WO2018084671A1 - Composition for preventing or treating pigmentation disorders - Google Patents

Abstract

The present invention relates to a composition for preventing or treating pigmentation disorders, the composition enabling the prevention or treatment of pigmentation disorders caused by hypopigmentation or hyperpigmentation of the skin.

Description

Composition for preventing or treating pigmented diseases

The present invention relates to a composition for preventing or treating pigmented diseases, and to a composition capable of preventing or treating pigmented diseases caused by hypopigmentation or hyperpigmentation of the skin.

Human skin is an organ that is exposed to various external environmental stresses, and various mechanisms exist to protect against potential damage caused by ultraviolet (UVR). Such defense mechanisms include DNA damage mechanisms, various enzymes such as catalase and peroxide scavengers, and skin pigmentation. Among these elements, pigmentation is the most important photo-protective element. Tyrosinase acts as an important regulator of pigmentation because it promotes two rate-limiting reactions in the synthesis pathway of melanin pigments. These rate-limiting reactions include: 1) tyrosine hydroxylation to produce 3,4-dihydroxyphenylalanine (DOPA), and 2) DOPA oxidation to produce dopaquinone. There is this.

Hypopigmentation is caused by melanin deficiency, which causes a 70-fold increase in skin cancer incidence. Genetic hypopigmentation leads to diseases such as albinism. In addition, low-pigmented lesions also make the exposed skin visually unsightly, resulting in psychosocial problems. Small molecules that can upregulate melanin biosynthesis are being investigated as potential agents in treating hypopigmentation disorders and reducing UV-induced skin damage. Thus, as pigmentation promoting substances having various photoprotective properties, for example, diacylglycerols, 3-isobutyl-1-methylxanthine (3-isobutyl-1-methylxanthine, IBMX) and dimethyl sulfoxide Said (dimethylsulfoxide, DMSO) has been developed. However, these compounds suffer from serious side effects of a tendency to promote tumor formation. Therefore, in recent years, there has been a demand for the development of a material that is superior in pigmentation effect but is nontoxic and economically effective.

On the other hand, dysregulation of melanin synthesis leads to excessive deposition of pigments and to diseases such as melasma, age spots and solar keratosis. Also, in the cosmetic industry, improvement of skin color has long been required, and various whitening products are sold worldwide. These whitening products or therapeutic agents include hydroquinones, retinoids and tyrosinase inhibitors, and the like, which are problematic because of side effects such as mutations, toxicity, and ochronosis and blue-black hyperpigmentation of skin. Therefore, in recent years, there is a continuous demand for research and development on a therapeutic agent or cosmetic having excellent skin whitening effect while not causing side effects and having low skin toxicity.

One object of the present invention is to provide a pharmaceutical composition that is excellent in promoting melanin formation and capable of preventing or treating various diseases due to low pigmentation.

Another object of the present invention is to provide a pharmaceutical composition that is excellent in inhibiting skin pigmentation and capable of preventing or treating various diseases due to hyperpigmentation.

Another object of the present invention is to provide a cosmetic or food composition that can prevent or improve pigment diseases.

The inventors of the present invention sacheolssuk extract, among others isopropyl proxy Dean 7- O of (Artemisia capillaris Thunberg) - (6'- O - p - one to Kumar) -β- glue nose Llano side (isofraxidin 7- O - ( 6'- O - p- coumaroyl) -β-glucopyranoside) has been found to have an excellent effect of promoting melanogenesis in melanocytes, leading to the present invention.

In addition, the inventors of the present invention is that the extract of Artemisia capillaris Thunberg, especially 4,5-O-dicaffeoylquinic acid (4,5-O-dicaffeoylquinic acid) has an excellent pigmentation inhibitory effect in melanocytes Discovered and led to the present invention.

According to one embodiment of the present invention, the extract comprising the extract of Artemisia capillaris Thunberg as an active ingredient, relates to a pharmaceutical composition for the prevention or treatment of pigment diseases.

In the present invention, the extract of cedars is low pigment because it contains a large amount of isoproxidine 7- O- (6'- O - p -coumaroyl) -β- glucopyranoside excellent melanin formation promoting effect in melanocytes Pigment disease caused by deposition can be effectively prevented or treated.

In addition, in the present invention, the extract of cedar mugwort contains a large amount of 4,5-O-dicafeoyl quinic acid excellent in the pigmentation inhibitory effect in melanocytes can effectively prevent or treat pigment diseases caused by hyperpigmentation.

In addition, in the present invention, the wormwood may use a site such as leaves, berries, bark, roots, and preferably may use a leaf, but is not limited thereto.

In the present invention, all the methods that can be extracted from a simple extraction method to a fat-soluble component in the method of extracting the cedar mugwort can be applied, it can be obtained by pulverizing to extract by extraction solvent, filtered and concentrated to facilitate the extraction .

Extraction solvents include water, ethanol, methanol, butanol, n-hexane, n-heptane or DMSO, and may be used as an extraction solvent by mixing two or more of them, but is not limited thereto. Those skilled in the art can appropriately select from known methods depending on the amount, extraction method, and the like. Extraction time and temperature can also be appropriately selected by those skilled in the art in consideration of extraction efficiency, extraction solvent and the like. Preferred extraction time is 10 minutes to 1 hour, and preferred extraction temperature is 40 to 100 ° C.

The extract extracted by the above extraction method can be concentrated by distillation or the like after filtering by a known filtration method such as vacuum filtration. Such extraction, filtration and concentration methods are well known in the art and therefore one skilled in the art can make appropriate selections to prepare ginkgo extracts.

In the present invention, but not particularly limited to the content of the extract of cedar mugwort, preferably in a concentration of 10 to 100 ㎍ / ml, or 20 to 75 ㎍ / ml, or 25 to 75 ㎍ / ml, or 25 to 50 ㎍ / ml May be included. In the present invention, when the extract of cedars is included in an amount less than 10 µg / ml may be a slight pigmentation inhibitory effect, when included in an amount exceeding 10 µg / ml toxicity may be a problem.

 In addition, the pigment disease due to the low pigmentation in the present invention may be all diseases caused by the loss of melanin cells or melanin production due to the inhibition of melanogenesis, such as vitiligo, Albinism, depigmentation nevus, white nasal ganglia, scrubbing, post-inflammatory bleaching, ecchymoses, partial leukemia, idiopathic red hypopigmentation, or mucinous leukemia.

According to another embodiment of the present invention and represented by the formula (1) isophthalic proxy Dean 7- O - (6'- O - p - Kumar In one) -β- glue nose Llano side (isofraxidin 7- O - (6 ' A pharmaceutical composition for the prophylaxis or treatment of pigment diseases originating in hypopigmentation comprising O - p- coumaroyl) -β-glucopyranoside) as an active ingredient:

[Formula 1]

In the present invention, the isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside may be isolated from the cedar extract of the present invention, but can be synthesized by a general synthetic method. It may be purchased or commercially available, and the source thereof is not particularly limited.

In addition, in the present invention, the content of the isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside is not particularly limited, but preferably 10 to 100 μΜ, or 10 to 50 μΜ, or 10-25 μΜ, or 12.5-25 μΜ. In the present invention, when the isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside is included in an amount of less than 10μΜ melanin enhancement promoting effect may be insignificant, 100μΜ If included in excess, toxicity may be a problem.

In the present invention, the pigment disease is a disease due to hypopigmentation, which may be caused by a loss of melanocytes or a inhibition of melanogenesis, and may be any disease caused by melanin production not reaching a normal level, for example vitiligo , Albinism, depigmentation nevus, white nasal cavities, scleroderma, post-inflammatory bleaching, ecchymoses, partial leukemia, idiopathic red hypopigmentation, or buccal leukemia.

According to another embodiment of the present invention, the skin comprising a 4,5-O-dicaffeoylquinic acid represented by the following formula (2,4,5-O-dicaffeoylquinic acid) as an active ingredient, hyperpigmentation skin A pharmaceutical composition for preventing or treating pigmented diseases:

[Formula 2]

In the present invention, the 4,5-O-dicafeoyl quinic acid may be isolated from the cedar extract of the present invention, can be synthesized by a general synthetic method, or may be used to buy a commercially available, and There is no particular restriction on the source.

In addition, the content of the 4,5-O-dicafeoylquinic acid in the present invention is not particularly limited, but may be included in a concentration of preferably 5 to 100 μΜ, or 5 to 50 μΜ, or 5 to 25 μΜ, or 6.25 to 25 μΜ. have. In the present invention, when the 4,5-O-dicafeoyl quinic acid is included in an amount of less than 5μM may be a slight pigmentation inhibitory effect, when included in an amount of more than 100μΜ toxicity may be a problem.

