KR20180078588A - Composition for whitening comprising an ethyl acetate fraction of Agastache rugosa Fisch. and C.A.Mey.Kuntze or an active compound isolated from the plant - Google Patents

Abstract

The present invention relates to a skin whitening cosmetic composition which contains an ethyl acetate fraction of an Agastache rugosa (Fisch. & C.A.Mey.) Kuntze extract and methyleugenol β-D-glucopyranoside isolated therefrom as an active ingredient. More particularly, according to the present invention, the skin whitening cosmetic composition is identified that the ethyl acetate fraction of the Agastache rugosa Kuntze extract inhibits melanogenesis, and a methyleugenol β-D-glucopyranoside compound isolated therefrom inhibits melanogenesis by down-regulating MITF, tyrosinase, TRP-1 and DCT which are genes involved in melanogenesis through inhibition of SOX9 expression. The composition which contains an ethyl acetate fraction of an Agastache rugosa (Fisch. & C.A.Mey.) Kuntze extract and a methyleugenol β-D-glucopyranoside compound isolated therefrom as an active ingredient, is a skin whitening cosmetic composition which can be useful as a skin whitening and lightening functional material.

Description

TECHNICAL FIELD The present invention relates to a composition for skin whitening comprising an ethyl acetate fraction or a compound isolated therefrom as an active ingredient, and to a composition for skin whitening comprising an ethyl acetate fraction of Agastache rugosa Fisch. and C.A.Mey.Kuntze or an active compound isolated from the plant}

The present invention relates to a skin whitening cosmetic composition containing an ethyl acetate fraction or a compound isolated therefrom as an active ingredient, a pharmaceutical composition for preventing or treating hypercholesterolemia, and a composition for skin whitening or hypercholesterolemia Health functional foods.

Skin is a very important tissue that protects the human body in direct contact with the external environment and has biochemical and physical functions. This tissue is divided into three major parts: epidermis, dermis and hypodermis. Melanin is a pigment present in the epidermis of the skin and is not essential for the growth or development of the individual, but is produced from melanocytes in the basal layer of the epidermis. In melanosome synthesis, tyrosine is first converted to DOPAquinone via DOPA by an enzyme called tyrosinase, and DOPAchrome is converted from dopaquinone into melanosome, To produce melanin as a dark brown copolymer. The resulting melanin is transferred to epidermal cells called keratinocytes, which not only determines the skin color of the individual, but also absorbs the light energy of UV to protect the subcutaneous skin organs from damage by UV, and harmful oxygen And free radicals to protect the skin from external harmful factors.

In spite of the abovementioned positive effects of melanin, an increase in the number or activity of melanocytes leads to melanin accumulation, which is caused by post inflammatory hyperpigmentation, melisma, solar lentigines, and geriatric pigment spots are frequently the causes of hyperpigmentary disorders. Therefore, there is a growing demand for the development of whitening agents with low cytotoxicity for the prevention and treatment of abnormal melanin production and abnormal hyperpigmentation, with an increasing interest in skin care.

Up to now, the control of melanogenesis enzymes or melanosome transfer has been the most common approach in the development of whitening agents. Among them, melanin such as tyrosinase, tyrosinase-related protein 1 (TRP-1), DCT (also called dopachrome tautomerase, tyrosinase-related protein 2 (TRP-2)) or MITF (microphthalmia associated transcription factor) There have been many reports on the whitening through the inhibition of the production enzyme. Tyrosinase, TRP-1 and DCT are well known as key enzymes in melanin biosynthesis, which work in the organelle called melanosome. Tyrosinase converts L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) and then converts it to dopaquinone. DCT and TRP-1 are involved in the conversion of dopachrome into a melanin polymer with subsequent steps. Melanogenesis enzymes such as tyrosinase, TRP-1 and DCT are also known to be regulated by SOX9 (SRY type HMF box 9), one of the SOX transcription factors, which plays an important role in the development of melanocytes There is no report on a whitening agent capable of controlling the synthesis of melanin through regulation of SOX9 itself.

