WO2015005512A1

WO2015005512A1 - Composition for antioxidation or skin whitening containing 21-o-angeloyltheasapogenol e3 ingredient derived from green tea seed

Info

Publication number: WO2015005512A1
Application number: PCT/KR2013/006209
Authority: WO
Inventors: 박준성; 심진섭; 황경환; 강영규; 박준호; 염명훈; 조준철
Original assignee: 주식회사 아모레퍼시픽
Priority date: 2013-07-11
Filing date: 2013-07-11
Publication date: 2015-01-15
Also published as: CN105377263B; JP6255493B2; CN105377263A; JP2016528204A

Abstract

The present invention relates to a composition for antioxidation or skin whitening containing, as an active ingredient, an 21-O-angeloyltheasapogenol E3 ingredient derived from a green tea seed, and more specifically, to a composition containing, as an active ingredient, a 21-O-angeloyltheasapogenol E3 ingredient derived from a green tea seed and is obtained from an extract of a seed having distinguishing characteristics through decomposition using an acid, a base, an enzyme, or a microorganism producing the enzyme, and thereby having excellent antioxidation and skin-whitening effects.

Description

Antioxidant or skin whitening composition containing 21-O-angeloyl deasapogenol E3 component derived from green tea seed

The present invention relates to an antioxidant or skin whitening composition containing 21-O-angeloyl deasapogenol E3 component derived from green tea seed as an active ingredient, and more specifically, to produce an acid, a base, an enzyme, or the enzyme. The present invention relates to a composition having excellent antioxidant and skin whitening efficacy by containing 21-O-angeloyl deaspazolol E3 component obtained from green tea seed extract through a decomposition reaction using microorganisms as an active ingredient.

Human skin is changed by a number of internal and external factors as it ages. That is, internally, the secretion of various hormones that regulate metabolism decreases, and the function of immune cells and the activity of cells decreases, thereby reducing the biosynthesis of immune proteins and constituent proteins necessary for living organisms. Due to the increase in the amount of ultraviolet rays that reach the surface of the sun's rays and further increase environmental pollution, the free radicals and active harmful oxygen increases, causing various problems on the skin. Superoxide anion radical _{^{(superoxide anion radical, O 2 -}} ) by the oxygen was present in a stable state such as enzyme, reduced metabolism, chemical, environmental, and biochemical factors such as pollutants, photochemical reaction, hydroxyl radicals (hydroxyl radical , OH˙), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen (single oxygen, ¹ O ₂ ), etc. When converted to reactive oxygen species (ROS) Proteins, lipids and DNA can be irreversibly destroyed. The action of free radicals are antioxidant enzymes such as superoxide dismutase (SOD), catalase, peroxidase, glutathione, and vitamin C (vitamin C, ascorbic). acid), vitamin E (tocopherol) can be minimized by the action of antioxidants. However, in the event of abnormal biological defenses or exposure to excessive free radicals, residual or excess free radicals cause lethal oxygen toxicity to the living body, and are non-selective and irreversible to cellular components such as lipids, proteins, sugars, and DNA. It is known to cause various diseases such as aging, cancer, stroke, brain disease such as Parkinson's disease, heart disease, ischemia, arteriosclerosis, skin disease, gastrointestinal disease, inflammation, rheumatism, autoimmune disease, and so on. (Cross et al., Ann. Intern. Med., 1987, 107, 526; Alderson et al., Proc. Natl. Acad. Sci. USA, 1988,85, 2706).

Many natural sources have been studied for the development of natural antioxidants. Most of the natural sources have been used in the form of simple extracts. It is a fact that it is used for cosmetics etc by word of mouth.

On the other hand, a number of factors are involved in determining the skin color of the human, including the activity of melanocytes (melanocytes) that make melanin pigment, the distribution of blood vessels, the thickness of the skin and the presence or absence of pigments, such as carotenoids, bilirubin, etc. Factors are important.

Especially, the most important factor is a black pigment called melanin which is produced by the action of various enzymes such as tyrosinase in melanocytes in the human body. The formation of this melanin pigment is influenced by genetic factors, physiological factors related to hormone secretion, stress, and environmental factors such as ultraviolet irradiation.

Melanin pigment, which is produced from melanocytes of the skin of the body, is a phenolic polymer material having a complex form of black pigment and protein. It protects skin organs below the dermis by blocking ultraviolet rays from the sun and free radicals generated in the skin body. It plays a useful role in protecting proteins and genes in the skin.

