KR101669359B1 - composition for promoting melanin synthesis - Google Patents

Abstract

The present invention relates to a composition comprising azalea extract, sari extract, oakwood extract or mixtures thereof, wherein melanin synthesis is induced upon treatment of the composition, thereby exhibiting an effect of improving vitiligo and white moth by melanin synthesis disorder, Since it is a composition for alleviating vitiligo or white moth, which contains only natural substances as an active ingredient without using chemicals, it can be usefully used as cosmetic, pharmaceutical composition or health functional food for alleviating vitiligo or white moth.

Description

[Composition for promoting melanin synthesis]

The present invention relates to a composition for promoting melanin synthesis, and more particularly, to a composition which induces melanin synthesis and has a therapeutic effect on vitiligo or white moth.

Vitiligo is a pigment deficiency in which melanin is not produced for a long time in melanocytes in skin epidermis, leading to white patches. Although the cause of vitiligo is not known precisely yet, hypotheses have been suggested for reducing the function of melanocytes such as genetic factors, abnormal action of nerve cells by stimulation of oxidative activity, and autoimmune destruction.

Recently, it has been noticed that these hypotheses are combined to cause vitiligo. In many patients with vitiligo, white spots are known to have a significant impact on quality of life, and recent reports have reported serious problems in employment and interpersonal problems in patients with vitiligo. This means that vitiligo can be seen as a small disorder, but it can affect psychological self-esteem and social activity for people with severe pigmented disease.

Baekmu, a disease similar to vitiligo, is also attracting attention due to the growing interest in appearance due to the recent improvement in the cultural level due to economic growth. White moths are more likely to be studied in health, beauty, and psychology because they appear more clearly in Asian people with black hair than in white ones. The more white moths are, the more psychological depression and stress increase like vitiligo, and feel discomfort in interpersonal relations and social activities.

It is important to activate melanocytes, which are pigment cells, in order to treat such vitiligo or white moth, and melanin synthesis due to activation of melanocytes plays a major role in pigment repigmentation. Therefore, the goal of treatment of vitiligo and white hair is to stimulate inactive melanocytes or pigment cells present in the hair follicles in the periphery or inside the lesion to promote differentiation, proliferation and migration and to produce melanin.

Melanin synthesis is a key enzyme in the synthesis of melanin in melanocytes. Tyrosinase, which is known to be an important enzyme, catalyzes the synthesis of melanin. It reacts with tyrosine to form dopa, while dopa reacts with tyrosinase to form dopa-quinone ) To finally produce melanin. Melanin is divided into black and brown eumelanin and pheomelanin with red and yellow. In the case of pheomelanin, melanin is known to be formed by the reaction with amino acid cysteine. The higher the production of black eumelanin in the treatment of vitiligo, the more effective it is.

Currently, treatment of vitiligo includes local corticosteroids, calcineurin inhibitors, vitamin D derivatives, UVA (narrow band UVB, photochemical treatment), surgical treatment to transplant epidermis with normal melanocytes, local treatment And phototherapy, but they have side effects such as increased incidence of non-melanoma skin cancer and melanoma.

In addition, isobutyl-methylxanthine (IBMX), which is used in studies on whitefly inhibition, promotes melanogenesis and increases protein levels of microphthalmia-associated transcription factors (MITF) and tyrosinase, Of the patients. Currently, various hair dyeing agents are being used as a solution to white hair. However, since this permanent hair dye is composed of a vegetable hair dye, a metallic hair dye, a synthetic hair dye and the like, it has side effects such as damage and stimulation to hair and scalp due to chemical action.

Therefore, there is a demand for research and development of natural products which have few side effects and are effective for the production of melanin.

1. Korean Patent No. 10-1282280.

Accordingly, it is an object of the present invention to provide a composition for promoting melanin synthesis, which comprises a natural substance having few side effects.

In order to attain the above object, the present invention provides a composition for promoting melanin synthesis comprising azalea extract.

In order to achieve the above object, the present invention provides a composition for promoting melanin synthesis, which comprises azalea extract, sari extract, oak oak extract or a mixture thereof.