Also known as ganoderma lucidum ( Agastache rugosa (Fisch. & CAMey.) Kuntze is a perennial plant of the dicotyledonous monocotyledonous plant. It is a plant called Korean mint. It grows in the gravel field of the Yangjiji and is 40 ~ 100cm in height. The stem stood straight and the branches split on the upper part and squared. Leaves are opposite, ovate, 5-10cm long and 3-7cm wide. It has a pointed end, rounded bottom, long petiole with a length of 1 ~ 4cm and dull serrations on the edge. Flowers are lips and bloom from July to September, purple, hanging on the inflorescence of umbellas, and fragrant. The inflorescence is in the shape of a spike, 5 to 15 cm long. Calyx is divided into 5 pieces, corolla is 8 ~ 10mm long, upper petal petal is small, lower lip is big, and it is divided into 5 pieces. Two out of four surgeries are long. Fruit is flattened and ovate with ovate. The young seeds are also used as ornamental plants. In China, it is written in the form of Gwakyeong (藿香). In Korea, mature ginseng is used as a medicinal product. It is distributed in Korea, Japan, Taiwan and China and has been reported to have antibacterial activity, antifungal activity and anti-atherogenic activity.

According to the present invention, it was confirmed that the ethyl acetate fraction of the pine needle extract significantly decreased the melanin synthesis and the dimethyl uregen β-D-glucopyranoside compound isolated from the fraction Confirming that the inhibition of melanin synthesis is inhibited by SOX9 inhibition, and confirming that the above compound can be used as a whitening agent, thereby completing the present invention.

It is an object of the present invention to provide a skin whitening cosmetic composition containing an ethyl acetate fraction or a compound isolated therefrom as an active ingredient, a pharmaceutical composition for preventing or treating hypercholesterolemia, and a skin whitening or hypercholesterolemia And a health functional food for improvement.

In order to achieve the above object, the present invention Agastache rugosa (Agastache rugosa (Fisch. ≪ / RTI >& CAMEY.) Kuntze) or a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

.

.

The invention also Agastache rugosa (Agastache rugosa (Fisch & CAMey.) Kuntze) as an active ingredient, or a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

.

.

Further, Agastache rugosa (Agastache rugosa (Fisch & CAMey.) Kuntze) or a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

.

.

The present invention relates to a process for producing rugosa (Fisch. & CAMey.) Kuntze) or Dimethylguanol β-D-glucopyranoside represented by the following formula (1) is cytotoxic and has a tie, which is a gene involved in melanogenesis through SOX9 inhibition Inhibits melanin production by inhibiting the expression of rhodinose, TRP-1, DCT and MITF, and thus can be usefully used as a composition for preventing, treating or improving a whitening or hyperchromatic disease.

[Chemical Formula 1]

.

.

1A is Agastache rugosa (Agastache rugosa (Fisch. & CAMey.) Kuntze) fractions of melanin-a cells.
Figure 1B is a graph showing the effect of aging on melanin- rugosa (Fisch. &Amp; CAMey.) Kuntze) extract fraction.
Figure 2 shows the melanin content of melanin-a cells treated with various structural analogues of dimethylugenol beta -D-glucopyranoside and cytotoxicity due to the treatment.
FIG. 3 is a graph showing the activity of human epidermal melanocyte cells treated with dimethylugenol? -D-glucopyranoside (Compound 1) NHEM) in situ tyrosinase activity.
4A is a graph showing the area of melanocytes stained in human epidermal melanocytes treated with dimethyl eugenol? -D-glucopyranoside (1)
4B is a graph showing cytotoxicity of dimethyl eugenol? -D-glucopyranoside (1) in human epidermal melanocytes after treatment
FIG. 5 shows the expression level of the melanin synthesis-related gene according to the treatment with dimethyl uangen β-D-glucopyranoside (Compound 1) by RT-PCR (FIG. 5A) 5B to 5F).
FIG. 6 is an immunofluorescence analysis of various melanin-related genes in human epidermal melanocytes treated with dimethyl ugenol? -D-glucopyranoside (Compound 1).
FIG. 7 shows the degree of color change to black due to melanin production in MelanoDerm (MatTek Ashland, MA) which is an artificial skin tissue treated with dimethyl ugenol? -D-glucopyranoside (Compound 1) ) Immunohistochemical analysis (Fig. 7B).

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for producing rugosa (Fisch. ≪ / RTI >& CAMEY.) Kuntze) or a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

.

.

The invention also Agastache rugosa (Agastache rugosa (Fisch & CAMey.) Kuntze) as an active ingredient, or a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

.

.

The extract or fraction thereof is preferably, but not exclusively, prepared by a process comprising the steps of:

1) extracting an extracting solvent by adding the extracting solvent to the extract;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to prepare an extract of the pomegranate; And

4) a step of extracting the extract from the step 3) with an organic solvent to prepare a fragrant fraction.

