KR20230099248A - Composition for skin whitening comprising extract or fraction of Catalpa ovata as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for skin whitening comprising an extract or fraction of a cypress tree as an active ingredient, and more particularly, to a cosmetic composition for skin whitening comprising a hot-water extract of a stem or twig of a cypress tree and an ethyl acetate fraction thereof as an active ingredient. will be.
In the present invention, it was confirmed that the hot-water extract of cypress stem or twig and its acetate fraction inhibit melanin production and tyrosinase activity in melanocytes to a significant level without cytotoxicity, so the composition of the present invention is skin whitening It can be usefully used as cosmetics for skin whitening, health functional foods for skin whitening, pharmaceuticals for preventing or treating skin pigmentation diseases, and quasi-drugs for preventing or improving skin pigmentation diseases.

Description

Composition for skin whitening comprising extract or fraction of Catalpa ovata as an active ingredient}

The present invention relates to a composition for skin whitening comprising an extract or fraction of a cypress tree as an active ingredient, and more particularly, to a cosmetic composition for skin whitening comprising a hot-water extract of a stem or twig of a cypress tree and an ethyl acetate fraction thereof as an active ingredient. will be.

Human skin color is determined by the amount of melanin, carotene and hemoglobin, of which melanin is the most decisive factor. Melanin is synthesized in melanocytes present in the basal layer of the skin and is transferred to surrounding keratinocytes to represent human skin color. Melanin has an important function of protecting the body from ultraviolet rays, but when it is overproduced, it is known to play an important role in promoting skin aging and inducing skin cancer as well as forming spots and freckles.

Melanin is produced through enzymatic and non-enzymatic oxidation of tyrosine in melanocyte cells. Enzymes involved in melanin synthesis include tyrosinase, tyrosinase related protein 1 (TRP) -1) and tyrosinase related protein 2 (TRP-2) are known. In addition, the major intracellular signaling pathway is the cAMP/PKA (cyclic monophosphate/protein kinase A) pathway, and cAMP promotes the expression of MITF (Microphthalmia-associated transcription factor) via PKA and CREB1 (cAMP resposive element binding protein 1) do. MITF is an important transcriptional regulator in the melanin synthesis process and is known to promote the transcription of tyrosinase, tyrosinase-related protein 1 and tyrosinase-related protein 2. In addition, α-MSH (alpha-melanocyte stimulating hormone) increases cAMP (cyclic AMP) and PKA (protein kinase A) through the melanocortin-1-receptor (MC1R), a cell membrane receptor, and inhibits tyrosinase by activated MITF. By regulating the expression, it is involved in the proliferation and pigmentation of melanocytes.

A lot of research has been conducted on cosmetic compositions for skin whitening using melanin synthesis inhibitory mechanisms, and in particular, plant-derived materials or herbal ingredients are excellent in terms of stability, and functional materials using them are being actively developed.

On the other hand, no tree ( Catalpa ovata ) is also called Gaodong tree as a flowering plant phylum dicotyledonous plant class Nephraceae. The fruit of the cypress tree is called fruiting, and has diuretic, anti-inflammatory, and insecticidal effects, and is used to treat chronic nephritis, edema, and proteinuria. It is used to treat , burns, skin itching, and window openings.

Nonamu mainly contains iridoid and naphthoquinone components, which have anticancer activity (Fujiwara et al. , Journal of natural products, 1998, 61, 629-632), antioxidant activity (Moon et al . al. , Toxicology letters, 2003, 145, 46-54) and anti-inflammatory effects (Oh et al. , Planta medica, 2002, 68, 685-689) have been reported a lot.

In the present invention, as a result of diligent efforts to select plant-derived extracts that are more effective and can stably reduce melanin production in the skin, hot water extracts of cypress stems or twigs and their acetate fractions produce melanin in melanocytes and tyrosina It was confirmed that the tyrosinase activity was inhibited to a significant level, and the present invention was completed.

Accordingly, an object of the present invention is to provide a cosmetic composition for skin whitening comprising a hot water extract of Japanese cypress and an ethyl acetate fraction thereof as an active ingredient.

Another object of the present invention is to provide a health functional food composition for skin whitening containing a hot water extract of Japanese quinoa and an ethyl acetate fraction thereof as an active ingredient, a pharmaceutical composition for preventing or treating skin pigmentation disease, and a quasi-drug composition for preventing or improving skin pigmentation disease. is to provide

To achieve the above purpose,

The present invention provides a cosmetic composition for skin whitening comprising a hot water extract of No tree ( Catalpa ovata ) and an ethyl acetate fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for skin whitening comprising a hot water extract of Nonamu and its ethyl acetate fraction as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of skin pigmentation diseases comprising a hot water extract of Nonamu and an ethyl acetate fraction thereof as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving skin pigmentation diseases, comprising a hot water extract of Japanese cypress and an ethyl acetate fraction thereof as an active ingredient.

In a specific embodiment of the present invention, the old tree ( Catalpa ovata ) may be a stem or a branch.

In another specific embodiment of the present invention, the cypress hot water extract or its ethyl acetate fraction may be included at a concentration of 10 to 500 μg/ml.

