KR101749497B1 - Composition for skin whitening, or preventing and improving skin hyper-pigmented diseases comprising compound separated from Sedum takesimense as active ingredient - Google Patents

Abstract

The present invention relates to a composition for prevention and improvement of skin whitening or skin hyperkeratotic disease, which comprises a compound isolated from an island giraffe, as an active ingredient, (4,6-di-O-galloylarbutin), 2,4,6-tri-O-galloyl-beta-glucose, , 2,4,6-tetra-O-galloyl-beta-glucose and pharmaceutically acceptable salts thereof. The present invention relates to a composition for prevention and improvement of skin whitening or skin hyperkeratotic diseases, which contains one or more compounds as an active ingredient.
The composition of the present invention can be safely applied to the human body by containing a compound isolated from the island girinchus as an active ingredient, exhibits excellent tylosinase inhibiting activity and is excellent in skin whitening effect, and also has a skin hyperkeratotic disease caused by excessive production of melanin Can be effectively prevented and improved.

Description

Technical Field [0001] The present invention relates to a composition for preventing and improving skin whitening or skin hyperkeratosis diseases,

The present invention relates to a composition for prevention and improvement of skin whitening or skin hyperkeratotic disease, which comprises a compound isolated from an island giraffe, as an active ingredient, (4,6-di-O-galloylarbutin), 2,4,6-tri-O-galloyl-beta-glucose, , 2,4,6-tetra-O-galloyl-beta-glucose and pharmaceutically acceptable salts thereof. The present invention relates to a composition for prevention and improvement of skin whitening or skin hyperkeratotic diseases, which contains one or more compounds as an active ingredient.

Skin is a human tissue that is physiologically and physiologically protective in direct contact with stimuli of various external environments. Its color is determined by the melanin contained in skin cells. The melanin pigment is produced in melanocytes of melanocytes called melanocytes. Melanin absorbs ultraviolet rays A, B, and C to prevent ultraviolet damage to other cells, and abnormal pigmentation of skin is caused by abnormal synthesis of melanin pigment. Lack of melanin pigment causes skin discoloration such as vitiligo, and melanin synthesis promotes spots and freckles.

The overproduction of melanin is mainly caused by nonspecific overexpression of tyrosinase due to external stimulation of the skin, which causes cosmetic problems such as blackening of the skin and may lead to skin diseases such as spots and freckles.

Ascorbic acid, hydroquinone, kojic acid, arbutin and some plant extracts having tyrosinase inhibitory activity as conventional skin whitening agents have been used in order to solve skin problems caused by melanin overproduction. However, these materials lack their chemical stability and their use is restricted due to natural decomposition and bad odor induction, in vitro efficacy, effects and uncertain human safety.

Accordingly, it is necessary to overcome the disadvantages of conventional whitening agents and to develop a highly effective natural whitening agent with low irritation and non-toxicity.

Sedum takesimense (NAKAI.) Is a perennial herbaceous plant belonging to the stamens of the genus Thistle. It is a native plant which grows in Ulleungdo and Soraksan in Kangwon Province in Korea. The lower part of the stem is about 30㎝ long in winter, but the stem shoots up laterally in the next spring. When grown in the wild, the height is about 50㎝, the yellow flower is about 13㎜ in diameter, and 20 ~ 30 flowers bloom around the mountain in July. It is known that fungi have been used in folk remedies for the treatment of diseases such as cancer, inflammation, scarring and hypotension, and have radioprotective effect and vasodilation effect.

The present invention relates to a compound having apoptosis inhibitory activity and a method for its isolation (Patent No. 10-0571582), an antimicrobial composition for bacterial pathogens of fish (Patent No. 10- 1298184), an antimicrobial and antioxidant composition containing an extract of an extract from the island rumulus (Patent No. 10-1333516), a composition having antimicrobial activity against a phytopathogenic bacterium containing an extract of the island girinchus as an active ingredient (Patent No. 10-1330591 However, there has been no report on the activity of whitening effect of single compounds derived from island giraffe.

