KR20160004688A

KR20160004688A - A cosmetic composition containing extracts of Trollius hondoensis

Info

Publication number: KR20160004688A
Application number: KR1020140083479A
Authority: KR
Inventors: 이범천; 백은주; 이아림
Original assignee: 이범천
Priority date: 2014-07-04
Filing date: 2014-07-04
Publication date: 2016-01-13

Abstract

The present invention relates to a cosmetic composition containing an extract of Trollius hondoensis as a main active component and, more specifically, to a cosmetic composition comprising 0.001-30.0 wt% of a Trollius hondoensis extract, and having excellent tyrosinase activity inhibiting effect, melanin biosynthesis inhibiting effect, anti-oxidation effect, skin wrinkle reducing effect, stimulation releasing effect, antibacterial effect, and hair loss preventing effect.

Description

A cosmetic composition containing extracts of Trollius hondoensis as an active ingredient,

The present invention relates to a cosmetic composition comprising as an active ingredient a gold plum extract as an active ingredient, and more specifically, to a cosmetic composition containing a gold plum extract in an amount of 0.001 to 30.0% by weight and having a tyrosinase inhibiting effect, a melanin biosynthesis inhibiting effect, An anti-irritation effect, an antibacterial effect and a hair-loss prevention effect.

The factors that determine skin color are skin color, freckles, sunburn, and overall pigmentation, as well as acne and scars, distribution of keratin and blood circulation, stress, And health status. The most important factor among these factors is pigmentation. Melanin, carotene and hemoglobin are the most important factors affecting skin color. Among them, melanin is the most important factor affecting the biosynthesis of melanin. The biosynthesis of melanin begins with the oxidation of tyrosine, a type of amino acid, in the melanosomes of melanocytes by tyrosinase to dihydroxyphenylalanine, followed by a series of oxidation processes to form pheomelanin, It is formed of a polymer of eumelanin. This biosynthesis process is carried out in a special form of melanocyte in the brown intracellular organelles. The melanosomes, including melanin granules, migrate from the periphery of the nucleus to the tip of the dendritic process, into the cytoplasm by the phagocytosis of keratinocytes, It accumulates around the nucleus of the site. The synthesis of melanin, the number of melanosomes, and the migration to keratinocytes in the periphery are primarily affected by genetic influences, and in part, by hormones and ultraviolet light. In addition, it is known that interleukin, prostaglandin, and histamine are involved in the expression of tyrosinase and cytokine, copper, zinc and iron, which are intracellular regulatory factors involved in the synthesis and transmission of melanin (Park, Soo Nam: , 25: 77-127, 1999). Therefore, the skin whitening effect can be expected by inhibiting the synthesis of melanin which determines the skin color and inhibiting the activity of tyrosinase, which is an enzyme involved therewith.

Next is the generation of wrinkles and the reduction of elasticity due to aging of the skin. The skin of a person is constantly changed by age and aging of the skin and pigmentation. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletries, 111, 31-37, 1996). Natural aging (endogenous aging) While control is difficult, external aging is relatively easy to manipulate because it is affected by environmental factors. The most prominent extrinsic aging factors are ultraviolet light and the most prominent external aging phenomenon is wrinkle formation (HM Daniel, Ann Intern Med, 75, 873-880, 1971, MJ Grove, J Am Acad Dermatol, 637, 1989, TS Griffiths et al, Hrch Dermatol, 128, 347-351, 1992).

One of the photoaging mechanisms caused by ultraviolet light is via free radical pathways (D. Harman, J. Gerontol, 11, 298-301, 1956). Free radicals are known to cause disruption of connective tissue such as skin collagen, inhibition of cell membrane function, promotion of DNA mutation, modification of protein action, intercellular energy transfer, and modification of molecules related to metabolism (AF Kligman and RM Lavker, J. Cutan, Aging Cosmet, Dermatol, 1, 5-12, 1988). The fact that free radicals are involved in aging means that antioxidants that inactivate free radicals or a variety of chemical scavengers can be used to delay the aging process.

The synthesis and degradation of extracellular matrix such as collagen in vivo is appropriately controlled, but the synthesis of extracellular matrix decreases as aging progresses and the matrix metalloproteinase (hereinafter referred to as MMP), an enzyme that degrades collagen Expression is promoted, skin elasticity is lowered, and wrinkles are formed. These enzymes are also activated by ultraviolet irradiation and free radicals. The substrate metalloproteinase (MMP) is secreted from various cells in the body including keratinocyte and fibroblast of human skin. It acts on neutral pH with calcium and zinc-dependent endopeptidase As a substrate, various extracellular substrates are used. MMPs are classified into MMP-1, MMP-2 and MMP-9 according to their substrate specificity. MMP-1 is a fibroblast type collagenase which is an enzyme of epilepsy and collagen type I, II, III, VII , VIII, X and gelatin, which are cut into peptides of 3/4 to 1/4 length of the original collagen length, facilitating the action of nonspecific gelatinase. MMP-2 belongs to 72 kD gelatinase (A), and substrates include gelatin, collagen type IV, V, VII, X, elastin and fibronectin. Metalloelastase also degrades elastin with MMP-13. Collagen type Ⅰ and Ⅲ, elastin, and fibronectin, which occupy most of the dermis of human skin, give strength and tension to the skin. When proteins are broken down by abnormally activated MMP by ultraviolet rays and free radicals, wrinkles, Aging phenomenon such as skin sagging is accelerated. In particular, it inhibits the activity and biosynthesis of MMP-1, which degrades type I collagen, which accounts for the largest proportion of binding proteins, thereby protecting skin binding proteins and preventing wrinkle formation and elasticity degradation.

