JP6289629B2 - Anti-aging skin external preparation composition containing 21-O-angeloylteasapogenol E3 component derived from green tea seeds - Google Patents

Description

  The present invention relates to an anti-aging skin external preparation composition containing 21-O-angeloylteasapogenol E3 component derived from green tea seeds as an active ingredient, and more specifically, an acid, a base, an enzyme, or the enzyme By containing 21-O-angeloylteasapogenol E3 component derived from green tea seeds obtained from green tea seed extract through a decomposition reaction using microorganisms that produce glycine as an active ingredient, excellent wrinkle improvement and skin elasticity It is related with the anti-aging skin external preparation composition with which the improvement effect is show | played.

  Skin aging is roughly divided into two types depending on the factor. One of these is natural aging, where the structure and physiological function of the skin continues to decline with age, and another, extrinsic aging, It is caused by accumulated external stress such as light rays. In particular, ultraviolet rays of sunlight (ultraviolet rays; UV) is one of the well-known causes of aging. Skin exposed to ultraviolet rays for a long time has a thick stratum corneum, and collagen and elastin which are main components of the skin. Is denatured and loses its elasticity.

  On the other hand, the elasticity of the skin is handled by the extracellular matrix (ECM) component of the dermal tissue, but the ECM is mainly composed of two types of components. One is elastic fiber that accounts for about 2-4% of the total ECM, and the other is collagen that accounts for about 70-80% of the total ECM. Elastic fibers have a form in which microfibrils are driven into an amorphous substrate called elastin, and elastin can only be produced from elastic fibers called desmocine and isodesmosine derived from lysine. It is a protein composed of very unique amino acids that cannot be seen. Such desmosine, isodesmosine and the like form cross-links in a long peptide chain, and such a structure gives elastin a rubber-like property.

  Elastic fibers made of elastin coexist with collagen fibers called collagen, but the elasticity of the skin can be maintained in a state where elastin and collagen are sufficiently present.

  The decrease in skin elasticity results from a decrease or destruction of collagen and elastin production caused by aging, ultraviolet rays, and the like. In particular, the expression of matrix metalloproteases such as collagenase and elastase degrades the elasticity of skin by degrading collagen and elastin normally produced in the skin.

  Various substances have been developed and used in order to suppress the decrease in collagen and elastin that cause such a decrease in elasticity. Among them, retinoids such as retinol and retinoic acid have an effect of improving elasticity (Dermatology). therapeutic, 1998, 16, 357-364), protein fractions obtained from leguminosa seeds also show an effect of increasing elasticity (US Pat. No. 5,322,839), malt extract (malt extract) and the like. The containing composition has been applied to suppress collagenase (Japanese Patent No. 5,105,693).

  However, these retinoids have the disadvantage that irritation appears when only a small amount is applied to the skin, and most natural sources are used in the form of extracts, and the effectiveness of these extracts is accurate. However, it is difficult to control and maintain the activity of the extract continuously.

  Accordingly, the present inventors have made extensive efforts to find a substance that exhibits an anti-aging effect and also exhibits an excellent skin elasticity improving effect, and generates an acid, a base, an enzyme or the enzyme from a green tea seed extract. It has been found that 21-O-angeloylteasapogenol E3 component obtained through a decomposition reaction using microorganisms has excellent skin wrinkle improvement and skin elasticity improvement effects, and has completed the present invention.

  Therefore, an object of the present invention is to provide an anti-aging skin external preparation composition containing 21-O-angeloylteasapogenol E3 component obtained from a green tea seed extract.

  In order to solve the above-described object, the present invention provides a skin external preparation composition containing 21-O-angeloylteasapogenol E3 as an active ingredient.

  The 21-O-angeloylteasapogenol E3 derived from green tea seeds used in the present invention suppresses the expression of elastase and collagenase while promoting the biosynthesis of collagen, effectively improving skin wrinkles, and skin elasticity Improves the anti-aging effect.

  The present invention provides a skin external preparation composition containing 21-O-angeloyltheasapogenol E3 represented by the following chemical formula 1 as an active ingredient.

  The 21-O-angeloylteasapogenol E3 is preferably derived from green tea seeds.