In the above method, the brewing of step 1) may be used without limitation such as grown or commercially available. The leaves may be leaves, stalks or roots, and mixtures of two or more of them may be used.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, a C ₁ to C ₄ lower alcohol is preferably used, and as the lower alcohol, ethanol, methanol or propyl alcohol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. The extraction solvent is preferably added by 1 to 10 times, more preferably 2 to 3 times, to the dried sieve. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. In addition, the extraction number is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the organic solvent in step 4) is preferably, but not limited to, n-hexane, chloroform, ethyl acetate, n-butanol or water. The fractions can be obtained by suspending a pine needle extract in water and then fractionating n-hexane fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction or water fraction obtained by sequential fractionation with n-hexane, chloroform, ethyl acetate, n- But it is more preferably an ethyl acetate fraction, but not limited thereto. The fraction can be obtained by repeating the fractionation process from the sphygmomanometer extract to 1 to 5 times, preferably 3 times, and after fractionation, concentration under reduced pressure is preferable, but it is not limited thereto.

The present invention includes not only the compounds represented by the above formula (1), but also prodrugs thereof, possible solvates, hydrates, racemates or stereoisomers thereof which can be prepared therefrom.

The ethyl acetate fraction of the bamboo shoot extract inhibits melanogenesis. The compound of Formula 1 inhibits the expression of tyrosinase, TRP-1, DCT, MITF and SOX9 genes, which are related to melanin production .

The hyperpigmentation disorder may be any one selected from the group consisting of spots, freckles, spots, daylight surplus, drug substance pigmentation, inflammatory pigmentation, senile pigmentation, and pregnancy pigmentation, but is not limited thereto.

In a particular embodiment of the present invention, the inventors Agastache rugosa (Agastache rugosa When the ethyl acetate fraction of Fisch. & CAMey. Kuntze extract and the methyl eugenol? -D-glucopyranoside isolated therefrom were treated, the ethyl acetate fraction of the brewer's extract inhibited melanin production, Methylugenol β-D-glucopyranoside compounds can inhibit melanogenesis by down-regulating MITF, tyrosinase, TRP-1 and DCT genes involved in melanogenesis through inhibition of SOX9 expression Respectively. By in experiments using the tissue with artificial skin melanocytes bushes (MelanoDerm, MatTek Ashland, MA) determined that the compound reducing the melanin in the epidermis without cytotoxicity, the Agastache rugosa (Agastache rugosa Ethyl acetate fraction of Fisch. & CAMey. Kuntze extract and methyl eugenol? -D-glucopyranoside isolated therefrom are useful as a skin whitening cosmetic composition or a pharmaceutical composition for the prevention or treatment of hyperpigmentation diseases Can be used.

The cosmetic composition may be in the form of a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in water, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) A cream, a skin, a lotion, a powder, an ointment, a spray, or a cone stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

The cosmetic composition may further contain, in addition to the ethyl acetate fraction of the herbal extract of the present invention and the methylugenol? -D-glucopyranoside isolated therefrom, a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, Antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, Oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics.

In a typical cosmetic composition, the ethyl acetate fraction of the leaf extract of the present invention of the present invention and methylugenol? -D-glucopyranoside isolated therefrom are contained in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight .

When the ethylacetate fraction of the herbal extract of the present invention and the methylugenol? -D-glucopyranoside isolated therefrom are used as external preparations for skin, they may further contain a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, , Antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, May contain adjuvants conventionally used in the field of dermatology, such as essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient conventionally used in external preparations for skin. In addition, the components can be introduced in amounts commonly used in the dermatology field.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which are prepared by dissolving at least the ethyl acetate fraction of the bamboo shoot extract and the methylugenol? -D-glucopyranoside One or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and the like can be prepared. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, Preferably 0.001 to 10 mg / kg, based on the amount of the methyluguanol β-D-glucopyranoside separated from the fraction or the methylergenol β-D-glucopyranoside isolated therefrom, and can be administered 1 to 6 times a day .

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The invention also Agastache rugosa (Agastache rugosa (Fisch & CAMey.) Kuntze) or a compound represented by the following formula (1) as an active ingredient:

[Chemical Formula 1]

.

.

In a specific example of the present invention, the present inventors have found that when the ethyl acetate fraction of bamboo shoot extract and methyl eugenol? -D-glucopyranoside isolated therefrom are treated, the ethyl acetate fraction of the bamboo extract inhibits melanin production, Methylugenol β-D-glucopyranoside compounds isolated from these compounds suppress melanogenesis by down-regulating MITF, tyrosinase, TRP-1 and DCT, which are involved in melanogenesis through inhibition of SOX9 expression The ethyl acetate fraction and the methylugenol? -D-glucopyranoside isolated therefrom may be useful as a health functional food for skin whitening or hypercholesterolemia.

