KR101865189B1 - Composition for preventing or treating pigmentary disorders caused by hypopigmentation - Google Patents

Abstract

The invention sacheolssuk of isopropyl proxy Dean 7- O extract or its active ingredients of (Artemisia capillaris Thunberg) - (6'- O - p - one to Kumar) -β- glue nose Llano side (isofraxidin 7- O - (6 ' -O - p- coumaroyl) -β-glucopyranoside, which is capable of preventing, ameliorating, and treating a pigment disease caused by hypopigmentation of skin as promoted by melanin formation.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating a pigment disease caused by low pigment deposition,

The present invention relates to a composition capable of preventing or treating a pigment disease caused by hypopigmentation of skin by promoting melanin formation.

Human skin is an organ that is exposed to various external environmental stresses, and there are various mechanisms to protect against potential damage by ultraviolet rays (UVR) and the like. Such defense mechanisms include DNA damage mechanisms, various enzymes such as catalase and peroxidase removal enzymes, and skin pigmentation. Among these factors, pigmentation is the most important photo-protective factor. Tyrosinase acts as an important regulator of pigmentation by promoting two rate - limiting reactions in the synthesis pathway of melanin pigment. These rate limiting reactions include 1) tyrosine hydroxylation to produce 3,4-dihydroxyphenylalanine (DOPA), and 2) DOPA oxidation to produce dopaquinone .

Hypopigmentation, on the other hand, is caused by a lack of melanin, which increases the incidence of skin cancer by 70 times or more at onset. Genetic hypopigmentation causes diseases such as albinism. In addition, hypochromic lesions can also create psychosocial problems by making exposed skin visually unsightly. Small molecules capable of up-regulating melanin biosynthesis are being studied as potential substances for treating hypochromic diseases and reducing UV-induced skin damage. Thus, various pigment-promoting substances having various photoprotective properties are known, for example, diacylglycerols, 3-isobutyl-1-methylxanthine (IBMX) and dimethyl sulfoxide Dimethylsulfoxide (DMSO) has been developed. However, these compounds have been challenged with serious side effects of the tendency to promote tumorigenesis. Therefore, in recent years, there is a demand for the development of a material which is superior in the effect of pigment deposition but is toxic and is economically effective.

It is an object of the present invention to provide a pharmaceutical composition which is excellent in promoting melanin formation and is capable of preventing or treating various diseases caused by hypochlorite deposition.

Another object of the present invention is to provide a cosmetic or food composition which is excellent in promoting melanin formation and is capable of preventing or improving various diseases caused by hypochlorite deposition.

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) -? - glucopyranoside) has an excellent effect of promoting melanin formation in melanocytes, leading to the present invention.

According to one embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating a pigment disease caused by hypochlorite deposition comprising an extract of Artemisia capillaris Thunberg as an active ingredient.

In the present invention, the black mugwort extract contains a large amount of isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside which has an excellent effect of promoting melanin formation in melanocytes, It is possible to effectively prevent or treat the pigment disease caused by the deposition.

In addition, in the present invention, the black mugwort can be a leaf, a fruit, a bark, a root, and the like, and preferably leaves can be used, but the present invention is not limited thereto.

In the present invention, in the method of extracting the black mugwort, all the methods capable of extracting from a simple extraction method to a lipid soluble component can be applied, and they can be obtained by grinding to facilitate extraction, extraction with an extraction solvent, filtration and concentration .

The extraction solvent may be water, ethanol, methanol, butanol, n-hexane, n-heptane or DMSO, and may be used as an extraction solvent by mixing two or more of these solvents. However, Amount, extraction method and the like, and those skilled in the art can appropriately select from known methods. The extraction time and temperature may be appropriately selected from those known to those skilled in the art in consideration of extraction efficiency, extraction solvent and the like. The preferred extraction time is 10 minutes to 1 hour, and the preferred extraction temperature is 40 to 100 占 폚.

In addition, the extract extracted by the above-mentioned extraction method can be filtered by a known filtration method such as vacuum filtration and then concentrated by distillation or the like. Such extraction, filtration and concentration methods are well known in the art, and one skilled in the art can appropriately select this to make bank extracts.

 In addition, in the present invention, the pigment disease caused by hypochromatosis may be all diseases caused by loss of melanocytes or melanin production due to inhibition of melanogenesis, It may be albinism, decolorizing nevus, white nasolacia, pestilence, post-inflammatory decolorization, anti-scleroderma, partial albinism, idiopathic hypochloremia, or papillary albinism.

