KR102453959B1 - Cosmetic composition comprising Castanopsis sieboldii leaves extract or its fraction as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition, and more specifically, to a cosmetic composition having antioxidant and whitening activity comprising a leaf extract of castanopsis sieboldii or a fraction thereof as active ingredients. The leaf extract of castanopsis sieboldii or the fraction thereof of the present invention has excellent antioxidant activity, and effectively suppresses melanin production through a mechanism of inhibiting expression of protein related to tyrosinase inhibitory activity and the melanin production. The composition of the present invention comprising a leaf extract of castanopsis sieboldii or a fraction thereof as active ingredients is usefully used as a functional cosmetic composition to provide an excellent whitening effect and an antioxidant effect by suppressing melanin pigments without irritation to skin. Especially, since the extract or the fraction thereof of the present invention is a natural material derived from leaves of the castanopsis sieboldii, the cosmetic composition of the present invention is safe even for long-term use.

Description

Cosmetic composition comprising Castanopsis sieboldii leaves extract or its fraction as an active ingredient}

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition having antioxidative and whitening activity comprising an extract of a cypress chestnut leaf or a fraction thereof as an active ingredient.

The skin is a very important tissue that protects the human body while in direct contact with the external environment and has biochemical and physical functions. This tissue is largely divided into three types, namely, the epidermis, dermis, and subcutaneous tissue. The color of human skin varies depending on the number, size, type, and distribution of melanosomes containing melanin in skin cells.

Melanosomes are produced by melanocytes, and melanin, a black pigment produced in the epidermis, is produced in cells called melanocytes. Melanin absorbs the light energy of ultraviolet rays in sunlight and plays a role in preventing the cells deeper in the sunlight from being damaged by ultraviolet rays. When such melanin is produced abnormally low, skin lesions such as vitiligo are induced. Conversely, when melanin is synthesized excessively by ultraviolet rays, it not only damages the skin, but also forms spots and freckles, and furthermore, it can cause skin cancer.

Tyrosinase enzyme is known to play the most important role in the formation of melanin, and this enzyme produces dopaquinone that acts on an amino acid called tyrosine present in tissues. Melanin, the black pigment, is produced. Therefore, a composition for whitening can be made in several ways as follows, depending on which process it acts during the step capable of inhibiting melanin production.

The first is to contain a component that can block ultraviolet rays, which is the main cause of melanin production. The composition by this method contains a light scattering agent or a light blocker, thereby reducing DNA damage or inflammation caused by ultraviolet rays, such as tyrosine such as glucosamine. It can inhibit melanin production by inhibiting the synthesis of core carbohydrates required to be active (acts on whitening A phase). The second is to use a substance that interferes with the function of tyrosinase involved in melanin production, such as enzymatic acid (kojic acid), leucinol, eric acid, or arbutin (acts on whitening stage B). Thirdly, there is a method that can prevent the progression to black melanin, Eu-Melanin, by stabilizing dopachrome (acting on whitening C step). Fourth, while tyrosine undergoes complex oxidation and condensation reactions through dopa and dopachrome, it uses a substance such as vitamin C that can produce a whitening effect by exerting a strong antioxidant effect (acting on the whitening stage D). Fifth, there is a method that can remove and reduce the generated melanin from the stratum corneum by using an enzyme or AHA (Alpha Hydroxy Acid) (acting on the whitening E step).

In the conventional cosmetic field, as a whitening component, for example, a substance that inhibits tyrosinase enzyme activity such as kojic acid, arbutin, etc., hydroquinone, vitamin C (L-Ascorbic acid) and derivatives thereof and various plant extracts have been used. However, the substance inhibiting the tyrosinase enzyme activity has low stability in the prescription system, so it is decomposed and colored, or its use is limited due to the occurrence of off-flavor, efficacy at the biological level, unclear effect, and safety issues. to be.

Accordingly, the present inventors were trying to find a material that is derived from a natural substance and that is stable to the human body and can exhibit an excellent whitening effect. The present invention by confirming that the leaf extract and its fractions not only have antioxidant activity, but can also effectively inhibit the synthesis of melanin by inhibiting the expression of proteins related to tyrosinase activity and melanin production in a concentration-dependent manner. completed.

Korean Patent Publication No. 10-2012-0054375

Accordingly, it is an object of the present invention to provide a cosmetic composition effective for skin whitening by being stable to the human body and having excellent antioxidant activity and melanin inhibitory activity.