In the present invention, the skin pigment disease is a disease caused by hyperpigmentation, and means a disease that becomes darker or darker than other areas due to excessive increase of melanin at a specific part of the skin or nail. Preferably, the skin pigment disease includes blemishes, freckles, senile plaques, blemishes, birthmarks, or solar lentigines, but is not limited thereto.

On the other hand, in the present invention, "prevention" can be used without limitation any action to block, suppress or delay the symptoms of pigment diseases due to hypopigmentation using the pharmaceutical composition of the present invention.

In addition, in the present invention, "treatment" may be used without limitation as long as the action of the symptoms of pigment disease due to hypopigmentation improve or benefit using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in that the capsule, tablets, granules, injections, ointments, powder or beverage form, the pharmaceutical composition may be characterized in that it is intended for humans.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc., and in the case of injections, buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.

Examples of suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. The dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

According to another embodiment of the present invention, the present invention relates to a cosmetic composition for preventing or ameliorating pigment diseases caused by hypopigmentation or hyperpigmentation, comprising the extract of cedars as an active ingredient.

According to another embodiment of the invention, a pigment due to low pigment deposition, comprising isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside as an active ingredient It relates to a cosmetic composition for the prevention or improvement of diseases.

In the cosmetic composition of the present invention, the description of the cedar extract and the isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside are overlapped with those described in the pharmaceutical composition. Hereafter, the specific description is omitted.

According to another embodiment of the present invention, the present invention relates to a cosmetic composition for preventing or ameliorating pigment diseases caused by hyperpigmentation, comprising 4,5-O-dicafeoylquinic acid as an active ingredient.

In the cosmetic composition of the present invention, the description of the extract of cedar wormwood and 4,5-O-dicafeoylquinic acid is duplicated with that described in the pharmaceutical composition, and the detailed description thereof will be omitted below.

On the other hand, in the present invention, "improvement" may be used without limitation if all the action to improve or beneficially change the symptoms of pigment disease due to hypopigmentation using the pharmaceutical composition of the present invention.

In the present invention, the cosmetic composition is a lotion, nutrition lotion, nutrition essence, massage cream, beauty bath additives, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, Suntan cream, skin lotion, skin cream, sunscreen cosmetic, cleansing milk, depilatory {cosmetic}, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, Bath Soap, Water Soap, Beauty Soap, Shampoo, Hand Cleanser (Hand Cleaner), Medicated Soap {Non-Medical}, Cream Soap, Facial Wash, Systemic Cleanser, Scalp Cleaner, Hairrin, Cosmetic Soap, Tooth Whitening Gel, Toothpaste It may be prepared in the form. To this end, the composition of the present invention may further comprise a solvent or a suitable carrier, excipient or diluent commonly used in the preparation of the cosmetic composition.

The kind of the solvent that can be further added in the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used, and as the carrier, excipient or diluent, purified water, oil, wax , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like. In addition, a whitening agent, a moisturizing agent, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents may be included as necessary.

The oil may be hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin Can be used.

Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as fatty acids, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as fatty acid alcohol. Isopropyl myristate, isopropyl palmitate, butyl stearate may be used as the fatty acid ester. As surfactants, cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as much as possible.

In addition, it may include a hygroscopic agent, a thickener, an antioxidant, and the like, which are widely known in the cosmetic field, and their types and amounts are known in the art.

According to still another embodiment of the present invention, the present invention relates to a food composition for preventing or ameliorating a pigment disease caused by hypopigmentation or hyperpigmentation, which contains an extract of cedars as an active ingredient.

According to another embodiment of the invention, a pigment due to low pigment deposition, comprising isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside as an active ingredient It relates to a food composition for preventing or ameliorating a disease.

In the food composition of the present invention, the description of the cedar extract and the isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside are overlapped with those described in the pharmaceutical composition. Hereafter, the specific description is omitted.

According to another embodiment of the present invention, the present invention relates to a food composition for preventing or improving pigment diseases caused by hyperpigmentation, comprising 4,5-O-dicafeoylquinic acid as an active ingredient.

In the food composition of the present invention, the description of the extract of cedars and 4,5-O-dicafeoylquinic acid is duplicated with that described in the pharmaceutical composition, and the detailed description thereof will be omitted below.

The food composition comprising the composition of the present invention as an active ingredient may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread. . Since the food composition of the present invention is composed of plant extracts having little toxicity and no side effects, it can be used with confidence even for long-term administration for the purpose of prevention.

When the composition of the present invention is included in a food composition, the amount may be added at a ratio of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in the form of a drink, there is no particular limitation other than the containing the food composition in the ratio indicated, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourants (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).

Other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.

These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

The composition provided by the present invention is excellent in promoting melanin formation without causing toxicity to the human body, and can effectively prevent, ameliorate, and treat various diseases caused by low pigmentation.

In addition, the composition provided in the present invention effectively inhibits melanin synthesis in the melanin pigment, and has an excellent skin whitening effect, such as blemishes, freckles, senile plaques, blemishes, birthmarks or solar lentigines caused by hyperpigmentation Pigment disease can be prevented or treated.

FIG. 1 (a) shows the chemical structure of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside isolated from cedar extract.

Figure 1 (b) shows the important HMBC correlations in isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside isolated from cedar extract.

Figure 1 (c) shows the results of HPLC chromatogram at 280nm of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside.

Figure 1 (d) shows the results of infrared (IR) spectrum analysis of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside.

Figure 1 (e) shows the results of absorption ultraviolet (UV) spectrum analysis of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside.

FIG. 2 (a) shows azelaic acid (AZ), isoproxidin or isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucoid in B16-F10 melanocytes. The cell image after the treatment of the lanoside (Compound 1) is taken with a digital camera (100X magnification) is shown (scale bar = 20㎛).

FIG. 2 (b) shows cells after treatment with azelaic acid, isoproxidine or isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside to B16-F10 melanin cells. It shows a picture taken of the pellets.

FIG. 2 (c) shows cells after treatment with azelaic acid, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside to B16-F10 melanocytes. The graph shows the change in melanin content.

FIG. 2 (d) shows tyrosine after treatment with azelaic acid, isoproxidine or isoproxidin 7- O- (6′- O - p -coumaroyl) -β-glucopyranoside to B16-F10 melanocytes. The change in sinease activity is shown graphically.

FIG. 3 shows untreated B16-F10 melanocytes after treatment with isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside, forskolin or IBMX The graph shows the change in the amount of melanin secreted from the cells compared to the control.

FIG. 4 (a) shows MIFT expression levels after B-F10 melanocytes treated with isoproxidine, isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside or IBMX. The graph shows the change in.

4B shows TRP-1 after B-F10 melanocytes treated with isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside or IBMX. The change in expression level is shown graphically.

4 (c) shows tyrosinase after B-F10 melanocytes treated with isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside or IBMX. The change in expression level is shown graphically.

5 shows tyrosine in melanocytes; Tyrosinase; Tyrosine and tyrosinase; Tyrosinase, tyrosine and isoproxidine; Tyrosinase, tyrosine and isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside; after treatment with a graph of the change in absorbance at 490 nm.

6 is a graph showing a photograph taken by a stereo microscope (100X magnification) after PTU treatment of zebrafish embryos by concentration (scale bar = 20 μm).

7 shows the heart rate after treatment of zebrafish embryos with different concentrations of PTU, isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside The change is graphed.

FIG. 8A shows zebrafish embryos treated with different concentrations of PTU, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside. The photograph taken by the stereo microscope (100X magnification) is shown (scale bar = 20 micrometers).

FIG. 8 (b) shows zebrafish embryos treated with different concentrations of PTU, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside. After the change in melanin content is shown in a graph.

8 (c) shows zebrafish embryos treated with different concentrations of PTU, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside. It is then shown graphically the change in tyrosinase activity.

Figure 9 (a) shows the number of melanocytes after treatment with varying concentrations of PTU or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside in zebrafish embryos. The graph shows the change in.

Figure 9 (b) is a zebrafish embryo of melanocytes after treatment with different concentrations of PTU or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside The change in area is shown graphically.

FIG. 10 (a) shows 48 hours of azelaic acid (AZ), cedar extract (A.capillaris), ACMF09 fraction, and 4,5-O-dicapoylquinic acid (4,5-diCQA) on B16-F10 melanocytes. After treatment for a while, it is shown a photograph observed with a phase contrast microscope (scale bar = 20㎛).

10 (b) shows the appearance of cell pellets after treatment with azelaic acid, cedar extract, ACMF09 fraction, and 4,5-O-dicafeylquinic acid on B16-F10 melanin cells.

10 (c) is a graph showing the change of melanin content in cells after treatment with azelaic acid, cedar extract, ACMF09 fraction and 4,5-O-dicafeylquinic acid in B16-F10 melanin cells.

Figure 10 (d) is a graph showing the change in tyrosinase activity after treatment with azelaic acid, cedar extract, ACMF09 fraction and 4,5-O-dicafeyl quinic acid on B16-F10 melanin cells.

