KR100561037B1 - Cosmetic composition containing Artemisia Anomala extract - Google Patents

Abstract

The present invention relates to a cosmetic composition containing an organic no extract, more specifically, a cosmetic composition containing 0.001-30.0% by weight of organic no extract, excellent in inhibiting melanin biosynthesis and tyrosinase activity, antioxidant effect and skin wrinkle improvement. It relates to a composition. The composition of the present invention can be confirmed the excellent whitening effect and stimulating effect, anti-wrinkle effect and to prepare a functional cosmetics containing an organic furnace extract.

Organic Nose Extract, Whitening, Anti-Aging, Irritation Relief, Artemisia Anomala

Description

Cosmetic composition containing Organic No. extract {Cosmetic composition containing Artemisia Anomala extract}

The present invention relates to a cosmetic composition containing an organic noh extract to whiten skin color or improve dull skin color, and to reduce skin aging due to wrinkle improvement and irritation-reducing effect. More specifically, organic noh extract 0.001 to 30.0 weight It relates to a cosmetic composition excellent in whitening effect, wrinkle improvement effect, stimulating alleviation effect containing%.

Factors that determine skin color include basically areas such as tanning due to blemishes, freckles and sun exposure, or overall pigmentation, other than acne, scars, and the distribution of keratin, Blood circulation, stress, and health. Among the above factors, the most important factor is pigmentation.

The pigments that affect skin color include melanin, carotene, and hemoglobin, the most important of which is melanin. The biggest factor affecting melanin biosynthesis is ultraviolet rays and the amount of hormones in the body. The biosynthesis of melanin begins with a series of oxidation processes, starting with the conversion of tyrosine, an amino acid, from melanocytes of melanocytes to tyrosinase and dihydroxy phenylalanine. It is formed of a polymer of eumelanin. This biosynthesis process is carried out in a special type of brown intracellular organelles called melanosomes. Melanosomes, including melanin granules, move from the periphery of the nucleus to the dendritic end, and then into the cytoplasm by the phagocytosis of keratinocytes. Accumulate around the nucleus of the site. The synthesis of melanin, the number of melanosomes, and the movement to surrounding keratinocytes are partially affected by hormones and ultraviolet rays, and primarily by genetic influences. In addition, cytokines (cytokine), which are involved in the expression of tyrosinase, synthesis and transfer of melanin, and metal ions such as copper, zinc and iron, as well as interferons, prostaglandins and histamines are known to be involved. Society of Cosmetic Scientists, 25: 77-127, 1999). Therefore, the skin whitening effect can be expected by inhibiting the synthesis of melanin, which determines the skin color, and the activity of tyrosinase, an enzyme involved therein.

Conventionally, ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione, etc. have been previously used to treat or alleviate abnormal pigmentation symptoms such as blemishes, freckles, pigmentation, and excessive melanin pigmentation caused by UV exposure. Or derivatives and substances having tyrosinase inhibitory activity have been used in cosmetics and pharmaceuticals, which are limited in use due to insufficient whitening effects, safety problems for skin, formulation and stability problems when formulated in cosmetics, etc. .

Meanwhile, in addition to these existing whitening efficacy substances, research is being conducted to find whitening active ingredients in natural products. Among them, Sangbaekpi (Japanese Patent Publication No. 55-44375, So64-26507, So64-83009, Pyeong1- 1) 25687, Hei 5-139950, Korean Laid-Open Patent Nos. 1992-002109, 1997-021273), Licorice (Japanese Laid-Open Patent Nos. 60-214721, 63-23809, 64-63506, 11-149706, Korea Publication Patent Nos. 1992-002109 and 1997-025601) have been found to have a number of plant extracts act on tyrosinase to inhibit melanin production, but they are also effective in cosmetics and pharmaceuticals in terms of safety, stability and discoloration potential. The use of more than the concentration has a lot of problems and does not produce a satisfactory effect.

Next, wrinkles are generated and skin elasticity is reduced due to aging of the skin. As a person ages, his skin is constantly changing due to skin aging and pigmentation. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories, 111, 31-37, 1996), and natural aging (endogenous aging) is artificial because it is influenced by genetic factors. While it is difficult to control, aging is relatively easy because it is influenced by environmental factors. Representative external aging factors include ultraviolet rays, 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, 21, 631-). 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 breakdown of connective tissue formation such as skin collagen, inhibition of cell membrane function, promotion of DNA mutation, modification of protein action, intercellular energy transfer, and modification of metabolic related molecules (AF Kligman and RM Lavker, J. Cutan.Aging Cosmet.Dermatol. 1, 5-12, 1988). The involvement of free radicals in aging means that antioxidants or various chemical scavengers can be used to delay the aging process.