In order to prevent aging and pigmentation by ultraviolet rays, a method of directly applying a product containing a sunscreen to the skin is the most common method. In 1988, retinoic acid was reported to be effective in relieving skin roughness and wrinkles of aged skin (KS Weiss et al., JAMA, 259, 527-532, 1988), researches have been actively conducted to develop substances that inhibit or improve skin aging or develop whitening effect globally. Among them, derivatives of vitamins such as vitamins A, C, and E and alpha -hydroxy acid (AHA) are representative materials showing the skin improvement effect so far known (Hermitte, Cosmetics & Toiletries, 107, 63-67, 1992, DR Rosenthal et al, J. Invest. Dermatol., 95, 510-515, 1990, TD Ditre et al, J. Invest. Dermatol, 34, 187-195, 1996). However, they are very unstable in the natural environment and have problems in use, or are somewhat insufficient to exhibit skin irritation or visual effects.

In addition, male-type alopecia are directly related to the amount of male hormone since male-type alopecia is dependent on male hormone, and thus many studies for prevention and treatment of alopecia through inhibition of male hormone activity have been reported recently. On the other hand, when the function of sebaceous glands is elevated by the increase of secretion of male hormone, the excess sebum produced in the hair follicle is stagnated in the hair follicle due to the overgrowth of the hair follicle. 5 alpha-Reductase exists in male hormone-responsive tissues such as sebaceous glands, hair follicles, prostate, and epididymis, and is one of the male hormones. Testosterone testosterone) to dihydrotestosterone, and NADPH is required for its conversion. In addition, testosterone is involved in male sexual dysfunction, skeletal muscle increase, male external genitalia, scrotum growth, spermatogenesis, and dihydrotestosterone is involved in the relevant tissues such as acne, increased sebum, hair loss and prostatome enlargement (Diane et al JID 1995). Therefore, studies for developing an inhibitor of sebum secretion and an antioxidant using an inhibitor of this enzyme have been actively conducted. After puberty, excessive secretion of male hormones causes acne and hair loss, and 5 alpha-reductase inhibitors are used to prevent excessive production of dihydrotestosterone, the active form of the male hormone, by 5 alpha-reductase Studies on the development of anti-acne agents have been actively conducted.

In order to solve such skin problems, many cosmetic products using natural products have been developed to reduce skin irritation caused by various chemical substances and the like. In addition to low adverse effects on skin, natural materials have recently become increasingly appreciated as a cosmetic raw material as consumers' response to cosmetics using natural materials has increased.

In recent years, many cosmetic products using natural products have been developed to reduce skin irritation. There have been increasing cases of developing various functional cosmetics by extracting various natural raw materials by a predetermined method and confirming the function of the extract. For example, it is known that mung bean has a function of cleansing the skin. In addition, its ginseng extract, mushroom extract and the like have been found to have effects such as anti-aging or whitening, which have been applied to various cosmetics. These natural materials have not only a small adverse effect on the skin but also have a high value for development as a raw material for cosmetics as consumers' response to cosmetics using natural materials has increased recently.

The present inventors have studied the applicability of various natural products as a skin condition improver. As a result, they have found that gold plum was selected and an extract was prepared therefrom, and the antioxidative effect and skin whitening effect, anti-aging effect, As a result, it has been found that an efficacy as a skin condition improving agent can be expected.

It is an object of the present invention to provide a cosmetic using a natural extract which exhibits excellent whitening effect by inhibiting the activity of melanin production and related enzymes.

It is another object of the present invention to provide a cosmetic using a natural extract which exhibits an anti-oxidative effect, a collagen synthesis effect, and an effect of improving wrinkles by acting on a substrate metalloproteinase and preventing skin wrinkles.

It is also an object of the present invention to provide a cosmetic using a natural extract having an excellent hair loss preventing effect and an anti-acne effect and exhibiting a 5 alpha -reduction inhibiting effect.

It is another object of the present invention to provide a cosmetic comprising a gold plum extract as a main active ingredient and a method for producing the same, since the objective efficacy is markedly increased according to the selection of a particular extraction solvent during the production of gold plum extract.

Accordingly, the inventors of the present invention have studied the applicability of natural materials not yet known as a skin condition improver. As a result, it has been confirmed that the gold plum extract has efficacy such as whitening, wrinkle reduction, irritation alleviating effect, antibacterial effect, To prepare a skin condition improving agent composition containing the same.

