KR101492074B1 - Compositions containing an extract of hydrangea macrophylla for. otaksa for skin whitening - Google Patents

Abstract

The skin whitening composition containing the hydrangea extract of the present invention as an active ingredient has safety to the skin and is superior in the effect of inhibiting tyrosinase activity, so that it can be usefully applied to external preparations for skin whitening.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for skin whitening comprising an extract of hydrangea as an active ingredient. FOR SKIN WHITENING}

The present invention relates to a skin whitening composition containing an extract of hydrangea as an active ingredient.

The skin color of a person is determined by various factors such as the activity of melanocyte making melanocyte, the distribution of blood vessels, the thickness of skin, and the presence or absence of pigment inside and outside the body such as carotenoid and bilirubin. Among them, tyrosinase And melanin, which is produced by the action of various enzymes, is most important. The formation of melanin pigment is affected by environmental factors such as genetic factors, hormonal secretion, physiological factors such as stress, and ultraviolet irradiation. Melanin has an important function of protecting the body from ultraviolet light while being present in the skin. However, when melanin is overproduced, it not only forms spots and freckles but also promotes skin aging and plays an important role in skin cancer induction

In order to treat or alleviate excessive melanin pigmentation caused by skin pigmentation abnormalities such as dullness, freckles and pigmentation, and ultraviolet ray exposure, it has been previously known that ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione, Substances having the same tyrosinase-inhibiting activity have been used in cosmetics or medicines, but they have been limited in their use due to insufficient whitening effect, safety to the skin, formulation in cosmetics, and stability within the formulation It is true.

On the other hand, there is little research and report on skin whitening effect on hydrangea.

Patent No. 10-0681638

An object of the present invention is to provide a composition for skin whitening which has safety against the skin and has excellent whitening effect by containing the extract of Hydrangea, which is a natural plant material, as an active ingredient.

In order to solve the above-mentioned problems, the present invention provides a skin whitening composition comprising an extract of hydrangea as an active ingredient.

In one embodiment of the present invention, the hydrangea extract may be at least one selected from the group consisting of stem, leaf and flower of hydrangea.

In one embodiment of the present invention, the hydrangea extract is selected from the group consisting of anhydrous and lower alcohol having 1 to 4 carbon atoms, acetone, butylene glycol, ethyl acetate, diethyl acetate, diethyl ether, benzene, chloroform and hexane And may be extracted using any one or more selected solvents.

In one embodiment of the present invention, the content of the hydrangea extract may be 0.0001 to 10% by weight based on the total weight of the composition.

In one embodiment of the present invention, the skin whitening composition may be one which whitens skin through inhibition of tyrosinase activity.

The skin whitening composition containing the hydrangea extract of the present invention as an active ingredient has safety to the skin and is superior in the effect of inhibiting tyrosinase activity, so that it can be usefully applied to external preparations for skin whitening.

1 is a graph showing the total phenolic content of a composition containing a hydrangea extract according to an embodiment of the present invention.
2 is a graph showing the total flavonoid content of a composition containing a hydrangea extract according to an embodiment of the present invention.
3 is a graph showing cell viability according to concentration of a composition containing a hydrangea extract according to an embodiment of the present invention.
4 is a graph showing the amount of melanin according to the concentration of a composition containing a hydrangea extract according to an embodiment of the present invention.
5 is a graph showing tyrosinase activity according to the concentration of a composition containing a hydrangea extract according to an embodiment of the present invention.
6 is a graph showing the results of western blotting of a composition containing a hydrangea extract according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail in order to facilitate the present invention by those skilled in the art.

The present invention provides a skin whitening composition comprising an extract of hydrangea as an active ingredient.

Hydrangea is a deciduous shrub of dicotyledonous roe-mallow, Hydrangea macrophylla for. otaksa and the like are known. Hydrangea is leaf-shaped, egg-shaped, thick, with sawtooth on the edge. The flower is bloomed in June ~ July with neutralization, 10 ~ 15cm in size, and runs on the flower of the mountain. In the private sector, flowers of hydrangea were used as an antipyretic. However, there have been no studies on the inhibition of tyrosinase activity and the whitening effect of hydrangeas.

In the present invention, although not particularly limited, it is preferable to use a ground portion of a hydrangea. The top part of the hydrangea may be at least one selected from the group consisting of stem, leaf and flower of hydrangea.