The present invention relates to a composition comprising azalea extract, sari extract, oakwood extract or mixtures thereof, wherein melanin synthesis is induced upon treatment of the composition, thereby exhibiting an effect of improving vitiligo and white moth by melanin synthesis disorder, Since it is a composition for alleviating vitiligo or white moth, which contains only natural substances as an active ingredient without using chemicals, it can be usefully used as cosmetic, pharmaceutical composition or health functional food for alleviating vitiligo or white moth.

1 is an analysis of the mushroom-type tyrosinase activity of ethanol extracts of azaleas, sari and oak wood of the present invention,
FIGS. 2 and 3 are graphs showing cytotoxicity of B16 melanoma cells of ethanol extracts of azaleas, sari and oak wood of the present invention,
FIG. 4 is a graph showing the effect of ethanol extracts of azaleas, sari and oakwood on the tyrosinase activity of B16 melanoma cells according to the present invention,
FIG. 5 is a graph showing the effect of ethanol extracts of azaleas, sari and oak oak wood on melanin production in B16 melanoma cells according to the present invention,
Fig. 6 shows the visual effects of melanin pigment deposition on the hair of the (C57 / BL6 x BALB) F1 mouse treated with the ethanol extract of azalea, sari and oak wood of the present invention,
FIG. 7 is a graph showing the effect of ethanolic extracts of Azalea, Sirius and Quercus serrata according to the present invention on melanin production in hair of (C57 / BL6xBALB) F1 mice,
FIG. 8 is a graphical representation of the effect of the ethanol extract of Azalea, Sari and Quercus serrata according to the present invention on the skin of (C57 / BL6 × BALB) F1 mice.

 The inventors of the present invention have investigated the functionality of natural extracts which have not suffered from resistance or safety problems, and melanin synthesis was induced when ethanol extract of azaleas, And thus the present invention has been completed.

Rhododendron schlippenbachii ( Rhododendron schlippenbachii ) is a plant belonging to the genus Rhododendron (Ericaceae). It has distinctive black spots on the flower bud and has a sticky solution on the calyx. Azaleas are known to have respiratory paralysis caused by toxic components such as grayanotoxin. However, leaf and throat of azaleas are used as research materials for the treatment of hypertension and inhibition of glucose metabolism related to diabetes come. Studies on various physiological activities of azalea spp. Are still insufficient, and only some cases of toxicity have been reported.

Lespedeza bicolor is a legume plant characterized by very straight stems and long peduncle. It has antioxidant activity and antimicrobial activity and contains various alkaloids, flavonoids, ascorbic acid, saponin, and tannin. Sri Lanka extract has been known to be effective in alleviating various diseases such as bruises, headache, heart disease, allergy, and atopy. Studies on electron donating ability, superoxide dismutase (SOD) - like activity, and nitrite scavenging ability using Sri Lanka extract have been carried out. However, the results of studies on the physiological activity using the thrips of the sorghum have not been reported, and many research results have been reported that have been applied to the medical field using sari roots and flowers.

The oak oak ( Quercus variabilis ) is a deciduous arboreous tree with oak tree oak and oak tree, and the bark is yellowish. Quercus mongolica has been used for food for a long time and it is very useful as a food as well as a medicinal plant because it contains various ingredients such as tannin, milk fat, quercetin, . In folk remedies, leaves and bark have been used for astringent hemostasis, diarrhea, desulfurization, hemorrhoids, genomes, and painkillers. Studies on the physical properties, growth characteristics, distribution and chemical properties of oak have been carried out for a long time, but development of the medical field using them has been insufficient.

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

The present invention provides a composition for promoting melanin synthesis, which comprises azalea extract.

In addition, the composition may further comprise sari extract, oakwood extract or a mixture thereof.

The composition comprises 5 to 90% by weight of azalea extract, 5 to 90% by weight of sari extract and 5 to 90% by weight of oak oak extract.

When the concentration is out of the above range, the effect of each component may not be exhibited properly.

The extract is extracted with a solvent selected from the group consisting of ethanol, methanol, distilled water and a mixture thereof, and ethanol is preferably used, but is not limited thereto.

The composition may be used as a composition for preventing or treating vitiligo or white moth caused by melanin-forming disorder.

The composition may be provided as a cosmetic composition, a pharmaceutical composition or a health functional food.