As described above, melanin produced by stress stimulation inside and outside the skin is a stable substance that does not disappear until it is discharged to the outside through skin keratinization even if the stress disappears. However, if more melanin is produced than necessary, it causes hyperpigmentation such as blemishes, freckles, and spots, resulting in cosmetically bad results.

In addition, as the leisure population increases, the number of people who enjoy being active outside increases the demand to prevent melanin pigmentation caused by ultraviolet rays.

In response to such demands, ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione or derivatives thereof, or substances having tyrosinase inhibitory activity have been used in combination with cosmetics or medicines. Its use is limited due to insufficient whitening effect, safety problems on skin, formulation and stability problems when formulated in cosmetics, and the like.

Thus, the present inventors have solved the above problems, and researched to find a material that exhibits an excellent antioxidant and skin lightening effect, the result obtained by the decomposition reaction using an acid, a base, an enzyme or a microorganism producing the enzyme from the green tea seed extract The present invention was completed by confirming that 21-O-angeloyl deasapogenol E3 component has excellent antioxidant and skin lightening effect.

Accordingly, an object of the present invention is to provide an antioxidant or skin whitening composition containing 21-O-angeloyl deaspazolol E3 component obtained from green tea seed extract.

In order to solve the above object, the present invention provides a topical skin composition for antioxidant containing 21-O-angeloyl de asafogenol E3 as an active ingredient.

In addition, the present invention provides a skin external preparation composition for skin whitening containing 21-O-angeloyl deasapogenol E3 as an active ingredient.

21-O-angeloyl deasapogenol E3 according to the present invention is effective in inhibiting melanin production and improving pigmentation produced by ultraviolet rays (UV) together with antioxidant effects such as DPPH and ROS production inhibition. Due to the excellent skin whitening effect, 21-O-angeloyl de asafogenol E3 according to the present invention can be very useful as an external skin composition or food composition such as antioxidant, skin whitening cosmetic composition or pharmaceutical composition. have.

The present invention provides a composition containing 21-O-angeloyl deasapogenol E3 (21-O-angeloyltheasapogenol E3) represented by Chemical Formula 1 as an active ingredient.

Formula 1

Said 21-O-angeloyl deasapogenol E3 is preferably derived from green tea seed.

21-O-angeloyl de asapogenol E3 used in the present invention is a first step of obtaining a crude saponin extract from a plant using water or an organic solvent; And a second step of hydrolyzing the extract using an acid, a base, an enzyme, or a microorganism producing the enzyme to separate 21-O-angeloyl deaspazogenol E3.

First Step: Obtaining the Saponin Crude Extract

A crude saponin extract is obtained from plants, especially green tea seeds. At this time, water or an organic solvent may be used as the extraction solvent, and at least one organic solvent selected from the group consisting of ethanol, methanol, butanol, ether, ethyl acetate and chloroform or a mixture thereof and water, preferably 50 % Ethanol can be used.

In order to obtain a crude saponin extract from the 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 by stirring 1 to 5 times at room temperature to degrease. Let's do it. About 1 to 8 times, preferably about 4 times, water or an organic solvent is added to the degreased plant, and the mixture is extracted at reflux 1 to 5 times and then deposited at 10 to 20 ° C. for 1 to 3 days. Then, the residue and the filtrate are separated by filtration and centrifugation, and the extract obtained by concentrating the separated filtrate under reduced pressure is suspended in water, and then the dye is removed using ether or the like. The aqueous layer was extracted 1-5 times with an organic solvent, and the obtained organic solvent layer was concentrated under reduced pressure to obtain an organic solvent extract, which was then dissolved in a small amount of methanol, ethanol, propanol, butanol, and the like. Subsequently, a large amount of ethyl acetate, acetonitrile and the like can be added to dry the resulting precipitate to obtain the saponin crude extract of the present invention.

Second Step: Separation of 21-O-angeloyl deasapogenol E3

The 21-O-angeloyl deaspazolol E3 may be separated by hydrolysis of the crude saponin extract obtained in the first step, and hydrolysis may be performed using an acid, a base, an enzyme, or a microorganism producing the enzyme. have.

The acid includes at least one acid selected from the group consisting of hydrochloric acid, sulfuric acid and nitric acid, or a mixed solvent of these acids with at least one alcohol selected from the group consisting of ethanol, methanol and butanol, preferably 50% ethanol Solvents may be used.