In order to achieve the object of the present invention as described above,

The present invention provides a cosmetic composition comprising, as an active ingredient, an extract of a cypress chestnut leaf or a fraction thereof.

In one embodiment of the present invention, the extract may be an ethanol solvent extract.

In one embodiment of the present invention, the fraction may be any one organic solvent fraction selected from the group consisting of hexane, chloroform, ethyl acetate and butanol.

In one embodiment of the present invention, the cosmetic composition may be for antioxidant.

In one embodiment of the present invention, the cosmetic composition may be for skin whitening.

In one embodiment of the present invention, the extract or fraction can inhibit melanin production through a mechanism of inhibiting the activity of tyrosinase and inhibiting the expression of proteins related to melanogenesis.

In one embodiment of the present invention, the extract or fraction may be included in the composition at a concentration of 0.0001 to 100 μg / ml.

In one embodiment of the present invention, the cosmetic composition is a flexible lotion, gel, water-soluble liquid, milk lotion, nourishing cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, face dry product, solid anhydrous product , oil dispersion in aqueous phase using globules, ionic lipid vesicles, non-ionic lipid vesicles, ointment, cleansing foam, cleansing water, pack, body oil, oil-in-water makeup base, water-in-oil makeup base, foundation, skin It may be one type of formulation selected from the group consisting of cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color and eyebrow pencil.

The leaf extract or fraction thereof of the present invention has excellent antioxidant activity, and can effectively inhibit melanin production through a mechanism of inhibiting the expression of proteins related to melanin production in addition to tyrosinase inhibitory activity. The composition of the present invention containing In particular, the extract of the present invention or a fraction thereof is a natural material derived from the leaves of the Quercus chestnut tree, and the cosmetic composition of the present invention comprising the same has the advantage of being safe for long-term use.

1 is a manufacturing process diagram schematically showing the extraction process of an extract of a cypress chestnut leaf and a fraction thereof of the present invention.
Figure 2 is a graph showing the results of the MTT assay showing the cytotoxicity of the acetate fractions of the leaves of the chestnut chestnut according to the concentration.
Figure 3 is a graph showing the tyrosinase inhibitory activity of extracts and fractions thereof from the leaves of Quercus chestnut.
Figure 4 is a western expression level of proteins (tyrosinase, TRP-1, TRP-2, p-PKA, p-CREB) related to melanin production after treatment by concentration in melanoma cells of the acetate fraction of the cypress chestnut leaf. These are the results measured by blot analysis.

The present invention is characterized in that it provides a cosmetic composition comprising an extract of a cypress chestnut leaf or a fraction thereof as an active ingredient.

' Castanopsis sieboldii ' referred to in the present specification is an evergreen broad-leaved tall tree growing at the foot of a mountain at the seaside in a genus of oak and buckwheat, 15m in height and 1m in diameter. The bark is black-gray, and the leaves are alternate. The leaf shape is lanceolate or long oval with a sharp tip, and there is a wavy serrate in the upper half. The front side of the leaf is glossy green and hairless, and the back side is covered with scale hairs, so it turns white, but there are some that are light brown. Flowers are male and female, bloom in June, and hang in tail-shaped inflorescences. The fruit is a nut, ovate, oval, and wrapped in a flower stalk with the lower part. It matures in October of the next year. This species is distinguished by its slightly larger leaves and long oval and oval shape compared to Buckwheat chestnut. It is native to the west and southern seas of Korea and Jeju Island, and is distributed in Japan, China, Taiwan, India, and Java. Plant it as a landscape tree, street tree, and green tree. The wood is used for building and furniture materials, the fruits are edible, and the bark is used as a dye.

To date, there have been no studies on the antioxidant and whitening activities of extracts of cypress chestnut leaves or fractions thereof.

In the present invention, it was confirmed for the first time that the extract of the cypress chestnut leaf or a fraction thereof can be used as a material for functional cosmetics because it has excellent whitening activity as well as antioxidant function.

The extract or fractions thereof according to the present invention may be those obtained by extraction and separation from nature using methods known in the art for extraction and separation, and the 'extract' as defined in the present invention is an appropriate solvent. It is extracted from the leaves of Castanopsis sieboldii leaves using

As a suitable solvent for extracting the extract from the leaves of the cypress chestnut tree, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but is not limited thereto, for example, purified water , methanol (methanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), alcohols having 1 to 4 carbon atoms, including butanol (butanol), acetone (acetone), ether (ether), benzene (benzene) Various solvents such as , chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. In consideration of safety, etc., preferably ethanol (ethanol) may be used, and more preferably 70-80% ethanol is used.