The present inventor has searched for a substance which is safe for human body and can effectively inhibit tyrosinase from the island giraffe so as to effectively use it for prevention, improvement or treatment of skin hypercholesterolemia due to skin whitening or excessive melanin production In this study,

Korean Patent No. 10-0571582 Korean Patent No. 10-1298184 Korean Patent No. 10-1330591 Korea Patent No. 10-1333516

Accordingly, it is a main object of the present invention to provide a novel skin whitening composition which is safe for human body and can effectively inhibit tyrosinase by containing a compound isolated from the island giraffe as an active ingredient.

It is another object of the present invention to provide a composition which can effectively prevent, ameliorate or treat skin hyperkeratotic diseases due to excessive production of melanin by containing the above compound as an active ingredient.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising 4,6-di-O-galloylarbutin, 2,4,6-tri-O-galloyl- (2,4,6-tri-O-galloyl-beta-glucose), 1,2,4,6-tetra-O-galloyl- galloyl-beta-glucose), and pharmaceutically acceptable salts thereof as an active ingredient. The present invention also provides a cosmetic composition for skin whitening, which comprises, as an active ingredient, any one or two or more compounds selected from the group consisting of galloyl-

In the cosmetic composition for skin whitening of the present invention, 1,2,4,6-tetra-O-galloyl-beta-glucose ) Or a pharmaceutically acceptable salt thereof.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising 4,6-di-O-galloylarbutin, 2,4,6-tri-O-galloyl- (2,4,6-tri-O-galloyl-beta-glucose), 1,2,4,6-tetra-O-galloyl- galloyl-beta-glucose, and pharmaceutically acceptable salts thereof as an active ingredient.

In the skin whitening food composition of the present invention, 1,2,4,6-tetra-O-galloyl-beta-glucose ) Or a pharmaceutically acceptable salt thereof.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising 4,6-di-O-galloylarbutin, 2,4,6-tri-O- (2,4,6-tri-O-galloyl-β-glucose), 1,2,4,6-tetra-O-galloyl-beta-glucose -galloyl-beta-glucose) and a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutical composition for preventing or ameliorating skin hypercholesterolemia due to excessive production of melanin, which comprises, as an active ingredient, any one or two or more compounds selected from the group consisting of .

In the pharmaceutical composition for prevention and improvement of skin hypercholesterolemia caused by excessive production of melanin according to the present invention, 1,2,4,6-tetra-O-galloyl-beta-glucose (1,2,4 , 6-tetra-O-galloyl-beta-glucose) or a pharmaceutically acceptable salt thereof.

In the pharmaceutical composition for prevention and improvement of skin hypercholesterolemia caused by excessive production of melanin according to the present invention, the skin hypercholesterolaemia caused by excessive production of melanin is a disease selected from the group consisting of spots, freckles, blotch and hyperpigmentation .

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising 4,6-di-O-galloylarbutin, 2,4,6-tri-O- (2,4,6-tri-O-galloyl-β-glucose), 1,2,4,6-tetra-O-galloyl-beta-glucose -galloyl-beta-glucose) and pharmaceutically acceptable salts thereof as an active ingredient. The present invention also relates to a food composition for prevention and improvement of skin hypercholesterolemia caused by excessive production of melanin comprising, as an active ingredient, one or two or more compounds selected from the group consisting of to provide.

In the food composition for prevention and improvement of skin hypercholesterolemia caused by excessive melanin production of the present invention, 1,2,4,6-tetra-O-galloyl-beta-glucose (1,2,4,6- 6-tetra-O-galloyl-beta-glucose) or a pharmaceutically acceptable salt thereof.

In the food composition for prevention and improvement of skin hypercholesterolemia caused by excessive production of melanin according to the present invention, the skin hypercholesterolaemia caused by excessive production of melanin may be a disease selected from the group consisting of spots, freckles, blotch and hyperpigmentation have.