The ethylacetate fraction and the methylugenol? -D-glucopyranose isolated therefrom according to the present invention may be added directly to the food or may be used in combination with other food or food ingredients, . The amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use. Generally, the amount of the ethyl acetate fraction of the bran extract and the amount of methylugenol? -D-glucopyranoside isolated therefrom is 0.01 to 15 And the health beverage composition may be added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food include functional mixed tea, dairy product, various kinds of soup, beverage, and vitamin complex, and include health functional foods in a conventional sense.

The health functional beverage composition of the present invention is not particularly limited to any other components other than the ethyl acetate fraction of the above-described bamboo shoot extract and the methylugenol? -D-glucopyranoside isolated therefrom as an essential ingredient at the indicated ratio And may contain various flavors or natural carbohydrates as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above-mentioned foods, the food of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the ethylacetate fraction of the extract of the present invention and the methylugenol? -D-glucopyranoside isolated therefrom may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of these additives is not so important, it is generally preferred to select from 0 to about 20 parts by weight per 100 parts by weight of the ethylacetate fraction of the bamboo shoot extract of the present invention and methyl eugenol? -D-glucopyranoside isolated therefrom to be.

Bamboo extract, Fraction  Produce

The dried leaves (4.0 kg) were ultrasonically extracted with 10 L of 100% methanol three times for 4 hours, and the extract was filtered and concentrated under reduced pressure using a vacuum concentrator to obtain 400.0 g of methanol crude extract.

100 g of the dried leaves were ultrasonically extracted with 500 mL of 100% ethanol and 100% propyl alcohol three times for 4 hours. The extract was filtered and concentrated under reduced pressure using a vacuum concentrator to obtain 11.0 g and 9.0 g of crude extract, respectively .

Fuller  Ethyl acetate From the fraction  Separation of compounds

The methanol extract of the bamboo leaves prepared by the method of Example 1 was resuspended in water and then sequentially fractionated with chloroform (CHCl3) and ethyl acetate (EtOAc) to obtain chloroform fraction (119.8 g, AR1), ethyl acetate fraction (Kieselgel 60, Merck, Darmstadt, Germany, CAS No. 7631-86-9) column chromatography with ethyl acetate fraction (AR2) A total of 5 fractions (11.4 g AR1A, 8.7 g AR1B, 3.5 g AR1C, 3.9 g AR1D and 55.8 g AR1E (1 ml) ). Three fractions (1.7 g AR1C1, 1.0 g AR1C2 and 0.8 g AR1C3) were obtained by carrying out the same procedure as above for the AR1C fractions.

The AR1C2 fractions were subjected to HPLC under the conditions shown in Table 3 to obtain Compound 5 (6.2 mg) and Compound 6 (7.5 mg).

Item Operating condition HPLC system column Jsphere ODS H-80 (250 mm × 20 mm, 4 μm, 8 nm) - YMC, Milford, MA menstruum 25% auqueous acetonitrile detection _? Flow rate 3 ml / min

The AR1C2 fractions were subjected to HPLC under the conditions shown in Table 3 to obtain Compound 2 (7.0 mg), 3 (6.1 mg) and 4 (2.9 mg).

Item Operating condition HPLC system column Jsphere ODS H-80 (250 mm × 20 mm, 4 μm, 8 nm) - YMC, Milford, MA menstruum 22% auqueous acetonitrile detection Flow rate 3 ml / min

Column chromatography (mobile phase: chloroform: methanol 6: 1, v / v, 2 mL / min) of silica gel (Kieselgel 60, Merck, Darmstadt, Germany, CAS No. 7631-86-9) was performed on the AR1E fraction To give compound 1 (3.3 mg) and compound 7 (4.8 mg).