The method for producing the hydrangea extract of the present invention is not particularly limited. For example, hydrants can be dried and crushed; The pulverized product obtained is mixed with at least one extraction solvent selected from the group consisting of anhydrous and lower alcohol having 1 to 4 carbon atoms, acetone, butylene glycol, ethyl acetate, diethyl acetate, diethyl ether, benzene, chloroform and hexane , Preferably with cooling or warming with methanol to extract at least once; The extract thus obtained can be prepared by concentrating the extract at a suitable temperature in a distillation apparatus equipped with a cooling condenser, preferably at 40 to 50 ° C under reduced pressure. Preferably, the extraction may be two extractions at room temperature for 48 hours.

As the extraction method, it may be carried out for about 12 to 96 hours in the case of cold beating, or aged at 4 to 25 ° C for 3 to 20 days at a temperature of 4 to 25 ° C with an anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, ethyl acetate or diethyl ether as a solvent The active ingredient can be extracted. In the case of warming, it is preferable that the heating is carried out at a temperature close to the reflux temperature of the solvent for about 5 to 24 hours although it depends on the kind and temperature of the extraction solvent.

The hydrangea extract is preferably contained in an amount of 0.0001 to 10% by weight based on the total weight of the composition. When the hydrangea extract is contained in an amount of less than 0.0001% by weight based on the total weight, the whitening effect is insignificant. When the hydrangea extract is contained in an amount of more than 10% by weight,

The skin whitening composition according to the present invention may be, for example, a pharmaceutical composition or a cosmetic composition.

The pharmaceutical composition for skin whitening may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt and / or a buffer for controlling osmotic pressure, and other therapeutically useful substances, And can be formulated into various parenteral dosage forms accordingly. Parenteral dosage forms, and may be, for example, but not limited to lotions, ointments, gels, creams, patches or spray formulations.

The dosage of the active ingredient is within the level of those skilled in the art, and the daily dose of the drug depends on various factors such as the degree of progress of the subject to be administered, the age of onset, age, health condition, As a standard, it is generally possible to administer the composition at a dose of 1 / / kg to 200 mg / kg, preferably 50 / / kg to 50 mg / kg, once or twice a day, Are not intended to limit the scope of the present invention.

The cosmetic composition for whitening skin is not particularly limited and may be appropriately selected according to the purpose. For example, skin lotion, skin softener, skin toner, astringent, lotion, milky lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing But the present invention is not limited thereto, and may be manufactured by any one or more formulations selected from the group consisting of a foam, a cleansing lotion, a cleansing cream, a body lotion and a body cleanser.

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 powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, 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, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

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 content of the active ingredient is not particularly limited, but may be in the range of 0.0001 to 10% by weight based on the total weight of the composition. When the active ingredient satisfies the above content, it can exhibit excellent efficacy without side effects.

In addition to the hydrangea extract prepared above, the cosmetic composition may further contain ingredients included in a functional additive and a general cosmetic composition. The functional additives may include water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides, sphingolipids and seaweed extracts.

The cosmetic composition of the present invention may further contain, in addition to the above-described functional additive, components contained in a general cosmetic composition as required. Examples of the other ingredients that can be included in the composition include humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbents, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, Accelerators, coolants, antiperspirants, purified water, and the like.

Further, the present invention relates to an external preparation for skin comprising the composition for skin whitening, wherein the external preparation for skin is a generic term that can be applied to any external skin, and cosmetics and medicines of various formulations may be included. The external preparation for skin is not particularly limited, but may be, for example, a skin external preparation for preventing skin aging or improving skin wrinkles.

The present invention has found an excellent skin whitening effect using a composition containing a hydrangea extract as a natural raw material. The composition for skin whitening according to the present invention has a tyrosinase activity inhibiting function and can improve skin whitening, as can be seen from the test examples to be described later.

The present invention will be described in more detail through the following examples. However, the examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Example 1] Preparation of hydrangea extract

Hydrangea macrophylla for. otaksa ), the ground part was dried, pulverized and extracted twice with methanol for 48 hours at room temperature. The extract was concentrated under reduced pressure at 45 캜 to obtain a hydrangea extract.

[Test Example 1] Effect of inhibiting tyrosinase activity

The hydronephrine extract of Example 1 was subjected to a test for measuring the inhibitory effect of tyrosinase activity. Add 40 μL of the sample according to the concentration and add 50 units / mL mushroom tyrosinase (Sigma) and 8.3 mM L-DOPA (Sigma) using 67 mM sodium phosphate buffer (pH 6.8) And 40 μL and 120 μL, respectively, were added to 96 wells. The hydrorea extract and enzyme were pretreated for 10 minutes at 37 ° C, L-DOPA was added as a substrate, and the reaction was further carried out at 37 ° C for 10 minutes. The absorbance was measured at 490 nm, which is the absorption wavelength of the reaction product, dopa chrome, and the enzyme inhibitory activity of the sample was determined by the following formula (1). In equation 1, Control is the absorbance measured without the sample, Blank-c is the absorbance measured without both L-DOPA and sample, and Blank-s is the absorbance measured without L-DOPA. As a comparative example, koji acid was used, and the results are shown in Table 1 below.

