WO2020241972A1 - Cosmetic composition comprising substance p for skin whitening - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising substance P as an active ingredient for skin whitening. More particularly, a composition comprising substance P, an antioxidant, a surfactant, and a thickener according to the present invention can be advantageously used as a cosmetic composition, a quasi-drug composition, a pharmaceutical composition, and a health functional food composition. The present invention relates to a cosmetic composition comprising substance P as an active ingredient for skin whitening, which provides a skin whitening effect through a reduction in melanocyte activity and the total production amount of melanin, in mRNA level of MITF transcriptional factor, and in mRNA level and activity of tyrosinase.

Description

Cosmetic composition for skin whitening containing substance P

The present invention relates to a cosmetic composition for skin whitening comprising a substance P (substance P) as an active ingredient, and more specifically, a composition comprising a substance P, an antioxidant, a surfactant and a thickener of the present invention, a cosmetic composition, It can be usefully used as quasi-drug composition, pharmaceutical composition and health functional food composition.

Human skin color is genetically determined according to the concentration and distribution of melanin in the skin, but is also affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, and stress.

The chemical action on the production of melanin, which determines the color of the skin, has been reported in a number of papers so far.Melanocytes, which are melanocytes, protect cells from ultraviolet rays in the intracellular organelle called melanosomes. After synthesizing melanin having a protective function, it is known that the melanin migrates from melanocytes to keratinocytes, and it is reported that the melanin transferred to the keratinocytes determines the skin color.

It is known that the precursor required in the melanin biosynthesis process is an amino acid called tyrosine, and this tyrosine is converted to dihydroxyphenylalanine (DOPA), and then finally converted to melanin through a substance called dopaquinone. .

In this regard, melanin-forming enzymes include tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (tyrosinase-related protein). 2, TRP-2/DOPA, chrome tautomerase) and the like are known, and the melanin-forming enzyme is known to be regulated by a transcription factor called a microphthalmia-associated transcription factor (MITF). Tyrosinase, which plays an important role in the melanin synthesis pathway, converts L-tyrosine to L-DOPA (L-3,4-dihydroxyphenylalanine), and rapidly converts the generated L-DOPA to L-dopaquinone, Through subsequent reactions, it eventually forms melanin. Melanin synthesized by tyrosinase darkens the skin color, and research on a skin whitening method to brighten the skin color by inhibiting the melanin synthesis due to health or beauty requirements is actively underway.

As a commonly known whitening ingredient, arbutin, kojic acid (kojic acid), azelaic acid, aloesin, 4-butylresorcinol, resveratrol, ceramide, sphingosine-1-phosphate, sphingosyl which inhibit tyrosinase enzyme activity There are substances such as phosphorylcholine and the like, and hydroquinone (Korean Patent Application Publication No. 2016-0014271). These whitening components inhibit the synthesis of melanin pigment, thereby brightening the skin tone to realize skin whitening, or to improve skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones, or genetics. However, when some whitening ingredients are applied to the skin, there is a problem in that the amount of use is limited due to safety problems such as irritation and redness, or the effect is insignificant and thus no practical effect can be expected. In particular, in the case of hydroquinone, its use was prohibited due to unwanted side effects such as irreversible bleaching (European Committee 24th Dir 2000/6/EC), and it was replaced with arbutin, a structural analog of hydroquinone. As described above, in the field of whitening, it is particularly important to not only have a whitening effect by having an effect of inhibiting tyrosinase activity, but also to secure stability, and development of a material that satisfies all of the above requirements is still required.

Substance P is a peptide consisting of 11 amino acids and is a neurotransmitter scattered in nervous tissue. It has been known for a long time that it is involved in the activity of various cells in the body such as nerve cells, blood cells, and epithelial cells, and recently, the scope of application is wide enough to be known to act on anti-inflammatory and angiogenesis. The present inventors confirmed the skin whitening effect of the substance P.

Under the above background, the present inventors have made diligent efforts to increase the stability of the substance P. As a result, the composition comprising the substance P, an antioxidant, a surfactant and a thickener has a better skin whitening effect compared to the use of substance P alone. It was confirmed that the present invention was completed.

One object of the present invention is to provide a cosmetic composition for skin whitening comprising a substance P, an antioxidant, a surfactant and a thickener.