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 ' -O - p- coumaroyl) -? - glucopyranoside as an active ingredient. The present invention relates to a pharmaceutical composition for the prevention or treatment of pigment diseases caused by hypochlorite deposition.

[Chemical Formula 1]

In the present invention, the isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside may be isolated from the safflower extract of the present invention, Or may be purchased and used on the market, and there is no particular limitation on the source.

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

In the present invention, the pigment disease is a disease caused by hypochromatosis, which may be caused by loss of melanocyte cells or melanin production inhibition, resulting in melanin production not reaching a normal level. For example, , Albinism, decolorizing nevus, white nasolacrimal gland, pestilence, post-inflammatory decolorization, anti-scleroderma, partial albinism, idiopathic hypochloremia, or papillary albinism.

In the present invention, the term "prevention" in the present invention can include, without limitation, any action that inhibits the symptoms of a pigment disease caused by hypochlorite deposition or inhibits or delays the symptom using the pharmaceutical composition of the present invention.

In the present invention, "treatment" can be used without limitation as long as the symptom of the pigment disease caused by hypochromatosis is improved or improved by using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be a human.

The pharmaceutical composition of the present invention may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterilized injection solutions according to a conventional method, have. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The route of administration of the pharmaceutical compositions according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Sublingual or rectal. Oral or parenteral administration is preferred.

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

The pharmaceutical composition of the present invention varies depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated. And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and may be 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

According to another embodiment of the present invention, there is provided a cosmetic composition for prevention or improvement of a pigment disease caused by hypochromatosis, which comprises an extract of Rhodochrosite as an active ingredient.

According to another embodiment of the present invention, there is provided a method for producing a colorant, which comprises isoproxcin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside as an active ingredient, And to a cosmetic composition for preventing or improving diseases.

The description of the safflower extract and the isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside in the cosmetic composition of the present invention is the same as that described in the above pharmaceutical composition , The detailed description thereof will be omitted.

In the present invention, the term "improvement" in the present invention can be used without limitation as long as the symptom of the pigment disease caused by low pigment deposition is improved or changed by using the pharmaceutical composition of the present invention.

The cosmetic composition according to the present invention may be used in cosmetics, nutritional lotions, nutritional essences, massage creams, cosmetic bath additives, body lotions, body milks, bath oils, baby oils, baby powders, shower gels, shower creams, sunscreen lotions, Face and Body Cream, Face and Body Cream, Skin Whitening Cream, Hand Lotion, Hair Lotion, Cosmetic Cream, Jasmine Oil, Face Cream, Skin Cream, Skin Cream, Sunscreen Cosmetics, Bath soap, water soap, soap, shampoo, hand cleanser, medicinal soap {non-medical use}, cream soap, facial wash, whole body cleanser, scalp cleaner, hair rinse, cosmetic soap, tooth whitening gel, toothpaste . ≪ / RTI > To this end, the composition of the present invention may further comprise a solvent commonly used in the production of a cosmetic composition, or a suitable carrier, excipient or diluent.

For example, water, saline solution, DMSO, or a combination thereof may be used. Examples of the carrier, excipient or diluent include purified water, oil, wax But are not limited to, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols and the like. Further, if necessary, it may contain a whitening agent, a moisturizing agent, a vitamin, an ultraviolet screening agent, a perfume, a dye, an antibiotic, an antibacterial agent, and an antifungal agent.

As the oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil and avocado oil may be used. Examples of the wax include wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, Can be used.

As the fatty acid, stearic acid, linoleic acid, linolenic acid and oleic acid may be used. As the fatty acid alcohol, cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol and hexadecanol may be used As fatty acid esters, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, a cationic surfactant, an anionic surfactant and a nonionic surfactant known in the art can be used, and a surfactant derived from a natural material is preferably used.

In addition, it may contain a hygroscopic agent, a thickening agent, an antioxidant and the like widely known in the field of cosmetics, and the kind and amount thereof are well known in the art.

According to another embodiment of the present invention, there is provided a food composition for prevention or improvement of a pigment disease caused by hypochromatosis, which comprises an extract of a black mugwort as an active ingredient.

According to another embodiment of the present invention, there is provided a method for producing a colorant, which comprises isoproxcin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside as an active ingredient, To food compositions for preventing or ameliorating diseases.

