KR100588830B1 - A cosmetic composition for anti-aging containing an extract of melothria heterophylla - Google Patents

Abstract

The present invention relates to a method for extracting and purifying an active ingredient from baekryeom, and to a skin cosmetic containing the extract or an active ingredient separated therefrom. According to the present invention, the ethanol extract obtained from baekryeom has excellent free radical scavenging effect, activity of inhibiting expression and expression of collagenase (MMP-1) and promoting collagen synthesis, and particularly, the active ingredient contained in this raw material is 1, 2,4,6-tetragaloylbetadiglucopyranose (1,2,4,6-Tetra-O-galloyl-beta-D-glucopyranose), which uses this substance to prevent skin aging and improve wrinkles It is possible to produce various functional cosmetic compositions excellent in the.

Whiteness, MELOTHRIA HETEROPHYLLA, extract, free radicals, wrinkle improvement, cosmetics.

Description

Anti-aging cosmetic composition containing baekryeom extract {A COSMETIC COMPOSITION FOR ANTI-AGING CONTAINING AN EXTRACT OF MELOTHRIA HETEROPHYLLA}

The present invention relates to a cosmetic containing an extract of baekryeom, in detail, a method for extracting and purifying the active ingredient from baekryeom excellent in preventing and improving skin wrinkles and containing the extract or an active ingredient separated therefrom It relates to skin cosmetics.

Human skin is constantly changing with age. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories, 1996; 111: 31-37), 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 (HW Daniell, Ann Intern Med, 1971; 75 (6): 873-880; GL Grove et al., J Am Acad Dermatol, 1989; 21 (3): 631-637; CE Griffiths et al., Arch Dermatol, 1992; 128: 347-351).

One of the photoaging mechanisms caused by ultraviolet light is via free radical pathways (D Harman, J Gerontol, 1956; 11 (3): 298-300). 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 function, intercellular energy transfer, and modification of metabolic related molecules (RM Lavker and AM Kligman, J Invest Dermatol, 1988; 90: 325-330). 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 collagen. The expression of collagenase (hereinafter referred to as 'MMP-1') and gelatinase (hereinafter referred to as 'MMP-2') is promoted to decrease the elasticity of the skin and to form wrinkles. Ultraviolet radiation and free radicals also activate these degrading enzymes.

MMPs are broadly classified into three groups according to their substrate specificity. Group I belongs to interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8). Types I, II, III, VIII, Gelatin and Gelatin are cut into peptides of 3/4 or 1/4 lengths of collagen length to facilitate the action of nonspecific enzymes (gelatinases). Group II includes 72kd gelatinases (MMP-2) and 92kd gelatinases (MMP-9), type IV collagenases, with gelatin, collagen types IV, V, VIII, VIII, elastin and fibro Decompose nectins. Group III includes stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and matrylysin (MMP-7). Decomposes gelatin, proteoglycans, fibronectin, laminin, elastin and the like.

MMPs of neutrophils and polymorphonuclear leukocytes are primarily activated by oxidation, which first generates hydrogen peroxide in the cell and is converted to hypochlorous acid by myeloperoxidase in the presence of chlorine ions. Activate (S Kondo, J Dermatol Sci, 2000; 23 (1): S30-S36).

Until now, the most common method was to apply sunscreen products directly to the skin to prevent aging due to ultraviolet rays.However, since 1988, retinoic acid was reported to be effective in reducing roughness and fine wrinkles of aged skin. JS Weiss et al., J Am Acad Dermatol, 1988; 19 (1): 169-175), research is 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 representative skin aging improving agents known to date (DS Rosenthal et al., J Invest Dermatol, 1990; 95: 510-515, CM Ditre et al., J Am Acad Dermatol, 1996; 34 (2): 187-195). However, they are very unstable in the natural environment, so there is a problem in use, or somewhat insufficient to show a visible effect. Therefore, there is an urgent need for the development of new skin anti-wrinkle substances that are safe for the living body, have the ability of scavenging free radicals, inhibiting the activity and expression of collagenase (MMP-1), and collagen synthesis.

The present invention has been made to solve the above problems, by selecting the baekryeom from a variety of herbal medicines, by confirming its efficacy as a cosmetic raw material and developing a method of using the same, to increase the selection range of various cosmetic raw materials, It is also an object of the present invention to provide a method for producing a cosmetic product having a predetermined functional effect without further irritation using this raw material.