(식 1)

(Equation 1)

IC ₅₀ Example 1 97.3 ± 3.2 μg / ml Comparative Example 1 (koji acid) 70.8 ± 2.6 μg / ml

[Test Example 2] Measurement of total phenolic content

The total phenolic content of the hydrangea extract of Example 1 was measured. Add 80 μl of Folin-Denis reagent to 40 μl of sample and mix well. After standing for 3 minutes, 80 μl of 10% Na ₂ CO ₃ was added, and the absorbance was measured at 725 nm within 1 hour. The amount was converted to a standard curve using gallic acid and tannic acid solution.

The results are shown in Fig. As can be seen in FIG. 1, the total polyphenol at 1000 μg / ml showed 68.68 ± 17.61 mg / g.

[Test Example 3] Measurement of total flavonoid content

The total flavonoid content of the hydrangea extract of Example 1 was measured. To measure the total flavonoid content, 80 80 of 80% ethanol was added to 20 100 of 100% methanol extract sample and 10 20 of 20 mg / mL aluminum trichloride was added. 10 μl of a 1M potassium acetate solution and 80 μl of 80% ethanol were added thereto, followed by mixing at room temperature for 40 minutes. Absorbance was measured at 415 nm with a UV-VIS spectrophotometer. Quercetin (SIGMA) was used as a reference material and a standard calibration curve was prepared to calculate the total flavonoid content.

The results are shown in Fig. As can be seen in Figure 2, the total flavonoid at 1000 μg / ml was 12.89 ± 3.08 mg / g.

[Test Example 4] Cell viability assay (Cell viability assay)

MTT [3- (4,5-dimethylthiazo-2-yl) -2,5-diphenyl tetrazolium bromide, Sigma] was performed using a B16F10 melanoma cell. Melanoma cells were plated on a 12-well plate at a density of 1 × 10 ⁵ cells / well and cultured in 10% fetal bovine serum / DMEM (GIBCO) medium for 24 hours. The diluted sample (hydrated extract of Example 1 and kojic acid) was added and again mixed with CO ₂ And cultured in an Incubator. After incubation, MTT solution (1 mg / mL) was added to each well, followed by further incubation for 2 hours. After the medium containing MTT was removed, isopropanol was added to each well and stirred. The absorbance was measured at 550 nm using a plate reader. The control group (α-MSH) was incubated with medium only without sample treatment, and the absorbance was compared.

The results are shown in Fig. As shown in FIG. 3, the concentration was found to be insignificant for B16F10 cells and the maximum permissible level (MPL) was found to be less than 3 μg / ml.

[Test Example 5] Measurement of melanin content

The melanin content of the hydrangea extract of Example 1 was measured. The B16 melanoma cells used in this experiment were cultured in DMEM supplemented with 10% FBS, placed in a 12-well plate at 4 × 10 ⁴ cells / well, and cultured for 24 hours at 37 ° C. in 5% CO ₂ Lt; / RTI > B16F10 cells were treated with kojic acid (200 μM) and hydronephrine in a concentration-dependent manner to determine melanin synthesis inhibition. After the culture, the culture medium was removed, and a fresh DMEM culture medium containing 10% FBS containing each sample was added, followed by incubation at 37 ° C for 3 days under 5% CO ₂ . After completion of the incubation, the culture medium was removed and washed with PBS. The cell pellet was recovered by treatment with trypsin-EDTA. The recovered pellet was centrifuged at 10,000 rpm for 10 minutes and then the supernatant was removed. The cell pellet was dried at 70 ° C, and 200 μL of 1 N NaOH containing 10% DMSO was added to dissolve the melanin in the cells at 70 ° C. 100 μL of this solution was placed in a 96-well plate and the absorbance was measured at 405 nm with an ELISA plate reader to determine the amount of melanin.

The results are shown in Fig. As can be seen from FIG. 4, the positive control group, kojic acid, significantly decreased (p <0.001) the melanin synthesis inhibitory effect by 13.3% at 200 μM compared to the control group (α-MSH) a-MSH alone: *** p < 0.001). The effect of 1, 3, and 3 μg / ml of hydrangea extract was better than that of 200 μM of kojic acid at 0.1, 0.5, 1, and 3 μg / ml, respectively, by 2.3, 12.6, 26.2, and 37.5%

[Test Example 6] Analysis of tyrosinase activity (Cellular tyrosinase assay)