When hydrolysis is carried out using an acid, after adding 0.1-2N, preferably 1N acid, or a mixed solvent of acid and alcohol to the saponin crude extract in an amount of 1-10%, 50-100 ° C, preferably Is heated to reflux for 0.5 to 8 hours, preferably 1 hour in an 80 ℃ water bath.

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

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

The enzyme is characterized by producing a 21-O-angeloyl de asafogenol E3 by removing the sugar portion of the green tea saponin as an enzyme that breaks down the sugar bond. This enzyme can be used commercially available or can be prepared and used as needed, in particular from the microorganisms producing the enzyme.

In addition, the enzyme is more preferably glucosidase, arabinosidase, rhamnosidase, xylosidase, cellulase, hesperidinase. (hesperidinase), naringinase, glucuronidase, glucuronidase, pectinase, galactosidase, and amyloglucosidase The above can be used.

In addition, the microorganisms producing the enzyme include Aspergillus genus, Bacillus genus, Penicillium genus, Rhizopus genus, Rhizomucor genus, Talarom Genus talomyces, bifidobacterium, genus mortierella, genus cryptoococcus, microbacterium, genus leuconostoc and lactobacillus One or more selected from the group consisting of) may be used.

When hydrolysis is performed using an enzyme, the crude saponin extract is dissolved in an acid buffer solution of 5 to 20 times, preferably about 10 times, and then the enzyme is added in an amount of 0.001 to 10%. While stirring this for about 40 to 55 hours, preferably about 48 hours in a water bath of about 25 ~ 37 ℃, by confirming the scavenging rate of the substrate by thin layer chromatography, if the substrate is completely lost, 5 to 5 in hot water (80 ~ 100 ℃) The reaction is terminated by heating for 15 minutes.

When hydrolysis is performed using microorganisms, the saponin crude extract is dissolved in 5 to 10 times, preferably about 10 times, ionized water, and then sterilized at 121 ° C for 30 minutes and cooled to 30 ° C. Then, inoculated with 5 to 10% by weight of the microorganisms incubated in advance, incubated for 2 to 5 days, preferably 5 days at 20 ~ 30 ℃, and then confirmed the scavenging rate of the substrate by thin layer chromatography When the substrate is completely lost, the precipitate obtained by centrifugation of the culture solution at 5,000 to 10,000 rpm is washed three times with distilled water, followed by centrifugation to obtain a precipitate.

After hydrolysis using an acid, a base, an enzyme, or a microorganism producing the enzyme as described above, the reaction solution is concentrated under reduced pressure to remove the solvent, and alcohol is added to the residue and stirred 1 to 5 times. Then, the precipitated salts were removed through filtration, and the filtrate was concentrated under reduced pressure to give a crude product, and the crude product obtained was separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1). 21-O-angeloyl deasapogenol E3 can be obtained.

The composition of the present invention contains 21-O-angeloyl deaspazolol E3, which can be prepared by the above method, in an amount of 0.0001 to 10% by weight based on the total weight of the composition. If the content is less than 0.0001% by weight, the antioxidant or skin lightening effect by the above-mentioned ingredient cannot be obtained, and even if the content exceeds 10% by weight, the increase in effect is not large compared to the increase in content.

The composition of the present invention can be used as an antioxidant composition and is excellent in inhibiting DPPH and ROS production.

In addition, the composition of the present invention can be used as a composition for skin whitening, inhibits melanin production, and is excellent in improving pigmentation produced by ultraviolet rays (UV).

The compositions of the present invention may be formulated into cosmetic or pharmaceutical compositions containing a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) and non-obtained by dispersing an oil phase in solution, gels, solids, pasty anhydrous products, aqueous phases. It may be provided in the form of an ionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. It may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. These compositions can be prepared according to conventional methods in the art.

In addition, the composition of the present invention is a fatty substance, organic solvent, solubilizer, thickening agent, gelling agent, softener, antioxidant, suspending agent, stabilizer, foaming agent, fragrance, surfactant, water, ionic or nonionic type Cosmetics such as emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredient commonly used in cosmetics Or an adjuvant commonly used in the field of dermatology. Such adjuvants are introduced in amounts generally used in the cosmetic or dermatological arts.

In addition, the composition of the present invention may contain a skin absorption promoting substance to increase the antioxidant or skin lightening effect.