Figure 11 (a) shows the chemical structure of 4,5-O- dicafeoyl quinic acid isolated from the cedars mugwort extract.

Figure 11 (b) shows the results of HPLC chromatogram of 4,5-O- dicafeoyl quinic acid isolated from the cedar mugwort extract.

FIG. 12 graphically shows changes in tyrosinase activity in acellular systems after treatment of B16-F10 melanocytes with AZ, ACMF09 fraction or 4,5-O-dicafeoylquinic acid.

FIG. 13 graphically shows changes in mRNA expression levels of MITF, tyrosinase and TRP-1 after treatment of AZ, ACMF09 fraction or 4,5-O-dicafeylquinic acid on B16-F10 melanocytes.

Figure 14 (a) shows a photograph taken with a stereomicroscope (100X magnification) after the zebrafish embryo treated with PTU, cedar mugwort extract, ACMF09 fraction or 4,5-O- dicafeyl quinic acid (scale bar = 20 μm).

Figure 14 (b) is a graph showing the change in melanin content after treatment with zebrafish embryos PTU, cephalococci extract, ACMF09 fraction or 4,5-O- dicafeoyl quinic acid at different concentrations.

Figure 14 (c) is a graph showing the change in tyrosinase activity after treatment of zebrafish embryos with different concentrations of PTU, cephalococci extract, ACMF09 fraction or 4,5-O-dicafeoylquinic acid.

Figure 15 (a) shows the change in the number of melanocytes after treatment of zebrafish embryos with different concentrations of PTU or 4,5-O-dicafeoylquinic acid.

Figure 15 (b) shows the change in the area of melanocytes after treatment with varying concentrations of PTU or 4,5-O- dicafeoyl quinic acid in zebrafish embryos.

16 is a graph showing the change in cell viability after treatment with varying concentrations of azelaic acid, cedar extract, ACMF09 fraction, or 4,5-O-dicafeylquinic acid in B16-F10 melanin cells.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1

Example 1 was carried out to determine the melanin forming activity of the cedars mugwort extract.

General experimental process

Visual rotation was measured with a JASCO DIP-1000 digital polarimeter. UV spectra were obtained using a JASCO V-530 UV / Vis spectrophotometer (Jasco Corp., Japan) and IR spectra were obtained using a JASCO FT / IR-300E spectrophotometer (Jasco Corp., Japan). ¹ H and ¹³ C NMR spectra were recorded in 1D and 2D NMR experiments with CD ₃ OD (tetramethylsilane (TMS) is used internationally) using a Varian VNMRS 600 MHz NMR spectrophotometer (Varian, Inc., USA). . Chemical shifts ( δ ) are expressed in parts per million (ppm) and coupling constants ( J ) are expressed in Hz. Weight spectra were measured using a JMS-700 (Jeol, Japan) and Varion 1200, Platform II (Varian, USA) spectrophotometer. High resolution mass (ESI-MS) and LCMS / MS spectra were measured using a negative electrospray ion mode by Water Synampt High Definition Mass Spectrometry / Time of a Flight Mass Spectrophotometer.

Preparation of plants

The leaves and stems of Astemisia capillaris Thunberg were obtained from Professor Lim Soon-ho of Dongshin University (Naju, Korea).

Preparation of Reagents

IBMX, forskolin, phenylthiourea (PTU), NaOH, dimethyl sulfoxide (DMSO), 3,4-dihydroxyphenylalanine (L-DOPA), caffeine powder, staurosporine, CellLytic ^TM buffer and MTT ( 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was purchased from Sigma (St Louis, MO, USA). Isofraxidin was purchased from ChemFaces Biochemical (Hubei, China).

Cell culture

Murine melanoma B16-F10 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). It was incubated in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin mixture (Gibco, USA). Cultured cells were maintained at 37 ° C. and humidified conditions in a 5% CO ₂ incubator.

Determination of Melanin Content in B16-F10 Melanocytes

Melanocytes were seeded in 6-well plates at a density of 2 × 10 ⁵ cells / well and treated with compound for 48 hours after 24 hours. Melanocytes were washed with phosphate buffered saline (PBS) and lysed with CellLytic buffer at 4 ° C. The cell extract was spun at 13,000 rpm for 10 minutes at 4 ° C. The remaining pellet was washed twice with ethanol: ether (1: 1) for melanin analysis and dissolved in 200 μl of 1N NaOH in 10% DMSO at 80 ° C. Absorbance was measured at 400 nm using a microplate reader (VersaMax ™; Molecular Devices Corporation, California, USA) for 100 μl aliquot of the resulting solution.

Determination of melanin secreted into the culture medium

B16-F10 melanocytes were seeded in a 24-well plate at a density of 5 × 10 ⁴ cells / well using 500 μl culture medium. After 24 hours the culture medium was supplemented and cells were treated with compound for 60 hours. The culture medium was recovered and the melanin level was measured for absorbance at 475 nm using a microplate reader (VersaMax ^™ ; Molecular Devices Corporation, California, USA).

Determination of Tyrosinase Activity in Melanocytes

B16-F10 melanocytes were seeded in 6-well plates at a density of 2 × 10 ⁵ cells / well. After 24 hours, the melanocytes were treated with the compound for 48 hours. Cells were washed with PBS and lysed with CellLytic buffer at 4 ° C. The cell extract was spun at 13,000 rpm for 10 minutes at 4 ° C. Protein concentration was adjusted using a Bradford assay and 100 μl cell lysate containing 40 μg protein was transferred to a 96-well plate. Thereafter, 100 μl of 5 mM L-DOPA was added thereto, followed by incubation at 37 ° C. for 60 minutes. Dopachrome formation was measured at 475 nm using a microplate reader.

Mushroom tyrosinase assay

Mushroom tyrosinase enzyme was dissolved in 50 mM potassium phosphate buffer (pH 6.5) at a concentration of 500 units / ml. 550 μl of 50 mM potassium phosphate and 50 μl of tyrosinase solution were mixed in an appropriate volume in a microfuse tube and incubated at room temperature for 5 minutes. 100 μL of 1.5 mM L-tyrosine was added to the solution and then loaded into a 96-well plate. The absorbance at 490 nm was measured using a microplate reader to determine the amount of dopachrome produced in the reaction mixture.

HPLC-based Activity Profiling of A. cappilaris Thunberg Extract

The dry powders of the cedars and leaves were extracted with 100% methanol and then concentrated in vacuo to produce methanol extract (6 g). HPLC was performed on a H ₂ O concentration gradient system containing MeCN and 0.1% HCOOH using a YMC-PAC Pro C18 (10 mm, 250 mm, 5 μm) column in the Agilent HP1100 series. However, the Agilent HP1100 series consists of a degasser, a secondary mixing pump, a column oven and a DAD detector. For activity profiling, a portion (6 g) of MeOH extract was prepared in semipreparative HPLC (Agilent 1100 Series, USA), starting with 80% H ₂ O with 0.1% HCOOH / 20% MeCN and starting with 0.1% HCOOH / 60% MeCN. Fractions of 40% H ₂ O containing 50 minutes were fractionated. The mobile phase moved at a flow rate of 6.0 ml / min and the eluate was detected at 280 nm. A total of 23 fractions were obtained and the biological activity for pigmentation in melanocytes by each fraction was evaluated. As a result, the ACMF09 fraction was selected, and the H ₂ O-MeOH was separated under a gradient starting from 60:40 (v: v) in the RP-18 column for further separation and kept constant for 50 minutes. The gradient system was then reduced to 0: 100 and held constant for 20 minutes to obtain purified compound (2 mg).