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but as aging progresses, its synthesis decreases and matrix metalloproteinase (MMP), an enzyme that breaks down collagen, especially The expression of collagenase (hereinafter referred to as 'MMP-1') and gelatinase (gelatinase (hereinafter referred to as 'MMP-2')) is accelerated to decrease the elasticity of the skin and form wrinkles. Ultraviolet irradiation and free radicals also activate these degrading enzymes.

MMPs are classified into MMP-1, MMP-2, and MMP-9 according to their substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and collagen type I, II, III, Ⅶ as substrate. There are, Ⅹ, Ⅹ and gelatin, cut into peptides of 3/4 or 1/4 length of the original collagen to facilitate the action of non-specific enzyme (gelatinase). MMP-2 belongs to 72 kD gelatinase (A) and degrades substrates such as gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin.

Substrate metalloproteinases (MMPs) of neutrophils and polymorphonuclear leukocytes are primarily activated by oxidative processes. First, when hydrogen peroxide is generated in cells, it is induced by myeloperoxidase in the presence of chlorine ions. It is converted to chloric acid, which activates the substrate metalloproteinase (MMP).

In order to prevent aging due to ultraviolet rays, the most common method is to apply sunscreen products directly to the skin.However, in 1988, retinoic acid was reported to be effective in relieving skin roughness and fine wrinkles of aged skin (KS Weiss). et al, JAMA, 259, 527-532, 1988), studies are being actively conducted to develop substances that inhibit or improve skin aging worldwide. Among them, derivatives of vitamins such as vitamins A, C, and E and AHA (alpha-hydroxy acid) are known representatives of aging skin improvement (Hermitte, Cosmetics & Toiletries, 107, 63-67, 1992, DR Rosenthal et. al, J. Invest.Dermatol., 95, 510-515, 1990, TDDitre et al, J. Invest.Dermatol, 34, 187-195, 1996). However, they are very unstable in the natural environment, so there is a problem in use, or somewhat insufficient to show a visible effect.

Meanwhile, the cosmetic composition using the Artemisia capillaris Thunb. Belonging to the genus Mugwort (artemisia) (JP2001181440), the skin external preparation and cosmetics using the Artemisia princeps Var. Orientalis (JP1997127775, JP1987216041) and Artemisia aplace A patent has been registered using plants belonging to the same genus (genus) as a cosmetic composition using a Hance (JP1998317841), but there is no example applied to cosmetics using an organic furnace ( Artemisia anomala ).

In general, herbal medicines have different effects depending on the place of birth and harvest time, and even if the plants of the same genus are different in species, the effects of course are different. In addition, organic oars are plants belonging to the genus Apothecary (compositae) and artemisia in terms of taxonomy and are classified as different plants because they have the same genus as the herbals used in cosmetics but different species.

Artemisia Anomala is an outpost of the compositae plant symbol Artemisia anomala S. Moore, whose shoots and stems resemble wormwood, and the leaves resemble green willows. Small flowers of color bloom and run like a millet. It is a kind of small mugwort. In July and August of the lunar calendar, care is sun-dried (herb) The nature is warm, the taste is bitter, and it is not poisonous. He smashes blood, lowers spears, makes menstruation well, and frees up the chastity. Gojo was a young boy named Song Kino when he was young. He was injured by iron and healed by bleeding with this grass. It is distributed in Korea, Japan, and China (Doosan World Encyclopedia, Doosan Dong-A, 1999).

Therefore, it is an object of the present invention to provide a natural organic furnace extract that is applicable to skin cosmetics and exhibits excellent whitening effect by inhibiting the production of melanin and the activity of enzymes related thereto when contained in cosmetics.                         

In addition, the object of the present invention is applicable to skin cosmetics and contains a natural organic noo extract exhibiting excellent anti-wrinkle effect, such as anti-wrinkle effect by acting on the antioxidant effect, collagen synthesis effect, substrate metalloproteinase when contained in cosmetics It is to provide a cosmetic composition.

The present inventors select an organic furnace among various herbal medicines, confirm its efficacy as a cosmetic raw material, and develop a method of using the same, thereby increasing the selection range of various cosmetic raw materials, and having a predetermined functional effect without skin irritation by using this raw material. An object of the present invention was to provide a method for preparing cosmetics.

The present invention relates to a cosmetic composition containing an organic furnace extract.