That is, an object of the present invention is a natural extract that can be applied to skin cosmetics and contains an anti-wrinkle effect such as antioxidant effect, collagen synthesis promoting effect, collagenase (MMP-1), and anti-wrinkle effect when contained in cosmetics. To provide.

In addition, the present invention reveals what is the active ingredient as a raw material for cosmetics from the point that the target efficacy is greater when selecting a specific extractant when preparing the extract of P. aeruginosa. It aims at providing the manufacturing method and its cosmetic composition.

In order to achieve the above object, the present inventors have studied to develop a material having excellent free radical scavenging effect, inhibitory activity and expression of collagenase (MMP-1) and collagen synthesis promoting effect on plants native to nature. As a result, the present inventors have found that there is a very powerful free radical scavenging effect on pneumonia, an activity and expression inhibitory effect of collagenase (MMP-1), and a collagen synthesis promoting effect, thereby completing the present invention.

The baekryeom used in the present invention is a perennial herbaceous herb of the gourd of Melothria heterophylla (Lour.) Cogn. There is an unbranched tendril and the root part is swollen to form fusiform muscle. The leaves are alternate with single leaves, have long petioles, and there are many variations, so they are divided into three branches shallowly or deeply into three to five branches, and further split, and the middle lobe is long and large, and the leaf bottom is multipolar.多 形). Flowers are monolithic, or several clusters are gathered from lobe, peduncles are thin, long, linear, and corolla is white, triangular bell-shaped, deeply divided into 5 branches, There are three stamens, and the female flower is the lower one. Fruits are mostly long round and 2 ~ 4㎝ long. It is distributed in the north of Hwanghae, North Korea, and in various places in China. In oriental medicine, the roots are dug up and washed in the fall, and fresh or sun-dried. They are prescribed for sore throat, conjunctivitis, lymphadenopathy, testicles, and skin eczema. The components of pneumonia are lignoceric acid, tricosanoic acid, calebasine B, cetrulline, arginine, glutamic acid, and beta-cytosterol. -sitosterol) and octacosanoic acid have been isolated (Chinese Herb, Shanghai Science and Technology Press, 1999).

Patents relating to such pneumonia include tyrosinase activity inhibitors, free radical scavengers, and antioxidants, which are commercially available from Melathria indica , M. heterophylla , and M. japonica extracts in Japanese Patent Application Laid-Open Nos. 10-114670 and 9-315398. The whitening effect and the whitening cosmetics containing Melothria genus plant extract as an active ingredient have been reported by adding one or two or more medicinal agents selected from cell activators, anti-inflammatory agents and moisturizers. There is no relationship between anti-oxidation and collagenase activity and expression inhibition and skin wrinkle improvement and prevention through promoting collagen synthesis.

The present invention is obtained by extracting 40 ~ 95% by weight ethanol aqueous solution containing baekryeom as an extraction solvent, characterized in that it contains 1,2,4,6-Tetra-O-galloyl-beta-D-glucopyranose 0.0001 ~ 2.0% by weight It provides a pneumonia extract.

The present invention also comprises the steps of (1) extracting baekryeom with an aqueous solution containing 40 to 95% by weight of ethanol at 70 ~ 85 ℃, (2) concentrating the filtrate obtained in the extraction step at 50 ℃ or less, ( 3) distilling off the solvent under reduced pressure with n-hexane, dichloromethane, ethyl acetate and saturated butanol, and (4) Sephadex L-20 with methanol (% by weight-40 to 100) as a mobile phase. Sephadex LH-20) isolating and purifying the ethanol extract containing 1,2,4,6-Tetra-O-galloyl-beta-D-glucopyranose as an active ingredient from the ethyl acetate fraction by column chromatography. It provides a method for producing baekryeom extract, characterized in that it comprises.

The present invention provides a white powder extract powder, characterized in that the extract is mixed with a film forming agent and powdered by a spray dryer.

In addition, 1,2,4,6-Tetra-O-galloyl-beta-D-glucopyranose is an ampelopsis japonica , Cynanchum auriculatum Royle, Cynanchum wilfordi , which contains many of the same components in addition to baekryeom. (Max.) Hemsl), Cornus (Cornus officinalis Sieb. et Zucc. ), pomegranate (Punica granatum L.), baekgyo (Liquidambar formosana Hance), Melothria indica , Melothria japonica, Ampelopsis serianaefolia Bunge, from the group consisting of Ampelopsis aconirifolia Bunge Can also be extracted.