Iso-proxy Dean 7- O - (6'- O - p - Kumar In one) - β - pyrano glue nose side (isofraxidin 7- O - (6'- O - p - coumaroyl) - β -glucopyranoside) of H- NMR Results

The isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside is a brown amorphous powder, UV, IR, ¹ H and ¹³ C NMR data are as follows, in particular ¹ H and ¹³ C NMR data are shown in Table 1 below (600 MHz, methanol- d ₄ ): UV λ _max (H ₂ O) (log ε) 300, 304, 322 nm; IR (KBr) λ _max : 3500, 1675, 1011 cm ⁻¹ ; ¹ H NMR (in CD ₃ OD, 600 MHz) δ 7.64 (1H, d, J = 9.6 Hz, H-4), 7.37 (1H, d, J = 15.9 Hz, H-7``), 7.32 (2H , d, J = 8.7 Hz, H-2`` / H-6 ''), 6.90 (1H, s, H-5), 6.80 (2H, d, J = 8.7 Hz, H-3 '' / H -5 ''), 6.16 (1H, d, J = 9.6 Hz, H-3), 6.10 (1H, d, J = 15.9 Hz, H-8``), 5.20 (1H, d, J = 7.5 Hz , H-1 '), 4.39 (1H, dd, J = 11.4, 7.2 Hz, H-6'), 4.34 (1H, dd, J = 11.4, 2.0 Hz, H-6 '), 3.99 (3H, s , 8-OMe), 3.88 (3H, s, 6-OMe), 3.55 (1H, dd, J = 9.0, 7.5 Hz, H-2 '), 3.50 (1H, m, H-5'), 3.47 ( 1H, t, J = 9.0 Hz, H-3 '), 3.40 (1H, t, J = 9.0 Hz, H-4'); ¹³ C NMR (in CD ₃ OD, 125 MHz) δ 168.7 (C-9), 162.7 (C-2), 161.5 (C-4 ″), 151.8 (C-6), 146.5 (C-7 ″), 145.7 (C-4), 144.0 (C-8a), 143.1 (C-7), 142.7 (C-8), 131.3 (C-2 ″ / C-6 ″), 127.1 (C-1 ″), 117.1 (C-3 ″ / C-5 ″), 116.9 (C-4a), 115.7 (C-3), 114.9 (C-8 ″), 105.9 (C-5), 103.9 (C-1 '), 77.9 (C-3 '), 75.9 (C-5'), 75.7 (C-2 '), 72.2 (C-4'), 64.4 (C-6 '), 62.5 (8-OMe), 57.2 (6- OMe); HRESI-MS m / z 529.1346 [M ⁻ H] ⁻ (C ₂₆ H ₂₆ O ₁₂ ).

Position	δ H ( J in Hz)	δ C	HMBC a
2	-	162.7
3	6.16 1 H, d (9.6)	115.7	C-2, 4a
4	7.64 1 H, d (9.6)	145.7	C-2, 5
4a	-	116.9
5	6.90 1H, s	105.9	C-4, 6, 7, 8a
6	-	151.8
7	-	143.1
8	-	142.7
8a	-	144.0
One'	5.20 1 H, d (7.5)	103.9	C-7
2'	3.55 1H, dd (7.5, 9.0)	75.7	C-1 ', 3',
3 '	3.47 1H, t (9.0)	77.9	C-2 ', 4'
4'	3.40 (1H, t, 9.0)	72.2	C-3 ', 5'
5 '	3.50 1H, m	75.9	C-4 '
6 '	4.39 1H, dd (11.4, 7.2) 4.34 1H, dd (11.4, 2.0)	64.4	C-4 ', 5'
One	-	127.1
2/6	7.32 2H, d (8.7)	131.3	C-4
3/5	6.80 2H, d (8.7)	117.1	C-1, 4
4	-	161.5
7	7.37 1 H, d (15.9)	146.5	C-1, 2/6, 9
8	6.10 1 H, d (15.9)	114.9	C-7, 9
9	-	168.7
6-OMe	3.88 3H, s	57.2	C-6
8-OMe	3.99 3H, s	62.5	C-8

Quantitative Real-Time PCR Analysis

B16-F10 melanocytes were treated with isoproxidin, isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside and IBMX for 48 hours. Total RNA was extracted using TRI-Solution ^™ and quantified using NanoDrop 2000 spectrophotometer (NanoDrop Technologies). CDNA was synthesized from 1 μg RNA using AccuPower PCR premix (Bioneer). MRNA expression levels of the MITF gene, tyrosinase gene and TRP-1 were measured using a Power SYBT Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized to β-actin and fold changes were calculated using the ΔΔCT method. Primer sequences are as follows: mouse tyrosinase forward 5′-TACTTGGAACAAGCCAGTCGTATC-3 ′, reverse 5′-ATAGCCTACTGCTAAGCC CAGAGA-3 ′; Mouse TRP-1 forward 5'-AAACCCATTTGTCTCCCAA-TGA-3 ', reverse 5'-CGTTTTCCAACGG-GAAGGT A-3'; Mouse MITF forward 5'-GGACTTTCCCTTATCCCATCCA-3 ', reverse 5'-GCCGAGGTTGTTGGTAAAG-GT-3'. After performing PCR for 2 minutes at 95 ° C., 40 cycles were performed at 95 ° C. for 30 seconds, at 60 ° C. for 1 minute, and at 72 ° C. for 1 minute, and at 72 ° C., the last 30 seconds of extension was performed. It was. Data was analyzed using Stepone ^™ software v2.3 (Applied Biosystems).

MTT assay for cell viability

B16-F10 melanocytes were seeded in 96-well plates for 12 hours at a concentration of 5 × 10 ³ cells / well. Cells were treated with injection mugwort extract or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside for 48 hours. Thereafter, the survival rate of the cells was evaluated using the MTT assay.

Maintenance of Zebrafish

An adult zebrafish ( Danio rerio ) was purchased at a general store (Lotte Mart, South Korea). 10-15 zebrafish were confined in each of the 5L acrylic tanks and maintained under the following conditions: day / night cycle of 28.5 ° C., 14/10 hours. The zebrafish was fed with live brine shrimp (Artemia salina) twice a day for 7 days. Embryos were obtained from natural scattering induced at 9:30 in the morning by changing the cycle with light. Embryos were secured within 30 minutes.

Phenotype-based evaluation of test compounds using zebrafish

The zebrafish embryos were maintained in the embryonic medium of the petri dishes at a density of 70-80 embryos per 100 m ² of petri dishes. Simultaneously generated embryos were harvested and three pipettes were dispensed for each well of a 96-well plate using a pipette. However, 200 μl of the embryo medium containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl ₂ H ₂ O, and 0.33 mM MgSO ₄ 7H ₂ O per well was injected into the wells. Tetanus extract or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside was dissolved in 0.1% DMSO and added to the embryo medium in an amount of 9-72hpf (63 Time exposure). Stir from time to time for even distribution of test compounds and change medium every 24 hours. 75 μM PTU was used to form clear zebrafish in all experiments. Phenotypic-based evaluation of body pigments was performed at 72 hpf. Embryos were removed using forceps, anesthetized with tricaine methanesulfonate solution, and fixed in 3% methyl cellulose. Stereomicroscopy (LEICA DFC425C) was used to observe the effect on the pigmentation of zebrafish. Melanin pigmented areas were measured using the Image J program (National Institutes of Health, USA).

Evaluation of Melanin Content and Tyrosinase Activity in Zebrafish

Approximately 40 zebrafish embryos were treated with 9-48 hpf of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside followed by sonication using CellLytic buffer. (Pulse on: 10 s, pulse off: 5 s, 8 min in 150 μL buffer). The lysate was centrifuged at 13,000 rpm for 10 minutes to recover the supernatant and the protein was quantified using the Bradford assay. 100 μl of lysis buffer containing 250 μg total protein was transferred to a 96-well plate and then 100 μl of 5 mM L-3,4-dihydroxyphenylalanine (L-DOPA) was added. Control wells contained 100 μl lysis buffer and 100 μl 5 mM L-DOPA. After incubation at 37 ° C. for 60 minutes, the absorbance was measured at 475 nm using a microplate reader. Blanks were removed at each absorbance value and the final activity was expressed as a percentage of the water control. Melanin content in zebrafish was measured using the following method. Specifically, protein extracts were prepared at 48 hpf using the same methodology as Tyrosinase assays. The pellet was recovered after centrifugation, dissolved in 200 μl 1N NaOH with 10% DMSO for 60 minutes and then vortexed. Absorbance was measured at 475 nm using a microplate reader.

Zebrafish Heart Rate Measurement

Atrial and ventricular heart rates were measured at 48 hpf to assess the toxicity of the compounds. Zeiss Stemi 2000-C stereomicroscopy was used to measure and record the number of atrial and ventricular contractions for 3 minutes. The results are expressed as average heart rate per minute.

Melanin cell count analysis

To count melanocytes, the embryos were exposed to indoor lamps to induce melanin contraction in melanocytes. Embryos were fixed in 4% paraformaldehyde and imaged using stereomicroscopy. The number of melanocytes in the head region of the embryo was measured.

Statistical analysis

Data were statistically evaluated by Student's t-test. P <0.05 was evaluated as statistically significant. All data are presented as mean ± standard deviation in three independent experiments.