The composition of the present invention can be safely applied to the human body by containing a compound isolated from the island girinchus as an active ingredient, exhibits excellent tylosinase inhibiting activity and is excellent in skin whitening effect, and also has a skin hyperkeratotic disease caused by excessive production of melanin Can be effectively prevented and improved.

1 is a flowchart illustrating an aqueous formulation of an extract of an island giraffe extract according to an embodiment of the present invention.
FIG. 2 is a chromatogram of high-performance liquid chromatography to separate the compound of the present invention from the island giraffe extract according to an embodiment of the present invention.
Fig. 3 shows the results of ¹ H-NMR spectrum analysis of the compounds of the present invention isolated from the island of giraffe.
Figure 4 shows the structure of the compounds of the present invention isolated from the island giraffe.
FIG. 5 is a graph showing inhibitory activity of tyrosinase of an island giraffe extract according to an embodiment of the present invention.
FIG. 6 is a graph showing the tyrosinase inhibiting activity of the compounds of the present invention isolated from the islet girin.
FIG. 7 is a graph showing a comparison of the tyrosinase inhibiting activity of kojic acid with the compounds of the present invention isolated from the islet girin.
FIG. 8 is a graph showing melanin production inhibitory activity of an island giraffe extract according to an embodiment of the present invention.

The present invention relates to a compound of 4,6-di-O-galloylarbutin, 2,4,6-tri-O-galloyl-beta-glucose (2,4,6-tri-O-galloyl-beta-glucose), 1,2,4,6-tetra-O-galloyl-beta-glucose -β-glucose) inhibits melanin production by inhibiting tyrosinase activity.

Tyrosinase is the most important factor involved in the production of melanin. If it can inhibit the expression or activity of this tyrosinase, it may inhibit the production of melanin and may exhibit a skin whitening effect. Ascorbic acid, hydroquinone, kojic acid and arbutin, which are known as skin whitening agents, have also been developed through their inhibitory activity against these tyrosinases.

When tyrosinase is overexpressed due to external stimuli, melanin is overproduced, resulting in skin hyperkeratotic diseases such as spots, freckles, black spots, and hyperpigmentation. Since the compounds of the present invention exhibit inhibitory activity against tyrosinase, it is possible to effectively prevent and improve such skin hypercholesterolemia.

In the present invention, it was demonstrated through experiments using mushroom tyrosinase that the compounds of the present invention exhibit tyrosinase inhibitory activity. This experimental method using mushroom tyrosinase is one of the most widely used methods for measuring skin whitening activity. According to the results of the experiments, the compounds of the present invention exhibit an inhibitory activity against tyrosinase that is superior to kojic acid, which has been used as a whitening agent. In particular, the activity of 1,2,4,6-tetra-O-galloyl-beta-glucose was excellent.

Compounds of the present invention can be prepared by drying an islet giraffe outpost, preparing an extract using a 20 to 80% (v / v) ethanol solution as a solvent, and then using a silica gel (Kiesel gel 60) resin in chloroform, methanol methanol, distilled water and acetic acid as the eluent, and the resulting solution was subjected to secondary fractionation using Sephadex LH-20 resin and methanol as elution solvents. Can be obtained through high performance liquid chromatography using a gradient of water and acetone nitrile concentration.

The islet giraffe used to isolate the compound of the present invention is preferably, but not limited to, an outpost. It can also be used without restrictions on cultivated or marketed products, and dried and non-dried.

The compound of the present invention can be used for medicines, foods, cosmetics and the like.

At this time, the compound of the present invention can be formulated based on a formulation standard of a conventional pharmaceutical formulation of KFDA or a formulation standard of a health supplement.