As a result of spectroscopic analysis such as 1D NMR, 2D NMR and MS data on the above-mentioned compounds, Compound 1 (Formula 1) was found to contain dimethylguanol β-D-glucopyranoside, Compound 2 ) Is a 5?, 6? -Dihydroxy-3? - (? - glucopyranosyloxy) -7-megestigmn-9 -one), and the compound 3 (Formula 3) was obtained in the same manner as in Example 1, except that E ) -4- [3 '- (? - glucopyranosyloxy) butyridene] -3,5,5-trimethyl-2-cyclohexene- (E) -4- [3 '- (β- glucopyranosyloxy) butylidene] -3,5,5-trimethyl-2-cyclohexen-1-one), compound 4 (formula 4) (6 R, 9 R) - 3-oxo -α- EO play -9- O -β- glue nose Llano side ((6 R, 9 R) -3-oxo-α-ionol-9- O -β-glucopyranoside), compound 5 (formula (5) Benzyl β-glucopyranoside, compound 6 and salicylic acid, respectively, and compound 7 (formula 7) was identified as citrusin C, respectively.

Demethyleugenol beta -D-glucopyranoside;

5β, 6α-dihydroxy-3β- (β-glucopyranosyloxy) -7-megestigmn-9-one );

(E) -4- [3 '- (β- glucopyranosyl-oxy) butylidene] -3,5,5- trimethyl-2-cyclohexene-1-circle ((E) -4- [3'- (β-glucopyranosyloxy) butylidene] -3,5,5-trimethyl-2-cyclohexen-1-one);

(6 R, 9 R) -3- oxo -α- EO play -9- O -β- glue nose Llano side ((6 R, 9 R) -3-oxo-α-ionol-9- O -β- glucopyranoside);

Benzyl? -Glucopyranoside;

Salidroside;

Citrusin C.

< Experimental Example  1> The fraction  Effect on melanin production

Melanin-a cells were treated with extracts and fractions prepared by the method of the above example to investigate the antimelanin-producing effect.

Melanin-a cells were obtained from Dr. Byeong-Gon Lee, skin researcher of AMOREPACIFIC Co., Ltd. and were treated with 10% FBS, penicillin / streptomycin, 1% PS and 200 nM TPA (phorbol 12-myristate 13-acetate, sigma aldrich) In a RPMI1640 (company, cat #) medium. For the determination of melanin content, the cells were firstly plated at a density of 1 × 10 ⁵ / well in a 24-well plate, and after 24 hours, the cells were replaced with fresh medium, and the fraction of brewer's extract was treated at a concentration of 1 μg / ml. After 24 hours, the medium was replaced with fresh medium and each fraction was treated in the same manner as above. The total amount of melanin was measured for 3 days. Determination of melanin content in Kumar et al (Kumar, KJ; . Yang, JC; Chu, FH; Chang, ST;. Wang, SY, Lucidone, a novel melanin inhibitor from the fruit of Lindera erythrocarpa Makino Phytother Res 2010, 24, 1158-65.). Briefly, the medium was removed from the cells, washed twice with PBS and then treated with H ₂ O ₂ (1 ml, 1 N) to dissolve the melanin. The absorbance was then measured with an ELISA microplate reader (Molecular Devices, USA) at 405 nm. Cytotoxicity was confirmed by MTT analysis. Specifically, the medium was removed from the cells and washed with PBS, followed by the addition of 400 μl of 0.1% 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (3- 5-dimethylthiazol-2yl) -2,5-diphenyl-2H-tetrazolium bromide, MTT; sigma) was treated and incubated for 1 hour. After the solution was removed, DMSO was added, and the absorbance was measured at 570 nm with an ELISA microplate reader (Molecular Devices, USA). Data are presented as means ± SD. Statistical comparisons between the groups were performed using one-way analysis of variance (ANOVA) with the Tukey 'multiple comparisons test in Turkey. The levels of statistical significance were * P <0.05, ** P <0.01, and *** P <0.001.

As a result, it was confirmed that the ethanol extract, propyl alcohol extract or ethylacetate fraction of the leaf of Baekjeolji showed remarkable melanin synthesis inhibitory activity without cytotoxicity at a concentration of 10 mg / ml (FIGS. 1A and 1B).

< Experimental Example  2> ethyl acetate From the fraction  Effect of Separated Compounds on Melanogenesis

From the results of <Experimental Example 1>, the effect of the seven compounds isolated by the method of <Example 2> on the melanin synthesis in order to identify compounds involved in the inhibition of melanin synthesis in the ethyl acetate fraction Was confirmed by the same method as described in < Experimental Example 1 >. Phenylthiourea (PTU) was purchased from Sigma-Aldrich (St. Louis, MO, USA) as a positive control.

As a result, it was confirmed that the compound 1 dimethyl uregen β-D-glucopyranoside significantly inhibited melanin synthesis at a concentration of 10 μM compared to the untreated group without cytotoxicity (Table 3).