In addition, the composition of the present invention may be formulated into a food composition, for example in the form of various foods, beverages, gums, tea vitamin complexes, health functional foods and various food additives and the like.

The food composition of the present invention is not particularly limited to other ingredients except for containing 21-O-angeloyl deasapogenol E3 as an essential ingredient in the indicated ratios, and various flavors or natural carbohydrates are added as in general drinks. It may contain as a component. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The ratio of the natural carbohydrate is not limited thereto, but is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

Moreover, the food composition of this invention can contain the other component etc. which can give a synergistic effect to a main effect in the range which does not impair the main effect aimed at by this invention. For example, it may further include additives such as perfumes, pigments, fungicides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers and synthetic polymer materials to improve physical properties. In addition, supplementary ingredients such as water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides and seaweed extract may be further included. The components may be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use, and the amount of the additives may be selected within a range that does not impair the object and effect of the present invention. For example, the addition amount of the components may be in the range of 0.01 to 5% by weight, more specifically 0.01 to 3% by weight relative to the total weight of the composition.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited only to these examples.

Example 1 Preparation of Green Tea Seed Extract

6 kg of hexane was added to 2 kg of green tea seeds, and extracted by stirring three times at room temperature. Then, 4 l of 50% ethanol was added to 1 kg of degreased green tea seeds, and refluxed three times, followed by immersion at 15 ° C. for 1 day. Thereafter, the residue and the filtrate were separated through filter cloth filtration and centrifugation, and the extract obtained by concentrating the separated filtrate under reduced pressure was suspended in water, extracted five times with 1 L of ether to remove the pigment, and the aqueous layer was 1-butanol. Extracted three times with 500 ml. The whole 1-butanol layer obtained therefrom was concentrated under reduced pressure to obtain 1-butanol extract, which was dissolved in a small amount of methanol, and then added to a large amount of ethyl acetate, followed by drying the resulting precipitate, thereby extracting green tea seed extract (crude saponin). 300 g were obtained.

Example 2 Preparation of 21-O-angeloyl deasapogenol E3 by Acid Hydrolysis Method

[2-1] 20 times (v / w) 1N HCl-50% methanol solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, and heated to reflux for 8 hours in an 80 ° C water bath. The sugars bound to green tea seed crude saponin were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 mL) was added to the residue, followed by stirring three times, and the precipitated salts were removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 7: 1 to 3: 1) to give 21-O-angeloyl deasapogenol E3 0.55. g was obtained.

[2-2] 20 g (v / w) of 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, followed by 8 hours in a 90 ° C water bath. By heating to reflux, the sugars bound to the green tea seed crude saponin were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 mL) was added to the residue, followed by stirring three times, and the precipitated salts were removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 7: 1 to 3: 1) to give 21-O-angeloyl deaspazolol E3 0.59. g was obtained.

[2-3] 20 g (v / w) of 1M HNO ₃ -10% aqueous solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, and heated to reflux for 8 hours in a 90 ° C water bath. The sugars bound to green tea seed crude saponin were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, ethanol (200 mL) was added to the residue, followed by stirring three times, and the precipitated salts were removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 7: 1 to 3: 1) to give 21-O-angeloyl deaspazolol E3 0.39. g was obtained.

Example 3 Preparation of 21-O-angeloyl deasapogenol E3 by Base Hydrolysis Method

[3-1] 10 g of the green tea seed extract obtained in Example 1 was dissolved in dried pyridine (500 ml), and sodium methoxide (powder, 10 g) was added thereto to reflux for 8 hours on an oil bath. The sugars bound to green tea seed saponin were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, washed three times with purified water to obtain a filtrate through filtration, and ethanol (200 mL) was added to the residue, followed by stirring three times. The precipitated salts were removed by filtration. It was. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deasapogenol E3 0.35. g was obtained.

[3-2] 20 g (v / w) 1M NaOH-20% aqueous solution (v / v) was added to 10 g of the green tea seed extract obtained in Example 1, followed by reflux reaction at 80 ° C. for 8 hours. Sugars bound to seed saponins were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, washed three times with purified water to obtain a filtrate through filtration, and ethanol (200 mL) was added to the residue, followed by stirring three times. The precipitated salts were removed by filtration. It was. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deaspazolol E3 0.31. g 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, followed by reflux reaction at 80 ° C. for 8 hours. Sugars bound to seed saponins were hydrolyzed. The reaction solution was concentrated under reduced pressure to remove the solvent, washed three times with purified water to obtain a filtrate through filtration, and ethanol (200 mL) was added to the residue, followed by stirring three times. The precipitated salts were removed by filtration. It was. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deaspazolol E3 0.25. g was obtained.