Isoproxidine 7- O -(6'- O - p -Cumaroyl) -β-Glucopyranoside Isolation and Purification

The 23 fractions obtained by HPLC were treated with B16-F10 melanocytes to determine tyrosinase activity and melanin content to find novel compounds that modulate melanin synthesis. Treatment of two fractions, ACMF09 and ACMF13, significantly reduced melanin content and tyrosinase activity in melanocytes. In particular, treatment with fraction ACMF09 further reduced tyrosinase activity. This was further fractionated ACMF09 (69.3 mg) using silica gel column and reverse-phase HPLC column. The linear gradient solvent conditions used for HPLC separation were detected at 280 nm with a flow rate of 6.0 ml / min for a total of 70 minutes: water-methanol 60: 40 → 60: 40 (50 minutes) → 0: 100 (20 minutes). As a result, two kinds of compounds were obtained from ACMF09 (2.0 mg and 7.2 mg). 7.2 mg of compound was 4,5-O-dicafeolquinic acid, which reduced tyrosinase activity. On the other hand, 2.0 mg compound was purified using reverse-phase HPLC prepared from fraction ACMF09 (Figures 1 (a) and 1 (b)). Figure 1 (c) shows a chromatogram of the extracted compound maintaining a retention time of 54.429 minutes. Compounds were defined based on supplementary information obtained using an ESI-MS detector. Spectroscopic anaylsis,^OneH NMR) was used to confirm the structure of the isolate. 1 (d) and 1 (e) show the IR and UV spectra of the purified compounds. The compound is a light brown amorphous powder. Voice ion mode (m / z 529.1346 [M-H]^-. calcd for C₂₆H₂₆O₁₂Based on the HREI-MS spectra of the compound obtained from), the molecular formula of the compound is C₂₆H₂₆O₁₂It was confirmed. The compound was found to absorb UV at 300, 304 ALC 322 nm, hydroxyl group (3500 cm)^-One) And carbonyl (1675 cm)^-One), The IR absorption band was confirmed. The 1D NMR spectra of the compounds showed signals of coumarin moieties, p-coumaroyl moieties, and β-glucopyranose units. Compound is δ_H Two doublets at 7.64, δ_C Carbonyl ester by coumarin moiety was shown at 162.7 (C-2). In addition, the coupling constants (J= 9.0 Hz)_H Δ including hydroxyl methine signals from 3.40 to 3.55_H 5.20 (1 H, d,J= 7.8 Hz, H-1 ') From the anomer signal, the presence of one β-glucopyranose unit was confirmed. The signals of the p-coumaroyl moiety are δ_H 7.32 (2H, d,J= 8.7 Hz, H-2`` / H-6 '') and 6.80 (2H, d,J= 8.7 Hz, symmetric doublet of H-3`` / H-5 ''), δ_H 7.37 (1 H, d,J= 15.9 Hz, H-7``) and 6.10 (1H, d,JQuantum doublet showing trans-array at 15.9 Hz, H-8``). δ_C At 161.5 (C-4 '') and 127.1 (C-1 ''), the two aromatic carbon signals were transformed into two symmetric doublet quantum signals and the translocation of the p-coumaroyl moiety in the hetero nuclear multiple bond connectivity (HMBC) spectrum. It appeared to be associated with quantum doublets with the array. In addition, a wide range of HMBC correlations are shown with the anomer quantum (H-1 ') and δ._C Between the oxygenated aromatic carbons at 143.1 (C-7), δ_H 3.88 (3H, s) and δ with methoxy quantum signals at C-6 carbons_H It was observed between methoxy both in C-8 aromatic carbon at 3.99 (3H, s) (FIG. 1 (b)). Besides δ_H 4.39, 4.43 (H-6 ') and δ_C The two oxygenated methylene signals shifted down at 64.4 (C-6 ′) showed that the carbonyl ester of the p-coumaroyl moiety was attached to C-6 ′ of the glucose unit. From these results, the compound isolated from ACMF09 in the amount of 2.0 mg wasoproxidine 7-O-(6'-O-p-Coumaryl) -β-glucopyranoside (isofraxidin 7-O-(6'-O-p-coumaroyl) -β-glucopyranoside).

In B16-F10 melanocytes Isoproxidine  7- O -(6'- O - p - Kumaroyl ) -β- Of glucopyranoside  Melanin formation effect

In order to confirm that isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside modulates melanogenesis, the compound of the above and its concept in B16-F10 melanocytes The melanin formation effect of phosphorous isoproxidine (7-hydroxy-6,8-dimethoxycoumarin) was confirmed. Melanin inhibitor azelaic acid (AZelaic acid, AZ) was used as a control. Treatment with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside (Compound 1) on B16-F10 melanocytes stimulated melanin formation. 2 (a) and 2 (b)). In addition, the compound was found to significantly increase melanin formation in a dose dependent manner (FIG. 2 (c)). In addition, it was seen that tyrosinase activity was also dose dependently promoted in melanocytes (FIG. 2 (d)). In addition, isoproxidine was also found to significantly increase the amount of melanin formation in B16-F10 cells (Fig. 2 (c) and 2 (d)).

In general, the treatment of melanin compounds with B16-F10 cells is known to increase the secretion of melanin in the culture medium. In this experiment, melanocytes were also treated with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside (Compound 1), which is equivalent to IBMX, a common pigmentation inducer. As can be seen that the melanin secretion increased, the increase was significantly greater than the case of treatment with isoproxidine or forskolin (Fig. 3).

In melanocytes Isoproxidine  7- O -(6'- O - p - Kumaroyl ) -β- Of glucopyranoside MITF  And Tyrosinase  Up-regulation effect of expression level

Three regulatory genes involved in melanogenesis include microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1) and tyrosinase. have. When B16-F10 melanocytes were treated with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside for 48 hours, MITF and tyrosinase expression levels were not treated or It was confirmed that the remarkably increased compared with the treatment with isoproxidine (Fig. 4 (a) to 4 (c)).

Isoproxidine 7- O -(6'- O - p -Cumaroyl) -β-Glucopyranoside Increases Mushroom Type Tyrosinase Activity

To determine the mechanism by which isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside increases pigmentation in melanocytes, a cell-free mushroom tyrosinase assay is used. It was. Tyrosinase activity was significantly increased after 15 min incubation with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside. It was confirmed that there was no change in sinease activity (FIG. 5).

Zebrafish  Skin pigmentation, melanin content and Tyrosinase  Active Isoproxidine  7- O -(6'- O - p - Kumaroyl Effect of) -β-glucopyranoside

To assess the effect of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside on pigmentation in vivo, a novel regulator of pigmentation was A zebrafish larvae system, which is suitable for identification, was used. Phenylthiourea (PTU), known as a tyrosinase inhibitor, was used to evaluate whether the zebrafish system can be used as a model for confirming in vivo changes in pigmentation. The PTU was confirmed to inhibit melanin formation in a dose dependent manner (FIG. 6).

In addition, the zebrafish larval system was also used to determine whether isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside induces toxicity in mammals. . To determine the safety and effective concentrations of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside, lethality, a marker of toxicity in zebrafish embryos treated with the compound, Heart rate was evaluated. Untreated zebrafish embryos were treated with 75 μΜ PTU and isoproxidine and isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside, respectively, at 12.5 μΜ or 25 μΜ. It was confirmed that there is no change in heart rate compared to the case (Fig. 7). In addition, when 9hpf embryos were treated with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside, 90% or more embryos survived at the same level as untreated. Could see.

Based on these toxicological data, the effect of isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside on pigmentation in zebrafish at concentrations of 12.5 μΜ and 25 μΜ (A). Specifically, 40 9hpf zebrafish embryos were treated with 75μΜ PTU, or 12.5μΜ each of isoproxidine or isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside Or 25 μM. As a result, the amount of melanin was increased when isoproxidine and isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside were treated, especially isoproxidine 7- O. In the case of treatment with-(6'- O - p -coumaroyl) -β-glucopyranoside, the increase was higher than that with isoproxidine (Figs. 8 (a) and 8 (b). )).

On the other hand, tyrosinase activity was also evaluated in zebrafish larvae treated with the above compounds. As a result, the treatment with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside at concentrations of 12.5 μM and 25 μM was more effective than isoproxidine or PTU. It was confirmed that tyrosinase activity was significantly increased (Fig. 8 (c)).

Zebrafish  On pigmentation of melanocytes Isoproxidine  7- O -(6'- O - p - Kumaroyl ) -β- Glucopyranosides  Effect

To determine the effect of isoproxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside on the pigmentation of melanocytes, in zebrafish skin treated or untreated with the compound The development of melanocytes was measured. The zebrafish melanocytes develop from melanocyte germ cells at approximately 25 hpf, and larva pigment patterns are formed at 60 hpf. Zebrafish embryos treated with isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside showed an increase in the number of melanocytes compared to untreated controls. 9 (a), melanin cell area was also increased (Fig. 9 (b)).

Example 2

Example 2 was carried out to determine the melanin formation inhibitory activity of the cedars mugwort extract.

Preparation of Reagents

PTU, NaOH, DMSO (dimethyl sulfoxide ), L-DOPA (3,4-dihydroxyphenylalanine), CellLytic TM buffer, mushroom tyrosinase, MTT (3- (4,5-dimethylthiazol -2-yl) -2,5-diphenyltetrazolium bromide ) And tricaine methanesulfonate solution were purchased from Sigma (St Louis, MO, USA) and L-tyrosine was purchased from Duchefa Biochemie (Haarlem, Netherland). All experimental compounds were dissolved in DMSO and stored protected at -20 ° C until use. HPLC grade solution, acetonitrile and methanol were purchased from Merck (Darmstadt, Germany).