The present invention provides a cosmetic composition for improving skin wrinkles and preventing aging by inhibiting the activity and expression of collagenase (MMP-1) and antioxidant activity containing baekryeom extract.

In addition, the present invention is to provide a cosmetic composition, characterized in that extracted by using baekryeom 40 ~ 95% by weight ethanol as the extraction solvent.

The present invention also relates to a cosmetic composition comprising 1,2,4,6-Tetra-O-galloyl-beta-D-glucopyranose as an active ingredient.

The content of the baekryeom extract contained in the cosmetic composition according to the present invention is preferably included in an amount of 0.0001 to 20% by weight. If the content of baekryeom extract is less than 0.0001% by weight of the skin wrinkle improvement effect is insignificant, if it exceeds 20% by weight because the effect of increasing the content is insignificant because it is not economical.

The active ingredient is contained in the form of the baekryeom extract or baekryeom extract powder.

 Furthermore, the cosmetic composition according to the present invention is a base cosmetic formulation consisting of a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type 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, characterized in that any one of the formulation selected from 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 Pleurotus Extract

Three hundred and fifty grams of healthy pneumonia were refluxed three times for 3 hours with a hot 95% (V / V) ethanol aqueous solution, and then cooled and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C. or lower to obtain 81.5 g of ethanol extract. The baekryeom extract was prepared using at least one solvent selected from purified water, ethanol, butylene glycol, and propylene glycol so that the vacuum concentrate and the freeze-dried product contained 0.0001 to 20.0% by weight. In addition, the depressurized concentrate and the freeze-dried product are dissolved in an aqueous ethanol solution containing 0.0001 to 20.0% by weight, and then a film forming agent, sodium carboxymethylcellulose, crystalline cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, and sucrose It was mixed with lactose, glucose, galactose, HP-β-cyclodexterine and powdered with a spray dryer.

Example 2: Isolation and Structure Confirmation of Active Active Ingredients from Bacillus Extract

The dried pneumatic vulture extract was dispersed in water and extracted three times with n-hexane to obtain an n-hexane fraction. The mixture was washed with saturated sodium chloride solution, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 5.8 g of a fraction. . The remaining water layer was dehydrated and concentrated using dichloromethane, ethyl acetate, saturated butanol and the like in the same manner as the n-hexane fraction to obtain 0.8g, 10.7g and 16.3g fractions, respectively. For each fraction, the ethyl acetate fraction having an excellent antioxidant effect was eluted with methanol (40% by weight) as a mobile phase by Sephadex L--20 column chromatography through the experiments of Examples 3 to 4. Then, gradient column chromatography was performed gradually increasing the proportion of methanol until the final materials were separated and eluted to obtain 20 fractions (Fr. 1 to Fr. 20). Six fractions with excellent antioxidant effects were selected from the experiments of Examples 3 to 4 for 20 fractions (Fr. 10, 12, 14, 15, 18, 19). Fraction 15, which showed the best antioxidant effect among the six fractions selected, was recrystallized using ethyl acetate. After confirming the qualitative purity of the recrystallized compound through thin layer chromatography, the structure was confirmed by UV-VIS and ¹ H-NMR, ¹³ C, NMR, MS spectrum analysis.

Physicochemical properties of the isolated compounds and the structures of the active ingredients of the extracts analyzed by various spectroscopy are as follows.

Fraction 15 material was separated into brown amorphous powder, showed FeCl ₃ positive reaction to predict the presence of phenolic OH group, and UV absorption at 217 nm and 277.1 nm was presumed to be a kind of galloyl glucosides (BS Min et al. al., Arch Pharm Res, 2002; 24 (4): 441-445). ¹³ C-NMR (125 MHz, Acetone- d ₆ ) analysis showed five aromatic carbon peaks between 65.67 and 74.7 ppm, one peak at 102.93 ppm, and ¹ H-NMR (500 MHz, Acetone- d ₆ ) analysis. From the splitting pattern of 4.47-4.67 ppm, it could be assumed that it contains glucopyranose per component. The four singlets at 7.28 ~ 7.32 ppm showed the overlapping hydrogens of galloyl group 2 'and 6', respectively, and 4 peaks were observed, indicating that there were 4 galloyl groups. In addition, the carbon peaks appearing between 110.42 and 167.32 ppm of the ¹³ C-NMR spectrum are typical gallic acid peaks, indicating that there are four galloyl groups in the signal pairs with similar chemical shift values. Fraction 15 was compared to the results of this data analysis and the previously reported results of Tanaka et al. (T Tanaka et al., Phytochemistry, 1985; 24 (9); 2075). 1,2,4,6-Tetra-O-galloyl-β -D-gluco-pyranose (MW 788.6, melting point 211 ~ 213 ℃) was identified.