Gold plum ( Trollius hondoensis , gold plum flower , globeflower) are 20 kinds of perennial plants belonging to the butterfly millet butterfly and are also called gilt millet . This flower is a rare plant and a rare plant which is native to North Pyongan province, North Hamgyong province and Mt. Paektu in the northern part of Korea. Its height is about 40 to 80 centimeters. Leaves are divided into several branches, with serrations on the edges. Yellow flowers bloom at the end of the stem in July and August, and the fruit is sticky. The gold plum is very yellow and golden in color and is called gold plum. It can be seen in the northern alpine region of Korea including Baekdusan. The flower is smaller than common gold plum (Trollius ledebourii Rchb.) And small size plum Gold plum (Trollius japonicus Miq.) Is also a kind of gold plum. In some records, roots are used as medicines, which are said to be beneficial in respiratory infections, tonsillitis, sore throat, acute otitis media, acute keratitis, acute conjunctivitis, However, these contents are not about the research and discovery of cosmetic application using the improvement effect on the skin condition of gold plum.

The present invention relates to a cosmetic composition containing a gold plum extract.

In addition, the present invention is characterized in that the above-mentioned gold plum extract is contained in an amount of 0.001 to 30.0% by weight based on the freeze-dried weight of the entire cosmetic composition. If the content of the extract is less than 0.001% by weight, the efficacy is insufficient. If the content is more than 30.0% by weight, the efficacy does not increase due to the increase of the content.

The present invention also relates to a method for preparing a liquid extract obtained by cold filtration and heat filtration at room temperature using at least one solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane Water, or the liquid substance, and furthermore, the solvent is concentrated or lyophilized under reduced pressure.

The present invention also includes a method in which the gold plum extract is extracted by an extraction method using a supercritical fluid such as ultrasound, ultrahigh pressure, high frequency, or carbon dioxide in addition to the above extraction method.

In addition, the present invention is characterized in that the gold plum extract is extracted from gold plum using 40 to 95% by weight of ethanol as an extraction solvent.

Furthermore, the present invention relates to a cosmetic composition, wherein the cosmetic composition is at least one selected from the group consisting of cosmetic lotion, gel, water-soluble liquid, cream, essence and other essential cosmetic formulations of O / W type and W / O type, , A foundation, a skin cover, a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a cheek color, an eyebrow pencil, and a shampoo.

Further, the present invention is characterized in that the above-mentioned cosmetic composition has anti-aging effect, whitening effect, skin irritation alleviation effect, antibacterial effect and hair loss prevention effect.

As described above, the gold-plum extract according to the present invention has excellent free radical scavenging effect and wrinkle reducing effect such as inhibition of the activity and expression of matrix metalloproteinase (MMP), particularly collagenase (MMP-1) It turned out. It was also found that the whitening effect such as suppression of tyrosinase activity and inhibition of melanin biosynthesis was excellent. It has also been found that the skin irritation mitigating effect is excellent through the skin cell inflammation induction-suppressing effect caused by the cosmetic base material. In addition, the antimicrobial effect was excellent and the hair loss prevention effect was shown.

Therefore, cosmetic compositions such as lotion, cream, lotion, and pack containing such a gold plum extract have a skin antiaging effect, a whitening effect, an antibacterial effect, a hair loss prevention effect, and a stimulation relaxation effect.

Hereinafter, the constitution of the present invention will be described in more detail by way of examples. It is to be understood, however, that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to the description of these embodiments.

<Example 1> Preparation of gold plum extract

The gold plum purchased from Mt. Paektu was refluxed for 3 hours with a 95% (v / v) ethanol aqueous solution with hot water containing flowers and stems, cooled down, and filtered through Whatman # 10 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 DEG C or lower. The extract was prepared by using at least one solvent selected from purified water, ethanol, butylene glycol and propylene glycol so that the reduced pressure concentrate and the lyophilized product contained 0.001 to 30.0% by weight.

&Lt; Example 2 > Measurement of antioxidative effect using NBT method

In order to confirm the antioxidative effect of the gold plum extract obtained in Example 1, other antioxidants, namely, green tea extract and vitamin E were used as comparative samples in the laboratory conditions, and the antioxidative activity was measured using the NBT method.

Molecular oxygen in humans is essential for life, but oxygen is consumed to produce a small amount of free radicals in the body, and its strong oxidative action destroys cell membranes and cellular components. Since such active oxygen has a great influence on the damage and aging of skin cells, antioxidant active substances having an active oxygen scavenging function play an important role in the living body.

To measure the antioxidant effect, active oxygen produced by xanthine and xanthine oxidase is measured by the NBT method and the effect of the test substance on the removal of active oxygen, that is, the effect of active oxygen scavenging is evaluated. Xanthine and xanthine oxidase produce active oxygen. The active oxygen is reacted with Nitro Blue Tetrazolium (NBT), and the blue color produced thereby is measured at a wavelength of 560 nm to measure the active oxygen scavenging rate.

The reagent used is

1) 0.05 M Na ₂ CO ₃ buffer solution (MW = 105.99): A solution of 5.25 g (50 mM) of sodium carbonate (purified water) in purified water and 50 mM NaHCO ₃ (MW = 84.01) solution are mixed to a pH of 10.2.

2) 3 ml of xanthine solution: 45.6 mg of xanthine (MW = 152.11) is dissolved in water to make 10 ml.

3) 3 mM EDTA solution: Sodium ethylenediamine tetraacetate (manufactured by Dojin Chemical Co., Ltd., MW = 60.1) is dissolved in distilled water to make 3 mM.

4) 0.15% BSA (bovine serum albumin) solution: 15 mg of BSA (Fraction V, powder, Sigma) is dissolved in distilled water to make 10 ml.