Another object of the present invention is to provide a quasi-drug composition for skin whitening comprising a substance P, an antioxidant, a surfactant and a thickener.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of whitening-related diseases comprising a substance P, an antioxidant, a surfactant, and a thickener.

The present invention relates to a cosmetic composition for skin whitening comprising a substance P, an antioxidant, a surfactant, and a thickener as active ingredients, and reduces melanocyte activity and total melanin production, decreases MITF transcription factor mRNA, tyrosinase mRNA and activity It provides skin whitening effect through reduction.

1 is a photograph showing activated melanocytes under a fluorescence microscope through HMB45 antibody staining for gp100, a melanosome-specific protein, after co-culture with skin keratinocytes and melanocytes to treat a composition containing substance P.

Figure 2a is a graph of measuring the amount of extracellular melanin production after treating a composition containing a substance P by co-culturing skin keratinocytes and melanocytes.

Figure 2b is a graph of measuring the amount of melanin production in cells after treating a composition containing substance P by co-culturing skin keratinocytes and melanocytes.

3A is a photo of an agarose gel band confirming the amount of mRNA production of MITF, tyrosinase, and GAPDH through PCR after co-culture of skin keratinocytes and melanocytes to treat a composition containing substance P.

Figure 3b is a graph obtained by co-culturing skin keratinocytes and melanocytes to treat a composition including substance P, and then quantifying the amount of mRNA of MITF through PCR.

3C is a graph in which keratinocytes and melanocytes are co-cultured to treat a composition including substance P, and then the amount of tyrosinase mRNA produced is quantified through PCR.

FIG. 4 is a graph showing the quantification of tyrosinase activity by co-culture of skin keratinocytes and melanocytes to treat a composition containing substance P, and then treatment with L-DOPA, a substrate of tyrosinase.

One aspect for achieving the above object is to provide a cosmetic composition for skin whitening comprising a substance P, an antioxidant, a surfactant, and a thickener.

The present invention relates to a novel cosmetic composition for improving the insignificant effect of skin whitening effect due to the instability of the existing substance P. The present inventors have developed components and contents of the composition that enable the optimal skin whitening effect to appear when the substance P is applied.

The term "composition comprising substance P" as used herein means a composition comprising substance P, an antioxidant, a surfactant and a thickener.

Specifically, the present invention provides a cosmetic composition for skin whitening comprising a substance P, sodium thiosulfate, polysorbate-80, and hydroxyethylcellulose.

The present inventors found that the composition comprising the substance P, an antioxidant, a surfactant, and a thickener has a skin whitening effect by reducing the total amount of melanocytes and melanin production, reducing MITF transcription factor mRNA, reducing tyrosinase mRNA and activity. And completed the present invention.

In the composition of the present composition, the substance P refers to a neuropeptide consisting of the amino acid "Arg- Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH ₂ "of SEQ ID NO: 1.

The concentration of the substance P included in the composition of the present invention may be 1 to 5 μg/ml.

In one embodiment of the present invention, it was confirmed that the composition containing the substance P having a concentration of 1 to 5 μg/ml has excellent skin whitening effect (Fig. 1).

As described above, in the cosmetic composition of the present invention, when the concentration of the substance P is included in the range of 1 to 5 μg/ml, it was confirmed that the cosmetic composition of the present invention has an excellent skin whitening effect.

Specifically, in the present invention, when the substance P is contained at a high concentration, pigmentation may occur.

In the composition of the present invention, the "antioxidant" acts on free groups or peroxides generated in the process of oxidation of the active ingredient by oxygen in the air to stop the chain reaction of oxidation, prevent the progress of oxidation, and deteriorate the active ingredient. It means a substance added for the purpose of preventing.

In the present invention, the antioxidant can prevent deterioration of the skin whitening effect of the cosmetic composition containing the substance P.

The antioxidant can be used without limitation, conventional antioxidants that can be used in the art, specifically beta-mercaptoethanol (β-mercaptoethanol, β-ME), glutathione (Glutathione, GSH), ascorbic acid ( ascorbic acid), vitamin E, beta carotene, lycopene coenzyme Q-10, selenium, chromium, magnesium, taurine, hypotaurine, or trehalose, etc. It is not limited. In the present invention, the antioxidant may be sodium thiosulfate.

The content of the antioxidant is not particularly limited as long as the whitening function is not altered, but may be 0.01 to 1% by weight, and specifically 0.1 to 1% by weight, based on the total weight of the composition of the present invention.