As the extraction method, any one of methods such as hot water extraction, cold extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method. There is no limitation on the method for preparing the extract of the sycamore chestnut leaf of the present invention, and any known method may be used.

In addition, the fraction of the quince nutmeg leaf extract of the present invention can be obtained by fractionating the extract from the quince chestnut leaf extract extracted as described above from non-polar to polar organic solvents to obtain each solvent fraction.

A suitable solvent for fractionation of the fraction of the leaf extract of Prunus oleifera may be hexane, chloroform, ethyl acetate, butanol, or a mixed solvent thereof, in particular, obtained by sequential fractionation using hexane, chloroform, ethyl acetate and butanol. It is better to be a fraction.

The extract or fractions thereof according to the present invention can be obtained by cooling, heating and filtering at room temperature by a conventional method known in the art to obtain a liquid, or additionally evaporating the solvent, spray-drying, or freeze-drying. have.

For example, the extract or fraction thereof contained in the composition of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying the extract or fraction extracted and fractionated as described above. You may. In addition, the extract or fraction is further purified using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. you may get

Therefore, in the present invention, the syphilitic chestnut leaf extract or its fraction is a concept including all extracts, fractions and purified products obtained in each step of extraction, fractionation or purification, and dilutions, concentrates or dried products thereof.

The composition of the present invention corresponds to a cosmetic composition comprising an extract of a cypress chestnut leaf or a fraction thereof as an active ingredient.

The cosmetic composition of the present invention may have antioxidant activity and whitening activity.

The extract or a fraction thereof, which corresponds to the active ingredient of the cosmetic composition of the present invention, has excellent antioxidant activity and effectively stimulates melanin production through a mechanism of inhibiting the expression of proteins related to melanin production in addition to tyrosinase inhibitory activity. It can be inhibited, the composition of the present invention comprising it as an active ingredient may exhibit antioxidant activity and whitening activity.

In one embodiment of the present invention, the extract or a fraction thereof from the sycamore chestnut leaf extract may be included in the cosmetic composition at a concentration of 0.0001 to 100 μg/ml.

Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent lotion, softening lotion, nutrient lotion, various creams, essence, pack, foundation, and cleansing, face wash, soap, treatment, and serum. etc.

Specific formulations of the cosmetic composition of the present invention include flexible lotions, gels, water-soluble liquids, milk lotions, nourishing creams, massage creams, essences, oil-in-water emulsions, water-in-oils emulsions, face dry products, solid anhydrous products, and globules. Oil dispersion in the aqueous phase used, ionic lipid vesicles, non-ionic lipid vesicles, ointment, cleansing foam, cleansing water, pack, body oil, oil-in-water makeup base, water-in-oil makeup base, foundation, skin cover, lipstick, It contains one formulation selected from the group consisting of lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color and eyebrow pencils.

According to a preferred embodiment of the present invention, the content of the active ingredient of the present invention (P. nutmeg leaf extract or a fraction thereof) is 0.00001-40% by weight, preferably 0.0005-40%, more preferably based on the total weight of the composition. preferably 0.0005-20% by weight. If the content of the active ingredient (P. nutmeg leaf extract or a fraction thereof) is less than 0.00001% by weight, the antioxidant effect and skin whitening effect are greatly reduced, and if it exceeds 20% by weight, it may cause skin irritation. Problems may arise.

On the other hand, the cosmetic composition according to the present invention can also be formulated by stabilizing the active ingredient (extract of cypress chestnut leaf or a fraction thereof) inside the nanoliposome. When the active ingredient (Prunus oak leaf extract or a fraction thereof) is contained inside the nanoliposome, the active ingredient is stabilized to solve problems such as precipitation, discoloration, and odor during formulation, and to increase the solubility and percutaneous absorption of the ingredient. It is possible to express the expected efficacy from the extract to the maximum.

In the present invention, the nanoliposome refers to a liposome having an average particle diameter of 10 to 500 nm as having the form of a conventional liposome. According to a preferred embodiment of the present invention, the average particle diameter of the nanoliposome is 50 to 300 nm, more preferably 100 to 200 nm. When the average particle diameter of the nanoliposome exceeds 500 nm, improvement in skin penetration and formulation stability among the technical effects to be achieved in the present invention are very weak.