5) 0.75 mM NBT solution: 61.32 mg of nitro blue tetrazolium (MW = 817.65, Tokyo Chemical Industry Co., Ltd.) is dissolved in distilled water to make 100 ml.

6) Xanthine oxidase solution: Xanthin oxidase (Boehringer) is diluted to 100 times with distilled water. The absorbance of the blank test should be in the range of 0.2 to 0.23 in the measurement procedure. In other words, the change in absorbance is diluted with purified water to give A ₅₆₀ = 0.3 / 20 min.

7) 6 mM CuCl ₂ solution: 102.29 mg of copper chloride (CuCl ₂ -2H ₂ O, MW = 134.45) is dissolved in distilled water to make 100 ml.

As a measuring method

1. 0.05 M Na ₂ CO ₃ 2.4 ml

2. 3 mM xanthine solution - 0.1 ml

3. 3 mM EDTA solution - 0.1 ml

4. BSA solution - 0.1 ml

5. 0.72 mM NBT solution - 0.1 ml

6. Solution of xanthine oxidase 0.1 ml

7. 6 mM CuCl ₂ solution - 0.1 ml

① Add 1, 2, 3, 4, 5 to the Bayer bottle, add 0.1 ml of the sample solution, and allow to stand at 25 ℃ for 10 minutes.

② Add the above 6 solution, stir rapidly, and start culturing at 25 ° C for 20 minutes.

(3) The 7 solution is then added to stop the reaction, and the absorbance St at 560 nm is measured.

(4) The absorbance Bt is measured by using distilled water instead of the sample solution.

(5) The blank of the sample solution is measured by the same procedure using distilled water instead of the above 6 solution, and the absorbance Bo is measured.

The results are shown in Equation (1), and the results are shown in Table 1.

St: Absorbance at 560 nm after enzyme reaction of sample solution

Bt: Absorbance at 560 nm after enzyme reaction of blank test solution

So: the absorbance at 560 nm before the reaction in the absence of enzyme in the sample solution

Bo: absorbance at 560 nm before the reaction in the absence of enzyme in the blank test solution

The results showed that the antioxidative effect was excellent in all of the tested samples such as the active oxygen scavenging effect by NBT method, and that of the green tea extract was similar to that of the green tea extract, which is widely used as an antioxidant, .

Name of sample Antioxidative effect(%) Gold plum extract 94 Green tea extract 95 Vitamin E 90

&Lt; Example 3 > Collagen synthesis effect measurement experiment

In order to observe the collagen synthesis effect of the gold plum extract obtained in Example 1, it was directly sampled from humans or treated with commercially purchased human fibroblasts.

Add DMEM (Dulbecco's Modified Eagle's Media) medium containing 2.5% fetal bovine serum and human fibroblast (5000 cells / well) to a 96-well 96-well plate and incubate until 70-80% Respectively. Then, the gold plum extract was treated at 0.01% and 0.001% concentration for 1 day, and the cell culture liquid was collected. The amount of collagen synthesis was measured using a collagen protein measuring device (Catalog N. MK 101, Takara Shuzo Co. Ltd., Japan) with respect to the collected cell culture medium.

For the measurement, first, the cell culture fluid collected in a 96 well plate incubator in which the primary collagen antibody was uniformly applied was subjected to an antigen-antibody reaction for 3 hours. After 3 hours, the secondary collagen antibody bound to the chromophore was placed in a 96-well plate incubator and allowed to react for 15 minutes. After 15 minutes, the color development inducing substance was added, coloring was made at room temperature, 1M sulfuric acid was added, and color development was stopped. The color of the reaction color is yellow, and the degree of reaction depends on the degree of reaction. The degree of collagen synthesis was calculated from the following equation (2) by measuring a yellow 96-well plate culture incubator at 450 nm using a spectrophotometer. At this time, the reaction absorbance of the cell culture without treatment of the extract was used as a control.

The collagen synthesis effect was calculated by Equation (2), and the results are shown in Table 2.

As a result, as shown in Table 2, it can be seen that the gold plum extract of the present invention has an effect of promoting collagen production.

sample Collagen synthesis effect (%) Gold plum extract 0.05% 74 0.005% 46 Control group 0

Example 4 Experiment to Measure Inhibitory Activity of Substrate Metallic Protease (MMP-1)

The substrate metalloproteinase (MMP-1) inhibitory activity was measured in a screening-type biochemical model in a test tube, which is based on the use of purified collagenase and collagen conjugated to its substrate fluororesin ( EnzChek (TM) collagenase kit, Molecular Probes). The collagenase purified from Clostridium hemiacythem was supplied into the EnzChek collagenase kit. This enzyme possesses double functionality for collagen types I and IV. The reaction buffer consisting of DQ-collagen purified from porcine skin and conjugated to fluorescein and 0.05 M Tris-HCl, 0.15 M, NaCl, 5 mM CaCl ₂ and 0.02 mM sodium azide (pH 7.6) was diluted with EnzChek collagenase kit (Molecular Probes) was used. The gold plum extract prepared in Example 1 was dissolved in the reaction buffer. It was tested at 4: 2; 0.4: 0.2; 0.04% (v / v). The dilution of the sample was incubated at room temperature for 15 minutes, 45 minutes and 120 minutes with 25 占 퐂 / ml of DQ-collagen and 0.1 U / ml collagenase.