The compounds of the present invention may be used in their own form or in the form of pharmaceutically acceptable acid addition salts or metal complexes, for example, salts such as zinc, iron and the like. More specifically, the acid addition salt can be selected from the group consisting of hydrogen chloride, hydrogen bromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate and tartrate. The composition containing the compound of the present invention and a salt thereof as an active ingredient may be prepared by a conventional method, and the active ingredient may be mixed and diluted with a pharmaceutically acceptable carrier according to the administration method, dosage form and therapeutic purpose, To form a carrier.

When the carrier is used as a diluent, oral administration using at least one carrier selected from the group consisting of saline, buffer, dextrose, water, glycerol, Ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose and ethanol For parenteral administration, formulations such as powders, granules, injections, syrups, solutions, tablets, suppositories, pessaries, ointments, creams or aerosols may be prepared. However, the carrier of the present invention is not limited to the above carrier. In this case, parenteral administration means administration of the active ingredient through rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, etc. in addition to orally.

The formulations may further comprise a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, an antiseptic, etc. to formulate the composition so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The dosage of the present invention can be adjusted according to the patient's condition, route of administration, and dosage form, and is not limited, and any person skilled in the art will be able to use the dosage within a wide range, Generally, in the present invention, it is judged that an experimentally effective amount of 1 to 100 mg / kg of body weight can be administered continuously or intermittently per day.

Based on the effective amount of the compound of the present invention, the present invention provides a cosmetic composition comprising the compound of the present invention or a cosmetically acceptable carrier, wherein the cosmetic composition is a lotion, a nutritional lotion, a nutritional cream, a massage cream , A pack, and a nutritional essence.

In addition, the present invention provides a food composition comprising the compound of the present invention or a pharmaceutically acceptable carrier, wherein the food composition is selected from the group consisting of meat products, fish meat products, tofu, Dairy products such as noodles, porridge, noodles such as ramen noodles, seasoned foods such as soy sauce, miso, kochujang, mixed potatoes, sauces, confectionery, fermented milk and cheese, pickles such as kimchi and pickles, fruits, vegetables, soy milk, fermentation It can be used as a food for beverages such as beverages. Pharmaceutically acceptable carriers may also be used as pharmaceutically acceptable carriers.

Hereinafter, the present invention will be described in more detail with reference to Examples, Experimental Examples and Formulation Examples. These examples, experimental examples and formulation examples are only for illustrating the present invention, and therefore, the scope of the present invention is not construed as being limited thereto.

Example 1 Isolation and Structure Identification of Whitening Active Substance in Island Kirinchos

1-1. Production of island giraffe extract

To obtain a whitening active substance, the island kiwifruit plant outpouples were thoroughly dried in an oven at 70 to 90 ° C for 1 to 4 days, and then 20 to 80% of the dried island girinencho weight of 5 to 12 times the volume v / v) ethanol solution was used as a solvent and extracted at room temperature for 15 to 30 hours. The resulting extract was filtered through a filter paper, and the filtrate was concentrated under reduced pressure.

1-2. Isolation and structure identification of whitening active compounds

150 g of a silica gel (Kiesel gel 60) resin was added to the filtrate of the extract having a solid content of about 10 g in the above 1-1, and the filtrate was concentrated under reduced pressure to completely remove the solvent. Then, an open column (diameter: 35 mm, length: 600 mm) And a solution prepared by mixing chloroform, methanol, distilled water and acetic acid in a volume ratio of 55: 36: 8: 1 was used as an eluent to carry out the first fractionation. At this time, the volume of the elution solvent used was 2 times the volume of the column, and eluted between 20 and 30 minutes at a flow rate of 2 ml per minute. The solids content in the fraction separated was found to be 3.5 g.

The fraction obtained through the first fractionation was loaded on an open column (diameter 35 mm, length 600 mm) containing 150 g of Sephadex LH-20 resin, followed by secondary fractionation using methanol as an elution solvent. At this time, the volume of the eluting solvent used was twice the volume of the column, and the eluting solvent was fractionated between 40 and 60 minutes at a flow rate of 1 ml per minute. The solids content in the fraction separated was found to be 1.5 g.