In addition, the present invention is characterized in that the organic furnace extract is contained in 0.001 ~ 30.0% by weight based on the total weight of the total composition. When the content of the extract is less than 0.001% by weight, the efficacy is weak, and when the content of the extract exceeds 30.0% by weight, it is not economical because there is no apparent increase in the effect of the content increase.

In addition, the present invention, the organic furnace extract is cold water at room temperature by using at least one selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane as a solvent The liquid product obtained by filtration and further obtained by concentrating the solvent under reduced pressure or lyophilization.

In addition, the present invention, the cosmetic composition is a cosmetic lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) or water-in-oil (W / O) type cosmetic formulation and oil-in-water or water-in-oil makeup Base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, teak color and eyebrow pencils are selected from the color cosmetic formulations.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative description of the present invention and the scope of the present invention is not limited to these Examples.

Example 1 Preparation of Organic Furnace Extract

The organic furnace was chopped, refluxed with a hot 95% (V / V) ethanol aqueous solution for 3 hours, cooled, and filtered through Whatman # 10 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C or lower. The organic furnace extract was prepared using one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol, such that the vacuum concentrate and the lyophilisate contained 0.001 to 30.0 wt%.

<Example 2: Measurement of tyrosinase inhibitory effect using mushroom tyrosinase>

This Example is to determine the whitening effect by looking at the degree of inhibition of the function of the enzyme called tyrosinase (tyrosinase) to confirm the whitening effect of the organic furnace extract obtained in Example 1.

Tyrosinase is an enzyme that accelerates the oxidation of tyrosine in vivo and helps melanin production. In this example, a method of inhibiting the function of this enzyme to inhibit the oxidation of tyrosine to form a black polymer called melanin (Pomerantz SH: J. Biochem. , 24: 161-168, 1996) is applied. The whitening effect was determined.

The inhibitory effect of tyrosinase activity was investigated using kojic acid, arbutin, lettuce extract, oil-soluble licorice extract, and organic no. Extract prepared in Example 1 as samples.

Inhibitory activity of tyrosinase of each sample was placed in a 96-well plate, 15 µl of a sample, 150 µl of 50 mM phosphate buffer (pH 6.5) and 25 µl of 1.5 mM L-tyrosine solution, followed by mushroom tyrosinase. 10 μl (1,500 units / ml, Sigma) was added and reacted at 37 ° C. for 20 minutes, and then the absorbance was measured at 490 nm using an absorbance spectrometer (microplate reader, ELx800, USA) to measure the inhibition rate against tyrosinase. Inhibition rate for tyrosinase (%) was calculated by Equation 1, IC ₅₀ value is the concentration of a substance that inhibits the tyrosinase enzyme activity by 50%.

% Inhibition = [{(D-C)-(B-A)} / (D-C)]] X 100

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 the 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 value of the organic no extract was 0.02%, which was superior to the conventional whitening agents koji acid, arbutin, and lettuce extract, which are known to be excellent in inhibiting tyrosinase (Table 1). .

sample Mushroom tyrosinase inhibitory effect (IC ₅₀ ) Kojisan 0.037% Arbutin 0.4% Organic Furnace Extract 0.02% Lettuce extract 10% Soluble Licorice Extract 0.02%

Example 3 Measurement of Inhibitory Effect of Intracellular Tyrosinase Enzyme Synthesis Using B16F1 Melanosite

In this example, the degree of inhibition of tyrosinase enzyme synthesis in B16F1 melanocytes was determined by measuring the amount of tyrosinase enzyme synthesis in the cells in order to confirm the whitening effect of the organic furnace extract obtained in Example 1.

The B16F1 melanocytes used in this example are cell strains derived from mice and cells that synthesize an enzyme called tyrosinase. The degree of inhibition of tyrosinase enzyme synthesis was compared by evaluating the amount of enzyme synthesis by treating the sample during the artificial culture of the cells and by separating the tyrosinase from the cells.

The B16F1 melanocytes used in this example were used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323). The tyrosinase enzyme synthesis inhibitory effect of B16F1 melanocytes was measured as follows.

B16F1 melanocytes were dispensed at a concentration of 2 × 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with Trypsin-EDTA, and then the number of cells was measured and centrifuged to recover the cells. The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells, followed by centrifugation (3,000 rpm, 10 minutes) to recover the supernatant. Put 1.5 mM L-tyrosine, 0.06 mM L-DOPA, and cell supernatant into 50 mM sodium phosphate buffer (pH 6.5), and incubate at 37 ° C for 30 minutes and measure the absorbance at 490 nm with an absorbance photometer to inhibit tyrosinase enzyme synthesis. Measured. The inhibition rate of tyrosinase enzyme synthesis (%) of B16F1 melanocytes was calculated by Equation 2, and the IC ₅₀ value is the concentration of a substance that inhibits tyrosinase enzyme synthesis by 50%.