In each experimental condition, the control group corresponding to the collagenase and DQ-collagen mixture was similarly incubated. Also, in each experimental condition, a blank, hereinafter referred to as "enzyme-free blank", was incubated in the presence of DQ-collagen and in the absence of collagenase. The signal corresponding to the decomposition of DQ-collagen after 15 minutes, 45 minutes and 120 minutes was measured with a fluorescence detector (excitation: 485 nm, emission: 505 nm). The fluorescence values of each sample were measured based on the 'enzyme-free blank' fluorescence value. The results were expressed as fluorescence units per sample and variance (%) for the control group. The results are expressed as fluorescein units / sample. In all tests, a non-specific metal chelator inhibitor, 1,10-phenanthroline, was used as a control inhibitor. A concentration of 0.2 mM is appropriate for the inhibitor in collagenase 0.1 U / ml. In conclusion, the gold plum extract tested at 0.04 to 4% (v / v) under selected experimental conditions retained dose-dependent anti-collagenase activity. The results are shown in Table 3. The gold plum extract inhibited about 80% of the collagenolytic activity of Clostridium collagenase at 0.5% treatment, which was superior to the inhibitory effect of 2 mM 1,10-phenanthroline.

0.1U
Collagenase 2 mM
1,10-phenanthroline Gold plum extract (%) 1.0 0.5 0.1 0.05 0.01 Fluorescence value 232.6 57.5 15.8 46.8 66.5 97.7 169.1 Enzyme activity 100 24.7 6.8 20.1 28.6 42.0 72.7 Inhibition rate - 75.3 93.2 79.9 71.4 58.0 27.3

<Example 5> Evaluation of suppression of MMP-1 expression by the gold plum extract after UV irradiation

ELISA (enzyme linked immunosorbent assay) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the gold plum extract obtained in Example 1.

UVA is irradiated to human dermal fibroblasts at an energy of 5 J / cm ² using a UV chamber. Ultraviolet irradiation dose and incubation time were established by preliminary experiment to maximize MMP expression level in fibroblasts. Negative controls were wrapped in silver foil and kept in the UVA environment for the same amount of time. UVA emission was measured using a UV radiometer. Cells under UVA irradiation are maintained in previously dispensed medium, irradiated with UVA, exchanged with a medium containing the sample, cultured for 24 hours, and the medium is recovered and coated on a 96-well plate. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) is treated and reacted at 37 ° C for 60 minutes. After reacting with secondary antibody, mouse mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, alkaline phosphatase substrate solution (1 mg / ml p-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes, Absorbance is measured at 405 nm with a plate reader. As a control group, a sample to which no sample is added is used.

The MMP-1 expression induced by ultraviolet irradiation showed a 92% inhibition rate compared with the control without the sample, which is similar to the inhibition rate of retinol used as a control. Table 4 shows the effect of inhibiting the expression of MMP-1 in the gold plum extract.

Test group MMP-1 expression inhibition rate (%) Control group - Gold plum extract 0.1% 92 Retinol 93

Example 6: Measurement of inhibitory effect of tyrosinase using mushroom tyrosinase

In order to confirm the whitening effect of the gold plum extract obtained in Example 1, the degree of inhibition of the enzyme function of tyrosinase was examined to determine the whitening effect.

Tyrosinase is an enzyme that helps the production of melanin by stimulating the oxidation process of tyrosine in vivo. In this embodiment, a method (Pomerantz SH: J. Biochem . , 24: 161-168, 1996) in which the function of the enzyme is inhibited to inhibit the formation of a black polymer called melanin by oxidation of tyrosine is measured To determine the whitening effect.

The inhibitory activity of each sample against tyrosinase was determined by adding 15 μl of a sample to a 96-well plate, adding 150 μl of 50 mM phosphate buffer (pH 6.5) and 25 μl of 1.5 mM L-tyrosine solution, and then adding Mushroom tyrosinase (1,500 units / ) Was added thereto, followed by reaction at 37 ° C for 20 minutes. The inhibition rate against tyrosinase was measured by measuring the absorbance at 490 nm using a microplate reader (ELx800, USA). The inhibition rate (%) for tyrosinase was calculated according to Equation (3), and IC ₅₀ The value is the concentration of the substance that inhibits tyrosinase enzyme activity by 50%.

A: absorbance before reaction of the well containing the sample

B: absorbance after reaction of the well containing the sample

C: Absorbance before reaction of well without sample

D: absorbance after reaction of well without sample

As a result of testing the inhibitory effect of tyrosinase activity, the IC ₅₀ The value was 0.52%, which was similar to that of arbutin (Table 5), although it was lower than that of the previously known whitening agent kojic acid and the soluble licorice extract.

sample Mushroom tyrosinase inhibitory effect (IC ₅₀ ) Gold plum extract 0.52% Kojic acid 0.04% Arbutin 0.47% Eucalyptus extract 10.20% Usefulness Licorice extract 0.08%

<Example 7> Experiment to measure melanin formation inhibitory effect using B16F1 melanocyte

The present example is to determine the whitening effect of B16F1 melanocyte in view of the degree of inhibition of melanin formation to confirm the whitening effect of the gold plum extract obtained in Example 1.