The cosmetic composition may be used for external ointment for skin, foundation cosmetic, make-up cosmetic, body cosmetic or shaving cosmetic. Examples of the basic cosmetic include essence, lotion, cream, gel, lotion, Such as foundation, lipstick, eye shadow, eye liner, mascara, eyebrow pencil, make-up base, etc. The body cosmetics may include soap, liquid cleanser, bath powder, sunscreen Cream, sun oil, etc., and shaving cosmetics include aftershave lotion and shaving cream.

In addition to the above-mentioned active ingredients, such as azalea extract, sari extract, oak oak wood extract, and mixtures thereof, functional materials and excipients such as diluents for improving physical properties may be included.

For example, the cosmetic composition may further contain a perfume, a pigment, a bactericide, an antioxidant, an antiseptic, a moisturizer, an increasing agent, an inorganic salt or a synthetic high molecular substance to improve physical properties. An organic pigment, an organic pigment, an ultraviolet absorber, a pH adjuster, an alcohol, a blood circulation accelerator, a cold agent, a limiting agent, or purified water may be used as a blending component that may be added.

The compounding ingredient to be added other than the above-mentioned effective ingredient is not limited, and any of the above-mentioned ingredients can be compounded within a range that does not impair the purpose and effect of the present invention.

The cosmetic composition may be in the form of a solution, an emulsion, a viscous mixture, and the like, and may be any of the formulations conventionally produced in the art. Examples thereof include a spray, a milky lotion, a cream, a lotion, a pack, a foundation, , Hair cosmetics, and the like.

More particularly, the cosmetic composition of the present invention can be used as a cosmetic composition in the form of a spray, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a skin lotion, a skin softener, a skin toner, an astigmant, a lotion, , Massage cream, nutritional cream, moisturizing cream, hand cream, foundation, essence, nutritional essence and pack.

For example, when the formulation of the present invention is spray or powder, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, particularly when the formulation of the present invention is a spray In addition, propellants such as chlorofluorohydrocarbons, propane / butane or dimethyl ether may be included.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a solution or an emulsion, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, -Butyl glycol oil, glycerol aliphatic esters, polyethylene glycols or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

The pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions.

Examples of the carrier, excipient or diluent which can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The use amount of the azalea extract, the sari extract, the oakwood extract or the mixture thereof, which is an effective ingredient of the pharmaceutical composition according to the present invention, may vary depending on the age, sex, body weight or disease of the patient but is preferably 0.001 to 100 mg / kg, 0.01 to 10 mg / kg may be administered once to several times per day.

In addition, the dosage of the pharmaceutical composition may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.

The health food may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food may contain other ingredients such as azalea extract, sari extract, oakwood extract or mixtures thereof, And can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the azalea extract, the sari extract, the oakwood extract or the mixture thereof contained in the above health food can be used in accordance with the effective dose of the above pharmaceutical composition, but may be appropriately selected for the purpose of health and hygiene, It can be used in an amount of more than the above range because there is no problem in terms of safety of the active ingredient.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Production of ethanol extracts of azaleas, sari and oak wood

The azaleas, sari and quercus serrata used in the present invention were collected from the scientific forests of Gyeongsang National University, Jinju, Gyeongnam, and stored in a low temperature storage at -75 ° C.

Dried azaleas, sari and oak wood were cut into small pieces (about 3 cm), and then 200 g of 95% ethanol (500 mL) was mixed and extracted at room temperature for 7 days. The extract was filtered with a filter paper (No. 2, Whatman), and then the filtered extract was prepared as a powder using a freeze dryer.

< Example  2> Antioxidant Activity Analysis DPPH  Radical Scatters  Review)

The antioxidant activity of the extract was determined by the DPPH method using 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma, USA) (Monica et al. 2007). The extract prepared in Example 1 was diluted to a concentration of 100 ppm, and 50 쨉 l of the extract was added to each well of a 96-well plate (Falcon ^ⓡ , USA). Then, 200 μl of 0.2 mM DPPH solution was added, reacted at room temperature for 30 minutes, and the absorbance was measured at 517 nm using an enzyme-linked immunosorbent assay (ELISA, Anthos ^ⓡ , Austria) reader. Control group was determined by the same method using butylated hydroxytoluene (BHT, Sigma, USA) and 95% ethanol.