The description of the safflower extract and the isoprochidin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside in the food composition of the present invention is the same as that described in the above pharmaceutical composition , The detailed description thereof will be omitted.

The food composition containing the composition of the present invention as an active ingredient may be prepared in the form of various foods such as beverage, gum, tea, vitamin complex, powder, granule, tablet, capsule, . Since the food composition of the present invention is composed of a plant extract having little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes.

When the composition of the present invention is contained in the food composition, the amount thereof may be added in a proportion of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in a beverage form, there are no particular limitations other than those containing the food composition at the indicated ratios and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and sugar sugars such as polysaccharide, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol can do. Examples of the above-mentioned flavors include natural flavors (such as tau martin and stevia extract (for example, rebaudioside A and glycyrrhizin) and synthetic flavors (for example, saccharine and aspartame).

In addition, the food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like.

These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally 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 of the present invention is excellent in promoting melanin formation without causing toxicity to human body, and can effectively prevent, ameliorate, and treat various diseases caused by hypochromatosis.

Figure 1 (a) shows the chemical structure of isoproxidine 7- O- (6'- O - p -coumaroyl) - [beta] -glucopyranoside isolated from Sapporo extract.
FIG. 1 (b) shows the significant HMBC correlation in isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside isolated from the black mugwort extract.
1 (c) shows HPLC chromatogram results of isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside at 280 nm.
1 (d) shows the infrared (IR) spectrum analysis of isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside.
FIG. 1 (e) shows the absorption spectrum (UV) spectrum of isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside.
FIG. 2 (a) is a graph showing the results of immunoprecipitation of B16-F10 melanocytes with azelaic acid (AZ), isofraxidin or isoproxidine 7- O- (6'- O - p -coumaroyl) (Scale bar = 20 탆) of the cell image after the treatment of the compound 1 with a digital camera (magnification of 100X).
FIG. 2 (b) is a graph showing the results of the treatment of B16-F10 melanocytes with cells treated with azelaic acid, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) Of the pellet of the present invention.
FIG. 2 (c) is a graph showing the results of the treatment of B16-F10 melanocytes with cells treated with azelaic acid, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) It is a graphical representation of changes in myelinin content.
Figure 2 (d) shows the results of treatment of melanocytes B16-F10 with azelaic acid, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside It is a graphical representation of the change in synease activity.
FIG. 3 is a graph showing the effect of B16-F10 melanocytes on isoprocedil, isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside, forskolin, And the change in the content of melanin secreted from the cells to the control group.
Figure 4 (a) shows the expression level of MIFT after treatment with B16-F10 melanocytes with isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside or IBMX As shown in FIG.
FIG. 4 (b) is a graph showing the fluorescence intensity of B16-F10 melanocytes treated with isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside, A graphical representation of changes in expression levels.
FIG. 4 (c) is a graph showing the results of the binding of B16-F10 melanocytes to isoproxidine, isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside, A graphical representation of changes in expression levels.
FIG. 5 is a graph showing the effect of tyrosine on melanocytes; Tyrosinase; Tyrosine and tyrosinase; Tyrosine, tyrosine and isoproxidine; Tire tyrosinase, tyrosine and iso proxy Dean 7- O - (6'- O - p - Kumar days) -β- glue nose Llano side; shows a change in absorbance at 490nm and then treated with a graph.
FIG. 6 is a graph showing a photograph of a zebrafish embryo taken by a stereomicroscope (magnification of 100X) after treating PTU by concentration in a graph (scale bar = 20 μm).
FIG. 7 shows the results of treatment of zebrafish embryos with different concentrations of PTU, isoflactidine and isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside The change is a graphical representation.
Figure 8 (a) shows the results of treatment of zebrafish embryos with PTU, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside at different concentrations (Scale bar = 20 탆) taken by a post-stereomicroscope (magnification of 100X).
Figure 8 (b) shows the results of treatment of zebrafish embryos with PTU, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside at different concentrations The graph shows changes in the content of postmelanin.
Figure 8 (c) shows the results of treatment of zebrafish embryos with PTU, isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside at different concentrations The graph shows the change in the activity of posttrosinase.
9 (a) shows the results of treatment of zebrafish embryos with different concentration of PTU or isoproxcin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside, As shown in FIG.
FIG. 9 (b) is a graph showing the effect of the PTU or isoprochidane 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside in zebrafish embryos at different concentrations, The graph shows the change in area.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

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 (using tetramethylsilane (TMS) as an international reference) using a Varian VNMRS 600 MHz NMR spectrophotometer (Varian, Inc., USA) . The chemical shifts ( δ ) are expressed in parts per million (ppm) and the coupling constants ( J ) in Hz. The weight spectra were measured using a JMS-700 (Jeol, Japan) and Varion 1200, Platform II (Varian, USA) spectrophotometer. Mass spectrometry (ESI-MS) and LCMS / MS spectra were measured using a negative electrospray ion mode with Water Synampt High Definition Mass Spectrometry / Time of a Flight mass spectrometer.