In another specific embodiment of the present invention, the hot-water extract of cypress can be extracted by adding 0.5 to 1.5 ℓ of water per 100 parts by weight of cypress and then applying heat for 1 to 3 hours.

In another specific embodiment of the present invention, the ethyl acetate fraction is

(a) adding a hexane solvent to the hot water extract of Japanese cypress, and then obtaining an aqueous phase;

(b) adding a chloroform solvent to the aqueous phase and then obtaining an aqueous phase; and

(c) adding an ethyl acetate solvent to the aqueous phase and then obtaining an ethyl acetate fraction;

In another specific embodiment of the present invention, the hot water extract of Japanese cypress or its ethyl acetate fraction can inhibit melanin production or tyrosinase activity.

In another specific embodiment of the present invention, the cypress hot water extract or its ethyl acetate fraction inhibits cAMP (cyclic AMP) production; inhibition of microphthalmia-associated transcription factor (MITF) production; Alternatively, melanin production or tyrosinase activity may be inhibited through regulation of the MAPK (mitogen-activated protein kinases) pathway by increasing ERK phosphorylation and inhibiting p38 or JNK phosphorylation.

In the present invention, it was confirmed that the hot-water extract of cypress stem or twig and its acetate fraction inhibit melanin production and tyrosinase activity in melanocytes to a significant level without cytotoxicity, so the composition of the present invention is skin whitening It can be usefully used as cosmetics for skin whitening, health functional foods for skin whitening, pharmaceuticals for preventing or treating skin pigmentation diseases, and quasi-drugs for preventing or improving skin pigmentation diseases.

1 is a schematic diagram showing a method for preparing a fraction of a hot water extract of Japanese cypress.
Figure 2 shows that when melanoma cells (B16F1 cells) were pretreated with (A) hot water extract (CO-WE) and (B) ethanol extract (CO-EE), α- melanocyte stimulating hormone (α-MSH) This is the data confirming the degree of melanin production according to the treatment. Kojic acid is a positive control, and data are presented as mean ± SD values (n = 3) ( ^### P < 0.001, compared to control; **p <0.01, ***p <0.001, compared with α-MSH alone treatment group).
Figure 3 is data confirming the cell viability according to the treatment of the hot water extract of Japanese cypress and its fractions. 3 to 5, Kojic acid is a positive control, WE is a hot water extract, HF is a hexane fraction, CF is a chloroform fraction, EF is an ethyl acetate fraction, BF is an n-butanol fraction, and WF is a water fraction do. Data are presented as mean ± standard deviation values (n = 3) ( ^*** P < 0.001 compared to control).
Figure 4 is data confirming (A) extracellular melanin content and (B) intracellular melanin content according to α-MSH treatment when melanoma cells were treated with a hot water extract of Japanese cypress and its fractions. Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001 compared to control; **p < 0.05, ***p < 0.001 compared to α-MSH alone treated group).
Figure 5 is data confirming the tyrosinase activity according to α-MSH treatment when melanoma cells were treated with hot water extract and fractions thereof. Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001, compared to control; ***p < 0.001, compared to α-MSH alone treated group).
Figure 6 shows that when melanoma cells were treated with the ethyl acetate fraction (EF) of the hot water extract of Japanese radish, α-MSH treatment (A) cell viability, (B) extracellular melanin content, (C) intracellular melanin content, (D) Data confirming tyrosinase activity. Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001, compared to control; ***p < 0.001, compared to α-MSH alone treated group).
7 is data confirming the concentration of cAMP according to α-MSH treatment when melanoma cells were treated with the ethyl acetate fraction (EF) of the hot-water extract of Japanese cypress. Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001, compared to control; ***p < 0.001, compared to α-MSH alone treated group).
Figure 8 shows the protein expression levels of tyrosinase, TRP-1, and TRP-2, which are melanin production-related proteins, according to α-MSH treatment, when melanoma cells were treated with the ethyl acetate fraction (EF) of the hot water extract of Japanese quinoa (A and B), and the data confirming the protein expression level (C and D) of MITF (A and C: western blot data, B and D: confirming the relative expression level). Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001, compared to control; ***p < 0.001, compared to α-MSH alone treated group).
9 is data confirming the protein expression levels and phosphorylation levels of ERK, p38, and JNK according to α-MSH treatment when melanoma cells were treated with the ethyl acetate fraction (EF) of the hot water extract of Japanese cypress (A: Western blot data , B: Check the relative expression level). Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001, compared to control; ***p < 0.001, compared to α-MSH alone treated group).
10 is data confirming the mRNA expression levels of (A) MITF and (B) tyrosinase according to α-MSH treatment when melanoma cells were treated with the ethyl acetate fraction (EF) of the hot water extract of Japanese cypress. Data are presented as mean ± standard deviation values (n = 3) ( ^### P < 0.001, compared to control; ***p < 0.001, compared to α-MSH alone treated group).

Hereinafter, the present invention will be described in detail.

From one point of view, the present invention relates to a cosmetic composition for skin whitening comprising a hot water extract of Catalpa ovata and an ethyl acetate fraction thereof as an active ingredient.

In the present invention, the old tree ( Catalpa ovata ) may be a stem or a branch.