% Inhibition = [(A-B) / A] x 100

A: Synthesis amount of tyrosinase enzyme in wells without sample

B: Tyrosinase Enzyme Synthesis of Wells Added Samples

As a result of measuring the inhibitory effect of tyrosinase enzyme synthesis of B16F1 melanocytes, the IC ₅₀ value of the organic no extract was 0.07%, which is effective in synthesizing tyrosinase at low concentrations compared to the conventional whitening agents, koji acid, arbutin, oil-soluble licorice extract, and lettuce extract. It was shown to inhibit (Table 2). As a result, it can be seen that the organic no extract of the present invention inhibits the synthesis of tyrosinase enzyme and shows a whitening effect.

sample Inhibitory effect of tyrosinase synthesis (IC ₅₀ ) Kojisan 0.8% Arbutin 0.4% Organic Furnace Extract 0.07% Lettuce extract One% Soluble Licorice Extract 0.4%

<Example 4: Measurement of melanin production inhibitory effect using B16F1 melanocytes>

This Example was to determine the whitening effect according to the degree of inhibition of melanin production on B16F1 melanocytes in order to confirm the whitening effect of the organic furnace extract obtained in Example 1.

B16F1 melanocytes used in this example are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin black pigment reduction. The B16F1 melanocytes used in this example were used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed at a concentration of 2 x 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Quantification of intracellular melanin was carried out with a slight modification of the method of Rotan: Cancer Res. , 40: 3345-3350, 1980. The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and then measure the absorbance of melanin at 405 nm with an absorbance photometer. Percent inhibition of production was measured. Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the equation (3), IC ₅₀ value is the concentration of the substance inhibits 50% melanin production.

% Inhibition = {(A-B) / A} X 100

A: amount of melanin in wells without added sample

B: Melanin amount in the wells to which the sample was added

The test result of B16F1 melanocytes suppresses melanin production, and the IC ₅₀ value of the organic no extract is 0.02%, which shows similar or superior effect to the existing whitening agents such as hydroquinone, arbutin, oil-soluble licorice extract, and lettuce extract. 3).

sample Melanin synthesis inhibitory effect (IC ₅₀ ) Hytroquinone 0.03% Arbutin 0.5% Organic Furnace Extract 0.02% Lettuce extract 5% Soluble Licorice Extract 0.04%

Example 5: Determination of Antioxidant Effect Using NBT Method

This Example is a Nitro Blue Tetrazolium (NBT) method by comparing a sample of other antioxidants, that is, green tea extract and vitamin E under laboratory conditions to confirm the antioxidant effect of the organic furnace extract obtained in Example 1 Antioxidant activity was measured by.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase was measured by NBT method, and the effect of removing the active oxygen by the test substance, that is, the effect of scavenging the active oxygen, was evaluated. After generation of active oxygen by xanthine and xanthine oxidase, the active oxygen scavenging rate was measured by measuring the blue color produced by reaction of this active oxygen with NBT at a wavelength of 565 nm.

As the reagent used

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

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

3) 3mM EDTA solution: Sodium ethylenediamine tetraacetate (MW = 60.1) is dissolved in distilled water to make 3mM.

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

5) 0.75 mM NBT solution: 61.32 mg of nitroblue tetrazolium (MW = 817.65, Tokyo University) is dissolved in distilled water to make 100 ml.

6) xanthine oxidase solution: Dilute the xanthine oxidase (Boehringer) with distilled water about 100 times and make the absorbance of the blank test within the range of 0.2 ~ 0.23 in the measurement operation. In other words, dilute with purified water so that the change in absorbance is A ₅₆₀ = 0.3 / 20 minutes.

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

As a measuring method

0.05M Na ₂ CO ₃ ------------------------------------ 2.4ml

2.3mM xanthine solution ---------------------------------- 0.1ml

3.3mM EDTA Solution ----------------------------------- 0.1ml

4.BSA solution ---------------------------------------- 0.1ml

5.0.72mM NBT solution ---------------------------------- 0.1ml

6. Xanthine oxidase solution ---------------------------- 0.1ml

7.6 mM CuCl ₂ solution ---------------------------------- 0.1 ml

① Add 1, 2, 3, 4, 5 to Bayer's bottle, and add 0.1 ml of sample solution to this and leave at 25 ℃ for 10 minutes.

② Add 6 and stir quickly, and start incubation for 20 minutes at 25 ℃.