Preparation of plants

Leaves and stems of Astemisia capillaris Thunberg were obtained from professor Imsoonho of Dongshin University (Naju, Korea).

Preparation of reagents

(Such as IBMX, forskolin, phenylthiourea (PTU), NaOH, dimethyl sulfoxide, 3,4-dihydroxyphenylalanine, 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.). The cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco, USA). The cultured cells were maintained in a 5% CO ₂ incubator at 37 ° C and humidified conditions.

B16-F10 Measurement of melanin content in melanocytes

Melanocytes were inoculated into 6-well plates at a density of 2 X 10 ⁵ cells / well and treated for 24 hours with compounds for 48 hours. Melanocytes were washed with phosphate buffered saline (PBS) and lysed in CellLytic buffer at 4 ° C. Cell extracts were 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 1 N NaOH in 10% DMSO at 80 ° C. The absorbance of the obtained solution was measured at 400 nm using a microplate reader (VersaMax (Molecular Devices Corporation, California, USA) for 100 앨 aliquots.

Measurement of melanin secreted in culture medium

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

Measurement of tyrosinase activity in melanocytes

The B16-F10 melanocytes in 6-well plates were seeded at a density of 2 X 10 ⁵ cells / well. After 24 hours, the compounds were treated with melanocytes for 48 hours. Cells were washed with PBS and lysed with CellLytic buffer at 4 ° C. Cell extracts were spun at 13,000 rpm for 10 minutes at 4 ° C. Protein concentration was adjusted using the Bradford assay, and 100 [mu] l cell lysate containing 40 [mu] g protein was transferred to 96-well plates. Then, 100 쨉 l of 5 mM L-DOPA was added, followed by incubation at 37 째 C for 60 minutes. Dopachrome formation was measured at 475 nm using a microplate reader.

Analysis of mushroom type tyrosinase

The mushroom tyrosinase enzyme was dissolved in 50 mM potassium phosphate buffer (pH 6.5) at a concentration of 500 units / ml. 550 [mu] l of 50 mM potassium phosphate and 50 [mu] l of tyrosinase solution were mixed in a microfuse tube in an appropriate volume and incubated at room temperature for 5 minutes. 100 [mu] L of 1.5 mM L-tyrosine was added to the solution and loaded onto a 96-well plate. Absorbance was measured at 490 nm using a microplate reader to determine the amount of dopa chromium produced in the reaction mixture.

HPLC-based active profiling of A. cappilaris Thunberg extract

Dried powder of rhizome leaves and stems was extracted with 100% methanol and concentrated in vacuo to produce methanol extract (6 g). HPLC was performed on a H ₂ O gradient system containing MeCN and 0.1% HCOOH using a YMC-PAC Pro C18 (10 mm, 250 mm, 5 μm) column on an Agilent HP1100 series. However, the Agilent HP1100 series is composed of a degasser, a binary mixing pump, a column oven and a DAD detector. For active profiling, a portion of the MeOH extract (6 g) was purified on a preparative HPLC (Agilent 1100 Series, USA) starting with 80% H ₂ O containing 0.1% HCOOH / 20% MeCN and eluting with 0.1% HCOOH / 60% MeCN Lt; RTI ID = 0.0 > 40% < / _RTI > The mobile phase migrated 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 of pigmentation on melanocytes of each fraction was evaluated. As a result, the ACMF09 fraction was selected and kept in a RP-18 column for further separation, separated under a gradient starting with 60:40 (v: v) H ₂ O-MeOH and kept constant for 50 minutes. The gradient system then decreased to 0: 100 and was kept constant for 20 minutes to obtain the 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 proxy Dean isopropyl 7- O - (6'- O - p - Kumar In one) -β- glue nose Llano side is a brown amorphous powder, UV, IR, ¹ H and ¹³ C NMR data are as follows, and in particular ¹ H and ¹³ C NMR data are shown in Table 1 below (600 MHz, methanol- d ₄ ): UV? _Max (H ₂ O) (log ε) 300, 304, 322 nm; IR (KBr) [lambda] _max : 3500, 1675, 1011 cm < ^{-1 &} gt ;; _{^{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 = 9.6Hz, 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 = , 8-OMe), 3.88 (3H, s, 6-OMe), 3.55 (1H, dd, J = 9.0, 7.5 Hz, H-2 '), 3.50 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"), C-12), 117.1 (C-4), 144.0 (C-8a), 143.1 (C-7), 142.7 (C-3), 115.9 (C-4), 115.9 (C-3) (C-5 '), 75.7 (C-2'), 72.2 (C-4 '), 64.4 OMe); HRESI-MS m / z 529.1346 [MH] ^- (C ₂₆ H ₂₆ O ₁₂ ).