Example 4 Preparation of 21-O-angeloyl deaspazogenol E3 by enzymatic digestion

[4-1] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of 0.1 M acetic acid buffer solution (pH 4.5), and 2.5 g of enzyme (0.5 g of hesperidinase and 0.5 naringinase 0.5) was added thereto. g, cellulase 0.5 g, β-glucuronidase 0.2 g, β-galactosidase 0.5 g, amyloglucosidase 0.3 g; manufactured by Sigma) were added and stirred for 48 hours in a 37 ° C. water bath. After periodic confirmation by thin layer chromatography, when green tea saponin was lost, 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, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1-4: 1) to obtain 21-O-angeloyl deaspazolol E3 1.02. g 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 acetic acid buffer solution (pH 6.5), and 3.5 g of enzyme (1 g of glucosidase and 0.5 g of arabinosidase) was added thereto. , 1 g of rhamnosidase, 0.5 g of xylosidase, 0.5 g of pectinase, was stirred for 48 hours in a 27 ° C. water bath, periodically checked by thin layer chromatography, and hot water (80 The reaction was terminated by heating in ˜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, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was separated by silica gel column chromatography (chloroform: methanol = 8: 1-4: 1) to obtain 21-O-angeloyl deaspazolol E3 1.53. g was obtained.

Example 5 Preparation of 21-O-angeloyl deaspazogenol E3 using microorganisms

[5-1] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 30 ° C., and pre-cultured Aspergillus niger KCCM After inoculating 11885 at 5-10% of the liquid amount and incubating for 5 days at 30 ° C., the substrate was removed by thin layer chromatography to confirm that the substrate was completely lost, and the culture solution was centrifuged at 5,000 to 10,000 rpm. The recovered precipitate was washed three times with distilled water and centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deasapogenol E3 0.72. g was obtained.

[5-2] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then pre-cultivated lyzopus oryzae (rhizopus oryzae) was obtained. After inoculating 5-10% of the liquid amount and incubating for 5 days at 27 ° C., the substrate was removed by thin layer chromatography to confirm that the substrate was completely lost. The culture was recovered by centrifugation at 5,000 to 10,000 rpm. The precipitate was washed three times with distilled water and centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1-4: 1) to give 21-O-angeloyl deaspazolol E3 0.92. g was obtained.

[5-3] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then incubated with Bacillus subtilis. After inoculating 5-10% of the liquid amount and incubating for 2 days at 27 ° C., the substrate was removed by thin layer chromatography to confirm that the substrate was completely lost. The culture was recovered by centrifugation at 5,000 to 10,000 rpm. The precipitate was washed three times with distilled water and centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deasapogenol E3 0.72. g was obtained.

[5-4] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then incubated with leuconostoc mesenteroides. ) Was inoculated at 5 to 10% of the liquid amount and incubated at 27 ° C. for 2 days, and then the substrate was removed by thin layer chromatography to confirm that the substrate was completely lost. The culture solution was centrifuged at 5,000 to 10,000 rpm. The recovered precipitate was washed three times with distilled water and centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deaspazolol E3 0.52. g was obtained.

[5-5] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then incubated with Bifidobacterium longum. Was inoculated at 5 to 10% of the liquid amount and incubated at 27 ° C. for 2 days, and then the substrate was removed by thin layer chromatography to confirm that the substrate was completely lost. The culture was recovered by centrifugation at 5,000 to 10,000 rpm. One precipitate was washed three times with distilled water and centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deaspazolol E3 0.52. g was obtained.

[5-6] 10 g of the green tea seed extract obtained in Example 1 was dissolved in 100 ml of ionized water, sterilized at 121 ° C. for 30 minutes, cooled to 27 ° C., and then incubated with Lactobacillus plantarum. Was inoculated at 5 to 10% of the liquid amount and incubated at 27 ° C. for 2 days, and then the substrate was removed by thin layer chromatography to confirm that the substrate was completely lost. The culture was recovered by centrifugation at 5,000 to 10,000 rpm. One precipitate was washed three times with distilled water and centrifuged to obtain a precipitate. Ethanol (200 mL) was added to the precipitate, followed by stirring three times, and then the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to give a crude product, which was then separated by silica gel column chromatography (chloroform: methanol = 8: 1 to 4: 1) to give 21-O-angeloyl deaspazolol E3 0.42. g was obtained.