③ After that, add 7 to stop the reaction and measure the absorbance St at 560nm.

④ For the blank test, measure the absorbance Bt by using distilled water instead of the sample solution as above.

⑤ Also, the blank of the sample solution is operated in the same manner using distilled water instead of 6 to measure the absorbance Bo.

The result of the effect was calculated by the equation (4), the results are shown in Table 4.

% Inhibition = {1- (St-So) / (Bt-Bo)} X 100

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

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme in sample solution

Bo: absorbance at 560 nm before reaction without enzyme in blank test solution

The results showed excellent antioxidant effect in all the samples tested as shown in Table 4, it was confirmed that the organic furnace extract also had similar antioxidant power as the green tea extract and vitamin E which are widely used as antioxidants.

 Sample Name  Antioxidative effect(%)  Organic Furnace Extract  92  Green Tea Extract  95  Vitamin E  90

Example 6: Collagen Synthesis Effect Measurement Experiment

In order to observe the collagen synthesis effect of the organic furnace extract obtained in Example 1, the experiment was performed by directly treating human fibroblasts obtained from humans or purchased commercially.

In a 96-well plate, add DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal bovine serum and human fibroblasts (5000 cells / well) and incubate until 70-80% growth. It was. Then, the organic o extract was treated with 0.01% by weight and 0.001% by weight of the cell culture solution after 1 day, and the collagen was collected using a collagen protein measuring instrument (Catalog N.: MK 101, Takara Shuzo Co. Ltd, Japan). Synthesis amount was measured.

In the measurement method, first, a cell culture medium collected in a 96-hole plate incubator uniformly coated with primary collagen antibody was subjected to antigen-antibody reaction for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-hole plate incubator and allowed to react for 15 minutes. After 15 minutes, the color development material was added and developed at room temperature, and 1M sulfuric acid was added to stop the color development. The color of the reaction color is yellow, depending on the intensity of the reaction. A yellowish 96-hole plate incubator was measured at 450 nm using an absorbance spectrometer, and the degree of collagen synthesis was calculated by Equation 5 below. At this time, the reaction absorbance of the cell culture solution without the extract was used as a control.

Collagen synthesis effect was calculated by the equation (5), the results are shown in Table 5.

Collagen Synthesis Effect (%) = (Reaction Absorbance of Cell Culture Treated with Organic Extracts / Reaction Absorption of Control) X 100

As shown in Table 5, it can be seen that the organic furnace extract has a collagen production promoting effect.

 sample  Collagen Synthesis Effect (%)  Organic Furnace Extract  0.01 wt%  80  0.001% by weight  52  Control  0

Example 7: Determination of Inhibitory Activity of Matrix Metalloproteinase (MMP-1)

Matrix metalloproteinase (MMP-1) inhibitory activity was measured in vitro in a screening type biochemical model based on the use of purified collagenase and collagen conjugated to its substrate, fluorescein (EnzChek). (Trade name) collagenase kit, Molecular Probe). Collagenase purified from Clostridium histories was fed into the EnzChek collagenase kit. This enzyme possesses dual functionality against collagen types I and IV. A reaction buffer consisting of DQ-collagen purified from porcine skin and conjugated to fluorescein and 0.05M Trs-HCl, 0.15M, NaCl, 5mM CaCl ₂ , and 0.02mM NaN ₃ (pH 7.6) was obtained from the EnzChek collagenase kit ( Molecular Probes) was used. The organic furnace extract prepared in Example 1 was tested after dissolving at a concentration (4, 2, 0.4, 0.2, 0.04% (v / v)) in the reaction buffer. Dilutions of the samples were incubated with 25 μg / ml of DQ-collagen and 0.1 U / ml of collagenase for 15 minutes, 45 minutes, and 120 minutes at room temperature.

In each experimental condition, the control corresponding to the collagenase and DQ-collagen mixture was also incubated in the same manner. Also, in each experimental condition, a sample, hereinafter referred to as 'enzyme-free blank', was incubated in the presence of DQ-collagen and in the absence of collagenase. After 15, 45 and 120 minutes, the signal corresponding to the degradation of DQ-collagen was measured with a fluorometer (excitation; 485 nm, emission; 505 nm). The fluorescence value of each sample was measured based on the 'enzyme free blank' fluorescence value. Results are expressed as percent variance for fluorescence units per sample and control. The results are shown in fluorescein units / sample. All tests used 1,10-phenanthroline, a nonspecific metal chelator inhibitor, as a control inhibitor. A concentration of 0.2 mM of inhibitor is appropriate for 0.1 U / ml of collagenase. In conclusion, the organo extract tested at 0.04 to 4% (v / v) under selected experimental conditions retained dose dependent anticollagenase activity. The results are shown in Table 6. The organo-no extract inhibited 70% of the collagen degrading activity of Clostridium collagenase when treated with 0.04%, which was similar to the inhibitory effect of 2mM 1,10-phenanthroline.