As a result, as shown in Table 1, the ethanol extracts of rhodopsin, sari, oak, and ethanol extracts of mixed (rhodamine ethanol extract: sorly ethanol extract: oak oak ethanol extract, 1: 1: 1 (w / w / w) , 82 or 90%, similar to the positive control BHT (95%). Antioxidant activity was proportional to polyphenol contents, so it could be expected that high polyphenol content and physiological activity effect could be expected.

extract Antioxidant activity % ) Total polyphenol content (mg / g) * Total flavonoid
Content (mg / g) ** Cari ethanol extract 87.3 ± 0.5 106.9 ± 0.7 33.5 ± 0.5 Azalea ethanol extract 91.9 ± 0.1 252.1 ± 0.0 74.0 ± 0.4 Oak oak ethanol
extract 82.7 ± 0.1 116.4 ± 0.7 27.7 ± 0.1 Extract blend 90.9 ± 0.5 200.2 ± 0.1 51.4 ± 0.1 BHT 95.5 ± 0.0 - -

*: Mg / g of galvanic equivalent of powder

**: Equivalent quercetin equivalent of powder mg / g

< Example  3> Analysis of polyphenol contents

The polyphenol content of the extract was determined according to the Folin-Ciocalteu method (see Luis et al., 2007). (Sigma, USA) was added to each well. After the addition of 100 μl of Folin-Ciocalteu (Sigma, USA) reagent, the mixture was allowed to stand for 7 minutes and 300 μl of sodium carbonate Mixed and allowed to react for 30 minutes. Then using a UV- spectrophotometer (UV-spectrophotometer, U-3000 ⓡ, Hitachi, Japan) to prepare a calibration curve by measuring the absorbance at 765nm. The absorbance of the extract powder prepared in Example 1 was measured in the same manner and then the polyphenol content of the extract was measured by comparing with the calibration curve.

As a result, the total polyphenol contents of the cari ethanol extract, the azalea ethanol extract and the oak oak ethanol extract were 106.9 mg / g, 252.1 mg / g or 116.4 mg / g, respectively, as shown in Table 1 of Example 2, The 1: 1: 1 (w / w / w) mixture of oak woody ethanol extract was 200.2 mg / g.

< Example  4> Analysis of flavonoid content

The flavonoid content of the extracts was determined according to the Davis's method (see Monica et al., 2007). 0.5 g of the extract powder prepared in Example 1 was mixed with 1 ml of methanol, mixed with 10 ml of diethylene glycol, and 1 ml of 1N NaOH was added. This was reacted for 1 hour in a 37 ° C water bath and absorbance was measured at 420 nm on a UV-spectrophotometer (U-3000 ^ⓡ , Hitachi, Japan). Quercetin was used as a reference material.

As a result, the content of flavonoids of the cari ethanol extract, azalea ethanol extract and oak oak ethanol extract were 33.5 mg / g, 74.4 mg / g or 27.7 mg / g, respectively, as shown in Table 1 of Example 2, 1: 1: 1 (w / w / w) mixture of the extract and oyster oak ethanol extract showed 51.4 mg / g.

< Example  5> L- Dopa (L-DOPA)  Used Mushroom type  Mushroom tyrosinase Bow performance  analysis

In order to confirm the melanin production ability of ethanol extract of sari, azalea, and oak oak, direct action was observed on tyrosinase, which plays an important role in the production of melanin. Tyrosinase converts L-tyrosine to L-DOPA and then L-DOPA to L-DOPA quinone. Therefore, the activity of mushroom tyrosinase purified using L-DOPA as a tyrosinase substrate was measured (see Chang et al., 2007). After adding 100 μl of 0.1 M phosphate buffer (pH 6) and 20 μl of extract (1 mg / ml, 2 mg / ml), add 20 μl of mushroom tyrosinase (2000 U / ml in phosphate buffer, pH 6) Lt; 0 &gt; C for 10 minutes. The reactants and the absorbance at 475nm was measured using a ^{UV-spectrophotometer (U-3000 ⓡ} , Hitachi, Japan). The tyrosinase activity was derived from the following equation (1).