In the composition of the present invention, the "surfactant" refers to a substance that uses a lipophilic oil component to maintain a uniform liquid composition.

The surfactant may be a general surfactant used in the manufacture of cosmetic compositions such as anionic, cationic, nonionic, or amphoteric surfactants. Specifically, in the present invention, the surfactant may be polysorbate 80.

The content of the surfactant may be 0.001 to 0.1% by weight based on the total weight of the composition of the present invention, and specifically 0.006 to 0.1% by weight, may be 0.01 to 0.1% by weight.

In the composition of the present invention, the "thickener" is a thickener added to impart viscosity. Specifically, the thickener of the present invention may be hydroxyethylcellulose.

The content of the thickener may be 1 to 5% by weight based on the total weight of the composition of the present invention, and specifically 1.5 to 5% by weight, 2 to 5% by weight, and 3 to 5% by weight. If the content of the thickener is 1% by weight or less with respect to the total weight of the composition, there may be a problem in the stability of the cosmetic composition, and if it is 5% by weight or more, the viscosity of the cosmetic composition may be too high, resulting in a composition not suitable for mixing. .

In the present invention, the term "skin whitening" refers to brightening skin tone by inhibiting the synthesis of melanin pigment and/or inhibiting tyrosinase activity. In addition, the skin whitening includes improving the deposition of skin hyperpigmentation such as spots, freckles, age spots, blemishes or melanoma caused by ultraviolet rays, hormones or genetics.

The melanin is a pigment of dark brown particles, and is produced by an organelle called melanosomes in melanocytes. Melanin exists in the skin, hair, and eyes, and the skin color is determined by the amount of melanin. The higher the amount of melanin, the darker the skin color is.

Accordingly, the composition may be characterized by a skin whitening effect that suppresses melanin production.

In one embodiment of the present invention, it was confirmed that the activity of melanin was significantly reduced by treating the composition containing the substance P by co-culturing skin keratinocytes and melanocytes (FIG. 1), and the amount of extracellular melanin production was reduced ( Figure 2a), it was confirmed that the amount of melanin production in the cell decreases (Figure 2b).

In addition, in the present invention, the skin whitening may be achieved by a tyrosinase inhibitory effect, and the tyrosinase inhibitory effect may be an effect exhibited by a composition containing the substance P.

In one embodiment of the present invention, it was confirmed that the amount of MITF and tyrosinase mRNA was reduced by co-culturing skin keratinocytes and melanocytes to treat a composition containing substance P (Figs. 3a to c), tyro When treated with L-DOPA, which is a synase substrate, it was also confirmed that tyrosinase activity was inhibited (FIG. 4).

The term "cosmetic composition" of the present invention can be prepared in the form of a general emulsified formulation and a solubilized formulation. The emulsified formulation includes nutrient lotion, cream, essence, and the like, and the solubilized formulation includes softening lotion. The cosmetic composition is a formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant including cleansing, an oil, an ampoule, a powder foundation, an emulsion foundation, a wax foundation, and a spray. It can be manufactured by, but is not limited thereto. Specifically, hypoallergenic cosmetic skin protectant, flexible lotion, nutrient lotion, nutrition cream, massage cream, essence, eye cream, serum, cleansing cream, cleansing foam, cleansing water, pack, cream, essence, stray or powder formulation Can be.

In addition, the cosmetic composition may additionally include one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, A chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately blended, but the present invention is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition varies depending on the formulation.

When the formulation of the cosmetic composition is an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide Or a mixture thereof may be used.

When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in particular, in the case of a spray, additionally Propellants such as chlorofluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizing agent or an 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-butylglycol oil may be used, and in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used. I can.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the cosmetic composition is soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. are used as a carrier component. Can be.

Another aspect of the present invention is to provide a quasi-drug composition for skin whitening comprising a substance P, an antioxidant, a surfactant, and a thickener.

In the present invention, the substance P, antioxidant, surfactant, and thickener are as described above.

In the present invention, the term "quasi-drug" refers to items that are less effective than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals. For example, according to the Korean Pharmacological Law Quasi-drugs exclude items used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans and animals, and products with minor or no direct action on the human body.

The quasi-drug composition of the present invention may be prepared in a form selected from the group consisting of a body cleanser, foam, soap, mask, ointment, cream, lotion, essence, and spray, but is not limited thereto.