According to the present invention, the nanoliposome used for stabilizing the active ingredient (Prunus chestnut leaf extract or a fraction thereof) may be prepared by a mixture containing a polyol, an oily ingredient, a surfactant, a phospholipid, a fatty acid, and water.

The polyol used in the nanoliposome of the present invention is not particularly limited, and preferably propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isopropylene glycol, pentylene glycol, erythritol, xylitol , at least one selected from the group consisting of sorbitol and mixtures thereof. The amount used is 10 to 80% by weight, preferably 30 to 70% by weight, based on the total weight of the nanoliposome.

As the oil component used in the preparation of the nanoliposome of the present invention, various oils known in the art may be used, preferably hydrocarbon-based oil such as hexadecane and paraffin oil, ester-based synthetic oil, Silicone oils such as dimethicone and cyclomethicone, animal and vegetable oils such as sunflower oil, corn oil, soybean oil, avocado oil, sesame oil and fish oil, ethoxylated alkyl ether oil, propoxylated alkyl ether oil , phytosphingosine, _{sphingonoid lipids such as sphingosine and sphinganine, cerebroside cholesterol, sitosterol cholesteryl sulfate, sitosteryl sulfate, C 10-40 fatty} alcohols and mixtures thereof. The amount may be 1.0 to 30.0% by weight based on the total weight of the nanoliposome, preferably 3.0 to 20.0% by weight.

The surfactant used in the preparation of the nanoliposome of the present invention may be any known in the art. For example, anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants can be used. Preferred are anionic surfactants and nonionic surfactants. Specific examples of the anionic surfactant include alkylacylglutamate, alkylphosphate, alkyllactylate, dialkylphosphate and trialkylphosphate. Specific examples of the nonionic surfactant include alkoxylated alkyl ethers, alkoxylated alkyl esters, alkyl polyglycosides, polyglyceryl esters and sugar esters. Particularly preferred surfactants are polysorbates belonging to nonionic surfactants. The amount may be 0.1 to 10% by weight based on the total weight of the nanoliposome, preferably 0.5 to 5.0% by weight.

Amphiphilic lipids are used as phospholipids, which are another component used in the preparation of the nanoliposomes of the present invention, natural phospholipids (eg, egg yolk lecithin or soybean lecithin, sphingomyelin) and synthetic phospholipids (eg, dipalmitoylphosphatidylcholine or hydrogenated lecithin), preferably lecithin. In particular, naturally-derived unsaturated lecithin or saturated lecithin extracted from soybean or egg yolk is preferable. In general, naturally-derived lecithin has an amount of phosphatidylcholine of 23-95%, and an amount of phosphatidylethanolamine of 20% or less. In the preparation of the nanoliposome of the present invention, the amount of phospholipid used is 0.5 to 20.0% by weight, preferably 2.0 to 8.0% by weight, based on the total weight of the nanoliposome.

The fatty acid used in the preparation of the nanoliposome of the present invention is a higher fatty acid, preferably a saturated or unsaturated fatty acid having a C ₁₂ - ₂₂ alkyl chain, for example, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid. includes The amount may be 0.05 to 3.0% by weight based on the total weight of the nanoliposome, preferably 0.1 to 1.0% by weight.

Water used in the preparation of the nanoliposome of the present invention is generally deionized distilled water, and the amount used may be 5.0 to 40% by weight based on the total weight of the nanoliposome.

The preparation of nanoliposomes may be made through various methods known in the art, but most preferably, a mixture including the above components is applied to a high-pressure homogenizer. Preparation of nanoliposomes by a high-pressure homogenizer can be carried out under various conditions (eg, pressure, number of times, etc.) depending on the desired particle size, preferably 1-5 times under a pressure of 600-1200 bar. By allowing it to pass through, it is possible to prepare nanoliposomes.

The cosmetic composition of the present invention may be used alone or in overlapping application, or may be used in overlapping application with other cosmetic compositions other than the present invention. In addition, the cosmetic composition having excellent antioxidant and whitening effects according to the present invention can be used according to a conventional method of use, and the number of times of use can be varied according to the user's skin condition or taste.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these Examples.