In the present invention, the hot-water extract of No tree can be extracted by adding 0.5 to 1.5 ℓ of water per 100 parts by weight of No tree and then applying heat for 1 to 3 hours.

In the present invention, the ethyl acetate fraction is prepared by (a) adding a hexane solvent to the hot water extract of Japanese cypress, and then obtaining an aqueous phase;

(b) adding a chloroform solvent to the aqueous phase and then obtaining an aqueous phase; and

(c) adding an ethyl acetate solvent to the aqueous phase and then obtaining an ethyl acetate fraction;

In a specific embodiment of the present invention, the hot water extract of Japanese cypress was prepared by hot water extraction for 2 hours by adding 1 liter of distilled water per 100 g of cypress stem or twig. Hexane, chloroform, ethyl acetate and butanol were sequentially added to the hot water extract to obtain a hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction and a water fraction (FIG. 1).

In another specific embodiment of the present invention, as a result of confirming the cytotoxicity and melanin production inhibitory effect of the hot water extract and 70% ethanol extract of Nonamu tree, it was confirmed that both the hot water extract and the ethanol extract had no cytotoxicity, but the melanin production inhibitory effect The hot water extract was found to be superior (Fig. 2).

In addition, as a result of confirming the cytotoxicity and melanin production inhibitory effect of the hexane fraction, the chloroform fraction, the ethyl acetate fraction, the butanol fraction, and the water fraction isolated from the hot-water extract of Japanese quinoa, strong cytotoxicity was observed in the hexane fraction and the chloroform fraction (FIG. 3 ), the ethyl acetate fraction was found to be superior in melanin production inhibitory effect and tyrosinase activity inhibitory effect to that of kojic acid as a positive control group (FIG. 4 and FIG. 5).

In the present invention, the cypress hot water extract or its ethyl acetate fraction may be included in a concentration of 10 to 500 μg/ml. If the concentration of the extract is less than 10 μg / ml, the whitening effect may be weak, and if it exceeds 500 μg / ml, the degree of effect increase according to the increase in the content is insignificant, and problems such as cytotoxicity may appear.

In the present invention, the cypress hot water extract or its ethyl acetate fraction can inhibit melanin production or tyrosinase activity.

In the present invention, the cypress hot water extract or its ethyl acetate fraction is MAPK (mitogen-activated protein kinases) through increased phosphorylation of cAMP (cyclic AMP), MITF (Microphthalmia-associated transcription factor), or ERK phosphorylation, and inhibition of p38 or JNK phosphorylation Through pathway modulation, tyrosinase production or activity can be inhibited.

In the present invention, the cypress hot water extract or its ethyl acetate fraction can inhibit melanin production by inhibiting the expression or activity of tyrosinase.

Various signaling substances are involved in the melanin biosynthesis signaling system. Among them, the cAMP/PKA pathway is the main pathway for melanin synthesis. Intracellular cAMP is increased by a-MSH, PKA, a lower signaling substance, is activated, and MITF expression is increased through CREB. MITF is known to promote the transcription of tyrosinase, TRP-1 and TRP-2 as an important transcriptional regulator in the melanin synthesis process.

In another specific embodiment of the present invention, as a result of confirming the effect of inhibiting melanin production and tyrosinase activity according to the concentration of the nonamu ethyl acetate fraction, concentration-dependently, α-MSH-stimulated melanoma cells released extracellular melanin and It was confirmed that it inhibited both intracellular melanin production as well as tyrosinase activity (FIG. 7).

In another specific embodiment of the present invention, as a result of confirming the effect of inhibiting the expression of proteins related to melanin production and tyrosinase activity in relation to the whitening activity of Nomu tree extract, melanoma stimulated with α-MSH by Nomu tree ethyl acetate fraction It was confirmed that cAMP production was suppressed in the cells (FIG. 8), and the expression of tyrosinase and MITF, which are key enzymes in the melanin production process, were inhibited (FIG. 9).

In addition, to confirm the mechanism of tyrosinase inhibition, the protein expression and phosphorylation levels of p38, JNK, and ERK related to the regulation of the MAPK (mitogen-activated protein kinases) pathway were confirmed. ERK phosphorylation was activated in melanoma cells, and phosphorylation of p38 and JNK was inhibited to regulate the MAPK pathway. When the phosphorylation of ERK is activated, the expression of MITF is inhibited, and as a result, it is judged to reduce melanin production by inhibiting tyrosinase expression.

Furthermore, in FIG. 9, for tyrosinase and MITF, which were the most effective in inhibiting protein expression by the nona tree ethyl acetate fraction, in melanoma cells stimulated with α-MSH, the degree of gene expression according to the treatment with a nona tree ethyl acetate fraction was confirmed. , It was confirmed that gene expression was inhibited in a concentration-dependent manner for both tyrosinase and MITF (FIG. 10).

That is, the hot water extract of Japanese quinoa and its ethyl acetate fraction of the present invention not only exhibit melanin production inhibition and tyrosinase activity inhibition efficacy at a significant level in melanin-producing cells, but also inhibit the expression of proteins related to melanin production and tyrosinase activity. inhibition was confirmed. Therefore, the hot water extract of Japanese quinoa and its ethyl acetate fraction of the present invention are cosmetic compositions for skin whitening, health functional food compositions for skin whitening, pharmaceutical compositions for preventing or treating skin pigmentation diseases, and quasi-drug compositions for preventing or improving skin pigmentation diseases. can be utilized as

The cosmetic composition for skin whitening of the present invention is a formulation of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray may be, but is not limited thereto.