  The 21-O-angeloylteasapogenol E3 used in the present invention is a first step of obtaining a saponin crude extract from a plant using water or an organic solvent, and the extract is converted into an acid, a base, an enzyme or the above It can be produced by a method comprising a second step of separating 21-O-angeloylteasapogenol E3 by hydrolysis using an enzyme-producing microorganism.

[First stage: Acquisition of crude saponin extract]
A crude saponin extract is obtained from plants, in particular green tea seeds. At this time, as the extraction solvent, water or an organic solvent can be used, and as the organic solvent, any one or more organic solvents selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and chloroform, or Mixtures of these with water, preferably 50% ethanol can be used.

  In order to obtain a crude saponin extract from a plant using water or an organic solvent, about 1 to 6 times, preferably about 3 times, water or an organic solvent is added to the plant and extracted while stirring at room temperature 1 to 5 times And degrease. About 1 to 8 times, preferably about 4 times, water or an organic solvent is added to the defatted plant, and after reflux extraction 1 to 5 times, it is immersed at 10 to 20 ° C. for 1 to 3 days. Next, the residue and the filtrate are separated using filtration and centrifugation, and the extract obtained by concentrating the separated filtrate under reduced pressure is suspended in water, and then the pigment is removed using ether or the like. After the aqueous layer is extracted 1 to 5 times using an organic solvent, the obtained organic solvent layer is concentrated under reduced pressure to obtain an organic solvent extract, which is then dissolved in a small amount of methanol, ethanol, propanol, butanol and the like. Then, a precipitate formed by adding a large amount of ethyl acetate, acetonitrile, or the like is dried to obtain the crude saponin extract of the present invention.

[Second stage: Separation of 21-O-angeloylteaasapogenol E3]
The crude saponin extract obtained in the first step is hydrolyzed to separate 21-O-angeloylteasapogenol E3, and hydrolysis uses an acid, a base, an enzyme, or a microorganism that produces the enzyme. Can be done.

  As the acid, any one or more acids selected from the group consisting of hydrochloric acid, sulfuric acid and nitric acid, or a mixed solvent of these acids and any one or more alcohols selected from the group consisting of ethanol, methanol and butanol, Preferably, a mixed solvent with 50% ethanol can be used.

  When hydrolyzing with an acid, after adding an acid having a concentration of 0.1 to 2N, preferably 1N, or a mixed solvent of an acid and an alcohol to the saponin crude extract in an amount of 1 to 10%, The mixture is heated to reflux in a water bath at 50 to 100 ° C., preferably 80 ° C. for 0.5 to 8 hours, preferably 1 hour.

  As the base, any one or more bases selected from the group consisting of sodium methoxide, sodium hydroxide and potassium hydroxide, or any one or more selected from the group consisting of these bases and ethanol, methanol and butanol A mixed solvent with alcohol, preferably a mixed solvent with 50% butanol can be used.

  When hydrolysis is performed using a base, the saponin crude extract is dissolved in water or a mixed solvent of water and ethanol, and then 0.1 to 2N, preferably 1N concentration of the base or base and alcohol. After adding the mixed solvent in an amount of 1 to 10%, the mixture is heated to reflux in a water bath at 50 to 100 ° C, preferably 100 ° C for 0.5 to 24 hours, preferably 12 hours.

  The enzyme is an enzyme that breaks down a sugar bond, and is characterized by removing the sugar moiety of green tea saponin to produce 21-O-angeloylteasapogenol E3. As this enzyme, a commercially available one is used, or it is produced and used as necessary. In particular, it is obtained from a microorganism that produces the enzyme.

  More preferably, the enzyme is glucosidase, arabinosidase, rhamnosidase, xylosidase, cellulase, hesperidinase, naringinase, naringinase, naringinase, Any one or more selected from the group consisting of glucuronidase, pectinase, galactosidase, and amyloglucosidase can be used.

  Furthermore, the microorganisms that produce the enzyme include the genera Aspergillus, Bacillus, Penicillium, Rhizopus, Rhizomucor, Taralomyces, and Talaromyces. Selected from the group consisting of the genus Bifidobacterium, Mortierella, Cryptococcus, Microbacterium, Leuconostoc and Lactobacillus The above can be used.

