells while the treatment of the quercetin compound showed that the number of pigment cells is maintained as it is or further increased. In addition, as a result of comparing relative tyrosinase activity in zebrafish, it was found that in the quercetin compound treatment group, tyrosinase activity was increased depending on the treatment concentration. These results indicate that the quercetin compound itself has no whitening activity and, conversely, inhibits whitening activity.

<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION CHOSUN UNIVERSITY <120> NEW COMPOUNDS AND COSMETIC COMPOSITION INCLUDING THE SAME <130> 07002 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase foward primer <400> 1 ataacagctc ccaccagtgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase reverse primer <400> 2 cccagaagcc aatgcaccta 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TRP-1 foward primer <400> 3 gctgcaggag ccttctttct c 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRP-1 reverse primer <400> 4 gctgcaggag ccttctttct 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TRP-2 foward primer <400> 5 ggatgaccgt gagcaatggc c 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TRP-2 reverse primer <400> 6 cggttgtgac caatgggtgc c 21

Claims (11)

A compound represented by formula 3, 4, or 5, a pharmaceutically acceptable salt thereof, or solvate thereof:
[Formula 3]

[Formula 4]

[Formula 5]

.
delete delete delete delete Cosmetic composition comprising a compound of claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.
The cosmetic composition according to claim 6, wherein the cosmetic composition is for skin whitening.
delete A whitening skin external composition comprising the compound of claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof. delete delete

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