In the present invention of the present invention, when a composition containing a substance P, an antioxidant, a surfactant, and a thickener is used as a quasi-drug additive, the composition containing the substance P, an antioxidant, a surfactant and a thickener of the present invention is added as it is or It can be used together with other quasi-drugs or quasi-drug ingredients, and can be used appropriately according to conventional methods.

Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of whitening-related diseases, comprising a substance P, an antioxidant, a surfactant and a thickener.

In addition, another aspect of the present invention is to provide a use for the prevention or treatment of whitening-related diseases, including the substance P, an antioxidant, a surfactant, and a thickener.

The substance P, antioxidant, surfactant, and thickener are as described above.

In the present invention, the term "whitening-related disease" refers to a disease having symptoms in which pigment is excessively deposited on the skin, and specifically, the whitening-related disease may be spots, freckles, age spots, blemishes, or melanoma.

In the present invention, the term "prevention" means any action in which the composition of the present invention inhibits or delays the occurrence of whitening-related diseases.

In the present invention, the term "treatment" refers to any action that makes the pharmaceutical composition of the present invention ameliorate or benefit the symptoms of whitening-related diseases.

In the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the prophylactic or therapeutic activity and properties of whitening-related disease symptoms of the present composition. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible ones, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol And one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.

In addition, the pharmaceutically acceptable carrier of the present invention may include a non-natural carrier.

The pharmaceutical composition of the present invention may be administered singly or multiple times in a pharmaceutically effective amount.

In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical prevention or treatment, and the effective dose level is the severity of the disease, the activity of the drug , Including the patient's weight, health, sex, sensitivity to the patient's drugs, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion, the duration of treatment, and drugs used in combination or concurrent with the composition of the present invention used Factors and other factors well known in the medical field can be determined.

Another aspect of the invention provides a method of whitening skin comprising administering to a subject in need thereof a composition comprising Substance P, an antioxidant, a surfactant and a thickener.

In the method for whitening the skin of the present invention, the composition and whitening including the substance P, an antioxidant, a surfactant, and a thickener are as described above.

Administration of the present invention may be a method, including application to the skin.

In the present invention, a composition comprising Substance P, an antioxidant, a surfactant and a thickener can be administered to a subject in need thereof in an effective amount. The level of effective dose can be determined by a person skilled in the art based on common knowledge to the extent that the desired skin whitening effect is exhibited.

Another aspect of the present invention is to provide a health functional food for skin whitening comprising a substance P, an antioxidant, a surfactant, and a thickener.

The present invention is as described above with respect to the composition and whitening containing the substance P, an antioxidant, a surfactant and a thickener.

In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body.

Here, the term "functionality" means obtaining a useful effect for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food may be prepared without limitation as long as it is a formulation recognized as a health functional food.

The health functional food of the present invention can be prepared in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time by using food as a raw material, and is excellent in portability. Health functional foods can be consumed as a supplement to enhance the skin whitening effect.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, Carbonating agents used in carbonated beverages may be contained. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected from 0.000001 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention, but is not particularly limited thereto.

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

Example 1: Preparation of a cosmetic composition for whitening containing substance P

A novel formulation was prepared in which sodium thiosulfate as an antioxidant, polysorbate 80 as a surfactant, and hydroxyethylcellulose as a thickener were added to the substance P. Material P was synthesized through a solid/solution phase using Fmoc-chemistry, a peptide synthesis technology, and purified through high performance liquid chromatography, and finally, a purity of 85% or more was used.

Composition comprising substance P
Composition of the composition	content
Substance P	5 μg/ml
Sodium thiosulfate	0.1%
Polysorbate 80	0.006%
Hydroxyethyl cellulose	1.5%

Example 2: Confirmation of the activated melanocyte reduction effect of the composition containing substance P

The effect of substance P on reducing the production of activated melanocytes was analyzed through cell experiments. Skin keratinocytes and melanocytes were co-cultured in a 5:1 ratio for cell experiments in an environment similar to human skin tissue.