As a result, when L-DOPA was used as a substrate as shown in Fig. 1, the ethanol extract of sari, azalea and oak oak was increased by about 8%, 20%, or 8%, respectively, The mushroom tyrosinase activity of the mixture of 1: 1: 1 (w / w / w) ethanol extract of sari, azalea and oak oak was 12% higher than that of the control. High activity.

< Example 6> B16  Activity analysis in melanoma cells

1. B16 melanoma cell culture

B16 melanoma cells derived from C57BL / 6J mice were purchased from Ajou University Hospital and cultured in DMEM medium containing 10% fetal bovine serum (PBS) at 37 ° C and 0.5% CO ₂ .

2. Cytotoxicity analysis using B16 melanoma

MTT assay was performed using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide , MTT) After the reagent is absorbed into cells, the succinate dehydrogenase of mitochondria forms formazan. The intracellular accumulation of this substance means the activity of mitochondria, broadly, the activity of cells It is a representative method of measuring the growth rate of cells.

B16 melanoma cells were plated on 24-well plates (7 × 10 ³ cells / well) and cultured in a 0.5% CO ₂ incubator at 37 ° C. for 24 hours. The ethanol extracts of Sari, Azalea and Quercus variabilis 5, 10, 20 mg / ml), and the cells were incubated in a 0.5% CO ₂ incubator for 72 hours. After the culture, the plate medium was removed, and the plate was washed once with PBS. 50 μl of MTT (5 mg / ml) was added per 500 μl of the medium, followed by incubation at 37 ° C. in a 0.5% CO ₂ incubator for 1 hour. After the reaction, the medium was removed, 500 μl of dimethyl sulfoxide (DMSO) was added, and the cells were melted in a plate shaker for 15 minutes. Then, absorbance was measured at 540 nm wavelength with an ELISA reader. The cell growth rate was shown to be comparable to that of the untreated control group.

As a result, as shown in FIG. 2, the activity of B16 melanoma cells was found not to inhibit cell proliferation except 10, 20 μg / mL azelaic ethanol extract and 10, 20 μg / mL mixed treatment group. The cell viability of B16 melanoma cells treated with 20 ㎍ / mL of azelaic ethanol extract decreased to 49% at 72 hours. However, the differentiation status of cells is usually monitored by the formation of dendrite-like shaped dendritic cells. FIG. 3 shows gross cell changes in cells treated with ethanol extracts of sari, azalea and oak oak and control. In Fig. 2, azodiazoles exhibiting cell proliferation inhibition showed a typical dendritic cell phase as compared with control cells. These dendrite-like morphologies were gradually enlarged as the concentration increased in the sari, azalea and oak oak ethanol extract-treated groups. In order to confirm whether dendrite-like morphology is related to induction of cell differentiation in the existing articles, it was confirmed that dendritic-like morphology was clearly formed when cells were treated with arsenic trioxide as a positive control.

Therefore, the ethanol extract of sari, azalea, and oak oak extract effectively induced B16 melanoma cell differentiation.

 3. Using B16 melanoma tyrosinase Bow performance  analysis

Because tyrosinase promotes three stages of melanin biosynthesis, measurement of tyrosinase activity is very important. Therefore, the effect of tyrosinase activity on ethanol extracts of sari, azalea and oak oak on B16 melanoma cells was evaluated. B16 melanoma cells were cultured in 60ø plate at a concentration of 8 × 10 ⁴ cells / well and incubated at 37 ° C and 0.5% CO ₂ for 24 hours. The ethanol extracts of sari, 5, 10, and 20 mg / ml), and the cells were incubated for 72 hours at 37 ° C in a 0.5% CO ₂ incubator. After incubation, the medium was removed and the cells were transferred into trypticin and dissolved in 100 μl of a lysis buffer containing 1% tryptone X-100 solution and pH 6.8 PBS. . The supernatant was centrifuged at 10,000 rpm for 30 minutes at 4 ° C. The supernatant was used as the tyrosinase activity measurement solution. The absorbance was measured with protein analysis solution (Bio rad, USA) and the amount of protein was calculated. The cell extracts containing 20 μg of the protein and the test substance were mixed to make a total of 20 μl, and then 180 μl of L-DOPA was added thereto, followed by reaction at 37 ° C. for 1 hour in a 0.5% CO ₂ incubator. After the reaction, the change in absorbance at 490 nm was measured.