The supernatant obtained through the second fractionation was loaded on a high-performance liquid chromatography system of Waters Co., Ltd., using a preparative YMC ODS-A (250 x 20 mm, 5 m) reversed phase column and subjected to a gradient of water and acetone nitrile Lt; / RTI >

As a result of detection at an ultraviolet wavelength of 270 nm, it was confirmed that Compound 1 eluted at 14.33 minutes, Compound 2 at 14.67 minutes and Compound 3 at 15.12 minutes as shown in FIG. 2. These peaks were collected to obtain 25 mg and 20 mg , 215 mg of a single compound were obtained.

All the compounds thus separated were refrigerated for the next experiment.

The separated compounds were subjected to spectral analysis such as LC-MS, ¹ H-NMR, and ¹³ C-NMR. The results were compared with the analytical values reported in the literature, and as a result, The separated compounds belong to gallic acid and tannin hydrolyzable as a derivative thereof. The derivatives include galloyarbutin and galloylglucose which are galloylated with 2 to 4 derivatives. (See the structure of FIG. 4) as shown in Table 1 at the end.

division Compound name Compound 1  4,6-di-O-galloylarbutin (4,6-di-O-galloylarbutin) Compound 2  2,4,6-tri-O-galloyl-glucose (2,4,6-tri- Compound 3  1,2,4,6-tetra-O-galloyl-glucose (1,2,4,6-tetra-O-galloyl-

Example  2. Island Kirincho  For cosmetic use of extract Formulation

In order to decolorize and exfoliate the island giraffe extract of Example 1-1, decolorization and exfoliation tests were carried out using various kinds of activated carbon. SX Ultra, CGSP, SX 1G, KB-G, CASP, SX-plus and GAS 1240 + were used as the activated carbon.

The results are shown in Table 2.

<Discoloration and Degradation Test by Activated Carbon Type> Sample number Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Activated carbon
Kinds SX ULTRA CGSP SX 1G KB-G CASP SX-Plus GAS1240 + Decolorization degree 2 3 2 4 One One One Deflection 2 4 2 3 One 2 One

Decolorization / Degreasing: The higher the number, the better the decolorization and deodorization

CGSP was selected as the best activated carbon for cosmetic formulation of islet lipoprotein extracts, and as an excipient, butylene glycol (1 (1)) was used as the excipient, , 3-butyleneglycol), glycerin was selected as an excipient for cosmetic formulation of giraffin juice extract because it was easy to be water-soluble and no precipitate was formed even at a concentration exceeding the use concentration (10% aqueous solution).

Experimental Example 1. Cytotoxicity determination

To confirm the safety, human dermal fibroblast cells derived from adult skin and RAW 264.7 cell line derived from mouse macrophages were used. Human skin fibroblasts were inoculated at a density of 1 × 10 ⁶ cells / dish in a 100 mm 2 culture dish, and RAW 264.7 cell lines were inoculated at 3 × 10 ⁶ cells / dish. Penicillin (100 IU / ml), streptomycin / Ml) and Dulbecco's Modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum (FBS), and cultured in an incubator containing 5% carbon dioxide at 37 ° C. Cytotoxicity was measured by water-soluble tetrazolium salts (WST-1) assay. The WST-1 assay was carried out using a reddish water soluble substrate WST-1 obtained by succinatetetrazolium reductase, a dehydrogenase present in the intracellular mitochondrial electron transport system, as a formazan (3- (4,5-dimethylthizol-2-yl ) -2,5-diphenyltetrazolium bromide).

The experiment was repeated 3 times. The absorbance reduction rate of each experiment was calculated using the equation 1, and the concentration of the sample (Lethal dose 50) was calculated when the cytotoxicity was 50%.