  When performing hydrolysis using an enzyme, the saponin crude extract is dissolved in an acidic buffer solution 5 to 20 times, preferably about 10 times, and then the enzyme is added in an amount of 0.001 to 10%. While this was stirred on a water bath at about 25 to 37 ° C. for about 40 to 55 hours, preferably about 48 hours, the elimination rate of the substrate was confirmed by thin layer chromatography. 80 to 100 ° C.) for 5 to 15 minutes to complete the reaction.

  When hydrolyzing using microorganisms, the saponin crude extract is dissolved in ionic water 5 to 10 times, preferably about 10 times, then sterilized at 121 ° C. for 30 minutes and cooled to 30 ° C. Next, after inoculating 5 to 10% by weight of a pre-cultured microorganism with respect to the liquid amount and culturing at 20 to 30 ° C. for 2 to 5 days, preferably 5 days, the substrate elimination rate by thin layer chromatography When the substrate disappears completely, the culture solution is centrifuged at 5,000 to 10,000 rpm, and the collected precipitate is washed three times with distilled water, and then centrifuged to obtain a precipitate.

  As described above, after hydrolysis using an acid, a base, an enzyme, or a microorganism that produces the enzyme, the reaction solution is concentrated under reduced pressure to remove the solvent, alcohol is added to the residue, and the mixture is stirred 1 to 5 times. Next, the precipitated salt is removed by filtration, and the filtrate is concentrated under reduced pressure to obtain a crude product. The resulting crude product is subjected to silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1). ) To obtain 21-O-angeloylteasapogenol E3.

  The composition of the present invention contains 21-O-angeloylteasapogenol E3, which can be produced using the method described above, in an amount of 0.000001 to 5% by weight based on the total weight of the composition. If the content is less than 0.000001% by weight, the wrinkle improvement and skin elasticity effect by the above components cannot be obtained, and even if the content exceeds 5% by weight, the increase in the effect is larger than the increase in the content Absent.

  The composition of the present invention can be used as an anti-aging composition, promotes collagen biosynthesis, suppresses the expression of elastase and collagenase, and further improves skin elasticity by the combined synergistic action of these two types of activities. In addition, the skin wrinkle improving effect is exhibited.

  The composition of the present invention is formulated with a cosmetically or dermatologically acceptable medium or base. This is any dosage form suitable for topical application, such as solutions, gels, solids, anhydrous kneaded products, emulsions obtained by dispersing the oil phase in the aqueous phase, suspensions, microemulsions, microcapsules, It is provided in the form of fine granulocytes or ionic (liposomes) and non-ionic vesicle dispersions, or in the form of creams, skins, lotions, powders, ointments, sprays or concealed sticks. It can be used in the form of a foam or an aerosol composition further containing a compressed propellant. These compositions are produced by conventional methods in the art.

  In addition, the composition of the present invention comprises a fatty substance, an organic solvent, a solubilizer, a thickener, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, and a fragrance. , Surfactant, water, ionic or non-ionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic or parent Adjuvants commonly used in the cosmetics or dermatological field such as oily active agents, lipid vesicles or any other ingredient commonly used in cosmetics can be included. The adjuvant is incorporated in an amount commonly used in the cosmetics or dermatological fields.

  Furthermore, the composition of the present invention can contain a skin absorption promoting substance in order to increase the effect of improving skin aging.

EXAMPLES Hereinafter, although an Example and a test example are given and this invention is explained in full detail, this invention is not limited to these examples.
[Example 1] Manufacture of green tea seed extract 6 kg of hexane was added to 2 kg of green tea seeds, extracted and stirred three times at room temperature, and then 4 l of 50% ethanol was added to 1 kg of defatted green tea seeds three times. After extraction under reflux, it was immersed at 15 ° C. for 1 day. Next, filtration and centrifugation using a filter cloth are performed to separate the residue and the filtrate. The extract obtained by concentrating the separated filtrate under reduced pressure is suspended in water, and then extracted 5 times with 1 l of ether. The dye was removed and the aqueous layer was extracted 3 times with 500 ml of 1-butanol. The whole 1-butanol layer obtained from this was concentrated under reduced pressure to obtain 1-butanol extract, which was dissolved in a small amount of methanol, added to a large amount of ethyl acetate, and the generated precipitate was dried. 300 g of green tea seed extract (crude saponin extract) was obtained.