Test Example 1 DPPH Production Inhibition Test (DPPH Test)

The method of evaluating the antioxidant activity through the change of absorbance generated by the reduction of the organic radical DPPH (1,1-diphenyl-2-picryl hydrazyl) (an antioxidant is oxidized) was used. The antioxidant degree was measured by the degree that the oxidation of DPPH was inhibited and the absorbance was reduced compared to the control, and the concentration (IC ₅₀ ) showing an absorbance of 50% or less compared to the absorbance of the control was evaluated as the effective antioxidant concentration.

190 μl of 100 μM (in ethanol) DPPH solution was mixed with 10 μl of the green tea seed extract of Example 1, 21-O-angeloyl deaspazolol E3 and the control sample of Examples 2 to 5, respectively, to prepare a reaction solution. After the reaction mixture was reacted for 30 minutes at 37 ℃ absorbance was measured at 540nm. As a control sample, Trolox, which is widely used as a synthetic antioxidant, was used. DPPH analysis results for each case are shown in Table 1 below.

Table 1

sample	IC ₅₀ (ppm)
Example 1	450.24
Example 2-1	7.73
Example 3-1	7.52
Example 4-1	7.47
Example 5-1	7.53
Trolox	8.64

From the results of Table 1, it can be seen that 21-O-angeloyl de asafogenol E3 has a very excellent antioxidant capacity, and is superior to the known antioxidant Trolox.

[Test Example 2] Test for inhibiting the generation of reactive oxygen species (ROS) using fluorescent materials

The cell line used in the test was human keratinocytes HaCaT cell line (Dubeccos Modification of Eagles Medium, DMEM), which was divided into ^four 2.0x10 cells per hole in a 96-hole black plate for fluorescence measurement and added penicillin / streptomycin. The test sample was treated after incubation for 1 day at 37 ° C. and 5% CO ₂ using FBS 10%) medium. As a test sample, green tea seed extract of Example 1, 21-O-angeloyl deaspazolol E3 and control samples of Examples 2 to 5 were used each 50 ppm, and the control sample was widely used as a trolox (synthetic antioxidant). Trolox) was used. Then, using a serum-free DMEM (FBS free) to which penicillin / streptomycin was added as a medium, it was incubated for 1 day at 37 ℃, 5% CO ₂ conditions.

After the test sample was added and incubated for 24 hours, the remaining medium was removed by washing with HCSS (HEPES-buffered control salt solution) and DCFH-DA (2 ', 7'-dichlorodihydro-fluorescein diacetate, Molecular Probes prepared with 20μM in HCSS). , Inc) was added thereto, followed by incubation at 37 ° C. and 5% CO ₂ for 20 minutes and washing with HCSS. Subsequently, 100 μl of HCSS treated as a sample was added, and then, the fluorescence intensity of DCF (dichlorofluorescein) oxidized to ROS was measured by a fluorescence plate reader (Ex = 485 nm, Em = 530 nm). Thereafter, UVB (30 mJ / cm ² ) was irradiated, and the fluorescence intensity immediately after the treatment and 3 hours after the treatment was measured with a fluorescence plate reader (Ex = 485 nm, Em = 530 nm).

In the case of using DMSO as a control, the ROS production inhibitory ability of each test substance was determined based on this (% of the control), and the experimental results are shown in Table 2 below.

TABLE 2

Sample (50 ppm)	Production Inhibition (%)
Control (DMSO)	00
Example 1	25
Example 2-1	61.7
Example 3-1	61.5
Example 4-1	61.2
Example 5-1	62.1
Trolox	51.7

From the results of Table 2, it can be seen that 21-O-angeloyl de asafogenol E3 is excellent in inhibiting the ROS production, the ROS production inhibitory ability than the known antioxidant Trolox.