Example 2 is a skin wrinkle improvement effect, such as the effect of the present inventors required in the fraction of the extract of baekryeom, that is, free radical scavenging effect, collagen synthesis promoting effect, the activity and expression suppression of collagenase (MMP-1) The active active material was traced by selecting only the fractions with excellent skin irritation-releasing effect. As a result of this study, 1,2,4,6-Tetra-O-galloyl-β-D It is significant that it is identified as -glucopyranose.

Example 3: Antioxidant Effect Measurement Experiment Using DPPH Radical

This example is a slight modification of the method used by Yoshida et al. (1989) to measure the antioxidant effect of the materials obtained in Examples 1 and 2, and the scavenging effect on 1,1-diphenyl-2-picryl hydrazyl (DPPH) radicals. Was measured. Since DPPH is purple as a stable free radical, it is a measure of the extent to which purple is removed as the sample scavenges radicals.

First, DPPH was dissolved in methanol at a concentration of 1 × 10 ⁻⁴ M and 0.15 ml of this solution was placed in a test tube. 0.15 ml of each concentration sample was added thereto, and the absorbance was measured at 565 nm after 20 minutes of reaction at room temperature. As a positive control, green tea extract and vitamin C were used instead of the material of the example. The free radical scavenging effect of each sample was expressed as SC ₅₀ by calculating the concentration of 50% scavenging of radicals using Equation 1. At this time, the negative control was taken as the reaction absorbance of the liquid not treated with the sample.

% Free radical scavenging ratio = [(absorbance of control-absorbance of each sample) / absorbance of control] x 100

Free Radical Scavenging Effect of Extract Experimental substance SC ₅₀ (μg / ml) Example 1 13 Example 2 3.1 Green tea extract 400 Vitamin c 10.6

As shown in Table 1, the baekryeom extract of the present invention and 1,2,4,6-Tetra-O-galloyl-β-D-glucopyranose, the active ingredient of the present invention, compared to the powerful antioxidant vitamin C and green tea extract, baekryeom extract Compared to the green tea extract in the form of silver extract, 30 times, the active ingredient showed a very strong activity of about 3 times compared to vitamin C, a single component.

Example 4 Antioxidant Effect Measurement Experiment Using Active Oxygen Radicals

This example was produced by the xanthine-xanthine oxidase system, with a slight modification of the method used by Furuno et al. (2002) to measure the antioxidant effects of the materials obtained in Examples 1-2. The scavenging effect on the superoxide radicals was measured. The free radicals produced by the xanthine and xanthine oxidase reactions react with Nitro Blue Tetrazolium (NBT) to give a blue color, so the amount of blue reduced as the sample eliminates free radicals is measured. It is.

First, 3 × 10 ^-3 M xanthine, 3 × 10 ^-4 M EDTA, 7.5 × 10 ^-4 M nitroblue tetrazolium (NBT), and 0.15 mg / mL bovine serum albumin (BSA) solution were added to each concentration sample. After mixing, the mixture was reacted at room temperature for 10 minutes. Thereafter, xanthine oxysidase (0.25 U / ml) solution was added to each test tube and reacted at room temperature for 20 minutes, and then absorbance was measured at 565 nm. Green tea extract, BHA, was used instead of the material of the example as a positive control. The active oxygen radical scavenging effect of each sample was expressed as SC ₅₀ by calculating the concentration of 50% scavenging radicals using Equation 2. At this time, the negative control was taken as the reaction absorbance of the liquid not treated with the sample.