[Example 2] Production of 21-O-angeloylteasapogenol E3 by acid hydrolysis method [2-1] 20 times (v / w) 1N of 10 g of green tea seed extract obtained in Example 1 above. HCl-50% methanol solution (v / v) was added and heated to reflux in a water bath at 80 ° C. for 8 hours to hydrolyze the sugar bound to the green tea seed crude saponin. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 ml) was added to the residue and stirred three times, and then the precipitated salt was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 7: 1 to 3: 1) to obtain 0.55 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

[2-2] 20 times (v / w) 1M H ₂ SO ₄ -30% aqueous solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, and the mixture was added to a 90 ° C. water bath The mixture was heated to reflux for 8 hours to hydrolyze the sugar bound to the green tea seed crude saponin. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 ml) was added to the residue and stirred three times, and then the precipitated salt was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 7: 1 to 3: 1) to obtain 0.59 g of 21-O-Angeloylteaasapogenol E3 was obtained.

[2-3] 20 times (v / w) 1M HNO ₃ -10% aqueous solution (v / v) is added to 10 g of the green tea seed extract obtained in Example 1, and the mixture is placed in a 90 ° C. water bath for 8 hours. The sugar bound to the green tea seed crude saponin was hydrolyzed by heating to reflux. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 ml) was added to the residue and stirred three times, and then the precipitated salt was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 7: 1 to 3: 1) to obtain 0.39 g of 21-O-Angeloylteaasapogenol E3 was obtained.

[Example 3] Production of 21-O-angeloylteasapogenol E3 by a base hydrolysis method [3-1] 10 g of the green tea seed extract obtained in Example 1 above was dissolved in dry pyridine (500 ml). 10 g of sodium methoxide powder was added to the mixture and refluxed for 8 hours in an oil bath to hydrolyze the sugar bound to the green tea seed saponin. The reaction solution was concentrated under reduced pressure to remove the solvent, washed with purified water three times, and then filtered to obtain a filtrate. After adding ethanol (200 ml) to the residue and stirring three times, the precipitated salt was filtered. Removed. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.35 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [3-2] 20 g (v / w) of 1M NaOH-20% aqueous solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, and the mixture was refluxed at 80 ° C. for 8 hours. The sugar bound to green tea seed saponin was hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, washed with purified water three times, and then filtered to obtain a filtrate. After adding ethanol (200 ml) to the residue and stirring three times, the precipitated salt was filtered. Removed. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.31 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [3-3] 20 g (v / w) of 1M KOH-20% aqueous solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, and the mixture was refluxed at 80 ° C. for 8 hours. The sugar bound to green tea seed saponin was hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, washed with purified water three times, and then filtered to obtain a filtrate. After adding ethanol (200 ml) to the residue and stirring three times, the precipitated salt was filtered. Removed. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.25 g of 21-O-Angeloylteaasapogenol E3 was obtained.

[Example 4] Production of 21-O-angeloylteasapogenol E3 by enzymatic degradation method [4-1] 10 g of the green tea seed extract obtained in Example 1 was added to 100 ml of 0.1 M acetate buffer solution ( dissolved in pH 4.5) where 2.5 g of enzyme (0.5 g of hesperidinase, 0.5 g of naringinase, 0.5 g of cellulase, 0.2 g of β-glucuronidase, 0.5 g of β-galactosidase, 0.3 g of amyloglucosidase; Sigma) was added and stirred on a water bath at 37 ° C. for 48 hours, and periodically confirmed by thin layer chromatography. When the green tea saponin disappeared, it was in hot water (80-100 ° C.) for 10 minutes. The reaction was terminated by heating. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 ml) was added to the residue and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 1.02 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [4-2] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of 0.1 M acetate buffer solution (pH 6.5), and 3.5 g of enzyme (glucosidase 1 g, arabinosidase 0) was dissolved therein. 0.5 g, rhamnosidase 1 g, xylosidase 0.5 g, pectinase 0.5 g) and stirring on a water bath at 27 ° C. for 48 hours while periodically checking by thin layer chromatography, when the green tea saponin disappears, The reaction was terminated by heating in hot water (80-100 ° C.) for 10 minutes. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 ml) was added to the residue and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 1.53 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