2 mM 1,10-phenanthroline Organic Furnace Extract (% by volume) 4 2 0.4 0.2 0.04  Inhibition rate  72  95  90  85  77  70

<Example 8: Evaluation of inhibition of MMP-1 expression by organic furnace extract after UV irradiation>

In this example, an ELISA (enzyme linked immunosorbent assay) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the organic furnace extract obtained in Example 1.

Human dermal fibroblasts were irradiated with UVA at an energy of 5 J / cm 2 using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. Negative controls were wrapped in tinfoil and maintained at the same time in the environment of UVA. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact with the previously dispensed medium, and then irradiated with UVA and exchanged with the medium containing the sample, followed by culture for 24 hours, and then the medium was recovered and coated in a 96-hole plate incubator. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was treated and reacted at 37 ° C. for 60 minutes. After reacting the secondary antibody anti-mouse immunoglobulin (anti-mouse IgG, whole mouse, alkaline phosphatase conjugated) for 60 minutes, the alkaline phosphatase substrate solution (1mg / ml p-nitrophenyl phosphate in diethanolamine buffer solution) ) Was reacted at room temperature for 30 minutes and absorbance was measured at 405 nm using an absorbance spectrometer. At this time, the reaction absorbance of the cell culture solution without the extract was used as a control.

MMP-1 expression inhibitory effect was calculated by the equation (6), the results are shown in Table 7.

% Inhibition of MMP-1 expression = (response absorbance of extract cell culture / response absorbance of control) X 100

As shown in Table 7, it was confirmed that the organo-no extract according to the present invention had an excellent MMP-1 expression inhibitory effect similar to the inhibition rate (96%) of the retinol used as a positive control.

Test group % Inhibition of MMP-1 expression Control - Organic Furnace Extract 0.1 wt% 94 Retinol 96

Example 9 Measurement of Inhibitory Effect of Inflammation-Related Enzyme (hyaluronidase)

This example is to determine the anti-inflammatory effect of the organic furnace extract obtained in Example 1 by determining the enzyme activity of the hyaluroniadiaze enzyme, an inflammation-associated enzyme.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and has a function of causing inflammation. In this example, the anti-inflammatory effect was determined by applying a method (Kakegawa Y., Japanese J. of Inflammantion , 4: 437-438, 1984) to inhibit the activity of this enzyme to measure the anti-inflammatory effect.

The inhibitory effect of hyaluroniadiase activity was investigated using comfrey, seesaw, triticale, oil-soluble licorice extract, and organo extract. Inhibitory activity of hyaluronidiases of each sample was performed as follows.

100 μl of sample and 50 μl of hyaluronidiase solution (type IV-S, Sigma, 400 U / mL) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl ₂ ㆍ 2H ₂ O). , 100 mg of Sigma, 0.1 mg / ml) was added and reacted again at 37 ° C. for 20 minutes. 250 μl of a hyaluronic acid solution (0.4 mg / ml) was added thereto and reacted at 37 ° C. for 40 minutes, and 100 μl of 0.4N NaOH was added to terminate the reaction. 100 μl of potassium borate solution was added thereto, reacted at 95 ° C. for 3 minutes, cooled, and 3 ml of ρ-dimethylaminobenzaldehyde solution was added thereto. Absorbance was measured at 565 nm to determine the inhibition rate for hyaluronidiases. Inhibition rate (%) for the hyaluronia diase was calculated by the equation (7), IC ₅₀ value is the concentration of a substance that inhibits the hyaluronia diase activity by 50%.

% Inhibition = {(A-B) / A} X 100

A: Enzyme activity of wells without sample

B: Enzyme Activity of Well Added Samples

As a result of testing the inhibitory effect of hyaluronidiaze enzyme activity, the IC ₅₀ value of the organo extract was 0.04%. It showed an excellent effect compared to the back (Table 8).

 sample Inhibitory effect of hyaluronidiases (IC ₅₀ )  Comfrey Extract  0.22%  Seesaw extract  0.58%  Soluble Licorice Extract  0.08%  Triticale extract  0.3%  Organic Furnace Extract  ] 0.04%

Example 10 Evaluation of Stimulation Relaxation Effect Using Cell Culture Technology

This Example is apoptosis by Sodium Lauryl Sulfate (SLS) which is a substance causing skin irritation by using cell culture technology to evaluate the stimulating relaxation effect of the organic o extract obtained in Example 1 The degree of stimulation alleviation was evaluated to the extent that it inhibits.