<Example 1>

Manufacture of cedar chestnut leaf extract and fractions thereof

<1-1> Preparation of ethanol extract from cypress chestnut leaves

The leaves of Castanopsis sieboldii obtained from the Wando Arboretum in Jeollanam-do were used. First, after drying the leaves of the cypress chestnut tree, 2 kg were immersed in 20L of 70% ethanol for 2 weeks to obtain an ethanol extract of the cypress chestnut tree leaves through the process of repeated extraction twice.

<1-2> Preparation of hot-water extract of cypress chestnut leaves

The leaves of Castanopsis sieboldii obtained from the Wando Arboretum in Jeollanam-do were used. First, after drying the leaves of the chestnut tree, 100 g of it was immersed in 1 L of distilled water and extracted using an autoclave at 121° C. for 90 minutes to obtain a hot water extract of the leaves of the chestnut tree.

<1-3> Preparation of fractions of ethanol extracts from the leaves of Prunus sycamore

135.03 g of the ethanol extract obtained in Example <1-1> was dissolved in 500 mL of 50% methanol, and then 3,000 mL of hexane was added to separate the hexane and water layers. The hexane layer thus obtained was concentrated under reduced pressure to obtain a hexane fraction (13.03 g) of the leaves of the quince chestnut tree in powder form. Chloroform, ethyl acetate, and n-butanol were sequentially added to the remaining water layer and fractionated in the same manner as above. After concentration under reduced pressure, freeze-drying was carried out. g), an ethyl acetate fraction (51.22 g), a butanol fraction (44.74 g) and a water fraction (15.93 g) were obtained, respectively.

The manufacturing process of the ethanol extract and its fractions of the leaves of the quince chestnut tree of the present invention is shown in detail in FIG.

<Experimental Example 1>

Antioxidative activity evaluation of Quercus chestnut leaf extract and its fractions

In order to evaluate the antioxidant activity of the leaf extract and fractions thereof of the present invention prepared in <Example 1>, DPPH free radical scavenging ability and ABTS radical scavenging ability were investigated.

<1-1> DPPH free radical scavenging ability

The DPPH method is widely used as a method for measuring the antioxidant activity of water-soluble or organic solvent extracts of natural products. In this experiment, the radical scavenging ability of the sample was measured by modifying the method of Blois (1958) among the methods for measuring antioxidant activity.

DPPH free radicals are a widely used method to measure the antioxidant activity of natural extracts. In this experiment, the radical scavenging ability of the sample was measured by modifying the method of Blois among the methods for measuring antioxidant activity. Specifically, 800 μL of 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, USA) dissolved in methanol and 200 μL of each sample were placed in an EP tube, vortexed, and reacted in the dark for 15 minutes. The concentration of the remaining radicals was measured at 517 nm using a UV-Visible spectrophotometer (SCINCO, Seoul, Korea). Ascorbic acid (Sigma, USA) was used as a positive control, and the scavenging ability (IC ₅₀ ) of the extracts and fractions on DPPH was expressed as the concentration required to reduce the absorbance of the control group using only the solvent by 50%.

As a result, as shown in Table 1 below, it was confirmed that the scavenging ability for DPPH was the highest in the chloroform fraction of the leaves of Quercus chestnut, and the ethyl acetate fraction and the butanol fraction of the leaves of Quercus chestnut also showed a high scavenging ability for DPPH. .

<1-2> ABTS radical scavenging ability

ABTS radical scavenging ability has the advantage of being able to measure both the radical scavenging activity of hydrophilic and hydrophobic samples, and thus a wide range of applications. In this experiment, the method of Jeong et al. was modified and measured. Specifically, a 7 mM ABTS solution and 2.45 mM potassium persulfate dissolved in distilled water were added at a 1:1 ratio and left in the dark for 12-16 hours. Radical stock solution was diluted with PBS (pH 7.4) so that the absorbance value was 0.70 ± 0.02. After adding 200 μL of each sample prepared by concentration to 800 μL of the diluted solution, the reaction was carried out in the dark for 15 minutes, and then absorbance was measured. Ascorbic acid (Sigma, USA) was used as a positive control group, and the scavenging ability (IC ₅₀ ) of the extracts and fractions against ABTS was expressed at the concentration required to reduce the absorbance of the control group using only the solvent by 50%.

As a result, as shown in Table 1 below, it was confirmed that the scavenging ability for ABTS was the highest in the ethyl acetate fraction of the leaves of cypress chestnut.