Free radical radical scavenging rate (%) = [(absorbance of the control group-absorbance of each sample) / absorbance of the control group] × 100

Free radical radical scavenging effect of baekryeom extract Experimental substance SC ₅₀ (μg / ml) Example 1 20 Example 2 3.4 Green tea extract 340 BHA 32.4

As shown in Table 2, the baekryeom extract of the present invention and 1,2,4,6-Tetra-O-galloyl-β-D-glucopyranose, the active ingredient of the present invention, are compared with the powerful antioxidants BHA and green tea extract, Compared to the green tea extract in the same extract form 17 times, the active ingredient showed a very strong activity of about 9 times compared to the single ingredient BHA.

Example 5: Experiment to measure collagen synthesis promoting effect

In order to observe the collagen synthesis promoting effect of the samples obtained in Examples 1 to 2, the experiments were 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 cells were collected for 1 day after treatment for each concentration. The collected cell culture solution was measured for collagen synthesis using a collagen protein measuring instrument (Catalog N .: MK 101, Takara Shuzo Co. Ltd, Japan).

In the measurement method, first, a cell culture solution 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 developing agent was added to develop color 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 for absorbance at 450 nm using an absorbance spectrometer, and the degree of collagen synthesis was calculated by Equation 3 below. At this time, the reaction absorbance of the cell culture without the sample was used as a control.

Collagen Synthesis Effect (%) = [(Reaction Absorbance of Control-Response Absorbance of Cell Culture of Sample) / Reaction Absorbance of Control] × 100

Promoting Collagen Synthesis of Bacillus Extract Test substance (㎍ / ㎖) Collagen Synthesis Effect (%) Example 1 100 80 10 52 Example 2 10 60 One 40

As shown in Table 3, the baekryeom extract of the present invention and the active ingredient 1,2,4,6-Tetra-O-galloyl-β-D-glucopyranose can be seen that the excellent collagen synthesis promoting effect.

Example 6: activity inhibition assay of collagenase (MMP-1)

This example was measured in a biochemical model of the screening type in vitro to observe the inhibitory effect of collagenase (MMP-1) of the samples obtained in Examples 1 to 2, which was purified collagenase and its It is based on the use of collagen conjugated to the substrate, fluorescein (EnzChek, Collagenase Kit, Molar Proof, USA).

First, 20 µl of 0.25 mg / ml DQ collagen lysis solution and 40 µl of the concentration sample in 100 µl of reaction buffer (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl ₂ , 0.02 mM NaN ₃ , pH7.3) After the addition, the mixture was well mixed with 40 µl of 0.5 Unit of collagenase and reacted for 20 minutes at dark and room temperature. Thereafter, the fluorescence value was measured at an absorption wavelength of 495 nm and an emission wavelength of 515 nm using a fluorescence spectrophotometer. Green tea extract was used instead of the material of the example as a positive control. The inhibitory effect of collagenase activity of each sample was expressed as IC ₅₀ by calculating the concentration of 50% scavenging enzyme activity using Equation 4. At this time, the negative control was used as the fluorescence value of the liquid without the sample.

Collagenase (MMP-1) activity inhibitory effect (%) = [(response fluorescence value of control-response fluorescence value of sample) / response fluorescence value of control group] × 100

Inhibitory Activity of Collagenase Activity of Bacillus Extract Experimental substance IC ₅₀ (μg / ml) Example 1 100 Example 2 10 Green tea extract 200

As shown in Table 4, the baekryeom extract of the present invention and the active ingredient 1,2,4,6-Tetra-O-galloyl-β-D-glucopyranose are very potent compared to the green tea extract, a potent collagenase inhibitor Inhibitory activity was shown.

Example 7: Experiment to measure the inhibitory effect of collagenase (MMP-1)

This example was performed by slightly modifying the enzyme linked immunosorbent assay (ELISA) used by Petersen et al. (1992) to inhibit the expression of collagenase, which is increased by UVA irradiation of the samples obtained in Examples 1 and 2. .