SLS is a substance that is used as a criterion for evaluation of skin irritation based on cosmetics, and is a substance that binds to cell membranes to inhibit cell metabolism and destroys cell membranes to cause skin irritation.

This example evaluates the effect of reducing SLS apoptosis when SLS and organo extracts are added to human fibroblasts at the same time.

The Hs68 fibroblasts used in this example were used as a dermal dermal cell in humans, sold at ATCC (American Type Culture Collection, Accession No. 1635).

Stimulation relaxation test using the cell culture technology was carried out as follows.

Distribute human-derived fibroblasts at a concentration of 1 × 10 ⁵ per well in a 96-well plate incubator, incubate for 24 hours, and simultaneously treat 0.01% SLS alone, 0.01% SLS, and 0.01% by weight organico extract, and add for 24 hours. Incubated. After incubation, MTT (Sigma) reagent was added and incubated for 4 hours, the culture medium was removed, 1N NaOH / isopropanol solution was added, the mixture was stirred for 20 minutes, and the absorbance was measured at 565 nm with an absorbance spectrometer. The effect of inhibiting apoptosis was measured.

As a result of the experiment, it was found that the organo extract is a stimulating substance that reduces skin irritation that may occur when applied with the cosmetic substrate by inhibiting apoptosis caused by SLS (Table 9).

 sample  Fibroblast survival rate (%)  0.01% SLS  23  0.01% SLS + 0.01% organic organo extract  60

Experimental Example 1

In this Experimental Example, the cosmetics containing the organic furnace extract obtained in Example 1 were prepared, and the skin whitening effect of humans was evaluated by comparative experiments with Comparative Example 1.

The cosmetics used in the comparative experiments are in the form of a cream, the composition of which is shown in Table 10. First, the phase b) recorded in Table 10 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Examples 11 to 13, Comparative Example 1).

Raw material Example 11 Example 12 Example 13 Comparative Example 1  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 mol addition) alcohol ester 3 3 3 3 Glyceryl Monostearic Acid Ester 2 2 2 2  I Organic Furnace Extract 10 One 0.1 - Propylene glycol 5 5 5 5 Purified water Quantity Quantity Quantity Quantity

Note) Unit: Weight%

For 20 experimenters (women aged 20-35 years), the creams prepared in Examples 11 to 13 on the right side of the face and the creams prepared in Comparative Example 1 on the left side of the face were applied twice a day for 2 consecutive months. Applied. After completion of the test, the skins of the application areas on both sides of the face were compared by using an image analyzer and a color difference meter. Table 11 compares the facial skin color of the experimenter using the cream prepared in Example 13 with the facial skin color of the experimenter using the cream made in Comparative Example 1.

As shown in Table 11, it can be seen that the whitening effect is excellent in the facial skin of the experimenter applying the cream containing the organic furnace extract.

Product used Example 13 Comparative Example 1 Experiment number Right skin color Left skin color One 1.5 2 2 2 2.5 3 1.5 2 4 2 2.5 5 3 3 6 1.5 2 7 3 3.5 8 2 2 9 1.5 2 10 1.5 2 11 2.5 3 12 1.5 2 13 1.5 2.5 14 2 2 15 2 2.5 16 1.5 2 17 2 3 18 2.5 3.5 19 1.5 2 20 1.5 2.5

Experimental Example 2

In this experimental example, the cream form cosmetics prepared in Examples 11 to 13 and Comparative Example 1 were applied to humans to evaluate the effect of improving skin wrinkles.

For 20 test subjects (20-35 year old female), the cream prepared in Examples 11 to 13 was applied to the right side of the face and the cream prepared in Comparative Example 1 was applied twice a day for 2 consecutive months to the left side of the face. It was.

After the experiment was completed, the wrinkles around the skin were collected with a silicone resin copy (replica) before and after using the product for two months, and the skin fine wrinkle device and the skin image analyzer were used to compare the condition of the eye wrinkles.

Table 12 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using the creams prepared in Examples 11 to 13 with the experimenter using the cream of Comparative Example 1. As shown in Table 12, it can be seen that the skin fine wrinkle improvement effect is excellent in the skin around the face of the experimenter applying the cream containing the organic furnace extract.