Antioxidant activity of extracts and fractions thereof sample DPPH IC ₅₀ (μg/mL) ABTS IC ₅₀ (μg/mL) ethanol extract 138.33±10.67 60.02±2.37 hot water extract 161.94±7.12 133.79±5.74 hexane fraction 1558.61±27.96 1101.06±14.28 chloroform fraction 93.92±1.96 79.33±1.19 ethyl acetate fraction 187.58±4.61 71.45±1.42 butanol fraction 169.18±1.36 136.71±1.69 water fraction 904.83±20.71 601.58±4.89

As a result of synthesizing the results of DPPH and ABTS radical scavenging activity along with the yield of the extracted extract, it was judged that the antioxidant activity of the ethyl acetate fraction of the leaves of the quince chestnut tree was the best. Thus, cytotoxicity evaluation and whitening related experiments were performed.

<Experimental Example 2>

Cytotoxicity evaluation of ethyl acetate fractions from cedar chestnut leaves

An MTT assay was performed to evaluate the cytotoxicity of the ethyl acetate fraction of the leaves of the present invention.

In the MTT assay, the MTT [3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl tetrazolium bromide] reagent is absorbed into the cells and then formsazan (formazan) by mitochondrial succinate dehydrogenase ), the intracellular accumulation of this substance means mitochondrial activity, broadly, cell activity, and is a representative method for measuring cell growth rate.

B16F10 cells were purchased from the Korean Cell Line Bank, and Dulbecco's modified Eagle's medium (DMEM; Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, USA) and 0.2% sodium bicarbonate was used as a medium. and cultured at 37° C. in 5% CO ₂ state, and subcultured in a state in which the cells grew to about 90% of the floor area, and the experiment was carried out.

The B16F10 cell suspension was aliquoted into 6 wells at a concentration of 1×10 ⁴ cells/mL by 2 mL, stabilized in an incubator for 24 hours, and samples were treated with each concentration (25, 50, 100 μg/ml). Then incubated for 24 hours. According to Choi's method, 100 μL of 2.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added per well and left in the incubator for 1 hour. . After removing the supernatant, 200 μL of DMSO was added per well, and the formazan was completely dissolved by shaking for 1 minute. Then, 540 using ELISA Bio-Tek instrument Inc (Winooski, VT, USA) Absorbance was measured in nm. MTT and DMSO used in this experiment were purchased from Sigma-Aldrich (St. Louis, MO, USA).

As a result, as shown in FIG. 2 , the ethyl acetate fraction of the leaves of the present invention prepared in Example <1-2> did not show cytotoxicity up to a concentration of 100 μg/mL. Through these results, it was confirmed that the ethyl acetate fraction of the leaves of the sycamore chestnut tree of the present invention was safe when used as a material for a cosmetic composition.

<Experimental Example 3>

Evaluation of the whitening activity of the ethyl acetate fraction of the quince leaf

In order to evaluate the whitening efficacy of the ethyl acetate fraction of the cypress chestnut leaf of the present invention prepared in Example <1-2>, tyrosinase inhibitory activity and intracellular whitening activity related proteins (tyrosinase, TRP) -1, TRP-2, p-PKA, p-CREB) expression levels were confirmed.

<3-1> Tyrosinase inhibitory activity

The main function of melanin is to protect the skin from damage by removing harmful radicals. Melanin is produced through complex oxidation and condensation reactions after tyrosine is converted into dopa and dopachrome by an enzyme called tyrosinase in melanin-forming cells by ultraviolet light. The generated melanin is delivered to the skin cells, and the melanin is lost and disappears along with epidermal peeling. This melanin production process is a natural phenomenon, and the excessive production of melanin does not occur in normal human skin. However, when the skin responds to an external stimulus, for example, ultraviolet rays, environmental pollution, or stress, melanin is excessively produced. When the excessively produced melanin is not discharged out of the skin and remains in the skin, pigmentation occurs. The biggest feature of this melanin production mechanism is that only one enzyme called tyrosinase is involved, and when melanin production is prevented by inhibiting the tyrosinase activity, a whitening effect can be expected.