Melanin content cell Survival (cell viability) Negative control group 100.0 + - 3.4 100.0 + - 0.8 Compound 1 61.6 ± 0.1 103.6 ± 3.8 Compound 2 114.1 ± 4.5 92.4 ± 0.4 Compound 3 105.8 ± 4.7 75.9 ± 5.3 Compound 4 107.4 ± 0.2 79.7 ± 2.9 Compound 5 109.9 ± 2.7 96.0 ± 3.2 Compound 6 98.0 + - 6.8 87.4 ± 3.0 Compound 7 120.8 ± 2.7 97.8 ± 3.1 PTU 68.8 ± 5.2 97.9 ± 0.6

< Experimental Example  3> Dimethylguanol  β-D- Glucopyranoside  Effect of analogs on melanin production

The results of Experimental Example 2 show that dimethylguanol β-D-glucopyranoside significantly inhibits melanin synthesis without cytotoxicity compared with the untreated group. As a result, It was confirmed in the same manner as in Experimental Example 2 above that various analogues of dimethylugenol? -D-glucopyranoside have the same activity.

2-hydroxy-5- (2-hydroxyethyl) phenyl -? - D-glucopyranoside;

(E) -4- hydroxy or when wheat alcohol 4-β-DD- glucopyranoside ((E) -4-Hydroxycinnamyl alcohol 4-β-D-glucopyranoside);

2- (4-hydroxyphenyl) -ethyl- beta -D-glucopyranoside; 2- (4-hydroxyphenyl) -ethyl- beta -D-glucopyranoside;

Benzyl [beta] -D-glucopyranoside;

Eugenyl-O-ss-D-glucopyranoside. &Lt; / RTI &gt;

As a result, it was confirmed that only dimethyl υgenol β-D-glucopyranoside significantly inhibited melanin synthesis. The eugenyl-O-β-D-glucopyranoside represented by the above formula (12) (Fig. 2). As shown in Fig.

< Experimental Example  4> Human epidermal melanocytes (human epidermal melanocyte )in Dimethylguanol  of β-D-glucopyranoside Tairosineiz  Inhibition of activity

Whether the dimethylguanol β-D-glucopyranoside has a pigmentation-reducing activity in normal cells is examined in normal human epidermal melanocytes, NHEM).

NHEM purchased from CEFObio was cultured in melanocyte growth medium (CEFObio corporation, Seoul, Korea), treated with L-DOPA to observe tyrosinase activity in situ , and dimethyl igenol β-D-Glue J Invest Dermatol (RA), Post-transcriptional regulation of melanin biosynthetic enzymes by cAMP and resveratrol in human melanocytes. J Invest Dermatol , 2007, 127 , 2216-27). Specifically, NHEM cells were attached to cover slips and treated with 5 μM and 10 μM dimethylguanol β-D-glucopyranoside. After 48 hours, the cells were washed with PBS and fixed with 4% paraformaldehyde (in PBS) for 15 minutes at room temperature. After the cover slip was placed on the slide, the cell membrane was treated with 0.1% Triton-X-100 and washed with PBS. Then, 0.1% L-DOPA was treated at 37 占 폚 for 3 hours and analyzed under a microscope. Cytotoxicity was measured in the same manner as described in <Experimental Example 1>. Phenylthiourea (PTU) was used as a positive control. Statistical comparisons between the groups were performed using one-way analysis of variance (ANOVA) with the Tukey 'multiple comparisons test in Turkey. The levels of statistical significance were * P <0.05, ** P <0.01, and *** P <0.001.

As a result, dimethylguanol β-D-glucopyranoside inhibited the activity of tyrosinase without cytotoxicity at the concentrations of 5 μM and 10 μM (FIGS. 3, 4A and 4B). This result suggests that the reduction of melanin synthesis by dimethylugenol? -D-glucopyranoside occurs through the modulation of tyrosinase enzyme.

< Experimental Example  5> in melanin-a cells Dimethylguanol  β-D- Glucopyranoside  Down regulation of the melanin synthesis gene by

Tyrosinase, TRP-1 and DCT are well known as key enzymes in melanin biosynthesis, which function in the organelle called melanosome. Tyrosinase first converts L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) and then converts it to dopaquinone. DCT and TRP-1 are involved in the conversion of dopachrome into a melanin polymer with subsequent steps. MITF (microphthalmia transcription factor) is well known as a transcription factor that regulates the expression of the above three types of melanin producing enzymes. Recent studies have shown that SOX family proteins are involved in melanogenesis and melanogenesis through modulation of MITF. For example, SOX5 is involved in the elaborate regulation of MITF in human melanocytes, and SOX10 directly activates transcription of MITF as a well-known gene for melanogenesis. In SOX10 mutant animals, pigmentary binding was observed. Recently, it has also been reported that, even though SOX9 does not regulate SOX10, it also decreases the expression of MITF when knocking down the expression of SOX9 with siRNA.