The B16F1 melanocyte used in this example is a cell strain derived from a mouse, and is a cell that secretes a melanin pigment called melanin. During the artificial culture of these cells, samples were treated to compare the degree of reduction of melanin pigment. The B16F1 melanocyte used in this example was purchased from ATCC (American Type Culture Collection, Accession No. 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocyte was measured as follows. B16F1 melanocytes were dispensed into 6-well plates at a concentration of 2 × 10 ⁶ cells per well, and the cells were incubated at 72 ° C. for 72 hours at a concentration that did not induce toxicity. After incubation for 72 hours, cells were detached with trypsin-EDTA, and the number of cells was measured and centrifuged to recover the cells. Quantification of intracellular melanin was carried out with a slight modification of the method of Lotan ( Cancer Res . , 40: 3345-3350, 1980). Cell pellets were washed once with PBS, and 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt the cells. After centrifugation (3,000 rpm, 10 min), 1N NaOH (10% DMSO) was added to the cell filtrate to dissolve the extracted melanin, and the absorbance of melanin was measured at 405 nm using a microplate reader. Melanin was quantitated to determine the melanin The percent inhibition of production was measured. Melanin formation inhibition rate (%) of B16F1 melanocyte was calculated as shown in Equation 4, and IC ₅₀ The value is the concentration of the substance that inhibits melanin production by 50%.

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

The inhibitory effect of B16F1 melanocyte on melanin formation was tested. The IC ₅₀ The value was 0.52%, which was similar to that of arbutin (Table 6), although it was weaker than the conventional whitening agent, hydroquinone and the soluble licorice extract.

sample Melanin synthesis inhibitory effect (IC ₅₀ ) Gold plum extract 0.50% Hydroquinone 0.03% Arbutin 0.46% Eucalyptus extract 5.32% Usefulness Licorice extract 0.04%

&Lt; Example 8 > Evaluation of effect of stimulation relaxation using cell culture technique

In order to evaluate the stimulation-relieving effect of the gold-plum extract of the present invention by ultraviolet irradiation, the following experiment was conducted to confirm the inhibitory effect of the inflammatory cytokine expression.

Fibroblasts isolated from human epidermal tissue were placed in a 24-well test plate in an amount of 5 × 10 ⁴ and adhered for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. After irradiating the fibroblasts with 10 mJ / cm 2 of ultraviolet light using an ultraviolet B (UVB) lamp (Model: F15T8, UVB 15 W, Sankyo Dennki, Japan), PBS was removed and the cells were cultured in DMEM supplemented with FBS &Lt; / RTI > medium). Here, the gold plum extract to be evaluated was treated and cultured for 5 hours. 150 [mu] l of the culture supernatant was measured to quantitate IL-1 [alpha], and the effect of inhibiting the expression of inflammatory cytokines was examined. The amount of IL-1.alpha. Was quantitated using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1.alpha. Was calculated by Equation 5, and the results are shown in Table 7 below.

Bo: IL-1? Production in wells without UV irradiation

Bt: IL-1? Production in wells irradiated with ultraviolet light and not treated with the sample

St: IL-1α production in wells irradiated with ultraviolet light and treated with the sample

Test group Inhibition rate of cytokine expression (%) Control group - Gold plum extract 0.1% 90 TNF-a 5 uM 93

As shown in Table 7, it was found that the gold plum extract showed an inhibitory rate of inflammatory cytokine expression, thereby effectively preventing inflammation caused by ultraviolet rays.

<Example 9> Measurement of antibacterial activity

As a test for the antimicrobial activity of the gold plum extract of the present invention, an antimicrobial test was conducted using a solid culture dilution method and a paper disc method. The test strains were distributed from the Korea Research Institute of Bioscience and Biotechnology. Staphylococcus aureus KCTC 6910), Pseudomonas aeruginosa by Gram-negative bacteria (Pseudomonas aeruginosa KCTC 1637), Escherichia coli (E. Coli KCTC 1039), a yeast Candida yeast (Candida albicans KCTC 7965), black Aspergillus (Aspergillus fungi as niger KCTC 6910) were used. The experimental methods and results are as follows.

The minimum inhibitory concentration was determined using the Agar Serial Dilution Method in order to measure the antibacterial activity of the gold plum extract. Tritic soy broth was used as the fungi, and the broth was inoculated on the medium and pre-cultured for 24 hours in a 37 ° C shaking incubator. Potato dextrose culture medium was used for culture of yeast, and the bacteria were inoculated on the medium and cultured for 2 days in a 25 ° C shaking incubator. Potato dextrose agar medium was used for culture of molds, and the bacteria were inoculated on the medium and pre-cultured for 7 days in a 25 ° C incubator.

More specifically, in the case of bacteria, bacteria are inoculated on a tryptic soy medium and cultured at 37 ° C for 24 hours. In the case of yeast, bacteria are inoculated on a potato dextrose medium and cultured for 2 days at 25 ° C. The filamentous fungus was inoculated on a potato dextrose agar medium and pre-cultured for 7 days in a 25 ° C. incubator. Spores of filamentous fungus formed on the surface of the medium were recovered by using a smear rod and diluted in sterilized saline.