As a result, as shown in Fig. 4, when treated with 20 μg / ml of the ethanol extract of Chenxianum, the maximum increase was 14% as compared with that of the control, and the ethanol extract of Azalea japonica increased by 13% when treated with 10 μg / ml The ethanol extract of Quercus mongolica showed a maximum increase of 14% at 20 ㎍ / ml compared to the control. (1: 1: 1 (w / w / w)) of ethanolic extracts of Silla, Azalea and Quercus variabilis were increased by 7% when treated with 10 μg / ml and the analysis of variance (ANOVA) Except for the extracts of Saffi, Azalea, and Quercus variabilis showed a significant P <0.05 significance. Therefore, the ethanol extracts of sari, azalea and oak wood were determined to affect tyrosinase activation in B16 melanoma cells.

4. Melanin using B16 melanoma Generation  analysis

B16 melanoma cells were plated on 60ø plate at a density of 8 × 10 ⁴ cells / well and incubated for 24 hours at 37 ° C and 0.5% CO ₂ in an incubator. The ethanol extracts of sari, azalea and oak 5, 10, 20 mg / ml), and the cells were incubated in a 0.5% CO ₂ incubator for 72 hours. After incubation, the medium was removed and the cells were removed with trypsin. The cells were transferred to an e-tube and lysed by adding lysis buffer containing 1% triton X-100 and pH 6.8 PBS. The supernatant was centrifuged at 10,000 rpm for 30 minutes at 4 ° C, and the supernatant was removed. The remaining pellet was used for measuring the melanin content. Melanin was dissolved in 1N NaOH solution and the absorbance was measured at 490 nm. The content was calculated using the melanin calibration curve (Y = 0.00075X + 0.0348).

As a result, as shown in FIG. 5, the melanin production of the ethanol extracts of Sacri, Azalea and Quercus variabilis from B16 melanoma cells was evaluated. As a result, ethanol extracts of Sari, Azalea and Quercus variabilis were effective in the melanin production of B16 melanoma cells . The ethanol extracts of 5, 10 and 20 μg / ml were increased by 39, 81 and 89%, respectively, and the melanin production was higher than that of the control. 78 or 98%, respectively. The extracts of oak oak ethanol increased by 14, 58 or 61% at 5, 10 or 20 ㎍ / ml, respectively. The melanin production of ethanol extracts of Silla, Azalea and Quercus variabilis increased depending on the concentration of extracts. The ANOVA statistical analysis showed that the ethanol extract of Quercus variabilis and the ethanol extract of Quercus, Azalea and Quercus variabilis (1: 1: 1 (w / w / w)) of 5 / / ml.

< Example  7> Analysis of melanin production effect of mouse hair and epidermis

One. Experimental animal  Ready

Positive control C57 / BL6 mice and test mice (C57 / BL6 × BALB) F1 mice were purchased from a central laboratory animal at 6 weeks of age and raised in an experimental animal control room of Gyeongsang National University. Animals were fed diets and drinking water freely at a temperature of 23 ± 2 ° C and relative humidity of 35 to 60% for 12 hours. The animals were fed a 7-day adaptation period and then were tested using a 7-week-old mouse.

2. Treatment of sari, azalea, oak oak ethanol extract or mixture thereof

The hair of the selected part of the mouse was first removed, and the ethanol extract was gently applied (0.2 mL / cm ² ) to the pinkish skin of the resting paw using a cotton swab. The control group was maintained with 60% ethanol applied to the skin on the opposite side of the same mouse. The extract treatment was carried out for 6 months and treated once a day. Changes in color of mouse fur associated with skin color were regularly observed.

3. Mouse hair of  Gross observation and measurement of melanin content

Melanin content was measured using a spectrophotometric assay, which can be conveniently used to determine the level of melanin in the hair compared to analytical HPLC analysis with a complex pretreatment process and expensive equipment (Lee et al., 2007). The melanin content in the hair after the application of the extract was measured spectrophotometrically to quantitatively evaluate the antioxidant effect of the material.