[Equation 1]

In Example 2, the extract formulated into cosmetics after discoloration and exfoliation was suspended in PBS and used as an experimental sample.

As a result, as shown in Table 3, human dermal fibroblast cells showed a cytotoxicity of 87.7% in the treatment group of 10 ㎎ / ㎖ and 13.78% of cytotoxicity in the concentration of 1 ㎍ / ㎖. The LD50 value was 4.51 mg / ml.

In RAW 264.7 cells derived from mouse macrophages, the cytotoxicity was 93.11% at 10 ㎎ / ㎖ and 0.77% at 1 ㎍ / ㎖. The LD50 value was 5.49 mg / ml.

As described above, since the LD50 values for both cells are all at a concentration of 4,000 ppm or more, it is considered to be very safe in terms of cytotoxicity.

<Cytotoxicity test of formulated island giraffe extract> 셀 라인 Sample concentration 10 mg / ml 1 mg / ml 100 mg / ml 10 mg / ml 1 mg / ml 100 ng / ml 10 ng / ml Human dermal fibroblast 87.70 25.85 10.36 9.37 13.78 12.02 20.01 RAW 264.7 93.11 7.07 -7.61 14.57 0.77 -1.97 -3.02

Experimental Example 2. Confirmation of tyrosinase inhibitory activity

The inhibitory activity against tyrosinase, which is involved in the main steps of melanin synthesis, was measured according to the KFDA guidelines.

200 쨉 l of 0.1 M phosphate buffer solution (pH 6.5), 20 쨉 l of each of the three compounds finally separated in Example 1 or 20 쨉 l of the extract formulated in cosmetics after decolorizing and defatting in Example 2, And 20 μl of mushroom tyrosinase (1500 U / ㎖ to 2000 U / ml) were added in this order, and 40 μl of a 1.5 mM tyrosine solution was added thereto, followed by reaction at 37 ° C. for 10 to 15 minutes. The reacted reaction solution was measured for absorbance at 490 nm using an ELISA reader, and the tyrosinase inhibitory activity was calculated according to the formula 2, and the inherent dissociation of the test substance according to the Lineweaver-Burk's equation The constant (Km value) was calculated and the affinity with the mushroom tyrosinase was measured.

0.1 M phosphate buffer (pH 6.5) was used as a control and kojic acid was used as a positive control. The concentration of the sample (IC ₅₀ ) when the inhibitory activity of tyrosinase activity was 50% was calculated to express the degree of inhibitory activity of tyrosinase.

[Equation 2]

a: Absorbance after reaction of blank sample

b: Absorbance after reaction of the sample solution

a ', b': absorbance measured by replacing with buffer instead of tyrosine

The results of evaluating the inhibitory effect of tyrosinase enzyme by the above method are shown in Table 4 and FIGS. 5 to 7.

&Lt; Three compounds of tyrosinase inhibitory activity isolated from islet girinchus > kojic acid Island giraffe extract Compound 1 Compound 2 Compound 3 IC50 27 ppm
136.3 uM 1,000 ppm 110.4 uM 155.2 uM 25.3 uM Km (uM) 242.0 uM N.D. 52.7 uM 29.5 uM 11.9 uM

The inhibitory activities of the extracts of the island giraffe extracts were 24.48% at 50 ppm, 21.55% at 5 ppm and 18.61% at 0.5 ppm, and the IC ₅₀ value was more than 1000 ppm. The inhibitory activity of kojic acid used as a positive control was 77.42% at 50 ppm, 15.4% at 5 ppm and 1.47% at 0.5 ppm, and the IC ₅₀ value was 27 ppm (136.3 uM). The IC ₅₀ values of the three compounds isolated from the island giraffe extract were 25.3 uM for 1,2,4,6-tetra-O-galloyl- beta -glucose, 2,4,6-tri-O Galoisyl-beta-glucose was 155.2 uM, and 4,6-di-O-galoisyl-arbutin was 110.4 uM. The tyrosinase inhibitory activity of these three compounds was confirmed to be a major active ingredient of the island giraffe extract.