Position 隆 _H ( J in Hz) δ _C HMBC ^a 2 - 162.7 3 6.16 1H, d (9.6) 115.7 C-2, 4a 4 7.64 1H, 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 1H, 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 1H, d (15.9) 146.5 C-1, 2/6, 9 8 6.10 1H, 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( Quantitative Real - Time PCR 분석 )

B16-F10 melanocytes were treated with isofraxidin, isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside and IBMX for 48 hours. Total RNA was extracted using TRI-Solution ^TM and quantified using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). CDNA was synthesized from 1 占 퐂 RNA using AccuPower 占 PCR premix (Bioneer). MRNA expression levels of MITF gene, tyrosinase gene and TRP-1 were measured using Power SYBT Green PCR Master Mix (Applied Biosystems). mRNA levels were normalized to β-actin and fold changes were calculated using the ΔΔ CT method. The primer sequences are: 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'. PCR was carried out at 95 ° C for 2 minutes, followed by 40 cycles of 95 ° C for 30 seconds, 60 ° C for 1 minute and 72 ° C for 1 minute, and extension at 72 ° C for 30 seconds Respectively. Data were analyzed using Stepone ^TM software v2.3 (Applied Biosystems).

For cell viability MTT  analysis

B16-F10 melanocytes were inoculated into 96-well plates at a concentration of 5 X 10 ³ cells / well for 12 hours. Cells were treated with mugwort extract or isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside for 48 hours. The survival rate of the cells was then evaluated using MTT assay.

Zebra fish  maintain

Adult zebrafish ( Danio rerio ) was purchased at a regular store (Lotte Mart, Korea). Ten to fifteen zebrafishs were kept in each of the 5 L acrylic tanks and the following conditions were maintained: 28.5 ° C, day / night cycle of 14/10 hours. Twice a day for seven days, a live brine shrimp (Artemia salina) was fed to the zebrafish. At 9:30 am, the embryos were obtained from the natural spawning induced by changing the light cycle. Securing the embryo was done within 30 minutes.

Zebra Fish  Phenotype-based evaluation of test compounds used phenotype - based  evaluation)

The zebrafish embryos at a density of between 70 and 80 embryos per petri dish of 100m ² was maintained in the embryo culture medium of the Petri dish. The co-generated embryos were harvested and dispensed three embryos per well of a 96-well plate using a pipette. However, 200 쨉 l of an 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 was injected into each well. Sacheolssuk extract or iso proxy Dean 7- O - (6'- O - p - one to Kumar) -β- glue the nose side pyrano was added in an amount of 9 to 72hpf embryos after the medium was dissolved in 0.1% DMSO (63 Time exposure). Stir occasionally for even distribution of the test compound and replace the medium every 24 hours. In all experiments 75 μM PTU was used to form transparent zebrafish. Expression - based evaluation of body pigment was performed at 72 hpf. The embryonic membranes were removed using forceps, anesthetized with a tricine methanesulfonate solution, and fixed in 3% methylcellulose. We observed the effect of zebrafish on pigmentation using stereo microscopy (LEICA DFC425C). The melanin pigment area was 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 isoprochidin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside and sonicated 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 quantitated using the Bradford assay. 100 μl of a dissolution buffer containing 250 μg of 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 [mu] l dissolution buffer and 100 [mu] l 5 mM L-DOPA. After incubation at 37 ° C for 60 minutes, the absorbance was measured at 475 nm using a microplate reader. The blank was removed at each absorbance and the final activity was expressed as a percentage of the water control. The melanin content in zebrafish was measured using the following method. Specifically, the protein extract was prepared at 48 hp using the same methodology as tyrosinease assay. After centrifugation, the pellet was recovered and dissolved in 200 μl of 1N NaOH with 10% DMSO for 60 minutes and vortexed. Absorbance was measured at 475 nm using a microplate reader.