The hair of the positive control group C57 / BL6 and the test group (C57 / BL6 × BALB) F1 mice were collected using a razor and then reacted with 1 mL of 1 M NaOH per 20 mg of hair for 24 hours at room temperature. Absorbance was measured at wavelengths A500 (total melanin) and A650 (eumelanin) in both the control and test groups. Melanin and eumelanin were dissolved in 1 M NaOH in concentrations ranging from 0.05 to 0.4 mg / ml, respectively, and melanin was added to the calibration curve to calculate the melanin content.

As a result, as shown in FIG. 6, when compared with the control group using 60% ethanol, the extracts of Cari ethanol, Azalea ethanol extract, Quercus oak ethanol extract and mixture (Cari ethanol extract: Azalea ethanol extract: Quercy oak ethanol extract, 1: 1: (w / w / w)), respectively.

The melanin content of the hair was measured by spectrophotometric method and quantitatively evaluated the anti-alopecia effect of the material. As a result, the melanin content of the mouse hair was measured as shown in Fig. 7. As a result, Melanin And eumelanin content were increased. The content of melanin was 40%, that of azalea ethanol extract was 47%, that of oak oak ethanol extract was 36%, that of sorghum azalea and oak oak ethanol was 1: 1: 1 (w / w / w)) was increased by 39%, and the content of yelmelinone showing black pigment was also the same as the content of ethanol extract, azalea ethanol extract and mixture of ethanol extract: azalea ethanol extract: oak oak ethanol extract (1: 1: 1 (w 29%, 29%, 21% and 27%, respectively), indicating that the ethanol extracts of sari, azalea and quercetin play a positive role in the production of melanin.

4. L- DOPA  Observation of melanocytes through staining

Melanocytes are positive by DOPA and show black color, so that the position of melanocytes and the changes of the treatment of extracts can be glanced. When a clean piece of mouse skin or pro-malignant tissue is cultured in a solution of 3: 4: dihydroxyphenylalanine (Dopa) at pH 7.4, it is oxidized with Dopa-melanin in the cells to be stained black . These changes in cells are called dopa-positive. In the mouse epidermis, this reaction usually occurs in melanocytes that undergo pigmentation activity.

To this end, we used the standard procedure of Laidlaw & Blackberg and prepared clean sheet and mouse skin to perform the reaction. First, ethylenediaminetetraacetic acid (EDTA) solution was used as a method for separating the epidermis and dermis of the skin isolated from mice and the like. After reacting with EDTA solution in a 6-well plate for 2 hours, the epidermis was separated using a microforcep. Separated epidermis was washed with saline for about 1 minute and incubated in L-DOPA solution for 1 hour at 37 ° C. After 1 hour, the L-DOPA solution was replaced and incubated at 37 ° C for 8 hours. After incubation, the cells were washed with saline for about 1 minute and fixed with 10% formalin for 20 minutes. After the fixation, it was cleaned using distilled water for 3 minutes, and the air in the epidermis was removed by sequentially reacting with 95% ethanol and 100% ethanol for 20 minutes. Finally, xylene was reacted for 2 minutes, and a cover glass was carefully covered with a cadana blasm solution so as to prevent air bubbles from forming. Then, stained melanocytes were observed using an optical microscope.

After 5 months of treatment with ethanol extract of sari, azalea and oak oak for 5 months, epidermis was separated and observed. As shown in Fig. 8, it was confirmed that melanocytes were observed around the hair follicles as compared with the control group. Melanocytes, which were not observed in the skin of 60% ethanol treated group, were observed around the hair follicles after ethanol treatment of sari, azalea and oak oak. It has been previously reported that the activation of melanocytes causes an increase in melanocytes around the roots of mouse hair follicles. Therefore, it is concluded that the ethanol extracts of sari, azalea and oak oak induce the pigmentation of vitiligo.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Wherein the composition comprises 5 to 90% by weight of azalea extract, 5 to 90% by weight of sari extract and 5 to 90% by weight of oak oak extract. delete delete The method according to claim 1,
Wherein the extract is extracted with a solvent selected from the group consisting of ethanol, methanol, distilled water, and mixtures thereof. delete 5 to 90% by weight of azalea extract, 5 to 90% by weight of sari extract and 5 to 90% by weight of oak oak extract. delete 5 to 90% by weight of azalea extract, 5 to 90% by weight of sari extract and 5 to 90% by weight of oak oak extract.

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