In another aspect, the present invention relates to a health functional food composition for skin whitening comprising a hot water extract of Japanese cypress and an ethyl acetate fraction thereof as an active ingredient.

The health functional food composition for skin whitening containing the hot water extract of No tree and its ethyl acetate fraction as an active ingredient may additionally contain various flavoring agents or natural carbohydrates like conventional food compositions.

Formulations of the health functional food may be tablets, capsules, powders, granules, liquids, pills, and the like. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inactive carrier such as ethanol, glycerol, or water. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include 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 if necessary.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating skin pigmentation diseases, comprising a hot water extract of Japanese cypress and an ethyl acetate fraction thereof as an active ingredient.

In another aspect, the present invention relates to a quasi-drug composition for preventing or improving skin pigmentation diseases, comprising a hot water extract of Japanese cypress and an ethyl acetate fraction thereof as an active ingredient.

In the present invention, the skin pigmentation disease may include at least one selected from the group consisting of melasma, freckles, and melanoma, but is not limited thereto.

The pharmaceutical composition of the present invention may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions. Depending on each formulation, pharmaceutically acceptable carriers, excipients and diluents may be further included. In addition, it can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injection solutions according to conventional methods.

The carriers, excipients and diluents include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like. When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the patient's age, weight, and general health condition , gender and diet, administration time, route of administration and secretion rate of the composition, treatment period, can be adjusted according to various factors including drugs used simultaneously. The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, intravenous, intraperitoneal, intramuscular, intraarterial, buccal, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration may be administered, but is not limited thereto. The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or divided into several times.

The term "quasi-drugs" used in the present invention refers to products whose action is milder than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, mitigating, treating or preventing human or animal diseases. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are items excluding items used for pharmaceutical purposes, and include products used for the treatment or prevention of human or animal diseases, products with minor or no direct action on the human body, etc.

The quasi-drug composition of the present invention is used for the purpose of improving skin pigmentation, and the formulation is not particularly limited. For example, softening lotion, nutrient lotion, massage cream, nutrient cream, pack, mask pack, mask sheet , It may be a cosmetic composition having a formulation of a gel or skin-adhesive cosmetic, and may also be a transdermal formulation such as a lotion, ointment, gel, cream, patch, or spray.

In addition, in each dosage form, the quasi-drug composition may arbitrarily select and mix other ingredients according to the formulation or purpose of use of other quasi-drugs. The mixing amount of the active ingredient can be suitably determined depending on the purpose of use (suppression or alleviation). For example, conventional adjuvants such as thickeners, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers may be included.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Preparation of nonamu extract and confirmation of whitening efficacy

1-1: Manufacture of No tree extract

The no tree used in the present invention was purchased from the herbal medicine market (www.hanyakjae.net) as a stem or branch.

Nonamu prepared two types of extracts using water (hot water extraction) and 70% ethanol. First, the hot water extract was boiled for 2 hours using an electric water heater (Daewoong Yaktang, Daewoong Bio Appliances, Korea) by adding 1 liter of distilled water per 100 g of No tree sample, and then filtered using filter paper (advantec, No.41) and reduced pressure. It was prepared by concentration and then freeze-drying. The yield of the hot-water extract of cypress cypress was confirmed to be 6.03%.

The 70% ethanol extract was firstly extracted by adding 1 liter of solvent per 100 g of rona tree sample, sonicating for 1 hour, and then reacting overnight at room temperature. It was added and extracted a second time in the same way. The first extract and the second extract were collected, filtered, concentrated under reduced pressure, and then freeze-dried. The yield of the 70% ethanol extract of Nonamu was confirmed to be 9.04%.

1-2: Confirmation of cytotoxicity

The cytotoxicity evaluation of the extract of Japanese cypress was confirmed by the MTT assay method.

B16F1 cells were inoculated in a 24-well plate at a density of 1Х10 ⁴ , and the next day, hot-water extracts of Japanese cypress (25 μg/ml, 50 μg/ml, 100 μg/ml), ethanol extract (25 μg/ml, 50 μg/ml, 100 μg/ml) [mu]g/ml), positive control kojic acid (kojic acid, 1 mM), respectively, and then α-melanocyte stimulating hormone (α-MSH, 200 nM), followed by incubation at 37°C for 72 hours.

After 72 hours, the medium was replaced with an MTT reagent medium and further incubated at 37°C for 1 hour. The medium containing the MTT reagent was removed, and 400 μl of dimethyl sulfoxide (DMSO) was added to dissolve the purple-colored formazan. After transferring 150 μl of the lysate to a 96-well plate, absorbance was measured at 540 nm. The cell viability was calculated by taking the viability of the control group not treated with α-MSH as 100%.

As a result, it was confirmed that both the hot water extract and the ethanol extract had no toxicity to B16F1 cells, with a cell viability of 95% or more at a concentration of 100 μg/ml or less.