Measure heart rate of zebrafish

To evaluate toxicity of compounds, heart rate of atria and ventricles were measured at 48hpf. The number of atrial and ventricular contractions was measured and recorded using a Zeiss Stemi 2000-C stereo microscope for 3 minutes. The results are expressed as mean heart rate per minute.

Melanocyte counting analysis

To quantify melanocytes, the embryos were exposed to indoor light to induce the contraction of melanin in melanocytes. The embryos were fixed in 4% paraformaldehyde and imaged using stereo microscopy. The number of melanocytes in the head area of the embryo was measured.

Statistical analysis

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

From black mugwort extract Isoproxidine  7- O - (6'- O - p - Kumaroto ) -? - Glucopyranos Separation and purification of the side

The 23 fractions obtained by the HPLC were treated with B16-F10 melanocytes to determine tyrosinase activity and melanin content to discover melanin synthesis. When the two fractions, ACMF09 and ACMF13 were treated, melanin content and tyrosinase activity were significantly decreased in melanocytes. In particular, the activity of tyrosinase was further reduced when the fraction ACMF09 was treated. The ACMF09 (69.3 mg) was further fractionated using a silica gel column and a 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 with 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 the compound was 4,5-O-dicapheolquinic acid, which reduces tyrosinase activity. On the other hand, the 2.0 mg compound was purified using reverse-phase HPLC prepared from fraction ACMF09 (Fig. 1 (a) and 1 (b)). Figure 1 (c) shows the chromatogram of the extracted compound with a retention time of 54.429 min. Compounds were defined based on supplemental information obtained using ESI-MS detectors. The structure of the separated product was confirmed by spectroscopic analysis ( ¹ H NMR). Figures 1 (d) and 1 (e) show the IR and UV spectra of the purified compound. The compound is a pale brown amorphous powder. Based on the HREI-MS spectra of the compound obtained from negative ion mode ( m / z 529.1346 [MH] ^- . Calcd for C ₂₆ H ₂₆ O ₁₂ ), the molecular formula of the compound was identified as C ₂₆ H ₂₆ O ₁₂ . The compound was found to absorb UV at 300, 304 ALC 322 nm and the IR absorption band could be confirmed at the portion corresponding to the hydroxyl group (3500 cm ^-1 ) and the carbonyl group (1675 cm ^-1 ). The 1D NMR spectra of the compounds showed signals of coumarin moiety, p-coumaroyl moiety, and beta -glucopyranose units. Compound showed a doublet two (doublets) showed a, carbonyl ester of thienyl coumarin moiety at δ _C 162.7 _(C-2) at δ _H 7.64. In addition, δ _H containing hydroxyl methine signal with coupling constant (J = 9.0 Hz) between adjacent two axes hydrogen at δ _H 3.40 ~ 3.55 5.20 (1H, d, J = 7.8 Hz, H-1 ') The presence of one β-glucopyranose unit from the anomer signal was confirmed. signal of one moiety to Kumar are p- _{δ H 7.32 (2H, d,} J = 8.7 Hz, H-2 '' / H-6 '') and 6.80 (2H, d, J = 8.7 Hz, H-3 and symmetrical doublet of '' / H-5 '' ), δ H 7.37 (1H, d, J = 15.9 Hz, H-7 '') and 6.10 (1H, d, J = 15.9 Hz, H-8 '') As a quantum doublet showing the trans-arrangement. δ _C The two aromatic carbon signals at 161.5 (C-4 '') and 127.1 (C-1 '') are the two symmetrical DoubleRet quantum signals and the trans nuclear of the p-coumaryl moiety in the HMBC spectrum - array with quantum doublet. In addition, the broad range of HMBC correlations is related to the anomeric proton (H-1 ') and δ _C Methoxy quantum signals at δ _H 3.88 (3H, s) and C-6 carbon and methoxy quantities at C-8 aromatic carbon at δ _H 3.99 (3H, s) between the oxygenated aromatic carbons at 143.1 (C- (Fig. 1 (b)). Furthermore, from the two oxygenated methylene signals shifted down in δ _H 4.39, 4.43 (H-6 ') and δ _C 64.4 (C-6'), the carbonyl ester of the p-coumaroyl moiety reacted with C- ', Respectively. The ACMF09 from Through this separation results in an amount of 2.0mg compound iso proxy Dean 7- O - (6'- O - p - Kumar In one) -β- glue nose Llano side (isofraxidin 7- O - (6 ' -O - p- coumaroyl) -? - glucopyranoside).