Test Example 3 Measurement of Melanin Inhibitory Effect Using Pigment Cells

Mel-Ab cells derived from C57BL / 6 mice (Dooley, TP et al, Skin pharmacol, 7, pp 188-200) in DMEM with 10% fetal placental serum, 100nM 2-O-tetradecanoyl Incubated at 37 ° C. and 5% CO ₂ in medium containing phorbol (tetradecanoyphorbol) -13-ate, 1 nM cholera toxin. Cultured Mel-Ab cells were detached with 0.25% trypsin-EDTA, and the cells were incubated in 24-well plates at a concentration of 10 ⁵ cells / well, 10 ppm each test sample for 2 consecutive days from day 2 Added freshly. Green tea seed extract of Example 1, 21-O-angeloyl deaspazolol E3 and the control sample of Examples 2 to 5 and a control sample were used as a test sample, and a positive sample was used as a hygroquinone, a known whitening agent. As a control, the test sample was not treated as a negative control. After addition of the test sample, the cells were incubated for 1 day at 37 ° C. and 5% CO ₂ , and then the culture solution was removed and washed with PBS. The cells were dissolved with 1N sodium hydroxide and absorbance was measured at 400 nm. The melanin production inhibition rate was calculated according to the following Equation 1, and the results are shown in Table 3 below (Dooley's method).

Equation 1

TABLE 3

Test substance	Melanin production inhibition rate (%)
Untreated group (negative control group)	00.0
Example 1	75.3
Example 2-1	75.2
Example 3-1	75.7
Example 4-1	74.9
Example 5-1	75.1
Hydroquinone (positive control)	41.1

From the results of Table 3, it can be seen that 21-O-angeloyl deasapogenol E3 shows excellent melanin production inhibition rate, and melanin production inhibition rate better than hydroquinone which is a known whitening agent.

Test Example 4 Skin Whitening Effect Test on Human Skin

In order to determine the skin whitening effect on the human skin on 21-O-angeloyl deasapogenol E3 obtained in Example 4, the following experiment was performed.

First, a opaque tape with a diameter of 1.5 cm was attached to the upper arm of 12 healthy men, and then 1.5 ~ 2 times of ultraviolet light (UVB) of each subject's minimum erythema (Minimal Erythema Dose). Was irradiated to induce skin blackening.

After ultraviolet irradiation, 1% of 21-O-angeloyl deaspazogenol E3 of Example 4-1 (solvent was 1,3-butylene glycol: ethanol = 7: 3), 1% hydroquinone, and a solvent (Negative control) 1% each was applied, and one state was observed for 10 weeks in a state where nothing was applied. Skin color was measured on a weekly basis with a colorimeter CR2002 (Minolta, Japan).

Then, the difference in skin color (ΔL *) between the start point of application and the end point of application of each test substance was calculated according to the following Equation 2, which is shown in Table 4 below. On the other hand, the whitening effect is determined by comparing ΔL * between the sample application site and the control site. When ΔL * value is about 2, the whitening effect of the deposited pigment is clear, and when about 1.5 or more, the whitening effect is determined. Can be.

Equation 2

Table 4

Test substance	Skin color brightness degree (△ L *)
Example 4-1	1.80 ± 0.21
Hydroquinone (positive control)	1.90 ± 0.11
Vehicle (negative control)	0.50 ± 0.15

From the results of Table 4, 21-O-angeloyl de asafogenol E3 brightened the skin color, it can be seen that the skin color brightness degree similar to the known whitening agent hydroquinone. This can be judged to be because 21-O-angeloyl deasapogenol E3 used in the present invention improves the pigmentation produced by ultraviolet light to brighten the skin color.

Claims

A skin external composition for antioxidant containing 21-O-angeloyl deasapogenol E3 represented by Chemical Formula 1 as an active ingredient.

[Formula 1]
A skin external preparation composition for skin whitening containing 21-O-angeloyl deasapogenol E3 represented by Chemical Formula 1 as an active ingredient.

[Formula 1]
The composition for external application for skin according to claim 1 or 2, wherein the 21-O-angeloyl deasapogenol E3 is contained in an amount of 0.0001 to 10% by weight based on the total weight of the composition.
The external preparation composition for skin according to claim 1 or 2, wherein the 21-O-angeloyl deaspazolol E3 is derived from green tea seed.
The external preparation composition for skin according to claim 4, wherein the 21-O-angeloyl deasapogenol E3 is obtained by decomposing the green tea seed extract using an acid, a base, an enzyme, or a microorganism producing the enzyme.
Use as an antioxidant for skin external composition containing 21-O-angeloyl deasapogenol E3 represented by the following formula (1) as an active ingredient.

[Formula 1]
Use as a skin whitening composition of the external preparation composition for skin containing 21-O-angeloyl deasapogenol E3 represented by the following formula (1) as an active ingredient.

[Formula 1]