RT-PCR was performed on tyrosinase, TRP-1, DCT, MITF and SOX9 genes in order to confirm the mechanism of inhibition of tyrosinase activity by dimethylguanol β-D-glucopyranoside. Specifically, RNA was isolated from melanin-a cells using TRIZOL Reagent (Invitrogen) according to the manual, and cDNA was synthesized from 1 μg of total RNA using MMLV reverse transcriptase (Invitrogen) RT-PCR was performed. The tyrosinase, TRP-1, DCT, MITF and SOX9 cDNAs were amplified using cDNA and the primers shown in Table 4 below, and electrophoresed on agarose gel to obtain the result . Statistical comparisons between the groups were performed using one-way analysis of variance (ANOVA) with the Tukey 'multiple comparisons test in Turkey. The levels of statistical significance were * P <0.05, ** P <0.01, and *** P <0.001.

Tairosineiz
Forward 5'-GGC CAG CTT TCA GGC AGA GGT-3 '(SEQ ID NO: 1) Reverse 5'-TGG TGC TTC ATG GGC AAA ATC-3 '(SEQ ID NO: 2) TRP-1
Forward 5'-GCT GCA GGA GCC TTC TTT CTC-3 '(SEQ ID NO: 3) Reverse 5'-AAG ACG CTG GTC T-3 '(SEQ ID NO: 4) DCT
Forward 5'-TAC CAT CTG TTG TGG CTG GA-3 '(SEQ ID NO: 5) Reverse 5'-TGG CAC TGC TGG TCA TCT TGT CTT GCT G-3 '(SEQ ID NO: 6) MITF
Forward 5'-CTA GAG CGC ATG GAC TTT CC-3 '(SEQ ID NO: 7) Reverse 5'-AAG TTG GAG CCC ATC TTC CT-3 '(SEQ ID NO: 8) SOX9
Forward 5'-AGC TCA CCA GAC CCT GAG AA-3 '(SEQ ID NO: 9) Reverse 5'-CTC CAG CAA TCG TTA CCT TC-3 '(SEQ ID NO: 10)

As a result, it was confirmed that treatment with dimethyl eugenol β-D-glucopyranoside at a concentration of 10 μM inhibited the expression of key melanogenic genes including SOX9 (FIG. 5).

< Experimental Example  6> Human epidermal melanocytes (human epidermal melanocyte )in Dimethylguanol  by β-D-glucopyranoside treatment SOX9  Down regulation

Similar to Experimental Example 2, dimethylguanol β-D-glucopyranoside was also found to regulate the expression of tyrosinase, TRP-1, DCT (TRP-2), MITF and SOX9 And analyzed by immunofluorescence using normal human epidermal melanocytes (NHEM).

Specifically, NHEM cells were attached to cover slips and treated with 10 μM dimethyl ugenol β-D-glucopyranoside. After 48 hours, they were washed with PBS and fixed with 4% paraformaldehyde (in PBS) for 15 minutes. After the cover slip was placed on the slide, the membrane was incubated with PBS containing 0.1% Triton-X-100 and 1% horse serum to allow the antibody to permeabilize and inhibit nonspecific binding of the antibody at room temperature And treated for 30 minutes. Then, the antibody was reacted with the primary antibody and reacted with the secondary antibody conjugated with fluorescence. The slides were then analyzed by confocal microscopy.

As a result, SOX9 and MITF were originally expressed high in the nucleus, but their expression level was decreased upon treatment with dimethyl eugenol? -D-glucopyranoside (FIG. 6). In addition, dimethylguanol β-D-glucopyranosid inhibited the expression of tyrosinase, TRP-1, DCT (TRP-2) in the cytosolic region (FIG. 6). This result shows that the pigmenting reducing activity of dimethyl-ugenol? -D-glucopyranoside is also caused by the downregulation of not only SOX9 but also its subordinate pathway, tyrosinase, TRP-1 and DCT.