1-3: Confirmation of melanin production inhibitory effect

In order to measure the melanin content, B16F1 cells were inoculated into a 60 mm culture vessel at a density of 8Х10 ⁴ , and then hot water extracts of Japanese quinoa (25 μg/ml, 50 μg/ml, 100 μg/ml), ethanol extract (25 μg/ml) , 50 μg/ml, 100 μg/ml), positive control kojic acid (kojic acid, 1 mM), respectively, and then α-melanocyte stimulating hormone (α-MSH, 200 nM), followed by treatment for 72 hours Melanin production was induced at 37°C. After 72 hours, 150 μl of the cell culture medium was transferred to a 96-well plate to measure the extracellular melanin content. After the cells were recovered, 1M sodium hydroxide (NaOH) containing 10% dimethyl sulfoxide (DMSO) was added and put at 80 ° C. for 1 hour to dissolve melanin, and then the content of melanin in the cells was measured at an absorbance of 405 nm. .

As a result, as shown in FIG. 2A, when melanin synthesis was induced with α-MSH, the amount of intracellular melanin released increased by 157%, whereas when the concentration of 25, 50, and 100 μg/ml of the Japanese cypress extract was treated, the cell My melanin production was 142%, 131%, and 115%, respectively.

When the 70% ethanol extract of No tree was treated at concentrations of 25, 50, and 100 μg/ml, the amount of intracellular melanin production was 149%, 144%, and 142%, respectively, compared to the α-MSH alone treatment group. It was confirmed that the effect of inhibiting melanin production was lower than that of

Therefore, in the present invention, a whitening activity test was performed using hot water extracts and fractions thereof having excellent melanin production inhibitory effects.

Preparation of nona hot water extract fractions and confirmation of whitening efficacy

2-1: Preparation of fractions

As for the fractions, a hexane fraction, a chloroform fraction, an ethyl acetate fraction, a butanol fraction, and a water fraction were obtained from the hot water extract of Nonamu trees in the same manner as in the schematic diagram shown in FIG. 1 .

Specifically, 130 ml of each of the fractionation solvents was mixed with 1.3 g of the hot water extract of Japanese cypress, and then the mixture was strongly mixed and allowed to stand to separate the layers three times. Fractions for each solvent were concentrated under reduced pressure and lyophilized to be used as samples.

The yield of the hexane fraction using the hot water extract of No tree was 0.92%, the chloroform fraction was 3.30%, the ethyl acetate fraction was 13.615%, the butanol fraction was 27.923%, and the water fraction was 33.69%. The yield of the water fraction was found to be the highest.

2-2: Confirmation of cytotoxicity

Cytotoxicity was evaluated for the fractions using the hot-water extract of the Japanese cypress of Example 1-1 and the hot-water extract of the cypress of Example 2-1.

After treatment with 100 μg/ml of hot-water extract of Japanese primrose, 100 μg/ml of each fraction, and kojic acid (1 mM) as a positive control group, α-melanocyte stimulating hormone (α-MSH, 200 nM) was treated. , and incubated at 37° C. for 72 hours. Then, the degree of cytotoxicity was measured using the MTT assay in the same manner as in Example 1-2.

As a result, as shown in FIG. 3, strong toxicity was observed in the hexane extract and the chloroform extract, but no toxicity was observed in the hot-water extract of Nonamu tree and the remaining fractions.

2-3: Confirmation of melanin production inhibitory effect

After treatment with 100 μg/ml of hot-water extract of Japanese primrose, 100 μg/ml of each fraction, and kojic acid (1 mM) as a positive control group, α-melanocyte stimulating hormone (α-MSH, 200 nM) was treated. , and incubated at 37° C. for 72 hours. Then, extracellular melanin release and intracellular melanin production were measured in the same manner as in Examples 1-3.

In the case of extracellular melanin release, as shown in FIG. 4A, when melanin synthesis was induced with α-MSH, the extracellular melanin release increased to 249%, whereas the quinoa hot water extract, hexane fraction, chloroform fraction, ethyl acetate fraction, butanol It was confirmed that the extracellular melanin release was reduced by 175%, 97%, 90%, 151%, 242%, and 233%, respectively, when the fraction and the water fraction were treated. In particular, when the hexane fraction and the chloroform fraction were treated, the amount of extracellular melanin released was lower than that of the control group, which was confirmed to be due to strong cytotoxicity.

In addition, the hot water extract and the ethyl acetate fraction were found to have the most excellent melanin production inhibitory effects, and the ethyl acetate fraction was found to have better melanin production inhibitory effects than the positive control kojic acid (160%).

The amount of intracellular melanin production showed a similar trend to the amount of extracellular melanin release, and as shown in FIG. 4B, when melanin synthesis was induced with α-MSH, the amount of intracellular melanin release increased to 162%, while the amount of melanin released in the cells was increased by 162%. It was confirmed that the intracellular melanin production decreased by 124%, 30%, 38%, 105%, 134%, and 153% when the fraction, chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction were treated, respectively. In particular, the melanogenesis inhibitory effect of the ethyl acetate fraction was the best, which was confirmed to be superior to that of kojic acid (112%) as a positive control.