More specifically, a composition containing a substance P prepared as shown in Table 1 in the co-cultured cells (substance P, 5 ㎍/㎖), a composition obtained by diluting it five times (substance P, 1 ㎍/㎖) and a phosphoric acid substance A composition in which P was dissolved in a buffer solution (PBS) (material P, 5 µg/ml) was treated on the co-cultured cells, respectively. After 48 hours, the degree of reduction of activated melanocytes was confirmed through HMB45 antibody staining for gp100, a melanosome-specific protein inside the activated melanin. For the activation of melanocytes, a melanocyte stimulating hormone (α-MSH) 150 nM and a phosphate buffer (PBS) as a negative control were treated with 10 ㎍/㎖ of arbutin as a positive control to reduce fluorescence. The degree was observed through a fluorescence microscope.

As a result, as shown in FIG. 1, it was confirmed that the number of fluorescently stained melanocytes was small in the group treated with the phosphate buffer solution, whereas the number of fluorescently stained melanocytes increased in the α-MSH-treated group. . In addition, in the case of the group treated with the substance P dissolved in the phosphate buffer solution together with α-MSH alone, it was confirmed that the number of fluorescently stained melanocytes slightly decreased compared to the group treated with only α-MSH. On the other hand, it was confirmed that the number of activated melanocytes significantly decreased in the group treated with the composition containing the substance P of the present invention together with α-MSH (substance P, 5 µg/ml and substance P, 5 µg/ml). In particular, it was confirmed that the composition of the present invention containing 5 μg/ml of substance P had the most excellent effect of reducing melanin production, which was similar to the group treated with arbutin, a positive control (FIG. 1).

Example 3: Confirmation of the effect of the composition containing the substance P on the production of extracellular and intracellular melanin

In order to confirm the whitening effect of the composition containing the substance P, changes in intracellular and extracellular melanin production were measured through the co-cultured cell experiment.

More specifically, a composition containing a substance P prepared as shown in Table 1 under the conditions of 5:1 co-cultivation of skin keratinocytes and melanocytes constructed for an environment similar to human skin tissue (substance P, 5 μg/ml) , A composition obtained by diluting it 5 times (substance P, 1 μg/mL) and as a comparative example, substance P dissolved in phosphate buffer solution (PBS) (substance P, 5 μg/mL) was applied to cells under co-culture conditions for up to 48 hours. After that, the supernatant of the cells was collected and the amount of melanin outside the cells was measured at 405 nm absorbance. In addition, the amount of melanin inside the cells was measured at 405 nm absorbance after washing the cells with PBS and centrifuging, dissolving the cells with 1N sodium hydroxide containing 10% DMSO for 1 hour at 80°C.

As a result, as can be seen in Figure 2a, when the amount of melanin produced in the group treated with only the phosphate buffer solution outside the cells was 100%, it was confirmed that the amount of melanin produced in the group treated with α-MSH increased to 229%. On the other hand, when the group treated with the substance P dissolved in the phosphate buffer solution with α-MSH (substance P, 5 ㎍/㎖) was treated, the amount of melanin production decreased by 60%p compared to the group treated with only α-MSH. %. In addition, when the composition containing substance P (substance P, 1 μg/ml) was treated, it was confirmed that the amount of melanin production decreased by about 77%p compared to the group treated with only α-MSH, which was about 152%. In addition, the group treated with the substance P-containing composition (substance P, 5 ㎍/㎖) with α-MSH confirmed that the melanin production amount was 125% reduced by 104%p. , It was confirmed that this is a similar result to the group treated with the positive control, arbutin (Fig. 2a).

In addition, as shown in FIG. 2B, when the amount of melanin produced in the group treated with only the phosphate buffer solution was 100%, it was confirmed that the amount of melanin produced in the group treated with α-MSH was 154%. On the other hand, when the group treated with the substance P dissolved in the phosphate buffer solution with α-MSH (substance P, 5 ㎍/㎖) was treated, the amount of melanin production decreased by 16%p compared to the group treated with only α-MSH. %. In addition, it was confirmed that when the composition containing substance P (substance P, 1 μg/ml) was treated, the amount of melanin production decreased by 24%p to 130% compared to the group treated with only α-MSH. In addition, the group treated with the substance P-containing composition (substance P, 5 ㎍/㎖) with α-MSH confirmed that the amount of melanin production was reduced by 54%p to 100%, which was 3 times higher than that of treatment with substance P alone. It was found that it showed an effect of reducing abnormal melanin production. In addition, this showed a decrease in the amount of melanin production to the same level as when not treated with α-MSH, and it was confirmed that the effect was superior to that of the group treated with arbutin, a positive control (FIG. 2B).