The tyrosinase activity of the hydrangea extract of Example 1 was measured. Mouse melanoma B16F10 was added to a culture dish at a density of 1 × 10 ⁵ cells / dish and cultured in DMEM medium containing 5% CO ₂ and 10% FBS at 37 ° C for 24 hours. Then, 10 nM of α- MSH and diluted samples were added for 3 days. Cells were washed twice with PBS, treated with trypsin, and transferred to ependorf tubes for centrifugation. Cell suspensions were washed with 100 μL of lysis buffer (1% NP-40, 0.25% sodium 1 mM sodium orthovanadate, 1 mM sodium fluoride, 150 mM NaCl, 1 μg / ml leupeptin, 50 mM Tris-Cl (pH 7.5), 1 mM EDTA and 1 mM PMSF (phenyl methyl sulfonyl fluoride). The mixture was allowed to stand at 4 DEG C for 1 hour to elute the protein, centrifuged, and the supernatant was taken and used as the enzyme solution. After addition of 5 mM L-DOPA, changes in absorbance were observed at 490 nm after a certain period of time.

The results are shown in Fig. 5 (Significant differences compare with the a-MSH alone: ** p < 0.05, *** p < 0.001). As shown in FIG. 5, when the melanocytes were treated with hydrangea extract and kojic acid, the activity of the hydrorephrine extract was 100.1, 86, 85.6, and 77.5% at 0.1, 0.5, 1, and 3 μg / , Indicating that tyrosinase activity was inhibited at a concentration lower than that of 200 μM (89.4%) of kojic acid

[Test Example 7] Western blotting assay [

The hydrangea extract of Example 1 was analyzed by Western blotting. B16F10 cells cultured and treated with a sample (hydrangea extract of Example 1 and kojic acid) were suspended in lysis buffer (1% NP-40, 0.25% sodium deoxychlolate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 150 mM 1 mM EDTA, 1 mM PMSF (phenyl methyl sulfonyl fluoride)) at 4 ° C for 1 hour and centrifuged at 13,500 rpm for 15 minutes. Respectively. Protein quantification was quantitated using bradford reagent, and 30 μg protein was separated by 10% SDS-PAGE and blotted on a nitrocellulose membrane.

Antibodies were detected by chemiluminescence using ECL western blotting detection reagents from BIO-RAD using SantaCruz products. In other words, to further confirm the mechanism of inhibition of melanin synthesis in hydrangea extract, the degree of protein expression, which plays an important role in the synthesis of melanin, was confirmed by Western blotting.

The results are shown in Fig. As shown in FIG. 6, the hydrangea extract contains tyrosinase, tyrosinase related protien-1 (TRP-1), tyrosinase related protien-2 (TRP-2) , And MITF (microphthalmia-associated transcription factor) protein expression in a concentration-dependent manner.

Formulation examples of the composition containing the hydrangea extract of the present invention are described below, but the composition of the present invention is not limited to this example.

[Formulation Example 1] Lotion-type formulation

The hydrangea extract of Example 1 3.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Water soluble collagen (1% aqueous solution) 1.00

Sodium citrate 0.10

Citric acid 0.05

Licorice extract 0.20

1,3-butylene glycol 3.00

Purified water balance

(Unit: wt%)

[Formulation Example 2]

The hydrangea extract of Example 1 1.00

Polyethylene glycol monostearate 2.00

Self emulsifying monostearate glycerin 5.00

Cetyl alcohol 4.00

Squalane 6.00

Tri-2-ethylhexane glyceryl 6.00

Sphingo glycolipid 1.00

1,3-butylene glycol 7.00

Purified water balance

(Unit: wt%)

[Formulation Example 3] Packed preparation

The hydrangea extract of Example 1 5.00

Polyvinyl alcohol 13.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Lauroylhydroxyproline 1.00

Water soluble collagen (1% aqueous solution) 2.00

1,3-butylene glycol 3.00

Ethanol 5.00

Purified water balance

(Unit: wt%)

[Formulation Example 4] Cosmetic liquid preparations

Hydrangea extract of Example 1 2.00

Hydroxyethylene Cellulose (2% aqueous solution) 12.00

Xanthan gum (2% aqueous solution) 2.00

1,3-butylene glycol 6.00

Dark glycerin 4.00

Sodium hyaluronate (1% aqueous solution) 5.00

Purified water balance

(Unit: wt%)

Claims (5)

Wherein the hydrangea extract comprises at least one selected from the group consisting of stem, leaf and flower of hydrangea as an active ingredient, and the hydrangea extract is extracted using methanol as a solvent. delete delete The method according to claim 1,
Wherein the content of the hydrangea extract is 0.0001 to 10% by weight based on the total weight of the composition. The method according to claim 1,
Wherein the skin whitening composition whitens the skin through inhibition of tyrosinase activity.

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