2-4: Confirmation of Tyrosinase activity inhibitory effect

B16F1 cells were inoculated into a 60 mm culture vessel at a density of 8 Х 10 ⁴ , and then treated with 100 μg/ml of hot water extract from Japanese cypress, 100 μg/ml of each fraction, and kojic acid (1 mM) as a positive control. , treated with α-melanocyte stimulating hormone (α-MSH, 200 nM), and cultured at 37° C. for 72 hours.

After culturing for 72 hours, the cells were washed with PBS (phosphate buffered saline) to obtain cells, and then proteins were extracted using a lysis buffer (PRO-PREP, iNtRON, Korea). After quantifying the extracted protein, the same amount of protein was added to a 96-well plate, L-dopa (2 mg/mL) was added, reacted at 37° C. for 1 hour, and absorbance was measured at 450 nm. The activity of tyrosinase was calculated as a percentage of the absorbance of the control group not treated with α-MSH.

As a result, as shown in FIG. 5, when α-MSH was treated, the activity of tyrosinase increased to 335%, while the hot water extract of Japanese cypress, the hexane fraction, the chloroform fraction, the ethyl acetate fraction, the butanol fraction, and the water fraction were treated. tyrosinase activity decreased by 280%, 233%, 171%, 243%, 275%, and 297%, respectively.

The hexane fraction and the chloroform fraction seem to have tyrosinase activity numerically, but this is considered to be due to cytotoxicity.

That is, it was confirmed that the hot water extract of Japanese cypress also showed excellent melanin production inhibitory effect and tyrosinase activity inhibitory effect, and in particular, the ethyl acetate fraction showed excellent whitening activity compared to the hot water extract or other fractions.

Confirmation of whitening efficacy of Nonamu ethyl acetate fraction

3-1: Confirmation of cytotoxicity

The cytotoxicity of the ethyl acetate fraction of the hot water extract of Japanese cypress of Example 2-1 was evaluated.

After treatment with 25 μg/ml, 50 μg/ml, and 100 μg/ml of Nonamu ethyl acetate fraction, kojic acid (1 mM) as a positive control, respectively, α-melanocyte stimulating hormone (α-MSH, 200 nM) and incubated at 37° C. for 72 hours. Then, the degree of cytotoxicity was measured using the MTT assay in the same manner as in Example 1-2.

As a result, as shown in FIG. 6A, the nonamu ethyl acetate fraction did not affect the viability of B16F1 melanoma cells at a concentration of 100 μg/ml or less, and thus was judged to be non-toxic. Therefore, in subsequent experiments, the experiment was performed with 100 μg/ml, which is a non-toxic concentration, as the highest concentration.

3-2: Confirmation of melanin production inhibitory effect

After treatment with 25 μg/ml, 50 μg/ml, and 100 μg/ml of Nonamu ethyl acetate fraction, kojic acid (1 mM) as a positive control, respectively, α-melanocyte stimulating hormone (α-MSH, 200 nM) and incubated at 37° C. for 72 hours. Then, extracellular melanin release and intracellular melanin production were measured in the same manner as in Examples 1-3.

In the case of the extracellular melanin release amount, as shown in FIG. 6B, when melanin synthesis was induced with α-MSH, the extracellular melanin release amount increased to 173% compared to the untreated group, while the nona tree ethyl acetate fraction was 25 μg/ml, When treated at concentrations of 50 μg/ml and 100 μg/ml, the amount of extracellular melanin released was decreased in a concentration-dependent manner by 143%, 130%, and 115%, respectively.

In addition, in the case of intracellular melanin production, as shown in FIG. 6C, when melanin synthesis was induced with α-MSH, intracellular melanin production increased by 165% compared to the untreated group, while 25 μg / When treated at concentrations of ㎖, 50 ㎍/㎖, and 100 ㎍/㎖, intracellular melanin production was decreased in a concentration-dependent manner by 145%, 144%, and 118%, respectively.

When treated with kojic acid used as a positive control, intracellular melanin production was reduced by 128%, and it was confirmed that treatment with nonamu ethyl acetate fraction at a concentration of 100 μg/ml inhibited melanin production more effectively than kojic acid.

3-4: Confirmation of Tyrosinase activity inhibitory effect

After treatment with 25 μg/ml, 50 μg/ml, and 100 μg/ml of Nonamu ethyl acetate fraction, kojic acid (1 mM) as a positive control, respectively, α-melanocyte stimulating hormone (α-MSH, 200 nM) and incubated at 37° C. for 72 hours. Then, the tyrosinase activity inhibitory effect was confirmed in the same manner as in Examples 2-4.

As a result, as shown in FIG. 6D, it was confirmed that the tyrosinase activity was inhibited in a concentration-dependent manner when the nona ethyl acetate fraction was treated. In particular, it was shown to inhibit tyrosinase activity at a level similar to that of kojic acid, a positive control group treated with nonamu ethyl acetate fraction at a concentration of 100 μg/ml.

Confirmation of cAMP inhibitory effect of ethyl acetate fractions of Nonamu tree

It is known that the central mechanism of melanin pigmentation is that excessive expression of cAMP induces tyrosinase gene expression and increases melanin production (Jung et al ., Food Chem. Toxicol. , 47:2436-2440, 2009).