[Example 5] Production of 21-O-angeloylteasapogenol E3 utilizing microorganisms [5-1] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionic water, and 121 ° C. After sterilizing for 30 minutes and cooling to 30 ° C., inoculate 5-10% of the pre-cultured Aspergillus niger KCCM 11885 with respect to the liquid amount and incubate at 30 ° C. for 5 days. After confirming the elimination rate of the substrate by chromatography, it was confirmed that the substrate had disappeared completely, and the culture solution was centrifuged at 5,000 to 10,000 rpm, and the collected precipitate was washed with distilled water three times. The precipitate was obtained by centrifugation. Ethanol (200 ml) was added to the precipitate and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.72 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [5-2] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionic water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then pre-cultured Rhizopus oryzae. ) Was inoculated with 5 to 10% of the liquid amount and cultured at 27 ° C. for 5 days. Then, the substrate elimination rate was confirmed by thin layer chromatography to confirm that the substrate had completely disappeared. The precipitate collected by centrifugation at 5,000 to 10,000 rpm was washed three times with distilled water, and then centrifuged to obtain a precipitate. Ethanol (200 ml) was added to the precipitate and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.92 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [5-3] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionic water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then pre-cultured Bacillus subtilis (bacillus). Subtilis) was inoculated with 5-10% of the liquid volume and cultured at 27 ° C. for 2 days, and then the substrate elimination rate was confirmed by thin layer chromatography to confirm that the substrate had completely disappeared. The precipitate collected by centrifugation at 5,000 to 10,000 rpm was washed three times with distilled water, and then centrifuged to obtain a precipitate. Ethanol (200 ml) was added to the precipitate and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.72 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [5-4] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionic water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then cultured in advance. Inoculate 5-10% of leuroidos mesenteroides with liquid and incubate at 27 ° C for 2 days, then check substrate elimination rate by thin layer chromatography and confirm that substrate has completely disappeared The precipitate collected by centrifuging the culture at 5,000 to 10,000 rpm was washed three times with distilled water and then centrifuged to obtain a precipitate. Ethanol (200 ml) was added to the precipitate and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.52 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [5-5] 10 g of the green tea seed extract obtained in Example 1 is dissolved in 100 ml of ionic water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then pre-cultured Bifidobacterium ron After inoculating 5-10% gum (Bifidobacterium longum) with respect to the amount of liquid and culturing at 27 ° C. for 2 days, the substrate elimination rate was confirmed by thin layer chromatography to confirm that the substrate had completely disappeared. The precipitate collected by centrifuging the culture broth at 5,000 to 10,000 rpm was washed three times with distilled water and then centrifuged to obtain a precipitate. Ethanol (200 ml) was added to the precipitate and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.52 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

  [5-6] 10 g of the green tea seed extract obtained in Example 1 above was dissolved in 100 ml of ionic water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then cultivated Lactobacillus plantarum ( Lactobacillus plantarum) is inoculated with 5-10% of the liquid amount and cultured at 27 ° C. for 2 days, and then the substrate elimination rate is confirmed by thin layer chromatography to confirm that the substrate has completely disappeared. The precipitate collected by centrifuging the liquid at 5,000 to 10,000 rpm was washed three times with distilled water, and then centrifuged to obtain a precipitate. Ethanol (200 ml) was added to the precipitate and stirred three times, and then the precipitate was removed by filtration. The filtered filtrate was concentrated under reduced pressure to obtain a crude product, and then the obtained crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to obtain 0.42 g of a crude product. 21-O-Angeloylteaasapogenol E3 was obtained.