Example 12 Comparative Example 1 Before use 2 months later Before use 2 months later Average fine wrinkle depth (AU) 0.201 0.180 0.199 0.195 Wrinkle Reduction (%) 10.45% 2.01%

     n = 20, p <0.01

Experimental Example 3

This Example evaluated the irritation-relieving effect of the cosmetic containing the organic furnace extract obtained in Example 1 by human patch experiment.

This evaluation is the result of evaluation obtained by directly applying the result obtained in Example 10 to the human body. Stimulation index by stimulating 24 hours, 48 hours, 72 hours by mixing SLS (sodium lauryl sulfate) causing irritation in general cosmetic prescription (cream, lotion, skin, essence) and the product prepared in Examples 11 to 13 The stimulation-releasing effect was evaluated based on the results.

FINN CHAMBER (FINLAND) was used to coat 0.2 mg of each product on the upper arm of 50 healthy men and women aged 20-50 years, and the acute irritation index was evaluated after 24 hours. After evaluation, the same amount of product was applied to the same site again, and the delayed stimulation index after 48 and 72 hours was evaluated.

The test resulted in no skin redness even after 24 hours, 48 hours, 72 hours of patching with the product containing the organo extract.

The results of this evaluation confirmed that there is a significant effect that can reduce the skin irritation caused by the substrate (surfactant, fragrance, alcohol) that causes irritation when the organo extract is used in cosmetics.

Other examples are shown below. That is, the organic o extract of the present invention and the composition containing the same showed an excellent effect on skin improvement such as whitening effect, stimulating effect, wrinkle improvement effect as before.

Example 14 Preparation of Cosmetic Water Containing Organic Furnace Extract

To 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleic alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoic acid methyl ester, a small amount of antioxidant, and a small amount of pigment are dissolved. . 0.05 g of the organic furnace extract obtained in Example 1 and 5 g of glycerine were dissolved in 85.33 g of purified water, and the mixed solution was added thereto, followed by stirring to prepare a lotion having a skin improvement effect.

Example 15 Preparation of Milky Milk Containing Organic Furnace Extract

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of petroleum jelly, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteraryide, 1 g of polyoxyethylene (20 mole addition) monooleate and 0.1 g of fragrance at 70 ° C Heat-dissolve and dissolve, and heat-dissolve 0.05 g of the organic furnace extract obtained in Example 2, 5 g of dipropylene glycol, 2 g of polyethylene glycol 1500, 0.2 g of triethanolamine, and 76.2 g of purified water at 75 ° C. Both were emulsified by mixing and then cooled to prepare an emulsion having a water / oil-based skin improvement effect.

Example 16: Preparation of Cosmetic Liquid Containing Organic Furnace Extract

To 5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of kitulose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium licorice, 0.1 g of paraoxybenzoic acid ester, Each 1 g of the organic furnace extract obtained in Example 1 and an appropriate amount of pigments were mixed to obtain a cosmetic liquid having a skin improvement effect.

 As described above, the organic o extract according to the present invention has a whitening effect and a free radical scavenging effect, such as a tyrosinase inhibitory effect, a melanin synthesis inhibitory effect, which inhibit melanin production, which is a substance causing blemishes, freckles and skin pigmentation. , Wrinkle improvement effect such as the activity and expression suppression of collagenase (MMP-1) was confirmed, and the effect of inhibiting skin cell death to reduce skin irritation caused by the cosmetics substrate, inhibitory effect on inflammation-induced enzyme activity It has been found that the skin irritation effect is excellent.

Therefore, the present invention may prepare a cosmetic composition such as a lotion, cream, milky lotion, pack, etc. containing an organic furnace extract having the above-described efficacy can provide a functional cosmetic composition having a skin whitening effect wrinkle improvement effect, irritation relief effect. .

Claims (4)

Cosmetic composition for improving skin whitening and wrinkles containing an organic extract (Artemisia Anomala). The cosmetic composition for improving skin whitening and wrinkles of claim 1, wherein the organic o extract is contained in an amount of 0.001 to 30.0 wt% based on the total weight of the total composition. According to claim 1 or claim 2, wherein the organic furnace extract is at least one selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane as a solvent A cosmetic composition for improving skin whitening and wrinkles, comprising an organic furnace extract, which is obtained by cooling at room temperature, heating and filtration to obtain a liquid, and further, by concentrating the solvent under reduced pressure or lyophilization. The cosmetic composition according to claim 1 or 2, wherein the cosmetic composition comprises a lotion, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water (O / W) type or a water-in-oil (W / O) type, and an oil-in-water type or Water-in-oil type makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, teak color and eyebrow pencils containing an organic no extract characterized in that it is selected from Cosmetic composition for skin whitening and wrinkle improvement.