In order to confirm the tyrosinase inhibitory activity, it proceeded according to the Macrini method. Experimental method is 0.1 M sodium phosphate buffer (sodium phosphate buffer, pH 6.5) 220 μL and 50-1000 μg/mL concentration of 20 μL of ethyl acetate fraction of cypress leaves prepared in a test tube, L-tyrosine (Sigma, USA) 1.5 mM 40 μL, 20 μL of mushroom tyrosinase (T3824, Sigma, USA) prepared in 1500 units was added and reacted at 37° C. for 15 minutes. After the reaction was completed, the amount of dopachrome formed from 3,4-dihydroxyphenylalanine (DOPA) by tyrosinase was measured at 490 nm. As a positive control, ascorbic acid, a hydrophilic component, and p-coumaric acid, a lipophilic component, were used. The degree of inhibition of tyrosinase activity was calculated as a percentage of the absorbance of the sample and distilled water. On the other hand, the tyrosinase inhibitory activity of the ethanol extract, the hot water extract and the chloroform fraction of the quince leaf was also measured to compare.

As a result, as shown in FIG. 3, the IC ₅₀ values of the 70% ethanol extract, hot water extract, chloroform fraction and ethyl acetate fraction of the leaves of the present invention are 294.74 μg/mL, 652.61 μg/mL, 359.20 μg/ mL, 96.77 μg/mL. On the other hand, the IC ₅₀ value of ascorbic acid used as a positive control was 38.71 μg/mL and that of p-coumaric acid was 87.17 μg/mL.

Tyrosinase inhibitory activity of extracts and fractions thereof Sample IC ₅₀ value Ethanol extract from the leaves of cypress chestnut 294.74 μg/mL Hot-water extract from the leaves of cypress chestnut 652.61 μg/mL Chloroform fractions of quince leaves 359.20 μg/mL Ethyl acetate fraction from the leaves of cypress chestnut 96.77 μg/mL ascorbic acid 38.71 μg/mL p-coumaric acid 87.17 μg/mL

Through these results, it was confirmed that the ethyl acetate fraction of the leaves of the sycamore chestnut tree of the present invention showed very good tyrosinase inhibitory activity.

<3-2> Effect of inhibiting the expression of proteins related to melanin production

In the above experimental example, the tyrosinase inhibitory activity of the ethyl acetate fraction of the leaves of the present invention was confirmed. , B16F10 tyrosinase involved in melanin synthesis by inducing α-MSH (α-Melanocyte-stimulating hormone) in melanoma cells and treating the ethyl acetate fractions of the quince leaves at different concentrations (5, 10, 20 μg/mL) , TRP-1, TRP-2, p-PKA, and p-CREB protein expression levels were analyzed by Western blot method. Primary antibodies of tyrosinase, TRP-1, TRP2, and actin and secondary antibodies of anti-goat, anti-rabbit, and anti-mouse were purchased from Santa Cruz Biotechnology (CA, USA) and used.

B16F10 cell suspension was added to a 100 π tissue culture dish at 1×10 ⁵ cells/ml cells per well and cultured for 24 hours to stabilize the cells, and then treated with α-MSH (1 μg/ml) as a stimulant. to induce pigmentation, and then the ethyl acetate fraction of the leaves of the present invention was treated with different concentrations (5, 10, 20 μg/mL). At this time, tyrosinase, TRP-1, TRP-2 was treated for 24 hours, p-PKA, p-CREB was treated for 3 hours. After culturing cells were washed 2-3 times with PBS, lysis buffer was added to lyse cells, followed by centrifugation at 13,000 rpm for 10 minutes. Quantification of protein was measured using the Bradford method. Briefly, 50 μg of protein was electrophoresed by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis), blotted on a nitrocellulose membrane, and then hybridized with a 1:1000 diluted primary antibody. After washing the membrane, it was reacted with a secondary antibody (1:1000) attached with horse radish peroxidase for 1 hour, and protein expression was analyzed using a chemiluminescence detection system (FluoChem FC2, AlphaInnotech, USA). Meanwhile, the arbutin-treated group (arbutin 100 μg/ml) was used as a positive control group.

As a result, as shown in FIG. 4 , when the cells were treated with the ethyl acetate fraction of the leaves of the present invention, tyrosinase, TRP-1, TRP-2, p-PKA, p, which are key protein markers for whitening activity. -CREB confirmed that the protein expression level was decreased depending on the treatment concentration.

Through these results, it was confirmed that the ethyl acetate fraction of the leaves of the cypress chestnut tree of the present invention had a very excellent melanin production inhibitory effect. showed

Formulation Example 1: Softening lotion (skin lotion)

As shown in the table below, softening lotion was prepared according to a conventional method.