Galactosyl-β-glucosidase and a combination of 1,2,4,6-tetra-O-galloyl-β-glucose and 2,4,6-tri- The Km value of Km was measured to be 242 uM of Koji acid and 11.9 uM of 1,2,4,6-tetra-O-galloyl- beta -glucose, and 2,4,6-tri-O- It was confirmed that the three compounds isolated from the island giraffin extract with 29.5 uM of glucose and 52.7 uM of 4,6-di-O-galloyl-arbutin had higher affinity for tyrosinase enzyme than mushroom acid than kojic acid.

Taken together, the tyrosinase inhibitory activity of the island giraffin extract can be determined by measuring the activity of 1,2,4,6-tetra-O-galloyl- beta -glucose and 2,4,6-tri-O -Glyoyl- beta -glucose and 4,6-di-O-galloyl-arbutin. These three compounds have a higher affinity for mash-tyrosinase enzyme than kojic acid and have a tyrosinase activity . &Lt; / RTI &gt;

Experimental Example 3. Confirmation of melanin formation inhibitory activity

Measurement of inhibitory activity of melanin formation was carried out in accordance with the guidelines of the Pharmaceutical Affairs Division.

Melanoma cells (murine melanoma B16F10) were dispensed into a 24-well plate at 2 × 10 ⁴ cells / well and melanogenesis was induced by 3-isobutyl-1-methylxanthine (IBMX). In Example 2, The extracts formulated into cosmetics were treated by concentration and cultured at 37 ° C for 48 hours under 5% CO ₂ .

After culturing, the cells were collected, and the number of cells was measured and centrifuged at 1,200 rpm for 5 minutes to precipitate. After removing the supernatant, 1 ml of homogenization buffer (50 mM phosphate buffer pH 6.5, 1% triton X-100, 2 mM PMSF) , 200 μl of 1 N sodium hydroxide solution containing 10% DMSO was added and the absorbance was measured at 405 nm after vortexing.

The inhibitory activity of melanin production was compared with the control group of the sample without the extract and the arbutin - treated sample as the positive control. The concentration of the sample when the inhibition rate of melanin formation was 50% (IC ₅₀ ) was calculated to express the degree of inhibition of melanin formation.

As shown in FIG. 8, the inhibitory effect of melanin on murine melanoma B16F10 cell line induced by IBMX was 82.27% at 100 ppm, 28.79% at 50 ppm, and 0.32% at 10 ppm compared to the control group, And the IC ₅₀ value was found to be 70 ppm.

In addition, the arbutin used as a positive control showed an inhibitory activity of 32.03% at 100 ppm, 10.52% at 50 ppm and 0.16% at 10 ppm, and the IC value was 240 ppm.

As a result of comparing the inhibitory effects of melanin on the islet giraffin extract and arbutin, it was confirmed that the melanin formation inhibitory activity was 2.5 times higher at 100 ppm concentration.

Formulation Example 1. Preparation of cosmetic composition

1-1. Manufacture of cream

4.6 parts by weight of the three compounds finally isolated in Example 1 or a pharmaceutically acceptable salt thereof, the substance selected from the extract formulated into cosmetics through decolorization and exfoliation in Example 2, 2.8 parts by weight of cetostearyl alcohol 2.6 parts by weight of beeswax, 1.4 parts by weight of stearic acid, 2 parts by weight of glycerin monostearate, 2 parts by weight of fat-100 stearate, 1.4 parts by weight of sesquioleate sorbitol, 4 parts by weight of jojoba oil, 3.8 parts by weight of squalane , 1.1 parts by weight of polysorbate 60, 2 parts by weight of macadia oil, 0.2 parts by weight of tocopheryl acetate, 0.4 parts by weight of methylpolysiloxane, 0.1 part by weight of ethylparaben, 0.1 parts by weight of propylparaben, 0.1 part by weight of Euxyl K- 7 parts by weight of 3-butylene glycol, 0.05 part by weight of methylparaben, 6 parts by weight of glycerin, 0.2 parts by weight of d-pandenol, 0.2 parts by weight of triethanolamine, 0.2 parts by weight of pt 41891 and 46.05 parts by weight of p- To prepare a cream.