2 ml of each extract diluted to the appropriate concentration in 5% DMSO (Dimethylsulfoxide) physiological saline solution was added to the sterilized Patry Dish according to the sample and the experimental species, and the control group was added with each sample 2 diluted to a proper concentration in 5% physiological saline solution Ml of a 5% DMSO physiological saline solution, sterilized in each of the Petri dishes, and 18 ml each of a tryptic soy agar medium and a potato dextrose agar medium cooled to 48 ° C were added, stirred and allowed to stand Coagulate.

Each of the pre-cultured bacteria was applied to each petri dish at a final concentration of about 1 × 10 ⁶ CFU / ml in the case of bacteria, 1 × 10 ⁵ CFU / ml for yeast, about 1 Spread each petri dish at a bacterial concentration of X 10 ⁴ CFU / ml. For each petri dish, the bacteria were cultured at 37 ° C. for 24 hours, the yeast was cultured at 25 ° C. for 3 days, the filamentous bacteria were cultured in a 25 ° C. incubator for 7 days, and colonies were observed in each compartment to determine the minimum The sample concentration was determined as the minimum inhibitory concentration (MIC), and the results are shown in Table 8. At this time, the minimum inhibitory concentration means that the smaller the value, the higher the antibacterial effect.

division Minimum inhibitory concentration (% by weight) B. subtilis S. aureus E. coli P. aeruginosa C. albicans A. niger Gold plum extract 0.1 0.05 0.025 0.04 1.0 or higher 0.2

<Example 10> Test for antibacterial activity (Paper Disc Test)

In this Example, a paper disc test was conducted to confirm the antibacterial activity against the acne bacterium of the gold plum extract obtained in Example 1. In order to activate Propionibacterium acnes, which is an over-the-skin acne causing acne, BHI broth (Brain-Heart Infusion Broth; 3.7%) was pre-cultured for 48 hours. The bacterial culture thus prepared was plated in 0.1 ml of BHI solid medium (Brain-Heart Infusion Broth; 3.7%; agar 1.5%) and dried. The gold plum extract obtained in Example 1 was diluted to 12% (w / v) in an aqueous 95% ethanol solution, and 50 쨉 l of the diluted plum extract was dropped on a paper disk having a diameter of 8 mm, and then placed on the solid medium prepared above. For 3 days. The antimicrobial activity was evaluated by observing the growth inhibition zone around the paper disk and measuring the size of the inhibition zone. As a result, it was found that the size of the inhibition of bacterial growth of the gold plum extract was 21 mm.

<Example 11> Evaluation test of 5 alpha-reductase activity inhibitory activity of gold leaf extract

5 Alpha-reductase activity inhibition The 5 alpha-reductase used in the experiment was the enzyme produced by the fibroblast-derived fibroblasts.

Fibroblasts are inoculated so that 10,000 cells are inserted per microplate hole and cultured. Add radio-labeled testosterone (0.1 MμCi) to each hole with ³ H (tritium), and measure whether the fibroblasts use it. As a control group, those not containing the gold plum extract are used. After 24 hours of incubation, the supernatant is obtained and steroids are obtained with 1 ml of ethyl acetate-cyclohexane (1: 1) extraction solvent. The obtained steroid is placed on a thin layer chromatography plate and developed with a chloroform / methanol mixture (98/2 (v / v)). The radioactivity of the points corresponding to testosterone and dihydrotestosterone was measured using a densitometer to calculate the conversion rate, and the result was compared with the control group (the conversion rate when the extract was not added) Alpha-reductase activity was evaluated.

A = conversion of testosterone to dihydrotestosterone (when no extract is added)

B = conversion of testosterone to dihydrotestosterone (upon addition of extract)

Experimental results show that the gold plum extract has 5 alpha-reductase inhibitory effects (Table 9).

Gold plum extract
density(%) One 0.1 0.05 0.01 0.005 0.001 0.0001 Inhibition rate (%) 100.0 97.0 92.0 78.5 71.5 42.9 34.0

Example 12 Effect of Hair Growth Test

This Example is a 30% aqueous ethanol solution containing 2% of the dried gold extract of Example 1 and used for the test. Hair growth test was carried out by using mouse (ICR) and 47 ~ 53 days of age to remove the hairs from the dorsal area, and to select the back area clean. Ten dogs were used for each test group, Respectively. The length of hair and the degree of hair growth over time were removed and the scores were added according to the degree of restoration. In order to compare the degree of hair growth, a 30% alcohol solution was applied to each individual as a control group and the growth state was observed.

As a result, it was found that the gold plum extract had excellent hair growth promoting effect (Table 10).

Elapsed days / sample
5 10 15
Gold plum extract
0.25 0.85 1.73 ± 0.36
Control group
0.08 0.24 1.01 + - 0.25

&Lt; Examples 13 to 15 and Comparative Example 1 >

In this Example, a cosmetic containing the gold plum extract obtained in Example 1 was prepared. The cosmetics were prepared in the form of a cream, and the composition thereof is shown in Table 11. First, the b) image recorded in Table 11 is heated and stored at 70 ° C. A) is added to the mixture to preliminarily emulsify, homogeneously emulsified with a homomixer, and then slowly cooled to prepare a cream (Examples 13 to 15, Comparative Example 1).