First, UVA was irradiated to human dermal fibroblasts with an energy of 6.3 J / cm ² using an ultraviolet chamber, and then exchanged with the medium to which the sample was added, followed by culturing for 24 hours, and then the medium was recovered and coated in a 96-hole plate incubator. The primary antibody (MMP-1 monoclonal antibody) was treated and reacted at 37 ° C. for 90 minutes, and then treated with 3% BSA for 1 hour to block unbound sites. After reacting the secondary antibody anti-mouse immunoglobulin (anti-mouse IgG, whole mouse, alkaline phosphatase conjugated) for about 90 minutes, alkaline phosphatase substrate solution (1 mg / ml p-nitrophenyl phosphate in diethanolamine buffer solution) Was added and reacted at room temperature for 30 minutes. Absorbance was measured at 405 nm using an absorbance spectrometer. The degree of inhibition of MMP-1 activity was calculated by Equation 5 below. At this time, the reaction absorbance of the cell culture without the sample was used as a control.

MMP-1 expression inhibition rate (%) = [(response absorbance of control-reaction absorbance of cell culture of sample) / control absorbance of control] x 100

Inhibitory Effects of Bacillus Extracts on Collagenase Expression Test substance (㎍ / ㎖) % Inhibition of expression Example 1 100 78.3 Example 2 5 45 Green tea extract 100 75

As shown in Table 5, the baekryeom extract of the present invention and 1,2,4,6-Tetra-O-galloyl-β-D-glucopyranose of the present invention are similar or very potent to the expression of collagenase in green tea extract. Appeared to inhibit.

Example 8: Evaluation of Stimulation Relaxation Effect Using Cell Culture Technology

In this example, the cell death by Sodium Lauryl Sulfate (SLS), a substance that causes skin irritation using cell culture technology, in order to evaluate the stimulating relaxation effect of the samples obtained in Examples 1 and 2 The degree of stimulation alleviation was evaluated to the extent that it inhibited.

SLS is a substance that is used as a criterion for evaluation of skin irritation based on cosmetics. It is bound to cell membranes to inhibit cell metabolism and destroys cell membranes to cause skin irritation. This example evaluates the effect of reducing cell death by SLS when SLS and a sample 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 evaluation experiment using the cell culture technology was carried out as follows.

First, human-derived fibroblasts were dispensed at a concentration of 1 × 10 ⁵ per well in a 96-well plate incubator, and cultured for 24 hours, followed by treatment with 0.01% SLS alone, 0.01% SLS and a sample (0.01%) at the same time for 24 hours. While further cultured. After incubation, MTT (Sigma) reagent was added and incubated for 4 hours, and then the culture solution was removed, stirred for 20 minutes by addition of 1 N NaOH / isopropanol solution, and the absorbance was measured at 565 nm with a spectrophotometer. The effect of inhibiting apoptosis was measured.

Stimulation Relaxation Effect Using Cell Culture Technology Test substance (㎍ / ㎖) Fibroblast survival rate (%) Example 1 (100) + SLS (100) 65 Example 2 (100) + SLS (100) 60 SLS (100) 23

As shown in Table 6, it was found that the baekryeom extract is an irritant that reduces skin irritation that may occur when applied with the cosmetic substrate by inhibiting apoptosis caused by SLS.

Examples 9 to 11 and Comparative Examples: skin wrinkle improvement

In this example, the cosmetic composition containing the baekryeom extract obtained in Example 1 was prepared to evaluate the effect of improving the fine wrinkles of the skin in a comparative experiment 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 7. First, the phase b) recorded in Table 7 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 9 to 11 and Comparative Example 1).

Raw material Example Comparative Example 1 9 10 11 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 Aster 2 2 2 2 I Baekryeom extract 10 One 0.1 - Propylene glycol 5 5 5 5 Purified water Quantity Quantity Quantity Quantity

Note) Unit: Weight%

For 20 experimenters (20-35 year old female), the creams prepared in Examples 9-11 were applied to the right side of the face and the creams prepared in Comparative Example 1 were applied twice a day for 2 consecutive months to the left side of the face. It was.

After completion of the experiment, the effect of improving skin wrinkles is obtained by using a silicone resin copy (replica) before and after using the product for 2 months, using a skin fine wrinkle device (SV600, C + K, Germany) and a skin imaging analyzer. The state of the fine wrinkles was compared.

Skin wrinkle improvement effect Evaluation item Example 10 Comparative Example 1 Before use 2 months later Before use 2 months later Average fine wrinkle depth 0.201 0.150 0.200 0.196 Wrinkle Reduction (%) 25.4% 2.0%

Table 8 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using the creams prepared in Examples 9 to 11 with the experimenter using the cream of Comparative Example 1. As shown in Table 8, it can be seen that the skin fine wrinkles improvement effect is excellent in the facial periphery of the experimenter who applied the cream containing baekryeom extract.