B16-F10 in melanocytes Isoproxidine  7- O - (6'- O - p - Kumaroto ) -? - Glucopia Melanin Formation Effect of Ranoside

To confirm that isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside regulates melanogenesis, B16-F10 melanocytes The effect of isofraxidin (7-hydroxy-6,8-dimethoxycoumarin) on melanin formation was confirmed. The melanin inhibitor, azelaic acid (AZ), was used as a control. Melanin formation was stimulated when B16-F10 melanocytes were treated with isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside (Compound 1) 2 (a) and 2 (b)). In addition, it was found that the compound remarkably increased melanin formation in a dose-dependent manner (Fig. 2 (c)). In addition, tyrosinase activity in melanocytes was also stimulated in a dose-dependent manner (Fig. 2 (d)). In addition, it was confirmed that isoproxidine also significantly increased the amount of melanin formation in B16-F10 cells (Fig. 2 (c) and 2 (d)).

It is generally known that when melanin compounds are treated with B16-F10 cells, the secretion of melanin increases in the culture medium. In this experiment, melanocytes were also treated with isoproxidine 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside (Compound 1) to produce an equivalent level of IBMX , And the amount of melanin secretion was increased. The increase of the amount of melanin was remarkably larger than that of isoproxidine or Forskolin treatment (FIG. 3).

In melanocytes Isoproxidine  7- O - (6'- O - p - Kumaroto ) -? - Glucopyranosyl De MITF  And Tyrosine  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) -? - glucopyranoside for 48 hours, the expression levels of MITF and tyrosinase were untreated (Fig. 4 (a) to Fig. 4 (c)).

Isoproxidine  7- O - (6'- O - p - Kumaroto ) -? - Glucopyranoside Mushroom type Tiro Cinease activity increase effect

Isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside To confirm the mechanism of increasing pigmentation in imelanin cells, a cell-free mushroom-type tyrosinase is used Respectively. The activity of tyrosinase was remarkably increased after treatment with isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside and incubation for 15 minutes, but when isoproxidine was treated, It was confirmed that there was no change in synechia activity (Fig. 5).

Zebra fish  Skin pigmentation, melanin content and Tyrosinase  On active Isoproxy Dean-7- O - (6'- O - p - Kumaroto ) -? - Glucopyranoside  effect

To evaluate the effect of isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside on pigmentation in vivo, a novel modulator of pigmentation A zebrafish larvae system, suitable for identification, was used. Phenylthiourea (PTU), known as a tyrosinase inhibitor, was used to evaluate whether a zebrafish system could be used as a model to identify in vivo changes in pigmentation. It was confirmed that the PTU inhibited melanin formation in a dose-dependent manner (FIG. 6).

The zebrafish larval system was also used in this experiment to determine whether isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside causes toxicity to mammals . In order to confirm the safety and effective concentration of isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside, the lethal rate as a marker of toxicity in zebrafish embryos treated with the compound The heart rate was evaluated. Treatment of zebrafish embryos with 75 μM of PTU and 12.5 μM or 25 μM of isoproxidine and isoproxcin 7- O- (6'- O - p -coumaroyl) - β-glucopyranoside (Fig. 7). As shown in Fig. In addition, when 9-hpf embryos were treated with isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside, at least 90% of embryos survived to the same level I could see that.

Based on this toxicity data, the effect of isoproxcin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside on pigmentation in zebrafish was evaluated at concentrations of 12.5 μM and 25 μM (Fig. 8 (a)). Specifically, treatment with 75 μM PTU in 40 9hpf zebrafish embryos or treatment with 12.5 μM each of isoproxidine or isoproxydine 7- O- (6'- O - p -coumaroyl) - β-glucopyranoside Or 25 [mu] M. As a result, the isobutane and isobutylene proxy proxy Dean Dean 7- O - (6'- O - p - Kumar In one) -β- glue nose when the handle side pyrano increased amount of melanin formation, especially isobutyl proxy Dean 7- O - (6'- O - p -coumaroyl) -? - glucopyranoside was found to be higher than in the case of isoproxidine treatment (FIGS. 8 (a) and 8 )).