In order to measure the amount of cAMP production by cells, B16F1 cells were placed in a 60 mm culture vessel at a density of 30Х10 ⁴ and cultured for 24 hours. Nonamu ethyl acetate fraction 25 μg/ml, 50 μg/ml, 100 μg/ml, kojic acid as a positive control (kojic acid, 1 mM) was pretreated for 24 hours, respectively, and then treated with α-melanocyte stimulating hormone (α-MSH, 200 nM), followed by incubation for 20 minutes.

Thereafter, the medium was removed, the cells were washed with PBS, the cells were removed with lysis buffer and frozen for 25 minutes in a freezer, and the supernatant was separated from the lysed cell lysate using a centrifuge at 13,000 rpm for 10 minutes. Then, the cAMP concentration in the supernatant was quantified using a cAMP ELISA kit (R&D Systems, Minneapolis, USA).

As a result, as shown in FIG. 7, the concentration of cAMP when treated with α-MSH was 189 pmol, which was more than 20 times higher than that of the untreated group at 7.2 pmol.

When the Nonamu ethyl acetate fraction was treated at concentrations of 25 μg/ml, 50 μg/ml, and 100 μg/ml, the concentrations of cAMP were 87 pmol, 73 pmol, and 56 pmol, respectively. In particular, at a concentration of 100 μg/ml, about It showed a 70% reduction rate, and it was found that the cAMP production inhibitory effect was superior to that of kojic acid, which was a positive control group.

Confirmation of whitening-related protein expression control effect of Nonamu ethyl acetate fraction

5-1: Confirmation of melanin synthesis inhibition mechanism

To identify the melanin synthesis inhibitory mechanism of the ron tree ethyl acetate fraction, tyrosinase, tyrosinase related protein 1 (TRP-1), tyrosinase related protein 2 (tyrosinase related protein 2), which are the main enzymes in the melanin production process 2, TRP-2) and MITF, a transcription factor inducing their expression, were observed.

First, B16F1 cells were inoculated into a 60 mm culture vessel at a density of 8Х10 ⁴ , and then 25 μg/ml, 50 μg/ml, and 100 μg/ml of Nonamu ethyl acetate fraction and kojic acid (1 mM) as a positive control, respectively. After treatment, α- melanocyte stimulating hormone (α-MSH, 200 nM) was treated, and cultured at 37° C. for 72 hours.

After 72 hours, cells were washed with PBS and proteins were extracted using lysis buffer. The extracted protein was quantified by Bradford assay, and the quantified protein was separated by 10% SDS-PAGE (SDS polyacrylamide gel electrophoresis) and then transferred to a PVDF (polyvinylidene difluoride) membrane. The membrane was blocked with 5% skim milk dissolved in TBS-T (Tris-buffered saline-Tween 20 solutions) for 1 hour, washed with TBS-T, and reacted with primary antibody overnight at 4°C. The next day, the membrane was washed with TBS-T and reacted with secondary antibodies (anti-mouse and anti-rabbit IgG) at room temperature for 1 hour. Protein bands were identified through HRP substrate and ImageQuant LAS 500 (GE Healthcare Life Science, USA).

As a result, as shown in FIG. 9, tyrosinase, MITF, TRP-1 and TRP-2 proteins increased when α-MSH was treated, whereas 25 μg/ml, 50 μg/ml, As a result of treatment at a concentration of 100 μg/ml, TRP-1 and TRP-2 proteins were not affected, but tyrosinase and MITF protein expressions were decreased in a concentration-dependent manner. Therefore, it was confirmed that the nona tree ethyl acetate fraction inhibits melanin production through inhibition of tyrosinase and MITF protein expression.

5-2: Confirmation of tyrosinase inhibition mechanism

In order to confirm the mechanism of tyrosinase inhibition, the protein expression and phosphorylation levels of p38, JNK, and ERK related to the regulation of MAPK (mitogen-activated protein kinases) pathway were examined.

As a result, as shown in FIG. 10, it was confirmed that phosphorylation of ERK (p-ERK) was activated when the nonamu ethyl acetate fraction or kojic acid was treated. In addition, it was confirmed that p38 phosphorylation (p-p38) and JNK phosphorylation (p-JNK) activated by a-MSH treatment were inhibited by treatment with ethyl acetate fraction or kojic acid.

When p-ERK is activated, MITF is inhibited and tyrosinase expression is further down-regulated. Therefore, the mechanism of whitening effect of Nona tree ethyl acetate extract is to reduce melanin production by suppressing tyrosinase expression through regulation of MAPK pathway. judged

Confirmation of whitening-related gene expression regulating efficacy of nona tree ethyl acetate fraction

Among the proteins related to whitening, gene expression of tyrosinase and MITF, which have the most excellent inhibitory effect by the ethyl acetate fraction of rona tree, were analyzed through real-time PCR.

First, B16F1 cells were inoculated into a 60 mm culture vessel at a density of 1×10 ⁵ and cultured for 24 hours. After treatment with 25 μg/ml, 50 μg/ml, and 100 μg/ml of Nonamu ethyl acetate fraction, kojic acid (1 mM) as a positive control, respectively, α-melanocyte stimulating hormone (α-MSH, 200 nM) and incubated for 4 hours (MITF) or 24 hours (Tyrosinase).