Example 12: Experiment for stimulating alleviation of cosmetics containing baekryeom extract

This Example is to evaluate the stimulating alleviation effect of the cosmetics containing the baekryeom extract obtained in Example 1 by human patch experiment. This evaluation is a result of evaluation obtained by directly applying the result obtained in Example 8 to the human body. SLS (sodium lauryl sulfate), which causes irritation to general cosmetic prescriptions (cream, lotion, skin, essence), is mixed with the products prepared in Examples 9 to 11 and patched for 24 hours, 48 hours, and 72 hours. The stimulation-relaxation effect was evaluated based on this.

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 the product containing the baekryeom extract.

The results of this evaluation indicate that when the chlorosis extract is mixed with cosmetics, there is a beneficial effect of reducing skin irritation by the substrate (surfactant, fragrance, alcohol) that causes irritation.

Other examples are shown below. That is, the baekryeom extract of the present invention and the composition containing the same showed an excellent effect on skin improvement such as stimulation relief effect, wrinkle improvement effect as described above.

Example 13: Preparation of Cosmetic Water Containing White Flower Extract

To 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of polyol 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 chlorosis extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water, and then the mixed solution was added and stirred to obtain a lotion having a skin improving effect.

Example 14 Preparation of Latex Containing a Pleurotus Extract

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of vaseline, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoester acetate, 1 g of polyoxyethylene (20 mole addition) monooleate and 0.1 g of fragrance at 70 ° C The mixture was dissolved by heating, followed by heating and dissolving 0.05 g of the white worm extract, 5 g of dipropylene glycol, 2 g of polyethylene glycol 1500, 0.2 g of triethanolamine, and 76.2 g of purified water to 75 ° C. Both mixtures were emulsified and then cooled to obtain an emulsion having a water / medium-based skin improvement effect.

Example 15 Preparation of a Cosmetic Solution Containing a Pleurotus Extract

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 licoate, 0.1 g of paraoxybenzoic acid ester, 1 g of each of the baekryeom extract obtained in Example 1 and a suitable amount of pigments were mixed to obtain a cosmetic solution having a skin improving effect.

As described above, the baekryeom extract according to the present invention has excellent skin wrinkle improvement effects such as free radical scavenging effect, collagen synthesis promoting effect, collagenase (MMP-1) activity and expression suppression, It is excellent in inhibiting skin cell death and alleviating skin irritation which reduces skin irritation.

In addition, the cosmetic composition containing the baekryeom extract according to the present invention or the active ingredient isolated therefrom can be applied as a component of the skin cosmetics excellent in terms of safety as well as effects when used in humans.

Accordingly, the present invention may provide a cosmetic composition including a lotion, cream, milky lotion, pack, etc. containing a baekryeom extract having the above-mentioned efficacy to provide a functional cosmetic composition having a skin wrinkle improvement effect and a stimulating alleviation effect.

In the above description of the preferred embodiment of the present invention, those skilled in the art will be able to perform various modifications without departing from the gist of the present invention, and such modifications are within the protection scope of the present invention. The protection scope of the present invention should be construed as described in the claims.

Claims (9)

delete (1) extracting baekryeom with an aqueous solution containing 40 to 95% by weight of ethanol at 70-85 ° C .; (2) concentrating the filtrate obtained in the extraction step under reduced pressure at 50 ° C or less; (3) solvent fractionating the reduced pressure concentrate with n-hexane, dichloromethane, ethyl acetate and saturated butanol; And (4) 1,2,4,6- active active ingredient from the ethyl acetate fractions by Sepadex LH-20 column chromatography using methanol having a weight percent ratio of 40 to 100 as a mobile phase. Separation and purification of the ethanol extract containing Tetra-O-galloyl-β-D-glucopyranose. delete A cosmetic composition comprising 1,2,4,6-Tetra-O-galloyl-β-D-glucopyranose as an active ingredient. The method of claim 4, wherein Cosmetic composition, characterized in that used for preventing skin aging. The method of claim 4, wherein Cosmetic composition, characterized in that used for alleviating skin irritation. The method of claim 4, wherein The active ingredient is a cosmetic composition, characterized in that it is included in the form of baekryeom extract prepared by claim 2. delete delete