Through this, the composition containing the substance P of the present invention shows a better effect of reducing melanin production outside and inside the cell, even when the substance P is used in a smaller dose than when the substance P is treated alone, and the same amount is used. In the case, it was confirmed that there is a better effect of reducing melanin production.

Example 4: Confirmation of the effect of reducing MITF transcription factor and tyrosinase mRNA of a composition containing substance P

In order to confirm the whitening effect of the composition containing the substance P, the amount of reduction in the production of MITF transcription factor and tyrosinase mRNA was confirmed through the co-cultured cell experiment.

More specifically, the co-cultured cell conditions and material treatment were the same as those mentioned in Example 1, and the experiment was a composition containing the substance P of Table 1 under the co-culture conditions of skin keratinocytes and melanocytes 5:1 (substance P , 5 µg/ml), a composition obtained by diluting it 5 times (material P, 1 µg/ml) and material P dissolved in a phosphate buffer solution (PBS) as a comparative example (substance P, 5 µg/ml) under co-culture conditions. After applying to the cells for up to 48 hours, the cells were extracted and proceeded. The cells were extracted by directly treating the cells with the RiboEx (cell lysate) reagent in the GeneAll Hybrid-R kit (Cat no. 305-101) and then suspending them. After separating the RNA inside the extracted cells with RNases-free water in the Hybrid-R kit, cDNA is synthesized using reverse transcriptase, and the amount of mRNA produced by MITF transcription factor and tyrosinase is reverse transcriptase chain It was confirmed through the reaction (RT-PCR: reverse transcription PCR) (Fig. 3a).

As a result, as can be seen in Figure 3a, it was confirmed through agarose gel that the mRNA of MITF and tyrosinase increased in the group treated with α-MSH compared to the group treated with only the phosphate buffer solution. It was confirmed that the reducing effect of MITF and tyrosinase mRNA was large in the group treated with the containing composition (substance P, 5 µg/ml) and the group treated with arbutin. More specifically, in order to confirm the amount of mRNA production, the band brightness of the sample loaded on a 1.5% agarose gel was quantified and graphed through the image J program.

As a result, as shown in FIG. 3b, when the mRNA level of the generated MITF transcription factor in the group treated with only phosphate buffer solution was 1, the mRNA production amount of the MITF transcription factor increased to 3.5 in the group treated with α-MSH. Confirmed that. On the other hand, in the case of the group treated with the substance P dissolved in the phosphate buffer solution with α-MSH, it was confirmed that the amount of mRNA production of the MITF transcription factor decreased to 3.1. In addition, when the composition containing the substance P (substance P, 1 ㎍/㎖) was treated, it was confirmed that the amount of mRNA produced by the MITF transcription factor was reduced to 2.9. In addition, in the case of the group treated with the composition containing substance P (substance P, 5 μg/ml) with α-MSH, the amount of mRNA production of the MITF transcription factor decreased to 1.3, similar to that of the case without treatment with α-MSH. It was confirmed that the mRNA reduction effect of the MITF transcription factor was shown. In addition, it was confirmed that this was similar to the group treated with the positive control, arbutin (Fig. 3b).

In addition, as can be seen in Fig. 3c, when the mRNA level of the generated tyrosinase in the group treated with only the phosphate buffer solution was 1, the mRNA production amount of tyrosinase in the group treated with α-MSH was 1.39. It was confirmed that it increased. On the other hand, it was confirmed that the mRNA production amount of tyrosinase decreased to 1.05 compared to the case of the group treated with the substance P dissolved in the phosphate buffer solution with α-MSH. In addition, when the composition containing the substance P (substance P, 1 μg/ml) was treated, it was confirmed that the amount of tyrosinase mRNA produced was reduced to 1.06. In addition, in the case of the group treated with the composition containing substance P (substance P, 5 μg/ml) together with α-MSH, it was confirmed that the amount of mRNA production of tyrosinase decreased to 0.88. This was confirmed that the amount of mRNA production of tyrosinase was reduced compared to the case where α-MSH was not treated, and the result was similar to that of the group treated with arbutin, a positive control (FIG. 3C).

Through this, the composition containing the substance P of the present invention has a similar or superior level of MITF transcription factor and tyrosinase mRNA reduction effect, even when a dose of 1/5 of the substance P is used than when the substance P is treated alone. When using the same amount, it was confirmed that there is a better effect of reducing MITF transcription factor and tyrosinase mRNA.