After culturing, the cells were washed twice with PBS, and then the cells were obtained and total RNA was isolated using trizol reagent (Invitrogen, USA). The isolated RNA was synthesized into cDNA using a cDNA reverse transcription kit (Applied Biosystems, USA). After adding the SYBR qPCR pre-mix (Dyne Bio, KOREA) and the primer set shown in Table 1 to the cDNA, PCR was performed using Light Cycler® Switzerland equipment.

primer sequence Gene Primer Sequence (5' 3') sequence number MITF Forward ATG CTG GAA ATG CTA GAA TAC AGT SEQ ID NO: 1 Reverse ATC ATC CAT CTG CAT GCA SEQ ID NO: 2 Tyrosinase Forward CCT CCT GGC AGA TCA TTT GT SEQ ID NO: 3 Reverse GGC AAA TCC TTC CAG TGT GT SEQ ID NO: 4 36B4 Forward TGG GCT CCA AGC AGA TGC SEQ ID NO: 5 Reverse GGC TTC GCT GGC TCC CAC SEQ ID NO: 6

As a result, as shown in FIG. 10, it was confirmed that the increased tyrosinase and MITF gene expression due to α-MSH treatment decreased in a concentration-dependent manner with the nona tree ethyl acetate fraction, similar to the tyrosinase and MITF protein expression inhibitory effects. showed a trend.

statistical processing

The experiment conducted in the present invention was repeated three times or more, and the statistical significance of the data was verified by the Student t-test and ANOVA method using the GraphPad Prism 5.0 program. The resultant value was expressed as mean ± standard deviation (SD), and a case where the p value was less than 0.05 was judged to be statistically significant.

<110> GLOCAL Industry-Academic Cooperation Foundation Konkuk University <120> Composition for skin whitening comprising extract or fraction of Catalpa ovata as an active ingredient <130> PDPC214581 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> artificial sequence <220> <223> MITF_forward primer <400> 1 atgctggaaa tgctagaata cagt 24 <210> 2 <211> 18 <212> DNA <213> artificial sequence <220> <223> MITF_reverse primer <400> 2 atcatccatc tgcatgca 18 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> Tyrosinase_forward primer <400> 3 ccctcctggca gatcatttgt 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> Tyrosinase_reverse primer <400> 4 ggcaaatcct tccagtgtgt 20 <210> 5 <211> 18 <212> DNA <213> artificial sequence <220> <223> 36B4_forward primer <400> 5 tgggctccaa gcagatgc 18 <210> 6 <211> 18 <212> DNA <213> artificial sequence <220> <223> 36B4_reverse primer <400> 6 ggcttcgctg gctcccac 18

Claims (11)

No tree ( Catalpa ovata ) Containing a hot water extract and its ethyl acetate fraction as an active ingredient, a cosmetic composition for skin whitening.
The cosmetic composition for skin whitening according to claim 1, wherein the no tree ( Catalpa ovata ) is a stem or a branch.
The cosmetic composition for skin whitening according to claim 1, wherein the nona hot water extract or its ethyl acetate fraction is contained in a concentration of 10 to 500 μg/ml.
The cosmetic composition for skin whitening according to claim 1, wherein the hot-water extract of cypress is extracted by adding 0.5 to 1.5 ℓ of water per 100 parts by weight of cypress and then applying heat for 1 to 3 hours.
The method of claim 1, wherein the ethyl acetate fraction is
(a) adding a hexane solvent to the hot water extract of Japanese cypress, and then obtaining an aqueous phase;
(b) adding a chloroform solvent to the aqueous phase and then obtaining an aqueous phase; and
(c) adding an ethyl acetate solvent to the aqueous phase, and then obtaining an ethyl acetate fraction; characterized in that it is prepared by a method comprising a, cosmetic composition for skin whitening.
The cosmetic composition for skin whitening according to claim 1, wherein the nona hot water extract or its ethyl acetate fraction inhibits melanin production or tyrosinase activity.
The method of claim 1, wherein the nona hot water extract or its ethyl acetate fraction inhibits cAMP (cyclic AMP) production; inhibition of microphthalmia-associated transcription factor (MITF) production; Or, through the regulation of the MAPK (mitogen-activated protein kinases) pathway through the increase of ERK phosphorylation, inhibition of p38 or JNK phosphorylation, suppression of melanin production or tyrosinase activity, skin whitening cosmetics composition.
A health functional food composition for skin whitening comprising a hot water extract of Japanese cypress and an ethyl acetate fraction thereof as an active ingredient.
A pharmaceutical composition for preventing or treating skin pigmentation diseases, comprising a hot-water extract of Nonamu tree and an ethyl acetate fraction thereof as an active ingredient.
A quasi-drug composition for preventing or improving skin pigmentation diseases, comprising a hot water extract of Nonamu and an ethyl acetate fraction thereof as an active ingredient.
The composition according to claim 9 or 10, wherein the skin pigmentation disease is melasma, freckle or melanoma.

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