[Test Example 1] Measurement of collagen biosynthesis promotion effect The collagen biosynthesis promotion effect of 21-O-angeloylteasapogenol E3 obtained from Examples 2 to 5 was measured in comparison with tocopherol and EGCG.

First, human fibroblasts (fibroblast) (Puromoseru Co., Germany) were cultured to each 10 ⁵ seeded (seeding) growing about 90 percent to 1 per well in a 24 well (well). This was cultured in serum-free Dulbecco's modified Eagle medium (DMEM) for 24 hours, and then the 21-O-angeloylteasapogenol E3, tocopherol and EGCG of Examples 2 to 5 dissolved in serum-free medium were each 10 ^−. Treated at a ⁴ molar concentration and cultured in a CO ₂ incubator for 24 hours. These supernatants were scooped up and examined for increase or decrease in procollagen using a procollagen type (I) ELISA kit, and the results are shown in Table 1 below. Here, the synthetic ability is a comparison with the untreated group as 100.

  As apparent from Table 1, it is confirmed that 21-O-angeloylteasapogenol E3 obtained in Examples 2 to 5 effectively increases the biosynthesis of collagen.

[Test Example 2] Measurement of Collagenase Expression Inhibitory Effect The 21-O-angeloylteasapogenol E3 obtained in Examples 2-5 was compared with tocopherol and EGCG in terms of the collagenase expression inhibitory effect (production inhibition ability). It measured by the expression level of collagenase as follows.

First, 5 human fibroblasts were placed in a 96-well microtiter plate containing Dulbecco's Modified Eagle's Medium (DMEM) containing 2.5% fetal bovine serum (Dulbecco's Modified Eagle's Media). The cells were cultured at about 2,000 cells / well and grown to about 90%. Next, after culturing in serum-free Dulbecco's modified Eagle's medium (DMEM) for 24 hours, the 21-O-angeloyltea sapogen of Examples 2-5 dissolved in serum-free Dulbecco's modified Eagle's medium (DMEM) as a test substance. After treatment of nor E3, tocopherol and EGCG (positive control group) at a concentration of 10 ⁻⁴ mol for 24 hours, cell culture medium was collected.

  Next, the degree of collagenase production was measured using a commercially available collagenase measuring instrument (Amersham Pharmacia Biotech, USA).

  First, the collected cell culture solution was put into a 96-well plate uniformly coated with the primary collagenase antibody, and the antigen-antibody reaction was performed in a thermostatic bath for 3 hours. After 3 hours, the secondary collagen antibody to which the chromophore was bound was placed in a 96-well plate and allowed to react for an additional 15 minutes. After 15 minutes, when a color-inducing substance is added and color development is induced at room temperature for 15 minutes, and further 1M sulfuric acid is added to stop the reaction (color development), the hue of the reaction solution becomes yellow, and the yellowness depends on the progress of the reaction. Come different.

  The absorbance of a yellowish 96-well plate (96-well plate) was measured at 405 nm using an absorptiometer, and the expression level of collagenase was calculated according to the following formula 1. At this time, the reaction absorbance of the cell culture solution collected from the group not treated with the test substance was set to the absorbance of the control group. The expression level of collagenase is shown in Table 2 below, which is a comparison with the expression level of collagenase in the untreated group as 100.

[Formula 1]
Collagenase expression level (%) = A / B × 100
A: Absorbance of the test substance-treated cell group B: Absorbance of the control group

  As is apparent from Table 2, when the 21-O-angeloylteasapogenol E3 obtained in Examples 2 to 5 is treated, the expression level of collagenase is known to suppress the expression of collagenase. It is lower than the existing tocopherol and shows the same level of collagenase expression as EGCG. For this reason, it turns out that 21-O-angeloyl theasapogenol E3 effectively suppresses the expression of collagenase.

Test Example 3 Measurement of Elastase Expression Inhibitory Efficacy The elastase expression inhibitory effect (production inhibition ability) of 21-O-angeloylteasapogenol E3 obtained in Examples 2 to 5 was compared with tocopherol and EGCG. It measured by the expression level of elastase as follows.