 < Experimental Example  7> In artificial skin Dimethylguanol  β-D- In the glucopyranoside treatment  by Reduced pigmentation

In order to confirm whether dimethyl igenol β-D-glucopyranoside has indeed a pigment-reducing activity in a human in vivo model, Melanoderm (MatTek Ashland, MA), an artificial skin tissue containing melanocytes, was used . Melanodum is a highly differentiated model of the human epidermis consisting of normal human epidemal keratinocytes and melanocytes.

Specifically, melanoderm was cultured in 5% CO ₂ in 6-well and PI-100-NMM (company name, Cat # medium 5 ml) at 37 ° C. The medium was changed once every two days. Dimethylguanol β-D Glucopyranoside was dissolved in a 1: 1 (v / v) mixture of PBS and propylene glycol and treated twice a day for 14 days. After the drug treatment, the tissues were harvested, washed with PBS, The fixed tissue was embedded with paraffin and cut to a thickness of 4 쨉 m. The cut tissue sections were subjected to hematoxylin-eosin staining and Fontana-Masson (PTU) as described above was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and the stained slides were observed under an optical microscope. Louis, MO, USA). And it was used.

As a result, the treatment with dimethyl ugenol β-D-glucopyranoside for 14 days in artificial skin tissue inhibited the degree of color change to black due to melanin production compared to the control, It was confirmed that this was similar to the widely used drug PTU (Fig. 7A). In addition, histological analysis showed that dimethylguanol β-D-glucopyranoside showed no toxicity to the tissues and decreased melanin in the epidermis (FIG. 7B).

<110> Gachon University of Industry-Academic cooperation Foundation <120> Composition for whitening is an ethyl acetate fraction of          Agastache rugosa Fisch. and C.A.Mey.Kuntze or an active compound          isolated from the plant <130> 2016P-05-021 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase F <400> 1 ggccagcttt caggcagagg t 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase R <400> 2 tggtgcttca tgggcaaaat c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TRP-1 F <400> 3 gctgcaggag ccttctttct c 21 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> TRP-1 R <400> 4 aagacgctgg tct 13 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DCT F <400> 5 taccatctgt tgtggctgga 20 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> DCT R <400> 6 tggcactgct ggtcatcttg tcttgctg 28 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MITF F <400> 7 ctagagcgca tggactttcc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MITF R <400> 8 aagttggagc ccatcttcct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9 F <400> 9 agctcaccag accctgagaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOX9 R <400> 10 ctccagcaat cgttaccttc 20

Claims (14)

Agastache rugosa (Fisch. &Lt; / RTI &gt;&amp; CAMEY.) Kuntze) or a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

.
The cosmetic composition for skin whitening according to claim 1, wherein the extract is extracted from leaves, stems, roots, or a mixture thereof.
The cosmetic composition for skin whitening according to claim 1, wherein the extract is extracted with water, a C ₁ to C ₄ lower alcohol or a mixture thereof.
2. The cosmetic composition for skin whitening according to claim 1, wherein the ethyl acetate fraction is an ethyl acetate fraction obtained by sequentially fractionating the extract with an extract of n-hexane, chloroform, ethyl acetate and water.
The cosmetic composition for skin whitening according to claim 1, wherein the ethylacetate fraction inhibits melanin production.
The cosmetic composition for skin whitening according to claim 1, wherein the compound inhibits the expression of tyrosinase, TRP-1, DCT, MITF and SOX9 genes.
Agastache rugosa (Fisch. &Amp; CAMEY.) Kuntze) as an active ingredient, or a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

.
The pharmaceutical composition according to claim 7, wherein the extract is extracted from leaves, stems, roots, or a mixture thereof of a bamboo plant.
The pharmaceutical composition according to claim 7, wherein the extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof.
8. The pharmaceutical composition according to claim 7, wherein the ethyl acetate fraction is an ethyl acetate fraction obtained by sequentially fractionating the extract with a mixture of n-hexane, chloroform, ethyl acetate and water.
The pharmaceutical composition according to claim 7, wherein said ethyl acetate fraction inhibits melanin production.
The pharmaceutical composition according to claim 7, wherein the compound inhibits the expression of tyrosinase, TRP-1, DCT, MITF and SOX9 genes.
8. The method according to claim 7, wherein the hyperpigmentation disease is selected from the group consisting of spots, freckles, spots, daylight surges, drug-induced pigmentation, inflammatory pigmentation, senile pigmentation and pregnancy pigmentation. A pharmaceutical composition for preventing or treating.
Agastache rugosa (Fisch. & CAMey.) Kuntze) extract or a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

.

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