Raw material Example
Comparative Example 1 13 14 15 end

Stearyl alcohol 8 8 8 8 Stearic acid 2 2 2 2 Stearic acid cholesterol 2 2 2 2 Squalane 4 4 4 4 2-octyldodecyl alcohol 6 6 6 6 Polyoxyethylene (25 mole addition)
Alcohol ester 3 3 3 3 Glyceryl monostearate Aster 2 2 2 2 I

Gold plum extract 10 3 0.5 - Propylene glycol 5 5 5 5 Purified water Suitable amount Suitable amount Suitable amount Suitable amount

Note) Unit; weight%

In this experimental example, the skin anti-aging effect was evaluated by applying the cream form cosmetics prepared in Examples 13 to 15 and Comparative Example 1 to humans.

The cream prepared in Examples 13 to 15 was applied to the right side of the subject and the cream prepared in Comparative Example 1 was applied to the left side of the face for 2 consecutive months for 2 consecutive months, respectively, for 40 persons (20 to 35 year old female) Respectively.

After the completion of the experiment, the skin wrinkle reducing effect was evaluated by using a silicone resin copy (replica) to collect the wrinkles around the facial surface of the eye before and after the use of the product for 2 months, and compare the state of the eye wrinkles with the skin wrinkle device and skin image analyzer.

Table 12 compares the average fine wrinkle depth and wrinkle reduction ratio of the eyes of the experimenter using the cream prepared in Examples 13 to 15 with those using the cream of Comparative Example 1. [ As shown in Table 12, it can be seen that the skin anti-aging effect is excellent in the skin around the eyes of the user who applied the cream containing the gold plum extract.

Example 14 Comparative Example 1 Before use Two months later Before use Two months later Average fine wrinkle depth 0.218 0.192 0.214 0.206 Wrinkle reduction rate before use (%) 11.93% 3.74%

n = 40, p < 0.01

In this Experimental Example, the stimulation relaxation effect of the cosmetic composition containing the gold plum extract obtained in Example 1 was evaluated by a human skin patch test.

This evaluation is the result of the evaluation obtained by directly applying the results obtained in Example 11 to the human body. The stimulation index was obtained by applying SLS (sodium lauryl sulfate), which stimulates general cosmetic formulations (cream, lotion, skin, essence), with the products prepared in Examples 13 to 15 for 24 hours, 48 hours and 72 hours, And the effect of stimulation relaxation was evaluated.

Each of the products was applied with 0.3 mg of FINN CHAMBER (FINLAND) on the upper arm of a healthy male and female of 20 to 50 years old, and the acute irritation index was evaluated after 24 hours. After the evaluation, the same amount of product was applied again to the same site again, and the delayed irritation index after 48 hours and 72 hours was evaluated.

As a result of the test, the product containing the gold plum extract was applied, and no skin episodes were observed after 24 hours, 48 hours and 72 hours.

The results of this evaluation indicate that there is a significant effect of reducing the skin irritation caused by stimulants (surfactant, fragrance, alcohol) when the gold plum extract is mixed with cosmetics.

Other examples are shown below. That is, the gold leaf extract of the present invention and the composition containing the same exhibit excellent effects on skin improvement such as antioxidant effect, wrinkle reduction effect, and stimulation relaxation effect as described above.

Example 16 Production of lotion containing the gold plum extract obtained in Example 1

0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of p-hydroxybenzoic acid methyl ester, a small amount of antioxidant and a small amount of dye are mixed and dissolved in 8 g of 95% ethanol . 0.1 g of the gold plum extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water. The mixture was added to the mixture, followed by stirring to obtain a skin lotion having skin-improving effect.

Example 17 Preparation of emulsion containing the gold plum extract obtained in Example 1

1.2 g of setyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of p-hydroxybenzoic acid ethyl ester, 1 g of glycerin monoesteraride, 1 g of polyoxyethylene (20 mol added) monooleate, 0.5 g of the gold plum extract obtained in Example 1, 5 g of dipropylene glycol, 2 g of polyethylene glycol, 0.2 g of triethanolamine and 76.2 g of purified water were heated and dissolved by heating at 75 캜. The two were mixed and emulsified and then cooled to obtain a milky lotion having a skin / milky type skin improving effect.

Example 18: Preparation of a serum containing the gold plum extract obtained in Example 1

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of chitoolose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium lacquinic acid, 0.1 g of paraoxybenzoic acid ester, 1 g each of the gold plum extract obtained in Example 1 and an appropriate amount of pigment were mixed to obtain a serum having skin-improving effect.

Claims

Gold plum ( Trollius hondoensis ) extract as a main active ingredient.

The method according to claim 1,
Wherein the gold plum extract is 0.001 to 30.0% by weight based on the total amount of the cosmetic composition.

The method according to claim 1,
Wherein the solvent of the extract is at least one solvent selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane. .

The method according to claim 1 or 2,
The cosmetic composition according to any one of claims 1 to 3, wherein the gold plum extract is a liquid product obtained by freezing, stirring, heating and filtering at room temperature, and furthermore, the solvent is concentrated under reduced pressure or lyophilized.

The method according to any one of claims 1 to 3,
The cosmetic composition may be in the form of a cosmetic, a gel, a water-soluble liquid, a cream, an essence, an O / W type or a w / o type cosmetics, a makeup base, a foundation, a skin cover, a lipstick, Face powder, two-way cake, eye shadow, and shampoo composition.