Formulation example of softening lotion of the present invention Ingredients Content (wt%) Pinus nutmeg leaf extract or fractions thereof 1-5 1.3-Butylene Glycol 6.0 glycerin 4.0 DL-Panthenol 0.3 allantoin 0.1 Polysorbate 20 0.5 ethanol 15.0 Benzophenone-9 0.05 fragrance, preservative a very small amount Purified water to 100

Formulation Example 2: Nutrient lotion (milk lotion)

Nutrient lotion was prepared according to a conventional method as shown in the table below.

Nutrient lotion formulation example of the present invention Ingredients Content (wt%) Pinus nutmeg leaf extract or fractions thereof 1-5 Glyceryl Stearate SE 1.5 cetearyl alcohol 1.5 lanolin 1.5 Polysorbate 60 1.3 sorbitastearate 0.5 hydrogenated vegetable oil 4.0 mineral oil 5.0 trioctanoin 2.0 dimethicone 0.8 tocopherol acetate 0.5 carboxyvinyl polymer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium Hyaluronate 5.0 triethanolamine 0.12 Uniside-You 13 0.02 incense a very small amount Distilled water to 100

Formulation Example 3: Nourishing Cream

As shown in the table below, a nourishing cream was prepared according to a conventional method.

Nutritional cream formulation example of the present invention Ingredients Content (wt%) Pinus nutmeg leaf extract or fractions thereof 1-5 lipophilic monostearate glycerin 1.5 cetearyl alcohol 1.5 stearic acid 1.0 Polysorbate 60 1.5 sorbitastearate 0.6 isostearyl isosterate 5.0 squalane 5.0 mineral oil 35.0 dimethicone 0.5 Hydroxyethyl Cellulose 0.12 glycerin 6.0 triethanolamine 0.7 Uniside-You 13 0.02 incense a very small amount Distilled water to 100

Formulation Example 4: Massage Cream

As shown in the table below, massage cream was prepared according to a conventional method.

Formulation example of massage cream of the present invention Ingredients Content (wt%) Pinus nutmeg leaf extract or fractions thereof 1-5 propylene glycol 6 glycerin 4.0 triethanolamine 0.5 beeswax 2.0 Tocopheryl Acetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 cetearyl alcohol 2.0 liquid paraffin 30.0 Carboxyvinyl Polymer 0.5 incense a very small amount Distilled water to 100

Formulation Example 5: Pack

As shown in the table below, packs were prepared according to a conventional method.

Pack formulation example of the present invention Ingredients Content (wt%) Pinus nutmeg leaf extract or fractions thereof 1-5 glycerin 10.0 betaine 5.0 REG 1500 2.0 allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Benzophenone-9 0.04 Hydroxyethyl Cellulose 0.1 Sodium Hyaluronate 80. Carboxyvinyl Polymer 0.2 triethanolamine 0.18 Octyldodecanol 0.3 Octyldodeces-16 0.4 ethanol 6.0 Uniside-You 13 0.01 incense a very small amount Distilled water to 100

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (8)

A cosmetic composition for skin whitening, comprising:
The composition is a cosmetic composition for skin whitening, characterized in that it comprises the ethyl acetate fraction of the ethanol extract of the leaves of the sycamore chestnut tree as an active ingredient. delete delete delete delete According to claim 1,
The ethyl acetate fraction inhibits the activity of tyrosinase and a cosmetic composition for skin whitening, characterized in that it inhibits melanin production through a mechanism of inhibiting the expression of proteins related to melanin production. According to claim 1,
The ethyl acetate fraction is a cosmetic composition for skin whitening, characterized in that it is included in the composition at a concentration of 0.0001 to 100 μg/ml. 8. The method of any one of claims 1, 6 and 7,
The cosmetic composition for skin whitening is a flexible lotion, gel, water-soluble liquid, milk lotion, nourishing cream, massage cream, essence, oil-in-water emulsion, water-in-oil emulsion, face-type anhydrous product, solid anhydrous product, aqueous using globules Oil dispersion, ionic lipid vesicles, non-ionic lipid vesicles, ointment, cleansing foam, cleansing water, pack, body oil, oil-in-water makeup base, water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss , face powder, two-way cake, eye shadow, mascara, cheek color, and a cosmetic composition for skin whitening, characterized in that one type selected from the group consisting of eyebrow pencils.

Images