Preparation of plants

It is the same as the preparation of the plant of Example 1.

Cell culture

Murine melanoma B16-F10 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). It was incubated in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin mixture (Gibco, USA). Cultured cells were maintained at 37 ° C. and humidified conditions in a 5% CO ₂ incubator.

Determination of Melanin Content in B16-F10 Melanocytes

Melanocytes were washed with phosphate buffered saline (PBS) and lysed with CellLytic buffer at 4 ° C. The cell extract was spun at 13,000 rpm for 10 minutes at 4 ° C. The remaining pellet was washed twice with ethanol: ether (1: 1) for melanin analysis and dissolved in 200 μl of 1N NaOH in 10% DMSO at 80 ° C. Absorbance was measured at 400 nm using a microplate reader (VersaMax ™; Molecular Devices Corporation, California, USA) for 100 μl aliquot of the resulting solution.

Measurement of in-vitro tyrosinase activity

B16-F10 melanocytes were seeded in 6-well plates at a density of 2 × 10 ⁵ cells / well. Melanocytes were treated with 4,5-O-dicaffeoylquinic acid for 48 hours. Thereafter, melanocytes were lysed with lysis buffer and centrifuged at 13,000 rpm for 10 minutes. Each lysate containing the same amount of protein (250 μg) was placed in a 96-well plate and 100 μl of 5 mM L-DOPA was added to each well. After incubation at 37 ° C. for 60 minutes, dopachrome formation was measured at 475 nm using a microplate reader.

HPLC-based Activity Profiling of A. capillaris Thunberg Extract

The dry powders of the cedars and leaves were extracted with 100% methanol and then concentrated in vacuo to produce methanol extract (6 g). HPLC was performed on a H ₂ O concentration gradient system containing MeCN and 0.1% HCOOH using a YMC-PAC Pro C18 (10 mm, 250 mm, 5 μm) column in the Agilent HP1100 series. However, the Agilent HP1100 series consists of a degasser, a secondary mixing pump, a column oven and a DAD detector. For activity profiling, a portion (6 g) of the MeOG extract was converted to semi-preparative HPLC (Agilent 1100 Series, USA) into a gradient eluent system consisting of acetonitrile (MeCN) and water containing 0.1% formic acid, ie Fractionation was performed between 20% MeCN and 60% MeCN for 50 minutes. The mobile phase moved at a flow rate of 6.0 ml / min and the eluate was detected at 280 nm. A total of 23 fractions were obtained and concentrated to assess each biological activity. To obtain potential bioactive compounds, the ACMF09 fraction was selected on the basis of inhibiting melanin synthesis. Since the ACMF09 fractions from RP-18 column to further separate the components H ₂ O-MeOH 60:40: separated under gradient starting with (v v) it was kept constant for 50 minutes. The gradient system was then reduced to 0: 100 and held constant for 20 minutes to obtain purified compound (7.2 mg).

H-NMR Results of 4,5-O-Dicapeoylquinic Acid

¹ H-NMR (in CD ₃ OD, 600 MHz) δ 7.59 (1H, d, = 15.9 Hz, H-7 'or H-7 "), 7.51 (1H, d, = 15.9 Hz, H-7' or H -7 "), 7.02 (1H, d, = 1.8 Hz, H-2 'or H-2"), 7.00 (1H, d, = 1.8 Hz, H-2' or H-2 "), 6.92 (1H , dd, = 8.1,1.8 Hz, H-6 'or H-6 "), 6.90 (1H, dd, = 8.1,1.8 Hz, H-6' or H-6"), 6.75 (1H, d, = 8.1 Hz, H-5 'or H-5 "), 6.74 (1H, d, = 8.1 Hz, H-5' or H-5"), 6.28 (1H, d, = 15.9 Hz, H-8 'or H-8 "), 6.19 (1H, d, = 15.9 Hz, H-8 'or H-8"), 5.62 (1H, brs, H-5), 5.11 (1H, brs, H-4), 4.37 (1H, brs, H-3), 2.40 (2H, H-6), 1.99 (2H, m, H-2); ¹³ C-NMR (inCD ₃ OD, 125MHz) 168.7 (C-9 'or 9 "), 168.4 (C-9' or 9"), 149.8 (C-4 'or 4 "), 147.8 (C-7' or 7 "), 147.7 (C-7 'or 7"), 146.9 (C-3' or 3 "), 146.8 (C-3 'or 3"), 127.8 (C-1' or 1 "), 127.7 (C-1 'or 1 "), 123.3 (C-6' or 6"), 116.6 (C-5 'or 5 "), 115.3 (C-2' or 2"), 115.2 (C-2 'or 2 "), 114.8 (C-8 'or 8"), 117.7 (C-8' or 8 "), 75.8 (C-1), 75.7 (C-2), 69.8 (C-3), 68.6 (C -5), 39.7 (C-6), 38.5 (C-14); ESI-MS m / z 515.1 [M−H] − (C ₂₅ H ₂₄ O ₁₂ ).

Mushroom tyrosinase assay

Mushroom tyrosinase enzyme was dissolved in 50 mM potassium phosphate buffer (pH 6.5) at a concentration of 500 units / ml. 550 μl of 50 mM potassium phosphate and 50 μl of tyrosinase solution were mixed in an appropriate volume in a microfuse tube and incubated at room temperature for 5 minutes. 100 μL of 1.5 mM L-tyrosine was added to the solution and then loaded into a 96-well plate. The absorbance at 490 nm was measured using a microplate reader to determine the amount of dopachrome produced in the reaction mixture.

Quantitative Real-Time PCR Analysis

B16-F10 melanocytes were treated with azelaic acid (AZ) or 4,5-O-dicafeoylquinic acid for 48 hours. Total RNA was extracted using TRI-Solution ^™ and quantified using NanoDrop 2000 spectrophotometer (NanoDrop Technologies). CDNA was synthesized from 1 μg RNA using AccuPower PCR premix (Bioneer). MRNA expression levels of the MITF gene, tyrosinase gene and TRP-1 were measured using a Power SYBT Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized to β-actin and fold changes were calculated using the ΔΔCT method. Primer sequences are as follows: mouse tyrosinase forward 5′-TACTTGGAACAAGCCAGTCGTATC-3 ′, reverse 5′-ATAGCCTACTGCTAAGCC CAGAGA-3 ′; Mouse TRP-1 forward 5'-AAACCCATTTGTCTCCCAA-TGA-3 ', reverse 5'-CGTTTTCCAACGG-GAAGGT A-3'; Mouse MITF forward 5'-GGACTTTCCCTTATCCCATCCA-3 ', reverse 5'-GCCGAGGTTGTTGGTAAAG-GT-3'. After performing PCR for 2 minutes at 95 ° C., 40 cycles were performed at 95 ° C. for 30 seconds, at 60 ° C. for 1 minute, and at 72 ° C. for 1 minute, and at 72 ° C. for the last 30 seconds, the reaction was performed. It was. Data was analyzed using Stepone ^™ software v2.3 (Applied Biosystems).

Origin and Maintenance of Parental Zebrafish

Adult zebrafish were purchased from a commercial vendor and 10-15 zebrafish in each 5L acrylic tank were kept under the following conditions: 28.5 ° C., 14/10 hours day / night cycle. The zebrafish was fed with live brine shrimp (Artemia salina) twice a day for 7 days. Embryos were obtained from natural scattering induced at 9:30 in the morning by changing the cycle with light. Embryos were secured within 30 minutes.

Phenotype-based evaluation of test compounds using zebrafish

The zebrafish embryos were maintained in the embryonic medium of the petri dishes at a density of 70-80 embryos per 100 m ² of petri dishes. Simultaneously generated embryos were harvested and three pipettes were dispensed for each well of a 96-well plate using a pipette. However, 200 μl of the embryo medium containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl ₂ H ₂ O, and 0.33 mM MgSO ₄ 7H ₂ O per well was injected into the wells. Mugwort extract was dissolved in 0.1% DMSO and added to the embryo medium in an amount of 9 to 72 hpf (63 hours exposure). Stir from time to time for even distribution of test compounds and change medium every 24 hours. 75 μM PTU was used to form clear zebrafish in all experiments. Phenotypic-based evaluation of body pigments was performed at 72 hpf. Embryos were removed using forceps, anesthetized with tricaine methanesulfonate solution, and fixed in 3% methyl cellulose. Stereomicroscopy (LEICA DFC425C) was used to observe the effect on the pigmentation of zebrafish. Melanin pigmented areas were measured using the Image J program (National Institutes of Health, USA).