On the other hand, tyrosinase activity was also evaluated in zebrafish larvae treated with the above compounds. As a result, when isoprocedin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside was treated at a concentration of 12.5 μM and 25 μM, It was confirmed that tyrosinase activity was significantly increased (Fig. 8 (c)).

Zebra fish  On the pigmentation of melanocytes Isoproxidine  7- O - (6'- O - p - Kumaro Yl) -? - glucopyranoside

In order to confirm the effect of isoproxcin 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside on pigmentation of melanocytes, the compound was treated or untreated on zebrafish skin The development of melanocytes was measured. Melanocytes in zebrafish develop from melanocyte cells at approximately 25 hpf, forming a larval pigment pattern at 60 hpf. Treatment of zebrafish embryos with isoproxidine or isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside resulted in an increase in the number of melanocytes compared to the untreated control (Fig. 9 (a)), indicating that the melanocyte cell area also increased (Fig. 9 (b)).

Claims (13)

O- (6'- O - p -coumaroyl) -? - glucopyranoside (isofraxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside as an active ingredient, which is a pharmaceutical composition for preventing or treating a pigment disease caused by hypochromatosis,
The pigment diseases caused by the hypochromatosis include, but are not limited to, vitiligo, albinism, decolorization nodule, white nasolacrimal duct, hypochromatosis due to papillary thrush, post-inflammatory decolorization, hypopigmentation due to scleroderma, partial albinism, Wherein the pharmaceutical composition is any one selected from the group consisting of paclitaxel and paclitaxel. delete The method according to claim 1,
Wherein the safflower is at least one of leaves, fruits, bark and roots of black tea mugwort. The method according to claim 1,
Wherein the extract of the black mugwort is extracted from the black mugwort using an extraction solvent. 5. The method of claim 4,
Wherein the extraction solvent is at least one selected from the group consisting of water, ethanol, methanol, butanol, n-hexane, n-heptane and DMSO. delete 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 A pharmaceutical composition for prevention or treatment of a pigment disease caused by hypochromatosis,
The pigment diseases caused by the hypochromatosis include, but are not limited to, vitiligo, albinism, decolorization nodule, white nasolacrimal duct, hypochromatosis due to papillary thrush, post-inflammatory decolorization, hypopigmentation due to scleroderma, partial albinism, Wherein the pharmaceutical composition is any one selected from the group consisting of paclitaxel and paclitaxel. 8. The method of claim 7,
Wherein said isoproxidine 7- O- (6'- O - p -coumaroyl) -? - glucopyranoside is included at a concentration of 10 to 100 μM. delete O- (6'- O - p -coumaroyl) -? - glucopyranoside (isofraxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside as an active ingredient, which is a cosmetic composition for prevention or improvement of a pigment disease caused by hypochromatosis,
The pigment diseases caused by the hypochromatosis include, but are not limited to, vitiligo, albinism, decolorization nodule, white nasolacrimal duct, hypochromatosis due to papillary thrush, post-inflammatory decolorization, hypopigmentation due to scleroderma, partial albinism, Wherein the cosmetic composition is any one selected from the group consisting of paclitaxel and paclitaxel. 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 , Which is a cosmetic composition for prevention or improvement of a pigment disease caused by low pigment deposition,
The pigment diseases caused by the hypochromatosis include, but are not limited to, vitiligo, albinism, decolorization nodule, white nasolacrimal duct, hypochromatosis due to papillary thrush, post-inflammatory decolorization, hypopigmentation due to scleroderma, partial albinism, Wherein the cosmetic composition is any one selected from the group consisting of paclitaxel and paclitaxel. O- (6'- O - p -coumaroyl) -? - glucopyranoside (isofraxidin 7- O- (6'- O - p -coumaroyl) -β-glucopyranoside as an active ingredient, which is a food composition for prevention or improvement of a pigment disease caused by hypochromatosis,
The pigment diseases caused by the hypochromatosis include, but are not limited to, vitiligo, albinism, decolorization nodule, white nasolacrimal duct, hypochromatosis due to papillary thrush, post-inflammatory decolorization, hypopigmentation due to scleroderma, partial albinism, And spotty white flour. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 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 Which is a food composition for prevention or improvement of a pigment disease caused by hypochromatosis,
The pigment diseases caused by the hypochromatosis include, but are not limited to, vitiligo, albinism, decolorization nodule, white nasolacrimal duct, hypochromatosis due to papillary thrush, post-inflammatory decolorization, hypopigmentation due to scleroderma, partial albinism, And spotty white flour. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;

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