First, the test was performed on human fibroblasts in a 96-well microtiter plate containing Dulbecco's Modified Eagle's Media containing 2.5% fetal bovine serum. Cells were added at 5,000 cells / well and cultured until approximately 90% growth. Subsequently, the 21-O-angeloylteasapogenol E3, tocopherol and EGCG (positive control group) of Examples 2 to 5 were cultured in a serum-free medium for 24 hours and dissolved in the serum-free medium as test substances. After treatment at ^-4 molar concentration for 24 hours, the cell culture was harvested.

  Next, the degree of elastase production was measured using a commercially available elastase measuring instrument (Amersham Pharmacia Biotech, USA).

  First, the collected cell culture solution was put into a 96-well plate (96-well plate) uniformly coated with the primary elastase antibody, and the antigen-antibody reaction was performed in a thermostatic bath for 3 hours. After 3 hours, the secondary elastin antibody to which the chromophore was bound was placed in a 96-well plate (96-well plate) and allowed to react for another 15 minutes. After 15 minutes, when a color-inducing substance is added and color development is induced at room temperature for 15 minutes, and further 1M sulfuric acid is added to stop the reaction (color development), the hue of the reaction solution becomes yellow, and the yellowness depends on the progress of the reaction. Come different.

  The absorbance of a 96-well plate with a yellowish color was measured at 405 nm using an absorptiometer, and the expression level of elastase was calculated according to the following formula 2. At this time, the reaction absorbance of the cell culture solution collected from the group not treated with the test substance was set to the absorbance of the control group. The expression level of elastase is shown in Table 3 below, which is a comparison with the expression level of elastase in the untreated group being 100.

[Formula 2]
Elastase expression level (%) = A / B × 100
A: Absorbance of the test substance-treated cell group B: Absorbance of the control group

  As is apparent from Table 3, when 21-O-angeloylteasapogenol E3 obtained in Examples 2 to 5 is treated, the expression level of elastase is known to suppress the expression of elastase. This indicates that 21-O-angeloylteasapogenol E3 effectively suppresses the expression of elastase, which is lower than that of tocopherol and EGCG.

[Test Example 4] Confirmation of skin elasticity improvement effect In order to confirm the skin elasticity improvement effect of 21-O-angeloylteasapogenol E3, a cosmetic composition having the components and contents shown in Table 4 below was used. The lotions of dosage form example 1 and comparative example 1 were produced.

  In order to compare the skin elasticity improvement effect of the lotions of the dosage form example 1 and the comparative example 1, the skin elasticity improvement degree was measured for 20 women in their 30s and 40s. The experimental method is as follows.

  First, the skin lotion of the dosage form example 1 and the comparative example 1 was provided to the subject, and a certain amount was applied to the corner of the eye for 12 weeks once a day. At this time, the lotion of the formulation example 1 was applied to the left corner of the subject and the lotion of the comparative example 1 was applied to the right corner of the subject. Skin elasticity of a subject using a cutometer (cutometer; SEM474, manufactured by College & Kazaka Electronic Co., Germany) before starting the application of the skin lotion and 12 weeks after finishing the application Was measured and the average was obtained. The experimental results are shown in Table 5 below.

  As apparent from Table 5, the skin elasticity improvement degree when using the cosmetic composition of Formulation Example 1 containing 21-O-angeloylteasapogenol E3 is the same as that of Comparative Example 1. It was confirmed that it was more than 3 times higher than that of the case. Therefore, it was confirmed that the cosmetic composition containing 21-O-angeloylteasapogenol E3 of the present invention is very effective for improving skin elasticity.

Claims (3)

A cosmetic composition for external use for skin wrinkle improvement and skin elasticity improvement containing 21-O-angeloylteasapogenol E3 represented by the following chemical formula 1 as an active ingredient.

The skin external makeup for skin wrinkle improvement and skin elasticity improvement according to claim 1, wherein the 21-O-angeloylteasapogenol E3 is contained in an amount of 0.000001 to 5% by weight based on the total weight of the composition. charge composition. Use of an external skin cosmetic composition containing 21-O-angeloylteasapogenol E3 represented by the following chemical formula 1 as an active ingredient for improving skin wrinkles and skin elasticity .