Evaluation of Melanin Content and Tyrosinase Activity in Zebrafish

Approximately 40 zebrafish embryos were treated with PTU, cedar extract, ACMF09 fraction or 4,5-O-dicafeylquinic acid at 9-48 hpf and sonicated using CellLytic buffer. The absorbance of the supernatant was measured at 400 nm to determine the melanin level. To assess tyrosinase activity, 100 μl of lysis buffer containing 250 μg total protein was transferred to a 96-well plate and then 100 μl of 5 mM L-3,4-dihydroxyphenylalanine (L-DOPA) was added. It was. Control wells contained 100 μl lysis buffer and 100 μl 5 mM L-DOPA. After incubation at 37 ° C. for 60 minutes, the absorbance was measured at 475 nm using a microplate reader. Blanks were removed at each absorbance value and the final activity was expressed as a percentage of the water control. PTU-treated embryos were used as positive controls.

Melanin cell count analysis

To count melanocytes, the embryos were exposed to indoor lamps to induce melanin contraction in melanocytes. Embryos were fixed in 4% paraformaldehyde and imaged using stereomicroscopy. The number of melanocytes in the head region of the embryo was measured.

Embryo Heart Rate Measurement

Atrial and ventricular heart rates were measured at 48 hpf to assess the toxicity of the compounds. Stereomicroscopy was used to measure and record the number of atrial and ventricular contractions for 3 minutes. The results are expressed as average heart rate per minute.

MTT assay for cell viability

B16-F10 melanocytes were seeded in 96-well plates for 12 hours at a concentration of 5 × 10 ³ cells / well. Cells were treated with azelaic acid, cedar extract, ACMF09 fraction or 4,5-O-dicafeylquinic acid for 48 hours. Thereafter, the survival rate of the cells was evaluated using the MTT assay.

Statistical analysis

Data were statistically evaluated by Student's t-test. P <0.05 was evaluated as statistically significant. All data are presented as mean ± standard deviation in three independent experiments.

Screening of Mugwort for Regulating Activity of Pigmentation Using Melanin Cell-based Screening System

In order to confirm the melanin formation regulating activity of cedar wormwood using murine melanin cells, methanol extracts of the aerial parts of wormwood were screened. Melanin pigment was treated with cedar extracts at different concentrations of 25 μg / ml and 50 μg / ml for 48 hours. Treatment of cedar extracts with B16-F10 melanin cells showed visually less dark than untreated cells. It can be seen that the melanin formation of the cell was reduced (Fig. 10 (a)). This could be confirmed again by observing weakly colored crude extracts of the treated cell pellets (FIG. 10 (b)). When the cedar extracts were treated at concentrations of 50 μg / ml and 25 μg / ml, the melanin formation rate was reduced to 91.92 ± 8.88% and 85.81 ± 10.12% compared to the untreated (Fig. 10 (c)). In order to evaluate the effect of cedar wormwood on melanin amount and tyrosinase activity, azelaic acid, known as a tyrosinase inhibitor, was used as a positive control. Although azelaic acid has been reported to be effective at concentrations of 40 mM and 20 mM in previous studies (Journal of Biomedical Science, vol. 17, supplement 1, article S45, 2010), B16-F10 when treated for 48 hours at the above concentrations Immediate separation was observed in more than 50% of melanocytes. In addition, 5 mM azelaic acid treatment was found to reduce the melanin content by 15-20% without imparting toxicity to melanocytes (Figs. 10 (a) to 10 (c)). After that, the tyrosinase inhibitory activity of cedars was evaluated as 87.82 ± 0.065% and tyrosinase activity was compared to when the cedar mugwort extract was treated with melanocytes at a concentration of 25 μg / ml or 50 μg / ml for 48 hours. It can be seen that the decrease to 75.68 ± 0.68% (Fig. 10 (d)).

Isolation of Anti-Melanin-forming Compounds from the Extracts of Fermented Artichoke

High performance liquid chromatography (HPLC) was performed to isolate the compound that inhibits melanin formation from the cedar extract. Twenty-three fractions of various weights were separated from the cedar extracts by activity-guided separation. Fractions were recovered, dried, dissolved in DMSO and evaluated for melanin formation inhibitory activity at the same concentration. All four fractions (ACMF09, ACMF13, ACMF14 and ACMF23) all showed melanin formation inhibitory activity. Thus, zebrafish embryos were used to confirm the pigmentation inhibition activity of these four magnetic fractions. The ACMF09 fraction showed an excellent melanin formation inhibitory effect and was selected for analysis in melanocytes of mammals. In melanocytes, ACMF09 fraction was found to reduce the pigmentation (Fig. 10 (a) and 10 (b)). In addition, the ACMF09 fraction was found to effectively inhibit melanin formation and tyrosinase activity (Fig. 10 (c) and 10 (d)). To isolate potential melanin forming regulatory compounds, ACMF09 fractions were fractionated on silica gel columns and reversed phase HPLC columns. The linear gradient solvent conditions applied for HPLC separation were H ₂ 0-methanol, started at 60:40 and remained constant for 50 minutes. The gradient system was then reduced to 0: 100 and remained constant for 20 minutes. The mobile phase was transferred at a moving speed of 6.0 ml / min and the eluate was detected at 280 nm. As a result, 4,5-O-dicaffeoylquinic acid (4,5-diCQA) was isolated through ¹ H-NMR and ESI-MS detection analysis (FIG. 11 (a)). And 11 (b)). The compound was observed as a yellow amorphous powder. Only one spot was observed at 280 nm UV. ¹ H- and ¹³ C-NMR spectra showed the presence of two caffeoyl moieties in the compound; 6 aromatic protons [δ _H 7.02 (1H, d, = 1.8 Hz, H-2 'or H-2 "), 7.00 (1H, d, = 1.8 Hz, H-2' or H-2"), 6.92 (1H, dd, = 8.1,1.8 Hz, H-6 'or H-6 "), 6.90 (1H, dd, = 8.1,1.8 Hz, H-6' or H-6"), 6.75 (1H, d, = 8.1 Hz, H-5 'or H-5 "), 6.74 (1H, d, = 8.1 Hz, H-5' or H-5")], trans doublets [δ _H 7.59 (1H, d, = 15.9 Hz, H-7 'or H-7 "), 7.51 (1H, d, = 15.9 Hz, H-7' or H-7"), 6.28 (1H, d, = 15.9 Hz , H-8 'or H-8 "), 6.19 (1H, d, = 15.9 Hz, H-8' or H-8")], two carboxyl groups (δ _C 168.7 and 168.4) and three hides Roxy groups (δ _C 149.8, 146.9 and 146.8). ¹ H- and ¹³ C-NMR spectra showed the presence of a quinic acid moiety in the compound; [δ _H 5.62 (1H, brs, H-5), 5.11 (1H, brs, H-4), 4.37 (1H, brs, H-3), 2.40 (2H, H-6), 1.99 (2H, m , H-2)]. The LC / UV / MS profile of the compound confirmed the UV absorption band at 328 mm, 292 mm and 245 mm and the ESI-MS [MH] ⁻ peak at m / z 515.11. These results showed that the structure of the compound was 4,5-O-dicafeoylquinic acid (Food Chemistry, vol. 120, no. 1, pp. 134-139, 2010). As a result of confirming the effect of 4,5-O-dicafeoylquinic acid on the pigmentation in mammalian melanocytes, it was confirmed that the pigmentation of melanocytes was reduced when treated with the compound through microscopic examination (Fig. 10 (a)). Cell pellets treated with the compound were found to be markedly brighter in color than the untreated control (FIG. 10 (b)). As a result of quantitative analysis, it was seen that the compound reduced melanin levels in melanocytes (FIG. 10 (c)). In addition, when the compound was treated with melanocytes at 50 μM, tyrosinase activity was reduced (FIG. 10 (d)).

Effects of ACMF09 Fraction and 4,5-O-Dicafeoylquinate on Mushroom Tyrosinase Activity in Cell-free Systems

To confirm whether 4,5-O-dicafeoylquinic acid directly inhibits the activity of tyrosinase enzyme, a cell-free mushroom tyrosinase assay was used. As a result, it was confirmed that 4,5-O-decaffeoyl quinic acid inhibited mushroom tyrosinase in a dose-dependent manner (FIG. 12). When the 4,5-O-dicafeoyl quinic acid was treated at a concentration of 50 µM, it was confirmed that the activity of the enzyme was inhibited by 32% compared to the untreated. Positive control (5 mM AZ) was shown to decrease the enzyme activity by 77%.