Example 5: Confirmation of the effect of reducing tyrosinase activity of a composition comprising substance P

In order to confirm the whitening effect of the composition containing the substance P, it was confirmed that the tyrosinase activity of the composition containing the substance P decreased through the co-cultured cell experiment.

More specifically, a composition containing the substance P of Table 1 (substance P, 5 µg/ml) and a composition obtained by diluting it 5 times (substance P, 1 µg/mL) under the conditions of 5:1 co-culture of skin keratinocytes and melanocytes. Ml) or as a comparative example, substance P (substance P, 5 µg/ml) dissolved in a phosphate buffer solution (PBS) was applied to cells under co-culture conditions for up to 48 hours, and then the cells were extracted and proceeded. Cells were extracted by suspension 1 minute after treatment with Trypsine-EDTA solution, and the supernatant was removed through centrifugation (1,200 rpm, 5 minutes). The remaining cells were lysed using a buffer mixed with 1% sodium dodecyl sulfate and 0.5% Triton x-100, and the lysed cells were centrifuged at 12,000 rpm for 30 minutes, and a supernatant was obtained. For the obtained supernatant, the total amount of protein was adjusted to 50 µg, and 2 mg/ml of L-DOPA, a substrate of tyrosinase, was mixed for 1 hour under dark conditions, and the amount of melanin changed in L-DOPA was measured at 475 nm. The activity of tyrosinase was quantified.

As a result, as shown in FIG. 4, when the activity level of tyrosinase in the group treated with only phosphate buffer solution was 100%, the amount of tyrosinase activity in the group treated with α-MSH increased to 253%. Confirmed that. On the other hand, in the case of the group treated with the substance P dissolved in the phosphate buffer solution together with α-MSH, it was confirmed that the tyrosinase activity decreased to 166%. In addition, it was confirmed that the amount of tyrosinase activity decreased to 170.6% when the composition containing the substance P (substance P, 1 µg/ml) was treated. In addition, in the case of the group treated with the composition containing substance P (substance P, 5 μg/ml) together with α-MSH, it was confirmed that the amount of tyrosinase activity decreased to about 117.8%. This is similar to that of the case where α-MSH was not treated, and it was confirmed that the result was similar to that of the group treated with the positive control arbutin (FIG. 4).

Through this, the composition containing the substance P of the present invention shows a better effect of reducing tyrosinase activity even when the amount of substance P is used 1/5 than when the substance P is treated alone, and when the same amount is used, It was confirmed that there is a better effect of reducing tyrosinase activity.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description, and equivalent concepts thereof, are included in the scope of the present invention.

Claims (12)

1. A cosmetic composition for skin whitening comprising a substance P (substance P) consisting of the amino acid sequence of SEQ ID NO: 1, an antioxidant, a surfactant, and a thickener.
2. The composition of claim 1, wherein the concentration of the substance P is 1 to 5 μg/ml.
3. The composition of claim 1, wherein the antioxidant is sodium thiosulfate.
4. The composition of claim 1, wherein the surfactant is polysorbate 80.
5. The composition of claim 1, wherein the thickener is hydroxyethylcellulose.
6. The cosmetic composition for skin whitening according to claim 1, wherein the composition is for preventing or improving at least one selected from the group consisting of spots, freckles, age spots and blemishes.
7. The cosmetic composition for skin whitening according to claim 1, wherein the whitening effect of the composition is due to a decrease in melanin activity and production.
8. The cosmetic composition for skin whitening according to claim 1, wherein the whitening effect of the composition is due to a decrease in the MITF transcription factor.
9. The cosmetic composition for skin whitening according to claim 1, wherein the whitening effect of the composition is due to decrease in mRNA and activity of tyrosinase.
10. A quasi-drug composition for skin whitening comprising a substance P (substance P) consisting of the amino acid sequence of SEQ ID NO: 1, an antioxidant, a surfactant, and a thickener.
11. A pharmaceutical composition for the prevention or treatment of whitening-related diseases, including a substance P (substance P) consisting of the amino acid sequence of SEQ ID NO: 1, antioxidants, surfactants, and thickeners.
12. The pharmaceutical composition according to claim 11, wherein the whitening-related disease is melasma, freckles, age spots, blemishes or melanoma.

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