1-2: Manufacture of Lotion

3.5 parts by weight of the three compounds finally isolated in Example 1 or a pharmaceutically acceptable salt thereof, an extract from the extract formulated into cosmetics through decolorization and exfoliation in Example 2, 1.6 parts by weight of cetostearyl alcohol 1.6 1.4 parts by weight of stearic acid, 1.8 parts by weight of glycerin monostearate, 0.6 parts by weight of sesquioleate, 4.8 parts by weight of squalene, 2 parts by weight of macadia oil, 2 parts by weight of jojoba oil 2 0.4 parts by weight of tocopheryl acetate, 0.2 parts by weight of methylpolysiloxane, 0.1 parts by weight of ethylparaben, 0.1 parts by weight of propylparaben, 4 parts by weight of 1,3-butylene glycol, 0.1 parts by weight of methylparaben, 4 parts by weight of glycerin, 0.15 part by weight of d-pandinol, 0.1 part by weight of allantoin, 4 parts by weight of carrageenan (2% aq. Sol), 0.15 part by weight of triethanolamine, 3 parts by weight of ethanol, , and 46.05 parts by weight of p-H2O are mixed to prepare a lotion.

Claims (10)

Tetra-O-galloyl-beta-glucose, 2,4,6-tri-O- -Glucose (1,2,4,6-tetra-O-galloyl-beta-glucose) and salts thereof as an active ingredient. The method according to claim 1,
Characterized by containing as an active ingredient 1,2,4,6-tetra-O-galloyl-beta-glucose or a salt thereof Wherein the cosmetic composition is a cosmetic composition for skin whitening. Tetra-O-galloyl-beta-glucose, 2,4,6-tri-O- -Glucose (1,2,4,6-tetra-O-galloyl-beta-glucose) and salts thereof as an active ingredient. The method of claim 3,
Characterized by containing as an active ingredient 1,2,4,6-tetra-O-galloyl-beta-glucose or a salt thereof Wherein the composition for skin whitening is a composition for skin whitening. Tetra-O-galloyl-beta-glucose, 2,4,6-tri-O- -Glucose (1,2,4,6-tetra-O-galloyl-beta-glucose) and salts thereof as an active ingredient, A pharmaceutical composition for the prevention and improvement of diseases. 6. The method of claim 5,
Characterized by containing as an active ingredient 1,2,4,6-tetra-O-galloyl-beta-glucose or a salt thereof Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and / or amelioration of hypercholesterolemia. 6. The method of claim 5,
The pharmaceutical composition for prevention and improvement of skin hypercholesterolemia due to excessive melanin production, characterized in that the skin hypercholesterolemia due to the excessive production of melanin is a disease selected from the group consisting of spots, freckles, blotch and hypercholesterolemia. Tetra-O-galloyl-beta-glucose, 2,4,6-tri-O- -Glucose (1,2,4,6-tetra-O-galloyl-beta-glucose) and salts thereof as an active ingredient, A food composition for prevention and improvement of diseases. 9. The method of claim 8,
Characterized by containing as an active ingredient 1,2,4,6-tetra-O-galloyl-beta-glucose or a salt thereof Wherein the melanin-induced excessive production of melanin is caused by the production of melanin. 9. The method of claim 8,
Wherein the skin hyperkeratosis disease caused by the excessive production of melanin is a disease selected from the group consisting of spots, freckles, blotch and hyperpigmentation, and a food composition for preventing and improving skin hypercholesterolemia caused by excessive production of melanin.

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