Effect of 4,5-O-Dicafeoylquinic Acid on Expression of MITF, Tyrosinase and TRP-1

To identify possible mechanisms of the pigmentation inhibitory effect of 4,5-O-dicafeoylquinic acid, three regulatory genes involved in melanogenesis are the microphthalmia-associated transcription factor (MITF) and tyrosine. The expression levels of tyrosinase-related protein-1 (TRP-1) and tyrosinase were confirmed using quantitative real-time PCR. As shown in Figure 4, it was confirmed that the mRNA level of tyrosinase was not related to the treatment of 4,5-O-dicafeyl quinic acid. However, it can be seen that the expression of TRP-1 was significantly reduced due to the treatment of the compound.

Zebrafish  Mugwort Extract from Vertebrate Model System, ACMF09  And 4,5-O- Decaf oil  Pigmentation inhibitory effect

Zebrafish is a noted animal model for pigmentation (Seminars in Cell and Developmental Biology, vol. 20, no. 1, pp. 90-104, 2009). Methanol extract and ACMF09 active fractions were tested in zebrafish larvae based in vivo system. As a well known pigmentation inhibitor, 1-phenyl 2-thiourea (PTU) inhibits tyrosinase activity, which was used as a positive control. However, at the 28-somite stage, PTU can slow hatching and have a lethal effect at 120 hpf. Thus, the effect on the pigmentation of zebrafish was confirmed by varying the concentration of PTU (25, 50, 75, 100 and 200 μΜ). As a result, when PTU was treated at a concentration of 75 μM, zebrafish pigmentation was reduced without adversely affecting fatality or malformation.

Methanol extract of A. capillaris, ACMF09 fraction, and 4,5-O-dicafeoylquinic acid were found to inhibit pigmentation in zebrafish (Fig. 14 (a)). In addition, depigmentation was observed in melanocytes by shrinking melanocytes (Fig. 14 (a)). In addition, it was confirmed that the cedar extract, ACMF09 fraction and 4,5-O- dicafeoyl quinic acid reduces the synthesis of melanin in a dose-dependent manner in zebrafish embryos (Fig. 14 (b)). In addition, partial inhibition of tyrosinase activity was also confirmed (Fig. 14 (c)), indicating that melanin formation in zebrafish was reduced by partial inhibition of tyrosinase activity of cells.

Zebrafish  4,5-O- for melanocyte survival in larvae head Decaf oil  effect

In zebrafish embryos, melanocytes begin to form melanin at approximately 24 hpf. There are approximately 460 melanocytes in approximately head, torso, tail and yolk sac that form a pattern of pigmentation at 60 hpf. Melanocytes were visualized in the head region of the entire embryo. 9hpf larvae were incubated with 25 μM of 4,5-O-dicafeoylquinic acid, and the number of melanocytes was lower than in untreated embryos (Fig. 15 (a)). A dose of 12.5 μM of 4,5-O-dicafeoylquinic acid did not affect the number of melanocytes. However, it was confirmed that the melanocyte area was significantly reduced at all doses treated with 4,5-O-dicafeoylquinic acid. For example, when 4,5-O-dicafeoyl quinic acid was treated at a dose of 25 μM, the area of melanocytes was reduced by 4 times compared to untreated embryos (FIG. 15 (b)).

In-vitro and In-Vivo vivo Cedar extract, ACMF09 Fraction  And 4,5-O- Decaf oil  Toxicity Assessment

To determine the effect of 4,5-O-dicafeoylquinic acid on melanogenesis, B16-F10 melanocytes were treated with various concentrations of 4,5-O-dicafeoylquinic acid and then cytotoxicity using MTT assay. Was evaluated. As shown in FIG. 16, even when treated with 4,5-O-dicafeoylquinic acid, the cell viability was not changed when compared with the untreated control group. It was found that the compound, 4,5-O-dicafeoylquinate, isolated from the cedar extract, was not cytotoxic to B16-F10 melanocytes. In order to identify the potential toxicity of cedar extract, ACMF09 fraction, and 4,5-O-dicafeylquinic acid in zebrafish, we observed external malformations, embryonic mortality, and heart rate impairment at 24 hpf, 48 hpf, and 72 hpf. Red and physiological changes could not be observed.

Claims (21)

1. A pharmaceutical composition for preventing or treating pigmented diseases, comprising an extract of Artemisia capillaris Thunberg as an active ingredient.
2. The method of claim 1,

   The extract is sacheolssuk isopropyl proxy Dean 7- O - (6'- O - p - Kumar In one) -β- glue nose Llano side (isofraxidin 7- O - (6'- O - p -coumaroyl) -β-glucopyranoside ) As a active ingredient, a pharmaceutical composition for preventing or treating pigmented diseases.
3. The method of claim 1,

   The cedar extracts include 4,5-O-dicafeoylquinic acid (4,5-O-dicaffeoylquinic acid) as an active ingredient, a pharmaceutical composition for preventing or treating pigmented diseases.
4. The method of claim 1,

   The cedar mugwort is one or more of the leaves, berries, bark and root of the cedar mugwort, pharmaceutical composition for the prevention or treatment of pigment diseases.
5. The method of claim 1,

   The cedar mugwort extract is that extracted from the cedar mugwort using an extraction solvent, a pharmaceutical composition for the prevention or treatment of pigment diseases.
6. The method of claim 5,

   The extraction solvent is at least one selected from the group consisting of water, ethanol, methanol, butanol, n-hexane, n-heptane and DMSO, pharmaceutical composition for preventing or treating pigmented diseases.
7. The method of claim 1,

   The pigment disease is low pigmented, including any one selected from the group consisting of vitiligo, albinism, depigmentation nevus, white nasal gangrene, stinging, post-inflammatory depigmentation, ecchymosis, partial leukemia, idiopathic red pigment hypopigmentation and pointy leukemia A pharmaceutical composition for the prevention or treatment of pigment diseases, which is a pigment disease due to deposition.
8. The method of claim 1,

   The cedar extract is contained in a concentration of 10 to 100㎍ / ml, pharmaceutical composition for the prevention or treatment of pigment diseases.
9. The method of claim 1,

   The pigment disease is a pigment disease due to hyperpigmentation, including any one selected from the group consisting of blemishes, freckles, senile plaques, blemishes, birthmarks and solar lentigines, for the prevention or treatment of pigment diseases Pharmaceutical composition.
10. Iso-proxy Dean 7- O - (6'- O - p - one to Kumar) -β- glue nose Llano side (isofraxidin 7- O - (6'- O - p -coumaroyl) -β-glucopyranoside) the active ingredient Included as, pharmaceutical composition for the prevention or treatment of pigment diseases.
11. The method of claim 10,

    The isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside is contained in a concentration of 10 to 100μΜ, the pharmaceutical composition for the prevention or treatment of pigment diseases.
12. The method of claim 10,

    The pigmented disease is low pigmented, including any one selected from the group consisting of vitiligo, albinism, depigmentation nevus, white nasal gangrene, stinging, post-inflammatory bleaching, ecchymosis, partial leukemia, idiopathic red hypopigmentation and pointy leukemia A pharmaceutical composition for the prevention or treatment of pigment diseases, which is a pigment disease due to deposition.
13. 4,5-O-dicaffeoylquinic acid (4,5-O-dicaffeoylquinic acid) as an active ingredient, a pharmaceutical composition for the prevention or treatment of pigment diseases.
14. The method of claim 13,

    The 4,5-O-dicafeoyl quinic acid is contained in a concentration of 5 to 100μΜ, the pharmaceutical composition for the prevention or treatment of pigment diseases.
15. The method of claim 13,

    The pigment disease is a pigment disease due to hyperpigmentation, including any one selected from the group consisting of blemishes, freckles, senile plaques, blemishes, birthmarks and solar lentigines, for the prevention or treatment of pigment diseases Pharmaceutical composition.
16. A cosmetic composition for preventing or improving pigment diseases, comprising an extract of Artemisia capillaris Thunberg as an active ingredient.
17. Iso-proxy Dean 7- O - (6'- O - p - one to Kumar) -β- glue nose Llano side (isofraxidin 7- O - (6'- O - p -coumaroyl) -β-glucopyranoside) the active ingredient Containing, cosmetic composition for the prevention or improvement of pigment diseases.
18. A 4,5-O-dicaffeoylquinic acid (4,5-O-dicaffeoylquinic acid) as an active ingredient, a cosmetic composition for preventing or improving pigment diseases.
19. Food composition for the prevention or improvement of pigment diseases, comprising the extract of Artemisia capillaris Thunberg as an active ingredient.
20. Iso-proxy Dean 7- O - (6'- O - p - one to Kumar) -β- glue nose Llano side (isofraxidin 7- O - (6'- O - p -coumaroyl) -β-glucopyranoside) the active ingredient Including, food composition for the prevention or improvement of pigment diseases.
21. 4,5-O-dicaffeoylquinic acid (4,5-O-dicaffeoylquinic acid) comprising as an active ingredient, a food composition for preventing or improving pigment diseases.

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